Embryo-specific expression of a visual reporter gene as a selection system for citrus transformation

PubMed Central

Zambon, Flavia T.; Erpen, Lígia; Soriano, Leonardo; Grosser, Jude

2018-01-01

The embryo-specific Dc3 gene promoter driving the VvMybA1 anthocyanin regulatory gene was used to develop a visual selection system for the genetic transformation of citrus. Agrobacterium-mediated transformation of cell suspension cultures resulted in the production of purple transgenic somatic embryos that could be easily separated from the green non-transgenic embryos. The somatic embryos produced phenotypically normal plants devoid of any visual purple coloration. These results were also confirmed using protoplast transformation. There was minimal gene expression in unstressed one-year-old transgenic lines. Cold and drought stress did not have any effect on gene expression, while exogenous ABA and NaCl application resulted in a minor change in gene expression in several transgenic lines. When gas exchange was measured in intact leaves, the transgenic lines were similar to controls under the same environment. Our results provide conclusive evidence for the utilization of a plant-derived, embryo-specific visual reporter system for the genetic transformation of citrus. Such a system could aid in the development of an all-plant, consumer-friendly GM citrus tree. PMID:29293649

Transcriptional profiles of bovine in vivo pre-implantation development.

PubMed

Jiang, Zongliang; Sun, Jiangwen; Dong, Hong; Luo, Oscar; Zheng, Xinbao; Obergfell, Craig; Tang, Yong; Bi, Jinbo; O'Neill, Rachel; Ruan, Yijun; Chen, Jingbo; Tian, Xiuchun Cindy

2014-09-04

During mammalian pre-implantation embryonic development dramatic and orchestrated changes occur in gene transcription. The identification of the complete changes has not been possible until the development of the Next Generation Sequencing Technology. Here we report comprehensive transcriptome dynamics of single matured bovine oocytes and pre-implantation embryos developed in vivo. Surprisingly, more than half of the estimated 22,000 bovine genes, 11,488 to 12,729 involved in more than 100 pathways, is expressed in oocytes and early embryos. Despite the similarity in the total numbers of genes expressed across stages, the nature of the expressed genes is dramatically different. A total of 2,845 genes were differentially expressed among different stages, of which the largest change was observed between the 4- and 8-cell stages, demonstrating that the bovine embryonic genome is activated at this transition. Additionally, 774 genes were identified as only expressed/highly enriched in particular stages of development, suggesting their stage-specific roles in embryogenesis. Using weighted gene co-expression network analysis, we found 12 stage-specific modules of co-expressed genes that can be used to represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the bovine expressed gene networks. Their vast association with other embryonic genes suggests that they may have important regulatory roles in embryo development; yet, the majority of the hub genes are relatively unknown/under-studied in embryos. We also conducted the first comparison of embryonic expression profiles across three mammalian species, human, mouse and bovine, for which RNA-seq data are available. We found that the three species share more maternally deposited genes than embryonic genome activated genes. More importantly, there are more similarities in embryonic transcriptomes between bovine and humans than between humans and mice, demonstrating that bovine embryos are better models for human embryonic development. This study provides a comprehensive examination of gene activities in bovine embryos and identified little-known potential master regulators of pre-implantation development.

Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

PubMed Central

Baurand, Pierre-Emmanuel; Pedrini-Martha, Veronika; de Vaufleury, Annette; Niederwanger, Michael; Capelli, Nicolas; Scheifler, Renaud; Dallinger, Reinhard

2015-01-01

The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene. PMID:25706953

Trichostatin A specifically improves the aberrant expression of transcription factor genes in embryos produced by somatic cell nuclear transfer

PubMed Central

Inoue, Kimiko; Oikawa, Mami; Kamimura, Satoshi; Ogonuki, Narumi; Nakamura, Toshinobu; Nakano, Toru; Abe, Kuniya; Ogura, Atsuo

2015-01-01

Although mammalian cloning by somatic cell nuclear transfer (SCNT) has been established in various species, the low developmental efficiency has hampered its practical applications. Treatment of SCNT-derived embryos with histone deacetylase (HDAC) inhibitors can improve their development, but the underlying mechanism is still unclear. To address this question, we analysed gene expression profiles of SCNT-derived 2-cell mouse embryos treated with trichostatin A (TSA), a potent HDAC inhibitor that is best used for mouse cloning. Unexpectedly, TSA had no effect on the numbers of aberrantly expressed genes or the overall gene expression pattern in the embryos. However, in-depth investigation by gene ontology and functional analyses revealed that TSA treatment specifically improved the expression of a small subset of genes encoding transcription factors and their regulatory factors, suggesting their positive involvement in de novo RNA synthesis. Indeed, introduction of one of such transcription factors, Spi-C, into the embryos at least partially mimicked the TSA-induced improvement in embryonic development by activating gene networks associated with transcriptional regulation. Thus, the effects of TSA treatment on embryonic gene expression did not seem to be stochastic, but more specific than expected, targeting genes that direct development and trigger zygotic genome activation at the 2-cell stage. PMID:25974394

Effect of Embryo Density on In Vitro Development and Gene Expression in Bovine In Vitro-fertilized Embryos Cultured in a Microwell System

PubMed Central

SUGIMURA, Satoshi; AKAI, Tomonori; HASHIYADA, Yutaka; AIKAWA, Yoshio; OHTAKE, Masaki; MATSUDA, Hideo; KOBAYASHI, Shuji; KOBAYASHI, Eiji; KONISHI, Kazuyuki; IMAI, Kei

2012-01-01

Abstract To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 µl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density. PMID:23154384

Characterization of embryo-specific genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, Z.R.

1988-01-01

The objective of the proposed research is to characterize the structure and function of a set of genes whose expression is regulated in embryo development, and that are not expressed in mature tissues -- the embryogenic genes. In order to isolate these genes, we immunized a rabbit with total extracts of somatic embryos of carrot, and enriched the anti-embryo antiserum for antibodies reacting with extracts of carrot somatic embryos. Using this enriched antiserum, we screened a lambda gt11 cDNA library constructed from embryo poly A{sup +} RNA, and isolated 10 cDNA clones that detect embryogenic mRNAs. Monospecific antibodies have beenmore » purified for proteins corresponding to each cDNA sequence. Four cDNA clones were further characterized in terms of the expression of their corresponding mRNA and protein in somatic embryos of carrot. In some cases, comparable gene sequences or products have been detected in somatic and zygotic embryos of other plant species. The characteristics of these 4 cDNA clones -- clone Nos. 8, 59, and 66 -- are described in this report. 3 figs.« less

High-resolution gene expression data from blastoderm embryos of the scuttle fly Megaselia abdita

PubMed Central

Wotton, Karl R; Jiménez-Guri, Eva; Crombach, Anton; Cicin-Sain, Damjan; Jaeger, Johannes

2015-01-01

Gap genes are involved in segment determination during early development in dipteran insects (flies, midges, and mosquitoes). We carried out a systematic quantitative comparative analysis of the gap gene network across different dipteran species. Our work provides mechanistic insights into the evolution of this pattern-forming network. As a central component of our project, we created a high-resolution quantitative spatio-temporal data set of gap and maternal co-ordinate gene expression in the blastoderm embryo of the non-drosophilid scuttle fly, Megaselia abdita. Our data include expression patterns in both wild-type and RNAi-treated embryos. The data—covering 10 genes, 10 time points, and over 1,000 individual embryos—consist of original embryo images, quantified expression profiles, extracted positions of expression boundaries, and integrated expression patterns, plus metadata and intermediate processing steps. These data provide a valuable resource for researchers interested in the comparative study of gene regulatory networks and pattern formation, an essential step towards a more quantitative and mechanistic understanding of developmental evolution. PMID:25977812

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

PubMed

Aberkane, A; Essahib, W; Spits, C; De Paepe, C; Sermon, K; Adriaenssens, T; Mackens, S; Tournaye, H; Brosens, J J; Van de, Velde H

2018-05-26

What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in-vitro implantation model. Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. Six-days post fertilisation (6dpf) human embryos were co-cultured with Ishikawa cells for 12 h, 24 h (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 115 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV, and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expression was validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. None. This in-vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. A.A. was supported by a grant from the "Instituut voor Innovatie door Wetenschap en Technologie" (IWT, 121716, Flanders, Belgium). This work was supported by the "Wetenschappelijk Fonds Willy Gepts" (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

Pre-gastrula expression of zebrafish extraembryonic genes

PubMed Central

2010-01-01

Background Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL). Results We hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent. Conclusion We have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning. PMID:20423468

Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media.

PubMed

Kleijkers, Sander H M; Eijssen, Lars M T; Coonen, Edith; Derhaag, Josien G; Mantikou, Eleni; Jonker, Martijs J; Mastenbroek, Sebastiaan; Repping, Sjoerd; Evers, Johannes L H; Dumoulin, John C M; van Montfoort, Aafke P A

2015-10-01

Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment? Six days of in vitro culture of human preimplantation embryos resulted in medium-dependent differences in expression level of genes involved in apoptosis, protein degradation, metabolism and cell-cycle regulation. Several human studies have shown an effect of culture medium on embryo development, pregnancy outcome and birthweight. However, the underlying mechanisms in human embryos are still unknown. In animal models of human development, it has been demonstrated that culture of preimplantation embryos in vitro affects gene expression. In humans, it has been found that culture medium affects gene expression of cryopreserved embryos that, after thawing, were cultured in two different media for 2 more days. In a multicenter trial, women were randomly assigned to two culture medium groups [G5 and human tubal fluid (HTF)]. Data on embryonic development were collected for all embryos. In one center, embryos originating from two pronuclei (2PN) zygotes that were not selected for transfer or cryopreservation on Day 2 or 3 because of lower morphological quality, were cultured until Day 6 and used in this study, if couples consented. Ten blastocysts each from the G5 and HTF study groups, matched for fertilization method, maternal age and blastocyst quality, were selected and their mRNA was isolated and amplified. Embryos were examined individually for genome-wide gene expression using Agilent microarrays and PathVisio was used to identify the pathways that showed a culture medium-dependent activity. Expression of 951 genes differed significantly (P < 0.01) between the G5 and HTF groups. Eighteen pathways, involved in apoptosis, metabolism, protein processing and cell-cycle regulation, showed a significant overrepresentation of differentially expressed genes. The DNA replication, G1 to S cell-cycle control and oxidative phosphorylation pathways were up-regulated in the G5 group compared with the HTF group. This is in agreement with the morphological assessment of the 1527 embryos (originating from 2PN zygotes), which showed that embryos consisted of more cells on Day 2 (3.73 ± 1.30 versus 3.40 ± 1.35, P < 0.001) and Day 3 (7.00 ± 2.41 versus 5.84 ± 2.36, P < 0.001) in the G5 group when compared with the HTF group. Furthermore, the implantation rate was significantly higher in the G5 group compared with the HTF group (26.7% versus 14.7%, P = 0.002) after transfer on the second or the third day after fertilization. Despite careful matching of the embryos, it cannot be excluded that the differences observed between the study groups are caused by factors that we did not investigate. Extrapolation of these results to embryos used for transfer demands caution as in the present study embryos that were not selected for either embryo transfer or cryopreservation have been used for the culture experiment until Day 6. This study shows that gene expression in human preimplantation embryos is altered by the culture medium used during IVF treatment and provides insight into the biological pathways that are affected. Whether these changes in gene expression have any long-term effects on children born after IVF remains unknown. However, it is possible that early adaptations of the preimplantation embryo to its environment persist during fetal and post-natal development. No funding and no competing interests declared. Not applicable. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Early bovine embryos regulate oviduct epithelial cell gene expression during in vitro co-culture.

PubMed

Schmaltz-Panneau, Barbara; Cordova, Amanda; Dhorne-Pollet, Sophie; Hennequet-Antier, Christelle; Uzbekova, Sveltlana; Martinot, Emmanuelle; Doret, Sarah; Martin, Patrice; Mermillod, Pascal; Locatelli, Yann

2014-10-01

In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

Reduced O2 concentration during CAM development--its effect on angiogenesis and gene expression in the broiler embryo CAM.

PubMed

Druyan, S; Levi, E

2012-01-01

Hypoxia during embryogenesis may induce changes in the development of some physiological regulatory systems, thereby causing permanent phenotypic changes in the embryo. Various levels of hypoxia at different time points during embryogenesis were found to affect both anatomical and physiological morphogenesis. These changes and adaptations depended on the timing, intensity, and duration of the hypoxic exposure and, moreover, were regulated by differential expression of developmentally important genes, mostly expressed in a stage- and time-dependent manner. Eggs incubated in a 17%-oxygen atmosphere for 12 h/d from E5 through E12 exhibited a clear and significant increase in the vascular area of the chorioallantoic membrane (CAM); an increase that was already significant within 12 h after the end of the 1st hypoxic exposures (E6). We used the combination of the genes, β-actin, RPLP0 and HPRT as a reference for gene expression profiling, in studying the expression levels of hypoxia-inducible factor 1-alpha (HIF1α), vascular endothelial growth factor alpha-2 (VEGF α 2), vascular endothelial growth factor receptor 2 (KDR), matrix metalloproteinase-2 (MMP2), and fibroblast growth factor 2 (FGF2), under normal and hypoxic conditions. In general, expression of all five investigated genes throughout the embryonic day of development had similar patterns of hypoxia-induced alterations. In E5.5 embryos, expression of HIF1α, MMP2, VEGFα2, and KDR was significantly higher in hypoxic embryos than in controls. In E6 embryos expression of HIF1α, VEGFα2, and FGF2 was significantly higher in hypoxic embryos than in controls. From E6.5 onward expression levels of the examined genes did not show any differences between hypoxic and control embryos. It can be concluded that in this experimental model, exposing broiler embryos to 17% O(2) from E5 to E7 induced significant angiogenesis, as expressed by the above genes. Further studies to examine whether this early exposure to hypoxic condition affects the chick's ability to withstand a post-hatch hypoxic environment is still required. Copyright © 2012 Elsevier B.V. All rights reserved.

Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis.

PubMed

Liu, Na; Enkemann, Steven A; Liang, Ping; Hersmus, Remko; Zanazzi, Claudia; Huang, Junjiu; Wu, Chao; Chen, Zhisheng; Looijenga, Leendert H J; Keefe, David L; Liu, Lin

2010-12-01

Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.

A mathematical model of in vivo bovine blastocyst developmental to gestational Day 15.

PubMed

Shorten, P R; Donnison, M; McDonald, R M; Meier, S; Ledgard, A M; Berg, D

2018-06-20

Bovine embryo growth involves a complex interaction between the developing embryo and the growth-promoting potential of the uterine environment. We have previously established links between embryonic factors (embryo stage, embryo gene expression), maternal factors (progesterone, body condition score), and embryonic growth to 8 d after bulk transfer of Day 7 in vitro-produced blastocysts. In this study we recovered blastocysts on Days 7 and 15 after artificial insemination to test the hypothesis that in vivo and in vitro embryos follow a similar growth program. We conducted our study using 4 commercial farms and repeated our study over 2 yr (2014, 2015), with data available from 2 of the 4 farms in the second year. Morphological and gene expression measurements (196 candidate genes) of the Day 7 embryos were measured and the progesterone concentration of the cows were measured throughout the reproductive cycle as a reflection of the state of the uterine environment. These data were also used to assess the interaction between the uterine environment and the developing embryo and to examine how well Day 7 embryo stage can be predicted from the Day 7 gene expression profile. Progesterone was not a strong predictor of in vivo embryo growth to Day 15. This contrasts with a range of Day 7 embryo transfer studies which demonstrated that progesterone is a very good predictor of embryo growth to Day 15. Our analysis demonstrates that in vivo embryos are 3 times less sensitive to progesterone than in vitro-transferred embryos (up to Day 15). This highlights that caution must be applied when extrapolating the results of in vitro embryo transfer studies to the in vivo situation. The similar variance in measured and predicted (based on Day 15 length) Day 7 embryo stage indicate low stochastic perturbations for in vivo embryo growth (large stochastic growth effects would generate a significantly larger standard deviation in measured embryo length on Day 15). We also identified that Day 7 embryo stage could be predicted based on the Day 7 gene expression profile (58% overall success rate for classification of 5 embryo stages). Our analysis also associated genes with each developmental stage and demonstrates the high level of temporal regulation of genes that occurs during early embryonic development. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The maternal genes Ci-p53/p73-a and Ci-p53/p73-b regulate zygotic ZicL expression and notochord differentiation in Ciona intestinalis embryos.

PubMed

Noda, Takeshi

2011-12-01

I isolated a Ciona intestinalis homolog of p53, Ci-p53/p73-a, in a microarray screen of rapidly degraded maternal mRNA by comparing the transcriptomes of unfertilized eggs and 32-cell stage embryos. Higher expression of the gene in eggs and lower expression in later embryonic stages were confirmed by whole-mount in situ hybridization (WISH) and quantitative reverse transcription-PCR (qRT-PCR); expression was ubiquitous in eggs and early embryos. Knockdown of Ci-p53/p73-a by injection of antisense morpholino oligonucleotides (MOs) severely perturbed gastrulation cell movements and expression of notochord marker genes. A key regulator of notochord differentiation in Ciona embryos is Brachyury (Ci-Bra), which is directly activated by a zic-like gene (Ci-ZicL). The expression of Ci-ZicL and Ci-Bra in A-line notochord precursors was downregulated in Ci-p53/p73-a knockdown embryos. Maternal expression of Ci-p53/p73-b, a homolog of Ci-p53/p73-a, was also detected. In Ci-p53/p73-b knockdown embryos, gastrulation cell movements, expression of Ci-ZicL and Ci-Bra in A-line notochord precursors, and expression of notochord marker gene at later stages were perturbed. The upstream region of Ci-ZicL contains putative p53-binding sites. Cis-regulatory analysis of Ci-ZicL showed that these sites are involved in expression of Ci-ZicL in A-line notochord precursors at the 32-cell and early gastrula stages. These results suggest that p53 genes are maternal factors that play a crucial role in A-line notochord differentiation in C. intestinalis embryos by regulating Ci-ZicL expression. Copyright © 2011 Elsevier Inc. All rights reserved.

RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

PubMed

Zhu, Hai-Ying; Jin, Long; Guo, Qing; Luo, Zhao-Bo; Li, Xiao-Chen; Zhang, Yu-Chen; Xing, Xiao-Xu; Xuan, Mei-Fu; Zhang, Guang-Lei; Luo, Qi-Rong; Wang, Jun-Xia; Cui, Cheng-Du; Li, Wen-Xue; Cui, Zheng-Yun; Yin, Xi-Jun; Kang, Jin-Dan

2017-05-01

To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2-4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2-4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant. RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

Expression of microRNAs in bovine and human pre-implantation embryo culture media

PubMed Central

Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

2014-01-01

MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development1[W

PubMed Central

Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

2007-01-01

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159

Sodium butyrate improves the cloned yak embryo viability and corrects gene expression patterns.

PubMed

Xiong, Xian-rong; Lan, Dao-liang; Li, Jian; Wang, Yong; Zhong, Jin-cheng

2015-02-01

Interspecies somatic cell nuclear transfer (iSCNT), a powerful tool in basic scientific research, has been used widely to increase and preserve the population of endangered species. Yak (Bos grunniens) is one of these species. Development to term of interspecies cloned yak embryos has not been achieved, possibly due to abnormal epigenetic reprogramming. Previous studies have demonstrated that treatment of intraspecies cloned embryos with (NaBu) significantly improves nuclear-cytoplasmic reprogramming and viability in vitro. Therefore, in this study, we evaluated the effect of optimal NaBu concentration and exposure time on preimplantation development of yak iSCNT embryos and on the expression patterns of developmentally important genes. The results showed that 8-cell rate, blastocyst formation rate and total cell number increased significantly compared with their untreated counterparts when yak iSCNT embryos were treated with 5 nM NaBu for 12 h after activation, but that the 2-cell stage embryo rate was not significantly different. The treatment of NaBu also increased significantly the expression levels of Oct-4 and decreased the expression levels of HDAC-2, Dnmt-1 and IGF-1; the expression patterns of these genes were more similar to that of their bovine-yak in vitro fertilization (BY-IVF) counterparts. The results described above indicated that NaBu treatment improved developmental competence in vitro and 'corrected' the gene expression patterns of yak iSCNT embryos.

Sex Bias and Maternal Contribution to Gene Expression Divergence in Drosophila Blastoderm Embryos

PubMed Central

Paris, Mathilde; Villalta, Jacqueline E.; Eisen, Michael B.; Lott, Susan E.

2015-01-01

Early embryogenesis is a unique developmental stage where genetic control of development is handed off from mother to zygote. Yet the contribution of this transition to the evolution of gene expression is poorly understood. Here we study two aspects of gene expression specific to early embryogenesis in Drosophila: sex-biased gene expression prior to the onset of canonical X chromosomal dosage compensation, and the contribution of maternally supplied mRNAs. We sequenced mRNAs from individual unfertilized eggs and precisely staged and sexed blastoderm embryos, and compared levels between D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. First, we find that mRNA content is highly conserved for a given stage and that studies relying on pooled embryos likely systematically overstate the degree of gene expression divergence. Unlike studies done on larvae and adults where most species show a larger proportion of genes with male-biased expression, we find that transcripts in Drosophila embryos are largely female-biased in all species, likely due to incomplete dosage compensation prior to the activation of the canonical dosage compensation mechanism. The divergence of sex-biased gene expression across species is observed to be often due to lineage-specific decrease of expression; the most drastic example of which is the overall reduction of male expression from the neo-X chromosome in D. pseudoobscura, leading to a pervasive female-bias on this chromosome. We see no evidence for a faster evolution of expression on the X chromosome in embryos (no “faster-X” effect), unlike in adults, and contrary to a previous study on pooled non-sexed embryos. Finally, we find that most genes are conserved in regard to their maternal or zygotic origin of transcription, and present evidence that differences in maternal contribution to the blastoderm transcript pool may be due to species-specific divergence of transcript degradation rates. PMID:26485701

Expression analysis of some genes regulated by retinoic acid in controls and triadimefon-exposed embryos: is the amphibian Xenopus laevis a suitable model for gene-based comparative teratology?

PubMed

Di Renzo, Francesca; Rossi, Federica; Bacchetta, Renato; Prati, Mariangela; Giavini, Erminio; Menegola, Elena

2011-06-01

The use of nonmammal models in teratological studies is a matter of debate and seems to be justified if the embryotoxic mechanism involves conserved processes. Published data on mammals and Xenopus laevis suggest that azoles are teratogenic by altering the endogenous concentration of retinoic acid (RA). The expression of some genes (Shh, Ptch-1, Gsc, and Msx2) controlled by retinoic acid is downregulated in rat embryos exposed at the phylotypic stage to the triazole triadimefon (FON). In order to propose X. laevis as a model for gene-based comparative teratology, this work evaluates the expression of Shh, Ptch-1, Gsc, and Msx2 in FON-exposed X. laevis embryos. Embryos, exposed to a high concentration level (500 µM) of FON from stage 13 till 17, were examined at stages 17, 27, and 47. Stage 17 and 27 embryos were processed to perform quantitative RT-PCR. The developmental rate was never affected by FON at any considered stage. FON-exposed stage 47 larvae showed the typical craniofacial malformations. A significant downregulation of Gsc was observed in FON-exposed stage 17 embryos. Shh, Ptch-1, Msx2 showed a high fluctuation of expression both in control and in FON-exposed samples both at stages 17 and 27. The downregulation of Gsc mimics the effects of FON on rat embryos, showing for this gene a common effect of FON in the two vertebrate classes. The high fluctuation observed in the gene expression of the other genes, however, suggests that X. laevis at this stage has limited utility for gene-based comparative teratology. © 2011 Wiley-Liss, Inc.

Characterization of the altered gene expression profile in early porcine embryos generated from parthenogenesis and somatic cell chromatin transfer.

PubMed

Zhou, Chi; Dobrinsky, John; Tsoi, Stephen; Foxcroft, George R; Dixon, Walter T; Stothard, Paul; Verstegen, John; Dyck, Michael K

2014-01-01

The in vitro production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from in vitro Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than in vivo-derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of in vivo (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The "eIF2 signalling", "mitochondrial dysfunction", "regulation of eIF4 and p70S6K signalling", "protein ubiquitination", and "mTOR signalling" pathways were down-regulated in PA HB. Dysregulation of notch signalling-associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.

Expression and characterization of constitutive heat shock protein 70.1 (HSPA-1A) gene in in vitro produced and in vivo-derived buffalo (Bubalus bubalis) embryos.

PubMed

Sharma, G T; Nath, A; Prasad, S; Singhal, S; Singh, N; Gade, N E; Dubey, P K; Saikumar, G

2012-12-01

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA-1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA-1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo-derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA-1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA-1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8-16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA-1A revealed that it shares 91-98% identity with other mammalian sequences. It can be concluded that higher level of HSPA-1A mRNA in IVP embryos in comparison with in vivo-derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA-1A gene could be used as a stress biomarker during pre-implantation development. © 2012 Blackwell Verlag GmbH.

Effect of timing of development on total cell number and expression profile of HSP-70.1 and GLUT-1 in buffalo (Bubalus bubalis) oocytes and preimplantation embryos produced in vitro.

PubMed

Rajhans, Rajib; Kumar, G Sai; Dubey, Pawan K; Sharma, G Taru

2010-03-29

The present study was designed to compare the expression profile of two developmentally important genes (HSP-70.1 and GLUT-1) and TCN (total cell number) count in fast (group A) and slow (group B) cleaved buffalo embryos to access their in vitro developmental competence. Buffalo COCs (cumulus oocyte complexes) were collected from local abattoir ovaries and subjected to in vitro maturation in: TCM-199 supplemented with 10% FBS (fetal bovine serum), BSA (3 mg/ml), sodium pyruvate (0.25 mM) and 20 ng/ml EGF (epidermal growth factor) at 38.5 degrees C under 5% CO2. In vitro derived embryos were collected at 4-8, 8-16 cell, morula and blastocyst stages at specific time points for gene expression analysis and total cell count. A semiquantitative RT-PCR (reverse transcriptase-PCR) assay was used to determine the HSP-70.1 and GLUT-1 transcripts. Results showed that developmental competence and TCN count in fast (group A)-cleaving embryos was significantly (P<0.05) higher than in the slow group (group B). The gene transcript of HSP-70.1 and GLUT-1 was expressed in oocytes (immature and mature) and throughout the embryonic developmental stages in the fast group (group A), while in the slow (group B) cleaving embryos, the expression of HSP-70.1 was absent in all the embryonic developmental stages, and expression of GLUT-1 was absent after 8-16 cell stage. In conclusion, TCN count and expression profile of HSP-70.1 and GLUT-1 genes in buffalo embryos are different taking into account the cleavage rate. Quality of such embryos for research purposes, TCN and expression profiling of developmentally important genes should be employed to optimize the in vitro culture system to produce superior quality of embryos.

Adaptation of video game UVW mapping to 3D visualization of gene expression patterns

NASA Astrophysics Data System (ADS)

Vize, Peter D.; Gerth, Victor E.

2007-01-01

Analysis of gene expression patterns within an organism plays a critical role in associating genes with biological processes in both health and disease. During embryonic development the analysis and comparison of different gene expression patterns allows biologists to identify candidate genes that may regulate the formation of normal tissues and organs and to search for genes associated with congenital diseases. No two individual embryos, or organs, are exactly the same shape or size so comparing spatial gene expression in one embryo to that in another is difficult. We will present our efforts in comparing gene expression data collected using both volumetric and projection approaches. Volumetric data is highly accurate but difficult to process and compare. Projection methods use UV mapping to align texture maps to standardized spatial frameworks. This approach is less accurate but is very rapid and requires very little processing. We have built a database of over 180 3D models depicting gene expression patterns mapped onto the surface of spline based embryo models. Gene expression data in different models can easily be compared to determine common regions of activity. Visualization software, both Java and OpenGL optimized for viewing 3D gene expression data will also be demonstrated.

Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

PubMed Central

Inoue, Kimiko; Ogura, Atsuo

2013-01-01

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT. PMID:24146866

Paternal poly (ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression.

PubMed

Ihara, Motomasa; Meyer-Ficca, Mirella L; Leu, N Adrian; Rao, Shilpa; Li, Fan; Gregory, Brian D; Zalenskaya, Irina A; Schultz, Richard M; Meyer, Ralph G

2014-05-01

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo.

Paternal Poly (ADP-ribose) Metabolism Modulates Retention of Inheritable Sperm Histones and Early Embryonic Gene Expression

PubMed Central

Leu, N. Adrian; Rao, Shilpa; Li, Fan; Gregory, Brian D.; Zalenskaya, Irina A.; Schultz, Richard M.; Meyer, Ralph G.

2014-01-01

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo. PMID:24810616

Controlled hydrostatic pressure stress downregulates the expression of ribosomal genes in preimplantation embryos: a possible protection mechanism?

PubMed

Bock, I; Raveh-Amit, H; Losonczi, E; Carstea, A C; Feher, A; Mashayekhi, K; Matyas, S; Dinnyes, A; Pribenszky, C

2016-04-01

The efficiency of various assisted reproductive techniques can be improved by preconditioning the gametes and embryos with sublethal hydrostatic pressure treatment. However, the underlying molecular mechanism responsible for this protective effect remains unknown and requires further investigation. Here, we studied the effect of optimised hydrostatic pressure treatment on the global gene expression of mouse oocytes after embryonic genome activation. Based on a gene expression microarray analysis, a significant effect of treatment was observed in 4-cell embryos derived from treated oocytes, revealing a transcriptional footprint of hydrostatic pressure-affected genes. Functional analysis identified numerous genes involved in protein synthesis that were downregulated in 4-cell embryos in response to hydrostatic pressure treatment, suggesting that regulation of translation has a major role in optimised hydrostatic pressure-induced stress tolerance. We present a comprehensive microarray analysis and further delineate a potential mechanism responsible for the protective effect of hydrostatic pressure treatment.

Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

PubMed

de Vega-Bartol, José J; Santos, Raquen Raissa; Simões, Marta; Miguel, Célia M

2013-05-01

Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

3D confocal reconstruction of gene expression in mouse.

PubMed

Hecksher-Sørensen, J; Sharpe, J

2001-01-01

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.

Transcriptomic and proteomic analysis reveals mechanisms of embryo abortion during chrysanthemum cross breeding.

PubMed

Zhang, Fengjiao; Wang, Zhiquan; Dong, Wen; Sun, Chunqing; Wang, Haibin; Song, Aiping; He, Lizhong; Fang, Weimin; Chen, Fadi; Teng, Nianjun

2014-10-07

Embryo abortion is the main cause of failure in chrysanthemum cross breeding, and the genes and proteins associated with embryo abortion are poorly understood. Here, we applied RNA sequencing and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic profiles of normal and abortive embryos. More than 68,000 annotated unigenes and 700 proteins were obtained from normal and abortive embryos. Functional analysis showed that 140 differentially expressed genes (DEGs) and 41 differentially expressed proteins (DEPs) were involved in embryo abortion. Most DEGs and DEPs associated with cell death, protein degradation, reactive oxygen species scavenging, and stress-response transcriptional factors were significantly up-regulated in abortive embryos relative to normal embryos. In contrast, most genes and proteins related to cell division and expansion, the cytoskeleton, protein synthesis and energy metabolism were significantly down-regulated in abortive embryos. Furthermore, abortive embryos had the highest activity of three executioner caspase-like enzymes. These results indicate that embryo abortion may be related to programmed cell death and the senescence- or death-associated genes or proteins contribute to embryo abortion. This adds to our understanding of embryo abortion and will aid in the cross breeding of chrysanthemum and other crops in the future.

Epigallocatechin gallate promotes the development of mouse 2-cell embryos in vitro by regulating mitochondrial activity and expression of genes related to p53 signalling pathway.

PubMed

Zhang, Weiyu; Lv, Junjie; Zhang, Yanqin; Jiang, Yufei; Chu, Chenfeng; Wang, Shie

2014-11-01

Preliminary studies have found that the epigallocatechin gallate (EGCG) at proper concentration could promote development of pre-implantation mouse embryos in vitro. However, the underlying mechanisms have not been well understood. In this study, we collected 1-cell embryos from Kunming (KM) mice, cultured them in M16 medium or M16 medium supplemented with 10 μg/mL EGCG and investigated the effects of EGCG on mitochondrial activity and reactive oxygen species (ROS) level of 2-cell embryos. Furthermore, we explored expression differences of genes related to p53 signalling pathway in 2-cell embryos using a PCR array. The results showed that ROS level and mitochondrial membrane potential were significantly lower in embryos cultured in the EGCG group than in the M16 group (p < 0.05), while the adenosine triphosphate content was slightly lower than in the M16 group (p > 0.05). PCR array test results showed that 18 genes were differentially expressed, among which eight genes involving cell growth, cell cycle regulation and mRNA transcription were up-regulated and 10 genes involving apoptosis, cell cycle arrest and DNA repair were down-regulated in the EGCG groups. It is concluded that EGCG could promote the development of 1-cell embryos in vitro possibly due to its ability to scavenge ROS and regulate mitochondrial activity. In addition, EGCG could influence expression of genes related to p53 signalling pathway in 2-cell embryos and promote cell cycle progression. © 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

The first murine zygotic transcription is promiscuous and uncoupled from splicing and 3′ processing

PubMed Central

Abe, Ken-ichiro; Yamamoto, Ryoma; Franke, Vedran; Cao, Minjun; Suzuki, Yutaka; Suzuki, Masataka G; Vlahovicek, Kristian; Svoboda, Petr; Schultz, Richard M; Aoki, Fugaku

2015-01-01

Initiation of zygotic transcription in mammals is poorly understood. In mice, zygotic transcription is first detected shortly after pronucleus formation in 1-cell embryos, but the identity of the transcribed loci and mechanisms regulating their expression are not known. Using total RNA-Seq, we have found that transcription in 1-cell embryos is highly promiscuous, such that intergenic regions are extensively expressed and thousands of genes are transcribed at comparably low levels. Striking is that transcription can occur in the absence of defined core-promoter elements. Furthermore, accumulation of translatable zygotic mRNAs is minimal in 1-cell embryos because of inefficient splicing and 3′ processing of nascent transcripts. These findings provide novel insights into regulation of gene expression in 1-cell mouse embryos that may confer a protective mechanism against precocious gene expression that is the product of a relaxed chromatin structure present in 1-cell embryos. The results also suggest that the first zygotic transcription itself is an active component of chromatin remodeling in 1-cell embryos. PMID:25896510

Embryonic genotype and inbreeding affect preimplantation development in cattle.

PubMed

Lazzari, G; Colleoni, S; Duchi, R; Galli, A; Houghton, F D; Galli, C

2011-05-01

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences.

PubMed

Dudczig, Stefanie; Currie, Peter D; Poggi, Lucia; Jusuf, Patricia R

2017-03-22

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments. In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

The dorsoventral patterning of Musca domestica embryos: insights into BMP/Dpp evolution from the base of the lower cyclorraphan flies.

PubMed

Hodar, Christian; Cambiazo, Verónica

2018-01-01

In the last few years, accumulated information has indicated that the evolution of an extra-embryonic membrane in dipterans was accompanied by changes in the gene regulatory network controlled by the BMP/Dpp pathway, which is responsible for dorsal patterning in these insects. However, only comparative analysis of gene expression levels between distant species with two extra-embryonic membranes, like A. gambiae or C. albipunctata , and D. melanogaster, has been conducted. Analysis of gene expression in ancestral species, which evolved closer to the amnioserosa origin, could provide new insights into the evolution of dorsoventral patterning in dipterans. Here we describe the spatial expression of several key and downstream elements of the Dpp pathway and show the compared patterns of expression between Musca and Drosophila embryos, both dipterans with amnioserosa. Most of the analyzed gene showed a high degree of expression conservation, however, we found several differences in the gene expression pattern of M. domestica orthologs for sog and tolloid . Bioinformatics analysis of the promoter of both genes indicated that the variations could be related to the gain of several binding sites for the transcriptional factor Dorsal in the Md.tld promoter and Snail in the Md.sog enhancer . These altered expressions could explain the unclear formation of the pMad gradient in the M. domestica embryo, compared to the formation of the gradient in D. melanogaster. Gene expression changes during the dorsal-ventral patterning in insects contribute to the differentiation of extra-embryonic tissues as a consequence of changes in the gene regulatory network controlled by BMP/Dpp. In this work, in early M. domestica embryos, we identified the expression pattern of several genes members involved in the dorsoventral specification of the embryo. We believe that these data can contribute to understanding the evolution of the BMP/Dpp pathway, the regulation of BMP ligands, and the formation of a Dpp gradient in higher cyclorraphan flies.

Development of a transgenic zebrafish model expressing GFP in the notochord, somite and liver directed by the hfe2 gene promoter.

PubMed

Bian, Yue-Hong; Xu, Cheng; Li, Junling; Xu, Jin; Zhang, Hongwei; Du, Shao Jun

2011-08-01

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

Transcriptome analysis of zebrafish embryos exposed to deltamethrin.

PubMed

Chueh, Tsung-Cheng; Hsu, Li-Sung; Kao, Chin-Ming; Hsu, Tung-Wei; Liao, Hung-Yu; Wang, Kuan-Yi; Chen, Ssu Ching

2017-05-01

Deltamethrin (DTM), a type II pyrethroid, is one of the most commonly used insecticides. The increased use of pyrethroid leads to potential adverse effects, particularly in sensitive populations such as children and pregnant women. None of the related studies was focused on the transcriptome responses in zebrafish embryos after treatment with DTM; therefore, RNA-seq, a high-throughput method, was performed to analyze the global expression of differential expressed genes (DEGs) in zebrafish embryos treated with DTM (40 and 80 μg/L) from fertilization to 48 h postfertilization (hpf) as compared with that in the control group (without DTM treatment). Two cDNA libraries were generated from treated embryos and one cDNA library from nontreated embryos, respectively. Over 92% of reads mapped to the reference in these three libraries. It was observed that many differential genes were expressed in comparison with embryos before and after DTM. The 20 most differentially expressed upregulated or downregulated genes were majorly involved in the signaling transduction. Validation of selected nine genes expression using qRT-PCR confirmed RNA-seq results. The transcriptome sequences were further subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, showing G-protein-coupled receptor signaling pathway and neuroactive ligand-receptor interaction, respectively, were most enriched. The data from this study contributed to a better understanding of the potential consequences of fish exposed to DTM, to an evaluation of the potential threat of DTM to fish populations in aquatic environments. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1548-1557, 2017. © 2016 Wiley Periodicals, Inc.

AmphiFoxQ2, a novel winged helix/forkhead gene, exclusively marks the anterior end of the amphioxus embryo

NASA Technical Reports Server (NTRS)

Yu, Jr-Kai; Holland, Nicholas D.; Holland, Linda Z.

2003-01-01

A full-length FoxQ-related gene (AmphiFoxQ2) was isolated from amphioxus. Expression is first detectable in the animal/anterior hemisphere at the mid blastula stage. The midpoint of this expression domain coincides with the anterior pole of the embryo and is offset dorsally by about 20 degrees from the animal pole. During the gastrula stage, expression is limited to the anterior ectoderm. By the early neurula stage, expression remains in the anterior ectoderm and also appears in the adjacent anterior mesendoderm. By the early larval stages, expression is detectable in the anteriormost ectoderm and in the rostral tip of the notochord. AmphiFoxQ2 is never expressed anywhere except at the anterior tip of amphioxus embryos and larvae. This is the first gene known that exclusively marks the anterior pole of chordate embryos. It may, therefore, play an important role in establishing and/or maintaining the anterior/posterior axis.

Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

PubMed

García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

2015-09-01

Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the blastocyst stage in the BMP5 group. Moreover, reverse transcription quantitative real-time polymerase chain reaction analysis showed a significant increase in the relative abundance of SOX2 in two-cell stage embryos, ID2 and OCT4 in eight-cell stage embryos, and NANOG and OCT4 in blastocysts derived from BMP5-treated embryos. In conclusion, our results report that early addition of BMP5 to the embryo culture medium had a positive effect on the blastocyst rate and affected the relative expression of BMP target and pluripotency genes, suggesting that BMP5 could play an important role in the preimplantation development of bovine embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

PubMed Central

Dahlberg, Olle; Shilkova, Olga; Tang, Min; Holmqvist, Per-Henrik; Mannervik, Mattias

2015-01-01

Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos. PMID:25679530

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression.

PubMed

Moro, L N; Hiriart, M I; Buemo, C; Jarazo, J; Sestelo, A; Veraguas, D; Rodriguez-Alvarez, L; Salamone, D F

2015-07-01

The aim of this study was to evaluate the capacity of domestic cat (Dc, Felis silvestris) oocytes to reprogram the nucleus of cheetah (Ch, Acinonyx jubatus) cells by interspecies SCNT (iSCNT), by using embryo aggregation. Dc oocytes were in vitro matured and subjected to zona pellucida free (ZP-free) SCNT or iSCNT, depending on whether the nucleus donor cell was of Dc or Ch respectively. ZP-free reconstructed embryos were then cultured in microwells individually (Dc1X and Ch1X groups) or in couples (Dc2X and Ch2X groups). Embryo aggregation improved in vitro development obtaining 27.4, 47.7, 16.7 and 28.3% of blastocyst rates in the Dc1X, Dc2X, Ch1X and Ch2X groups, respectively (P<0.05). Moreover, aggregation improved the morphological quality of blastocysts from the Dc2X over the Dc1X group. Gene expression analysis revealed that Ch1X and Ch2X blastocysts had significantly lower relative expression of OCT4, CDX2 and NANOG than the Dc1X, Dc2X and IVF control groups. The OCT4, NANOG, SOX2 and CDX2 genes were overexpressed in Dc1X blastocysts, but the relative expression of these four genes decreased in the Dc2X, reaching similar relative levels to those of Dc IVF blastocysts. In conclusion, Ch blastocysts were produced using Dc oocytes, but with lower relative expression of pluripotent and trophoblastic genes, indicating that nuclear reprogramming could be still incomplete. Despite this, embryo aggregation improved the development of Ch and Dc embryos, and normalized Dc gene expression, which suggests that this strategy could improve full-term developmental efficiency of cat and feline iSCNT embryos. © 2015 Society for Reproduction and Fertility.

Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance

PubMed Central

2013-01-01

Background While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. Results Using 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT. Conclusions Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage. PMID:23320502

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

PubMed

Bai, Guang-Yu; Song, Si-Hang; Wang, Zhen-Dong; Shan, Zhi-Yan; Sun, Rui-Zhen; Liu, Chun-Jia; Wu, Yan-Shuang; Li, Tong; Lei, Lei

2016-04-01

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study. © 2016 Japanese Society of Developmental Biologists.

l-Ergothioneine improves the developmental potential of in vitro sheep embryos without influencing OCTN1-mediated cross-membrane transcript expression.

PubMed

Mishra, A; Reddy, I J; Dhali, A; Javvaji, P K

2018-04-02

SummaryThe objective of the study was to investigate the effect of l-ergothioneine (l-erg) (5 mM or 10 mM) supplementation in maturation medium on the developmental potential and OCTN1-dependant l-erg-mediated (10 mM) change in mRNA abundance of apoptotic (Bcl2, Bax, Casp3 and PCNA) and antioxidant (GPx, SOD1, SOD2 and CAT) genes in sheep oocytes and developmental stages of embryos produced in vitro. Oocytes matured with l-erg (10 mM) reduced their embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH in matured oocytes that in turn improved developmental potential, resulting in significantly (P < 0.05) higher percentages of cleavage (53.72% vs 38.86, 46.56%), morulae (34.36% vs 20.62, 25.84%) and blastocysts (14.83% vs 6.98, 9.26%) compared with other lower concentrations (0 mM and 5 mM) of l-erg without change in maturation rate. l-Erg (10 mM) treatment did not influence the mRNA abundance of the majority of apoptotic and antioxidant genes studied in the matured oocytes and developmental stages of embryo. A gene expression study found that the SLC22A4 gene that encodes OCTN1, an integral membrane protein and specific transporter of l-erg was not expressed in oocytes and developmental stages of embryos. Therefore it was concluded from the study that although there was improvement in the developmental potential of sheep embryos by l-erg supplementation in maturation medium, there was no change in the expression of the majority of the genes studied due to the absence of the SLC22A4 gene in oocytes and embryos that encode OCTN1, which is responsible for transportation of l-erg across the membrane to alter gene expression.

Expression of the homeotic gene mab-5 during Caenorhabditis elegans embryogenesis.

PubMed

Cowing, D W; Kenyon, C

1992-10-01

mab-5 is a member of a complex of homeobox-containing genes evolutionarily related to the Antennapedia and bithorax complexes of Drosophila melanogaster. Like the homeotic genes in Drosophila, mab-5 is required in a particular region along the anterior-posterior body axis, and acts during postembryonic development to give cells in this region their characteristic identities. We have used a mab-5-lacZ fusion integrated into the C. elegans genome to study the posterior-specific expression of mab-5 during embryogenesis. The mab-5-lacZ fusion was expressed in the posterior of the embryo by 180 minutes after the first cleavage, indicating that the mechanisms responsible for the position-specific expression of mab-5-lacZ act at a relatively early stage of embryogenesis. In embryos homozygous for mutations in the par genes, which disrupt segregation of factors during early cleavages, expression of mab-5-lacZ was no longer localized to the posterior. This suggests that posterior-specific expression of mab-5 depends on the appropriate segregation of developmental factors during early embryogenesis. After extrusion of any blastomere of the four-cell embryo, descendants of the remaining three cells could still express the mab-5-lacZ fusion. In these partial embryos, however, the fusion was often expressed in cells scattered throughout the embryo, suggesting that cell-cell interactions and/or proper positioning of early blastomeres are required for mab-5 expression to be localized to the posterior.

Identification of MicroRNAs and their Targets Associated with Embryo Abortion during Chrysanthemum Cross Breeding via High-Throughput Sequencing.

PubMed

Zhang, Fengjiao; Dong, Wen; Huang, Lulu; Song, Aiping; Wang, Haibin; Fang, Weimin; Chen, Fadi; Teng, Nianjun

2015-01-01

MicroRNAs (miRNAs) are important regulators in plant development. They post-transcriptionally regulate gene expression during various biological and metabolic processes by binding to the 3'-untranslated region of target mRNAs to facilitate mRNA degradation or inhibit translation. Chrysanthemum (Chrysanthemum morifolium) is one of the most important ornamental flowers with increasing demand each year. However, embryo abortion is the main reason for chrysanthemum cross breeding failure. To date, there have been no experiments examining the expression of miRNAs associated with chrysanthemum embryo development. Therefore, we sequenced three small RNA libraries to identify miRNAs and their functions. Our results will provide molecular insights into chrysanthemum embryo abortion. Three small RNA libraries were built from normal chrysanthemum ovules at 12 days after pollination (DAP), and normal and abnormal chrysanthemum ovules at 18 DAP. We validated 228 miRNAs with significant changes in expression frequency during embryonic development. Comparative profiling revealed that 69 miRNAs exhibited significant differential expression between normal and abnormal embryos at 18 DAP. In addition, a total of 1037 miRNA target genes were predicted, and their annotations were defined by transcriptome data. Target genes associated with metabolic pathways were most highly represented according to the annotation. Moreover, 52 predicted target genes were identified to be associated with embryonic development, including 31 transcription factors and 21 additional genes. Gene ontology (GO) annotation also revealed that high-ranking miRNA target genes related to cellular processes and metabolic processes were involved in transcription regulation and the embryo developmental process. The present study generated three miRNA libraries and gained information on miRNAs and their targets in the chrysanthemum embryo. These results enrich the growing database of new miRNAs and lay the foundation for the further understanding of miRNA biological function in the regulation of chrysanthemum embryo abortion.

Developmental Toxicity of Diclofenac and Elucidation of Gene Regulation in zebrafish (Danio rerio)

NASA Astrophysics Data System (ADS)

Chen, Jia-Bin; Gao, Hong-Wen; Zhang, Ya-Lei; Zhang, Yong; Zhou, Xue-Fei; Li, Chun-Qi; Gao, Hai-Ping

2014-05-01

Environmental pollution by emerging contaminants, e.g. pharmaceuticals, has become a matter of widespread concern in recent years. We investigated the membrane transport of diclofenac and its toxic effects on gene expression and the development of zebrafish embryos. The association of diclofenac with the embryos conformed to the general partition model at low concentration, the partition coefficient being 0.0033 ml per embryo. At high concentration, the interaction fitted the Freundlich model. Most of the diclofenac remained in the extracellular aqueous solution with less than 5% interacting with the embryo, about half of which was adsorbed on the membranes while the rest entered the cytoplasm. Concentrations of diclofenac over 10.13 μM were lethal to all the embryos, while 3.78 μM diclofenac was teratogenic. The development abnormalities at 4 day post treatment (dpt) include shorter body length, smaller eye, pericardial and body edema, lack of liver, intestine and circulation, muscle degeneration, and abnormal pigmentation. The portion of the diclofenac transferred into the embryo altered the expression of certain genes, e.g. down-regulation of Wnt3a and Gata4 and up-regulation of Wnt8a. The alteration of expression of such genes or the regulation of downstream genes could cause defects in the cardiovascular and nervous systems.

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

PubMed Central

Urrego, Rodrigo; Rodriguez-Osorio, Nélida; Niemann, Heiner

2014-01-01

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders. PMID:24709985

Gene expression analysis in zebrafish embryos: a potential approach to predict effect concentrations in the fish early life stage test.

PubMed

Weil, Mirco; Scholz, Stefan; Zimmer, Michaela; Sacher, Frank; Duis, Karen

2009-09-01

Based on the hypothesis that analysis of gene expression could be used to predict chronic fish toxicity, the zebrafish (Danio rerio) embryo test (DarT), developed as a replacement method for the acute fish test, was expanded to a gene expression D. rerio embryo test (Gene-DarT). The effects of 14 substances on lethal and sublethal endpoints of the DarT and on expression of potential marker genes were investigated: the aryl hydrocarbon receptor 2, cytochrome P450 1A (cypla), heat shock protein 70, fizzy-related protein 1, the transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene family protein g (avian) 1 and NF-E2-p45-related factor, and heme oxygenase 1 (hmox1). After exposure of zebrafish embryos for 48 h, differential gene expression was evaluated using reverse transcriptase-polymerase chain reaction, gel electrophoresis, and densitometric analysis of the gels. All tested compounds significantly affected the expression of at least one potential marker gene, with cyp1a and hmox1 being most sensitive. Lowest-observed-effect concentrations (LOECs) for gene expression were below concentrations resulting in 10% lethal effects in the DarT. For 10 (3,4- and 3,5-dichloroaniline, 1,4-dichlorobenzene, 2,4-dinitrophenol, atrazine, parathion-ethyl, chlorotoluron, genistein, 4-nitroquinoline-1-oxide, and cadmium) out of the 14 tested substances, LOEC values derived with the Gene-DarT differ by a factor of less than 10 from LOEC values of fish early life stage tests with zebrafish. For pentachloroaniline and pentachlorobenzene, the Gene-DarT showed a 23- and 153-fold higher sensitivity, respectively, while for lindane, it showed a 13-fold lower sensitivity. For ivermectin, the Gene-DarT was by a factor of more than 1,000 less sensitive than the acute fish test. The results of the present study indicate that gene expression analysis in zebrafish embryos could principally be used to predict effect concentrations in the fish early life stage test.

Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos.

PubMed

Arias, María E; Ross, Pablo J; Felmer, Ricardo N

2013-01-01

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

Developmental toxicity in flounder embryos exposed to crude oils derived from different geographical regions.

PubMed

Jung, Jee-Hyun; Lee, Eun-Hee; Choi, Kwang-Min; Yim, Un Hyuk; Ha, Sung Yong; An, Joon Geon; Kim, Moonkoo

2017-06-01

Crude oils from distinct geographical regions have distinct chemical compositions, and, as a result, their toxicity may be different. However, developmental toxicity of crude oils derived from different geographical regions has not been extensively characterized. In this study, flounder embryos were separately exposed to effluents contaminated by three crude oils including: Basrah Light (BLO), Pyrenees (PCO), and Sakhalin Vityaz (SVO), in addition to a processed fuel oil (MFO-380), to measure developmental toxicity and for gene expressions. Each oil possessed a distinct chemical composition. Edema defect was highest in embryos exposed to PCO and MFO-380 that both have a greater fraction of three-ring PAHs (33% and 22%, respectively) compared to BLO and SVO. Observed caudal fin defects were higher in embryos exposed to SVO and MFO-380, which are both dominated by naphthalenes (81% and 52%, respectively). CYP1A gene expressions were also highest in embryos exposed to SVO and MFO-380. Higher incidence of cardiotoxicity and lower nkx 2.5 expression were detected in embryos exposed to PCO. Unique gene expression profiles were observed in embryos exposed to crude oils with distinct compositions. This study demonstrates that crude oils of different geographical origins with different compositional characteristics induce developmental toxicity to different degrees. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of choriogenin cis-regulatory elements and production of estrogen-inducible, liver-specific transgenic Medaka.

PubMed

Ueno, Tetsuro; Yasumasu, Shigeki; Hayashi, Shinji; Iuchi, Ichiro

2004-07-01

Choriogenins (chg-H, chg-L) are precursor proteins of egg envelope of medaka and synthesized in the spawning female liver in response to estrogen. We linked a gene construct chg-L1.5 kb/GFP (a 1.5 kb 5'-upstream region of the chg-L gene fused with a green fluorescence protein (GFP) gene) to another construct emgb/RFP (a cis-regulatory region of embryonic globin gene fused with an RFP gene), injected the double fusion gene construct into 1- or 2-cell-stage embryos, and selected embryos expressing the RFP in erythroid cells. From the embryos, we established two lines of chg-L1.5 kb/GFP-emgb/RFP-transgenic medaka. The 3-month-old spawning females and estradiol-17beta (E2)-exposed males displayed the liver-specific GFP expression. The E2-dependent GFP expression was detected in the differentiating liver of the stage 37-38 embryos. In addition, RT-PCR and whole-mount in situ hybridization showed that the E2-dependent chg expression was found in the liver of the stage 34 embryos of wild medaka, suggesting that such E2-dependency is achieved shortly after differentiation of the liver. Analysis using serial deletion mutants fused with GFP showed that the region -426 to -284 of the chg-L gene or the region -364 to -265 of the chg-H gene had the ability to promote the E2-dependent liver-specific GFP expression of its downstream gene. Further analyses suggested that an estrogen response element (ERE) at -309, an ERE half-site at -330 and a binding site for C/EBP at -363 of the chg-L gene played important roles in its downstream chg-L gene expression. In addition, this transgenic medaka may be useful as one of the test animals for detecting environmental estrogenic steroids.

Transcriptional regulators TRIM28, SETDB1, and TP53 are aberrantly expressed in porcine embryos produced by in vitro fertilization in comparison to in vivo- and somatic-cell nuclear transfer-derived embryos

PubMed Central

Hamm, Jennifer; Tessanne, Kim; Murphy, Clifton N; Prather, Randall S

2014-01-01

In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos. Mol. Reprod. Dev. 81: 552–556, 2014. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:24659575

Effect of chilling and cryopreservation on expression of Pax genes in zebrafish (Danio rerio) embryos and blastomeres.

PubMed

Lin, C; Spikings, E; Zhang, T; Rawson, D M

2009-08-01

Cryopreservation is now common practice in the fields of aquaculture, conservation and biomedicine. However, there is a lack of information on the effect of chilling and cryopreservation at the molecular level. In the present study, we used real-time RT-PCR analysis to determine the effect of chilling and cryopreservation on expression of Pax2a, Pax2b, Pax5 and Pax8 which constitute one subgroup of the Pax gene family. As intact embryos of zebrafish have not yet been successfully cryopreserved, we have used two alternatives: chilling of intact embryos and cryopreservation of isolated blastomeres. Cryopreservation was found to affect the normal pattern of gene expression in zebrafish embryonic blastomeres. The trends, profile changes, in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryonic blastomeres which could have a detrimental effect on embryo development.

Isolation and expression of the human gametocyte-specific factor 1 gene (GTSF1) in fetal ovary, oocytes, and preimplantation embryos.

PubMed

Huntriss, John; Lu, Jianping; Hemmings, Karen; Bayne, Rosemary; Anderson, Richard; Rutherford, Anthony; Balen, Adam; Elder, Kay; Picton, Helen M

2017-01-01

Gametocyte-specific factor 1 has been shown in other species to be required for the silencing of retrotransposons via the Piwi-interacting RNA (piRNA) pathway. In this study, we aimed to isolate and assess expression of transcripts of the gametocyte-specific factor 1 (GTSF1) gene in the human female germline and in preimplantation embryos. Complementary DNA (cDNA) libraries from human fetal ovaries and testes, human oocytes and preimplantation embryos and ovarian follicles isolated from an adult ovarian cortex biopsy were used to as templates for PCR, cloning and sequencing, and real time PCR experiments of GTSF1 expression. GTSF1 cDNA clones that covered the entire coding region were isolated from human oocytes and preimplantation embryos. GTSF1 mRNA expression was detected in archived cDNAs from staged human ovarian follicles, germinal vesicle (GV) stage oocytes, metaphase II oocytes, and morula and blastocyst stage preimplantation embryos. Within the adult female germline, expression was highest in GV oocytes. GTSF1 mRNA expression was also assessed in human fetal ovary and was observed to increase during gestation, from 8 to 21 weeks, during which time oogonia enter meiosis and primordial follicle formation first occurs. In human fetal testis, GTSF1 expression also increased from 8 to 19 weeks. To our knowledge, this report is the first to describe the expression of the human GTSF1 gene in human gametes and preimplantation embryos.

Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis.

PubMed

Ji, Qianqian; Zhu, Kongju; Liu, Zhiguo; Song, Zhenwei; Huang, Yuankai; Zhao, Haijing; Chen, Yaosheng; He, Zuyong; Mo, Delin; Cong, Peiqing

2013-03-15

Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality. Copyright © 2013 Elsevier Inc. All rights reserved.

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi Xiongjie; Graduate School of the Chinese Academy of Sciences, Beijing 100039; Du Yongbing

2008-07-01

Perfluorooctanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure tomore » PFOS concentrations of 1 mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity.« less

Additional mitochondrial DNA influences the interactions between the nuclear and mitochondrial genomes in a bovine embryo model of nuclear transfer.

PubMed

Srirattana, Kanokwan; St John, Justin C

2018-05-08

We generated cattle embryos using mitochondrial supplementation and somatic cell nuclear transfer (SCNT), named miNT, to determine how additional mitochondrial DNA (mtDNA) modulates the nuclear genome. To eliminate any confounding effects from somatic cell mtDNA in intraspecies SCNT, donor cell mtDNA was depleted prior to embryo production. Additional oocyte mtDNA did not affect embryo development rates but increased mtDNA copy number in blastocyst stage embryos. Moreover, miNT-derived blastocysts had different gene expression profiles when compared with SCNT-derived blastocysts. Additional mtDNA increased expression levels of genes involved in oxidative phosphorylation, cell cycle and DNA repair. Supplementing the embryo culture media with a histone deacetylase inhibitor, Trichostatin A (TSA), had no beneficial effects on the development of miNT-derived embryos, unlike SCNT-derived embryos. When compared with SCNT-derived blastocysts cultured in the presence of TSA, additional mtDNA alone had beneficial effects as the activity of glycolysis may increase and embryonic cell death may decrease. However, these beneficial effects were not found with additional mtDNA and TSA together, suggesting that additional mtDNA alone enhances reprogramming. In conclusion, additional mtDNA increased mtDNA copy number and expression levels of genes involved in energy production and embryo development in blastocyst stage embryos emphasising the importance of nuclear-mitochondrial interactions.

Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes.

PubMed

Khosla, S; Dean, W; Brown, D; Reik, W; Feil, R

2001-03-01

Culture of preimplantation mammalian embryos and cells can influence their subsequent growth and differentiation. Previously, we reported that culture of mouse embryonic stem cells is associated with deregulation of genomic imprinting and affects the potential for these cells to develop into normal fetuses. The purpose of our current study was to determine whether culture of preimplantation mouse embryos in a chemically defined medium (M16) with or without fetal calf serum (FCS) can affect their subsequent development and imprinted gene expression. Only one third of the blastocysts that had been cultured from two-cell embryos in M16 medium complemented with FCS developed into viable Day 14 fetuses after transfer into recipients. These M16 + FCS fetuses were reduced in weight as compared with controls and M16 fetuses and had decreased expression of the imprinted H19 and insulin-like growth factor 2 genes associated with a gain of DNA methylation at an imprinting control region upstream of H19. They also displayed increased expression of the imprinted gene Grb10. The growth factor receptor binding gene Grb7, in contrast, was strongly reduced in its expression in most of the M16 + FCS fetuses. No alterations were detected for the imprinted gene MEST: Preimplantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes, thus leading to aberrant fetal growth and development.

Bovine embryo induces an anti-inflammatory response in uterine epithelial cells and immune cells in vitro: possible involvement of interferon tau as an intermediator

PubMed Central

TALUKDER, Anup K.; YOUSEF, Mohamed S.; RASHID, Mohammad B.; AWAI, Kensuke; ACOSTA, Tomas J.; SHIMIZU, Takashi; OKUDA, Kiyoshi; SHIMADA, Masayuki; IMAKAWA, Kazuhiko; MIYAMOTO, Akio

2017-01-01

Recent observations suggest that the bovine uterus starts to react to the early embryo immediately after its arrival from the oviduct. The present study aimed to investigate the effect of the early developing embryo on the immune-related gene profile in bovine uterine epithelial cells (BUECs) in vitro, and to further examine the impact of conditioned media (CM), either from embryo-BUEC co-culture or embryo culture alone, on gene expression in peripheral blood mononuclear cells (PBMCs). First, BUECs were co-cultured with morulae (n = 10) for D5-D9 (D0 = IVF), and gene expression in BUECs was analyzed. Subsequently, PBMCs were cultured in CM from embryo-BUEC co-culture or D5-D9 embryo culture, and gene expression was evaluated. In BUECs, the embryo induced interferon (IFN)-stimulated genes (ISGs: ISG15, OAS1, and MX2), a key factor for IFN-signaling (STAT1), and type-1 IFN receptors (IFNAR1 and IFNAR2), with suppression of NFkB2, NFkBIA and pro-inflammatory cytokines (TNFA and IL1B). The embryo also stimulated PTGES and PGE2 secretion in BUECs. In PBMCs, both CM from embryo-BUEC co-culture and embryo culture alone induced ISGs, STAT1 and TGFB1, while suppressing TNFA and IL17. Similarly, interferon tau (IFNT) at 100 pg/ml suppressed NFkB2, TNFA and IL1B in BUECs, and also stimulated TGFB1 and suppressed TNFA in PBMCs. Our findings suggest that the bovine embryo, in the first four days in the uterus (D5-D9), starts to induce an anti-inflammatory response in epithelial cells and in immune cells. IFNT is likely to act as one of the intermediators for induction of the anti-inflammatory response in the bovine uterus. PMID:28603222

Aberrant Expression of Xist in Aborted Porcine Fetuses Derived from Somatic Cell Nuclear Transfer Embryos

PubMed Central

Yuan, Lin; Wang, Anfeng; Yao, Chaogang; Huang, Yongye; Duan, Feifei; Lv, Qinyan; Wang, Dongxu; Ouyang, Hongsheng; Li, Zhanjun; Lai, Liangxue

2014-01-01

Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos. PMID:25429426

GENE EXPRESSION PATTERNS OF CD-1 DAY-8 EMBRYO CULTURES EXPOSED TO BROMOCHLORO ACETIC ACID

EPA Science Inventory

Gene expression patterns of CD-1 day-8 embryo cultures exposed to bromochloro acetic acid

Edward D. Karoly?*, Judith E. Schmid* and E. Sidney Hunter III*
?Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina and *Reproductiv...

Triazole induced concentration-related gene signatures in rat whole embryo culture.

PubMed

Robinson, Joshua F; Tonk, Elisa C M; Verhoef, Aart; Piersma, Aldert H

2012-09-01

Commonly used as antifungal agents in agriculture and medicine, triazoles have been shown to cause teratogenicity in a diverse set of animal models. Here, we evaluated the dose-dependent impacts of flusilazole, cyproconazole and triadimefon, on global gene expression in relation to effects on embryonic development using the rat whole embryo culture (WEC) model. After 4 h exposure, we identified changes in gene expression due to triazole exposure which preceded morphological alterations observed at 48 h. In general, across the three triazoles, we observed similar directionality of regulation in gene expression and the magnitude of effects on gene expression correlated with the degree of induced developmental toxicity. Significantly regulated genes included key members of steroid/cholesterol and retinoic acid metabolism and hindbrain developmental pathways. Direct comparisons with previous studies suggest that triazole-gene signatures identified in the WEC overlap with zebrafish and mouse, and furthermore, triazoles impact gene expression in a similar manner as retinoic acid exposures in rat embryos. In summary, we further differentiate pathways underlying triazole-developmental toxicity using WEC and demonstrate the conservation of these response-pathways across model systems. Copyright © 2012 Elsevier Inc. All rights reserved.

Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies Black-footed cat cloned embryos

USGS Publications Warehouse

Gómez, M. C.; Biancardi, M.N.; Jenkins, J.A.; Dumas, C.; Galiguis, J.; Wang, G.; Earle Pope, C.

2012-01-01

Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423.

PubMed

Liu, Beibei; Su, Shengzhong; Wu, Ying; Li, Ying; Shan, Xiaohui; Li, Shipeng; Liu, Hongkui; Dong, Haixiao; Ding, Meiqi; Han, Junyou; Yuan, Yaping

2015-07-01

Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Precision matters for position decoding in the early fly embryo

NASA Astrophysics Data System (ADS)

Petkova, Mariela D.; Tkacik, Gasper; Wieschaus, Eric F.; Bialek, William; Gregor, Thomas

Genetic networks can determine cell fates in multicellular organisms with precision that often reaches the physical limits of the system. However, it is unclear how the organism uses this precision and whether it has biological content. Here we address this question in the developing fly embryo, in which a genetic network of patterning genes reaches 1% precision in positioning cells along the embryo axis. The network consists of three interconnected layers: an input layer of maternal gradients, a processing layer of gap genes, and an output layer of pair-rule genes with seven-striped patterns. From measurements of gap gene protein expression in hundreds of wild-type embryos we construct a ``decoder'', which is a look-up table that determines cellular positions from the concentration means, variances and co-variances. When we apply the decoder to measurements in mutant embryos lacking various combinations of the maternal inputs, we predict quantitative changes in the output layer such as missing, altered or displaced stripes. We confirm these predictions by measuring pair-rule expression in the mutant embryos. Our results thereby show that the precision of the patterning network is biologically meaningful and a necessary feature for decoding cell positions in the early fly embryo.

Expression profiling of the mouse early embryo: Reflections and Perspectives

PubMed Central

Ko, Minoru S. H.

2008-01-01

Laboratory mouse plays important role in our understanding of early mammalian development and provides invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. Comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over last two decades, have provided even more advantages to mouse models. Here the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells are reviewed and the future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, which include: bird’s-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks. PMID:16739220

Sexually Dimorphic Expression of Secreted Frizzled-Related (SFRP) Genes in the Developing Mouse Müllerian Duct

PubMed Central

COX, SAM; SMITH, LEE; BOGANI, DEBORA; CHEESEMAN, MICHAEL; SIGGERS, PAM; GREENFIELD, ANDY

2007-01-01

In developing male embryos, the female reproductive tract primordia (Müllerian ducts) regress due to the production of testicular anti-Müllerian hormone (AMH). Because of the association between secreted frizzled-related proteins (SFRPs) and apoptosis, their reported developmental expression patterns and the role of WNT signaling in female reproductive tract development, we examined expression of Sfrp2 and Sfrp5 during development of the Müllerian duct in male (XY) and female (XX) mouse embryos. We show that expression of both Sfrp2 and Sfrp5 is dynamic and sexually dimorphic. In addition, the male-specific expression observed for both genes prior to the onset of regression is absent in mutant male embryos that fail to undergo Müllerian duct regression. We identified ENU-induced point mutations in Sfrp5 and Sfrp2 that are predicted to severely disrupt the function of these genes. Male embryos and adults homozygous for these mutations, both individually and in combination, are viable and apparently fertile with no overt abnormalities of reproductive tract development. PMID:16700072

Resveratrol-cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

PubMed

Torres, V; Hamdi, M; Millán de la Blanca, M G; Urrego, R; Echeverri, J; López-Herrera, A; Rizos, D; Gutiérrez-Adán, A; Sánchez-Calabuig, M J

2018-03-26

Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol-methyl β-cyclodextrin (RV-CD) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV-CD during in vitro oocyte maturation (IVM) or in vitro embryo culture (IVC) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV-CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT-qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV-CD and embryo development and blastocysts gene expression by RT-qPCR were studied. A group without RV-CD (control - ) and a group with cyclodextrin (control + ) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p < .05). Blastocysts produced by IVC with resveratrol showed that RV-CD could modify the expression of genes related to lipid metabolism (CYP51A1, PNPLA2 and MTORC1) compared with control groups (p < .05). RV-CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts. © 2018 Blackwell Verlag GmbH.

In ovo trace element supplementation enhances expression of growth genes in embryo and immune genes in post-hatch broiler chickens.

PubMed

Goel, Akshat; Bhanja, Subrat K; Mehra, Manish; Mandal, Asitbaran; Pande, Veena

2016-06-01

Differential expression of growth- and immunity-related genes and post-hatch performances were evaluated in in ovo zinc (Zn), iodine (I) or selenium (Se) supplemented chicken embryos. There was about 9-18% reduction in hatchability of Zn, I or Se supplemented eggs. In ovo trace element supplementation did not improve post-hatch growth. Two-way analysis of data revealed significant effect (P > 0.01) of period, trace elements and their interactions. Expression of hepatic somatotropin, insulin-like growth factor-II and mucin gene was highest at 20(th) embryonic day but decreased during post-hatch periods. In ovo Zn or I supplemented embryos had higher expression of growth-related genes compared to the Se or un-injected control group. Expression of interleukin-6 was higher (P < 0.01) in in ovo I supplemented chicks (2.5-fold) but lower in the Zn and Se groups than in the un-injected control group. However, Zn and Se supplemented chicks had higher cellular immune gene expression. In vivo response to mitogen phytohaemaglutinin was also higher (P < 0.01) in Zn or Se supplemented chicks In ovo supplementation of Zn, I and Se did not improve the post-hatch growth, but increased growth-related gene expression. Iodine improved humoral immune gene expression whereas Zn and Se enhanced cell-mediated immune gene expression in broiler chickens. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Mutation of the XIST gene upregulates expression of X-linked genes but decreases the developmental rates of cloned male porcine embryos.

PubMed

Yang, Yang; Wu, Dan; Liu, Dewu; Shi, Junsong; Zhou, Rong; He, Xiaoyan; Quan, Jianping; Cai, Gengyuan; Zheng, Enqin; Wu, Zhenfang; Li, Zicong

2017-06-01

XIST is an X-linked, non-coding gene responsible for the cis induction of X-chromosome inactivation (XCI). Knockout of the XIST allele on an active X chromosome abolishes erroneous XCI and enhances the in vivo development of cloned mouse embryos by more than 10-fold. This study aimed to investigate whether a similar manipulation would improve cloning efficiency in pigs. A male, porcine kidney cell line containing an EGFP insert in exon 1 of the XIST gene, resulting in a knockout allele (XIST-KO), was generated by homologous recombination using transcription activator-like effector nucleases (TALENs). The expression of X-linked genes in embryos cloned from the XIST-KO kidney cells was significantly higher than in male embryos cloned from wild-type (WT) kidney cells, but remained lower than that of in vivo fertilization-produced counterparts. The XIST-KO cloned embryos also had a significantly lower blastocyst rate and a reduced full-term development rate compared to cloned WT embryos. These data suggested that while mutation of a XIST gene can partially rescue abnormal XCI, it cannot improve the developmental efficiency of cloned male porcine embryos-a deficiency that may be caused by incomplete rescue of abnormal XCI and/or by long-term drug selection of the XIST-KO nuclear donor cells, which might adversely affect the developmental efficiency of embryos created from them. © 2017 Wiley Periodicals, Inc.

Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

PubMed Central

2010-01-01

Background Infection by infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 × 44 K Agilent chicken custom oligo microarrays. Results Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi). Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB), cell cycle regulation (cyclin B2, CDK1, and CKI3), matrix metalloproteinases (MMPs) and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death. Conclusion The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections. PMID:20663125

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre-Implantation Stage.

PubMed

Camargo, L S A; Paludo, F; Pereira, M M; Wohlres-Viana, S; Gioso, M M; Carvalho, B C; Quintao, C C R; Viana, J H M

2016-02-01

Oocyte has been considered the major contributor for embryo thermo-tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo-tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo-sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post-insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat-shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat-shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock. © 2015 Blackwell Verlag GmbH.

Endo-β-mannanase and β-tubulin gene expression during the final phases of coffee seed maturation.

PubMed

Santos, F C; Clemente, A C S; Caixeta, F; Rosa, S D V F

2015-10-02

Coffee seeds begin to develop shortly after fertilization and can take 6 to 8 months to complete their formation, a period during which all the characteristics of the mature seed are determined, directly influencing physiological quality. However, little is known about the molecular mechanisms that act during coffee seed maturation. The objective of the current study was to analyze expression of the β-tubulin (TUB) and endo-β-mannanase (MAN) genes during different phases at the end of development and in different tissues of Coffea arabica seeds. The transcription levels of the TUB and MAN genes were quantified in a relative manner using qRT-PCR in whole seeds, and dissected into embryos and endosperms at different developmental stages. Greater expression of MAN was observed in whole seeds and in endosperms during the green stage, and in the embryo during the over-ripe stage. High TUB gene expression was observed in whole seeds during the green stage and, in the embryos, there were peaks in expression during the over-ripe stage. In endosperms, the peak of expression occurred in both the green stage and in the cherry stage. These results suggest participation of endo-β-mannanase during the initial seed developmental stages, and in the stages of physiological maturity in the embryo tissues. TUB gene expression varied depending on the developmental stage and section of seed analyzed, indicating the participation of β-tubulin during organogenesis and coffee seed maturation.

Ftx is dispensable for imprinted X-chromosome inactivation in preimplantation mouse embryos

PubMed Central

Soma, Miki; Fujihara, Yoshitaka; Okabe, Masaru; Ishino, Fumitoshi; Kobayashi, Shin

2014-01-01

X-chromosome inactivation (XCI) equalizes gene expression between the sexes by inactivating one of the two X chromosomes in female mammals. Xist has been considered as a major cis-acting factor that inactivates the paternally derived X chromosome (Xp) in preimplantation mouse embryos (imprinted XCI). Ftx has been proposed as a positive regulator of Xist. However, the physiological role of Ftx in female animals has never been studied. We recently reported that Ftx is located in the cis-acting regulatory region of the imprinted XCI and expressed from the inactive Xp, suggesting a role in the imprinted XCI mechanism. Here we examined the effects on imprinted XCI using targeted deletion of Ftx. Disruption of Ftx did not affect the survival of female embryos or expression of Xist and other X-linked genes in the preimplantation female embryos. Our results indicate that Ftx is dispensable for imprinted XCI in preimplantation embryos. PMID:24899465

Ftx is dispensable for imprinted X-chromosome inactivation in preimplantation mouse embryos.

PubMed

Soma, Miki; Fujihara, Yoshitaka; Okabe, Masaru; Ishino, Fumitoshi; Kobayashi, Shin

2014-06-05

X-chromosome inactivation (XCI) equalizes gene expression between the sexes by inactivating one of the two X chromosomes in female mammals. Xist has been considered as a major cis-acting factor that inactivates the paternally derived X chromosome (Xp) in preimplantation mouse embryos (imprinted XCI). Ftx has been proposed as a positive regulator of Xist. However, the physiological role of Ftx in female animals has never been studied. We recently reported that Ftx is located in the cis-acting regulatory region of the imprinted XCI and expressed from the inactive Xp, suggesting a role in the imprinted XCI mechanism. Here we examined the effects on imprinted XCI using targeted deletion of Ftx. Disruption of Ftx did not affect the survival of female embryos or expression of Xist and other X-linked genes in the preimplantation female embryos. Our results indicate that Ftx is dispensable for imprinted XCI in preimplantation embryos.

Targeted gene expression without a tissue-specific promoter: creating mosaic embryos using laser-induced single-cell heat shock

NASA Technical Reports Server (NTRS)

Halfon, M. S.; Kose, H.; Chiba, A.; Keshishian, H.

1997-01-01

We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of beta-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.

Oocyte developmental failure in response to elevated nonesterified fatty acid concentrations: mechanistic insights.

PubMed

Van Hoeck, V; Leroy, J L M R; Arias Alvarez, M; Rizos, D; Gutierrez-Adan, A; Schnorbusch, K; Bols, P E J; Leese, H J; Sturmey, R G

2013-01-01

Elevated plasma nonesterified fatty acid (NEFA) concentrations are associated with negative energy balance and metabolic disorders such as obesity and type II diabetes. Such increased plasma NEFA concentrations induce changes in the microenvironment of the ovarian follicle, which can compromise oocyte competence. Exposing oocytes to elevated NEFA concentrations during maturation affects the gene expression and phenotype of the subsequent embryo, notably prompting a disrupted oxidative metabolism. We hypothesized that these changes in the embryo are a consequence of modified energy metabolism in the oocyte. To investigate this, bovine cumulus oocyte complexes were matured under elevated NEFA conditions, and energy metabolism-related gene expression, mitochondrial function, and ultrastructure evaluated. It was found that expression of genes related to REDOX maintenance was modified in NEFA-exposed oocytes, cumulus cells, and resultant blastocysts. Moreover, the expression of genes related to fatty acid synthesis in embryos that developed from NEFA-exposed oocytes was upregulated. From a functional perspective, inhibition of fatty acid β-oxidation in maturing oocytes exposed to elevated NEFA concentrations restored developmental competence. There were no clear differences in mitochondrial morphology or oxygen consumption between treatments, although there was a trend for a higher mitochondrial membrane potential in zygotes derived from NEFA-exposed oocytes. These data show that the degree of mitochondrial fatty acid β-oxidation has a decisive impact on the development of NEFA-exposed oocytes. Furthermore, the gene expression data suggest that the resulting embryos adapt through altered metabolic strategies, which might explain the aberrant energy metabolism previously observed in these embryos originating from NEFA-exposed maturing oocytes.

Evolutionary conservation of vertebrate notochord genes in the ascidian Ciona intestinalis.

PubMed

Kugler, Jamie E; Passamaneck, Yale J; Feldman, Taya G; Beh, Jeni; Regnier, Todd W; Di Gregorio, Anna

2008-11-01

To reconstruct a minimum complement of notochord genes evolutionarily conserved across chordates, we scanned the Ciona intestinalis genome using the sequences of 182 genes reported to be expressed in the notochord of different vertebrates and identified 139 candidate notochord genes. For 66 of these Ciona genes expression data were already available, hence we analyzed the expression of the remaining 73 genes and found notochord expression for 20. The predicted products of the newly identified notochord genes range from the transcription factors Ci-XBPa and Ci-miER1 to extracellular matrix proteins. We examined the expression of the newly identified notochord genes in embryos ectopically expressing Ciona Brachyury (Ci-Bra) and in embryos expressing a repressor form of this transcription factor in the notochord, and we found that while a subset of the genes examined are clearly responsive to Ci-Bra, other genes are not affected by alterations in its levels. We provide a first description of notochord genes that are not evidently influenced by the ectopic expression of Ci-Bra and we propose alternative regulatory mechanisms that might control their transcription. Copyright 2008 Wiley-Liss, Inc.

Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo.

PubMed

Woda, Juliana M; Calzonetti, Teresa; Hilditch-Maguire, Paige; Duyao, Mabel P; Conlon, Ronald A; MacDonald, Marcy E

2005-08-18

Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdh(ex4/5) huntingtin deficient embryos. In the absence of huntingtin, expression of nutritive genes appears normal but E7.0-7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease.

DEFECTS IN CERVICAL VERTEBRAE IN BORIC ACID-EXPOSED RAT EMBRYOS ARE ASSOCIATED WITH ANTERIOR SHIFTS OF HOX GENE EXPRESSION DOMAINS

EPA Science Inventory

Defects in cervical vertebrae in boric acid-exposed rat embryos are associated with anterior shifts of hox gene expression domains

Nathalie Wery,1 Michael G. Narotsky,2 Nathalie Pacico,1 Robert J. Kavlock,2 Jacques J. Picard,1 AND Francoise Gofflot,1*
1Unit of Developme...

[The induction of neovascularization in chorioallantoic membrane of chicken embryos transfected by a recombinant plasmid containing human angiogenin gene].

PubMed

Avdeeva, S V; Khaĭdarova, N V; Logunov, D Iu; Neugodova, G L; Sevast'ianova, G A; Tarantul, V Z; Naroditskiĭ, B S

2003-01-01

A method was elaborated to evaluate the biological activity of expression products of gene in the plasmid vectors, which are crucial for the synthesis of growth factor of blood vessels. It was proven as possible that the chrioallantonic membrane (CAM) of chicken's embryos could be transfected by recombinant plasmids containing both the reporter and target genes. The efficiency of CAM transfection was assessed by a plasmid carrying the reporter gene of green fluorescent protein (GFP). Finally, it was demonstrated that, at an infiltration of the recombinant plasmid containing the human angiogenine gene, its expression products induce the neovascularization in the CAM cells of chicken's embryos and stimulate an accretion in vessels of the 1st, 2nd and 3d orders.

In Vivo Functional Genomic Studies of Sterol Carrier Protein-2 Gene in the Yellow Fever Mosquito

PubMed Central

Peng, Rong; Maklokova, Vilena I.; Chandrashekhar, Jayadevi H.; Lan, Que

2011-01-01

A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. The conventional method for delivery of DNA into insects is to inject the DNA directly into the embryos. Taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediates DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. Our method was able to introduce inducible gene expression vectors transiently into F0 mosquitoes to perform functional studies in vivo without transgenic lines. The high efficiency of expression knockdown was reproducible with more than 70% of the F0 individuals showed sufficient gene expression suppression (<30% of the controls' levels). At the cohort level, AeSCP-2 expression knockdown in early instar larvae resulted in detectable phenotypes of the expression deficiency such as high mortality, lowered fertility, and distorted sex ratio after induction of AeSCP-2 siRNA expression in vivo. The results further confirmed the important role of AeSCP-2 in the development and reproduction of A. aegypti. In this study, we proved that extrachromosaomal transient expression of an inducible gene from a DNA vector vertically delivered via vitellogenic females can be used to manipulate gene expression in F0 generation. This new method will be a simple and efficient tool for in vivo functional genomic studies in mosquitoes. PMID:21437205

The cellular and molecular biology of conifer embryogenesis.

PubMed

Cairney, John; Pullman, Gerald S

2007-01-01

Gymnosperms and angiosperms are thought to have evolved from a common ancestor c. 300 million yr ago. The manner in which gymnosperms and angiosperms form seeds has diverged and, although broad similarities are evident, the anatomy and cell and molecular biology of embryogenesis in gymnosperms, such as the coniferous trees pine, spruce and fir, differ significantly from those in the most widely studied model angiosperm Arabidopsis thaliana. Molecular analysis of signaling pathways and processes such as programmed cell death and embryo maturation indicates that many developmental pathways are conserved between angiosperms and gymnosperms. Recent genomics research reveals that almost 30% of mRNAs found in developing pine embryos are absent from other conifer expressed sequence tag (EST) collections. These data show that the conifer embryo differs markedly from other gymnosperm tissues studied to date in terms of the range of genes transcribed. Approximately 72% of conifer embryo-expressed genes are found in the Arabidopsis proteome and conifer embryos contain mRNAs of very similar sequence to key genes that regulate seed development in Arabidopsis. However, 1388 loblolly pine (Pinus taeda) embryo ESTs (11.4% of the collection) are novel and, to date, have been found in no other plant. The data imply that, in gymnosperm embryogenesis, differences in structure and development are achieved by subtle molecular interactions, control of spatial and temporal gene expression and the regulating agency of a few unique proteins.

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

PubMed

Trivedi, Vikas; Choi, Harry M T; Fraser, Scott E; Pierce, Niles A

2018-01-08

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization. © 2018. Published by The Company of Biologists Ltd.

Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene.

PubMed

Rao, Suryadevara S; Hildebrand, David

2009-10-01

The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al. in J Biol Chem 269:22156-22163, 1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.

Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos

PubMed Central

Fauque, Patricia; Jouannet, Pierre; Lesaffre, Corinne; Ripoche, Marie-Anne; Dandolo, Luisa; Vaiman, Daniel; Jammes, Hélène

2007-01-01

Background In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos. In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 promoter was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data. Results We show that: 1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium. 2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium. 3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16. Conclusion Compared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the H19 ICR and H19 promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the epigenetic disturbance of the utilized techniques. PMID:17949482

Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos.

PubMed

Fauque, Patricia; Jouannet, Pierre; Lesaffre, Corinne; Ripoche, Marie-Anne; Dandolo, Luisa; Vaiman, Daniel; Jammes, Hélène

2007-10-18

In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos. In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 promoter was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data. We show that: 1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium. 2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium. 3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16. Compared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the H19 ICR and H19 promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the epigenetic disturbance of the utilized techniques.

Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish.

PubMed

Ota, Satoshi; Taimatsu, Kiyohito; Yanagi, Kanoko; Namiki, Tomohiro; Ohga, Rie; Higashijima, Shin-Ichi; Kawahara, Atsuo

2016-10-11

The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes.

Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish

PubMed Central

Ota, Satoshi; Taimatsu, Kiyohito; Yanagi, Kanoko; Namiki, Tomohiro; Ohga, Rie; Higashijima, Shin-ichi; Kawahara, Atsuo

2016-01-01

The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes. PMID:27725766

HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yingying; State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080; Graduate University of Chinese Academy of Sciences, Beijing 100049

2010-07-02

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilizedmore » embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo.« less

Multicellular structures developing during maize microspore culture express endosperm and embryo-specific genes and show different embryogenic potentialities.

PubMed

Massonneau, Agnes; Coronado, Maria-José; Audran, Arthur; Bagniewska, Agnieszka; Mòl, Rafal; Testillano, Pilar S; Goralski, Grzegorz; Dumas, Christian; Risueño, Maria-Carmen; Matthys-Rochon, Elisabeth

2005-07-01

During maize pollen embryogenesis, a range of multicellular structures are formed. Using different approaches, the "nature" of these structures has been determined in terms of their embryogenic potential. In situ molecular identification techniques for gene transcripts and products, and a novel cell tracking system indicated the presence of embryogenic (embryo-like structures, ELS) and non-embryogenic (callus-like structures, CLS) structures that occurred for short periods within the cultures. Some multicellular structures with a compact appearance generated embryos. RT-PCR and fluorescence in situ hybridization (FISH) with confocal microscopy techniques using specific gene markers of the endosperm (ZmESR2, ZmAE3) and embryo (LTP2 and ZmOCL1, ZmOCL3) revealed "embryo" and "endosperm" potentialities in these various multicellular structures present in the cultures. The results presented here showed distinct and specific patterns of gene expression. Altogether, the results demonstrate the presence of different molecules on both embryonic and non-embryonic structures. Their possible roles are discussed in the context of a parallel between embryo/endosperm interactions in planta and embryonic and non-embryonic structure interrelations under in vitro conditions.

Dissection of Arabidopsis NCED9 promoter regulatory regions reveals a role for ABA synthesized in embryos in the regulation of GA-dependent seed germination.

PubMed

Seo, Mitsunori; Kanno, Yuri; Frey, Anne; North, Helen M; Marion-Poll, Annie

2016-05-01

Nine-cis-epoxycarotenoid dioxygenase (NCED) catalyzes the key step of abscisic acid (ABA) biosynthesis. There are five genes encoding NCED in Arabidopsis, which differentially regulate ABA biosynthesis in a spatiotemporal manner in response to endogenous and environmental stimuli. Previous studies have shown that NCED9 is expressed in testa and embryos during seed development. In the present study, we have identified promoter regions required for the expression of NCED9 in testa and embryos, respectively. Electrophoretic mobility shift assays (EMSA) and yeast one-hybrid (Y1H) assays showed that several homeodomain-leucine zipper (HD-Zip) proteins, namely ATHBs, bound to the sequence required for expression of NCED9 in testa, suggesting that they redundantly regulate NCED9 expression. By expressing the NCED9 gene under the control of a deleted NCED9 promoter in an nced9 mutant expression was limited to embryos. Transformants were complemented for the paclobutrazol resistant germination phenotype of the mutant, suggesting that the ABA synthesis mediated by NCED9 in embryos plays an important role in the regulation of gibberellin (GA)-dependent seed germination. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.

PubMed

Hribal, R; Jewgenow, K; Braun, B C; Comizzoli, P

2013-04-01

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. © 2012 Blackwell Verlag GmbH.

Expression of putative sex-determining genes during the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina.

PubMed

Rhen, T; Metzger, K; Schroeder, A; Woodward, R

2007-01-01

Modes of sex determination are quite variable in vertebrates. The developmental decision to form a testis or an ovary can be influenced by one gene, several genes, environmental variables, or a combination of these factors. Nevertheless, certain morphogenetic aspects of sex determination appear to be conserved in amniotes. Here we clone fragments of nine candidate sex-determining genes from the snapping turtle Chelydra serpentina, a species with temperature-dependent sex determination (TSD). We then analyze expression of these genes during the thermosensitive period of gonad development. In particular, we compare gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature. Expression of Dmrt1 and Sox9 mRNA increased gradually at the male-producing temperature, but was suppressed at the female-producing temperature. This finding suggests that Dmrt1 and Sox9 play a role in testis development. In contrast, expression of aromatase, androgen receptor (Ar), and Foxl2 mRNA was constant at the male-producing temperature, but increased several-fold in embryos at the female-producing temperature. Aromatase, Ar, and Foxl2 may therefore play a role in ovary development. In addition, there was a small temperature effect on ER alpha expression with lower mRNA levels found in embryos at the female-producing temperature. Finally, Dax1, Fgf9, and SF-1 were not differentially expressed during the sex-determining period, suggesting these genes are not involved in sex determination in the snapping turtle. Comparison of gene expression profiles among amniotes indicates that Dmrt1 and Sox9 are part of a core testis-determining pathway and that Ar, aromatase, ER alpha, and Foxl2 are part of a core ovary-determining pathway. 2007 S. Karger AG, Basel

High-Throughput Sequencing of Small RNA Transcriptomes in Maize Kernel Identifies miRNAs Involved in Embryo and Endosperm Development.

PubMed

Xing, Lijuan; Zhu, Ming; Zhang, Min; Li, Wenzong; Jiang, Haiyang; Zou, Junjie; Wang, Lei; Xu, Miaoyun

2017-12-14

Maize kernel development is a complex biological process that involves the temporal and spatial expression of many genes and fine gene regulation at a transcriptional and post-transcriptional level, and microRNAs (miRNAs) play vital roles during this process. To gain insight into miRNA-mediated regulation of maize kernel development, a deep-sequencing technique was used to investigate the dynamic expression of miRNAs in the embryo and endosperm at three developmental stages in B73. By miRNA transcriptomic analysis, we characterized 132 known miRNAs and six novel miRNAs in developing maize kernel, among which, 15 and 14 miRNAs were commonly differentially expressed between the embryo and endosperm at 9 days after pollination (DAP), 15 DAP and 20 DAP respectively. Conserved miRNA families such as miR159, miR160, miR166, miR390, miR319, miR528 and miR529 were highly expressed in developing embryos; miR164, miR171, miR393 and miR2118 were highly expressed in developing endosperm. Genes targeted by those highly expressed miRNAs were found to be largely related to a regulation category, including the transcription, macromolecule biosynthetic and metabolic process in the embryo as well as the vitamin biosynthetic and metabolic process in the endosperm. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that these miRNAs displayed a negative correlation with the levels of their corresponding target genes. Importantly, our findings revealed that members of the miR169 family were highly and dynamically expressed in the developing kernel, which will help to exploit new players functioning in maize kernel development.

Toxoplasma Gondii Infection of Chicken Embryos Causes Retinal Changes and Modulates HSP90B1 Gene Expression: A Promising Ocular Toxoplasmosis Model.

PubMed

Nasaré, Alex M; Tedesco, Roberto C; Cristovam, Priscila C; Cenedese, Marcos A; Galisteo, Andrés J; Andrade, Heitor F; Gomes, José Álvaro P; Guimarães, Érik V; Barbosa, Helene S; Alonso, Luis G

2015-12-01

HSP90B1 is a gene that codifies heat shock protein 108 (HSP108) that belongs to a group of proteins induced under stress situation, and it has close relation with the nervous system, especially in the retina. Toxoplasma gondii causes ocular toxoplasmosis that has been associated with a late manifestation of the congenital toxoplasmosis although experimental models show that morphological alterations are already present during embryological development. Here, we used 18 eyes of Gallus domesticus embryos in 7th and 20th embryonic days to establish a model of congenital ocular toxoplasmosis, experimentally infected in its fifth day correlating with HSP90B1 gene expression. Embryos' eyes were histologically evaluated, and gene expression was performed by real-time polymerase chain reaction (PCR). Our data showed parasite present in the choroid, unusual migration of retinal pigment epithelium, and chorioretinal scars, and a tendency to a lower expression of the HSP90B1 gene upon experimental infection. This is a promising model to better understand T. gondii etiopathogeny.

Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6.

PubMed

Liu, Wan-Ju; Reece-Hoyes, John S; Walhout, Albertha J M; Eisenmann, David M

2014-05-13

Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, we aimed to identify transcription factors necessary for lin-39 expression in the VPCs, and in particular sought factors that initiate lin-39 expression in the embryo. We used the yeast one-hybrid (Y1H) method to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and we characterized several factors by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations in lin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site we identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs. These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust.

Gestation-related gene expression and protein localization in endometrial tissue of Suffolk and Cheviot ewes at gestation Day 19, after transfer of Suffolk or Cheviot embryos.

PubMed

Sequeira, M; Pain, S J; de Brun, V; Meikle, A; Kenyon, P R; Blair, H T

2016-10-01

The objective of this study was to investigate the gene expression of progesterone and estrogen receptor α (PR, ERα), insulin-like growth factor (IGF) 1, IGF-2, their receptor (IGFR1), IGF-binding proteins (BP) 1 to 6, insulin receptor, adiponectin receptors (AdipoR1/2), cyclooxygenase 2 (PTGS2), mucin 1 and to localize PR, ERα, IGF-1, IGFR1, PTGS2, and proliferating cellular nuclear antigen (PCNA) in the endometrium of pregnant (Day 19) Suffolk and Cheviot ewes carrying Suffolk and Cheviot embryos transferred within and reciprocally between breeds. Gene expression was determined by real-time quantitative polymerase chain reaction (RT-qPCR), and antigen determination was measured by immunohistochemistry in the luminal epithelium (LE), superficial and deep glands (SG, DG, respectively) and superficial and deep stroma. Gene expression of PR, IGF-1, IGFBP2, and IGFBP5 was higher in Suffolk than that in Cheviot ewes (P < 0.05). Greater abundance of IGF-2 and IGBP3 expression was found in Cheviot ewes carrying Cheviot embryos than Cheviot ewes carrying Suffolk embryos (P < 0.05). No staining for PR and ERα was observed in the LE, very scarce staining in SG and DG, whereas positive staining was observed in both superficial and deep stroma. No differences were found for PR staining, but Cheviot ewes had higher ERα staining intensity than Suffolk ewes (P < 0.05). Positive staining for IGF-1 was observed in all cell types except DG, and staining of IGFR1 was observed in all cell types. No differences among groups in staining were found for IGF-1 or IGFR1 in any cell type. Positive staining of PTGS2 was observed in LE and SG in all groups. An interaction between ewe and embryo breed affected PTGS2 staining (P < 0.05), whereby Cheviot ewes carrying Suffolk embryos had a lower PTGS2 staining than Suffolk ewes carrying Suffolk embryos. Positive staining of PCNA was found in LE and SG. Suffolk ewes carrying Suffolk embryos showed lower PCNA immunostaining than Cheviot ewes carrying Suffolk embryos (P < 0.05), whereas no differences were observed in ewes carrying Cheviot embryos. This study showed that gestation-related protein expression in the endometrium of Suffolk and Cheviot ewes is affected by both ewe and embryo breed at Day 19 of pregnancy. Copyright © 2016 Elsevier Inc. All rights reserved.

Genome and epigenome engineering CRISPR toolkit for in vivo modulation of cis-regulatory interactions and gene expression in the chicken embryo.

PubMed

Williams, Ruth M; Senanayake, Upeka; Artibani, Mara; Taylor, Gunes; Wells, Daniel; Ahmed, Ahmed Ashour; Sauka-Spengler, Tatjana

2018-02-23

CRISPR/Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present an optimised, adaptable toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise novel efficient guide-RNA mini expression vectors utilising chick U6 promoters, provide a strategy for rapid somatic gene knockout and establish a protocol for evaluation of mutational penetrance by targeted next-generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo . © 2018. Published by The Company of Biologists Ltd.

A homeobox gene involved in node, notochord and neural plate formation of chick embryos.

PubMed

Stein, S; Kessel, M

1995-01-01

We have isolated a chicken cDNA clone, Cnot, resembling in sequence and expression pattern the Xenopus homeobox gene Xnot. The major, early transcription domains of Cnot are the node, the notochord and prenodal and postnodal neural plate caudal from the prospective hindbrain level. All these cell populations appear to be descendants of the Cnot-expressing cells of the node, suggesting a cell lineage relationship. After the onset of somitogenesis, a second, independent expression domain appears in the neural folds at the prospective mid- and forebrain levels, and further transcripts are found in the epiphysis, the ventral diencephalon, the preoral gut and the limb buds. Transplantation of nodes from extended streak embryos leads to the formation of ectopic notochords, which express Cnot in the typical, cranially decreasing gradient. Transplantation of young nodes to young hosts has previously been described to induce secondary embryos. We observed that secondary chick embryos express Cnot in node derived, notochord-like structures and in the anterior neural plate, similar to the domains seen in primary embryos. However, expression was absent from the posterior neural plate, which in the induction experiments is excluded from the node lineage. This finding corroborates our initial conclusion about a cell lineage relationship between node, notochord, and neural plate defined by Cnot expression. The midline mesoderm of vertebrate embryos consists of two tissues, the prechordal mesoderm and the notochord. The anterior notochord, the head process, may represent an intermediate form. The transition from prechordal to chordal mesoderm can be followed by the expression of the two marker homeobox genes goosecoid and Cnot, first in the primitive streak, and then in the head process. We suggest that expression of goosecoid or Cnot is involved in the specification of a prechordal or notochordal identity, respectively. A transition from goosecoid to Cnot expression may proceed, while cells are still in the epiblast, but not after becoming mesodermal. A molecular coding of axial positions in the midline mesoderm may occur by specific homeobox genes, similar to the situation in the neural tube and the somitic mesoderm.

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

PubMed Central

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF. PMID:26053554

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

PubMed

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

Transcriptional Innate Immune Response of the Developing Chicken Embryo to Newcastle Disease Virus Infection

PubMed Central

Schilling, Megan A.; Katani, Robab; Memari, Sahar; Cavanaugh, Meredith; Buza, Joram; Radzio-Basu, Jessica; Mpenda, Fulgence N.; Deist, Melissa S.; Lamont, Susan J.; Kapur, Vivek

2018-01-01

Traditional approaches to assess the immune response of chickens to infection are through animal trials, which are expensive, require enhanced biosecurity, compromise welfare, and are frequently influenced by confounding variables. Since the chicken embryo becomes immunocompetent prior to hatch, we here characterized the transcriptional response of selected innate immune genes to Newcastle disease virus (NDV) infection in chicken embryos at days 10, 14, and 18 of embryonic development. The results suggest that the innate immune response 72 h after challenge of 18-day chicken embryo is both consistent and robust. The expression of CCL5, Mx1, and TLR3 in lung tissues of NDV challenged chicken embryos from the outbred Kuroiler and Tanzanian local ecotype lines showed that their expression was several orders of magnitude higher in the Kuroiler than in the local ecotypes. Next, the expression patterns of three additional innate-immunity related genes, IL-8, IRF-1, and STAT1, were examined in the highly congenic Fayoumi (M5.1 and M15.2) and Leghorn (Ghs6 and Ghs13) sublines that differ only at the microchromosome bearing the major histocompatibility locus. The results show that the Ghs13 Leghorn subline had a consistently higher expression of all genes except IL-8 and expression seemed to be subline-dependent rather than breed-dependent, suggesting that the innate immune response of chicken embryos to NDV infection may be genetically controlled by the MHC-locus. Taken together, the results suggest that the chicken embryo may represent a promising model to studying the patterns and sources of variation of the avian innate immune response to infection with NDV and related pathogens. PMID:29535762

Crosstalk between AhR and wnt/β-catenin signal pathways in the cardiac developmental toxicity of PM2.5 in zebrafish embryos.

PubMed

Zhang, Hang; Yao, Yugang; Chen, Yang; Yue, Cong; Chen, Jiahong; Tong, Jian; Jiang, Yan; Chen, Tao

2016-04-29

Recent studies have shown an association between congenital heart defects and air fine particle matter (PM2.5), but the molecular mechanisms remain elusive. It is well known that a number of organic compounds in PM2.5 can act as AhR agonists, and activation of AhR can antagonize Wnt/β-catenin signaling. Therefore, we hypothesized that PM2.5 could activate AhR and then repress the expression of wnt/β-catenin targeted genes essential for cardiogenesis, resulting in heart defects. To test this hypothesis, we investigated the effects of extractable organic matter (EOM) from PM2.5 on AhR and Wnt/β-catenin signal pathways in zebrafish embryos. We confirmed that EOM could cause malformations in the heart and decreased heart rate in zebrafish embryos at 72hpf, and found that the EOM-induced heart defects were rescued in embryos co-exposed with EOM plus AhR antagonist CH223191 or β-catenin agonist CHIR99021. We further found that EOM had increased the expression levels of AhR targeted genes (Cyp1a1, Cyp1b1 and Ahrra) and reduced the mRNA levels of β-catenin targeted genes (axin2, nkx2.5 and sox9b). The mRNA expression level of Rspo2, a β-catenin upstream gene, was also decreased in embryos exposed to EOM. Supplementation with CH223191 or CHIR99021 attenuated most of the EOM-induced expression changes of genes involved in both AhR and wnt/β-catenin signal pathways. However, the mRNA expression level of AhR inhibitor Ahrrb, which did not change by EOM treatment alone, was increased in embryos co-exposed to EOM plus CH223191 or CHIR99021. We conclude that the activation of AhR by EOM from PM2.5 might repress wnt/β-catenin signaling, leading to heart defects in zebrafish embryos. Furthermore, our results indicate that the cardiac developmental toxicity of PM2.5 might be prevented by targeting AhR or wnt/β-catenin signaling. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

PubMed

Hodar, Christian; Zuñiga, Alejandro; Pulgar, Rodrigo; Travisany, Dante; Chacon, Carlos; Pino, Michael; Maass, Alejandro; Cambiazo, Verónica

2014-02-10

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-β superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica. Copyright © 2013 Elsevier B.V. All rights reserved.

Activation of the hexosamine pathway causes oxidative stress and abnormal embryo gene expression: involvement in diabetic teratogenesis.

PubMed

Horal, Melissa; Zhang, Zhiquan; Stanton, Robert; Virkamäki, Antti; Loeken, Mary R

2004-08-01

Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific biochemical pathways responsible for oxidative stress have not been fully elucidated. The hexosamine pathway is activated in many tissues during diabetes and could contribute to oxidative stress by inhibiting the pentose shunt pathway, thereby diminishing production of the cellular antioxidant, reduced glutathione (GSH). To test the hypothesis that activation of the hexosamine pathway might contribute to the teratogenic effects of diabetic pregnancy, pregnant mice were injected with glucose, to induce hyperglycemia, or glucosamine, to directly activate the hexosamine pathway. Embryo tissue fragments were also cultured in physiological glucose, high glucose, or physiological glucose plus glucosamine, to test effects on oxidative stress and embryo gene expression. Glucosamine increased hexosamine synthesis and inhibited pentose shunt activity. There was a trend for transient hyperglycemia to have the same effects, but they did not reach statistical significance. However, both glucose and glucosamine significantly decreased GSH, and increased oxidative stress, as indicated by 2',7'-dichloro-dihydrofluorescein fluorescence. Glucose and glucosamine inhibited expression of Pax-3, a gene required for neural tube closure both in vivo and in vitro, and increased neural tube defects (NTDs) in vivo; these effects were prevented by GSH ethyl ester. High glucose and glucosamine inhibited Pax-3 expression by embryo culture, but culture in glutamine-free media to block the hexosamine pathway prevented the inhibition of Pax-3 expression by high glucose. Activation of the hexosamine pathway causes oxidative stress through depletion of GSH and consequent disruption of embryo gene expression. Activation of this pathway may contribute to diabetic teratogenesis.

Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

PubMed

Yu, Dawei; Zhang, Shoufeng; Du, Weihua; Zhang, Jinxia; Fan, Zongxing; Hao, Haisheng; Liu, Yan; Zhao, Xueming; Qin, Tong; Zhu, Huabin

2014-01-01

Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α) (without secretory signal sequence) gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT). Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9%) became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR) and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS), which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

Studying the effects of genistein on gene expression of fish embryos as an alternative testing approach for endocrine disruption.

PubMed

Schiller, Viktoria; Wichmann, Arne; Kriehuber, Ralf; Muth-Köhne, Elke; Giesy, John P; Hecker, Markus; Fenske, Martina

2013-01-01

Assessment of endocrine disruption currently relies on testing strategies involving adult vertebrates. In order to minimize the use of animal tests according to the 3Rs principle of replacement, reduction and refinement, we propose a transcriptomics and fish embryo based approach as an alternative to identify and analyze an estrogenic activity of environmental chemicals. For this purpose, the suitability of 48 h and 7 days post-fertilization zebrafish and medaka embryos to test for estrogenic disruption was evaluated. The embryos were exposed to the phytoestrogen genistein and subsequently analyzed by microarrays and quantitative real-time PCR. The functional analysis showed that the genes affected related to multiple metabolic and signaling pathways in the early fish embryo, which reflect the known components of genistein's mode of actions, like apoptosis, estrogenic response, hox gene expression and steroid hormone synthesis. Moreover, the transcriptomic data also suggested a thyroidal mode of action and disruption of the nervous system development. The parallel testing of two fish species provided complementary data on the effects of genistein at gene expression level and facilitated the separation of common from species-dependent effects. Overall, the study demonstrated that combining fish embryo testing with transcriptomics can deliver abundant information about the mechanistic effects of endocrine disrupting chemicals, rendering this strategy a promising alternative approach to test for endocrine disruption in a whole organism in-vitro scale system. Copyright © 2012 Elsevier Inc. All rights reserved.

Chromosome doubling to overcome the chrysanthemum cross barrier based on insight from transcriptomic and proteomic analyses.

PubMed

Zhang, Fengjiao; Hua, Lichun; Fei, Jiangsong; Wang, Fan; Liao, Yuan; Fang, Weimin; Chen, Fadi; Teng, Nianjun

2016-08-09

Cross breeding is the most commonly used method in chrysanthemum (Chrysanthemum morifolium) breeding; however, cross barriers always exist in these combinations. Many studies have shown that paternal chromosome doubling can often overcome hybridization barriers during cross breeding, although the underlying mechanism has seldom been investigated. In this study, we performed two crosses: C. morifolium (pollen receptor) × diploid C. nankingense (pollen donor) and C. morifolium × tetraploid C. nankingense. Seeds were obtained only from the latter cross. RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) were used to investigate differentially expressed genes and proteins during key embryo development stages in the latter cross. A previously performed cross, C. morifolium × diploid C. nankingense, was compared to our results and revealed that transcription factors (i.e., the agamous-like MADS-box protein AGL80 and the leucine-rich repeat receptor protein kinase EXS), hormone-responsive genes (auxin-binding protein 1), genes and proteins related to metabolism (ATP-citrate synthase, citrate synthase and malate dehydrogenase) and other genes reported to contribute to embryo development (i.e., LEA, elongation factor and tubulin) had higher expression levels in the C. morifolium × tetraploid C. nankingense cross. In contrast, genes related to senescence and cell death were down-regulated in the C. morifolium × tetraploid C. nankingense cross. The data resources helped elucidate the gene and protein expression profiles and identify functional genes during different development stages. When the chromosomes from the male parent are doubled, the genes contributing to normal embryo developmentare more abundant. However, genes with negative functions were suppressed, suggesting that chromosome doubling may epigenetically inhibit the expression of these genes and allow the embryo to develop normally.

Combined lineage mapping and gene expression profiling of embryonic brain patterning using ultrashort pulse microscopy and image registration

NASA Astrophysics Data System (ADS)

Gibbs, Holly C.; Dodson, Colin R.; Bai, Yuqiang; Lekven, Arne C.; Yeh, Alvin T.

2014-12-01

During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.

Combined lineage mapping and gene expression profiling of embryonic brain patterning using ultrashort pulse microscopy and image registration.

PubMed

Gibbs, Holly C; Dodson, Colin R; Bai, Yuqiang; Lekven, Arne C; Yeh, Alvin T

2014-12-01

During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.

Brachyury expression in tailless Molgulid ascidian embryos.

PubMed

Takada, Norio; York, Jonathan; Davis, J Muse; Schumpert, Brenda; Yasuo, Hitoyoshi; Satoh, Nori; Swalla, Billie J

2002-01-01

The T-box transcription factor gene Brachyury is important for the differentiation of notochord in all chordates, including the ascidians Halocynthia roretzi and Ciona intestinalis. We isolated Brachyury from molgulid ascidians, which have evolved tailless larvae multiple times independently, and found the genes appear functional by cDNA sequence analyses. We then compared the expression of Mocu-Bra in tailed Molgula oculata embryos to two tailless species, Molgula occulta (Mocc-Bra) and Molgula tectiformis (Mt-Bra). Here we show that both tailless species express Brachyury in the notochord lineage during embryogenesis. Initial expression of Mocu-Bra is normal in tailed M. oculata embryos; 10 precursor notochord cells divide twice to result in 40 notochord cells that converge and extend to make a notochord down the center of the tail. In contrast, in tailless Molgula occulta, Mocc-Bra expression disappears prematurely, and there is only one round of division, resulting in 20 cells in the final notochord lineage that never converge or extend. In M. occulta x M. oculata hybrid embryos, expression of Mocu-Bra is prolonged, and the embryos form a tail with 20 notochord cells that converge and extend normally. However, in Molgula tectiformis, a different tailless ascidian, Mt-Bra was expressed only in the 10 notochord precursor cells, which never divide, converge, or extend. In summary, neither Brachyury function nor the early establishment of the notochord lineage appears to be impaired in tailless embryos. In light of these results, we are continuing to investigate how and why notochord development is lost in tailless molgulid ascidian embryos.

Placental alterations in structure and function in intra-uterine growth-retarded horses.

PubMed

Robles, M; Peugnet, P M; Valentino, S A; Dubois, C; Dahirel, M; Aubrière, M-C; Reigner, F; Serteyn, D; Wimel, L; Couturier-Tarrade, A; Chavatte-Palmer, P

2018-05-01

Following embryo transfer (ET), the size and breed of the recipient mare can affect fetal development and subsequent post natal growth rate and insulin sensitivity in foals. To investigate placental adaptation in pregnancies where increased or restricted fetal growth was induced through ET between Pony, Saddlebred and Draught horses. In vivo experiment. Control Pony (P, n = 21) and Saddlebred (S, n = 28) pregnancies were obtained by artificial insemination. Increased pregnancies were obtained by transferring Pony (P-D, n = 6) and Saddlebred (S-D, n = 8) embryos into Draught mares. Restricted pregnancies were obtained by transferring Saddlebred embryos into Pony mares (S-P, n = 6). Placental weight and surface were recorded and samples collected for stereology and analysis of expression of genes involved in placental growth, vascularisation and nutrient transport. Data were analysed by linear model. S-P foals were growth retarded when compared with controls despite increased gestational length. Placental weight was reduced but placental surface density and volume fraction were increased. Placental expression of genes involved in growth and development and nutrient transfer was strongly reduced. In contrast, placental size and weight were increased in enhanced growth P-D and S-D foals. The trophoblastic surface density and the allantoic vessels surface density were decreased in P-D and S-D, respectively, both with very few modifications in gene expression. Control embryos were produced by artificial insemination whereas experimental embryos were produced by ET. Placental structure and gene expression are modified after ET into a smaller or larger breed than that of the embryo. These adaptations contribute to the observed phenotype of foal growth restriction or enhanced growth at birth. © 2017 EVJ Ltd.

Effect of recipient breed on delivery rate of cloned miniature pig.

PubMed

Koo, Ok Jae; Park, Hee Jung; Kwon, Dae Kee; Kang, Jung Taek; Jang, Goo; Lee, Byeong Chun

2009-08-01

The miniature pig is regarded as a better organ donor breed for xenotransplantation than other pig breeds because the size of their organs is similar to that of humans. To improve efficiency of cloned miniature pig production, we analysed the effect of breed difference between donor cells and embryo recipients on pregnancy rate and delivery rate. Cloned porcine embryos derived from domestic or miniature pig donor cells were transferred to domestic or miniature recipient pigs. Delivery rate was significantly higher when embryos reconstructed with miniature pig donor cells were transferred to miniature pig recipients as compared with that of embryos transferred to domestic pig recipients. However, pregnancy rates were similar between the two groups. The breed of donor cells, but not of embryo recipients, seems likely to affect litter size. From a 13 610 gene cDNA microarray, 1551 (11.7%) genes showed significantly different levels of expression between the fetuses of the two breeds. Vascular endothelial growth factor and c-kit ligand genes related to implantation and maintenance of pregnancy were significantly down-regulated in miniature pigs. In conclusion, the differential gene expression in fetuses interferes with proper fetal/maternal interactions, and results in late-stage pregnancy loss. Our results indicate that the miniature pig is the preferred embryo recipient breed than domestic pig for producing cloned miniature piglets.

Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression

PubMed Central

QI, Xiaonan; LI, Huatao; CONG, Xia; WANG, Xin; JIANG, Zhongling; CAO, Rongfeng; TIAN, Wenru

2016-01-01

Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation. PMID:27478062

Parental vitamin deficiency affects the embryonic gene expression of immune-, lipid transport- and apolipoprotein genes

NASA Astrophysics Data System (ADS)

Skjærven, Kaja H.; Jakt, Lars Martin; Dahl, John Arne; Espe, Marit; Aanes, Håvard; Hamre, Kristin; Fernandes, Jorge M. O.

2016-10-01

World Health Organization is concerned for parental vitamin deficiency and its effect on offspring health. This study examines the effect of a marginally dietary-induced parental one carbon (1-C) micronutrient deficiency on embryonic gene expression using zebrafish. Metabolic profiling revealed a reduced 1-C cycle efficiency in F0 generation. Parental deficiency reduced the fecundity and a total of 364 genes were differentially expressed in the F1 embryos. The upregulated genes (53%) in the deficient group were enriched in biological processes such as immune response and blood coagulation. Several genes encoding enzymes essential for the 1-C cycle and for lipid transport (especially apolipoproteins) were aberrantly expressed. We show that a parental diet deficient in micronutrients disturbs the expression in descendant embryos of genes associated with overall health, and result in inherited aberrations in the 1-C cycle and lipid metabolism. This emphasises the importance of parental micronutrient status for the health of the offspring.

EXTRA-EMBRYONIC-SPECIFIC IMPRINTED EXPRESSION IS RESTRICTED TO DEFINED LINEAGES IN THE POST-IMPLANTATION EMBRYO

PubMed Central

Hudson, Quanah J.; Seidl, Christine I.M.; Kulinski, Tomasz M.; Huang, Ru; Warczok, Katarzyna E.; Bittner, Romana; Bartolomei, Marisa S.; Barlow, Denise P.

2011-01-01

A subset of imprinted genes in the mouse have been reported to show imprinted expression that is restricted to the placenta, a short-lived extra-embryonic organ. Notably these so-called 'placental-specific' imprinted genes are expressed from both parental alleles in embryo and adult tissues. The placenta is an embryonic-derived organ that is closely associated with maternal tissue and as a consequence, maternal contamination can be mistaken for maternal-specific imprinted expression. The complexity of the placenta, which arises from multiple embryonic lineages, poses additional problems in accurately assessing allele-specific repressive epigenetic modifications in genes that also show lineage-specific silencing in this organ. These problems require that extra evidence be obtained to support the imprinted status of genes whose imprinted expression is restricted to the placenta. We show here that the extra-embryonic visceral yolk sac (VYS), a nutritive membrane surrounding the developing embryo, shows a similar 'extra-embryonic-lineage-specific' pattern of imprinted expression. We present an improved enzymatic technique for separating the bilaminar VYS and show that this pattern of imprinted expression is restricted to the endoderm layer. Finally, we show that VYS 'extra-embryonic-lineage-specific' imprinted expression is regulated by DNA methylation in a similar manner as shown for genes showing multi-lineage imprinted expression in extra-embryonic, embryonic and adult tissues. These results show that the VYS is an improved model for studying the epigenetic mechanisms regulating extra-embryonic-lineage-specific imprinted expression. PMID:21354127

Identification of low Ca(2+) stress-induced embryo apoptosis response genes in Arachis hypogaea by SSH-associated library lift (SSHaLL).

PubMed

Chen, Hua; Zhang, Chong; Cai, Tie Cheng; Deng, Ye; Zhou, Shuangbiao; Zheng, Yixiong; Ma, Shiwei; Tang, Ronghua; Varshney, Rajeev K; Zhuang, Weijian

2016-02-01

Calcium is a universal signal in the regulation of wide aspects in biology, but few are known about the function of calcium in the control of early embryo development. Ca(2+) deficiency in soil induces early embryo abortion in peanut, producing empty pods, which is a general problem; however, the underlying mechanism remains unclear. In this study, embryo abortion was characterized to be caused by apoptosis marked with cell wall degradation. Using a method of SSH cDNA libraries associated with library lift (SSHaLL), 62 differentially expressed genes were isolated from young peanut embryos. These genes were classified to be stress responses, catabolic process, carbohydrate and lipid metabolism, embryo morphogenesis, regulation, etc. The cell retardation with cell wall degradation was caused by up-regulated cell wall hydrolases and down-regulated cellular synthases genes. HsfA4a, which was characterized to be important to embryo development, was significantly down-regulated under Ca(2+) -deficient conditions from 15 days after pegging (DAP) to 30 DAP. Two AhCYP707A4 genes, encoding abscisic acid (ABA) 8'-hydroxylases, key enzymes for ABA catabolism, were up-regulated by 21-fold under Ca(2+) -deficient conditions upstream of HsfA4a, reducing the ABA level in early embryos. Over-expression of AhCYP707A4 in Nicotiana benthamiana showed a phenotype of low ABA content with high numbers of aborted embryos, small pods and less seeds, which confirms that AhCYP707A4 is a key player in regulation of Ca(2+) deficiency-induced embryo abortion via ABA-mediated apoptosis. The results elucidated the mechanism of low Ca(2+) -induced embryo abortion and described the method for other fields of study. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo

PubMed Central

Woda, Juliana M; Calzonetti, Teresa; Hilditch-Maguire, Paige; Duyao, Mabel P; Conlon, Ronald A; MacDonald, Marcy E

2005-01-01

Background Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdhex4/5 huntingtin deficient embryos. Results In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. Conclusion Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease. PMID:16109169

Developmental Competence and Epigenetic Profile of Porcine Embryos Produced by Two Different Cloning Methods.

PubMed

Liu, Ying; Lucas-Hahn, Andrea; Petersen, Bjoern; Li, Rong; Hermann, Doris; Hassel, Petra; Ziegler, Maren; Larsen, Knud; Niemann, Heiner; Callesen, Henrik

2017-06-01

The "Dolly" based cloning (classical nuclear transfer, [CNT]) and the handmade cloning (HMC) are methods that are nowadays routinely used for somatic cloning of large domestic species. Both cloning protocols share several similarities, but differ with regard to the required in vitro culture, which in turn results in different time intervals until embryo transfer. It is not yet known whether the differences between cloned embryos from the two protocols are due to the cloning methods themselves or the in vitro culture, as some studies have shown detrimental effects of in vitro culture on conventionally produced embryos. The goal of this study was to unravel putative differences between two cloning methods, with regard to developmental competence, expression profile of a panel of developmentally important genes and epigenetic profile of porcine cloned embryos produced by either CNT or HMC, either with (D5 or D6) or without (D0) in vitro culture. Embryos cloned by these two methods had a similar morphological appearance on D0, but displayed different cleavage rates and different quality of blastocysts, with HMC embryos showing higher blastocyst rates (HMC vs. CNT: 35% vs. 10%, p < 0.05) and cell numbers per blastocyst (HMC vs. CNT: 31 vs. 23 on D5 and 42 vs. 18 on D6, p < 0.05) compared to CNT embryos. With regard to histone acetylation and gene expression, CNT and HMC derived cloned embryos were similar on D0, but differed on D6. In conclusion, both cloning methods and the in vitro culture may affect porcine embryo development and epigenetic profile. The two cloning methods essentially produce embryos of similar quality on D0 and after 5 days in vitro culture, but thereafter both histone acetylation and gene expression differ between the two types of cloned embryos.

MATER protein expression and intracellular localization throughout folliculogenesis and preimplantation embryo development in the bovine

PubMed Central

Pennetier, Sophie; Perreau, Christine; Uzbekova, Svetlana; Thélie, Aurore; Delaleu, Bernadette; Mermillod, Pascal; Dalbiès-Tran, Rozenn

2006-01-01

Background Mater (Maternal Antigen that Embryos Require), also known as Nalp5 (NACHT, leucine rich repeat and PYD containing 5), is an oocyte-specific maternal effect gene required for early embryonic development beyond the two-cell stage in mouse. We previously characterized the bovine orthologue MATER as an oocyte marker gene in cattle, and this gene was recently assigned to a QTL region for reproductive traits. Results Here we have analyzed gene expression during folliculogenesis and preimplantation embryo development. In situ hybridization and immunohistochemistry on bovine ovarian section revealed that both the transcript and protein are restricted to the oocyte from primary follicles onwards, and accumulate in the oocyte cytoplasm during follicle growth. In immature oocytes, cytoplasmic, and more precisely cytosolic localization of MATER was confirmed by immunohistochemistry coupled with confocal microscopy and immunogold electron microscopy. By real-time PCR, MATER messenger RNA was observed to decrease strongly during maturation, and progressively during the embryo cleavage stages; it was hardly detected in morulae and blastocysts. The protein persisted after fertilization up until the blastocyst stage, and was mostly degraded after hatching. A similar predominantly cytoplasmic localization was observed in blastomeres from embryos up to 8-cells, with an apparent concentration near the nuclear membrane. Conclusion Altogether, these expression patterns are consistent with bovine MATER protein being an oocyte specific maternal effect factor as in mouse. PMID:16753072

JAK/Stat signaling regulates heart precursor diversification in Drosophila

PubMed Central

Johnson, Aaron N.; Mokalled, Mayssa H.; Haden, Tom N.; Olson, Eric N.

2011-01-01

Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the Janus kinase (JAK) and Signal transducer and activator of transcription (Stat) pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, we performed a systematic characterization of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, we show that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. We show the E(spl)-C gene HLHm5 is expressed in a pattern complementary to tin during phase 3 and that this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain. PMID:21965617

Hedgehog participates in the establishment of left-right asymmetry during amphioxus development by controlling Cerberus expression.

PubMed

Hu, Guangwei; Li, Guang; Wang, Hui; Wang, Yiquan

2017-12-15

Correct patterning of left-right (LR) asymmetry is essential during the embryonic development of bilaterians. Hedgehog (Hh) signaling is known to play a role in LR asymmetry development of mouse, chicken and sea urchin embryos by regulating Nodal expression. In this study, we report a novel regulatory mechanism for Hh in LR asymmetry development of amphioxus embryos. Our results revealed that Hh -/- embryos abolish Cerberus ( Cer ) transcription, with bilaterally symmetric expression of Nodal , Lefty and Pitx In consequence, Hh -/- mutants duplicated left-side structures and lost right-side characters, displaying an abnormal bilaterally symmetric body plan. These LR defects in morphology and gene expression could be rescued by Hh mRNA injection. Our results indicate that Hh participates in amphioxus LR patterning by controlling Cer gene expression. Curiously, however, upregulation of Hh signaling failed to alter the Cer expression pattern or LR morphology in amphioxus embryos, indicating that Hh might not provide an asymmetric cue for Cer expression. In addition, Hh is required for mouth opening in amphioxus, hinting at a homologous relationship between amphioxus and vertebrate mouth development. © 2017. Published by The Company of Biologists Ltd.

EMAGE mouse embryo spatial gene expression database: 2010 update

PubMed Central

Richardson, Lorna; Venkataraman, Shanmugasundaram; Stevenson, Peter; Yang, Yiya; Burton, Nicholas; Rao, Jianguo; Fisher, Malcolm; Baldock, Richard A.; Davidson, Duncan R.; Christiansen, Jeffrey H.

2010-01-01

EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (∼19 000 gene) ‘EURExpress’ dataset into EMAGE. PMID:19767607

Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish.

PubMed

Gothilf, Yoav; Toyama, Reiko; Coon, Steven L; Du, Shao-Jun; Dawid, Igor B; Klein, David C

2002-11-01

Zebrafish serotonin-N-acetyltransferase-2 (zfAANAT-2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue-specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT-2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5'-flanking region, a regulatory sequence in the 3'-flanking region is required for pineal-specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5' and 3' regulatory regions of the zfAANAT-2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT-2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal-specific gene expression in the zebrafish. Published 2002 Wiley-Liss, Inc.

Transcriptome Analysis of Genes Involved in Lipid Biosynthesis in the Developing Embryo of Pecan (Carya illinoinensis).

PubMed

Huang, Ruimin; Huang, Youjun; Sun, Zhichao; Huang, Jianqin; Wang, Zhengjia

2017-05-24

Pecan (Carya illinoinensis) is an important woody tree species because of the high content of healthy oil in its nut. Thus far, the pathways and key genes related to oil biosynthesis in developing pecan seeds remain largely unclear. Our analyses revealed that mature pecan embryo accumulated more than 80% oil, in which 90% was unsaturated fatty acids with abundant oleic acid. RNA sequencing generated 84,643 unigenes in three cDNA libraries prepared from pecan embryos collected at 105, 120, and 165 days after flowering (DAF). We identified 153 unigenes associated with lipid biosynthesis, including 107 unigenes for fatty acid biosynthesis, 34 for triacylglycerol biosynthesis, 7 for oil bodies, and 5 for transcription factors involved in oil synthesis. The genes associated with fatty acid synthesis were the most abundantly expressed genes at 120 DAF. Additionally, the biosynthesis of oil began to increase while crude fat contents increased from 16.61 to 74.45% (165 DAF). We identified four SAD, two FAD2, one FAD6, two FAD7, and two FAD8 unigenes responsible for unsaturated fatty acid biosynthesis. However, FAD3 homologues were not detected. Consequently, we inferred that the linolenic acid in developing pecan embryos is generated by FAD7 and FAD8 in plastids rather than FAD3 in endoplasmic reticula. During pecan embryo development, different unigenes are expressed for plastidial and cytosolic glycolysis. Plastidial glycolysis is more relevant to lipid synthesis than cytosolic glycolysis. The 18 most important genes associated with lipid biosynthesis were evaluated in five stages of developing embryos using quantitative PCR (qPCR). The qPCR data were well consistent with their expression in transcriptomic analyses. Our data would be important for the metabolic engineering of pecans to increase oil contents and modify fatty acid composition.

Evaluation of reference genes in mouse preimplantation embryos for gene expression studies using real-time quantitative RT-PCR (RT-qPCR).

PubMed

Jeong, Jae-Kyo; Kang, Min-Hee; Gurunathan, Sangiliyandi; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

2014-09-25

Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) is the most sensitive, and valuable technique for rare mRNA detection. However, the expression profiles of reference genes under different experimental conditions, such as different mouse strains, developmental stage, and culture conditions have been poorly studied. mRNA stability of the actb, gapdh, sdha, ablim, ywhaz, sptbn, h2afz, tgfb1, 18 s and wrnip genes was analyzed. Using the NormFinder program, the most stable genes are as follows: h2afz for the B6D2F-1 and C57BL/6 strains; sptbn for ICR; h2afz for KOSOM and CZB cultures of B6D2F-1 and C57BL/6 strain-derived embryos; wrnip for M16 culture of B6D2F-1 and C57BL/6 strain-derived embryos; ywhaz, tgfb1, 18 s, 18 s, ywhaz, and h2afz for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst embryonic stages cultured in KSOM medium, respectively; h2afz, wrnip, wrnip, h2afz, ywhaz, and ablim for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in CZB medium, respectively; 18 s, h2afz, h2afz, actb, h2afz, and wrnip for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in M16 medium, respectively. These results demonstrated that candidate reference genes for normalization of target gene expression using RT-qPCR should be selected according to mouse strains, developmental stage, and culture conditions.

Defining the Genomic Signature of Totipotency and Pluripotency during Early Human Development

PubMed Central

Galan, Amparo; Diaz-Gimeno, Patricia; Poo, Maria Eugenia; Valbuena, Diana; Sanchez, Eva; Ruiz, Veronica; Dopazo, Joaquin; Montaner, David; Conesa, Ana; Simon, Carlos

2013-01-01

The genetic mechanisms governing human pre-implantation embryo development and the in vitro counterparts, human embryonic stem cells (hESCs), still remain incomplete. Previous global genome studies demonstrated that totipotent blastomeres from day-3 human embryos and pluripotent inner cell masses (ICMs) from blastocysts, display unique and differing transcriptomes. Nevertheless, comparative gene expression analysis has revealed that no significant differences exist between hESCs derived from blastomeres versus those obtained from ICMs, suggesting that pluripotent hESCs involve a new developmental progression. To understand early human stages evolution, we developed an undifferentiation network signature (UNS) and applied it to a differential gene expression profile between single blastomeres from day-3 embryos, ICMs and hESCs. This allowed us to establish a unique signature composed of highly interconnected genes characteristic of totipotency (61 genes), in vivo pluripotency (20 genes), and in vitro pluripotency (107 genes), and which are also proprietary according to functional analysis. This systems biology approach has led to an improved understanding of the molecular and signaling processes governing human pre-implantation embryo development, as well as enabling us to comprehend how hESCs might adapt to in vitro culture conditions. PMID:23614026

Gene delivery to the neurulating embryo during culture

EPA Science Inventory

Modulating expression of specific genes during embryogenesis will help elucidate their role in development. Transient overexpression of specific genes can be accomplished by adding additional copies, or else antisense transcripts can be used to block expression. Manipulation of g...

Growth and recovery of zebrafish embryos after developmental exposure to raw and ozonated oil sands process-affected water.

PubMed

Lyons, Danielle D; Morrison, Christie; Philibert, Danielle A; Gamal El-Din, Mohamed; Tierney, Keith B

2018-05-07

Due to the increasing volume of oil sands process-affect water (OSPW) and its toxicity to aquatic organisms, it is important to fully understand its effects and study remediation processes that will enable its release to the environment. Ozone treatment is currently being considered as a tool to expedite remediation, as it is known to degrade toxic organic compounds present in OSPW. In this study, we aimed to measure the effects of OSPW exposure on the growth, development and recovery of zebrafish (Danio rerio) embryos. We also used ozone-treated OSPW to determine whether ozonation negated any effects of raw OSPW exposure. As biomarkers of exposure, we assessed the expression of genes involved in neurodevelopment (ngn1, neuroD), estrogenicity (vtg), oxidative stress (sod1), and biotransformation (cyp1a, cyp1b). Our study found that exposure to both raw and ozonated OSPW did not impair growth of zebrafish embryos, however, otoliths of exposed embryos were smaller than those of control embryos. The expression levels of both cyp1a and cyp1b were induced by raw OSPW exposure. However, after the exposure period, expression levels of these genes returned to control levels within two days of residence in clean water. We found no changes in the expression levels of ngn1, neuroD and vtg genes with exposure to treated or untreated OSPW. Overall, our study found that raw OSPW exposure did not have many negative effects on zebrafish embryos and embryos appeared to recover relatively quickly after exposure ended. Furthermore, ozone treatment decreased the induction of cyp1a and cyp1b. Copyright © 2018 Elsevier Ltd. All rights reserved.

Species-specific considerations in using the fish embryo test as an alternative to identify endocrine disruption.

PubMed

Schiller, Viktoria; Zhang, Xiaowei; Hecker, Markus; Schäfers, Christoph; Fischer, Rainer; Fenske, Martina

2014-10-01

A number of regulations have been implemented that aim to control the release of potentially adverse endocrine disrupters into the aquatic environment based on evidence from laboratory studies. Currently, such studies rely on testing approaches with adult fish because reliable alternatives have not been validated so far. Fish embryo tests have been proposed as such an alternative, and here we compared two species (medaka and zebrafish) to determine their suitability for the assessment of substances with estrogenic and anti-androgenic activity. Changes in gene expression (in here the phrase gene expression is used synonymously to gene transcription, although it is acknowledged that gene expression is additionally regulated, e.g., by translation and protein stability) patterns between the two species were compared in short term embryo exposure tests (medaka: 7-day post fertilization [dpf]; zebrafish: 48 and 96h post fertilization [hpf]) by using relative quantitative real-time RT-PCR. The tested genes were related to the hypothalamic-gonadal-axis and early steroidogenesis. Test chemicals included 17α-ethinylestradiol and flutamide as estrogenic and anti-androgenic reference compounds, respectively, as well as five additional substances with endocrine activities, namely bisphenol A, genistein, prochloraz, linuron and propanil. Estrogenic responses were comparable in 7-dpf medaka and 48/96-hpf zebrafish embryos and included transcriptional upregulation of aromatase b, vitellogenin 1 as well as steroidogenic genes, suggesting that both species reliably detected exposure to estrogenic compounds. However, anti-androgenic responses differed between the two species, with each species providing specific information concerning the mechanism of anti-androgenic disruption in fish embryos. Although small but significant changes in the expression of selected genes was observed in 48-hpf zebrafish embryos, exposure prolonged to 96hpf was necessary to obtain a response indicative of anti-androgenic activity. In contrast, for medaka clear anti-androgenic response, e.g. transcriptional downregulation of 11β-hydroxylase, 3β-hydroxysteroid-dehydrogenase, gonadotropin-releasing hormone receptor 2, was already observed at the pre-hatch stage. Together, this data suggests that medaka and zebrafish embryos would provide a beneficial alternative testing platform for endocrine disruption that involves additive information on interspecies and exposure time variability when using both species. Copyright © 2014 Elsevier B.V. All rights reserved.

The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation.

PubMed

Zhang, Yufan; Clemens, Adam; Maximova, Siela N; Guiltinan, Mark J

2014-04-24

The Arabidopsis thaliana LEC2 gene encodes a B3 domain transcription factor, which plays critical roles during both zygotic and somatic embryogenesis. LEC2 exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network. An ortholog of the Arabidopsis Leafy Cotyledon 2 gene (AtLEC2) was characterized in Theobroma cacao (TcLEC2). TcLEC2 encodes a B3 domain transcription factor preferentially expressed during early and late zygotic embryo development. The expression of TcLEC2 was higher in dedifferentiated cells competent for somatic embryogenesis (embryogenic calli), compared to non-embryogenic calli. Transient overexpression of TcLEC2 in immature zygotic embryos resulted in changes in gene expression profiles and fatty acid composition. Ectopic expression of TcLEC2 in cacao leaves changed the expression levels of several seed related genes. The overexpression of TcLEC2 in cacao explants greatly increased the frequency of regeneration of stably transformed somatic embryos. TcLEC2 overexpressing cotyledon explants exhibited a very high level of embryogenic competency and when cultured on hormone free medium, exhibited an iterative embryogenic chain-reaction. Our study revealed essential roles of TcLEC2 during both zygotic and somatic embryo development. Collectively, our evidence supports the conclusion that TcLEC2 is a functional ortholog of AtLEC2 and that it is involved in similar genetic regulatory networks during cacao somatic embryogenesis. To our knowledge, this is the first detailed report of the functional analysis of a LEC2 ortholog in a species other then Arabidopsis. TcLEC2 could potentially be used as a biomarker for the improvement of the SE process and screen for elite varieties in cacao germplasm.

A stress-associated protein containing A20/AN1 zing-finger domains expressed in Medicago truncatula seeds.

PubMed

Gimeno-Gilles, Christine; Gervais, Marie-Laure; Planchet, Elisabeth; Satour, Pascale; Limami, Anis M; Lelievre, Eric

2011-03-01

MtSAP1 (Medicago truncatula stress-associated protein 1) was revealed as a down-regulated gene by suppressive subtractive hybridization between two mRNA populations of embryo axes harvested before and after radicle emergence. MtSAP1 is the first gene encoding a SAP with A20 and AN1 zinc-finger domains characterized in M. truncatula. MtSAP1 protein shares 54% and 62% homology with AtSAP7 (Arabidopsis thaliana) and OsiSAP8 (Oryza sativa) respectively, with in particular a strong homology in the A20 and AN1 conserved domains. MtSAP1 gene expression increased in the embryos during the acquisition of tolerance to desiccation, reached its maximum in dry seed and decreased dramatically during the first hours of imbibition. Abiotic stresses (cold and hypoxia), abscisic acid and desiccation treatments induced MtSAP1 gene expression and protein accumulation in embryo axis, while mild drought stress did not affect significantly its expression. This profile of expression along with the presence of anaerobic response elements and ABRE sequences in the upstream region of the gene is consistent with a role of MtSAP1 in the tolerance of low oxygen availability and desiccation during late stages of seed maturation. Silencing of MtSAP1 by RNA interference (RNAi) showed that the function of the encoded protein is required for adequate accumulation of storage globulin proteins, vicilin and legumin, and for the development of embryos able to achieve successful germination. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Molecular Mechanisms Underlying γ-Aminobutyric Acid (GABA) Accumulation in Giant Embryo Rice Seeds.

PubMed

Zhao, Guo-Chao; Xie, Mi-Xue; Wang, Ying-Cun; Li, Jian-Yue

2017-06-21

To uncover the molecular mechanisms underlying GABA accumulation in giant embryo rice seeds, we analyzed the expression levels of GABA metabolism genes and contents of GABA and GABA metabolic intermediates in developing grains and germinated brown rice of giant embryo rice 'Shangshida No. 5' and normal embryo rice 'Chao2-10' respectively. In developing grains, the higher GABA contents in 'Shangshida No. 5' were accompanied with upregulation of gene transcripts and intermediate contents in the polyamine pathway and downregulation of GABA catabolic gene transcripts, as compared with those in 'Chao2-10'. In germinated brown rice, the higher GABA contents in 'Shangshida No. 5' were parallel with upregulation of OsGAD and polyamine pathway gene transcripts and Glu and polyamine pathway intermediate contents and downregulation of GABA catabolic gene transcripts. These results are the first to indicate that polyamine pathway and GABA catabolic genes play a crucial role in GABA accumulation in giant embryo rice seeds.

Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf.

PubMed

Hornyak, T J; Hayes, D J; Chiu, L Y; Ziff, E B

2001-03-01

A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing beta-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants, Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitf(mi), with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of beta-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitf(mi)/+ embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quantification and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitf(mi)/Mitf(mi) embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration.

No turning, a mouse mutation causing left-right and axial patterning defects.

PubMed

Melloy, P G; Ewart, J L; Cohen, M F; Desmond, M E; Kuehn, M R; Lo, C W

1998-01-01

Patterning along the left/right axes helps establish the orientation of visceral organ asymmetries, a process which is of fundamental importance to the viability of an organism. A linkage between left/right and axial patterning is indicated by the finding that a number of genes involved in left/right patterning also play a role in anteroposterior and dorsoventral patterning. We have recovered a spontaneous mouse mutation causing left/right patterning defects together with defects in anteroposterior and dorsoventral patterning. This mutation is recessive lethal and was named no turning (nt) because the mutant embryos fail to undergo embryonic turning. nt embryos exhibit cranial neural tube closure defects and malformed somites and are caudally truncated. Development of the heart arrests at the looped heart tube stage, with cardiovascular defects indicated by ballooning of the pericardial sac and the pooling of blood in various regions of the embryo. Interestingly, in nt embryos, the direction of heart looping was randomized. Nodal and lefty, two genes that are normally expressed only in the left lateral plate mesoderm, show expression in the right and left lateral plate mesoderm. Lefty, which is normally also expressed in the floorplate, is not found in the prospective floor plate of nt embryos. This suggests the possibility of notochordal defects. This was confirmed by histological analysis and the examination of sonic hedgehog, Brachyury, and HNF-3 beta gene expression. These studies showed that the notochord is present in the early nt embryo, but degenerates as development progresses. Overall, these findings support the hypothesis that the notochord plays an active role in left/right patterning. Our results suggest that nt may participate in this process by modulating the notochordal expression of HNF-3 beta.

Changes in gravitational force induce alterations in gene expression that can be monitored in the live, developing zebrafish heart

NASA Astrophysics Data System (ADS)

Gillette-Ferguson, I.; Ferguson, D. G.; Poss, K. D.; Moorman, S. J.

2003-10-01

Little is known about the effect of microgravity on gene expression, particularly in vivo during embryonic development. Using transgenic zebrafish that express the gfp gene under the influence of a β-actin promoter, we examined the affect of simulated-microgravity on GFP expression in the heart. Zebrafish embryos, at the 18-20 somite-stage, were exposed to simulated-microgravity for 24 hours. The intensity of GFP fluorescence associated with the heart was then determined using fluorescence microscopy. Our measurements indicated that simulated-microgravity induced a 23.9% increase in GFP-associated fluorescence in the heart. In contrast, the caudal notochord showed a 17.5% increase and the embryo as a whole showed only an 8.5% increase in GFP-associated fluorescence. This suggests that there are specific effects on the heart causing the more dramatic increase. These studies indicate that microgravity can influence gene expression and demonstrate the usefulness of this in vivo model of "reporter-gene" expression for studying the effects of microgravity.

Mechanisms of gap gene expression canalization in the Drosophila blastoderm.

PubMed

Gursky, Vitaly V; Panok, Lena; Myasnikova, Ekaterina M; Manu; Samsonova, Maria G; Reinitz, John; Samsonov, Alexander M

2011-01-01

Extensive variation in early gap gene expression in the Drosophila blastoderm is reduced over time because of gap gene cross regulation. This phenomenon is a manifestation of canalization, the ability of an organism to produce a consistent phenotype despite variations in genotype or environment. The canalization of gap gene expression can be understood as arising from the actions of attractors in the gap gene dynamical system. In order to better understand the processes of developmental robustness and canalization in the early Drosophila embryo, we investigated the dynamical effects of varying spatial profiles of Bicoid protein concentration on the formation of the expression border of the gap gene hunchback. At several positions on the anterior-posterior axis of the embryo, we analyzed attractors and their basins of attraction in a dynamical model describing expression of four gap genes with the Bicoid concentration profile accounted as a given input in the model equations. This model was tested against a family of Bicoid gradients obtained from individual embryos. These gradients were normalized by two independent methods, which are based on distinct biological hypotheses and provide different magnitudes for Bicoid spatial variability. We showed how the border formation is dictated by the biological initial conditions (the concentration gradient of maternal Hunchback protein) being attracted to specific attracting sets in a local vicinity of the border. Different types of these attracting sets (point attractors or one dimensional attracting manifolds) define several possible mechanisms of border formation. The hunchback border formation is associated with intersection of the spatial gradient of the maternal Hunchback protein and a boundary between the attraction basins of two different point attractors. We demonstrated how the positional variability for hunchback is related to the corresponding variability of the basin boundaries. The observed reduction in variability of the hunchback gene expression can be accounted for by specific geometrical properties of the basin boundaries. We clarified the mechanisms of gap gene expression canalization in early Drosophila embryos. These mechanisms were specified in the case of hunchback in well defined terms of the dynamical system theory.

Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.

PubMed

Urrego, R; Bernal-Ulloa, S M; Chavarría, N A; Herrera-Puerta, E; Lucas-Hahn, A; Herrmann, D; Winkler, S; Pache, D; Niemann, H; Rodriguez-Osorio, N

2017-04-01

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

Comparison of the Structure and Expression of Odd-Skipped and Two Related Genes That Encode a New Family of Zinc Finger Proteins in Drosophila

PubMed Central

Hart, M. C.; Wang, L.; Coulter, D. E.

1996-01-01

The odd-skipped (odd) gene, which was identified on the basis of a pair-rule segmentation phenotype in mutant embryos, is initially expressed in the Drosophila embryo in seven pair-rule stripes, but later exhibits a segment polarity-like pattern for which no phenotypic correlate is apparent. We have molecularly characterized two embryonically expressed odd-cognate genes, sob and bowel (bowl), that encode proteins with highly conserved C(2)H(2) zinc fingers. While the Sob and Bowl proteins each contain five tandem fingers, the Odd protein lacks a fifth (C-terminal) finger and is also less conserved among the four common fingers. Reminiscent of many segmentation gene paralogues, the closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns; in contrast, the less-tightly linked bowl gene is expressed in a distinctly different pattern at the termini of the early embryo. Although our results indicate that odd and sob are more likely than bowl to share overlapping developmental roles, some functional divergence between the Odd and Sob proteins is suggested by the absence of homology outside the zinc fingers, and also by amino acid substitutions in the Odd zinc fingers at positions that appear to be constrained in Sob and Bowl. PMID:8878683

Selection of the in vitro culture media influences mRNA expression of Hedgehog genes, Il-6, and important genes regarding reactive oxygen species in single murine preimplantation embryos.

PubMed

Pfeifer, N; Baston-Büst, D M; Hirchenhain, J; Friebe-Hoffmann, U; Rein, D T; Krüssel, J S; Hess, A P

2012-01-01

The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

Selection of the In Vitro Culture Media Influences mRNA Expression of Hedgehog Genes, Il-6, and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

PubMed Central

Pfeifer, N.; Baston-Büst, D. M.; Hirchenhain, J.; Friebe-Hoffmann, U.; Rein, D. T.; Krüssel, J. S.; Hess, A. P.

2012-01-01

Background. The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Methods. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. Results. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. Conclusions. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies. PMID:22919324

Higher growth rate and gene expression in male zebra finch embryos are independent of manipulation of maternal steroids in the eggs.

PubMed

Lutyk, Dorota; Tagirov, Makhsud; Drobniak, Szymon; Rutkowska, Joanna

2017-12-01

Sexual dimorphism in prenatal development is widespread among vertebrates, including birds. Its mechanism remains unclear, although it has been attributed to the effect of maternal steroid hormones. The aim of this study was to investigate how increased levels of steroid hormones in the eggs influence early embryonic development of male and female offspring. We also asked whether maternal hormones take part in the control of sex-specific expression of the genes involved in prenatal development. We experimentally manipulated hormones' concentrations in the egg yolk by injecting zebra finch females prior to ovulation with testosterone or corticosterone. We assessed growth rate and expression levels of CDK7, FBP1 and GHR genes in 37h-old embryos. We found faster growth and higher expression of two studied genes in male compared to female embryos. Hormonal treatment, despite clearly differentiating egg steroid levels, had no effect on the sex-specific pattern of the embryonic gene expression, even though we confirmed expression of receptors of androgens and glucocorticoids at such an early stage of development. Thus, our study shows high stability of the early sex differences in the embryonic development before the onset of sexual differentiation and indicates their independence of maternal hormones in the egg. Copyright © 2017 Elsevier Inc. All rights reserved.

tortuga refines Notch pathway gene expression in the zebrafish presomitic mesoderm at the post-transcriptional level.

PubMed

Dill, Kariena K; Amacher, Sharon L

2005-11-15

We have identified the zebrafish tortuga (tor) gene by an ENU-induced mutation that disrupts the presomitic mesoderm (PSM) expression of Notch pathway genes. In tor mutants, Notch pathway gene expression persists in regions of the PSM where expression is normally off in wild type embryos. The expression of hairy/Enhancer of split-related 1 (her1) is affected first, followed by the delta genes deltaC and deltaD, and finally, by another hairy/Enhancer of split-related gene, her7. In situ hybridization with intron-specific probes for her1 and deltaC indicates that transcriptional bursts of expression are normal in tor mutants, suggesting that tor normally functions to refine her1 and deltaC message levels downstream of transcription. Despite the striking defects in Notch pathway gene expression, somite boundaries form normally in tor mutant embryos, although somitic mesoderm defects are apparent later, when cells mature to form muscle fibers. Thus, while the function of Notch pathway genes is required for proper somite formation, the tor mutant phenotype suggests that precise oscillations of Notch pathway transcripts are not essential for establishing segmental pattern in the presomitic mesoderm.

Role of GSK-3β in the Osteogenic Differentiation of Palatal Mesenchyme

PubMed Central

Sorkin, Michael; James, Aaron W.; Liu, Karen J.; Quarto, Natalina; Longaker, Michael T.

2011-01-01

Introduction The function of Glycogen Synthase Kinases 3β (GSK-3β) has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification. Methods Palates were harvested from GSK-3β, embryonic days 15.0–18.5 (e15.0–e18.5), and e15.5 Indian Hedgehog (Ihh) null embryos, and their wild-type littermates. The phenotype of GSK-3β null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3β and Ihh +/+ and −/− e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists. Results GSK-3β null embryos displayed a 48 percent decrease (*p<0.05) in palatine bone formation compared to wild-type littermates. GSK-3β null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3β −/− palate cultures were “rescued” with the Wnt inhibitor, Dkk-1. Conclusions Here, we identify a critical role for GSK-3β in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways involved in palatogenesis and may lead to novel molecular targets to prevent cleft palate formation. PMID:22022457

A transgene design for enhancing oil content in Arabidopsis and Camelina seeds

USDA-ARS?s Scientific Manuscript database

Increasing the oil yield is a major objective for oilseed crop improvement. Oil biosynthesis and accumulation are influenced by multiple genes involved in embryo and seed development. The LEAFY COTYLEDON1 (LEC1) is a master regulator of embryo development that also enhances the expression of genes i...

Ovary transcriptome profiling via artificial intelligence reveals a transcriptomic fingerprint predicting egg quality in striped bass, Morone saxatilis.

PubMed

Chapman, Robert W; Reading, Benjamin J; Sullivan, Craig V

2014-01-01

Inherited gene transcripts deposited in oocytes direct early embryonic development in all vertebrates, but transcript profiles indicative of embryo developmental competence have not previously been identified. We employed artificial intelligence to model profiles of maternal ovary gene expression and their relationship to egg quality, evaluated as production of viable mid-blastula stage embryos, in the striped bass (Morone saxatilis), a farmed species with serious egg quality problems. In models developed using artificial neural networks (ANNs) and supervised machine learning, collective changes in the expression of a limited suite of genes (233) representing <2% of the queried ovary transcriptome explained >90% of the eventual variance in embryo survival. Egg quality related to minor changes in gene expression (<0.2-fold), with most individual transcripts making a small contribution (<1%) to the overall prediction of egg quality. These findings indicate that the predictive power of the transcriptome as regards egg quality resides not in levels of individual genes, but rather in the collective, coordinated expression of a suite of transcripts constituting a transcriptomic "fingerprint". Correlation analyses of the corresponding candidate genes indicated that dysfunction of the ubiquitin-26S proteasome, COP9 signalosome, and subsequent control of the cell cycle engenders embryonic developmental incompetence. The affected gene networks are centrally involved in regulation of early development in all vertebrates, including humans. By assessing collective levels of the relevant ovarian transcripts via ANNs we were able, for the first time in any vertebrate, to accurately predict the subsequent embryo developmental potential of eggs from individual females. Our results show that the transcriptomic fingerprint evidencing developmental dysfunction is highly predictive of, and therefore likely to regulate, egg quality, a biologically complex trait crucial to reproductive fitness.

Ovary Transcriptome Profiling via Artificial Intelligence Reveals a Transcriptomic Fingerprint Predicting Egg Quality in Striped Bass, Morone saxatilis

PubMed Central

2014-01-01

Inherited gene transcripts deposited in oocytes direct early embryonic development in all vertebrates, but transcript profiles indicative of embryo developmental competence have not previously been identified. We employed artificial intelligence to model profiles of maternal ovary gene expression and their relationship to egg quality, evaluated as production of viable mid-blastula stage embryos, in the striped bass (Morone saxatilis), a farmed species with serious egg quality problems. In models developed using artificial neural networks (ANNs) and supervised machine learning, collective changes in the expression of a limited suite of genes (233) representing <2% of the queried ovary transcriptome explained >90% of the eventual variance in embryo survival. Egg quality related to minor changes in gene expression (<0.2-fold), with most individual transcripts making a small contribution (<1%) to the overall prediction of egg quality. These findings indicate that the predictive power of the transcriptome as regards egg quality resides not in levels of individual genes, but rather in the collective, coordinated expression of a suite of transcripts constituting a transcriptomic “fingerprint”. Correlation analyses of the corresponding candidate genes indicated that dysfunction of the ubiquitin-26S proteasome, COP9 signalosome, and subsequent control of the cell cycle engenders embryonic developmental incompetence. The affected gene networks are centrally involved in regulation of early development in all vertebrates, including humans. By assessing collective levels of the relevant ovarian transcripts via ANNs we were able, for the first time in any vertebrate, to accurately predict the subsequent embryo developmental potential of eggs from individual females. Our results show that the transcriptomic fingerprint evidencing developmental dysfunction is highly predictive of, and therefore likely to regulate, egg quality, a biologically complex trait crucial to reproductive fitness. PMID:24820964

The Use of Whole-Mount "in Situ" Hybridization to Illustrate Gene Expression Regulation

ERIC Educational Resources Information Center

Llamusí, Beatriz; Muñoz-Soriano, Verónica; Paricio, Nuria; Artero, Rubén

2014-01-01

"In situ" hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in "Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the…

Three enhancer regions regulate gbx2 gene expression in the isthmic region during zebrafish development.

PubMed

Islam, Md Ekramul; Kikuta, Hiroshi; Inoue, Fumitaka; Kanai, Maiko; Kawakami, Atsushi; Parvin, Mst Shahnaj; Takeda, Hiroyuki; Yamasu, Kyo

2006-12-01

In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the regions flanking the gene.

The Ca2+-induced methyltransferase xPRMT1b controls neural fate in amphibian embryo.

PubMed

Batut, Julie; Vandel, Laurence; Leclerc, Catherine; Daguzan, Christiane; Moreau, Marc; Néant, Isabelle

2005-10-18

We have previously shown that an increase in intracellular Ca2+ is both necessary and sufficient to commit ectoderm to a neural fate in Xenopus embryos. However, the relationship between this Ca2+ increase and the expression of early neural genes has yet to be defined. Using a subtractive cDNA library between untreated and caffeine-treated animal caps, i.e., control ectoderm and ectoderm induced toward a neural fate by a release of Ca2+, we have isolated the arginine N-methyltransferase, xPRMT1b, a Ca2+-induced target gene, which plays a pivotal role in this process. First, we show in embryo and in animal cap that xPRMT1b expression is Ca2+-regulated. Second, overexpression of xPRMT1b induces the expression of early neural genes such as Zic3. Finally, in the whole embryo, antisense approach with morpholino oligonucleotide against xPRMT1b impairs neural development and in animal caps blocks the expression of neural markers induced by a release of internal Ca2+. Our results implicate an instructive role of an enzyme, an arginine methyltransferase protein, in the embryonic choice of determination between epidermal and neural fate. The results presented provide insights by which a Ca2+ increase induces neural fate.

Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis.

PubMed

Aït-Azzouzene, D; Langkopf, A; Cohen, J; Bleux, C; Gendron, M C; Kanellopoulos-Langevin, C

1998-05-01

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class la gene H-2Kb under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2Kb heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-Kb construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models.

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

PubMed

de Vega-Bartol, José J; Simões, Marta; Lorenz, W Walter; Rodrigues, Andreia S; Alba, Rob; Dean, Jeffrey F D; Miguel, Célia M

2013-08-30

It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species. This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms.

Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster

PubMed Central

2013-01-01

Background It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Results Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species. Conclusions This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms. PMID:23987738

Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription.

PubMed

Ellerström, M; Stålberg, K; Ezcurra, I; Rask, L

1996-12-01

The promoter region (-309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5' as well as internal deletions fused to the reporter gene GUS (beta-glucuronidase). The expression in the two main tissues of the seed, the endosperm and the embryo, was shown to be differentially regulated. This tissue-specific regulation within the seed was found to affect the developmental expression during seed development. The region between -309 to -152, which has a large effect on quantitative expression, was shown to harbour four elements regulating embryo and one regulating endosperm expression. This region also displayed enhancer activity. Deletion of eight bp from position -152 to position -144 totally abolished the activity of the napA promoter. This deletion disrupted a cis element with similarity to an ABA-responsive element (ABRE) overlapping with an E-box, demonstrating its crucial importance for quantitative expression. An internal deletion of the region -133 to -120, resulted in increased activity in both leaves and endosperm and a decreased activity in the embryo. Within this region, a cis element similar to the (CA)n element, found in other storage protein promoters, was identified. This suggest that the (CA)n element is important for conferring seed specificity by serving both as an activator and a repressor element.

Post-transcriptional regulation of myotube elongation and myogenesis by Hoi Polloi

PubMed Central

Johnson, Aaron N.; Mokalled, Mayssa H.; Valera, Juliana M.; Poss, Kenneth D.; Olson, Eric N.

2013-01-01

Striated muscle development requires the coordinated expression of genes involved in sarcomere formation and contractility, as well as genes that determine muscle morphology. However, relatively little is known about the molecular mechanisms that control the early stages of muscle morphogenesis. To explore this facet of myogenesis, we performed a genetic screen for regulators of somatic muscle morphology in Drosophila, and identified the putative RNA-binding protein (RBP) Hoi Polloi (Hoip). Hoip is expressed in striated muscle precursors within the muscle lineage and controls two genetically separable events: myotube elongation and sarcomeric protein expression. Myotubes fail to elongate in hoip mutant embryos, even though the known regulators of somatic muscle elongation, target recognition and muscle attachment are expressed normally. In addition, a majority of sarcomeric proteins, including Myosin Heavy Chain (MHC) and Tropomyosin, require Hoip for their expression. A transgenic MHC construct that contains the endogenous MHC promoter and a spliced open reading frame rescues MHC protein expression in hoip embryos, demonstrating the involvement of Hoip in pre-mRNA splicing, but not in transcription, of muscle structural genes. In addition, the human Hoip ortholog NHP2L1 rescues muscle defects in hoip embryos, and knockdown of endogenous nhp2l1 in zebrafish disrupts skeletal muscle development. We conclude that Hoip is a conserved, post-transcriptional regulator of muscle morphogenesis and structural gene expression. PMID:23942517

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

PubMed

Pang, Yun-Wei; An, Lei; Wang, Peng; Yu, Yong; Yin, Qiu-Dan; Wang, Xiao-Hong; Xin-Zhang; Qian-Zhang; Yang, Mei-Ling; Min-Guo; Wu, Zhong-Hong; Tian, Jian-Hui

2013-05-01

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Plasticity of cardiovascular function in snapping turtle embryos (Chelydra serpentina): chronic hypoxia alters autonomic regulation and gene expression.

PubMed

Eme, John; Rhen, Turk; Tate, Kevin B; Gruchalla, Kathryn; Kohl, Zachary F; Slay, Christopher E; Crossley, Dane A

2013-06-01

Reptile embryos tolerate large decreases in the concentration of ambient oxygen. However, we do not fully understand the mechanisms that underlie embryonic cardiovascular short- or long-term responses to hypoxia in most species. We therefore measured cardiac growth and function in snapping turtle embryos incubated under normoxic (N21; 21% O₂) or chronic hypoxic conditions (H10; 10% O₂). We determined heart rate (fH) and mean arterial pressure (Pm) in acute normoxic (21% O₂) and acute hypoxic (10% O₂) conditions, as well as embryonic responses to cholinergic, adrenergic, and ganglionic pharmacological blockade. Compared with N21 embryos, chronic H10 embryos had smaller bodies and relatively larger hearts and were hypotensive, tachycardic, and following autonomic neural blockade showed reduced intrinsic fH at 90% of incubation. Unlike other reptile embryos, cholinergic and ganglionic receptor blockade both increased fH. β-Adrenergic receptor blockade with propranolol decreased fH, and α-adrenergic blockade with phentolamine decreased Pm. We also measured cardiac mRNA expression. Cholinergic tone was reduced in H10 embryos, but cholinergic receptor (Chrm2) mRNA levels were unchanged. However, expression of adrenergic receptor mRNA (Adrb1, Adra1a, Adra2c) and growth factor mRNA (Igf1, Igf2, Igf2r, Pdgfb) was lowered in H10 embryos. Hypoxia altered the balance between cholinergic receptors, α-adrenoreceptor and β-adrenoreceptor function, which was reflected in altered intrinsic fH and adrenergic receptor mRNA levels. This is the first study to link gene expression with morphological and cardioregulatory plasticity in a developing reptile embryo.

Evolutionary origin and functional divergence of totipotent cell homeobox genes in eutherian mammals.

PubMed

Maeso, Ignacio; Dunwell, Thomas L; Wyatt, Chris D R; Marlétaz, Ferdinand; Vető, Borbála; Bernal, Juan A; Quah, Shan; Irimia, Manuel; Holland, Peter W H

2016-06-13

A central goal of evolutionary biology is to link genomic change to phenotypic evolution. The origin of new transcription factors is a special case of genomic evolution since it brings opportunities for novel regulatory interactions and potentially the emergence of new biological properties. We demonstrate that a group of four homeobox gene families (Argfx, Leutx, Dprx, Tprx), plus a gene newly described here (Pargfx), arose by tandem gene duplication from the retinal-expressed Crx gene, followed by asymmetric sequence evolution. We show these genes arose as part of repeated gene gain and loss events on a dynamic chromosomal region in the stem lineage of placental mammals, on the forerunner of human chromosome 19. The human orthologues of these genes are expressed specifically in early embryo totipotent cells, peaking from 8-cell to morula, prior to cell fate restrictions; cow orthologues have similar expression. To examine biological roles, we used ectopic gene expression in cultured human cells followed by high-throughput RNA-seq and uncovered extensive transcriptional remodelling driven by three of the genes. Comparison to transcriptional profiles of early human embryos suggest roles in activating and repressing a set of developmentally-important genes that spike at 8-cell to morula, rather than a general role in genome activation. We conclude that a dynamic chromosome region spawned a set of evolutionarily new homeobox genes, the ETCHbox genes, specifically in eutherian mammals. After these genes diverged from the parental Crx gene, we argue they were recruited for roles in the preimplantation embryo including activation of genes at the 8-cell stage and repression after morula. We propose these new homeobox gene roles permitted fine-tuning of cell fate decisions necessary for specification and function of embryonic and extra-embryonic tissues utilised in mammalian development and pregnancy.

Diffusion and scaling during early embryonic pattern formation.

PubMed

Gregor, Thomas; Bialek, William; de Ruyter van Steveninck, Rob R; Tank, David W; Wieschaus, Eric F

2005-12-20

Development of spatial patterns in multicellular organisms depends on gradients in the concentration of signaling molecules that control gene expression. In the Drosophila embryo, Bicoid (Bcd) morphogen controls cell fate along 70% of the anteroposterior axis but is translated from mRNA localized at the anterior pole. Gradients of Bcd and other morphogens are thought to arise through diffusion, but this basic assumption has never been rigorously tested in living embryos. Furthermore, because diffusion sets a relationship between length and time scales, it is hard to see how patterns of gene expression established by diffusion would scale proportionately as egg size changes during evolution. Here, we show that the motion of inert molecules through the embryo is well described by the diffusion equation on the relevant length and time scales, and that effective diffusion constants are essentially the same in closely related dipteran species with embryos of very different size. Nonetheless, patterns of gene expression in these different species scale with egg length. We show that this scaling can be traced back to scaling of the Bcd gradient itself. Our results, together with constraints imposed by the time scales of development, suggest that the mechanism for scaling is a species-specific adaptation of the Bcd lifetime.

Expression profiling identifies novel Hh/Gli regulated genes in developing zebrafish embryos.

PubMed Central

Bergeron, Sadie A.; Milla, Luis A.; Villegas, Rosario; Shen, Meng-Chieh; Burgess, Shawn M.; Allende, Miguel L.; Karlstrom, Rolf O.; Palma, Verónica

2008-01-01

The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Mis-regulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants have been identified affecting different components of the Hh/Gli signaling system. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified several known (e.g. ptc1 and nkx2.2a) as well as a large number of novel Hh regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of mis-regulation during tumorogenesis. PMID:18055165

Gene expression of antioxidant enzymes and coffee seed quality during pre- and post-physiological maturity.

PubMed

Santos, F C; Caixeta, F; Clemente, A C S; Pinho, E V; Rosa, S D V F

2014-12-19

Seeds collected at different maturation stages vary in physiological quality and patterns of protective antioxidant systems against deterioration. In this study we investigated the expression of genes that codify catalase (CAT), dismutase superoxide (SOD), and polyphenol oxidase (PPO) during the pre- and post-physiological maturation phases in whole seeds and in endosperms and embryos extracted from the seeds. Coffea arabica L. berries were collected at the green, yellowish-green, cherry, over-ripe, and dry stages, and the seeds were examined physiologically. The transcription levels of the genes were quantified by quantitative real-time polymerase chain reaction using coffee-specific primers. The highest level of SOD expression was observed in the endosperm at the cherry and over-ripe stages; in addition, these seeds presented the greatest physiological quality (assessed via germination test). The highest CAT3 transcript expression was observed at the green stage in whole seeds, and at the green and over-ripe stages in the embryos and endosperms. High expression of the PPO transcript was observed at the green and yellowish-green stages in whole seeds. In embryos and endosperms, peak expression of the PPO transcript was observed at the green stage; subsequently, peaks at the cherry and over-ripe stages were observed. We concluded that the expression patterns of the SOD and CAT3 transcripts were similar at the more advanced maturation stages, which corresponded to enhanced physiological seed quality. High expression of the PPO transcript at the over-ripe stage, also observed in the embryos and endosperms at the cherry stage, coincided with the highest physiological seed quality.

Effects of KCNQ2 gene truncation on M-type Kv7 potassium currents.

PubMed

Robbins, Jon; Passmore, Gayle M; Abogadie, Fe C; Reilly, Joanne M; Brown, David A

2013-01-01

The KCNQ2 gene product, Kv7.2, is a subunit of the M-channel, a low-threshold voltage-gated K(+) channel that regulates mammalian and human neuronal excitability. Spontaneous mutations one of the KCNQ2 genes cause disorders of neural excitability such as Benign Familial Neonatal Seizures. However there appear to be no reports in which both human KCNQ2 genes are mutated. We therefore asked what happens to M-channel function when both KCNQ2 genes are disrupted. We addressed this using sympathetic neurons isolated from mice in which the KCNQ2 gene was truncated at a position corresponding to the second transmembrane domain of the Kv7.2 protein. Since homozygote KCNQ2-/- mice die postnatally, experiments were largely restricted to neurons from late embryos. Quantitative PCR revealed an absence of KCNQ2 mRNA in ganglia from KCNQ2-/- embryos but 100-120% increase of KCNQ3 and KCNQ5 mRNAs; KCNQ2+/- ganglia showed ∼30% less KCNQ2 mRNA than wild-type (+/+) ganglia but 40-50% more KCNQ3 and KCNQ5 mRNA. Neurons from KCNQ2-/- embryos showed a complete absence of M-current, even after applying the Kv7 channel enhancer, retigabine. Neurons from heterozygote KCNQ2+/- embryos had ∼60% reduced M-current. In contrast, M-currents in neurons from adult KCNQ2+/- mice were no smaller than those in neurons from wild-type mice. Measurements of tetraethylammonium block did not indicate an increased expression of Kv7.5-containing subunits, implying a compensatory increase in Kv7.2 expression from the remaining KCNQ2 gene. We conclude that mouse embryonic M-channels have an absolute requirement for Kv7.2 subunits for functionality, that the reduced M-channel activity in heterozygote KCNQ2+/- mouse embryos results primarily from a gene-dosage effect, and that there is a compensatory increase in Kv7.2 expression in adult mice.

Constraint of gene expression by the chromatin remodelling protein CHD4 facilitates lineage specification

PubMed Central

O'Shaughnessy-Kirwan, Aoife; Signolet, Jason; Costello, Ita; Gharbi, Sarah; Hendrich, Brian

2015-01-01

Chromatin remodelling proteins are essential for different aspects of metazoan biology, yet functional details of why these proteins are important are lacking. Although it is possible to describe the biochemistry of how they remodel chromatin, their chromatin-binding profiles in cell lines, and gene expression changes upon loss of a given protein, in very few cases can this easily translate into an understanding of how the function of that protein actually influences a developmental process. Here, we investigate how the chromatin remodelling protein CHD4 facilitates the first lineage decision in mammalian embryogenesis. Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE). In the absence of a functional TE, Chd4 mutant blastocysts do not implant and are hence not viable. By measuring transcript levels in single cells from early embryos, we show that CHD4 influences the frequency at which unspecified cells in preimplantation stage embryos express lineage markers prior to the execution of this first lineage decision. In the absence of CHD4, this frequency is increased in 16-cell embryos, and by the blastocyst stage cells fail to properly adopt a TE gene expression programme. We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed. This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment. PMID:26116663

Six post-implantation lethal knockouts of genes for lipophilic MAPK pathway proteins are expressed in preimplantation mouse embryos and trophoblast stem cells.

PubMed

Xie, Yufen; Wang, Yingchun; Sun, Tong; Wang, Fangfei; Trostinskaia, Anna; Puscheck, Elizabeth; Rappolee, Daniel A

2005-05-01

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in controlling embryonic proliferation and differentiation. It has been demonstrated that sequential lipophilic signal transduction mediators that participate in the MAPK pathway are null post-implantation lethal. It is not clear why the lethality of these null mutants arises after implantation and not before. One hypothesis is that the gene product of these post-implantation lethal null mutants are not present before implantation in normal embryos and do not have function until after implantation. To test this hypothesis, we selected a set of lipophilic genes mediating MAPK signal transduction pathways whose null mutants result in early peri-implantation or placental lethality. These included FRS2alpha, GAB1, GRB2, SOS1, Raf-B, and Raf1. Products of these selected genes were detected and their locations and functions indicated by indirect immunocytochemistry and Western blotting for proteins and RT-polymerase chain reaction (PCR) for mRNA transcription. We report here that all six signal mediators are detected at the protein level in preimplantation mouse embryo, placental trophoblasts, and in cultured trophoblast stem cells (TSC). Proteins are all detected in E3.5 embryos at a time when the first known mitogenic intercellular communication has been documented. mRNA transcripts of two post-implantation null mutant genes are expressed in mouse preimplantation embryos and unfertilized eggs. These mRNA transcripts were detected as maternal mRNA in unfertilized eggs that could delay the lethality of null mutants. All of the proteins were detected in the cytoplasm or in the cell membrane. This study of spatial and temporal expression revealed that all of these six null mutants post-implantation genes in MAPK pathway are expressed and, where tested, phosphorylated/activated proteins are detected in the blastocyst. Studies on RNA expression using RT-PCR suggest that maternal RNA could play an important role in delaying the presence of the lethal phenotype of null mutations. Copyright (c) 2005 Wiley-Liss, Inc.

The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during the first developmental cell lineage segregation.

PubMed

Huang, Daosheng; Guo, Guoji; Yuan, Ping; Ralston, Amy; Sun, Lingang; Huss, Mikael; Mistri, Tapan; Pinello, Luca; Ng, Huck Hui; Yuan, Guocheng; Ji, Junfeng; Rossant, Janet; Robson, Paul; Han, Xiaoping

2017-12-07

The first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and trophectoderm (TE). The transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4). However its downstream network in the developing embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify CDX2-regulated targets in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells. In addition, we examined the dynamics of gene expression changes using inducible CDX2 embryonic stem (ES) cells, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we identify putative novel regulatory elements that repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.

Impact of broiler egg storage on the relative expression of selected blastoderm genes associated with apoptosis, oxidative stress, and fatty acid metabolism.

PubMed

Bakst, M R; Welch, G R; Fetterer, R; Miska, K

2016-06-01

Cool temperature storage of eggs prior to incubation is a frequent practice by commercial broiler hatcheries. However, continued storage beyond 7 d leads to a progressive increase in the rate of early embryonic mortality. In this study, we examined the relative expression of 31 genes associated with fatty acid metabolism (8), apoptosis (7), and oxidative stress (16) pathways to better understand the basis of embryo mortality during egg storage. A total of 642 broiler eggs in 2 separate trials were subjected to the following egg treatments: stored 4 d (Control 1, C1); stored 21 d but subjected to short periods of incubation during egg storage (SPIDES); stored un-manipulated 21 d (NonSPIDES, NS); and stored 4 d then incubated for 10 h to advance the embryos to the same developmental stages as the SPIDES embryos (Control 2, C2). Hatchability trials (277 eggs) confirmed the efficacy of SPIDES compared to NS treatments in both trials. To determine relative expression of 31 selected genes, 365 blastoderms were isolated, staged, and flash frozen in batches of 5 to 10 blastoderms per vial (7 vials per egg treatment) prior to RNA extractions. Analysis of gene expression was performed using qRT-PCR and the results presented as relative expression normalized to C1. The relative expression of genes in which the SPIDES and C2 treatments were significantly up- or down-regulated in tandem indicated that the stage-specific expression of those genes was maintained by the SPIDES treatment. This study provides the relative gene expressions of blastodermal cells before and after prolonged egg storage as well as insight as to how SPIDES impacts blastodermal cell gene expression. Published by Oxford University Press on behalf of Poultry Science Association 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Maternal experience with predation risk influences genome-wide embryonic gene expression in threespined sticklebacks (Gasterosteus aculeatus).

PubMed

Mommer, Brett C; Bell, Alison M

2014-01-01

There is growing evidence for nongenetic effects of maternal experience on offspring. For example, previous studies have shown that female threespined stickleback fish (Gasterosteus aculeatus) exposed to predation risk produce offspring with altered behavior, metabolism and stress physiology. Here, we investigate the effect of maternal exposure to predation risk on the embryonic transcriptome in sticklebacks. Using RNA-sequencing we compared genome-wide transcription in three day post-fertilization embryos of predator-exposed and control mothers. There were hundreds of differentially expressed transcripts between embryos of predator-exposed mothers and embryos of control mothers including several non-coding RNAs. Gene Ontology analysis revealed biological pathways involved in metabolism, epigenetic inheritance, and neural proliferation and differentiation that differed between treatments. Interestingly, predation risk is associated with an accelerated life history in many vertebrates, and several of the genes and biological pathways that were identified in this study suggest that maternal exposure to predation risk accelerates the timing of embryonic development. Consistent with this hypothesis, embryos of predator-exposed mothers were larger than embryos of control mothers. These findings point to some of the molecular mechanisms that might underlie maternal effects.

Expression of a gymnosperm PIN homologous gene correlates with auxin immunolocalization pattern at cotyledon formation and in demarcation of the procambium during Picea abies somatic embryo development and in seedling tissues.

PubMed

Palovaara, Joakim; Hallberg, Henrik; Stasolla, Claudio; Luit, Bert; Hakman, Inger

2010-04-01

In seed plants, the body organization is established during embryogenesis and is uniform across gymnosperms and angiosperms, despite differences during early embryogeny. Evidence from angiosperms implicates the plant hormone auxin and its polar transport, mainly established by the PIN family of auxin efflux transporters, in the patterning of embryos. Here, PaPIN1 from Norway spruce (Picea abies [L.] Karst.), a gene widely expressed in conifer tissues and organs, was characterized and its expression and localization patterns were determined with reverse transcription polymerase chain reaction and in situ hybridization during somatic embryo development and in seedlings. PaPIN1 shares the predicted structure of other PIN proteins, but its central hydrophilic loop is longer than most PINs. In phylogenetic analyses, PaPIN1 clusters with Arabidopsis thaliana (L.) Heynh. PIN3, PIN4 and PIN7, but its expression pattern also suggests similarity to PIN1. The PaPIN1 expression signal was high in the protoderm of pre-cotyledonary embryos, but not if embryos were pre-treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This, together with a high auxin immunolocalization signal in this cell layer, suggests a role of PaPIN1 during cotyledon formation. At later stages, high PaPIN1 expression was observed in differentiating procambium, running from the tip of incipient cotyledons down through the embryo axis and to the root apical meristem (RAM), although the mode of RAM specification in conifer embryos differs from that of most angiosperms. Also, the PaPIN1 in situ signal was high in seedling root tips including root cap columella cells. The results thus suggest that PaPIN1 provides an ancient function associated with auxin transport and embryo pattern formation prior to the separation of angiosperms and gymnosperms, in spite of some morphological differences.

Quantitative developmental transcriptomes of the Mediterranean sea urchin Paracentrotus lividus.

PubMed

Gildor, Tsvia; Malik, Assaf; Sher, Noa; Avraham, Linor; Ben-Tabou de-Leon, Smadar

2016-02-01

Embryonic development progresses through the timely activation of thousands of differentially activated genes. Quantitative developmental transcriptomes provide the means to relate global patterns of differentially expressed genes to the emerging body plans they generate. The sea urchin is one of the classic model systems for embryogenesis and the models of its developmental gene regulatory networks are of the most comprehensive of their kind. Thus, the sea urchin embryo is an excellent system for studies of its global developmental transcriptional profiles. Here we produced quantitative developmental transcriptomes of the sea urchin Paracentrotus lividus (P. lividus) at seven developmental stages from the fertilized egg to prism stage. We generated de-novo reference transcriptome and identified 29,817 genes that are expressed at this time period. We annotated and quantified gene expression at the different developmental stages and confirmed the reliability of the expression profiles by QPCR measurement of a subset of genes. The progression of embryo development is reflected in the observed global expression patterns and in our principle component analysis. Our study illuminates the rich patterns of gene expression that participate in sea urchin embryogenesis and provide an essential resource for further studies of the dynamic expression of P. lividus genes. Copyright © 2015 Elsevier B.V. All rights reserved.

MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

PubMed

Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

2011-02-15

At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

PM19, a barley (Hordeum vulgare L.) gene encoding a putative plasma membrane protein, is expressed during embryo development and dormancy.

PubMed

Ranford, Julia C; Bryce, James H; Morris, Peter C

2002-01-01

A barley (Hordeum vulgare L.) cDNA, PM19, encoding a putative plasma membrane protein was isolated through differential screening of a dormant wild oat embryo library. PM19 is expressed in barley embryos from mid-embryogenesis up to maturity. PM19 mRNA levels decline upon germination, whereas dormant embryos retained high levels of message for up to 72 h of imbibition. PM19 mRNA levels also remained high or were reinduced in non-dormant embryos by treatments that prevented germination (250 mm NaCl, 10% sorbitol, or 50 microm ABA). The PM19 protein sequence is highly conserved in monocotyledonous and dicotyledonous plants.

The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

PubMed

Miyamoto, Kei; Suzuki, Ken-Ichi T; Suzuki, Miyuki; Sakane, Yuto; Sakuma, Tetsushi; Herberg, Sarah; Simeone, Angela; Simpson, David; Jullien, Jerome; Yamamoto, Takashi; Gurdon, J B

2015-01-01

Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

Developmentally regulated HEART STOPPER, a mitochondrially targeted L18 ribosomal protein gene, is required for cell division, differentiation, and seed development in Arabidopsis

PubMed Central

Zhang, Hongyu; Luo, Ming; Day, Robert C.; Talbot, Mark J.; Ivanova, Aneta; Ashton, Anthony R.; Chaudhury, Abed M.; Macknight, Richard C.; Hrmova, Maria; Koltunow, Anna M.

2015-01-01

Evidence is presented for the role of a mitochondrial ribosomal (mitoribosomal) L18 protein in cell division, differentiation, and seed development after the characterization of a recessive mutant, heart stopper (hes). The hes mutant produced uncellularized endosperm and embryos arrested at the late globular stage. The mutant embryos differentiated partially on rescue medium with some forming callus. HES (At1g08845) encodes a mitochondrially targeted member of a highly diverged L18 ribosomal protein family. The substitution of a conserved amino residue in the hes mutant potentially perturbs mitoribosomal function via altered binding of 5S rRNA and/or influences the stability of the 50S ribosomal subunit, affecting mRNA binding and translation. Consistent with this, marker genes for mitochondrial dysfunction were up-regulated in the mutant. The slow growth of the endosperm and embryo indicates a defect in cell cycle progression, which is evidenced by the down-regulation of cell cycle genes. The down-regulation of other genes such as EMBRYO DEFECTIVE genes links the mitochondria to the regulation of many aspects of seed development. HES expression is developmentally regulated, being preferentially expressed in tissues with active cell division and differentiation, including developing embryos and the root tips. The divergence of the L18 family, the tissue type restricted expression of HES, and the failure of other L18 members to complement the hes phenotype suggest that the L18 proteins are involved in modulating development. This is likely via heterogeneous mitoribosomes containing different L18 members, which may result in differential mitochondrial functions in response to different physiological situations during development. PMID:26105995

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

PubMed

Stowe, Heather M; Curry, Erin; Calcatera, Samantha M; Krisher, Rebecca L; Paczkowski, Melissa; Pratt, Scott L

2012-06-15

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of exogenous progesterone on embryo size and ewe uterine gene expression in an ovine 'dam size' model of maternal constraint.

PubMed

Fermin, Lisanne M; Pain, Sarah J; Morel, Patrick C H; Gedye, Kristene R; Kenyon, Paul R; Blair, Hugh T

2017-11-21

Progesterone (P4), acting via its receptor, regulates uterine function and histotroph production, which are crucial to embryo growth. This study aimed to examine exogenous P4 effects on embryo size and differential endometrial gene expression at Day 19 of gestation using a 'dam size' sheep model of maternal constraint. Purebred Suffolk (S, genotypically large) embryos were transferred into recipient groups of Cheviot (C, genotypically small) or Suffolk ewes that had, or had not, been pre-treated with P4 from Days 0 to 6 of pregnancy. At Day 19S embryos were collected from four experimental groups: P4 pretreated S ewes (SP4; n=5), untreated S ewes (SnP4; n=15), P4 pretreated C ewes (CP4; n=7) and untreated C ewes (CnP4; n=21). Day-19 embryos from CP4 ewes were larger (P<0.05) than those from CnP4 ewes and similar in size (P>0.05) to embryos from SnP4 and SP4 ewes. Expression of mucin 1 (MUC1) and prostaglandin-endoperoxide synthase 2 (PTGS2) was upregulated in uterine horns ipsilateral to the corpus luteum from CP4 ewes. Prostaglandin receptor (PGR), MUC1 and PTGS2 expression was upregulated, whilst cathepsin L (CTSL) and radical S-adenosyl methionine domain-containing 2 (RSAD2) expression was downregulated in the ipsilateral horn of SP4 ewes. This suggests that pretreating ewes with exogenous P4 may alleviate early pregnancy maternal constraint via mechanisms that alter uterine function. However, further research is required to investigate the timing of P4 administration and its impact on conception rates.

Regulation of notochord-specific expression of Ci-Bra downstream genes in Ciona intestinalis embryos.

PubMed

Takahashi, Hiroki; Hotta, Kohji; Takagi, Chiyo; Ueno, Naoto; Satoh, Nori; Shoguchi, Eiichi

2010-02-01

Brachyury, a T-box transcription factor, is expressed in ascidian embryos exclusively in primordial notochord cells and plays a pivotal role in differentiation of notochord cells. Previously, we identified approximately 450 genes downstream of Ciona intestinalis Brachyury (Ci-Bra), and characterized the expression profiles of 45 of these in differentiating notochord cells. In this study, we looked for cisregulatory sequences in minimal enhancers of 20 Ci-Bra downstream genes by electroporating region within approximately 3 kb upstream of each gene fused with lacZ. Eight of the 20 reporters were expressed in notochord cells. The minimal enchancer for each of these eight genes was narrowed to a region approximately 0.5-1.0-kb long. We also explored the genome-wide and coordinate regulation of 43 Ci-Bra-downstream genes. When we determined their chromosomal localization, it became evident that they are not clustered in a given region of the genome, but rather distributed evenly over 13 of the 14 pairs of chromosomes, suggesting that gene clustering does not contribute to coordinate control of the Ci-Bra downstream gene expression. Our results might provide Insights Into the molecular mechanisms underlying notochord formation in chordates.

Let-7b regulates the expression of the growth hormone receptor gene in deletion-type dwarf chickens.

PubMed

Lin, Shumao; Li, Hongmei; Mu, Heping; Luo, Wen; Li, Ying; Jia, Xinzheng; Wang, Sibing; Jia, Xiaolu; Nie, Qinghua; Li, Yugu; Zhang, Xiquan

2012-07-10

A deletion mutation in the growth hormone receptor (GHR) gene results in the inhibition of skeletal muscle growth and fat deposition in dwarf chickens. We used microarray techniques to determine microRNA (miRNA) and mRNA expression profiles of GHR in the skeletal muscles of 14-day-old embryos as well as 7-week-old deletion-type dwarf and normal-type chickens. Our aim was to elucidate the miRNA regulation of GHR expression with respect to growth inhibition and fat deposition. At the same developmental stages, different expression profiles in skeletal muscles of dwarf and normal chickens occurred for four miRNAs (miR-1623, miR-181b, let-7b, and miR-128). At different developmental stages, there was a significant difference in the expression profiles of a greater number of miRNAs. Eleven miRNAs were up-regulated and 18 down-regulated in the 7-week-old dwarf chickens when compared with profiles in 14-day-old embryos. In 7-week-old normal chickens, seven miRNAs were up-regulated and nine down-regulated compared with those in 14-day-old embryos. In skeletal muscles, 22 genes were up-regulated and 33 down-regulated in 14-day-old embryos compared with 7-week-old dwarf chickens. Sixty-five mRNAs were up-regulated and 108 down-regulated in 14-day-old embryos as compared with 7-week-old normal chickens. Thirty-four differentially expressed miRNAs were grouped into 18 categories based on overlapping seed and target sequences. Only let-7b was found to be complementary to its target in the 3' untranslated region of GHR, and was able to inhibit its expression. Kyoto Encyclopedia of Genes and Genomes pathway analysis and quantitative polymerase chain reactions indicated there were three main signaling pathways regulating skeletal muscle growth and fat deposition of chickens. These were influenced by let-7b-regulated GHR. Suppression of the cytokine signaling 3 (SOCS3) gene was found to be involved in the signaling pathway of adipocytokines. There is a critical miRNA, let-7b, involved in the regulation of GHR. SOCS3 plays a critical role in regulating skeletal muscle growth and fat deposition via let-7b-mediated GHR expression.

Let-7b regulates the expression of the growth hormone receptor gene in deletion-type dwarf chickens

PubMed Central

2012-01-01

Background A deletion mutation in the growth hormone receptor (GHR) gene results in the inhibition of skeletal muscle growth and fat deposition in dwarf chickens. We used microarray techniques to determine microRNA (miRNA) and mRNA expression profiles of GHR in the skeletal muscles of 14-day-old embryos as well as 7-week-old deletion-type dwarf and normal-type chickens. Our aim was to elucidate the miRNA regulation of GHR expression with respect to growth inhibition and fat deposition. Results At the same developmental stages, different expression profiles in skeletal muscles of dwarf and normal chickens occurred for four miRNAs (miR-1623, miR-181b, let-7b, and miR-128). At different developmental stages, there was a significant difference in the expression profiles of a greater number of miRNAs. Eleven miRNAs were up-regulated and 18 down-regulated in the 7-week-old dwarf chickens when compared with profiles in 14-day-old embryos. In 7-week-old normal chickens, seven miRNAs were up-regulated and nine down-regulated compared with those in 14-day-old embryos. In skeletal muscles, 22 genes were up-regulated and 33 down-regulated in 14-day-old embryos compared with 7-week-old dwarf chickens. Sixty-five mRNAs were up-regulated and 108 down-regulated in 14-day-old embryos as compared with 7-week-old normal chickens. Thirty-four differentially expressed miRNAs were grouped into 18 categories based on overlapping seed and target sequences. Only let-7b was found to be complementary to its target in the 3′ untranslated region of GHR, and was able to inhibit its expression. Kyoto Encyclopedia of Genes and Genomes pathway analysis and quantitative polymerase chain reactions indicated there were three main signaling pathways regulating skeletal muscle growth and fat deposition of chickens. These were influenced by let-7b-regulated GHR. Suppression of the cytokine signaling 3 (SOCS3) gene was found to be involved in the signaling pathway of adipocytokines. Conclusions There is a critical miRNA, let-7b, involved in the regulation of GHR. SOCS3 plays a critical role in regulating skeletal muscle growth and fat deposition via let-7b-mediated GHR expression. PMID:22781587

Effect of exogenous transforming growth factor β1 (TGF-β1) on early bovine embryo development.

PubMed

Barrera, Antonio D; García, Elina V; Miceli, Dora C

2018-06-08

SummaryDuring preimplantation development, embryos are exposed and have the capacity to respond to different growth factors present in the maternal environment. Among these factors, transforming growth factor β1 (TGF-β1) is a well known modulator of embryonic growth and development. However, its action during the first stages of development, when the embryo transits through the oviduct, has not been yet elucidated. The objective of the present study was to examine the effect of early exposure to exogenous TGF-β1 on embryo development and expression of pluripotency (OCT4, NANOG) and DNA methylation (DNMT1, DNMT3A, DNMT3B) genes in bovine embryos produced in vitro. First, gene expression analysis of TGF-β receptors confirmed a stage-specific expression pattern, showing greater mRNA abundance of TGFBR1 and TGFBR2 from the 2- to the 8-cell stage, before embryonic genome activation. Second, embryo culture for the first 48 h in serum-free CR1aa medium supplemented with 50 or 100 ng/ml recombinant TGF-β1 did not affect the cleavage and blastocyst rate (days 7 and 8). However, RT-qPCR analysis showed a significant increase in the relative abundance of NANOG and DNMT3A in the 8-cell stage embryos and expanded blastocysts (day 8) derived from TGF-β1 treated embryos. These results suggest an early action of exogenous TGF-β1 on the bovine embryo, highlighting the importance to provide a more comprehensive understanding of the role of TGF-β signalling during early embryogenesis.

The paracrine effect of exogenous growth hormone alleviates dysmorphogenesis caused by tbx5 deficiency in zebrafish (Danio rerio) embryos

PubMed Central

2012-01-01

Background Dysmorphogenesis and multiple organ defects are well known in zebrafish (Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome. Methods Using an oligonucleotide-based microarray analysis to study the expression of special genes in tbx5 morphants, we demonstrated that GH and some GH-related genes were markedly downregulated. Zebrafish embryos microinjected with tbx5-morpholino (MO) antisense RNA and mismatched antisense RNA in the 1-cell stage served as controls, while zebrafish embryos co-injected with exogenous growth hormone (GH) concomitant with tbx5-MO comprised the treatment group. Results The attenuating effects of GH in tbx5-MO knockdown embryos were quantified and observed at 24, 30, 48, 72, and 96 h post-fertilization. Though the understanding of mechanisms involving GH in the tbx5 functioning complex is limited, exogenous GH supplied to tbx5 knockdown zebrafish embryos is able to enhance the expression of downstream mediators in the GH and insulin-like growth factor (IGF)-1 pathway, including igf1, ghra, and ghrb, and signal transductors (erk1, akt2), and eventually to correct dysmorphogenesis in various organs including the heart and pectoral fins. Supplementary GH also reduced apoptosis as determined by a TUNEL assay and decreased the expression of apoptosis-related genes and proteins (bcl2 and bad) according to semiquantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis, respectively, as well as improving cell cycle-related genes (p27 and cdk2) and cardiomyogenetic genes (amhc, vmhc, and cmlc2). Conclusions Based on our results, tbx5 knockdown causes a pseudo GH deficiency in zebrafish during early embryonic stages, and supplementation of exogenous GH can partially restore dysmorphogenesis, apoptosis, cell growth inhibition, and abnormal cardiomyogenesis in tbx5 knockdown zebrafish in a paracrine manner. PMID:22776023

Distinct Spatiotemporal Expression of Serine Proteases Prss23 and Prss35 in Periimplantation Mouse Uterus and Dispensable Function of Prss35 in Fertility

PubMed Central

Diao, Honglu; Xiao, Shuo; Li, Rong; Zhao, Fei; Ye, Xiaoqin

2013-01-01

PRSS23 and PRSS35 are homologous proteases originally identified in mouse ovaries. In the periimplantation mouse uterus, Prss23 was highly expressed in the preimplantation gestation day 3.5 (D3.5) uterine luminal epithelium (LE). It disappeared from the postimplantation LE and reappeared in the stromal compartment next to the myometrium on D6.5. It was undetectable in the embryo from D4.5 to D6.5 but highly expressed in the embryo on D7.5. Prss35 became detectable in the uterine stromal compartment surrounding the embryo on D4.5 and shifted towards the mesometrial side of the stromal compartment next to the embryo from D5.5 to D7.5. In the ovariectomized uterus, Prss23 was moderately and Prss35 was dramatically downregulated by progesterone and 17β-estradiol. Based on the expression of Prss35 in granulosa cells and corpus luteum of the ovary and the early pregnant uterus, we hypothesized that PRSS35 might play a role in female reproduction, especially in oocyte development, ovulation, implantation, and decidualization. This hypothesis was tested in Prss35(−/−) mice, which proved otherwise. Between wild type (WT) and Prss35(−/−) mice, superovulation of immature females produced comparable numbers of cumulus-oocyte complexes; there were comparable numbers of implantation sites detected on D4.5 and D7.5; there were no obvious differences in the expression of implantation and decidualization marker genes in D4.5 or D7.5 uteri. Comparable mRNA expression levels of a few known protease-related genes in the WT and Prss35(−/−) D4.5 uteri indicated no compensatory upregulation. Comparable litter sizes from WT × WT and Prss35 (−/−)× Prss35 (−/−) crosses suggested that Prss35 gene was unessential for fertility and embryo development. Prss35 gene has been linked to cleft lip/palate in humans. However, no obvious such defects were observed in Prss35(−/−) mice. This study demonstrates the distinct expression of Prss23 and Prss35 in the periimplantation uterus and the dispensable role of Prss35 in fertility and embryo development. PMID:23451081

Human chorionic gonadotropin (hCG) in the secretome of cultured embryos: hyperglycosylated hCG and hCG-free beta subunit are potential markers for infertility management and treatment.

PubMed

Butler, Stephen A; Luttoo, Jameel; Freire, Maísa O T; Abban, Thomas K; Borrelli, Paola T A; Iles, Ray K

2013-09-01

Human chorionic gonadotropin (hCG) is produced by trophoblast cells throughout pregnancy, and gene expression studies have indicated that hCG-beta subunit (hCGβ) expression is active at the 2 blastomere stage. Here, we investigated the qualitative hCG output of developing embryos in culture and hCG isoforms expressed in the secretome as a novel sensitive method for detecting hCG. Culture media was collected from the culture plates of 118 embryos in culture (including controls and embryos at different stages of culture) from 16 patients undergoing routine fertility treatment. The hCGβ was detectable in media from 2 pronuclear (2PN) stage embryos through to the blastocyst stage. The hCGβ was absent in 1PN and arrested embryos as well as all media controls. Prior to hatching, hyperglycosylated hCG (hCGh) was observed selectively in 3PN embryos, but after hatching, along with hCG, became the dominant hCG molecule observed. We have reported at the 2PN stage the earliest evidence of hCGβ expression in embryos. There is a suggestion this may be indicative of quality in early embryos, and hCGh seen at the pronuclear stage may suggest triploid abnormality. The dominance of hCG, and hCGh expression, seen after blastocyst hatching may be indicative of potential implantation success. Thus, hCG isoforms have potential roles as biomarkers of embryo viability for embryo/blastocyst transfer.

Epigenetic analysis of bovine parthenogenetic embryonic fibroblasts.

PubMed

Kaneda, Masahiro; Takahashi, Masashi; Yamanaka, Ken-Ichi; Saito, Koji; Taniguchi, Masanori; Akagi, Satoshi; Watanabe, Shinya; Nagai, Takashi

2017-08-19

Although more than 100 imprinted genes have already been identified in the mouse and human genomes, little is known about genomic imprinting in cattle. For a better understanding of these genes in cattle, parthenogenetically activated bovine blastocysts were transferred to recipient cows to obtain parthenotes, and fibroblasts derived from a Day 40 (Day 0 being the day of parthenogenetic activation) parthenogenetic embryo (BpEFs) were successfully obtained. Bovine embryonic fibroblasts (BEFs) were also isolated from a normal fertilized embryo obtained from an artificially inseminated cow. The expression of imprinted genes was analyzed by RT-PCR. Paternally expressed genes (PEGs) in mouse (viz., IGF2, PEG3, ZAC1, NDN, DLK1, SGCE, and PEG10) were expressed in BEFs, but not in BpEFs, suggesting that these genes are also imprinted in cattle. However, other PEGs in mouse (viz., IMPACT, MAGEL2, SNRPN, and PEG1/MEST) were expressed in both BEFs and BpEFs. These genes may not be imprinted in BEFs. The expression of seven maternally expressed genes in mouse was also analyzed, and only CDKN1C was not expressed in BpEFs. The DNA methylation patterns of repetitive elements (Satellite I, Satellite II, alpha-satellite, and Art2) were not different between the BEFs and BpEFs; however, the differentially methylated region (DMR) of paternally methylated H19 was hypomethylated, whereas those of maternally methylated PEG3 and PEG10 were hypermethylated in BpEFs, as expected. The methylation of the SNRPN DMR was not different between the BEFs and BpEFs, in accordance with the SNRPN expression levels in both cell types. The XIST gene, which is essential for X chromosome inactivation in females, was expressed in BpEFs, whereas its DMR was half-methylated, suggesting that X chromosome inactivation is normal in these cells. Microarray analysis was also applied to identify novel PEGs that should be expressed only in BEFs but not in BpEFs. More than 300 PEG candidate genes, including IGF2, PEG3, and PEG10, were obtained. These results illustrate the epigenetic characteristic of bovine parthenogenetic embryos and contribute to the identification of novel imprinted genes in cattle.

Chlorpyrifos exposure affects fgf8, sox9, and bmp4 expression required for cranial neural crest morphogenesis and chondrogenesis in Xenopus laevis embryos.

PubMed

Tussellino, Margherita; Ronca, Raffaele; Carotenuto, Rosa; Pallotta, Maria M; Furia, Maria; Capriglione, Teresa

2016-10-01

Chlorpyrifos (CPF) is an organophosphate insecticide used primarily to control foliage and soil-borne insect pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce dose-related abnormalities such as slower brain growth and cerebral cortex thinning. In lower vertebrates, for example, fish and amphibians, teratogenic activity of this compound is correlated with several anatomical alterations. Little is known about the effects of CPF on mRNA expression of genes involved in early development of the anatomical structures appearing abnormal in embryos. This study investigated the effects of exposure to different CPF concentrations (10, 15 and 20 mg/L) on Xenopus laevis embryos from stage 4/8 to stage 46. Some of the morphological changes we detected in CPF-exposed embryos included cranial neural crest cell (NCC)-derived structures. For this reason, we analyzed the expression of select genes involved in hindbrain patterning (egr2), cranial neural crest chondrogenesis, and craniofacial development (fgf8, bmp4, sox9, hoxa2 and hoxb2). We found that CPF exposure induced a reduction in transcription of all the genes involved in NCC-dependent chondrogenesis, with largest reductions in fgf8 and sox9; whereas, in hindbrain, we did not find any alterations in egr2 expression. Changes in the expression of fgf8, bmp4, and sox9, which are master regulators of several developmental pathways, have important implications. If these changes are confirmed to belong to a general pattern of alterations in vertebrates prenatally exposed to OP, they might be useful to assess damage during vertebrate embryo development. Environ. Mol. Mutagen. 57:589-604, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Transcriptomic analysis in the developing zebrafish embryo after compound exposure: Individual gene expression and pathway regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hermsen, Sanne A.B., E-mail: Sanne.Hermsen@rivm.nl; Department of Toxicogenomics, Maastricht University, P.O. Box 616, 6200 MD, Maastricht; Institute for Risk Assessment Sciences

2013-10-01

The zebrafish embryotoxicity test is a promising alternative assay for developmental toxicity. Classically, morphological assessment of the embryos is applied to evaluate the effects of compound exposure. However, by applying differential gene expression analysis the sensitivity and predictability of the test may be increased. For defining gene expression signatures of developmental toxicity, we explored the possibility of using gene expression signatures of compound exposures based on commonly expressed individual genes as well as based on regulated gene pathways. Four developmental toxic compounds were tested in concentration-response design, caffeine, carbamazepine, retinoic acid and valproic acid, and two non-embryotoxic compounds, D-mannitol andmore » saccharin, were included. With transcriptomic analyses we were able to identify commonly expressed genes, which were mostly development related, after exposure to the embryotoxicants. We also identified gene pathways regulated by the embryotoxicants, suggestive of their modes of action. Furthermore, whereas pathways may be regulated by all compounds, individual gene expression within these pathways can differ for each compound. Overall, the present study suggests that the use of individual gene expression signatures as well as pathway regulation may be useful starting points for defining gene biomarkers for predicting embryotoxicity. - Highlights: • The zebrafish embryotoxicity test in combination with transcriptomics was used. • We explored two approaches of defining gene biomarkers for developmental toxicity. • Four compounds in concentration-response design were tested. • We identified commonly expressed individual genes as well as regulated gene pathways. • Both approaches seem suitable starting points for defining gene biomarkers.« less

Effects of the UV filter benzophenone-3 (oxybenzone) at low concentrations in zebrafish (Danio rerio)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blüthgen, Nancy; University of Basel, Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Klingelbergstrasse 50, CH-4056 Basel; Zucchi, Sara

Organic UV filters including benzophenone-3 (BP-3) are widely used to protect humans and materials from damage by UV irradiation. Despite the environmental occurrence of BP-3 in the aquatic environment, little is known about its effects and modes of action. In the present study we assess molecular and physiological effects of BP-3 in adult male zebrafish (Danio rerio) and in eleuthero-embryos by a targeted gene expression approach focusing on the sex hormone system. Fish and embryos are exposed for 14 days and 120 hours post fertilization, respectively, to 2.4–312 μg/L and 8.2–438 μg/L BP-3. Chemical analysis of water and fish demonstratesmore » that BP-3 is partly transformed to benzophenone-1 (BP-1) and both compounds are accumulated in adult fish. Biotransformation to BP-1 is absent in eleuthero-embryos. BP-3 exposure leads to similar alterations of gene expression in both adult fish and eleuthero-embryos. In the brain of adult males esr1, ar and cyp19b are down-regulated at 84 μg/L BP-3. There is no induction of vitellogenin expression by BP-3, both at the transcriptional and protein level. An overall down-regulation of the hsd3b, hsd17b3, hsd11b2 and cyp11b2 transcripts is observed in the testes, suggesting an antiandrogenic activity. No histological changes were observed in the testes after BP-3 treatment. The study leads to the conclusion that low concentrations of BP-3 exhibit similar multiple hormonal activities at the transcription level in two different life stages of zebrafish. Forthcoming studies should show whether this translates to additional physiological effects. Highlights: ► Activity of UV filter benzophenone-3 (BP-3) is assessed in zebrafish. ► BP-3 is partly metabolized to benzophenone-1 by adult fish but not embryos. ► Alterations of gene expression are similar in adult males and embryos. ► Gene expression alterations point to multiple hormonal activity of BP-3.« less

Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

PubMed Central

Dykes, Iain M.; Tempest, Lynne; Lee, Su-In; Turner, Eric E.

2011-01-01

The combinatorial expression of transcription factors frequently marks cellular identity in the nervous system, yet how these factors interact to determine specific neuronal phenotypes is not well understood. Sensory neurons of the trigeminal (TG) and dorsal root ganglia (DRG) co-express the homeodomain transcription factors Brn3a and Islet1, and past work has revealed partially overlapping programs of gene expression downstream of these factors. Here we examine sensory development in Brn3a/Islet1 double knockout mice (DKO mice). Sensory neurogenesis and the formation of the TG and DRG occur in DKO embryos, but the DRG are dorsally displaced, and the peripheral projections of the ganglia are markedly disturbed. Sensory neurons in DKO embryos show a profound loss of all early markers of sensory subtypes, including the Ntrk neurotrophin receptors, and the runt-family transcription factors Runx1 and Runx3. Examination of global gene expression in the E12.5 DRG of single and double mutant embryos shows that Brn3a and Islet1 are together required for nearly all aspects of sensory-specific gene expression, including several newly identified sensory markers. On a majority of targets Brn3a and Islet1 exhibit negative epistasis, in which the effects of the individual knockout alleles are less than additive in the DKO. Smaller subsets of targets exhibit positive epistasis, or are regulated exclusively by one factor. Brn3a/Islet1 double mutants also fail to developmentally repress neurogenic bHLH genes, and in vivo chromatin immunoprecipitation shows that Islet1 binds to a known Brn3a -regulated enhancer in the neurod4 gene, suggesting a mechanism of interaction between these genes. PMID:21734270

Applications of Tol2 Transposon-Mediated Gene Transfer for Stable Integration and Conditional Expression of Electroporated Genes in Chicken Embryos

NASA Astrophysics Data System (ADS)

Sato, Yuki; Takahashi, Yoshiko

Because of the high accessibility to developing embryos, avian embryos (chicken and quail) have long been used as a good model animal to study embryogenesis in vertebrates, especially amniotes (reviewed in Wolpert, 2004). The techniques used for “classical” avian embryology included tissue transplantations, tissue ablations, and cell-labeling by vital dye. At the end of the last century, the in ovo electropora tion technique was developed by Nakamura and his colleagues, and this modern method opened a way to study the roles of developmental genes directly in living embryos (Funahashi et al., 1999) reviewed in (Nakamura et al., 2004; Yasuda et al., 2000; Yasugi and Nakamura, 2000). This powerful technique allows us to introduce genes (DNA, RNA, morpholino) into embryos in a tissue-specific way by targeting a restricted area of embryonic tissues. Thus, the electroporation technique using chickens has provided numerous novel insights into the understanding of early development in vertebrates, making the chicken a unique model animal.

The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation

PubMed Central

2014-01-01

Background The Arabidopsis thaliana LEC2 gene encodes a B3 domain transcription factor, which plays critical roles during both zygotic and somatic embryogenesis. LEC2 exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network. Results An ortholog of the Arabidopsis Leafy Cotyledon 2 gene (AtLEC2) was characterized in Theobroma cacao (TcLEC2). TcLEC2 encodes a B3 domain transcription factor preferentially expressed during early and late zygotic embryo development. The expression of TcLEC2 was higher in dedifferentiated cells competent for somatic embryogenesis (embryogenic calli), compared to non-embryogenic calli. Transient overexpression of TcLEC2 in immature zygotic embryos resulted in changes in gene expression profiles and fatty acid composition. Ectopic expression of TcLEC2 in cacao leaves changed the expression levels of several seed related genes. The overexpression of TcLEC2 in cacao explants greatly increased the frequency of regeneration of stably transformed somatic embryos. TcLEC2 overexpressing cotyledon explants exhibited a very high level of embryogenic competency and when cultured on hormone free medium, exhibited an iterative embryogenic chain-reaction. Conclusions Our study revealed essential roles of TcLEC2 during both zygotic and somatic embryo development. Collectively, our evidence supports the conclusion that TcLEC2 is a functional ortholog of AtLEC2 and that it is involved in similar genetic regulatory networks during cacao somatic embryogenesis. To our knowledge, this is the first detailed report of the functional analysis of a LEC2 ortholog in a species other then Arabidopsis. TcLEC2 could potentially be used as a biomarker for the improvement of the SE process and screen for elite varieties in cacao germplasm. PMID:24758406

Rotatin is a novel gene required for axial rotation and left-right specification in mouse embryos.

PubMed

Faisst, Anja M; Alvarez-Bolado, Gonzalo; Treichel, Dieter; Gruss, Peter

2002-04-01

The genetic cascade that governs left-right (L-R) specification is starting to be elucidated. In the mouse, the lateral asymmetry of the body axis is revealed first by the asymmetric expression of nodal, lefty2 and pitx2 in the left lateral plate mesoderm of the neurulating embryo. Here we describe a novel gene, rotatin, essential for the correct expression of the key L-R specification genes nodal, lefty and Pitx2. Embryos deficient in rotatin show also randomized heart looping and delayed neural tube closure, and fail to undergo the critical morphogenetic step of axial rotation. The amino acid sequence deduced from the cDNA is predicted to contain at least three transmembrane domains. Our results show a novel key player in the genetic cascade that determines L-R specification, and suggest a causal link between this process and axial rotation.

The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28.

PubMed

Pla, M; Vilardell, J; Guiltinan, M J; Marcotte, W R; Niogret, M F; Quatrano, R S; Pagès, M

1993-01-01

The maize gene rab28 has been identified as ABA-inducible in embryos and vegetative tissues. It is also induced by water stress in young leaves. The proximal promoter region contains the conserved cis-acting element CCACGTGG (ABRE) reported for ABA induction in other plant genes. Transient expression assays in rice protoplasts indicate that a 134 bp fragment (-194 to -60 containing the ABRE) fused to a truncated cauliflower mosaic virus promoter (35S) is sufficient to confer ABA-responsiveness upon the GUS reporter gene. Gel retardation experiments indicate that nuclear proteins from tissues in which the rab28 gene is expressed can interact specifically with this 134 bp DNA fragment. Nuclear protein extracts from embryo and water-stressed leaves generate specific complexes of different electrophoretic mobility which are stable in the presence of detergent and high salt. However, by DMS footprinting the same guanine-specific contacts with the ABRE in both the embryo and leaf binding activities were detected. These results indicate that the rab28 promoter sequence CCACGTGG is a functional ABA-responsive element, and suggest that distinct regulatory factors with apparent similar affinity for the ABRE sequence may be involved in the hormone action during embryo development and in vegetative tissues subjected to osmotic stress.

Mechanisms that determine the internal environment of the developing brain: a transcriptomic, functional and ultrastructural approach.

PubMed

Liddelow, Shane A; Dziegielewska, Katarzyna M; Ek, C Joakim; Habgood, Mark D; Bauer, Hannelore; Bauer, Hans-Christian; Lindsay, Helen; Wakefield, Matthew J; Strazielle, Nathalie; Kratzer, Ingrid; Møllgård, Kjeld; Ghersi-Egea, Jean-François; Saunders, Norman R

2013-01-01

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

Breaking down pluripotency in the porcine embryo reveals both a premature and reticent stem cell state in the inner cell mass and unique expression profiles of the naive and primed stem cell states.

PubMed

Hall, Vanessa Jane; Hyttel, Poul

2014-09-01

To date, it has been difficult to establish bona fide porcine embryonic stem cells (pESC) and stable induced pluripotent stem cells. Reasons for this remain unclear, but they may depend on inappropriate culture conditions. This study reports the most insights to date on genes expressed in the pluripotent cells of the porcine embryo, namely the inner cell mass (ICM), the trophectoderm-covered epiblast (EPI), and the embryonic disc epiblast (ED). Specifically, we reveal that the early porcine ICM represents a premature state of pluripotency due to lack of translation of key pluripotent proteins, and the late ICM enters a transient, reticent pluripotent state which lacks expression of most genes associated with pluripotency. We describe a unique expression profile of the porcine EPI, reflecting the naive stem cell state, including expression of OCT4, NANOG, CRIPTO, and SSEA-1; weak expression of NrOB1 and REX1; but very limited expression of genes in classical pathways involved in regulating pluripotency. The porcine ED, reflecting the primed stem cell state, can be characterized by the expression of OCT4, NANOG, SOX2, KLF4, cMYC, REX1, CRIPTO, and KLF2. Further cell culture experiments using inhibitors against FGF, JAK/STAT, BMP, WNT, and NODAL pathways on cell cultures derived from day 5 and 10 embryos reveal the importance of FGF, JAK/STAT, and BMP signaling in maintaining cell proliferation of pESCs in vitro. Together, this article provides new insights into the regulation of pluripotency, revealing unique stem cell states in the different porcine stem cell populations derived from the early developing embryo.

Lmx1b is essential for Fgf8 and Wnt1 expression in the isthmic organizer during tectum and cerebellum development in mice.

PubMed

Guo, Chao; Qiu, Hai-Yan; Huang, Ying; Chen, Haixu; Yang, Rong-Qiang; Chen, Sheng-Di; Johnson, Randy L; Chen, Zhou-Feng; Ding, Yu-Qiang

2007-01-01

Secreted factors FGF8 and WNT1 are essential either for the inductive activity of the isthmus organizer or for the regionalization of the midbrain-hindbrain boundary (MHB). However, transcriptional regulation of these secreted factors during development remains to be elucidated. Here we show that the LIM homeobox gene Lmx1b is expressed in the anterior embryo as early as E7.5 and its expression becomes progressively restricted to the isthmus at E9.0. Analysis of gene expression in the MHB of the mutant embryos showed that many genes were lost by E9.5. In the MHB of Lmx1b-/- embryos, the expression of Fgf8, which normally occurs at the 4-somite stage, was completely absent, whereas Wnt1 was downregulated before the 4-somite stage. Moreover, transcription factors En1 and Pax2 were also downregulated prior to the 4-somite stage, whereas Gbx2 downregulation occurred at the 4-somite stage. By contrast, Otx2 and Pax6 expression was not affected in Lmx1b-/- embryos. The requirement of specific Lmx1b expression in the MHB was further confirmed by Wnt1-Cre-mediated region-specific conditional knockout of Lmx1b. As a result of these molecular defects, the development of the tectum and cerebellum was severely impaired in Lmx1b-/- mice. Taken together, our results indicate that Lmx1b plays an essential role in the development of the tectum and cerebellum by regulating expression of Fgf8, Wnt1 and several isthmus-related transcription factors in the MHB, and is a crucial component of a cross-regulatory network required for the induction activity of the isthmic organizer in the MHB.

Xmsx-1 modifies mesodermal tissue pattern along dorsoventral axis in Xenopus laevis embryo.

PubMed

Maeda, R; Kobayashi, A; Sekine, R; Lin, J J; Kung, H; Maéno, M

1997-07-01

This study analyzes the expression and the function of Xenopus msx-1 (Xmsx-1) in embryos, in relation to the ventralizing activity of bone morphogenetic protein-4 (BMP-4). Expression of Xmsx-1 was increased in UV-treated ventralized embryos and decreased in LiCl-treated dorsalized embryos at the neurula stage (stage 14). Whole-mount in situ hybridization analysis showed that Xmsx-1 is expressed in marginal zone and animal pole areas, laterally and ventrally, but not dorsally, at mid-gastrula (stage 11) and late-gastrula (stage 13) stages. Injection of BMP-4 RNA, but not activin RNA, induced Xmsx-1 expression in the dorsal marginal zone at the early gastrula stage (stage 10+), and introduction of a dominant negative form of BMP-4 receptor RNA suppressed Xmsx-1 expression in animal cap and ventral marginal zone explants at stage 14. Thus, Xmsx-1 is a target gene specifically regulated by BMP-4 signaling. Embryos injected with Xmsx-1 RNA in dorsal blastomeres at the 4-cell stage exhibited a ventralized phenotype, with microcephaly and swollen abdomen. Histological observation and immunostaining revealed that these embryos had a large block of muscle tissue in the dorsal mesodermal area instead of notochord. On the basis of molecular marker analysis, however, the injection of Xmsx-1 RNA did not induce the expression of alpha-globin, nor reduce cardiac alpha-actin in dorsal marginal zone explants. Furthermore, a significant amount of alpha-actin was induced and alpha-globin was turned off in the ventral marginal zone explants injected with Xmsx-1. These results indicated that Xmsx-1 is a target gene of BMP-4 signaling, but possesses a distinct activity on dorsal-ventral patterning of mesodermal tissues.

The TORMOZ Gene Encodes a Nucleolar Protein Required for Regulated Division Planes and Embryo Development in Arabidopsis[W

PubMed Central

Griffith, Megan E.; Mayer, Ulrike; Capron, Arnaud; Ngo, Quy A.; Surendrarao, Anandkumar; McClinton, Regina; Jürgens, Gerd; Sundaresan, Venkatesan

2007-01-01

Embryogenesis in Arabidopsis thaliana is marked by a predictable sequence of oriented cell divisions, which precede cell fate determination. We show that mutation of the TORMOZ (TOZ) gene yields embryos with aberrant cell division planes and arrested embryos that appear not to have established normal patterning. The defects in toz mutants differ from previously described mutations that affect embryonic cell division patterns. Longitudinal division planes of the proembryo are frequently replaced by transverse divisions and less frequently by oblique divisions, while divisions of the suspensor cells, which divide only transversely, appear generally unaffected. Expression patterns of selected embryo patterning genes are altered in the mutant embryos, implying that the positional cues required for their proper expression are perturbed by the misoriented divisions. The TOZ gene encodes a nucleolar protein containing WD repeats. Putative TOZ orthologs exist in other eukaryotes including Saccharomyces cerevisiae, where the protein is predicted to function in 18S rRNA biogenesis. We find that disruption of the Sp TOZ gene results in cell division defects in Schizosaccharomyces pombe. Previous studies in yeast and animal cells have identified nucleolar proteins that regulate the exit from M phase and cytokinesis, including factors involved in pre-rRNA processing. Our study suggests that in plant cells, nucleolar functions might interact with the processes of regulated cell divisions and influence the selection of longitudinal division planes during embryogenesis. PMID:17616738

DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

PubMed Central

Canovas, Sebastian; Ivanova, Elena; Romar, Raquel; García-Martínez, Soledad; Soriano-Úbeda, Cristina; García-Vázquez, Francisco A; Saadeh, Heba; Andrews, Simon; Kelsey, Gavin; Coy, Pilar

2017-01-01

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT. DOI: http://dx.doi.org/10.7554/eLife.23670.001 PMID:28134613

Evolution of vertebrate central nervous system is accompanied by novel expression changes of duplicate genes.

PubMed

Chen, Yuan; Ding, Yun; Zhang, Zuming; Wang, Wen; Chen, Jun-Yuan; Ueno, Naoto; Mao, Bingyu

2011-12-20

The evolution of the central nervous system (CNS) is one of the most striking changes during the transition from invertebrates to vertebrates. As a major source of genetic novelties, gene duplication might play an important role in the functional innovation of vertebrate CNS. In this study, we focused on a group of CNS-biased genes that duplicated during early vertebrate evolution. We investigated the tempo-spatial expression patterns of 33 duplicate gene families and their orthologs during the embryonic development of the vertebrate Xenopus laevis and the cephalochordate Brachiostoma belcheri. Almost all the identified duplicate genes are differentially expressed in the CNS in Xenopus embryos, and more than 50% and 30% duplicate genes are expressed in the telencephalon and mid-hindbrain boundary, respectively, which are mostly considered as two innovations in the vertebrate CNS. Interestingly, more than 50% of the amphioxus orthologs do not show apparent expression in the CNS in amphioxus embryos as detected by in situ hybridization, indicating that some of the vertebrate CNS-biased duplicate genes might arise from non-CNS genes in invertebrates. Our data accentuate the functional contribution of gene duplication in the CNS evolution of vertebrate and uncover an invertebrate non-CNS history for some vertebrate CNS-biased duplicate genes. Copyright © 2011. Published by Elsevier Ltd.

Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.

PubMed

Maconochie, M K; Nonchev, S; Studer, M; Chan, S K; Pöpperl, H; Sham, M H; Mann, R S; Krumlauf, R

1997-07-15

Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression.

PubMed

Lin, Che-Yi; Tsai, Ming-Yuan; Liu, Yu-Hsiu; Lu, Yu-Fen; Chen, Yi-Chung; Lai, Yun-Ren; Liao, Hsin-Chi; Lien, Huang-Wei; Yang, Chung-Hsiang; Huang, Chang-Jen; Hwang, Sheng-Ping L

2017-07-17

Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression. Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.

Functional analysis of sex-determination genes by gene silencing with LNA-DNA gapmers in the silkworm, Bombyx mori.

PubMed

Sakai, Hiroki; Sakaguchi, Honami; Aoki, Fugaku; Suzuki, Masataka G

2015-08-01

The sexual fate of B. mori is determined genetically; ZW, female and ZZ, male. Recently, we successfully identified a strong candidate gene at the top of the sex determination cascade in B. mori. This gene was termed Feminizer (Fem) and revealed to be a source of Fem-piRNA. Further, we found that B. mori doublesex (Bmdsx) splicing was markedly altered to produce the male-type isoform when a Fem-piRNA inhibitor was injected into ZW embryos. Moreover, knockdown of Masculinizer (Masc), a Fem-piRNA target gene, altered to produce the female-type isoform of Bmdsx in male embryos. However, it remains unclear as to whether Masc directly regulates the sex-specific expression of Bmdsx. In previous studies, we determined that the male-specific isoform of the Bombyx homolog of IGF-II mRNA-binding protein (Imp(M)) was involved in the male-specific splicing of Bmdsx. In an attempt to clarify the genetic relationship between Fem, Masc, Imp(M), and Bmdsx, knockdown experiments were performed. Knockdown of Fem shifted into male-type Bmdsx, Imp(M) and Masc in female embryos. Knockdown of Masc led to the production of the female-type Bmdsx and a dramatic reduction in Imp(M) expression in male embryos. Knockdown of Imp(M) shifted Bmdsx splice mode from the male-type into the female-type. Our results suggest that: (1) Fem reduces Masc expression, (2) Masc dramatically induces Imp(M) expression, and (3) Imp(M) shifting Bmdsx splice mode from the female-type into the male-type. Based on these findings, we propose a possible genetic cascade regulating sex determination in B. mori. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Making lineage decisions with biological noise: Lessons from the early mouse embryo.

PubMed

Simon, Claire S; Hadjantonakis, Anna-Katerina; Schröter, Christian

2018-04-30

Understanding how individual cells make fate decisions that lead to the faithful formation and homeostatic maintenance of tissues is a fundamental goal of contemporary developmental and stem cell biology. Seemingly uniform populations of stem cells and multipotent progenitors display a surprising degree of heterogeneity, primarily originating from the inherent stochastic nature of molecular processes underlying gene expression. Despite this heterogeneity, lineage decisions result in tissues of a defined size and with consistent proportions of differentiated cell types. Using the early mouse embryo as a model we review recent developments that have allowed the quantification of molecular intercellular heterogeneity during cell differentiation. We first discuss the relationship between these heterogeneities and developmental cellular potential. We then review recent theoretical approaches that formalize the mechanisms underlying fate decisions in the inner cell mass of the blastocyst stage embryo. These models build on our extensive knowledge of the genetic control of fate decisions in this system and will become essential tools for a rigorous understanding of the connection between noisy molecular processes and reproducible outcomes at the multicellular level. We conclude by suggesting that cell-to-cell communication provides a mechanism to exploit and buffer intercellular variability in a self-organized process that culminates in the reproducible formation of the mature mammalian blastocyst stage embryo that is ready for implantation into the maternal uterus. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Gene Expression and Transcriptional Hierarchies > Quantitative Methods and Models. © 2018 Wiley Periodicals, Inc.

Disruption of Msx-1 and Msx-2 reveals roles for these genes in craniofacial, eye, and axial development.

PubMed

Foerst-Potts, L; Sadler, T W

1997-05-01

In mouse embryos, the muscle segment homeobox genes, Msx-1 and Msx-2 are expressed during critical stages of neural tube, neural crest, and craniofacial development, suggesting that these genes play important roles in organogenesis and cell differentiation. Although the patterns of expression are intriguing, little is known about the function of these genes in vertebrate embryonic development. Therefore, the expression of both genes, separately and together, was disrupted using antisense oligodeoxynucleotides and whole embryo culture techniques. Antisense attenuation of Msx-1 during early stages of neurulation produced hypoplasia of the maxillary, mandibular, and frontonasal prominences, eye anomalies, and somite and neural tube abnormalities. Eye defects consisted of enlarged optic vesicles, which may ultimately result in micropthalmia similar to that observed in Small eye mice homozygous for mutations in the Pax-6 gene. Histological sections and SEM analysis revealed a thinning of the neuroepithelium in the diencephalon and optic vesicle and mesenchymal deficiencies in the craniofacial region. Injections of Msx-2 antisense oligodeoxynucleotides produced similar malformations as those targeting Msx-1, with the exception that there was an increase in number and severity of neural tube and somite defects. Embryos injected with the combination of Msx-1 + Msx-2 antisense oligodeoxynucleotides showed no novel abnormalities, suggesting that the genes do not operate in a redundant manner.

The unique and cooperative roles of the Grainy head-like transcription factors in epidermal development reflect unexpected target gene specificity.

PubMed

Boglev, Yeliz; Wilanowski, Tomasz; Caddy, Jacinta; Parekh, Vishwas; Auden, Alana; Darido, Charbel; Hislop, Nikki R; Cangkrama, Michael; Ting, Stephen B; Jane, Stephen M

2011-01-15

The Grainy head-like 3 (Grhl3) gene encodes a transcription factor that plays essential roles in epidermal morphogenesis during embryonic development, with deficient mice exhibiting failed skin barrier formation, defective wound repair, and loss of eyelid fusion. Despite sharing significant sequence homology, overlapping expression patterns, and an identical core consensus DNA binding site, the other members of the Grhl family (Grhl1 and -2) fail to compensate for the loss of Grhl3 in these processes. Here, we have employed diverse genetic models, coupled with biochemical studies, to define the inter-relationships of the Grhl factors in epidermal development. We show that Grhl1 and Grhl3 have evolved complete functional independence, as evidenced by a lack of genetic interactions in embryos carrying combinations of targeted alleles of these genes. In contrast, compound heterozygous Grhl2/Grhl3 embryos displayed failed wound repair, and loss of a single Grhl2 allele in Grhl3-null embryos results in fully penetrant eyes open at birth. Expression of Grhl2 from the Grhl3 locus in homozygous knock-in mice corrects the wound repair defect, but these embryos still display a complete failure of skin barrier formation. This functional dissociation is due to unexpected differences in target gene specificity, as both GRHL2 and GRHL3 bind to and regulate expression of the wound repair gene Rho GEF 19, but regulation of the barrier forming gene, Transglutaminase 1 (TGase1), is unique to GRHL3. Our findings define the mechanisms underpinning the unique and cooperative roles of the Grhl genes in epidermal development. Copyright © 2010 Elsevier Inc. All rights reserved.

Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers

PubMed Central

Field, Amanda; Xiang, Jie; Anderson, W. Ray; Graham, Patricia; Pick, Leslie

2016-01-01

The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern. PMID:27723822

Alteration of gene expression by alcohol exposure at early neurulation.

PubMed

Zhou, Feng C; Zhao, Qianqian; Liu, Yunlong; Goodlett, Charles R; Liang, Tiebing; McClintick, Jeanette N; Edenberg, Howard J; Li, Lang

2011-02-21

We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.

Effects of maternal dietary olive oil on pathways involved in diabetic embryopathy.

PubMed

Higa, Romina; Roberti, Sabrina Lorena; Musikant, Daniel; Mazzucco, María Belén; White, Verónica; Jawerbaum, Alicia

2014-11-01

Maternal diabetes induces a pro-oxidant/pro-inflammatory intrauterine environment related to the induction of congenital anomalies. Peroxisome proliferator activated receptors (PPARs) are transcription factors that regulate antioxidant and anti-inflammatory pathways. We investigated whether maternal diets supplemented with olive oil, enriched in oleic acid, a PPAR agonist, can regulate the expression of PPAR system genes, levels of lipoperoxidation and activity of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) in embryos and decidua from diabetic rats. The embryos and decidua from diabetic rats showed reduced expression of PPARs and increased concentration of lipoperoxidation, MMPs and TIMPs, whereas the maternal treatments enriched in olive oil increased PPARδ in embryos and PPARγ and PPARγ-coactivator-1α expression in decidua, and increased TIMPs concentrations and decreased lipoperoxidation and MMPs activity in both tissues. Thus, maternal diets enriched in olive oil can regulate embryonic and decidual PPAR system genes expression and reduce the pro-oxidant/pro-inflammatory environment during rat early organogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

Pax-3, a novel murine DNA binding protein expressed during early neurogenesis.

PubMed Central

Goulding, M D; Chalepakis, G; Deutsch, U; Erselius, J R; Gruss, P

1991-01-01

We describe the isolation and characterization of Pax-3, a novel murine paired box gene expressed exclusively during embryogenesis. Pax-3 encodes a 479 amino acid protein with an Mr of 56 kd containing both a paired domain and a paired-type homeodomain. The Pax-3 protein is a DNA binding protein that specifically recognizes the e5 sequence present upstream of the Drosophila even-skipped gene. Pax-3 transcripts are first detected in 8.5 day mouse embryos where they are restricted to the dorsal part of the neuroepithelium and to the adjacent segmented dermomyotome. During early neurogenesis, Pax-3 expression is limited to mitotic cells in the ventricular zone of the developing spinal cord and to distinct regions in the hindbrain, midbrain and diencephalon. In 10-12 day embryos, expression of Pax-3 is also seen in neural crest cells of the developing spinal ganglia, the craniofacial mesectoderm and in limb mesenchyme of 10 and 11 day embryos. Images PMID:2022185

Improved exogenous DNA uptake in bovine spermatozoa and gene expression in embryos using membrane destabilizing agents in ICSI-SMGT.

PubMed

Sánchez-Villalba, Esther; Arias, María Elena; Zambrano, Fabiola; Loren, Pía; Felmer, Ricardo

2018-02-01

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

Diffusion and scaling during early embryonic pattern formation

PubMed Central

Gregor, Thomas; Bialek, William; van Steveninck, Rob R. de Ruyter; Tank, David W.; Wieschaus, Eric F.

2005-01-01

Development of spatial patterns in multicellular organisms depends on gradients in the concentration of signaling molecules that control gene expression. In the Drosophila embryo, Bicoid (Bcd) morphogen controls cell fate along 70% of the anteroposterior axis but is translated from mRNA localized at the anterior pole. Gradients of Bcd and other morphogens are thought to arise through diffusion, but this basic assumption has never been rigorously tested in living embryos. Furthermore, because diffusion sets a relationship between length and time scales, it is hard to see how patterns of gene expression established by diffusion would scale proportionately as egg size changes during evolution. Here, we show that the motion of inert molecules through the embryo is well described by the diffusion equation on the relevant length and time scales, and that effective diffusion constants are essentially the same in closely related dipteran species with embryos of very different size. Nonetheless, patterns of gene expression in these different species scale with egg length. We show that this scaling can be traced back to scaling of the Bcd gradient itself. Our results, together with constraints imposed by the time scales of development, suggest that the mechanism for scaling is a species-specific adaptation of the Bcd lifetime. PMID:16352710

Expression of thyroid hormone regulator genes in the yolk sac membrane of the developing chicken embryo

PubMed Central

TOO, Hanny Cho; SHIBATA, Mitsuhiro; YAYOTA, Masato; DARRAS, Veerle M.; IWASAWA, Atsushi

2017-01-01

Thyroid hormones (THs) are essential for the correct development of nearly every structure in the body from the very early stages of development, yet the embryonic thyroid gland is not functional at these stages. To clarify the roles of the egg yolk as a source of THs, the TH content in the yolk and the expression of TH regulator genes in the yolk sac membrane were evaluated throughout the 21-day incubation period of chicken embryos. The yolk TH content (22.3 ng triiodothyronine and 654.7 ng thyroxine per total yolk on day 4 of incubation) decreased almost linearly along with development. Real-time PCR revealed gene expression of transthyretin, a principal TH distributor in the chicken, and of a TH-inactivating iodothyronine deiodinase (DIO3), until the second week of incubation when the embryonic pituitary-thyroid axis is generally thought to start functioning. The TH-activating deiodinase (DIO2) and transmembrane transporter of thyroxine (SLCO1C1) genes were expressed in the last week of incubation, which coincided with a marked increase of circulating thyroxine and a reduction in the yolk sac weight. DIO1, which can remove iodine from inactive THs, was expressed throughout the incubation period. It is assumed that the chicken yolk sac inactivates THs contained abundantly in the yolk and supplies the hormones to the developing embryo in appropriate concentrations until the second week of incubation, while THs may be activated in the yolk sac membrane in the last week of incubation. Additionally, the yolk sac could serve as a source of iodine for the embryo. PMID:28652559

Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation

PubMed Central

Liu, Yunlong; Balaraman, Yokesh; Wang, Guohua; Nephew, Kenneth P.; Zhou, Feng C.

2009-01-01

Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88 mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10 and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p < 0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes in gene expression, which together may contribute to the observed abnormal fetal development. PMID:20009564

Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation.

PubMed

Liu, Yunlong; Balaraman, Yokesh; Wang, Guohua; Nephew, Kenneth P; Zhou, Feng C

2009-10-01

Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10, and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p<0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes in gene expression, which together may contribute to the observed abnormal fetal development.

Zebrafish msxB, msxC and msxE function together to refine the neural-nonneural border and regulate cranial placodes and neural crest development.

PubMed

Phillips, Bryan T; Kwon, Hye-Joo; Melton, Colt; Houghtaling, Paul; Fritz, Andreas; Riley, Bruce B

2006-06-15

The zebrafish muscle segment homeobox genes msxB, msxC and msxE are expressed in partially overlapping domains in the neural crest and preplacodal ectoderm. We examined the roles of these msx genes in early development. Disrupting individual msx genes causes modest variable defects, whereas disrupting all three produces a reproducible severe phenotype, suggesting functional redundancy. Neural crest differentiation is blocked at an early stage. Preplacodal development begins normally, but placodes arising from the msx expression domain later show elevated apoptosis and are reduced in size. Cell proliferation is normal in these tissues. Unexpectedly, Msx-deficient embryos become ventralized by late gastrulation whereas misexpression of msxB dorsalizes the embryo. These effects appear to involve Distal-less (Dlx) protein activity, as loss of dlx3b and dlx4b suppresses ventralization in Msx-depleted embryos. At the same time, Msx-depletion restores normal preplacodal gene expression to dlx3b-dlx4b mutants. These data suggest that mutual antagonism between Msx and Dlx proteins achieves a balance of function required for normal preplacodal differentiation and placement of the neural-nonneural border.

The Armadillo Repeat Gene ZAK IXIK Promotes Arabidopsis Early Embryo and Endosperm Development through a Distinctive Gametophytic Maternal Effect[C][W][OA

PubMed Central

Ngo, Quy A.; Baroux, Celia; Guthörl, Daniela; Mozerov, Peter; Collinge, Margaret A.; Sundaresan, Venkatesan; Grossniklaus, Ueli

2012-01-01

The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin–dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of MINISEED3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways. PMID:23064319

The gap gene giant of Rhodnius prolixus is maternally expressed and required for proper head and abdomen formation.

PubMed

Lavore, Andrés; Pagola, Lucía; Esponda-Behrens, Natalia; Rivera-Pomar, Rolando

2012-01-01

The segmentation process in insects depends on a hierarchical cascade of gene activity. The first effectors downstream of the maternal activation are the gap genes, which divide the embryo in broad fields. We discovered a sequence corresponding to the leucine-zipper domain of the orthologue of the gene giant (Rp-gt) in traces from the genome of Rhodnius prolixus, a hemipteran with intermediate germ-band development. We cloned the Rp-gt gene from a normalized cDNA library and characterized its expression and function. Bioinformatic analysis of 12.5 kbp of genomic sequence containing the Rp-gt transcriptional unit shows a cluster of bona fide regulatory binding sites, which is similar in location and structure to the predicted posterior expression domain of the Drosophila orthologue. Rp-gt is expressed in ovaries and maternally supplied in the early embryo. The maternal contribution forms a gradient of scattered patches of mRNA in the preblastoderm embryo. Zygotic Rp-gt is expressed in two domains that after germ band extension are restricted to the head and the posterior growth zone. Parental RNAi shows that Rp-gt is required for proper head and abdomen formation. The head lacks mandibulary and maxillary appendages and shows reduced clypeus-labrum, while the abdomen lacks anterior segments. We conclude that Rp-gt is a gap gene on the head and abdomen and, in addition, has a function in patterning the anterior head capsule suggesting that the function of gt in hemipterans is more similar to dipterans than expected. Copyright © 2011. Published by Elsevier Inc.

Transcript Profiling of Individual Twin Blastomeres Derived by Splitting Two-Cell Stage Murine Embryos1

PubMed Central

Roberts, R. Michael; Katayama, Mika; Magnuson, Scott R.; Falduto, Michael T.; Torres, Karen E.O.

2010-01-01

In invertebrates and amphibians, informational macromolecules in egg cytoplasm are organized to provide direction to the formation of embryonic lineages, but it is unclear whether vestiges of such prepatterning exist in mammals. Here we examined whether twin blastomeres from 2-cell stage mouse embryos differ in mRNA content. mRNA from 26 blastomeres derived from 13 embryos approximately mid-way through their second cell cycle was subjected to amplification. Twenty amplified samples were hybridized to arrays. Of those samples that hybridized successfully, 12 samples in six pairs were used in the final analysis. Probes displaying normalized values >0.25 (n = 4573) were examined for consistent bias in expression within blastomere pairs. Although transcript content varied between both individual embryos and twin blastomeres, no consistent asymmetries were observed for the majority of genes, with only 178 genes displaying a >1.4-fold difference in expression across all six pairs. Although class discovery clustering showed that blastomere pairs separated into two distinct groups in terms of their differentially expressed genes, when the data were tested for significance of asymmetrical expression, only 39 genes with >1.4-fold change ratios in six of six blastomere pairs passed the two-sample t-test (P < 0.05). Transcripts encoding proteins implicated in RNA processing and cytoskeletal organization were among the most abundant, differentially distributed mRNA, suggesting that a stochastically based lack of synchrony in cell cycle progression between the two cells might explain at least some and possibly all of the asymmetries in transcript composition. PMID:21076082

A Conserved Developmental Patterning Network Produces Quantitatively Different Output in Multiple Species of Drosophila

PubMed Central

Meyer, Miriah; Wunderlich, Zeba; Simirenko, Lisa; Luengo Hendriks, Cris L.; Keränen, Soile V. E.; Henriquez, Clara; Knowles, David W.; Biggin, Mark D.; Eisen, Michael B.; DePace, Angela H.

2011-01-01

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3–4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells. PMID:22046143

Comparative analysis of gene expression between semigametic Pima 57-6 and non-semigametic Pima S-1 in cotton by differential display

USDA-ARS?s Scientific Manuscript database

Semigamy in cotton is a type of facultative apomixis controlled by a single incompletely dominant gene (Se) in which the sperm and egg nuclei fail to fuse after the sperm nucleus has entered the embryo sac, giving rise to diploid, haploid or even chimeral embryos comprised of paternal and maternal o...

Dose–response analysis of phthalate effects on gene expression in rat whole embryo culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robinson, Joshua F.; Department of Toxicogenomics, Maastricht University, Maastricht; Verhoef, Aart

2012-10-01

The rat postimplantation whole embryo culture (WEC) model serves as a potential screening tool for developmental toxicity. In this model, cultured rat embryos are exposed during early embryogenesis and evaluated for morphological effects. The integration of molecular-based markers may lead to improved objectivity, sensitivity and predictability of WEC in assessing developmental toxic properties of compounds. In this study, we investigated the concentration-dependent effects of two phthalates differing in potency, mono(2-ethylhexyl) phthalate (MEHP) and monomethyl phthalate (MMP, less toxic), on the transcriptome in WEC to examine gene expression in relation with dysmorphogenesis. MEHP was more potent than MMP in inducing genemore » expression changes as well as changes on morphology. MEHP induced significant enrichment of cholesterol/lipid/steroid (CLS) metabolism and apoptosis pathways which was associated with developmental toxicity. Regulation of genes within CLS metabolism pathways represented the most sensitive markers of MEHP exposure, more sensitive than classical morphological endpoints. As shown in direct comparisons with toxicogenomic in vivo studies, alterations in the regulation of CLS metabolism pathways has been previously identified to be associated with developmental toxicity due to phthalate exposure in utero. Our results support the application of WEC as a model to examine relative phthalate potency through gene expression and morphological responses. Additionally, our results further define the applicability domain of the WEC model for developmental toxicological investigations. -- Highlights: ► We examine the effect of two phthalates on gene expression and morphology in WEC. ► MEHP is more potent than MMP in inducing gene expression changes and dysmorphogenesis. ► MEHP significantly disrupts cholesterol metabolism pathways in a dose-dependent manner. ► Specific phthalate-related mechanisms in WEC are relevant to mechanisms in vivo.« less

The murine homeobox gene Msx-3 shows highly restricted expression in the developing neural tube.

PubMed

Shimeld, S M; McKay, I J; Sharpe, P T

1996-04-01

The mouse homeobox-genes Msx-1 and Msx-2 are expressed in several areas of the developing embryo, including the neural tube, neural crest, facial processes and limb buds. Here we report the characterisation of a third mouse Msx gene, which we designate Msx-3. The embryonic expression of Msx-3 was found to differ from that of Msx-1 and -2 in that it was confined to the dorsal neural tube. In embryos with 5-8 somites a segmental pattern of expression was observed in the hindbrain, with rhombomeres 3 and 5 lacking Msx-3 while other rhombomeres expressed Msx-3. This pattern was transient, however, such that in embryos with 18 or more somites expression was continuous throughout the dorsal hindbrain and anterior dorsal spinal cord. Differentiation of dorsal cell types in the neural tube can be induced by addition of members of the Tgf-beta family. Additionally, Msx-1 and -2 have been shown to be activated by addition of the Tgf-beta family member Bmp-4. To determine if Bmp-4 could activate Msx-3, we incubated embryonic hindbrain explants with exogenous Bmp-4. The dorsal expression of Msx-3 was seen to expand into more ventral regions of the neurectoderm in Bmp-4-treated cultures, implying that Bmp-4 may be able to mimic an in vivo signal that induces Msx-3.

Manipulation of gene expression by infrared laser heat shock and its application to the study of tracheal development in Drosophila.

PubMed

Miao, Guangxia; Hayashi, Shigeo

2015-03-01

Induction of gene expression in a specific cell and a defined time window is desirable to investigate gene function at the cellular level during morphogenesis. To achieve this, we attempted to introduce the infrared laser-evoked gene operator system (IR-LEGO, Kamei et al., 2009) in the Drosophila embryo. In this technique, infrared laser light illumination induces genes to be expressed under the control of heat shock promoters at the single cell level. We applied IR-LEGO to a transgenic fly stock, HS-eGFP, in which the enhanced green fluorescent protein (eGFP) gene is placed under the control of heat shock protein 70 promoter, and showed that eGFP expression can be induced in single cells within 1-2 hr after IR illumination. Furthermore, induction of HS-Branchless transgene encoding the Drosophila fibroblast growth factor (FGF) effectively altered the migration and branching patterns of the tracheal system. Our results indicated that IR-LEGO is a promising choice for the timely control of gene expression in a small group of cells in the Drosophila embryo. By using IR-LEGO, we further demonstrated that the tracheal terminal branching program is sensitive to localized expression of exogenous FGF. © 2014 Wiley Periodicals, Inc.

Conserved and divergent expression patterns of markers of axial development in the laboratory opossum, Monodelphis domestica.

PubMed

Yoshida, Michio; Kajikawa, Eriko; Yamamoto, Daisuke; Kurokawa, Daisuke; Yonemura, Shigenobu; Kobayashi, Kensaku; Kiyonari, Hiroshi; Aizawa, Shinichi

2016-12-01

Previous comparative studies suggest that the requirement for Nodal in epiblast and hypoblast development is unique to mammalians. Expression of anterior visceral endoderm (AVE) genes in the visceral endoderm and of their orthologs in the hypoblast may be unique to mammalians and avians, and is absent in the reptilian hypoblast. Axis formation in reptiles is signaled by the formation of the posterior marginal epiblast (PME), which expresses a series of primitive streak genes. To assess the phylogenetic origin of Nodal and AVE gene expression and axis formation in amniotes, we examined marker gene expression in gray short-tailed opossum, a metatherian. Nodal was expressed in neither epiblast nor hypoblast of opossum embryos. No AVE genes were expressed in the opossum hypoblast. Attainment of polarity in the embryonic disk was signaled by Nodal, Wnt3a, Fgf8, and Bra expression in the PME at 8.5 days post-coitus. Nodal expression in epiblast or hypoblast may be unique to eutherians. AVE gene expression in visceral endoderm and hypoblast may have been independently acquired in eutherian and avian lineages. PME formation appears to be the event that signals axis formation in reptilian and metatherian embryos, and thus may be an ancestral characteristic of basal amniotes. Developmental Dynamics 245:1176-1188, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos.

PubMed

Gui, Tao; Zhang, Meiling; Chen, Jianwen; Zhang, Yuanliang; Zhou, Naru; Zhang, Yu; Tao, Jia; Sui, Liucai; Li, Yunsheng; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2012-08-01

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.

Tissue-specific expression of FoxD reporter constructs in amphioxus embryos.

PubMed

Yu, Jr-Kai; Holland, Nicholas D; Holland, Linda Z

2004-10-15

Cephalochordates (amphioxus), the closest living invertebrate relatives of the vertebrates, are key to understanding the evolution of developmental mechanisms during the invertebrate-to-vertebrate transition. However, a major impediment to amphioxus as a model organism for developmental biology has been the inability to introduce transgenes or other macromolecules into the embryos. Here, we report the development of a reproducible method for microinjection of amphioxus eggs. Specifically, we show that expression of a LacZ reporter construct including 6.3 kb of AmphiFoxD upstream regulatory DNA recapitulates expression of the endogenous gene in the nerve cord, somites, and notochord. We have also identified the 1.6 kb at the 5' end of this region as essential for expression in the first two of these domains and the 4.7 kb at the 3' end as sufficient for expression in the notochord. This study, which is the first report of a method for introduction of large molecules such as DNA into amphioxus embryos, opens the way for studies of gene regulation and function in amphioxus and for comparative studies with vertebrates to understand the relationship between the extensive gene duplications that occurred within the vertebrate lineage and the evolution of vertebrate innovations such as neural crest.

A liver enhancer in the fibrinogen gene cluster.

PubMed

Fort, Alexandre; Fish, Richard J; Attanasio, Catia; Dosch, Roland; Visel, Axel; Neerman-Arbez, Marguerite

2011-01-06

The plasma concentration of fibrinogen varies in the healthy human population between 1.5 and 3.5 g/L. Understanding the basis of this variability has clinical importance because elevated fibrinogen levels are associated with increased cardiovascular disease risk. To identify novel regulatory elements involved in the control of fibrinogen expression, we used sequence conservation and in silico-predicted regulatory potential to select 14 conserved noncoding sequences (CNCs) within the conserved block of synteny containing the fibrinogen locus. The regulatory potential of each CNC was tested in vitro using a luciferase reporter gene assay in fibrinogen-expressing hepatoma cell lines (HuH7 and HepG2). 4 potential enhancers were tested for their ability to direct enhanced green fluorescent protein expression in zebrafish embryos. CNC12, a sequence equidistant from the human fibrinogen alpha and beta chain genes, activates strong liver enhanced green fluorescent protein expression in injected embryos and their transgenic progeny. A transgenic assay in embryonic day 14.5 mouse embryos confirmed the ability of CNC12 to activate transcription in the liver. While additional experiments are necessary to prove the role of CNC12 in the regulation of fibrinogen, our study reveals a novel regulatory element in the fibrinogen locus that is active in the liver and may contribute to variable fibrinogen expression in humans.

Part I. Embryonic surgery using femtosecond laser pulses for the delivery of exogenous materials and the analysis of gene expression

NASA Astrophysics Data System (ADS)

Kohli, V.; Elezzabi, A. Y.

2008-02-01

Herein, we demonstrate the application of high-intensity femtosecond (fs) laser pulses for performing laser surgery on the embryonic cells of developing zebrafish (Danio rerio). When fs laser pulses were focused onto individual blastomeres, transient pores were formed exposing the extracellular space to the intracellular environment. Utilizing the transient pores as a pathway for delivery of exogenous material, both chorionated and dechorionated zebrafish embryos were successfully loaded with a fluorescent reporter molecule (fluorescein isothiocyanate (FITC)). Streptavidin-conjugated quantum dots or plasmid DNA (Simian-CMV-EGFP). Both FITC and quantum dots were found to disperse throughout the blastomere cells as the embryo developed. Gene expression was seen in 24 hour post-fertilized embryos, with fluorescence observed in the notochord, floor plates, somites and tails of the larvae. We also determined the survivability of laser-manipulated embryos by rearing zebrafish from early to mid cleavage stage (2-cell to 8/16-cell) to pec-fin stage. Survival rates of 89 and 100 % were found for dechorionated and chorionated embryos, respectively.

Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize.

PubMed

Busk, P K; Jensen, A B; Pagès, M

1997-06-01

The rab17 gene from maize is transcribed in late embryonic development and is responsive to abscisic acid and water stress in embryo and vegetative tissues. In vivo footprinting and transient transformation of rab17 were performed in embryos and vegetative tissues to characterize the cis-elements involved in regulation of the gene. By in vivo footprinting, protein binding was observed to nine elements in the promoter, which correspond to five putative ABREs (abscisic acid responsive elements) and four other sequences. The footprints indicated that distinct proteins interact with these elements in the two developmental stages. In transient transformation, six of the elements were important for high level expression of the rab17 promoter in embryos, whereas only three elements were important in leaves. The cis-acting sequences can be divided in embryo-specific, ABA-specific and leaf-specific elements on the basis of protein binding and the ability to confer expression of rab17. We found one positive, new element, called GRA, with the sequence CACTGGCCGCCC. This element was important for transcription in leaves but not in embryos. Two other non-ABRE elements that stimulated transcription from the rab17 promoter resemble previously described abscisic acid and drought-inducible elements. There were differences in protein binding and function of the five ABREs in the rab17 promoter. The possible reasons for these differences are discussed. The in vivo data obtained suggest that an embryo-specific pathway regulates transcription of the rab genes during development, whereas another pathway is responsible for induction in response to ABA and drought in vegetative tissues.

Size-dependent regulation of dorsal-ventral patterning in the early Drosophila embryo

PubMed Central

Garcia, Mayra; Nahmad, Marcos; Reeves, Gregory T.; Stathopoulos, Angelike

2013-01-01

How natural variation in embryo size affects patterning of the Drosophila embryo dorsal-ventral (DV) axis is not known. Here we examined quantitatively the relationship between nuclear distribution of the Dorsal transcription factor, boundary positions for several target genes, and DV axis length. Data were obtained from embryos of a wild-type background as well as from mutant lines inbred to size select embryos of smaller or larger sizes. Our data show that the width of the nuclear Dorsal gradient correlates with DV axis length. In turn, for some genes expressed along the DV axis, the boundary positions correlate closely with nuclear Dorsal levels and with DV axis length; while the expression pattern of others is relatively constant and independent of the width of the Dorsal gradient. In particular, the patterns of snail (sna) and ventral nervous-system defective (vnd) correlate with nuclear Dorsal levels and exhibit scaling to DV length; while the pattern of intermediate neuroblasts defective (ind) remains relatively constant with respect to changes in Dorsal and DV length. However, in mutants that exhibit an abnormal expansion of the Dorsal gradient which fails to scale to DV length, only sna follows the Dorsal distribution and exhibits overexpansion; in contrast, vnd and ind do not overexpand suggesting some additional mechanism acts to refine the dorsal boundaries of these two genes. Thus, our results argue against the idea that the Dorsal gradient works as a global system of relative coordinates along the DV axis and suggest that individual targets respond to changes in embryo size in a gene-specific manner. PMID:23800450

Mono-2-ethylhexyl phthalate disrupts neurulation and modifies the embryonic redox environment and gene expression

PubMed Central

Sant, Karilyn E.; Dolinoy, Dana C.; Jilek, Joseph L.; Sartor, Maureen A.; Harris, Craig

2016-01-01

Mono-2-ethylhexl phthalate (MEHP) is the primary metabolite of di-2-ethylhexyl phthalate (DEHP), a ubiquitous contaminant in plastics. This study sought to determine how structural defects caused by MEHP in mouse whole embryo culture were related to temporal and spatial patterns of redox state and gene expression. MEHP reduced morphology scores along with increased incidence of neural tube defects. Glutathione (GSH) and cysteine (Cys) concentrations fluctuated spatially and temporally in embryo (EMB) and visceral yolk sac (VYS) across the 24h culture. Redox potentials (Eh) for GSSG/GSH were increased by MEHP in EMB (12h) but not in VYS. CySS/CyS Eh in EMB and VYS were significantly increased at 3h and 24h, respectively. Gene expression at 6h showed that MEHP induced selective alterations in EMB and VYS for oxidative phosphorylation and energy metabolism pathways. Overall, MEHP affects neurulation, alters Eh, and spatially alters the expression of metabolic genes in the early organogenesis-stage mouse conceptus. PMID:27167697

BMP signaling modulates hepcidin expression in zebrafish embryos independent of hemojuvelin.

PubMed

Gibert, Yann; Lattanzi, Victoria J; Zhen, Aileen W; Vedder, Lea; Brunet, Frédéric; Faasse, Sarah A; Babitt, Jodie L; Lin, Herbert Y; Hammerschmidt, Matthias; Fraenkel, Paula G

2011-01-21

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

Functional organization of DNA elements regulating SM30alpha, a spicule matrix gene of sea urchin embryos.

PubMed

Yamasu, K; Wilt, F H

1999-02-01

The SM30a gene encodes a protein in the embryonic endoskeleton of the sea urchin Strongylocentrotus purpuratus, and is specifically expressed in the skeletogenic primary mesenchyme cell lineage. To clarify the mechanism for the differentiation of this cell lineage, which proceeds rather autonomously in the embryo, regulation of the SM30alpha gene was investigated previously and it was shown that the distal DNA region upstream of this gene from - 1.6 to - 1.0 kb contained numerous negative regulatory elements that suppressed the ectopic expression of the gene in the gut. Here we study the influence of the proximal region from - 303 to + 104 bp. Analysis of the expression of reporter constructs indicated that a strong positive enhancer element existed in the region from -142 to -105bp. This element worked both in forward and reverse orientations and additively when placed tandemly upstream to the reporter gene. In addition, other weaker positive and negative regulatory sites were also detected throughout the proximal region. Electrophoretic gel mobility shift analyses showed that multiple nuclear proteins were bound to the putative strong enhancer region. One of the proteins binding to this region was present in ear y blastulae, a time when the SM30 gene was still silent, but it was not in prism embryos actively expressing the gene. The binding region for this blastula-specific protein was narrowed down to the region from - 132 to -122 bp, which included the consensus binding site for the mammalian proto-oncogene product, Ets. Two possible SpGCF1 binding sites were identified in the vicinity of the enhancer region. This information was used to make a comparison of the general regulatory architecture of genes that contribute to the formation of the skeletal spicule.

Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells

PubMed Central

Uzbekova, Svetlana; Roy-Sabau, Monica; Dalbiès-Tran, Rozenn; Perreau, Christine; Papillier, Pascal; Mompart, Florence; Thelie, Aurore; Pennetier, Sophie; Cognie, Juliette; Cadoret, Veronique; Royere, Dominique; Monget, Philippe; Mermillod, Pascal

2006-01-01

Background Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. Methods Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. Results We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. Conclusion Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes. PMID:16551357

Tris(1,3-dichloro-2-propyl) phosphate perturbs the expression of genes involved in immune response and lipid and steroid metabolism in chicken embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farhat, Amani; National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3; Buick, Julie K.

We previously demonstrated that in ovo exposure to the flame retardant tris(1,3-dichloro-2-propyl) phosphate (TDCPP) decreased plasma thyroxine levels, reduced growth parameters, and decreased gallbladder size in chicken embryos. In the current study DNA microarrays were used to evaluate global mRNA expression in liver tissue of male chicken embryos that exhibited the above mentioned effects. Injected doses were dimethyl sulfoxide vehicle control, 7.6 or 45 μg TDCPP/g egg. TDCPP caused significant changes in the expression of five genes at the low dose and 47 genes at the high dose (False Discovery Rate p ≤ 0.1, fold change ≥ 1.5). The genemore » expression analysis suggested a compromised immune function, a state of cholestatic liver/biliary fibrosis, and disrupted lipid and steroid metabolism. Circulating bile acid levels were elevated, which is an indication of liver dysfunction, and plasma cholesterol levels were reduced; however, hepatic bile acid and cholesterol levels were unaltered. Interactome analyses identified apolipoprotein E, hepatocyte nuclear factor 4 alpha, and peroxisome proliferator-activated receptor alpha as key regulatory molecules involved in the effects of TDCPP. Our results demonstrate a targeted effect of TDCPP toxicity on lipid metabolism, including cholesterol, that helps explain the aforementioned phenotypic effects, as chicken embryos are highly dependent on yolk lipids for growth and maintenance throughout development. Finally, our results are in concordance with the literature that describes TDCPP as a cancer-causing agent, since the majority of dysregulated genes were involved in cancer pathways. - Highlights: • TDCPP dysregulates genes involved in immune function and lipid metabolism. • A targeted effect of TDCPP toxicity on cholesterol metabolism is apparent. • A state of cholestatic liver fibrosis is suggested by the expression profile. • Elevated plasma bile acids suggest that TDCPP causes liver dysfunction.« less

Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos.

PubMed

Sussarellu, Rossana; Lebreton, Morgane; Rouxel, Julien; Akcha, Farida; Rivière, Guillaume

2018-03-01

Copper contamination is widespread along coastal areas and exerts adverse effects on marine organisms such as mollusks. In the Pacific oyster, copper induces severe developmental abnormalities during early life stages; however, the underlying molecular mechanisms are largely unknown. This study aims to better understand whether the embryotoxic effects of copper in Crassostrea gigas could be mediated by alterations in gene expression, and the putative role of DNA methylation, which is known to contribute to gene regulation in early embryo development. For that purpose, oyster embryos were exposed to 4 nominal copper concentrations (0.1, 1, 10 and 20 μg L -1 Cu 2+ ) during early development assays. Embryotoxicity was monitored through the oyster embryo-larval bioassay at the D-larva stage 24 h post fertilization (hpf) and genotoxicity at gastrulation 7 hpf. In parallel, the relative expression of 15 genes encoding putative homeotic, biomineralization and DNA methylation proteins was measured at three developmental stages (3 hpf morula stage, 7 hpf gastrula stage, 24 hpf D-larvae stage) using RT-qPCR. Global DNA content in methylcytosine and hydroxymethylcytosine were measured by HPLC and gene-specific DNA methylation levels were monitored using MeDIP-qPCR. A significant increase in larval abnormalities was observed from copper concentrations of 10 μg L -1 , while significant genotoxic effects were detected at 1 μg L -1 and above. All the selected genes presented a stage-dependent expression pattern, which was impaired for some homeobox and DNA methylation genes (Notochord, HOXA1, HOX2, Lox5, DNMT3b and CXXC-1) after copper exposure. While global DNA methylation (5-methylcytosine) at gastrula stage didn't show significant changes between experimental conditions, 5-hydroxymethylcytosine, its degradation product, decreased upon copper treatment. The DNA methylation of exons and the transcript levels were correlated in control samples for HOXA1 but such a correlation was diminished following copper exposure. The methylation level of some specific gene regions (HoxA1, Hox2, Engrailed2 and Notochord) displayed changes upon copper exposure. Such changes were gene and exon-specific and no obvious global trends could be identified. Our study suggests that the embryotoxic effects of copper in oysters could involve homeotic gene expression impairment possibly by changing DNA methylation levels. Copyright © 2018 Elsevier B.V. All rights reserved.

slc7a6os gene plays a critical role in defined areas of the developing CNS in zebrafish.

PubMed

Benini, Anna; Cignarella, Francesca; Calvarini, Laura; Mantovanelli, Silvia; Giacopuzzi, Edoardo; Zizioli, Daniela; Borsani, Giuseppe

2015-01-01

The aim of this study is to shed light on the functional role of slc7a6os, a gene highly conserved in vertebrates. The Danio rerio slc7a6os gene encodes a protein of 326 amino acids with 46% identity to human SLC7A6OS and 14% to Saccharomyces cerevisiae polypeptide Iwr1. Yeast Iwr1 specifically binds RNA pol II, interacts with the basal transcription machinery and regulates the transcription of specific genes. In this study we investigated for the first time the biological role of SLC7A6OS in vertebrates. Zebrafish slc7a6os is a maternal gene that is expressed throughout development, with a prevalent localization in the developing central nervous system (CNS). The gene is also expressed, although at different levels, in various tissues of the adult fish. To determine the functional role of slc7a6os during zebrafish development, we knocked-down the gene by injecting a splice-blocking morpholino. At 24 hpf morphants show morphological defects in the CNS, particularly the interface between hindbrain and midbrain is not well-defined. At 28 hpf the morpholino injected embryos present an altered somite morphology and appear partially or completely immotile. At this stage the midbrain, hindbrain and cerebellum are compromised and not well defined compared with control embryos. The observed alterations persist at later developmental stages. Consistently, the expression pattern of two markers specifically expressed in the developing CNS, pax2a and neurod, is significantly altered in morphants. The co-injection of embryos with synthetic slc7a6os mRNA, rescues the morphant phenotype and restores the wild type expression pattern of pax2a and neurod. Our data suggest that slc7a6os might play a critical role in defined areas of the developing CNS in vertebrates, probably by regulating the expression of key genes.

slc7a6os Gene Plays a Critical Role in Defined Areas of the Developing CNS in Zebrafish

PubMed Central

Benini, Anna; Cignarella, Francesca; Calvarini, Laura; Mantovanelli, Silvia; Giacopuzzi, Edoardo; Zizioli, Daniela; Borsani, Giuseppe

2015-01-01

The aim of this study is to shed light on the functional role of slc7a6os, a gene highly conserved in vertebrates. The Danio rerio slc7a6os gene encodes a protein of 326 amino acids with 46% identity to human SLC7A6OS and 14% to Saccharomyces cerevisiae polypeptide Iwr1. Yeast Iwr1 specifically binds RNA pol II, interacts with the basal transcription machinery and regulates the transcription of specific genes. In this study we investigated for the first time the biological role of SLC7A6OS in vertebrates. Zebrafish slc7a6os is a maternal gene that is expressed throughout development, with a prevalent localization in the developing central nervous system (CNS). The gene is also expressed, although at different levels, in various tissues of the adult fish. To determine the functional role of slc7a6os during zebrafish development, we knocked-down the gene by injecting a splice-blocking morpholino. At 24 hpf morphants show morphological defects in the CNS, particularly the interface between hindbrain and midbrain is not well-defined. At 28 hpf the morpholino injected embryos present an altered somite morphology and appear partially or completely immotile. At this stage the midbrain, hindbrain and cerebellum are compromised and not well defined compared with control embryos. The observed alterations persist at later developmental stages. Consistently, the expression pattern of two markers specifically expressed in the developing CNS, pax2a and neurod, is significantly altered in morphants. The co-injection of embryos with synthetic slc7a6os mRNA, rescues the morphant phenotype and restores the wild type expression pattern of pax2a and neurod. Our data suggest that slc7a6os might play a critical role in defined areas of the developing CNS in vertebrates, probably by regulating the expression of key genes. PMID:25803583

Impaired expression of importin/karyopherin {beta}1 leads to post-implantation lethality

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miura, Katsutaka; Yoshinobu, Kumiko; Imaizumi, Takashi

2006-03-03

Importin {beta}1 (Imp{beta})/karyopherin {beta}1 (Kpnb1) mediates the nuclear import of a large variety of substrates. This study aimed to investigate the requirement for the Kpnb1 gene in mouse development, using a gene trap line, B6-CB-Ayu8108 {sup GtgeoIMEG} (Ayu8108 {sup geo}), in which the trap vector was inserted into the promoter region of the Kpnb1 gene, but in reverse orientation of the Kpnb1 gene. Ayu8108 {sup geo/geo} homozygous embryos could develop to the blastocyst stage, but died before embryonic day 5.5, and expression of the Kpnb1 gene in homozygous blastocysts was undetectable. We also replaced the {beta}geo gene with Imp{beta} cDNAmore » through Cre-mediated recombination to rescue Imp{beta} expression. Homozygous mice for the rescued allele Ayu8108 {sup Imp{beta}}{sup /Imp{beta}} were born and developed normally. These results demonstrated that the cause of post-implantation lethality of Ayu8108 {sup geo/geo} homozygous embryos was impaired expression of the Kpnb1 gene, indicating indispensable roles of Imp{beta}1 in early development of mice.« less

Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo.

PubMed

Sakai, Kaori; Taconnat, Ludivine; Borrega, Nero; Yansouni, Jennifer; Brunaud, Véronique; Paysant-Le Roux, Christine; Delannoy, Etienne; Martin Magniette, Marie-Laure; Lepiniec, Loïc; Faure, Jean Denis; Balzergue, Sandrine; Dubreucq, Bertrand

2018-01-01

Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. We have developed a sensitive approach based on ultra-low RNA sequencing coupled to laser assisted microdissection for analyzing different tissues of the small Arabidopsis embryo. We first characterized the number of genes detected according to the quantity of tissue yield and total RNA extracted. Our results revealed that as low as 0.02 mm 2 of tissue and 50 pg of total RNA can be used without compromising the number of genes detected. The optimised protocol was used to compare the epidermal versus mesophyll cell transcriptomes of cotyledons at the torpedo-shaped stage of embryo development. The approach was validated by the recovery of well-known epidermal genes such AtML1 or AtPDF2 and genes involved in flavonoid and cuticular waxes pathways. Moreover, the interest and sensitivity of this approach were highlighted by the characterization of several transcription factors preferentially expressed in epidermal cells. This technical advance unlocks some current limitations of transcriptomic analyses and allows to investigate further and efficiently new biological questions for which only a very small amounts of cells need to be isolated. For instance, it paves the way to increasing the spatial accuracy of regulatory networks in developing small embryo of Arabidopsis or other plant tissues.

ING2 (inhibitor of growth protein-2) plays a crucial role in preimplantation development.

PubMed

Zhou, Lin; Wang, Pei; Zhang, Juanjuan; Heng, Boon Chin; Tong, Guo Qing

2016-02-01

ING2 (inhibitor of growth protein-2) is a member of the ING-gene family and participates in diverse cellular processes involving tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. As a subunit of the Sin3 histone deacetylase complex co-repressor complex, ING2 binds to H3K4me3 to regulate chromatin modification and gene expression. Additionally, ING2 recruits histone methyltransferase (HMT) activity for gene repression, which is independent of the HDAC class I or II pathway. However, the physiological function of ING2 in mouse preimplantation embryo development has not yet been characterized previously. The expression, localization and function of ING2 during preimplantation development were investigated in this study. We showed increasing expression of ING2 within the nucleus from the 4-cell embryo stage onwards; and that down-regulation of ING2 expression by endoribonuclease-prepared small interfering RNA (esiRNA) microinjection results in developmental arrest during the morula to blastocyst transition. Embryonic cells microinjected with ING2-specific esiRNA exhibited decreased blastulation rate compared to the negative control. Further investigation of the underlying mechanism indicated that down-regulation of ING2 significantly increased expression of p21, whilst decreasing expression of HDAC1. These results suggest that ING2 may play a crucial role in the process of preimplantation embryo development through chromatin regulation.

Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts.

PubMed

Hwang, Jae Yeon; Choi, Kwang-Hwan; Lee, Dong-Kyung; Kim, Seung-Hun; Kim, Eun Bae; Hyun, Sang-Hwan; Lee, Chang-Kyu

2015-01-01

X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts.

Microspore embryogenesis in wheat: new marker genes for early, middle and late stages of embryo development.

PubMed

Sánchez-Díaz, Rosa Angélica; Castillo, Ana María; Vallés, María Pilar

2013-09-01

Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation.

Gonadal morphogenesis and gene expression in reptiles with temperature-dependent sex determination.

PubMed

Merchant-Larios, H; Díaz-Hernández, V; Marmolejo-Valencia, A

2010-01-01

In reptiles with temperature-dependent sexual determination, the thermosensitive period (TSP) is the interval in which the sex is defined during gonadal morphogenesis. One-shift experiments in a group of eggs define the onset and the end of the TSP as all and none responses, respectively. Timing for sex-undetermined (UG) and -determined gonads (DG) differs at male- (MPT) or female-producing temperatures (FPT). During the TSP a decreasing number of embryos respond to temperature shifts indicating that in this period embryos with both UG and DG exist. Although most UG correspond to undifferentiated gonads, some embryos extend UG after the onset of histological differentiation. Thus, temperature affects gonadal cells during the process of morphogenesis, but timing of commitment depends on individual embryos. A correlation between gonadal morphogenesis, TSP, and gene expression suggests that determination of the molecular pathways modulated by temperature in epithelial cells (surface epithelium and medullary cords) holds the key for a unifying hypothesis on temperature-dependent sex determination. (c) 2010 S. Karger AG, Basel.

Uterine responses to early pre-attachment embryos in the domestic dog and comparisons with other domestic animal species†

PubMed Central

Graubner, Felix R.; Gram, Aykut; Kautz, Ewa; Bauersachs, Stefan; Aslan, Selim; Agaoglu, Ali R.; Boos, Alois

2017-01-01

Abstract In the dog, there is no luteolysis in the absence of pregnancy. Thus, this species lacks any anti-luteolytic endocrine signal as found in other species that modulate uterine function during the critical period of pregnancy establishment. Nevertheless, in the dog an embryo-maternal communication must occur in order to prevent rejection of embryos. Based on this hypothesis, we performed microarray analysis of canine uterine samples collected during pre-attachment phase (days 10-12) and in corresponding non-pregnant controls, in order to elucidate the embryo attachment signal. An additional goal was to identify differences in uterine responses to pre-attachment embryos between dogs and other mammalian species exhibiting different reproductive patterns with regard to luteolysis, implantation, and preparation for placentation. Therefore, the canine microarray data were compared with gene sets from pigs, cattle, horses, and humans. We found 412 genes differentially regulated between the two experimental groups. The functional terms most strongly enriched in response to pre-attachment embryos related to extracellular matrix function and remodeling, and to immune and inflammatory responses. Several candidate genes were validated by semi-quantitative PCR. When compared with other species, best matches were found with human and equine counterparts. Especially for the pig, the majority of overlapping genes showed opposite expression patterns. Interestingly, 1926 genes did not pair with any of the other gene sets. Using a microarray approach, we report the uterine changes in the dog driven by the presence of embryos and compare these results with datasets from other mammalian species, finding common-, contrary-, and exclusively canine-regulated genes. PMID:28651344

Developmental fate and lineage commitment of singled mouse blastomeres.

PubMed

Lorthongpanich, Chanchao; Doris, Tham Puay Yoke; Limviphuvadh, Vachiranee; Knowles, Barbara B; Solter, Davor

2012-10-01

The inside-outside model has been invoked to explain cell-fate specification of the pre-implantation mammalian embryo. Here, we investigate whether cell-cell interaction can influence the fate specification of embryonic blastomeres by sequentially separating the blastomeres in two-cell stage mouse embryos and continuing separation after each cell division throughout pre-implantation development. This procedure eliminates information provided by cell-cell interaction and cell positioning. Gene expression profiles, polarity protein localization and functional tests of these separated blastomeres reveal that cell interactions, through cell position, influence the fate of the blastomere. Blastomeres, in the absence of cell contact and inner-outer positional information, have a unique pattern of gene expression that is characteristic of neither inner cell mass nor trophectoderm, but overall they have a tendency towards a 'trophectoderm-like' gene expression pattern and preferentially contribute to the trophectoderm lineage.

Recombinant avian adeno-associated virus: transgene expression in vivo and enhancement of expression in vitro.

PubMed

Estevez, Carlos; Villegas, Pedro

2006-06-01

Recombinant avian adeno-associated viruses coding for the LacZ gene were used to inoculate embryonating chicken eggs, to assess the usefulness of the system for the expression of a transgene in vivo. The results obtained indicate significantly higher levels of expression of the reporter gene at various time intervals in the embryos inoculated with the recombinant virus in comparison with the mock-inoculated controls. At the embryo level, significant differences were evident at 120 hr postinoculation; hatched chicks showed transgene expression up to 14 days of age. In a second experiment, different cell-line cultures were transfected with plasmids encoding for a reporter gene flanked by the avian adeno-associated virus inverted terminal repeats (ITR), either alone or in the presence of the major nonstructural proteins of the virus (Rep 78/68) to assess the ability of these proteins and DNA elements to enhance gene expression. Results indicate that the inclusion of the viral ITR alone or during coexpression of the Rep proteins significantly enhances the expression of the transgene in all cell lines tested, as evidenced by the detection of the beta-galacrosidase protein through chemiluminescence reactions and staining of transfected monolayers.

Protective effects of puerarin against tetrabromobisphenol a-induced apoptosis and cardiac developmental toxicity in zebrafish embryo-larvae.

PubMed

Yang, Suwen; Wang, Shengrui; Sun, Fengchao; Zhang, Mengmeng; Wu, Fengchang; Xu, Fanfan; Ding, Zhishan

2015-09-01

Tetrabromobisphenol A (TBBPA), a brominated flame retardant, is detected commonly in aquatic environments, where it is thought to be highly toxic to the development of aquatic life. In this study, zebrafish embryos and larvae were used to investigate the protective effects of puerarin after exposure to TBBPA. Malformation, blood flow disorders, pericardial edema, and spawn coagulation rates increased, whereas survival decreased significantly after exposure to 0.5 and 1.0 mg L(-1) TBBPA. The measured indices of morphological toxicity improved after treatment with puerarin. TBBPA also induced reactive oxygen species (ROS) production in a dose-dependent manner. Acridine orange staining results revealed that TBBPA exposure caused cardiomyocyte apoptosis and induced the expression of three proapoptotic genes: P53, Bax, and Caspase9. In contrast, the expression of the antiapoptotic gene Bcl2 was down-regulated. When genes related to cardiac development were assessed, the expression of Tbx1, Raldh2, and Bmp2b changed after exposure to the combination of TBBPA and puerarin. These results suggest that TBBPA induces cardiomyocyte apoptosis and ROS production, resulting in cardiac developmental toxicity in zebrafish embryos or larvae. Therefore, puerarin regulates the expression of cardiac developmental genes, such as Tbx1, Bmp2b, and Raldh2 by inhibiting ROS production, and subsequently modulates cardiac development after the exposure of zebrafish larvae to TBBPA. © 2014 Wiley Periodicals, Inc.

Glutamine supplementation enhances development of in vitro-produced porcine embryos and increases leucine consumption from the medium.

PubMed

Chen, Paula R; Redel, Bethany K; Spate, Lee D; Ji, Tieming; Salazar, Shirley Rojas; Prather, Randall S

2018-05-31

Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with +Gln or +Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P < 0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism were corrected when embryos were cultured with +Gln or +Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with +Max and increased consumption of leucine by embryos cultured with +Gln or +Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with +Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with +Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.

Editor's Highlight: Hydroxyurea Exposure Activates the P53 Signaling Pathway in Murine Organogenesis-Stage Embryos

PubMed Central

El Husseini, Nazem; Schlisser, Ava E.; Hales, Barbara F.

2016-01-01

Hydroxyurea, an anticancer agent and potent teratogen, induces oxidative stress and activates a DNA damage response pathway in the gestation day (GD) 9 mouse embryo. To delineate the stress response pathways activated by this drug, we investigated the effect of hydroxyurea exposure on the transcriptome of GD 9 embryos. Timed pregnant CD-1 mice were treated with saline or hydroxyurea (400 mg/kg or 600 mg/kg) on GD 9; embryonic gene and protein expression were examined 3 h later. Microarray analysis revealed that the expression of 1346 probe sets changed significantly in embryos exposed to hydroxyurea compared with controls; the P53 signaling pathway was highly affected. In addition, P53 related family members, P63 and P73, were predicted to be activated and had common and unique downstream targets. Western blot analysis revealed that active phospho-P53 was significantly increased in drug-exposed embryos; confocal microscopy showed that the translocation of phospho-P53 to the nucleus was widespread in the embryo. Furthermore, qRT-PCR showed that the expression of P53-regulated genes (Cdkn1A, Fas, and Trp53inp1) was significantly upregulated in hydroxyurea-exposed embryos; the concentration of the redox sensitive P53INP1 protein was also increased in a hydroxyurea dose-dependent fashion. Thus, hydroxyurea elicits a significant effect on the transcriptome of the organogenesis stage murine embryo, activating several key developmental signaling pathways related to DNA damage and oxidative stress. We propose that the P53 pathway plays a central role in the embryonic stress response and the developmental outcome after teratogen exposure. PMID:27208086

Chondroitin sulfate effects on neural stem cell differentiation.

PubMed

Canning, David R; Brelsford, Natalie R; Lovett, Neil W

2016-01-01

We have investigated the role chondroitin sulfate has on cell interactions during neural plate formation in the early chick embryo. Using tissue culture isolates from the prospective neural plate, we have measured neural gene expression profiles associated with neural stem cell differentiation. Removal of chondroitin sulfate from stage 4 neural plate tissue leads to altered associations of N-cadherin-positive neural progenitors and causes changes in the normal sequence of neural marker gene expression. Absence of chondroitin sulfate in the neural plate leads to reduced Sox2 expression and is accompanied by an increase in the expression of anterior markers of neural regionalization. Results obtained in this study suggest that the presence of chondroitin sulfate in the anterior chick embryo is instrumental in maintaining cells in the neural precursor state.

Caspase activity and expression of cell death genes during development of human preimplantation embryos.

PubMed

Spanos, S; Rice, S; Karagiannis, P; Taylor, D; Becker, D L; Winston, R M L; Hardy, K

2002-09-01

It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.

Multiple, Distinct Isoforms of Sucrose Synthase in Pea1

PubMed Central

Barratt, D.H. Paul; Barber, Lorraine; Kruger, Nicholas J.; Smith, Alison M.; Wang, Trevor L.; Martin, Cathie

2001-01-01

Genes encoding three isoforms of sucrose synthase (Sus1, Sus2, and Sus3) have been cloned from pea (Pisum sativum). The genes have distinct patterns of expression in different organs of the plant, and during organ development. Studies of the isoforms expressed as recombinant proteins in Escherichia coli show that they differ in kinetic properties. Although not of great magnitude, the differences in properties are consistent with some differentiation of physiological function between the isoforms. Evidence for differentiation of function in vivo comes from the phenotypes of rug4 mutants of pea, which carry mutations in the gene encoding Sus1. One mutant line (rug4-c) lacks detectable Sus1 protein in both the soluble and membrane-associated fractions of the embryo, and Sus activity in the embryo is reduced by 95%. The starch content of the embryo is reduced by 30%, but the cellulose content is unaffected. The results imply that different isoforms of Sus may channel carbon from sucrose towards different metabolic fates within the cell. PMID:11598239

LlamaTags: A Versatile Tool to Image Transcription Factor Dynamics in Live Embryos.

PubMed

Bothma, Jacques P; Norstad, Matthew R; Alamos, Simon; Garcia, Hernan G

2018-06-14

Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. Here, we pioneer a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, we discovered stochastic bursts in the concentration of transcription factors that are correlated with bursts in transcription. We further used LlamaTags to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; we speculate that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos. Copyright © 2018 Elsevier Inc. All rights reserved.

Functional Characterization of G12, a Gene Required for Mitotic Progression during Gastrulation in Zebrafish

NASA Technical Reports Server (NTRS)

Reinsch, Sigrid; Conway, Gregory; Dalton, Bonnie P. (Technical Monitor)

2002-01-01

In a differential RNA display screen we have isolated a zebrafish gene, G12, for which homologs can only be found in DNA databases for vertebrates, but not invertebrates. This suggests that this is a gene required specifically in vertebrates. G12 expression is upregulated at mid-blastula transition (MBT). Morpholino inactivation of this gene by injection into 1-cell embryos results in mitotic defects and apoptosis shortly after MBT. Nuclei in morpholino treated embryos also display segregation defects. We have characterized the localization of this gene as a GFP fusion in live and fixed embryos. Overexpression of G12-GFP is non-toxic. Animals retain GFP expression for at least 7 days with no developmental defects, Interestingly in these animals G12-GFP is never detectable in blood cells though blood is present. In the deep cells of early embryos, G 12GFP is localized to nuclei and cytoskeletal elements in interphase and to the centrosome and spindle apparatus during mitosis. In the EVL, G12-GFP shows additional localization to the cell periphery, especially in mitosis. In the yolk syncytium, G12-GFP again localizes to nuclei and strongly to cytoplasmic microtubules of migrating nuclei at the YSL margin. Morpholinc, injection specifically into the YSL after cellularization blocks epiboly and nuclei of the YSL show mitotic defects while deep cells show no mitotic defects and continue to divide. Rescue experiments in which morpholino and G12-GFP RNA are co-injected indicate partial rescue by the G12-GFP. The rescue is cell autonomous; that is, regions of the embryo with higher G12-GFP expression show fewer mitotic defects. Spot 14, the human bomolog of G12, has been shown to be amplified in aggressive breast tumors. This finding, along with our functional and morphological data suggest that G12 and spot 14 are vertebrate-specific and may function either as mitotic checkpoints or as structural components of the spindle apparatus.

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

PubMed Central

Ochiai, Hiroshi; Sakamoto, Naoaki; Fujita, Kazumasa; Nishikawa, Masatoshi; Suzuki, Ken-ichi; Matsuura, Shinya; Miyamoto, Tatsuo; Sakuma, Tetsushi; Shibata, Tatsuo; Yamamoto, Takashi

2012-01-01

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B–GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos. PMID:22711830

Jmjd5 functions as a regulator of p53 signaling during mouse embryogenesis.

PubMed

Ishimura, Akihiko; Terashima, Minoru; Tange, Shoichiro; Suzuki, Takeshi

2016-03-01

Genetic studies have shown that aberrant activation of p53 signaling leads to embryonic lethality. Maintenance of a fine balance of the p53 protein level is critical for normal development. Previously, we have reported that Jmjd5, a member of the Jumonji C (JmjC) family, regulates embryonic cell proliferation through the control of Cdkn1a expression. Since Cdkn1a is the representative p53-regulated gene, we have examined whether the expression of other p53 target genes is coincidentally upregulated with Cdkn1a in Jmjd5-deficient embryos. The expression of a subset of p53-regulated genes was increased in both Jmjd5 hypomorphic mouse embryonic fibroblasts (MEFs) and Jmjd5-deficient embryos at embryonic day 8.25 without the induced expression of Trp53. Intercrossing of Jmjd5-deficient mice with Trp53 knockout mice showed that the growth defect of Jmjd5 mutant cells was significantly recovered under a Trp53 null genetic background. Chromatin immunoprecipitation analysis in Jmjd5 hypomorphic MEFs indicated the increased recruitment of p53 at several p53 target gene loci, such as Cdkn1a, Pmaip1, and Mdm2. These results suggest that Jmjd5 is involved in the transcriptional regulation of a subset of p53-regulated genes, possibly through the control of p53 recruitment at the gene loci. In Jmjd5-deficient embryos, the enhanced recruitment of p53 might result in the abnormal activation of p53 signaling leading to embryonic lethality.

The Krüppel-like factor 2 and Krüppel-like factor 4 genes interact to maintain endothelial integrity in mouse embryonic vasculogenesis

PubMed Central

2013-01-01

Background Krüppel-like Factor 2 (KLF2) plays an important role in vessel maturation during embryonic development. In adult mice, KLF2 regulates expression of the tight junction protein occludin, which may allow KLF2 to maintain vascular integrity. Adult tamoxifen-inducible Krüppel-like Factor 4 (KLF4) knockout mice have thickened arterial intima following vascular injury. The role of KLF4, and the possible overlapping functions of KLF2 and KLF4, in the developing vasculature are not well-studied. Results Endothelial breaks are observed in a major vessel, the primary head vein (PHV), in KLF2-/-KLF4-/- embryos at E9.5. KLF2-/-KLF4-/- embryos die by E10.5, which is earlier than either single knockout. Gross hemorrhaging of multiple vessels may be the cause of death. E9.5 KLF2-/-KLF4+/- embryos do not exhibit gross hemorrhaging, but cross-sections display disruptions of the endothelial cell layer of the PHV, and these embryos generally also die by E10.5. Electron micrographs confirm that there are gaps in the PHV endothelial layer in E9.5 KLF2-/-KLF4-/- embryos, and show that the endothelial cells are abnormally bulbous compared to KLF2-/- and wild-type (WT). The amount of endothelial Nitric Oxide Synthase (eNOS) mRNA, which encodes an endothelial regulator, is reduced by 10-fold in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. VEGFR2, an eNOS inducer, and occludin, a tight junction protein, gene expression are also reduced in E9.5 KLF2-/-KLF4-/- compared to KLF2-/- and WT embryos. Conclusions This study begins to define the roles of KLF2 and KLF4 in the embryonic development of blood vessels. It indicates that the two genes interact to maintain an intact endothelial layer. KLF2 and KLF4 positively regulate the eNOS, VEGFR2 and occludin genes. Down-regulation of these genes in KLF2-/-KLF4-/- embryos may result in the observed loss of vascular integrity. PMID:24261709

Misexpression of BRE gene in the developing chick neural tube affects neurulation and somitogenesis

PubMed Central

Wang, Guang; Li, Yan; Wang, Xiao-Yu; Chuai, Manli; Yeuk-Hon Chan, John; Lei, Jian; Münsterberg, Andrea; Lee, Kenneth Ka Ho; Yang, Xuesong

2015-01-01

The brain and reproductive expression (BRE) gene is expressed in numerous adult tissues and especially in the nervous and reproductive systems. However, little is known about BRE expression in the developing embryo or about its role in embryonic development. In this study, we used in situ hybridization to reveal the spatiotemporal expression pattern for BRE in chick embryo during development. To determine the importance of BRE in neurogenesis, we overexpressed BRE and also silenced BRE expression specifically in the neural tube. We established that overexpressing BRE in the neural tube indirectly accelerated Pax7+ somite development and directly increased HNK-1+ neural crest cell (NCC) migration and TuJ-1+ neurite outgrowth. These altered morphogenetic processes were associated with changes in the cell cycle of NCCs and neural tube cells. The inverse effect was obtained when BRE expression was silenced in the neural tube. We also determined that BMP4 and Shh expression in the neural tube was affected by misexpression of BRE. This provides a possible mechanism for how altering BRE expression was able to affect somitogenesis, neurogenesis, and NCC migration. In summary, our results demonstrate that BRE plays an important role in regulating neurogenesis and indirectly somite differentiation during early chick embryo development. PMID:25568339

Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis)

PubMed Central

Tate, Kevin B.; Rhen, Turk; Eme, John; Kohl, Zachary F.; Crossley, Janna; Elsey, Ruth M.

2016-01-01

During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50–70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70–90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered β-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator. PMID:27101296

Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules

PubMed Central

Tajima, Yosuke; Yoshida, Koki; Oikawa, Mami; Azuma, Rika; Allen, George E.; Tsujikawa, Tomomi; Tsukaguchi, Tomomasa; Bradshaw, Charles R.; Jullien, Jerome; Yamagata, Kazuo; Matsumoto, Kazuya; Anzai, Masayuki; Imai, Hiroshi; Gurdon, John B.; Yamada, Masayasu

2017-01-01

ABSTRACT Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency. PMID:28412714

The PR/SET Domain Zinc Finger Protein Prdm4 Regulates Gene Expression in Embryonic Stem Cells but Plays a Nonessential Role in the Developing Mouse Embryo

PubMed Central

Bogani, Debora; Morgan, Marc A. J.; Nelson, Andrew C.; Costello, Ita; McGouran, Joanna F.; Kessler, Benedikt M.

2013-01-01

Prdm4 is a highly conserved member of the Prdm family of PR/SET domain zinc finger proteins. Many well-studied Prdm family members play critical roles in development and display striking loss-of-function phenotypes. Prdm4 functional contributions have yet to be characterized. Here, we describe its widespread expression in the early embryo and adult tissues. We demonstrate that DNA binding is exclusively mediated by the Prdm4 zinc finger domain, and we characterize its tripartite consensus sequence via SELEX (systematic evolution of ligands by exponential enrichment) and ChIP-seq (chromatin immunoprecipitation-sequencing) experiments. In embryonic stem cells (ESCs), Prdm4 regulates key pluripotency and differentiation pathways. Two independent strategies, namely, targeted deletion of the zinc finger domain and generation of a EUCOMM LacZ reporter allele, resulted in functional null alleles. However, homozygous mutant embryos develop normally and adults are healthy and fertile. Collectively, these results strongly suggest that Prdm4 functions redundantly with other transcriptional partners to cooperatively regulate gene expression in the embryo and adult animal. PMID:23918801

Msx homeobox genes critically regulate embryo implantation by controlling paracrine signaling between uterine stroma and epithelium.

PubMed

Nallasamy, Shanmugasundaram; Li, Quanxi; Bagchi, Milan K; Bagchi, Indrani C

2012-01-01

The mammalian Msx homeobox genes, Msx1 and Msx2, encode transcription factors that control organogenesis and tissue interactions during embryonic development. We observed overlapping expression of these factors in uterine epithelial and stromal compartments of pregnant mice prior to embryo implantation. Conditional ablation of both Msx1 and Msx2 in the uterus resulted in female infertility due to a failure in implantation. In these mutant mice (Msx1/2(d/d)), the uterine epithelium exhibited persistent proliferative activity and failed to attach to the embryos. Gene expression profiling of uterine epithelium and stroma of Msx1/2(d/d) mice revealed an elevated expression of several members of the Wnt gene family in the preimplantation uterus. Increased canonical Wnt signaling in the stromal cells activated β-catenin, stimulating the production of a subset of fibroblast growth factors (FGFs) in these cells. The secreted FGFs acted in a paracrine manner via the FGF receptors in the epithelium to promote epithelial proliferation, thereby preventing differentiation of this tissue and creating a non-receptive uterus refractory to implantation. Collectively, these findings delineate a unique signaling network, involving Msx1/2, Wnts, and FGFs, which operate in the uterus at the time of implantation to control the mesenchymal-epithelial dialogue critical for successful establishment of pregnancy.

Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo.

PubMed

Oliveri, Paola; Walton, Katherine D; Davidson, Eric H; McClay, David R

2006-11-01

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks.

Roles of the first and second round of DNA replication in the regulation of zygotic gene activation in mice.

PubMed

Sonehara, Hiroki; Nagata, Masao; Aoki, Fugaku

2008-10-01

In the mouse embryo, expression of zygotic genes starts in the S/G2 phase of the 1-cell stage and greatly increases during the 2-cell stage. Although the timing of zygotic gene activation (ZGA) is thus established, the mechanism regulating ZGA is poorly understood. Previous studies using reporter genes have suggested that a transcriptionally repressive state is established during the 2-cell stage and that the first and second rounds of DNA replication are involved in this process. To further elucidate the respective roles of the two rounds of DNA replication in ZGA, we analyzed the expression of four ZGA genes (hsp70.1, eif-1a, muerv and zscan4d) in embryos whose DNA replication was inhibited by treatment with aphidicolin, an inhibitor of DNA polymerase. Inhibiting the first round increased the expression levels of hsp70.1, eif-1a and zscan4d but decreased that of muerv, while inhibiting the second round increased the expression levels of all four genes. These results suggest that the transcriptionally repressive state seems to be established after the second round of DNA replication.

Identification of an Imprinted Gene Cluster in the X-Inactivation Center

PubMed Central

Kobayashi, Shin; Totoki, Yasushi; Soma, Miki; Matsumoto, Kazuya; Fujihara, Yoshitaka; Toyoda, Atsushi; Sakaki, Yoshiyuki; Okabe, Masaru; Ishino, Fumitoshi

2013-01-01

Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed Xist, an imprinted gene, has been considered as a single cis-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than Xist expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs–miR-374-5p and miR-421-3p–mapped adjacent to Xist that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: Jpx, Ftx and Zcchc13. To our knowledge, this is the first identified cluster of imprinted genes in the cis-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development. PMID:23940725

Identification of an imprinted gene cluster in the X-inactivation center.

PubMed

Kobayashi, Shin; Totoki, Yasushi; Soma, Miki; Matsumoto, Kazuya; Fujihara, Yoshitaka; Toyoda, Atsushi; Sakaki, Yoshiyuki; Okabe, Masaru; Ishino, Fumitoshi

2013-01-01

Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed Xist, an imprinted gene, has been considered as a single cis-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than Xist expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs-miR-374-5p and miR-421-3p-mapped adjacent to Xist that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: Jpx, Ftx and Zcchc13. To our knowledge, this is the first identified cluster of imprinted genes in the cis-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.

Non-invasive assessment of culture media from goat cloned embryos associated with subjective morphology by gas chromatography - mass spectroscopy-based metabolomic analysis.

PubMed

Zhang, Yan-Li; Zhang, Guo-Min; Jia, Ruo-Xin; Wan, Yong-Jie; Yang, Hua; Sun, Ling-Wei; Han, Le; Wang, Feng

2018-01-01

Pre-implantation embryo metabolism demonstrates distinctive characteristics associated with the development potential of embryos. We aim to determine if metabolic differences correlate with embryo morphology. In this study, gas chromatography - mass spectroscopy (GC-MS)-based metabolomics was used to assess the culture media of goat cloned embryos collected from high-quality (HQ) and low-quality (LQ) groups based on morphology. Expression levels of amino acid transport genes were further examined by quantitative real-time PCR. Results showed that the HQ group presented higher percentages of blastocysts compared with the LQ counterparts (P < 0.05). Metabolic differences were also present between HQ and LQ groups. The culture media of the HQ group showed lower levels of valin, lysine, glutamine, mannose and acetol, and higher levels of glucose, phytosphingosine and phosphate than those of the LQ group. Additionally, expression levels of amino acid transport genes SLC1A5 and SLC3A2 were significantly lower in the HQ group than the LQ group (P < 0.05, respectively). To our knowledge, this is the first report which uses GC-MS to detect metabolic differences in goat cloned embryo culture media. The biochemical profiles may help to select the most in vitro viable embryos. © 2017 Japanese Society of Animal Science.

Analysis of the SWI/SNF chromatin-remodeling complex during early heart development and BAF250a repression cardiac gene transcription during P19 cell differentiation

PubMed Central

Singh, Ajeet Pratap; Archer, Trevor K.

2014-01-01

The regulatory networks of differentiation programs and the molecular mechanisms of lineage-specific gene regulation in mammalian embryos remain only partially defined. We document differential expression and temporal switching of BRG1-associated factor (BAF) subunits, core pluripotency factors and cardiac-specific genes during post-implantation development and subsequent early organogenesis. Using affinity purification of BRG1 ATPase coupled to mass spectrometry, we characterized the cardiac-enriched remodeling complexes present in E8.5 mouse embryos. The relative abundance and combinatorial assembly of the BAF subunits provides functional specificity to Switch/Sucrose NonFermentable (SWI/SNF) complexes resulting in a unique gene expression profile in the developing heart. Remarkably, the specific depletion of the BAF250a subunit demonstrated differential effects on cardiac-specific gene expression and resulted in arrhythmic contracting cardiomyocytes in vitro. Indeed, the BAF250a physically interacts and functionally cooperates with Nucleosome Remodeling and Histone Deacetylase (NURD) complex subunits to repressively regulate chromatin structure of the cardiac genes by switching open and poised chromatin marks associated with active and repressed gene expression. Finally, BAF250a expression modulates BRG1 occupancy at the loci of cardiac genes regulatory regions in P19 cell differentiation. These findings reveal specialized and novel cardiac-enriched SWI/SNF chromatin-remodeling complexes, which are required for heart formation and critical for cardiac gene expression regulation at the early stages of heart development. PMID:24335282

A reduced transcriptome approach to assess environmental toxicants using zebrafish embryo tests

EPA Science Inventory

This paper reports on the pilot testing of a new bioassay platform that monitors expression of 1600 genes in zebrafish embryos exposed to either single chemicals or complex water samples. The method provides a more cost effective, high throughput means to broadly evaluate the pot...

Heritable variation in heat shock gene expression: a potential mechanism for adaptation to thermal stress in embryos of sea turtles.

PubMed

Tedeschi, J N; Kennington, W J; Tomkins, J L; Berry, O; Whiting, S; Meekan, M G; Mitchell, N J

2016-01-13

The capacity of species to respond adaptively to warming temperatures will be key to their survival in the Anthropocene. The embryos of egg-laying species such as sea turtles have limited behavioural means for avoiding high nest temperatures, and responses at the physiological level may be critical to coping with predicted global temperature increases. Using the loggerhead sea turtle (Caretta caretta) as a model, we used quantitative PCR to characterise variation in the expression response of heat-shock genes (hsp60, hsp70 and hsp90; molecular chaperones involved in cellular stress response) to an acute non-lethal heat shock. We show significant variation in gene expression at the clutch and population levels for some, but not all hsp genes. Using pedigree information, we estimated heritabilities of the expression response of hsp genes to heat shock and demonstrated both maternal and additive genetic effects. This is the first evidence that the heat-shock response is heritable in sea turtles and operates at the embryonic stage in any reptile. The presence of heritable variation in the expression of key thermotolerance genes is necessary for sea turtles to adapt at a molecular level to warming incubation environments. © 2016 The Author(s).

Heritable variation in heat shock gene expression: a potential mechanism for adaptation to thermal stress in embryos of sea turtles

PubMed Central

Kennington, W. J.; Tomkins, J. L.; Berry, O.; Whiting, S.; Meekan, M. G.; Mitchell, N. J.

2016-01-01

The capacity of species to respond adaptively to warming temperatures will be key to their survival in the Anthropocene. The embryos of egg-laying species such as sea turtles have limited behavioural means for avoiding high nest temperatures, and responses at the physiological level may be critical to coping with predicted global temperature increases. Using the loggerhead sea turtle (Caretta caretta) as a model, we used quantitative PCR to characterise variation in the expression response of heat-shock genes (hsp60, hsp70 and hsp90; molecular chaperones involved in cellular stress response) to an acute non-lethal heat shock. We show significant variation in gene expression at the clutch and population levels for some, but not all hsp genes. Using pedigree information, we estimated heritabilities of the expression response of hsp genes to heat shock and demonstrated both maternal and additive genetic effects. This is the first evidence that the heat-shock response is heritable in sea turtles and operates at the embryonic stage in any reptile. The presence of heritable variation in the expression of key thermotolerance genes is necessary for sea turtles to adapt at a molecular level to warming incubation environments. PMID:26763709

Neurogenic gene regulatory pathways in the sea urchin embryo.

PubMed

Wei, Zheng; Angerer, Lynne M; Angerer, Robert C

2016-01-15

During embryogenesis the sea urchin early pluteus larva differentiates 40-50 neurons marked by expression of the pan-neural marker synaptotagmin B (SynB) that are distributed along the ciliary band, in the apical plate and pharyngeal endoderm, and 4-6 serotonergic neurons that are confined to the apical plate. Development of all neurons has been shown to depend on the function of Six3. Using a combination of molecular screens and tests of gene function by morpholino-mediated knockdown, we identified SoxC and Brn1/2/4, which function sequentially in the neurogenic regulatory pathway and are also required for the differentiation of all neurons. Misexpression of Brn1/2/4 at low dose caused an increase in the number of serotonin-expressing cells and at higher dose converted most of the embryo to a neurogenic epithelial sphere expressing the Hnf6 ciliary band marker. A third factor, Z167, was shown to work downstream of the Six3 and SoxC core factors and to define a branch specific for the differentiation of serotonergic neurons. These results provide a framework for building a gene regulatory network for neurogenesis in the sea urchin embryo. © 2016. Published by The Company of Biologists Ltd.

Embryotrophic factor-3 from human oviductal cells enhances proliferation, suppresses apoptosis and stimulates the expression of the beta1 subunit of sodium-potassium ATPase in mouse embryos.

PubMed

Xu, J S; Lee, Y L; Lee, K F; Kwok, K L; Lee, W M; Luk, J M; Yeung, W S B

2004-12-01

Embrytrophic factor-3 (ETF-3) from human oviductal cells enhanced the development of mouse preimplantation embryos. This report studied the embryotrophic mechanisms of the molecule. Mouse embryos were incubated with ETF-3 for 24 h at different stages of development. ETF-3 treatment between 96 and 120 h post-HCG increased the cell count of blastocysts, whilst treatment between 72 and 96 h post-HCG enhanced the expansion and hatching of the blastocysts. ETF-3 increased the cell number of the embryos by suppressing apoptosis and increasing proliferation as determined by TUNEL and bromodeoxyuridine uptake assays, respectively. Real-time quantitative PCR showed that the in vivo developed and ETF-3-treated blastocysts had a significantly higher mRNA copy number of Na/K-ATPase-beta1, but not of hepsin, than that of blastocysts cultured in medium alone. The former gene was associated with cavitation of blastocysts while the latter was related to hatching of blastocyst. The beneficial effect of ETF-3 on blastocyst hatching was also seen when ETF-3-supplemented commercially available sequential culture medium for human embryo culture was used to culture mouse embryos. ETF-3 improves embryo development by enhancing proliferation, suppressing apoptosis and stimulating expression of genes related to blastocyst cavitation. Supplementating human embryo culture medium with ETF-3 may improve the success rate in clinical assisted reproduction.

Mutual regulatory interactions of the trunk gap genes during blastoderm patterning in the hemipteran Oncopeltus fasciatus.

PubMed

Ben-David, Jonathan; Chipman, Ariel D

2010-10-01

The early embryo of the milkweed bug, Oncopeltus fasciatus, appears as a single cell layer - the embryonic blastoderm - covering the entire egg. It is at this blastoderm stage that morphological domains are first determined, long before the appearance of overt segmentation. Central to the process of patterning the blastoderm into distinct domains are a group of transcription factors known as gap genes. In Drosophila melanogaster these genes form a network of interactions, and maintain sharp expression boundaries through strong mutual repression. Their restricted expression domains define specific areas along the entire body. We have studied the expression domains of the four trunk gap gene homologues in O. fasciatus and have determined their interactions through dsRNA gene knockdown experiments, followed by expression analyses. While the blastoderm in O. fasciatus includes only the first six segments of the embryo, the expression domains of the gap genes within these segments are broadly similar to those in Drosophila where the blastoderm includes all 15 segments. However, the interactions between the gap genes are surprisingly different from those in Drosophila, and mutual repression between the genes seems to play a much less significant role. This suggests that the well-studied interaction pattern in Drosophila is evolutionarily derived, and has evolved from a less strongly interacting network. Copyright © 2010 Elsevier Inc. All rights reserved.

The evolving role of the orphan nuclear receptor ftz-f1, a pair-rule segmentation gene.

PubMed

Heffer, Alison; Grubbs, Nathaniel; Mahaffey, James; Pick, Leslie

2013-01-01

Segmentation is a critical developmental process that occurs by different mechanisms in diverse taxa. In insects, there are three common modes of embryogenesis-short-, intermediate-, and long-germ development-which differ in the number of segments specified at the blastoderm stage. While genes involved in segmentation have been extensively studied in the long-germ insect Drosophila melanogaster (Dm), it has been found that their expression and function in segmentation in short- and intermediate-germ insects often differ. Drosophila ftz-f1 encodes an orphan nuclear receptor that functions as a maternally expressed pair-rule segmentation gene, responsible for the formation of alternate body segments during Drosophila embryogenesis. Here we investigated the expression and function of ftz-f1 in the short-germ beetle, Tribolium castaneum (Tc). We found that Tc-ftz-f1 is expressed in stripes in Tribolium embryos. These stripes overlap alternate Tc-Engrailed (Tc-En) stripes, indicative of a pair-rule expression pattern. To test whether Tc-ftz-f1 has pair-rule function, we utilized embryonic RNAi, injecting double-stranded RNA corresponding to Tc-ftz-f1 coding or non-coding regions into early Tribolium embryos. Knockdown of Tc-ftz-f1 produced pair-rule segmentation defects, evidenced by loss of expression of alternate En stripes. In addition, a later role for Tc-ftz-f1 in cuticle formation was revealed. These results identify a new pair-rule gene in Tribolium and suggest that its role in segmentation may be shared among holometabolous insects. Interestingly, while Tc-ftz-f1 is expressed in pair-rule stripes, the gene is ubiquitously expressed in Drosophila embryos. Thus, the pair-rule function of ftz-f1 is conserved despite differences in expression patterns of ftz-f1 genes in different lineages. This suggests that ftz-f1 expression changed after the divergence of lineages leading to extant beetles and flies, likely due to differences in cis-regulatory sequences. We propose that the dependence of Dm-Ftz-F1 on interaction with the homeodomain protein Ftz which is expressed in stripes in Drosophila, loosened constraints on Dm-ftz-f1 expression, allowing for ubiquitous expression of this pair-rule gene in Drosophila. © 2013 Wiley Periodicals, Inc.

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

PubMed Central

Yamauchi, N; Kiessling, A A; Cooper, G M

1994-01-01

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos. Images PMID:7935384

Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta).

PubMed

Tedeschi, J N; Kennington, W J; Berry, O; Whiting, S; Meekan, M; Mitchell, N J

2015-01-01

The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34°C and 36°C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36°C, 3h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events. Copyright © 2014 Elsevier Ltd. All rights reserved.

Uterine responses to early pre-attachment embryos in the domestic dog and comparisons with other domestic animal species.

PubMed

Graubner, Felix R; Gram, Aykut; Kautz, Ewa; Bauersachs, Stefan; Aslan, Selim; Agaoglu, Ali R; Boos, Alois; Kowalewski, Mariusz P

2017-08-01

In the dog, there is no luteolysis in the absence of pregnancy. Thus, this species lacks any anti-luteolytic endocrine signal as found in other species that modulate uterine function during the critical period of pregnancy establishment. Nevertheless, in the dog an embryo-maternal communication must occur in order to prevent rejection of embryos. Based on this hypothesis, we performed microarray analysis of canine uterine samples collected during pre-attachment phase (days 10-12) and in corresponding non-pregnant controls, in order to elucidate the embryo attachment signal. An additional goal was to identify differences in uterine responses to pre-attachment embryos between dogs and other mammalian species exhibiting different reproductive patterns with regard to luteolysis, implantation, and preparation for placentation. Therefore, the canine microarray data were compared with gene sets from pigs, cattle, horses, and humans. We found 412 genes differentially regulated between the two experimental groups. The functional terms most strongly enriched in response to pre-attachment embryos related to extracellular matrix function and remodeling, and to immune and inflammatory responses. Several candidate genes were validated by semi-quantitative PCR. When compared with other species, best matches were found with human and equine counterparts. Especially for the pig, the majority of overlapping genes showed opposite expression patterns. Interestingly, 1926 genes did not pair with any of the other gene sets. Using a microarray approach, we report the uterine changes in the dog driven by the presence of embryos and compare these results with datasets from other mammalian species, finding common-, contrary-, and exclusively canine-regulated genes. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction.

Convergent evolution of hemoglobin switching in jawed and jawless vertebrates.

PubMed

Rohlfing, Kim; Stuhlmann, Friederike; Docker, Margaret F; Burmester, Thorsten

2016-02-01

During development, humans and other jawed vertebrates (Gnathostomata) express distinct hemoglobin genes, resulting in different hemoglobin tetramers. Embryonic and fetal hemoglobin have higher oxygen affinities than the adult hemoglobin, sustaining the oxygen demand of the developing organism. Little is known about the expression of hemoglobins during development of jawless vertebrates (Agnatha). We identified three hemoglobin switches in the life cycle of the sea lamprey. Three hemoglobin genes are specifically expressed in the embryo, four genes in the filter feeding larva (ammocoete), and nine genes correspond to the adult hemoglobin chains. During the development from the parasitic to the reproductive adult, the composition of hemoglobin changes again, with a massive increase of chain aHb1. A single hemoglobin chain is expressed constitutively in all stages. We further showed the differential expression of other globin genes: Myoglobin 1 is most highly expressed in the reproductive adult, myoglobin 2 expression peaks in the larva. Globin X1 is restricted to the embryo; globin X2 was only found in the reproductive adult. Cytoglobin is expressed at low levels throughout the life cycle. Because the hemoglobins of jawed and jawless vertebrates evolved independently from a common globin ancestor, hemoglobin switching must also have evolved convergently in these taxa. Notably, the ontogeny of sea lamprey hemoglobins essentially recapitulates their phylogeny, with the embryonic hemoglobins emerging first, followed by the evolution of larval and adult hemoglobins.

Effect of Sex of Embryo on Developmental Competence, Epigenetic Status, and Gene Expression in Buffalo (Bubalus bubalis) Embryos Produced by Hand-Made Cloning.

PubMed

Sandhu, Anjit; Mohapatra, Sushil K; Agrawal, Himanshu; Singh, Manoj K; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S

2016-10-01

Buffalo embryos were produced by hand-made cloning using skin fibroblasts from male and female buffaloes (n = 4 each) as donor cells for examining the effect of sex. Although the rate of blastocyst formation (43.8% ± 1.31% vs. 42.2% ± 1.22%) was similar, the total cell number (333 ± 10.4 vs. 270 ± 10.9) was higher (p < 0.05) whereas the apoptotic index (6.39 ± 0.25 vs. 8.52 ± 0.38) was lower (p < 0.05) for male than for female blastocysts. In the blastocysts, the global level of H3K18ac was found to be in the following order: male>female>IVF (in vitro fertilization) blastocysts (p < 0.05). The global level of H3K9me2 was not significantly different between male and female blastocysts and was higher (p < 0.05) compared with that in their IVF counterparts. The relative mRNA abundance of X-chromosome-linked (XIST, HPRT, PGK, and G6PD), apoptosis- (CASPASE3) and pregnancy-related genes (IFN-τ) was significantly higher (p < 0.05) whereas that of DNMT1 was significantly lower (p < 0.05) in female than in male blastocysts; however, in the case of apoptosis- (BCL-XL) and developmental competence-related genes (IGF1R and OCT4), the expression level was similar between the two groups. The gene expression level of OCT4 and IFN-τ but not of IGF1R was significantly lower (p < 0.05) in cloned than in IVF blastocysts. This study demonstrates that the epigenetic status, quality, and expression level of several genes but not the developmental competence are affected by the sex of cloned embryos.

Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts.

PubMed

Laskowski, Denise; Båge, Renée; Humblot, Patrice; Andersson, Göran; Sirard, Marc-André; Sjunnesson, Ylva

2017-10-01

Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL - 1 and 0.1 µg mL - 1 ), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation (ATP5D; CYP11A1; NDUFB7; NDUFB10; NDUFS8). Taken together, we hypothesise that the increased proliferation in the insulin-treated groups might impair the developmental potential of the embryos by inducing metabolic stress on the molecular level, which could be detrimental for the survival of the embryo. Copyright © 2017 Elsevier Inc. All rights reserved.

Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos

PubMed Central

Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li

2013-01-01

AIM: To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos. METHODS: Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting. RESULTS: In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages. We did not observe a significant change of mesodermal and endodermal marker gene expression, while after stage 40, about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil, but other structures outside the gastrointestinal tract were relatively normal. To test if the phenotypes were specifically caused by the knockdown of IRE1α, a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1α was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC, to investigate the functional domain of IRE1α. Injection of IRE1αΔC-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α. In order to investigate if IRE1α/XBP1 pathway was involved in gut development, 50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage. CONCLUSION: IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway. PMID:23345945

Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos.

PubMed

Guo, Jing; Li, Xin-Xin; Feng, Jiao-Jiao; Yin, Chen-Yang; Wang, Xue-Jun; Wang, Ning; Yuan, Li

2013-01-14

To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos. Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting. In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages. We did not observe a significant change of mesodermal and endodermal marker gene expression, while after stage 40, about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil, but other structures outside the gastrointestinal tract were relatively normal. To test if the phenotypes were specifically caused by the knockdown of IRE1α, a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1α was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC, to investigate the functional domain of IRE1α. Injection of IRE1αΔC-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α. In order to investigate if IRE1α/XBP1 pathway was involved in gut development, 50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage. IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.

Omics in Reproductive Medicine: Application of Novel Technologies to Improve the IVF Success Rate.

PubMed

Nerenz, R D

Treatment for many infertile couples often consists of in vitro fertilization (IVF) but an estimated 70% of IVF cycles fail to produce a live birth. In an attempt to improve the live birth rate, the vast majority of IVF cycles performed in the United States involve the transfer of multiple embryos, a practice that increases the risk of multiple gestation pregnancy. This is a concern because multiple gestation pregnancies are associated with an increased incidence of maternal and fetal complications and significant cost associated with the care of preterm infants. As the ideal outcome of each IVF cycle is the birth of a single healthy baby, significant effort has focused on identifying embryos with the greatest developmental potential. To date, selection of euploid embryos using comprehensive chromosome screening (CCS) is the most promising approach while metabolomic and proteomic assessment of spent culture medium have the potential to noninvasively assess embryo viability. Endometrial gene expression profiling may help determine the optimal time to perform embryo transfer. While CCS has been implemented in some clinics, further development and optimization will be required before analysis of spent culture medium and endometrial gene expression profiling make the transition to clinical use. This review will describe efforts to identify embryos with the greatest potential to result in a healthy, live birth, with a particular emphasis on detection of embryo aneuploidy and metabolic profiling of spent embryo culture medium. Assessment of endometrial receptivity to identify the optimal time to perform embryo transfer will also be discussed. © 2016 Elsevier Inc. All rights reserved.

m-carboxycinnamic acid bishydroxamide improves developmental competence, reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos.

PubMed

Agrawal, H; Selokar, N L; Saini, M; Singh, M K; Chauhan, M S; Palta, P; Singla, S K; Manik, R S

2018-05-07

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m-carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand-made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency-related genes OCT-4 and NANOG, and anti-apoptotic gene BCL-XL, and decreased (p < .05) that of pro-apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis-related genes p53 and CASPASE3 and epigenetics-related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern. © 2018 Blackwell Verlag GmbH.

Genome-wide identification, classification, and analysis of NADP-ME family members from 12 crucifer species.

PubMed

Tao, Peng; Guo, Weiling; Li, Biyuan; Wang, Wuhong; Yue, Zhichen; Lei, Juanli; Zhao, Yanting; Zhong, Xinmin

2016-06-01

NADP-dependent malic enzymes (NADP-MEs) play essential roles in both normal development and stress responses in plants. Here, genome-wide analysis was performed to identify 65 putative NADP-ME genes from 12 crucifer species. These NADP-ME genes were grouped into five categories of syntenic orthologous genes and were divided into three clades of a phylogenic tree. Promoter motif analysis showed that NADP-ME1 genes in Group IV were more conserved with each other than the other NADP-ME genes in Groups I and II. A nucleotide motif involved in ABA responses, desiccation and seed development was found in the promoters of most NADP-ME1 genes. Generally, the NADP-ME genes of Brassica rapa, B. oleracea and B. napus had less introns than their corresponding Arabidopsis orthologs. In these three Brassica species, the NADP-ME genes derived from the least fractionated subgenome have lost less introns than those from the medium fractionated and most fractionated subgenomes. BrNADP-ME1 showed the highest expression in petals and mature embryos. Two paralogous NADP-ME2 genes (BrNADP-ME2a and BrNADP-ME2b) shared similar expression profiles and differential expression levels. BrNADP-ME3 showed down-regulation during embryogenesis and reached its lowest expression in early cotyledonary embryos. BrNADP-ME4 was expressed widely in multiple organs and showed high expression during the whole embryogenesis process. Different NADP-ME genes of B. rapa showed differential gene expression profiles in young leaves after ABA treatment or cold stress. Our genome-wide identification and characterization of NADP-ME genes extend our understanding of the evolution or function of this family in Brassicaceae.

METscout: a pathfinder exploring the landscape of metabolites, enzymes and transporters.

PubMed

Geffers, Lars; Tetzlaff, Benjamin; Cui, Xiao; Yan, Jun; Eichele, Gregor

2013-01-01

METscout (http://metscout.mpg.de) brings together metabolism and gene expression landscapes. It is a MySQL relational database linking biochemical pathway information with 3D patterns of gene expression determined by robotic in situ hybridization in the E14.5 mouse embryo. The sites of expression of ∼1500 metabolic enzymes and of ∼350 solute carriers (SLCs) were included and are accessible as single cell resolution images and in the form of semi-quantitative image abstractions. METscout provides several graphical web-interfaces allowing navigation through complex anatomical and metabolic information. Specifically, the database shows where in the organism each of the many metabolic reactions take place and where SLCs transport metabolites. To link enzymatic reactions and transport, the KEGG metabolic reaction network was extended to include metabolite transport. This network in conjunction with spatial expression pattern of the network genes allows for a tracing of metabolic reactions and transport processes across the entire body of the embryo.

Ultraviolet filters differentially impact the expression of key endocrine and stress genes in embryos and larvae of Chironomus riparius.

PubMed

Ozáez, Irene; Morcillo, Gloria; Martínez-Guitarte, José-Luis

2016-07-01

Several organic UV filters have hormonal activity in vertebrates, as demonstrated in fishes, rodents and human cells. Despite the accumulation of filter contaminants in aquatic systems, research on their effects on the endocrine systems of freshwaters invertebrates is scarce. In this work, the effects of five frequently used UV filters were investigated in embryos and larvae of Chironomus riparius, which is a reference organism in ecotoxicology. LC50 values for larvae as well as the percentage of eclosion of eggs were determined following exposures to: octyl-p-methoxycinnamate (OMC) also known as 2-ethylhexyl-4-methoxycinnamate (EHMC); 4-methylbenzylidene camphor (4MBC); 4-hydroxybenzophenone (4HB); octocrylene (OC); and octyldimethyl-p-aminobenzoate (OD-PABA). To assess sublethal effects, expression levels of the genes coding for the ecdysone receptor (EcR) and heat shock protein HSP70 were investigated as biomarkers for endocrine and stress effects at the cellular level. Life-stage-dependent sensitivity was found. In embryos, all of the UV filters provoked a significant overexpression of EcR at 24h after exposure. OC, 4MBC and OD-PABA also triggered transcriptional activation of the hsp70 stress gene in embryos. In contrast, in larvae, only 4MBC and OMC/EHMC increased EcR and hsp70 mRNA levels and OD-PABA upregulated only the EcR gene. These results revealed that embryos are particularly sensitive to UV filters, which affect endocrine regulation during development. Most UV filters also triggered the cellular stress response, and thus exhibit proteotoxic effects. The differences observed between embryos and larvae and the higher sensitivity of embryos highlight the importance of considering different life stages when evaluating the environmental risks of pollutants, particularly when analyzing endocrine effects. Copyright © 2016 Elsevier B.V. All rights reserved.

Systems biology of embryonic development: Prospects for a complete understanding of the Caenorhabditis elegans embryo.

PubMed

Murray, John Isaac

2018-05-01

The convergence of developmental biology and modern genomics tools brings the potential for a comprehensive understanding of developmental systems. This is especially true for the Caenorhabditis elegans embryo because its small size, invariant developmental lineage, and powerful genetic and genomic tools provide the prospect of a cellular resolution understanding of messenger RNA (mRNA) expression and regulation across the organism. We describe here how a systems biology framework might allow large-scale determination of the embryonic regulatory relationships encoded in the C. elegans genome. This framework consists of two broad steps: (a) defining the "parts list"-all genes expressed in all cells at each time during development and (b) iterative steps of computational modeling and refinement of these models by experimental perturbation. Substantial progress has been made towards defining the parts list through imaging methods such as large-scale green fluorescent protein (GFP) reporter analysis. Imaging results are now being augmented by high-resolution transcriptome methods such as single-cell RNA sequencing, and it is likely the complete expression patterns of all genes across the embryo will be known within the next few years. In contrast, the modeling and perturbation experiments performed so far have focused largely on individual cell types or genes, and improved methods will be needed to expand them to the full genome and organism. This emerging comprehensive map of embryonic expression and regulatory function will provide a powerful resource for developmental biologists, and would also allow scientists to ask questions not accessible without a comprehensive picture. This article is categorized under: Invertebrate Organogenesis > Worms Technologies > Analysis of the Transcriptome Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics. © 2018 Wiley Periodicals, Inc.

Coordinated metabolic transitions during Drosophila embryogenesis and the onset of aerobic glycolysis.

PubMed

Tennessen, Jason M; Bertagnolli, Nicolas M; Evans, Janelle; Sieber, Matt H; Cox, James; Thummel, Carl S

2014-03-12

Rapidly proliferating cells such as cancer cells and embryonic stem cells rely on a specialized metabolic program known as aerobic glycolysis, which supports biomass production from carbohydrates. The fruit fly Drosophila melanogaster also utilizes aerobic glycolysis to support the rapid growth that occurs during larval development. Here we use singular value decomposition analysis of modENCODE RNA-seq data combined with GC-MS-based metabolomic analysis to analyze the changes in gene expression and metabolism that occur during Drosophila embryogenesis, spanning the onset of aerobic glycolysis. Unexpectedly, we find that the most common pattern of co-expressed genes in embryos includes the global switch to glycolytic gene expression that occurs midway through embryogenesis. In contrast to the canonical aerobic glycolytic pathway, however, which is accompanied by reduced mitochondrial oxidative metabolism, the expression of genes involved in the tricarboxylic cycle (TCA cycle) and the electron transport chain are also upregulated at this time. Mitochondrial activity, however, appears to be attenuated, as embryos exhibit a block in the TCA cycle that results in elevated levels of citrate, isocitrate, and α-ketoglutarate. We also find that genes involved in lipid breakdown and β-oxidation are upregulated prior to the transcriptional initiation of glycolysis, but are downregulated before the onset of larval development, revealing coordinated use of lipids and carbohydrates during development. These observations demonstrate the efficient use of nutrient stores to support embryonic development, define sequential metabolic transitions during this stage, and demonstrate striking similarities between the metabolic state of late-stage fly embryos and tumor cells. Copyright © 2014 Tennessen et al.

The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: potential role in temperature-dependent sex determination and testis development.

PubMed

Rhen, Turk; Jangula, Adam; Schroeder, Anthony; Woodward-Bosh, Rikki

2009-05-01

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-alpha and PdgfR-beta in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-alpha mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-beta mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-alpha and PdgfR-beta in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle.

The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: potential role in temperature-dependent sex determination and testis development

PubMed Central

Rhen, Turk; Jangula, Adam; Schroeder, Anthony; Woodward-Bosh, Rikki

2009-01-01

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-α and PdgfR-β in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-α mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-β mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-α and PdgfR-β in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle. PMID:19523392

Oxidative Damage to Rhesus Macaque Spermatozoa Results in Mitotic Arrest and Transcript Abundance Changes in Early Embryos1

PubMed Central

Burruel, Victoria; Klooster, Katie L.; Chitwood, James; Ross, Pablo J.; Meyers, Stuart A.

2013-01-01

ABSTRACT Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO2 in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development. PMID:23904511

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui Xiangshun; Li Xingyu; Kim, Nam-Hyung

To gain insights into the roles the paternal genome and chromosome number play in pre-implantation development, we cultured fertilized embryos and diploid and haploid parthenotes (DPs and HPs, respectively), and compared their development and gene expression patterns. The DPs and fertilized embryos did not differ in developmental ability but HPs development was slower and characterized by impaired compaction and blastocoel formation. Microarray analysis revealed that fertilized blastocysts expressed several genes at higher levels than DP blastocysts; these included the Y-chromosome-specific gene eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked (Eif2s3y) and the imprinting gene U2 small nuclear ribonucleoproteinmore » auxiliary factor 1, related sequence 1 (U2af1-rs1). We also found that when DPs and HPs were both harvested at 44 and 58 h of culture, they differed in the expression of 38 and 665 genes, respectively. However, when DPs and HPs were harvested at the midpoints of 4-cell stage (44 and 49 h, respectively), no differences in expression was observed. Similarly, when the DPs and HPs were harvested when they became blastocysts (102 and 138 h, respectively), only 15 genes showed disparate expression. These results suggest that while transcripts needed for early development are delayed in HPs, it does progress sufficiently for the generation of the various developmental stages despite the lack of genetic components.« less

Projecting 2D gene expression data into 3D and 4D space.

PubMed

Gerth, Victor E; Katsuyama, Kaori; Snyder, Kevin A; Bowes, Jeff B; Kitayama, Atsushi; Ueno, Naoto; Vize, Peter D

2007-04-01

Video games typically generate virtual 3D objects by texture mapping an image onto a 3D polygonal frame. The feeling of movement is then achieved by mathematically simulating camera movement relative to the polygonal frame. We have built customized scripts that adapt video game authoring software to texture mapping images of gene expression data onto b-spline based embryo models. This approach, known as UV mapping, associates two-dimensional (U and V) coordinates within images to the three dimensions (X, Y, and Z) of a b-spline model. B-spline model frameworks were built either from confocal data or de novo extracted from 2D images, once again using video game authoring approaches. This system was then used to build 3D models of 182 genes expressed in developing Xenopus embryos and to implement these in a web-accessible database. Models can be viewed via simple Internet browsers and utilize openGL hardware acceleration via a Shockwave plugin. Not only does this database display static data in a dynamic and scalable manner, the UV mapping system also serves as a method to align different images to a common framework, an approach that may make high-throughput automated comparisons of gene expression patterns possible. Finally, video game systems also have elegant methods for handling movement, allowing biomechanical algorithms to drive the animation of models. With further development, these biomechanical techniques offer practical methods for generating virtual embryos that recapitulate morphogenesis.

Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis.

PubMed

Sessions-Bresnahan, Dawn R; Heuberger, Adam L; Carnevale, Elaine M

2018-05-07

Maternal body composition can be an important determinant for development of obesity and metabolic syndrome in adult offspring. Obesity-related outcomes in offspring may include epigenetic alterations; however, mechanisms of fetal programming remain to be fully elucidated. This study was conducted to determine the impact of maternal obesity in the absence of a high fat diet on equine endometrium and preimplantation embryos. Embryos were collected from normal and obese mares at 8 and 16 d and a uterine biopsy at 16 d (0 d = ovulation). With the exception of 8 d embryos, each sample was divided into two pieces. One piece was analyzed for gene expression markers related to carbohydrate metabolism, lipid homeostasis, inflammation, endoplasmic reticulum stress, oxidative stress, mitochondrial stress, and components of the insulin-like growth factor (IGF) system. The second piece was analyzed for lipid content using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Obese mares had elevated concentrations of insulin, leptin and total cholesterol, and they tended to have increased triglycerides and decreased insulin sensitivity. Embryos from obese mares had altered transcript abundance in genes for inflammation and lipid homeostasis, as well as, endoplasmic reticulum, oxidative and mitochondrial stress and altered lipid fingerprints. Endometrium from obese mares had increased expression of inflammatory cytokines, lipid homeostasis regulation, mitochondrial stress, and the IGF2 system. This study demonstrates increased adiposity in mares alters the uterine environment, transcript abundance of genes for cellular functions, and lipid profiles of embryos. These alterations could affect prenatal programming, with potential long-term effects in offspring.

The expression of an immune-related phenoloxidase gene is modulated in Ciona intestinalis ovary, test cells, embryos and larva.

PubMed

Parrinello, Daniela; Sanfratello, Maria A; Vizzini, Aiti; Cammarata, Matteo

2015-03-01

Two distinct Ciona intestinalis phenoloxidases (CinPO1, 2) had previously been cloned and sequenced. The CinPO2 is involved in innate immunity and is expressed by inflammatory hemocytes that populate the tunic and pharynx vessels as a response to LPS inoculation. In situ hybridization and immunohistochemistry assays on histological section, showed that the expression of this gene and the produced protein are shared with oogenesis, embryogenesis and larval morphogenesis. Intriguingly, upregulation of gene transcription was found in the test cell layer that envelopes the ovary follicle, ovulated egg, and gastrula, as well as it was modulated in the zygotic nucleus of outer balstomers of 32-cell embryo, neurula presumptive epidermis tissue and larval mesenchyme. The anti-CinPO2 antibodies, specific for adult inflammatory cells, recognize epitopes in the cytoplasm of ovarian oocytes, ovulated eggs, development stages and larval mesenchyme. The overall findings disclose the precocious activation of the CinPO2 immunity-related gene, and show a developmentally programmed expression of this phenoloxidase. Furthermore, these findings support the multifunctional roles of immunity-related genes and allows us to explore new perspectives on ascidian development and immunity. © 2015 Wiley Periodicals, Inc.

Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

PubMed Central

Tu, Zhuchi; Yang, Weili; Yan, Sen; Yin, An; Gao, Jinquan; Liu, Xudong; Zheng, Yinghui; Zheng, Jiezhao; Li, Zhujun; Yang, Su; Li, Shihua; Guo, Xiangyu; Li, Xiao-Jiang

2017-01-01

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing. PMID:28155910

Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways.

PubMed

Rolfe, Rebecca A; Nowlan, Niamh C; Kenny, Elaine M; Cormican, Paul; Morris, Derek W; Prendergast, Patrick J; Kelly, Daniel; Murphy, Paula

2014-01-20

Mechanical stimulation is necessary for regulating correct formation of the skeleton. Here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. We use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle (Splotch-delayed) where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. We used Microarray and RNA sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic (TS23) humerus tissue. We found that 680 independent genes were down-regulated and 452 genes up-regulated in humeri from muscle-less Spd embryos compared to littermate controls (at least 2-fold; corrected p-value ≤0.05). We analysed the resulting differentially expressed gene sets using Gene Ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. Among cell signalling pathways, the most strongly disturbed was Wnt signalling, with 34 genes including 19 pathway target genes affected. Spatial gene expression analysis showed that both a Wnt ligand encoding gene (Wnt4) and a pathway antagonist (Sfrp2) are up-regulated specifically in the developing joint line, while the expression of a Wnt target gene, Cd44, is no longer detectable in muscle-less embryos. The identification of 84 genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a mechanical stimulus into a transcriptional response. This work identifies key developmental regulatory genes impacted by altered mechanical stimulation, sheds light on the molecular mechanisms that interpret mechanical stimulation during skeletal development and provides valuable resources for further investigation of the mechanistic basis of mechanoregulation. In particular it highlights the Wnt signalling pathway as a potential point of integration of mechanical and molecular signalling and cytoskeletal components as mediators of the response.

Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways

PubMed Central

2014-01-01

Background Mechanical stimulation is necessary for regulating correct formation of the skeleton. Here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. We use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle (Splotch-delayed) where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. We used Microarray and RNA sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic (TS23) humerus tissue. Results We found that 680 independent genes were down-regulated and 452 genes up-regulated in humeri from muscle-less Spd embryos compared to littermate controls (at least 2-fold; corrected p-value ≤0.05). We analysed the resulting differentially expressed gene sets using Gene Ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. Among cell signalling pathways, the most strongly disturbed was Wnt signalling, with 34 genes including 19 pathway target genes affected. Spatial gene expression analysis showed that both a Wnt ligand encoding gene (Wnt4) and a pathway antagonist (Sfrp2) are up-regulated specifically in the developing joint line, while the expression of a Wnt target gene, Cd44, is no longer detectable in muscle-less embryos. The identification of 84 genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a mechanical stimulus into a transcriptional response. Conclusions This work identifies key developmental regulatory genes impacted by altered mechanical stimulation, sheds light on the molecular mechanisms that interpret mechanical stimulation during skeletal development and provides valuable resources for further investigation of the mechanistic basis of mechanoregulation. In particular it highlights the Wnt signalling pathway as a potential point of integration of mechanical and molecular signalling and cytoskeletal components as mediators of the response. PMID:24443808

Transcriptome profiles of embryos before and after cleavage in Eriocheir sinensis: identification of developmental genes at the earliest stages

NASA Astrophysics Data System (ADS)

Hui, Min; Cui, Zhaoxia; Liu, Yuan; Song, Chengwen

2017-07-01

In crab, embryogenesis is a complicated developmental program marked by a series of critical events. RNA-Sequencing technology offers developmental biologists a way to identify many more developmental genes than ever before. Here, we present a comprehensive analysis of the transcriptomes of Eriocheir sinensis oosperms (Os) and embryos at the 2-4 cell stage (Cs), which are separated by a cleavage event. A total of 18 923 unigenes were identified, and 403 genes matched with gene ontology (GO) terms related to developmental processes. In total, 432 differentially expressed genes (DEGs) were detected between the two stages. Nine DEGs were specifically expressed at only one stage. These DEGs may be relevant to stage-specific molecular events during development. A number of DEGs related to `hedgehog signaling pathway', `Wnt signaling pathway' `germplasm', `nervous system', `sensory perception' and `segment polarity' were identified as being up-regulated at the Cs stage. The results suggest that these embryonic developmental events begin before the early cleavage event in crabs, and that many of the genes expressed in the two transcriptomes might be maternal genes. Our study provides ample information for further research on the molecular mechanisms underlying crab development.

Differential temporal control of Foxa.a and Zic-r.b specifies brain versus notochord fate in the ascidian embryo.

PubMed

Ikeda, Tatsuro; Satou, Yutaka

2017-01-01

In embryos of an invertebrate chordate, Ciona intestinalis, two transcription factors, Foxa.a and Zic-r.b, are required for specification of the brain and the notochord, which are derived from distinct cell lineages. In the brain lineage, Foxa.a and Zic-r.b are expressed with no temporal overlap. In the notochord lineage, Foxa.a and Zic-r.b are expressed simultaneously. In the present study, we found that the temporally non-overlapping expression of Foxa.a and Zic-r.b in the brain lineage was regulated by three repressors: Prdm1-r.a (formerly called BZ1), Prdm1-r.b (BZ2) and Hes.a. In morphant embryos of these three repressor genes, Foxa.a expression was not terminated at the normal time, and Zic-r.b was precociously expressed. Consequently, Foxa.a and Zic-r.b were expressed simultaneously, which led to ectopic activation of Brachyury and its downstream pathways for notochord differentiation. Thus, temporal controls by transcriptional repressors are essential for specification of the two distinct fates of brain and notochord by Foxa.a and Zic-r.b Such a mechanism might enable the repeated use of a limited repertoire of transcription factors in developmental gene regulatory networks. © 2017. Published by The Company of Biologists Ltd.

Hepatocyte growth factor is crucial for development of the carapace in turtles

PubMed Central

Kawashima-Ohya, Yoshie; Narita, Yuichi; Nagashima, Hiroshi; Usuda, Ryo; Kuratani, Shigeru

2011-01-01

Turtles are characterized by their shell, composed of a dorsal carapace and a ventral plastron. The carapace first appears as the turtle-specific carapacial ridge (CR) on the lateral aspect of the embryonic flank. Accompanying the acquisition of the shell, unlike in other amniotes, hypaxial muscles in turtle embryos appear as thin threads of fibrous tissue. To understand carapacial evolution from the perspective of muscle development, we compared the development of the muscle plate, the anlage of hypaxial muscles, between the Chinese soft-shelled turtle, Pelodiscus sinensis, and chicken embryos. We found that the ventrolateral lip (VLL) of the thoracic dermomyotome of P. sinensis delaminates early and produces sparse muscle plate in the lateral body wall. Expression patterns of the regulatory genes for myotome differentiation, such as Myf5, myogenin, Pax3, and Pax7 have been conserved among amniotes, including turtles. However, in P. sinensis embryos, the gene hepatocyte growth factor (HGF), encoding a regulatory factor for delamination of the dermomyotomal VLL, was uniquely expressed in sclerotome and the lateral body wall at the interlimb level. Implantation of COS-7 cells expressing a HGF antagonist into the turtle embryo inhibited CR formation. We conclude that the de novo expression of HGF in the turtle mesoderm would have played an innovative role resulting in the acquisition of the turtle-specific body plan. PMID:21535464

Establishing references for gene expression analyses by RT-qPCR in Theobroma cacao tissues.

PubMed

Pinheiro, T T; Litholdo, C G; Sereno, M L; Leal, G A; Albuquerque, P S B; Figueira, A

2011-11-17

Lack of continuous progress in Theobroma cacao (Malvaceae) breeding, especially associated with seed quality traits, requires more efficient selection methods based on genomic information. Reverse transcript quantitative PCR (RT-qPCR) has become the method of choice for gene expression analysis, but relative expression analysis requires various reference genes, which must be stable across various biological conditions. We sought suitable reference genes for various tissues of cacao, especially developing seeds. Ten potential reference genes were analyzed for stability at various stages of embryo development, leaves, stems, roots, flowers, and pod epicarp; seven of them were also evaluated in shoot tips treated either with hormones (salicylate; ethefon; methyl-jasmonate) or after inoculation with the fungus Moniliophthora perniciosa (Marasmiaceae sensu lato). For developing embryos, the three most stable genes were actin (ACT), polyubiquitin (PUB), and ribosomal protein L35 (Rpl35). In the analyses of various tissues, the most stable genes were malate dehydrogenase (MDH), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and acyl-carrier protein B (ACP B). GAPDH, MDH and tubulin (TUB) were the most appropriate for normalization when shoot apexes were treated with hormones, while ACT, TUB and Rpl35 were the most appropriate after inoculation with M. perniciosa. We conclude that for each plant system and biological or ontogenetical condition, there is a need to define suitable reference genes. This is the first report to define reference genes for expression studies in cacao.

Delayed blastocyst formation or an extra day culture increases apoptosis in pig blastocysts.

PubMed

Lin, Tao; Lee, Jae Eun; Oqani, Reza K; Kim, So Yeon; Cho, Eun Seok; Jeong, Yong Dae; Baek, Jun Jong; Jin, Dong Il

2017-10-01

In the present study, the timing was examined of blastocyst collection/formation or of how the duration of post-blastulation culture affected the quality and developmental competence of in vitro-produced pig parthenogenetic embryos. The earliest apoptotic signals were observed at the morula stage while the earliest cytoplasmic fragmentation was observed before the 4- to 8-cell stage of embryo development. Nuclear condensation was detected in morulae and blastocysts, but not all condensed nuclei were positive for the apoptotic signal (TUNEL staining). The mean blastocyst diameter increased with delayed blastocyst collection or extended post-blastulation culture, but decreased with delayed blastocyst formation. Delayed blastocyst collection/formation or an additional day of post-blastulation culture increased the frequencies of apoptosis, condensed nuclei, and low quality blastocysts (those showing a nuclear destruction that negated counting of the nuclei); increased the expression of the pro-apoptotic BAX gene; and reduced the ratio of ICM (inner cell mass) cells to TE (trophectoderm) cells. In addition, delayed blastocyst formation decreased POU5F1 gene expression. These results suggest that a delay in blastocyst collection/formation or an additional day of culture could increase the incidence of apoptosis, decrease the ICM:TE cell ratio, and influence the gene expression and diameter of blastocysts derived from in vitro-produced pig embryos. These findings provide a useful reference for improving the quality of in vitro-produced embryos. Copyright © 2017 Elsevier B.V. All rights reserved.

Cardiac-specific expression and hypertrophic upregulation of the feline Na(+)-Ca(2+) exchanger gene H1-promoter in a transgenic mouse model.

PubMed

Müller, Joachim G; Isomatsu, Yukihisa; Koushik, Srinagesh V; O'Quinn, Michael; Xu, Lin; Kappler, Christiana S; Hapke, Elizabeth; Zile, Michael R; Conway, Simon J; Menick, Donald R

2002-02-08

The NCX1 gene contains three promoters (H1, K1, and Br1), and as a result of alternative promoter usage and alternative splicing, there are multiple tissue-specific variants of the Na(+)-Ca(2+) exchanger. We have proposed that for NCX1, the H1 promoter regulates expression in the heart, the K1 promoter regulates expression in the kidney, and the Br1 promoter regulates expression in the brain as well as low-level ubiquitous expression. Here, using a transgenic mouse model, we test the role of the DNA region including -1831 to 67 bp of intron 1, encompassing exon H1 of the feline NCX1 gene (NCX1H1). The NCX1H1 promoter was sufficient for driving the normal spatiotemporal pattern of NCX1 expression in cardiac development. The luciferase reporter gene was expressed in a heart-restricted pattern both in early embryos (embryonic days 8 to 14) and in later embryos (after embryonic day 14), when NCX1 is also expressed in other tissues. In the adult, no luciferase activity was detected in the kidney, liver, spleen, uterus, or skeletal muscle; minimal activity was detected in the brain; and very high levels of luciferase expression were detected in the heart. Transverse aortic constriction-operated mice showed significantly increased left ventricular mass after 7 days. In addition, there was a 2-fold upregulation of NCX1H1 promoter activity in the left ventricle in animals after 7 days of pressure overload compared with both control and sham-operated animals. This work demonstrates that the NCX1H1 promoter directs cardiac-specific expression of the exchanger in both the embryo and adult and is also sufficient for the upregulation of NCX1 in response to pressure overload.

Loss of maternal CTCF is associated with peri-implantation lethality of Ctcf null embryos.

PubMed

Moore, James M; Rabaia, Natalia A; Smith, Leslie E; Fagerlie, Sara; Gurley, Kay; Loukinov, Dmitry; Disteche, Christine M; Collins, Steven J; Kemp, Christopher J; Lobanenkov, Victor V; Filippova, Galina N

2012-01-01

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5-E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16-32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

Parental genetic material and oxygen concentration affect hatch dynamics of mouse embryo in vitro.

PubMed

Zhan, Shaoquan; Cao, Shanbo; Du, Hongzi; Sun, Yuan; Li, Li; Ding, Chenhui; Zheng, Haiyan; Huang, Junjiu

2018-04-21

Hatching is crucial for mammalian embryo implantation, since difficulties during this process can lead to implantation failure, ectopic pregnancy and consequent infertility. Despite years of intensive researches, how internal and external factors affecting embryo hatch are still largely unclear. The effects of parental genetic material and oxygen concentration on hatch process were examined. Fertilized and parthenogenetic mouse preimplantation embryos were cultured in vitro under 5 and 20% oxygen for 120 h. Zona pellucida drilling by Peizo micromanipulation were performed to resemble the breach by sperm penetration. Firstly, parthenogenetic embryos had similarly high blastocyst developmental efficiency as fertilized embryos, but significantly higher hatch ratio than fertilized embryos in both O 2 concentrations. 5% O 2 reduced the hatch rate of fertilized embryos from 58.2 to 23.8%, but increased that of parthenogenetic embryos from 81.2 to 90.8% significantly. Analogously, 5% O 2 decreased the ratio of Oct4-positive cells in fertilized blastocysts, whereas increased that in parthenogenetic blastocysts. Additionally, 5% O 2 increased the total embryonic cell number in both fertilized and parthegenetic embryos, when compared to 20% O 2 , and the total cell number of fertilized embryos was also higher than that of parthegenetic embryos, despite O 2 concentration. Real-time PCR revealed that the expression of key genes involving in MAPK pathway and superoxide dismutase family might contribute to preimplantation development and consequent blastocyst hatch in vitro. Finally, we showed that fertilized and parthenogenetic embryos have diverse hatch dynamics in vitro, although the zona pellucida integrity is not the main reason for their mechanistic differences. Both parental genetic material and O 2 concentration, as the representative of intrinsic and extrinsic factors respectively, have significant impacts on mouse preimplantation development and subsequent hatch dynamics, probably by regulating the gene expression involving in MAPK pathway and superoxide dismutase family to control embryonic cell proliferation and allocation of ICM cells.

RNA-Seq Analysis of Developing Pecan (Carya illinoinensis) Embryos Reveals Parallel Expression Patterns among Allergen and Lipid Metabolism Genes.

PubMed

Mattison, Christopher P; Rai, Ruhi; Settlage, Robert E; Hinchliffe, Doug J; Madison, Crista; Bland, John M; Brashear, Suzanne; Graham, Charles J; Tarver, Matthew R; Florane, Christopher; Bechtel, Peter J

2017-02-22

The pecan nut is a nutrient-rich part of a healthy diet full of beneficial fatty acids and antioxidants, but can also cause allergic reactions in people suffering from food allergy to the nuts. The transcriptome of a developing pecan nut was characterized to identify the gene expression occurring during the process of nut development and to highlight those genes involved in fatty acid metabolism and those that commonly act as food allergens. Pecan samples were collected at several time points during the embryo development process including the water, gel, dough, and mature nut stages. Library preparation and sequencing were performed using Illumina-based mRNA HiSeq with RNA from four time points during the growing season during August and September 2012. Sequence analysis with Trinotate software following the Trinity protocol identified 133,000 unigenes with 52,267 named transcripts and 45,882 annotated genes. A total of 27,312 genes were defined by GO annotation. Gene expression clustering analysis identified 12 different gene expression profiles, each containing a number of genes. Three pecan seed storage proteins that commonly act as allergens, Car i 1, Car i 2, and Car i 4, were significantly up-regulated during the time course. Up-regulated fatty acid metabolism genes that were identified included acyl-[ACP] desaturase and omega-6 desaturase genes involved in oleic and linoleic acid metabolism. Notably, a few of the up-regulated acyl-[ACP] desaturase and omega-6 desaturase genes that were identified have expression patterns similar to the allergen genes based upon gene expression clustering and qPCR analysis. These findings suggest the possibility of coordinated accumulation of lipids and allergens during pecan nut embryogenesis.

The Fanconi anemia/BRCA gene network in zebrafish: embryonic expression and comparative genomics.

PubMed

Titus, Tom A; Yan, Yi-Lin; Wilson, Catherine; Starks, Amber M; Frohnmayer, Jonathan D; Bremiller, Ruth A; Cañestro, Cristian; Rodriguez-Mari, Adriana; He, Xinjun; Postlethwait, John H

2009-07-31

Fanconi anemia (FA) is a genetic disease resulting in bone marrow failure, high cancer risks, and infertility, and developmental anomalies including microphthalmia, microcephaly, hypoplastic radius and thumb. Here we present cDNA sequences, genetic mapping, and genomic analyses for the four previously undescribed zebrafish FA genes (fanci, fancj, fancm, and fancn), and show that they reverted to single copy after the teleost genome duplication. We tested the hypothesis that FA genes are expressed during embryonic development in tissues that are disrupted in human patients by investigating fanc gene expression patterns. We found fanc gene maternal message, which can provide Fanc proteins to repair DNA damage encountered in rapid cleavage divisions. Zygotic expression was broad but especially strong in eyes, central nervous system and hematopoietic tissues. In the pectoral fin bud at hatching, fanc genes were expressed specifically in the apical ectodermal ridge, a signaling center for fin/limb development that may be relevant to the radius/thumb anomaly of FA patients. Hatching embryos expressed fanc genes strongly in the oral epithelium, a site of squamous cell carcinomas in FA patients. Larval and adult zebrafish expressed fanc genes in proliferative regions of the brain, which may be related to microcephaly in FA. Mature ovaries and testes expressed fanc genes in specific stages of oocyte and spermatocyte development, which may be related to DNA repair during homologous recombination in meiosis and to infertility in human patients. The intestine strongly expressed some fanc genes specifically in proliferative zones. Our results show that zebrafish has a complete complement of fanc genes in single copy and that these genes are expressed in zebrafish embryos and adults in proliferative tissues that are often affected in FA patients. These results support the notion that zebrafish offers an attractive experimental system to help unravel mechanisms relevant not only to FA, but also to breast cancer, given the involvement of fancj (brip1), fancn (palb2) and fancd1 (brca2) in both conditions.

The Fanconi anemia/BRCA gene network in zebrafish: Embryonic expression and comparative genomics

PubMed Central

Titus, Tom A.; Yan, Yi-Lin; Wilson, Catherine; Starks, Amber M.; Frohnmayer, Jonathan D.; Canestro, Cristian; Rodriguez-Mari, Adriana; He, Xinjun; Postlethwait, John H.

2008-01-01

Fanconi anemia (FA) is a genic disease resulting in bone marrow failure, high cancer risks, and infertility, and developmental anomalies including microphthalmia, microcephaly, hypoplastic radius and thumb. Here we present cDNA sequences, genetic mapping, and genomic analyses for the four previously undescribed zebrafish FA genes (fanci, fancj, fancm, and fancn, and show that they reverted to single copy after the teleost genome duplication. We tested the hypothesis that FA genes are expressed during embryonic development in tissues that are disrupted in human patients by investigating fanc gene expression patterns. We found fanc gene maternal message, which can provide Fanc proteins to repair DNA damage encountered in rapid cleavage divisions. Zygotic expression was broad but especially strong in eyes, central nervous system and hematopoietic tissues. In the pectoral fin bud at hatching, fanc genes were expressed specifically in the apical ectodermal ridge, a signaling center for fin/limb development that may be relevant to the radius/thumb anomaly of FA patients. Hatching embryos expressed fanc genes strongly in the oral epithelium, a site of squamous cell carcinomas in FA patients. Larval and adult zebrafish expressed fanc genes in proliferative regions of the brain, which may be related to microcephaly in FA. Mature ovaries and testes expressed fanc genes in specific stages of oocyte and spermatocyte development, which may be related to DNA repair during homologous recombination in meiosis and to infertility in human patients. The intestine strongly expressed some fanc genes specifically in proliferative zones. Our results show that zebrafish has a complete complement of fanc genes in single copy and that these genes are expressed in zebrafish embryos and adults in proliferative tissues that are often affected in FA patients. These results support the notion that zebrafish offers an attractive experimental system to help unravel mechanisms relevant not only to FA, but also to breast cancer, given the involvement of fancj (brip1), fancn (palb2) and fancd1 (brca2) in both conditions. PMID:19101574

The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm.

PubMed

Mahlapuu, M; Ormestad, M; Enerbäck, S; Carlsson, P

2001-01-01

The murine Foxf1 gene encodes a forkhead transcription factor expressed in extra-embryonic and lateral plate mesoderm and later in splanchnic mesenchyme surrounding the gut and its derivatives. We have disrupted Foxf1 and show that mutant embryos die at midgestation due to defects in mesodermal differentiation and cell adhesion. The embryos do not turn and become deformed by the constraints of a small, inflexible amnion. Extra-embryonic structures exhibit a number of differentiation defects: no vasculogenesis occurs in yolk sac or allantois; chorioallantoic fusion fails; the amnion does not expand with the growth of the embryo, but misexpresses vascular and hematopoietic markers. Separation of the bulk of yolk sac mesoderm from the endodermal layer and adherence between mesoderm of yolk sac and amnion, indicate altered cell adhesion properties and enhanced intramesodermal cohesion. A possible cause of this is misexpression of the cell-adhesion protein VCAM1 in Foxf1-deficient extra-embryonic mesoderm, which leads to co-expression of VCAM with its receptor, alpha(4)-integrin. The expression level of Bmp4 is decreased in the posterior part of the embryo proper. Consistent with this, mesodermal proliferation in the primitive streak is reduced and somite formation is retarded. Expression of Foxf1 and the homeobox gene Irx3 defines the splanchnic and somatic mesodermal layers, respectively. In Foxf1-deficient embryos incomplete separation of splanchnic and somatic mesoderm is accompanied by misexpression of Irx3 in the splanchnopleure, which implicates Foxf1 as a repressor of Irx3 and as a factor involved in coelom formation.

Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

PubMed

Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

2009-09-01

Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

Precision of Readout at the hunchback Gene: Analyzing Short Transcription Time Traces in Living Fly Embryos

PubMed Central

Tran, Huy; Ferraro, Teresa; Lucas, Tanguy; Guillou, Aurelien; Coppey, Mathieu; Dostatni, Nathalie

2016-01-01

The simultaneous expression of the hunchback gene in the numerous nuclei of the developing fly embryo gives us a unique opportunity to study how transcription is regulated in living organisms. A recently developed MS2-MCP technique for imaging nascent messenger RNA in living Drosophila embryos allows us to quantify the dynamics of the developmental transcription process. The initial measurement of the morphogens by the hunchback promoter takes place during very short cell cycles, not only giving each nucleus little time for a precise readout, but also resulting in short time traces of transcription. Additionally, the relationship between the measured signal and the promoter state depends on the molecular design of the reporting probe. We develop an analysis approach based on tailor made autocorrelation functions that overcomes the short trace problems and quantifies the dynamics of transcription initiation. Based on live imaging data, we identify signatures of bursty transcription initiation from the hunchback promoter. We show that the precision of the expression of the hunchback gene to measure its position along the anterior-posterior axis is low both at the boundary and in the anterior even at cycle 13, suggesting additional post-transcriptional averaging mechanisms to provide the precision observed in fixed embryos. PMID:27942043

Medium-throughput processing of whole mount in situ hybridisation experiments into gene expression domains.

PubMed

Crombach, Anton; Cicin-Sain, Damjan; Wotton, Karl R; Jaeger, Johannes

2012-01-01

Understanding the function and evolution of developmental regulatory networks requires the characterisation and quantification of spatio-temporal gene expression patterns across a range of systems and species. However, most high-throughput methods to measure the dynamics of gene expression do not preserve the detailed spatial information needed in this context. For this reason, quantification methods based on image bioinformatics have become increasingly important over the past few years. Most available approaches in this field either focus on the detailed and accurate quantification of a small set of gene expression patterns, or attempt high-throughput analysis of spatial expression through binary pattern extraction and large-scale analysis of the resulting datasets. Here we present a robust, "medium-throughput" pipeline to process in situ hybridisation patterns from embryos of different species of flies. It bridges the gap between high-resolution, and high-throughput image processing methods, enabling us to quantify graded expression patterns along the antero-posterior axis of the embryo in an efficient and straightforward manner. Our method is based on a robust enzymatic (colorimetric) in situ hybridisation protocol and rapid data acquisition through wide-field microscopy. Data processing consists of image segmentation, profile extraction, and determination of expression domain boundary positions using a spline approximation. It results in sets of measured boundaries sorted by gene and developmental time point, which are analysed in terms of expression variability or spatio-temporal dynamics. Our method yields integrated time series of spatial gene expression, which can be used to reverse-engineer developmental gene regulatory networks across species. It is easily adaptable to other processes and species, enabling the in silico reconstitution of gene regulatory networks in a wide range of developmental contexts.

Expression of the cytokeratin endo A gene during early mouse embryogenesis.

PubMed Central

Duprey, P; Morello, D; Vasseur, M; Babinet, C; Condamine, H; Brûlet, P; Jacob, F

1985-01-01

Expression of cytokeratin endo A has been analyzed during mouse blastocyst formation and embryonal carcinoma cell differentiation. To study the regulation of endo A expression, nuclease S1 mapping experiments have been performed on RNA extracted from two-cell to 7.5-day embryos. Low levels of endo A mRNA begin to be detectable in eight-cell embryos. The amount of this mRNA increases at the blastocyst stage, suggesting that endo A expression is regulated at the mRNA level during blastocyst formation. At this stage, in situ hybridization studies show that endo A mRNA is present in the trophectoderm but not in the inner cell mass. In 7.5-day embryos, endo A mRNAs are also detectable in the endoderm layer and in the amnion. Images PMID:2417224

Dorsoventral patterning in hemichordates: insights into early chordate evolution.

PubMed

Lowe, Christopher J; Terasaki, Mark; Wu, Michael; Freeman, Robert M; Runft, Linda; Kwan, Kristen; Haigo, Saori; Aronowicz, Jochanan; Lander, Eric; Gruber, Chris; Smith, Mark; Kirschner, Marc; Gerhart, John

2006-09-01

We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called "dorsal." On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other.

Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution

PubMed Central

Lowe, Christopher J; Terasaki, Mark; Wu, Michael; Freeman, Robert M; Runft, Linda; Kwan, Kristen; Haigo, Saori; Aronowicz, Jochanan; Lander, Eric; Gruber, Chris; Smith, Mark; Kirschner, Marc; Gerhart, John

2006-01-01

We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called “dorsal.” On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other. PMID:16933975

Differential gene expression in notochord and nerve cord fate segregation in the Ciona intestinalis embryo.

PubMed

Kobayashi, Kenji; Yamada, Lixy; Satou, Yutaka; Satoh, Nori

2013-09-01

During early embryogenesis, embryonic cells gradually restrict their developmental potential and are eventually destined to give rise to one type of cells. Molecular mechanisms underlying developmental fate restriction are one of the major research subjects within developmental biology. In this article, this subject was addressed by combining blastomere isolation with microarray analysis. During the 6th cleavage of the Ciona intestinalis embryo, from the 32-cell to the 64-cell stage, four mother cells divide into daughter cells with two distinct fates, one giving rise to notochord precursor cells and the other to nerve cord precursors. Approximately 2,200 each of notochord and nerve cord precursor cells were isolated, and their mRNA expression profiles were compared by microarray. This analysis identified 106 and 68 genes, respectively, that are differentially expressed in notochord and nerve cord precursor cells. These included not only genes for transcription factors and signaling molecules but also those with generalized functions observed in many types of cells. In addition, whole-mount in situ hybridization showed dynamic spatial expression profiles of these genes during segregation of the two fates: partitioning of transcripts present in the mother cells into either type of daughter cells, and initiation of preferential gene expression in either type of cells. Copyright © 2013 Wiley Periodicals, Inc.

Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

PubMed

Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

2017-01-01

Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto

PubMed Central

Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

2017-01-01

Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3’UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3’UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3’UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo. PMID:28253363

Msx Homeobox Genes Critically Regulate Embryo Implantation by Controlling Paracrine Signaling between Uterine Stroma and Epithelium

PubMed Central

Nallasamy, Shanmugasundaram; Li, Quanxi; Bagchi, Milan K.; Bagchi, Indrani C.

2012-01-01

The mammalian Msx homeobox genes, Msx1 and Msx2, encode transcription factors that control organogenesis and tissue interactions during embryonic development. We observed overlapping expression of these factors in uterine epithelial and stromal compartments of pregnant mice prior to embryo implantation. Conditional ablation of both Msx1 and Msx2 in the uterus resulted in female infertility due to a failure in implantation. In these mutant mice (Msx1/2 d/d), the uterine epithelium exhibited persistent proliferative activity and failed to attach to the embryos. Gene expression profiling of uterine epithelium and stroma of Msx1/2 d/d mice revealed an elevated expression of several members of the Wnt gene family in the preimplantation uterus. Increased canonical Wnt signaling in the stromal cells activated β-catenin, stimulating the production of a subset of fibroblast growth factors (FGFs) in these cells. The secreted FGFs acted in a paracrine manner via the FGF receptors in the epithelium to promote epithelial proliferation, thereby preventing differentiation of this tissue and creating a non-receptive uterus refractory to implantation. Collectively, these findings delineate a unique signaling network, involving Msx1/2, Wnts, and FGFs, which operate in the uterus at the time of implantation to control the mesenchymal-epithelial dialogue critical for successful establishment of pregnancy. PMID:22383889

A Protocol for Epigenetic Imprinting Analysis with RNA-Seq Data.

PubMed

Zou, Jinfeng; Xiang, Daoquan; Datla, Raju; Wang, Edwin

2018-01-01

Genomic imprinting is an epigenetic regulatory mechanism that operates through expression of certain genes from maternal or paternal in a parent-of-origin-specific manner. Imprinted genes have been identified in diverse biological systems that are implicated in some human diseases and in embryonic and seed developmental programs in plants. The molecular underpinning programs and mechanisms involved in imprinting are yet to be explored in depth in plants. The recent advances in RNA-Seq-based methods and technologies offer an opportunity to systematically analyze epigenetic imprinting that operates at the whole genome level in the model and crop plants. We are interested using Arabidopsis model system, to investigate gene expression patterns associated with parent of origin and their implications to imprinting during embryo and seed development. Toward this, we have generated early embryo development RNA-Seq-based transcriptome datasets in F1s from a genetic cross between two diverse Arabidopsis thaliana ecotypes Col-0 and Tsu-1. With the data, we developed a protocol for evaluating the maternal and paternal contributions of genes during the early stages of embryo development after fertilization. This protocol is also designed to consider the contamination from other potential seed tissues, sequencing quality, proper processing of sequenced reads and variant calling, and appropriate inference of the parental contributions based on the parent-of-origin-specific single-nucleotide polymorphisms within the expressed genes. The approach, methods and the protocol developed in this study can be used for evaluating the effects of epigenetic imprinting in plants.

Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dimopoulou, Myrto, E-mail: myrto.dimopoulou@wur.nl

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48 h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4 h exposure at the ID{sub 10} (effectivemore » dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds. - Highlights: • Embryonic exposure to azoles revealed concentration-dependent malformations. • Transcriptomics could enhance the mechanistic knowledge of embryotoxicants. • Retinoic acid gene set identifies early embryotoxic responses to azoles. • Toxic versus pharmacologic potency determines functional efficacy.« less

Developing a Zebrafish Model of NF1 for Structure-Function Analysis and Identification of Modifier Genes

DTIC Science & Technology

2010-04-01

equipped with a spinning-disc confocal system ( Yokogawa ) was used. The statistical significance of changes to OPC cell numbers and migration upon nf1...that they are expressed in overlapping tissues. We examined the expression of both genes by whole mount in situ hybridization between the 4- cell stage...sorted cells confirmed expression, particularly in the vascular endothelium (Figure 4E-G), while RNA from 1- cell embryos indicate that both genes are

Embryo-Specific Gene Expression in Microspore-Derived Embryos of Brassica napus. An Interaction between Abscisic Acid and Jasmonic Acid1, 2

PubMed Central

Hays, Dirk B.; Wilen, Ronald W.; Sheng, Chuxing; Moloney, Maurice M.; Pharis, Richard P.

1999-01-01

The induction of napin and oleosin gene expression in Brassica napus microspore-derived embryos (MDEs) was studied to assess the possible interaction between abscisic acid (ABA) and jasmonic acid (JA). Napin and oleosin transcripts were detected sooner following treatment with ABA than JA. Treatment of MDEs with ABA plus JA gave an additive accumulation of both napin and oleosin mRNA, the absolute amount being dependent on the concentration of each hormone. Endogenous ABA levels were reduced by 10-fold after treatment with JA, negating the possibility that the observed additive interaction was due to JA-induced ABA biosynthesis. Also, JA did not significantly increase the uptake of [3H-ABA] from the medium into MDEs. This suggests that the additive interaction was not due to an enhanced carrier-mediated ABA uptake by JA. Finally, when JA was added to MDEs that had been treated with the ABA biosynthesis inhibitor fluridone, napin mRNA did not increase. Based on these results with the MDE system, it is possible that embryos of B. napus use endogenous JA to modulate ABA effects on expression of both napin and oleosin. In addition, JA could play a causal role in the reduction of ABA that occurs during late stages of seed development. PMID:10069845

Evidence that interferon-tau secreted from Day-7 embryo in vivo generates anti-inflammatory immune response in the bovine uterus.

PubMed

Rashid, Mohammad B; Talukder, Anup K; Kusama, Kazuya; Haneda, Shingo; Takedomi, Toshiro; Yoshino, Hitomi; Moriyasu, Satoru; Matsui, Motozumi; Shimada, Masayuki; Imakawa, Kazuhiko; Miyamoto, Akio

2018-06-12

Recent studies suggest that Day-7 bovine embryo starts to communicate with the uterine epithelium through interferon-tau (IFNT) signaling. However, immune modulatory role of IFNT in the uterus just after the embryo moves from the oviduct is unclear. We aimed to examine the hypothesis that Day-7 bovine embryo secretes IFNT in the uterus, which induces anti-inflammatory response in immune cells. The uterine flush (UF) with multiple embryos was collected from Day-7 donor pregnant cows and peripheral blood mononuclear cells (PBMCs) were then cultured in UF. Transcripts detected in PBMCs revealed that UF from pregnant cows down-regulated pro-inflammatory cytokines (TNFA, IL1B) and up-regulated anti-inflammatory cytokine (IL10) expression, with activation of interferon-stimulated genes (ISGs; ISG15, OAS1) as compared with UF from non-pregnant cows. An addition of specific anti-IFNT antibody to the UF inhibited the effect on PBMCs, indicating that IFNT is a major factor for such immune modulation. The observation that conditioned media from bovine uterine epithelial cells both stimulated with IFNT in vitro and supplemented with fresh IFNT induced similar PBMCs gene expression, confirming that IFNT directly acts on this immune crosstalk. This study shows that IFNT secreted from Day-7 embryo in vivo generates anti-inflammatory response in immune cells, which may provide immunological tolerance to accept the embryo. Copyright © 2018 Elsevier Inc. All rights reserved.

Formation of the spinal network in zebrafish determined by domain-specific Pax genes

PubMed Central

Ikenaga, Takanori; Urban, Jason M.; Gebhart, Nichole; Hatta, Kohei; Kawakami, Koichi; Ono, Fumihito

2012-01-01

In the formation of the spinal network, various transcription factors interact to develop specific cell types. Using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted in the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies using morpholinos. The pax8 homozygous embryos survived to adulthood in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP (+) neurons include Commissural Bifurcating Longitudinal (CoBL) interneurons, but other inhibitory neurons such as Commissural Local (CoLo) interneurons and Circumferential Ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP (+) cells undergo differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP (+) cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP (+) cells exhibited axons descending ipsilaterally: a morphology resembling that of V2a/V2b interneurons. PMID:21452218

Formation of the spinal network in zebrafish determined by domain-specific pax genes.

PubMed

Ikenaga, Takanori; Urban, Jason M; Gebhart, Nichole; Hatta, Kohei; Kawakami, Koichi; Ono, Fumihito

2011-06-01

In the formation of the spinal network, various transcription factors interact to develop specific cell types. By using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted into the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies with morpholinos. The pax8 homozygous embryos survived to adulthood, in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP(+) neurons include commissural bifurcating longitudinal (CoBL) interneurons, but other inhibitory neurons such as commissural local (CoLo) interneurons and circumferential ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP(+) cells underwent differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP(+) cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP(+) cells exhibited axons descending ipsilaterally, a morphology resembling that of V2a/V2b interneurons. Copyright © 2010 Wiley-Liss, Inc.

Morphological and transcriptomic effects of endocrine modulators on the gonadal differentiation of chicken embryos: The case of tributyltin (TBT).

PubMed

Scheider, Jessica; Afonso-Grunz, Fabian; Jessl, Luzie; Hoffmeier, Klaus; Winter, Peter; Oehlmann, Jörg

2018-03-01

Morphological malformations induced by tributyltin (TBT) exposure during embryonic development have already been characterized in various taxonomic groups, but, nonetheless, the molecular processes underlying these changes remain obscure. The present study provides the first genome-wide screening for differentially expressed genes that are linked to morphological alterations of gonadal tissue from chicken embryos after exposure to TBT. We applied a single injection of TBT (between 0.5 and 30 pg as Sn/g egg) into incubated fertile eggs to simulate maternal transfer of the endocrine disruptive compound. Methyltestosterone (MT) served as a positive control (30 pg/g egg). After 19 days of incubation, structural features of the gonads as well as genome-wide gene expression profiles were assessed simultaneously. TBT induced significant morphological and histological malformations of gonadal tissue from female embryos that show a virilization of the ovaries. This phenotypical virilization was mirrored by altered expression profiles of sex-dependent genes. Among these are several transcription and growth factors (e.g. FGF12, CTCF, NFIB), whose altered expression might serve as a set of markers for early identification of endocrine active chemicals that affect embryonic development by transcriptome profiling without the need of elaborate histological analyses. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Characterization of a type-A response regulator differentially expressed during adventitious caulogenesis in Pinus pinaster.

PubMed

Alvarez, José M; Cortizo, Millán; Ordás, Ricardo J

2012-12-15

The molecular cloning and characterization of PipsRR1, a type-A response regulator in Pinus pinaster, is reported here. Type-A response regulators mediate downstream responses to cytokinin and act as negative feedback regulators of the signal transduction pathway. Some type-A response regulators in Arabidopsis have been related to de novo meristem formation. However, little information exists in Pinus spp. The PipsRR1 gene contains 5 exons, as do all type-A response regulators in Arabidopsis, and the deduced protein contains a receiver domain with the conserved DDK residues and a short C terminal extension. Expression analysis showed that the PipsRR1 gene is differentially expressed during the first phases of adventitious caulogenesis induced by benzyladenine in P. pinaster cotyledons, suggesting that PipsRR1 plays a role in caulogenesis in conifers. Additionally, a binary vector carrying the PipsRR1 promoter driving GFP:GUS expression was constructed to analyze the promoter activity in P. pinaster somatic embryos. The results of genetic transformation showed GUS activity during somatic embryo mass proliferation and embryo maturation. Copyright © 2012 Elsevier GmbH. All rights reserved.

Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line

PubMed Central

2011-01-01

Background When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. Results A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. Conclusions The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells. PMID:22111699

Conservation of Pax gene expression in ectodermal placodes of the lamprey

NASA Technical Reports Server (NTRS)

McCauley, David W.; Bronner-Fraser, Marianne

2002-01-01

Ectodermal placodes contribute to the cranial ganglia and sense organs of the head and, together with neural crest cells, represent defining features of the vertebrate embryo. The identity of different placodes appears to be specified in part by the expression of different Pax genes, with Pax-3/7 class genes being expressed in the trigeminal placode of mice, chick, frogs and fish, and Pax-2/5/8 class genes expressed in the otic placode. Here, we present the cloning and expression pattern of lamprey Pax-7 and Pax-2, which mark the trigeminal and otic placodes, respectively, as well as other structures characteristic of vertebrate Pax genes. These results suggest conservation of Pax genes and placodal structures in basal and derived vertebrates.

Retinoic Acid Signaling Is Essential for Valvulogenesis by Affecting Endocardial Cushions Formation in Zebrafish Embryos.

PubMed

Li, Junbo; Yue, Yunyun; Zhao, Qingshun

2016-02-01

Retinoic acid (RA) plays important roles in many stages of heart morphogenesis. Zebrafish embryos treated with exogenous RA display defective atrio-ventricular canal (AVC) specification. However, whether endogenous RA signaling takes part in cardiac valve formation remains unknown. Herein, we investigated the role of RA signaling in cardiac valve development by knocking down aldh1a2, the gene encoding an enzyme that is mainly responsible for RA synthesis during early development, in zebrafish embryos. The results showed that partially knocking down aldh1a2 caused defective formation of primitive cardiac valve leaflets at 108 hpf (hour post-fertilization). Inhibiting endogenous RA signaling by 4-diethylaminobenzal-dehyde revealed that 16-26 hpf was a key time window when RA signaling affects the valvulogenesis. The aldh1a2 morphants had defective formation of endocardial cushion (EC) at 76 hpf though they had almost normal hemodynamics and cardiac chamber specification at early development. Examining the expression patterns of AVC marker genes including bmp4, bmp2b, nppa, notch1b, and has2, we found the morphants displayed abnormal development of endocardial AVC but almost normal development of myocardial AVC at 50 hpf. Being consistent with the reduced expression of notch1b in endocardial AVC, the VE-cadherin gene cdh5, the downstream gene of Notch signaling, was ectopically expressed in AVC of aldh1a2 morphants at 50 hpf, and overexpression of cdh5 greatly affected the formation of EC in the embryos at 76 hpf. Taken together, our results suggest that RA signaling plays essential roles in zebrafish cardiac valvulogenesis.

Zebrafish (Danio rerio) androgen receptor: sequence homology and up-regulation by the fungicide vinclozolin.

PubMed

Smolinsky, Amanda N; Doughman, Jennifer M; Kratzke, Liên-Thành C; Lassiter, Christopher S

2010-03-01

Steroid hormones regulate gene expression in organisms by binding to receptor proteins. These hormones include the androgens, which signal through androgen receptors (ARs). Endocrine disrupters (EDCs) are chemicals in the environment that adversely affect organisms by binding to nuclear receptors, including ARs. Vinclozolin, a fungicide used on fruit and vegetable crops, is a known anti-androgen, a type of EDC that blocks signals from testosterone and its derivatives. In order to better understand the effects of EDCs, further research on androgen receptors and other hormone signaling pathways is necessary. In this study, we demonstrate the evolutionary conservation between the genomic structure of the human and zebrafish ar genes and find that ar mRNA expression increases in zebrafish embryos exposed to vinclozolin, which may be evolutionarily conserved as well. At 48 and 72 h post-fertilization, vinclozolin-treated embryos express ar mRNA 8-fold higher than the control level. These findings suggest that zebrafish embryos attempt to compensate for the presence of an anti-androgen by increasing the number of androgen receptors available.

Temporal and tissue-specific regulation of a Brassica napus stearoyl-acyl carrier protein desaturase gene.

PubMed Central

Slocombe, S P; Piffanelli, P; Fairbairn, D; Bowra, S; Hatzopoulos, P; Tsiantis, M; Murphy, D J

1994-01-01

The nucleotide sequence of a Brassica napus stearoyl-acyl carrier protein desaturase gene (Bn10) is presented. This gene is one member of a family of four closely related genes expressed in oilseed rape. The expression of the promoter of this gene in transgenic tobacco was found to be temporally regulated in the developing seed tissues. However, the promoter was also particularly active in other oleogenic tissues such as the tapetum and pollen grains. This raises the interesting question of whether seed-expressed lipid synthesis genes are regulated by separate tissue-specific determinants or by a single factor common to all oleogenic tissues. Parts of the plants undergoing rapid development such as the components of immature flowers and seedlings also exhibited high levels of promoter activity. These tissues are likely to have an elevated requirement for membrane lipid synthesis. Stearoyl-acyl carrier protein desaturase transcript levels have previously been shown to be temporally regulated in the B. napus embryo (S.P. Slocombe, I. Cummins, R.P. Jarvis, D.J. Murphy [1992] Plant Mol Biol 20: 151-155). Evidence is presented demonstrating the induction of desaturase mRNA by abscisic acid in the embryo. PMID:8016261

Antagonistic interactions in the zebrafish midline prior to the emergence of asymmetric gene expression are important for left–right patterning

PubMed Central

Grimes, Daniel T.

2016-01-01

Left–right (L-R) asymmetry of the internal organs of vertebrates is presaged by domains of asymmetric gene expression in the lateral plate mesoderm (LPM) during somitogenesis. Ciliated L-R coordinators (LRCs) are critical for biasing the initiation of asymmetrically expressed genes, such as nodal and pitx2, to the left LPM. Other midline structures, including the notochord and floorplate, are then required to maintain these asymmetries. Here we report an unexpected role for the zebrafish EGF-CFC gene one-eyed pinhead (oep) in the midline to promote pitx2 expression in the LPM. Late zygotic oep (LZoep) mutants have strongly reduced or absent pitx2 expression in the LPM, but this expression can be rescued to strong levels by restoring oep in midline structures only. Furthermore, removing midline structures from LZoep embryos can rescue pitx2 expression in the LPM, suggesting the midline is a source of an LPM pitx2 repressor that is itself inhibited by oep. Reducing lefty1 activity in LZoep embryos mimics removal of the midline, implicating lefty1 in the midline-derived repression. Together, this suggests a model where Oep in the midline functions to overcome a midline-derived repressor, involving lefty1, to allow for the expression of left side-specific genes in the LPM. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821532

Effects of endocrine disruptors on imprinted gene expression in the mouse embryo

PubMed Central

Tran, Diana A; Rivas, Guillermo E; Singh, Purnima; Pfeifer, Gerd P

2011-01-01

Environmental endocrine disruptors (EDs) are synthetic chemicals that resemble natural hormones and are known to cause epigenetic perturbations. EDs have profound effects on development and fertility. Imprinted genes had been identified as candidate susceptibility loci to environmental insults because they are functionally haploid, and because the imprints undergo epigenetic resetting between generations. To screen for possible epigenetic perturbations caused by EDs at imprinted loci, we treated pregnant mice daily between 8.5 and 12.5 days post coitum (dpc) with di-(2-ethylhexyl)-phthalate (DEHP), bisphenol A (BPA), vinclozolin (VZ) or control oil vehicle. After isolating RNA from the placenta, yolk sac, amnion, head, body, heart, liver, lung, stomach and intestines of 13.5 dpc embryos we measured the allele-specific expression of 39 imprinted transcripts using multiplex single nucleotide primer extension (SNuPE) assays. In this representative data set we identified only a small number of transcripts that exhibited a substantial relaxation of imprinted expression with statistical significance: Slc22a18 with 10% relaxation in the embryo after BPA treatment; Rtl1as with 11 and 16% relaxation in the lung and placenta, respectively after BPA treatment; and Rtl1 with 12% relaxation in the yolk sac after DEHP treatment. Additionally, the standard deviation of allele-specificity increased in various organs after ED treatment for several transcripts including Igf2r, Rasgrf1, Usp29, Slc38a4 and Xist. Our data suggest that the maintenance of strongly biased monoallelic expression of imprinted genes is generally insensitive to EDs in the 13.5 dpc embryo and extra-embryonic organs, but is not immune to those effects. PMID:21636974

An Elk transcription factor is required for Runx-dependent survival signaling in the sea urchin embryo.

PubMed

Rizzo, Francesca; Coffman, James A; Arnone, Maria Ina

2016-08-01

Elk proteins are Ets family transcription factors that regulate cell proliferation, survival, and differentiation in response to ERK (extracellular-signal regulated kinase)-mediated phosphorylation. Here we report the embryonic expression and function of Sp-Elk, the single Elk gene of the sea urchin Strongylocentrotus purpuratus. Sp-Elk is zygotically expressed throughout the embryo beginning at late cleavage stage, with peak expression occurring at blastula stage. Morpholino antisense-mediated knockdown of Sp-Elk causes blastula-stage developmental arrest and embryo disintegration due to apoptosis, a phenotype that is rescued by wild-type Elk mRNA. Development is also rescued by Elk mRNA encoding a serine to aspartic acid substitution (S402D) that mimics ERK-mediated phosphorylation of a conserved site that enhances DNA binding, but not by Elk mRNA encoding an alanine substitution at the same site (S402A). This demonstrates both that the apoptotic phenotype of the morphants is specifically caused by Elk depletion, and that phosphorylation of serine 402 of Sp-Elk is critical for its anti-apoptotic function. Knockdown of Sp-Elk results in under-expression of several regulatory genes involved in cell fate specification, cell cycle control, and survival signaling, including the transcriptional regulator Sp-Runt-1 and its target Sp-PKC1, both of which were shown previously to be required for cell survival during embryogenesis. Both Sp-Runt-1 and Sp-PKC1 have sequences upstream of their transcription start sites that specifically bind Sp-Elk. These results indicate that Sp-Elk is the signal-dependent activator of a feed-forward gene regulatory circuit, consisting also of Sp-Runt-1 and Sp-PKC1, which actively suppresses apoptosis in the early embryo. Copyright © 2016 Elsevier Inc. All rights reserved.

Editor's Highlight: Hydroxyurea Exposure Activates the P53 Signaling Pathway in Murine Organogenesis-Stage Embryos.

PubMed

El Husseini, Nazem; Schlisser, Ava E; Hales, Barbara F

2016-08-01

Hydroxyurea, an anticancer agent and potent teratogen, induces oxidative stress and activates a DNA damage response pathway in the gestation day (GD) 9 mouse embryo. To delineate the stress response pathways activated by this drug, we investigated the effect of hydroxyurea exposure on the transcriptome of GD 9 embryos. Timed pregnant CD-1 mice were treated with saline or hydroxyurea (400 mg/kg or 600 mg/kg) on GD 9; embryonic gene and protein expression were examined 3 h later. Microarray analysis revealed that the expression of 1346 probe sets changed significantly in embryos exposed to hydroxyurea compared with controls; the P53 signaling pathway was highly affected. In addition, P53 related family members, P63 and P73, were predicted to be activated and had common and unique downstream targets. Western blot analysis revealed that active phospho-P53 was significantly increased in drug-exposed embryos; confocal microscopy showed that the translocation of phospho-P53 to the nucleus was widespread in the embryo. Furthermore, qRT-PCR showed that the expression of P53-regulated genes (Cdkn1A, Fas, and Trp53inp1) was significantly upregulated in hydroxyurea-exposed embryos; the concentration of the redox sensitive P53INP1 protein was also increased in a hydroxyurea dose-dependent fashion. Thus, hydroxyurea elicits a significant effect on the transcriptome of the organogenesis stage murine embryo, activating several key developmental signaling pathways related to DNA damage and oxidative stress. We propose that the P53 pathway plays a central role in the embryonic stress response and the developmental outcome after teratogen exposure. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Entire mitogen activated protein kinase (MAPK) pathway is present in preimplantation mouse embryos.

PubMed

Wang, Yingchun; Wang, Fangfei; Sun, Tong; Trostinskaia, Anna; Wygle, Dana; Puscheck, Elizabeth; Rappolee, Daniel A

2004-09-01

To understand how mitogenic signals are transduced into the trophoblasts in preimplantation embryos, the expression of mitogen-activated protein kinase (MAPK) pathway molecules was tested. We used immunocytochemical means and reverse transcriptase-polymerase chain reaction to test whether MAPK pathway molecule gene products exist at the protein and phosphoprotein level in the zygote and the RNA level in the egg and zygote. In addition, all antibodies detected the correct-sized major band in Westerns of placental cell lines representing the most prevalent cell type in preimplantation embryos. A majority of mRNA transcripts of MAPK pathway genes were detected in unfertilized eggs, and all were expressed in the zygote. We found that the MAPK pathway protein set consisting of the following gene products was present: FRS2 alpha, GRB2, GAB1, SOS1, Ha-ras, Raf1/RafB, MEK1,2,5, MAPK/ERK1,2, MAPK/ERK5, and RSK1,2,3 (see abbreviations). These proteins were detected in trophoblasts in embryonic day (E) 3.5 embryos when they could mediate mitogenic fibroblast growth factor signals from the embryo or colony stimulating factor-1 signals from the uterus. The phosphorylation state and position of the phosphoproteins in the cells suggested that they might function in mediating mitogenic signals. Interestingly, a subtle transition from maternal MAPK function to zygotic function was suggested by the localization for three MAPK pathway enzymes between E2.5 and E3.5, Raf1 phospho is largely cell membrane-localized at E2.5 and E3.5, and MEK1,2 phospho accumulates in the nucleus on E2.5 and E3.5. However, MAPK phospho shifts from nuclear accumulation at E2.5 to cytoplasmic accumulation at E3.5. This finding is similar to the cytoplasmic MAPK phospho localization reported in fibroblast growth factor signaling fields in postimplantation embryos (Corson et al. [2003] Development 130:4527-4537). This spatial and temporal expression study lays a foundation to plan and analyze perturbation studies aimed at understanding the role of the major mitogenic pathway in preimplantation mouse embryos.

The role of apoptosis in MCLR-induced developmental toxicity in zebrafish embryos.

PubMed

Zeng, Cheng; Sun, Hong; Xie, Ping; Wang, Jianghua; Zhang, Guirong; Chen, Nan; Yan, Wei; Li, Guangyu

2014-04-01

We previously demonstrated that cyanobacteria-derived microcystin-leucine-arginine (MCLR) is able to induce developing toxicity, such as malformation, growth delay and also decreased heart rates in zebrafish embryos. However, the molecular mechanisms by which MCLR induces its toxicity during the development of zebrafish remain largely unknown. Here, we evaluate the role of apoptosis in MCLR-induced developmental toxicity. Zebrafish embryos were exposed to various concentrations of MCLR (0, 0.2, 0.5, 2, and 5.0 mg L(-1)) for 96 h, at which time reactive oxygen species (ROS) was significantly induced in the 2 and 5.0 mg L(-1) MCLR exposure groups. Acridine orange (AO) staining and terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labelling (TUNEL) assay showed that MCLR exposure resulted in cell apoptosis. To test the apoptotic pathway, the expression pattern of several apoptotic-related genes was examined for the level of enzyme activity, gene and protein expression, respectively. The overall results demonstrate that MCLR induced ROS which consequently triggered apoptosis in the heart of developing zebrafish embryos. Our results also indicate that the p53-Bax-Bcl-2 pathway and the caspase-dependent apoptotic pathway play major roles in MCLR-induced apoptosis in the developing embryos. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes.

PubMed

Sugawara, Atsushi; Sugimura, Satoshi; Hoshino, Yumi; Sato, Eimei

2009-08-01

Cloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

Acquisition of the dorsal structures in chordate amphioxus.

PubMed

Morov, Arseniy R; Ukizintambara, Tharcisse; Sabirov, Rushan M; Yasui, Kinya

2016-06-01

Acquisition of dorsal structures, such as notochord and hollow nerve cord, is likely to have had a profound influence upon vertebrate evolution. Dorsal formation in chordate development thus has been intensively studied in vertebrates and ascidians. However, the present understanding does not explain how chordates acquired dorsal structures. Here we show that amphioxus retains a key clue to answer this question. In amphioxus embryos, maternal nodal mRNA distributes asymmetrically in accordance with the remodelling of the cortical cytoskeleton in the fertilized egg, and subsequently lefty is first expressed in a patch of blastomeres across the equator where wnt8 is expressed circularly and which will become the margin of the blastopore. The lefty domain co-expresses zygotic nodal by the initial gastrula stage on the one side of the blastopore margin and induces the expression of goosecoid, not-like, chordin and brachyury1 genes in this region, as in the oral ectoderm of sea urchin embryos, which provides a basis for the formation of the dorsal structures. The striking similarity in the gene regulations and their respective expression domains when comparing dorsal formation in amphioxus and the determination of the oral ectoderm in sea urchin embryos suggests that chordates derived from an ambulacrarian-type blastula with dorsoventral inversion. © 2016 The Authors.

Acquisition of the dorsal structures in chordate amphioxus

PubMed Central

Morov, Arseniy R.; Ukizintambara, Tharcisse; Sabirov, Rushan M.

2016-01-01

Acquisition of dorsal structures, such as notochord and hollow nerve cord, is likely to have had a profound influence upon vertebrate evolution. Dorsal formation in chordate development thus has been intensively studied in vertebrates and ascidians. However, the present understanding does not explain how chordates acquired dorsal structures. Here we show that amphioxus retains a key clue to answer this question. In amphioxus embryos, maternal nodal mRNA distributes asymmetrically in accordance with the remodelling of the cortical cytoskeleton in the fertilized egg, and subsequently lefty is first expressed in a patch of blastomeres across the equator where wnt8 is expressed circularly and which will become the margin of the blastopore. The lefty domain co-expresses zygotic nodal by the initial gastrula stage on the one side of the blastopore margin and induces the expression of goosecoid, not-like, chordin and brachyury1 genes in this region, as in the oral ectoderm of sea urchin embryos, which provides a basis for the formation of the dorsal structures. The striking similarity in the gene regulations and their respective expression domains when comparing dorsal formation in amphioxus and the determination of the oral ectoderm in sea urchin embryos suggests that chordates derived from an ambulacrarian-type blastula with dorsoventral inversion. PMID:27307516

Immunolocalization and expression of Na(+)/K(+) -ATPase in embryos, early larval stages and adults of the freshwater shrimp Palaemonetes argentinus (Decapoda, Caridea, Palaemonidae).

PubMed

Ituarte, Romina Belén; Lignot, Jehan-Hervé; Charmantier, Guy; Spivak, Eduardo; Lorin-Nebel, Catherine

2016-06-01

The euryhaline shrimp Palaemonetes argentinus exemplifies an evolutionary transition from brackish to freshwater habitats that requires adequate osmoregulatory capacities. Hyperosmoregulation is functional at hatching and it likely begins during the embryonic phase allowing this species to develop entirely in fresh water. Here, we investigated the Na(+)/K(+)-ATPase α-subunit gene (nka-α) expression using quantitative real-time PCR and localized Na(+)/K(+)-ATPase (NKA) in ion-transporting epithelia through immunofluorescence microscopy. We reared shrimps from spawning to juvenile stages at two salinities (1, 15 ‰) and maintained adults for 3 weeks at three salinity treatments (1, 15, 25 ‰). nka-α gene expression was measured in: (1) embryos at an early (SI), intermediate (SII) and late (SIII) stage of embryonic development; (2) newly hatched larvae (Zoea I, ZI); and (3) isolated gill tissue of adults. The nka-α expression was low in SI and SII embryos and reached maximum levels prior to hatching (SIII), which were similar to expression levels detected in the ZI. The nka-α expression in SIII and ZI was highest at 15 ‰, whereas salinity did not affect expression in earlier embryos. In SIII, in ZI and in a later zoeal stage ZIV, NKA was localized in epithelial cells of pleurae, in the inner-side epithelium of branchiostegite and in the antennal glands. Gills appeared in the ZIV but NKA immunolabeling of the cells of the gill shaft occurred in a subsequent developmental larval stage, the decapodid. Extrabranchial organs constitute the main site of osmoregulation in early ontogenetic stages of this freshwater shrimp.

The UV-absorber benzophenone-4 alters transcripts of genes involved in hormonal pathways in zebrafish (Danio rerio) eleuthero-embryos and adult males

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zucchi, Sara; Bluethgen, Nancy; University of Basel, Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Klingelbergstrasse 50, CH-4056 Basel

Benzophenone-4 (BP-4) is frequently used as UV-absorber in cosmetics and materials protection. Despite its frequent detection in the aquatic environment potential effects on aquatic life are unknown. In this study, we evaluate the effects of BP-4 in eleuthero-embryos and in the liver, testis and brain of adult male fish on the transcriptional level by focusing on target genes involved in hormonal pathways to provide a more complete toxicological profile of this important UV-absorber. Eleuthero-embryos and males of zebrafish were exposed up to 3 days after hatching and for 14 days, respectively, to BP-4 concentrations between 30 and 3000 {mu}g/L. Inmore » eleuthero-embryos transcripts of vtg1, vtg3, esr1, esr2b, hsd17ss3, cyp19b cyp19a, hhex and pax8 were induced at 3000 {mu}g/L BP-4, which points to a low estrogenic activity and interference with early thyroid development, respectively. In adult males BP-4 displayed multiple effects on gene expression in different tissues. In the liver vtg1, vtg3, esr1 and esr2b were down-regulated, while in the brain, vtg1, vtg3 and cyp19b transcripts were up-regulated. In conclusion, the transcription profile revealed that BP-4 interferes with the expression of genes involved in hormonal pathways and steroidogenesis. The effects of BP-4 differ in life stages and adult tissues and point to an estrogenic activity in eleuthero-embryos and adult brain, and an antiestrogenic activity in the liver. The results indicate that BP-4 interferes with the sex hormone system of fish, which is important for the risk assessment of this UV-absorber.« less

Reprogramming the Maternal Zebrafish Genome after Fertilization to Match the Paternal Methylation Pattern

PubMed Central

Potok, Magdalena E.; Nix, David A.; Parnell, Timothy J.; Cairns, Bradley R.

2014-01-01

SUMMARY Early vertebrate embryos must achieve totipotency and prepare for zygotic genome activation (ZGA). To understand this process, we determined the DNA methylation (DNAme) profiles of zebrafish gametes, embryos at different stages, and somatic muscle and compared them to gene activity and histone modifications. Sperm chromatin patterns are virtually identical to those at ZGA. Unexpectedly, the DNA of many oocyte genes important for germ-line functions (i.e., piwil1) or early development (i.e., hox genes) is methylated, but the loci are demethylated during zygotic cleavage stages to precisely the state observed in sperm, even in parthenogenetic embryos lacking a replicating paternal genome. Furthermore, this cohort constitutes the genes and loci that acquire DNAme during development (i.e., ZGA to muscle). Finally, DNA methyltransferase inhibition experiments suggest that DNAme silences particular gene and chromatin cohorts at ZGA, preventing their precocious expression. Thus, zebrafish achieve a totipotent chromatin state at ZGA through paternal genome competency and maternal genome DNAme reprogramming. PMID:23663776

Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos.

PubMed

Terashita, Yukari; Yamagata, Kazuo; Tokoro, Mikiko; Itoi, Fumiaki; Wakayama, Sayaka; Li, Chong; Sato, Eimei; Tanemura, Kentaro; Wakayama, Teruhiko

2013-01-01

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

Toxicogenomic analysis in the combined effect of tributyltin and benzo[a]pyrene on the development of zebrafish embryos.

PubMed

Huang, Lixing; Zuo, Zhenghong; Zhang, Youyu; Wang, Chonggang

2015-01-01

There is a growing recognition that the toxic effects of chemical mixtures are been an important issue in toxicological sciences. Tributyltin (TBT) and benzo[a]pyrene (BaP) are widespread pollutants that occur simultaneously in the aquatic environments. This study was designed to examine comprehensively the combined effects of TBT and BaP on zebrafish (Danio rerio) embryos using toxicogenomic approach combined with biochemical detection and morphological analysis, and tried to gain insight into the mechanisms underlying the combined effects of TBT and BaP. The results of toxicogenomic data indicated that: (1) TBT cotreatment rescued the embryos from decreased hatching ratio caused by BaP alone, while the alteration of gene expression (in this article the phrase gene expression is used as a synonym to gene transcription, although in is acknowledged that gene expression can also be regulated by, e.g., translation and mRNA or protein stability) relative to zebrafish hatching in the BaP groups was resumed by the cotreatment with TBT; (2) BaP cotreatment decreased TBT-mediated dorsal curvature, and alleviated the perturbation of Notch pathway caused by TBT alone; (3) cotreatment with TBT decreased BaP-mediated bradycardia, which might be due to that TBT cotreatment alleviated the perturbation in expression of genes related to cardiac muscle cell development and calcium handling caused by BaP alone; 4) TBT cotreatment brought an antagonistic effect on the BaP-mediated oxidative stress and DNA damage. These results suggested that toxicogenomic approach was available for analyzing combined toxicity with high sensitivity and accuracy, which might improve our understanding and predictability for the combined effects of chemicals. Copyright © 2014 Elsevier B.V. All rights reserved.

Loggerhead sea turtle embryos (Caretta caretta) regulate expression of stress response and developmental genes when exposed to a biologically realistic heat stress.

PubMed

Bentley, Blair P; Haas, Brian J; Tedeschi, Jamie N; Berry, Oliver

2017-06-01

Oviparous reptile embryos are expected to breach their critical thermal maxima if temperatures reach those predicted under current climate change models due to the lack of the maternal buffering processes and parental care. Heat-shock proteins (HSPs) are integral in the molecular response to thermal stress, and their expression is heritable, but the roles of other candidate families such as the heat-shock factors (HSFs) have not been determined in reptiles. Here, we subject embryonic sea turtles (Caretta caretta) to a biologically realistic thermal stress and employ de novo transcriptomic profiling of brain tissue to investigate the underlying molecular response. From a reference transcriptome of 302 293 transcripts, 179 were identified as differentially expressed between treatments. As anticipated, genes enriched in the heat-shock treatment were primarily associated with the Hsp families, or were genes whose products play similar protein editing and chaperone functions (e.g. bag3, MYOC and serpinh1). Unexpectedly, genes encoding the HSFs were not significantly upregulated under thermal stress, indicating their presence in unstressed cells in an inactive state. Genes that were downregulated under thermal stress were less well functionally defined but were associated with stress response, development and cellular organization, suggesting that developmental processes may be compromised at realistically high temperatures. These results confirm that genes from the Hsp families play vital roles in the thermal tolerance of developing reptile embryos and, in addition with a number of other genes, should be targets for evaluating the capacity of oviparous reptiles to respond adaptively to the effects of climate change. © 2017 John Wiley & Sons Ltd.

Transcriptomic profiling of genes in matured dimorphic seeds of euhalophyte Suaeda salsa.

PubMed

Xu, Yange; Zhao, Yuanqin; Duan, Huimin; Sui, Na; Yuan, Fang; Song, Jie

2017-09-13

Suaeda salsa (S. salsa) is a euhalophyte with high economic value. S. salsa can produce dimorphic seeds. Brown seeds are more salt tolerant, can germinate quickly and maintain the fitness of the species under high saline conditions. Black seeds are less salt tolerant, may become part of the seed bank and germinate when soil salinity is reduced. Previous reports have mainly focused on the ecophysiological traits of seed germination and production under saline conditions in this species. However, there is no information available on the molecular characteristics of S. salsa dimorphic seeds. In the present study, a total of 5825 differentially expressed genes were obtained; and 4648 differentially expressed genes were annotated based on a sequence similarity search, utilizing five public databases by transcriptome analysis. The different expression of these genes may be associated with embryo development, fatty acid, osmotic regulation substances and plant hormones in brown and black seeds. Compared to black seeds, most genes may relate to embryo development, and various genes that encode fatty acid desaturase and are involved in osmotic regulation substance synthesis or transport are upregulated in brown seeds. A large number of differentially expressed genes related to plant hormones were found in brown and black seeds, and their possible roles in regulating seed dormancy/germination were discussed. Upregulated genes involved in seed development and osmotic regulation substance accumulation may relate to bigger seed size and rapid seed germination in brown seeds, compared to black seeds. Differentially expressed genes of hormones may relate to seed dormancy/germination and the development of brown and black seeds. The transcriptome dataset will serve as a valuable resource to further understand gene expression and functional genomics in S. salsa dimorphic seeds.

Regulation of FA and TAG biosynthesis pathway genes in endosperms and embryos of high and low oil content genotypes of Jatropha curcas L.

PubMed

Sood, Archit; Chauhan, Rajinder Singh

2015-09-01

The rising demand for biofuels has raised concerns about selecting alternate and promising renewable energy crops which do not compete with food supply. Jatropha (Jatropha curcas L.), a non-edible energy crop of the family euphorbiaceae, has the potential of providing biodiesel feedstock due to the presence of high proportion of unsaturated fatty acids (75%) in seed oil which is mainly accumulated in endosperm and embryo. The molecular basis of seed oil biosynthesis machinery has been studied in J. curcas, however, what genetic differences contribute to differential oil biosynthesis and accumulation in genotypes varying for oil content is poorly understood. We investigated expression profile of 18 FA and TAG biosynthetic pathway genes in different developmental stages of embryo and endosperm from high (42%) and low (30%) oil content genotypes grown at two geographical locations. Most of the genes showed relatively higher expression in endosperms of high oil content genotype, whereas no significant difference was observed in endosperms versus embryos of low oil content genotype. The promoter regions of key genes from FA and TAG biosynthetic pathways as well as other genes implicated in oil accumulation were analyzed for regulatory elements and transcription factors specific to oil or lipid accumulation in plants such as Dof, CBF (LEC1), SORLIP, GATA and Skn-1_motif etc. Identification of key genes from oil biosynthesis and regulatory elements specific to oil deposition will be useful not only in dissecting the molecular basis of high oil content but also improving seed oil content through transgenic or molecular breeding approaches. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Transient expression of GUS in bombarded embryogenic longleaf, loblolly, and eastern white pine

Treesearch

Alex M. Diner; Allan Zipf; Rufina Ward; Yinghua Huang; George Brown

1999-01-01

Embryogenic tissue cultures derived from immature zygotic embryos of longleaf, loblolly, and eastern white pine were maintained in culture for up to 2 years, then bombarded with gold particles coated with a gene construct containing the GUS reporter gene fused to an adenine methyltransferase promoter from an algal virus. Physiological expression of GUS was observed in...

The organization and expression of the mdm2 gene.

PubMed

de Oca Luna, R M; Tabor, A D; Eberspaecher, H; Hulboy, D L; Worth, L L; Colman, M S; Finlay, C A; Lozano, G

1996-05-01

The mdm2 gene encodes a zinc finger protein that negatively regulates p53 function by binding and masking the p53 transcriptional activation domain. Two different promoters control expression of mdm2, one of which is also transactivated by p53. We cloned and characterized the mdm2 gene from a murine 129 library. It contained at least 12 exons and spanned approximately 25 kb of DNA. Sequencing of the mdm2 gene revealed three nucleotide differences that resulted in amino acid substitutions in the previously published mdm2 sequence. Sequencing of normal BalbC/J DNA and the original cosmid clone isolated from the 3T3DM cell line revealed that they are identical, suggesting that the published sequence is in error at these three positions. In addition, we analyzed the expression pattern of mdm2 and found ubiquitous low-level expression throughout embryo development and in adult tissues. Analysis of mRNA from numerous tissues for several mdm2 spliced variants that had been identified in the transformed 3T3DM cell line revealed that these variants could not be detected in the developing embryo or in adult tissues.

Dual mode of embryonic development is highlighted by expression and function of Nasonia pair-rule genes

PubMed Central

Rosenberg, Miriam I; Brent, Ava E; Payre, François; Desplan, Claude

2014-01-01

Embryonic anterior–posterior patterning is well understood in Drosophila, which uses ‘long germ’ embryogenesis, in which all segments are patterned before cellularization. In contrast, most insects use ‘short germ’ embryogenesis, wherein only head and thorax are patterned in a syncytial environment while the remainder of the embryo is generated after cellularization. We use the wasp Nasonia (Nv) to address how the transition from short to long germ embryogenesis occurred. Maternal and gap gene expression in Nasonia suggest long germ embryogenesis. However, the Nasonia pair-rule genes even-skipped, odd-skipped, runt and hairy are all expressed as early blastoderm pair-rule stripes and late-forming posterior stripes. Knockdown of Nv eve, odd or h causes loss of alternate segments at the anterior and complete loss of abdominal segments. We propose that Nasonia uses a mixed mode of segmentation wherein pair-rule genes pattern the embryo in a manner resembling Drosophila at the anterior and ancestral Tribolium at the posterior. DOI: http://dx.doi.org/10.7554/eLife.01440.001 PMID:24599282

Handmade Cloned Buffalo (Bubalus bubalis) Embryos Produced from Somatic Cells Isolated from Milk and Ear Skin Differ in Their Developmental Competence, Epigenetic Status, and Gene Expression.

PubMed

Jyotsana, Basanti; Sahare, Amol A; Raja, Anuj K; Singh, Karn P; Singla, Suresh K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat

2015-10-01

We compared the cloning efficiency of buffalo embryos produced by handmade cloning (HMC) using ear skin- and milk-derived donor cells. The blastocyst rate was lower (p < 0.05) for milk-derived than that for skin-derived embryos, whereas the total cell number and apoptotic index were similar. The global level of H3K9ac was higher (p < 0.05) in skin- than in milk-derived cells, whereas the level of H3K27me3 was similar in the two groups. The global level of H3K9ac was similar between milk-derived and in vitro-fertilized (IVF) blastocysts, which was higher (p < 0.05) than that in skin-derived blastocysts. The level of H3K27me3 was similar among the three groups. The expression level of IGF-1R and G6PD was higher (p < 0.05) in skin- than in milk-derived cells, whereas DNMT1, DNMT3a, and HDAC1 expression level was similar. In the blastocysts, the expression level of DNMT1, HDAC1, OCT4, and CDX2 was higher (p < 0.05) in skin-derived than that in IVF blastocysts. The expression level of DNMT3a and IGF-1R, was in the order (p < 0.05) skin-derived and IVF > milk-derived blastocysts and that of NANOG was (p < 0.05) IVF-> milk-derived > skin-derived blastocysts. The expression level of all these genes, except NANOG, was lower (p < 0.05) in milk- than in skin-derived or IVF blastocysts. In conclusion, milk-derived cells can be used for producing HMC embryos of quality similar to that of skin-derived embryos, although with a lower blastocyst rate.

Controversies Surrounding Segments and Parasegments in Onychophora: Insights from the Expression Patterns of Four “Segment Polarity Genes” in the Peripatopsid Euperipatoides rowelli

PubMed Central

Franke, Franziska Anni; Mayer, Georg

2014-01-01

Arthropods typically show two types of segmentation: the embryonic parasegments and the adult segments that lie out of register with each other. Such a dual nature of body segmentation has not been described from Onychophora, one of the closest arthropod relatives. Hence, it is unclear whether onychophorans have segments, parasegments, or both, and which of these features was present in the last common ancestor of Onychophora and Arthropoda. To address this issue, we analysed the expression patterns of the “segment polarity genes” engrailed, cubitus interruptus, wingless and hedgehog in embryos of the onychophoran Euperipatoides rowelli. Our data revealed that these genes are expressed in repeated sets with a specific anterior-to-posterior order along the body in embryos of E. rowelli. In contrast to arthropods, the expression occurs after the segmental boundaries have formed. Moreover, the initial segmental furrow retains its position within the engrailed domain throughout development, whereas no new furrow is formed posterior to this domain. This suggests that no re-segmentation of the embryo occurs in E. rowelli. Irrespective of whether or not there is a morphological or genetic manifestation of parasegments in Onychophora, our data clearly show that parasegments, even if present, cannot be regarded as the initial metameric units of the onychophoran embryo, because the expression of key genes that define the parasegmental boundaries in arthropods occurs after the segmental boundaries have formed. This is in contrast to arthropods, in which parasegments rather than segments are the initial metameric units of the embryo. Our data further revealed that the expression patterns of “segment polarity genes” correspond to organogenesis rather than segment formation. This is in line with the concept of segmentation as a result of concerted evolution of individual periodic structures rather than with the interpretation of ‘segments’ as holistic units. PMID:25470738

Cloning and Characterization of 5′ Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

PubMed Central

Tang, Guiying; Xu, Pingli; Liu, Wei; Liu, Zhanji; Shan, Lei

2015-01-01

LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5′ flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5′ terminal and/or 3′ terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65bp proximal promoter region and the 52bp 5′ UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements. PMID:26426444

SpSM30 gene family expression patterns in embryonic and adult biomineralized tissues of the sea urchin, Strongylocentrotus purpuratus.

PubMed

Killian, Christopher E; Croker, Lindsay; Wilt, Fred H

2010-01-01

The SpSM30 gene family of the sea urchin, Strongylocentrotus purpuratus, is comprised of six members, designated SpSM30A through SpSM30F (Livingston et al., 2006). The SpSM30 proteins are found uniquely in embryonic and adult mineralized tissues of the sea urchin. Previous studies have revealed that SpSM30 proteins are occluded within the embryonic endoskeleton and adult mineralized tissues (Killian and Wilt, 1996; Mann et al., 2008a,b; Urry et al., 2000). Furthermore, some of the SpSM30 proteins are among the most abundant of the approximately four-dozen integral matrix proteins of the larval spicule (Killian and Wilt, 1996). The amino acid sequence, protein domain architecture, and contiguity within the genome strongly support the supposition that the six genes constitute a gene family. Reverse transcription-polymerase chain reaction (RT-PCR) is used in the present study to describe the time course of expression of the family members during embryonic development, and their expression in adult tissues. SpSM30A, B, C and E are expressed, albeit at different levels, during overt spicule deposition in the embryo with some differences in the precise timing of expression. SpSM30D is not expressed in the embryo, and SpSM30F is expressed transiently and at low levels just prior to overt spicule formation. Whole mount in situ hybridization studies show that SpSM30A, B, C, and E are expressed exclusively in primary mesenchyme (PMC) cells and their descendants. In addition, tissue fractionation studies indicate that SpSM30F expression is highly enriched in PMCs. Each adult tissue examined expresses a different cohort of the SpSM30 family members at varying levels: SpSM30A mRNA is not expressed in adult tissues. Its expression is limited to the embryo. Conversely, SpSM30D mRNA is not expressed in the embryo, but is expressed in adult spines and teeth. SpSM30B and SpSM30C are expressed at modest levels in all mineralized adult tissues; SpSM30E is expressed highly in tooth and test; and SpSM30F is expressed in spine and at low levels in the other adult tissues except the test. Relative levels of expression of the several family members in these different tissues vary widely. It is likely SpSM30 proteins play a vital, but still unknown, role in biomineralization of these tissues during development. Copyright 2010 Elsevier B.V. All rights reserved.

Differential Responses to Wnt and PCP Disruption Predict Expression and Developmental Function of Conserved and Novel Genes in a Cnidarian

PubMed Central

Lapébie, Pascal; Ruggiero, Antonella; Barreau, Carine; Chevalier, Sandra; Chang, Patrick; Dru, Philippe; Houliston, Evelyn; Momose, Tsuyoshi

2014-01-01

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at “oral” and “aboral” poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes. PMID:25233086

Differential responses to Wnt and PCP disruption predict expression and developmental function of conserved and novel genes in a cnidarian.

PubMed

Lapébie, Pascal; Ruggiero, Antonella; Barreau, Carine; Chevalier, Sandra; Chang, Patrick; Dru, Philippe; Houliston, Evelyn; Momose, Tsuyoshi

2014-09-01

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at "oral" and "aboral" poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes.

The function and evolution of Msx genes: pointers and paradoxes.

PubMed

Davidson, D

1995-10-01

The Msx genes of vertebrates comprise a small family of chromosomally unlinked homeobox-containing genes related to the Drosophila gene muscle-segment homeobox (msh). Despite their ancient pedigree, the Msx genes are expressed in a range of vertebrate-specific tissues, including neural crest, cranial sensory placodes, bone and teeth. They are active in numerous systems, which have been used as models to study pattern formation and tissue interaction, and are, therefore, attracting a growing interest among developmental biologists. But beyond their presumed role as transcription factors, we do not know what their functions are in the cell or the embryo. Here, I review recent evidence that is beginning to address this problem and might eventually increase our understanding of how the vertebrate embryo has evolved.

Regulation of Msx genes by a Bmp gradient is essential for neural crest specification.

PubMed

Tribulo, Celeste; Aybar, Manuel J; Nguyen, Vu H; Mullins, Mary C; Mayor, Roberto

2003-12-01

There is evidence in Xenopus and zebrafish embryos that the neural crest/neural folds are specified at the border of the neural plate by a precise threshold concentration of a Bmp gradient. In order to understand the molecular mechanism by which a gradient of Bmp is able to specify the neural crest, we analyzed how the expression of Bmp targets, the Msx genes, is regulated and the role that Msx genes has in neural crest specification. As Msx genes are directly downstream of Bmp, we analyzed Msx gene expression after experimental modification in the level of Bmp activity by grafting a bead soaked with noggin into Xenopus embryos, by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and also through Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, we show that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. Our results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region. In addition, we have analyzed the role that msx1 plays on neural crest specification. As msx1 has a role in dorsoventral pattering, we have carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. We show that msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. It has been previously shown that snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and snail/slug we performed several rescue experiments using dominant negatives for these genes. The rescuing activity by snail and slug on neural crest development of the msx1 dominant negative, together with the inability of msx1 to rescue the dominant negatives of slug and snail strongly argue that msx1 is upstream of snail and slug in the genetic cascade that specifies the neural crest in the ectoderm. We propose a model where a gradient of Bmp activity specifies the expression of Msx genes in the neural folds, and that this expression is essential for the early specification of the neural crest.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm

PubMed Central

Elwell, Jennifer A.; Lovato, TyAnna L.; Adams, Melanie M.; Baca, Erica M.; Lee, Thai; Cripps, Richard M.

2015-01-01

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arise through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist expression in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. PMID:25704510

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.

PubMed

Damle, Sagar; Hanser, Bridget; Davidson, Eric H; Fraser, Scott E

2006-11-15

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.

Cloning and pattern of expression of the shiro-uo vasa gene during embryogenesis and its roles in PGC development.

PubMed

Miyake, Akimitsu; Saito, Taiju; Kashiwagi, Noboru; Ando, Daisuke; Yamamoto, Akitsugu; Suzuki, Tohru; Nakatsuji, Norio; Nakatsuji, Takako

2006-01-01

The vasa genes are expressed in the germ cell lineage in many organisms, but their expression patterns show large variations. Recent studies suggest that vasa transcripts are involved in germ cell lineage development. In this paper, we isolated the vasa cDNA clone from a teleost, shiro-uo, Leucopsarion petersii and examined its expression pattern during embryogenesis. Then, we examined the functional significance of vasa mRNA during the formation of primordial germ cells (PGCs). The amino acid sequence of shiro-uo VASA is 61.1% identical to that of zebrafish. In whole-mount in situ hybridization, vasa transcripts appeared at the 4- and 8-cell stages as four spots at both ends of two cleavage planes between the lower tier of blastomeres and the yolk cell mass. At the 16-cell stage, eight spots were observed. After the blastula stage, shiro-uo vasa transcripts showed similar localization as in the zebrafish. Ultrastructural analysis of 4-cell stage embryos revealed the presence of a subcellular organelle that resembled 'nuage' in the germ cell lineage observed in the embryos of various organisms. We carried out micromanipulation of 4- or 8-cell stage embryos to remove the vasa mRNA-containing spots and then measured the number of the vasa-expressing PGCs in the genital ridge of the manipulated embryos. The numbers decreased when all of the four spots were removed, indicating that the vasa-containing spots at early cleavage stages have important functions in the development of PGCs.

TBBPA induces developmental toxicity, oxidative stress, and apoptosis in embryos and zebrafish larvae (Danio rerio).

PubMed

Wu, Shengmin; Ji, Guixiang; Liu, Jining; Zhang, Shenghu; Gong, Yang; Shi, Lili

2016-10-01

Tetrabromobisphenol A (TBBPA) is currently one of the most frequently used brominated flame retardants and can be considered as a high production volume chemical. In this study, zebrafish embryos and larvae served as a biological model to evaluate TBBPA-induced developmental toxicity, oxidative stress, oxidant-associated gene expression, and cell apoptosis. Abnormalities, including hyperemia and pericardial edema, were induced in zebrafish larvae. The results showed that toxicity endpoints such as hatching rate, survival rate, malformation rate, and growth rate had a significant dose-response relationship with TBBPA. Further studies revealed that TBBPA did not alter the enzyme activities of Copper/Zinc Superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathioneperoxidase (GPx) at 0.10 mg/L, but decreased activities following exposure to 0.40, 0.70, and 1.00 mg/L. Despite the significantly decreased gene expression of Cu/Zn-SOD, CAT, and GPx1a in the 1.00 mg/L treatment group, other treatments (0.10, 0.40, 0.70 mg/L) did not alter gene expression. Moreover, Acridine orange staining results showed that apoptotic cells mainly accumulated in the brain, heart, and tail, indicating possible TBBPA-induced brain, cardiac, and blood circulation system impairment in zebrafish embryos and larvae. Histological analysis also showed evidence of obvious heart impairment in TBBPA-treated groups. This study provides new evidence on the developmental toxicity, oxidative stress, and apoptosis of embryos and zebrafish larvae, which is important for the evaluation of environmental toxicity and chemical risk. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1241-1249, 2016. © 2015 Wiley Periodicals, Inc.

Subtle Changes in Motif Positioning Cause Tissue-Specific Effects on Robustness of an Enhancer's Activity

PubMed Central

Erceg, Jelena; Saunders, Timothy E.; Girardot, Charles; Devos, Damien P.; Hufnagel, Lars; Furlong, Eileen E. M.

2014-01-01

Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, we are limited in our ability to relate information on motif occupancy to function from sequence alone. Here, we engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, we were able to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood. PMID:24391522

Biallelic expression of the L-arginine:glycine amidinotransferase gene with different methylation status between male and female primordial germ cells in chickens.

PubMed

Jang, H J; Lee, M O; Kim, S; Kim, T H; Kim, S K; Song, G; Womack, J E; Han, J Y

2013-03-01

The basic functions of DNA methylation include in gene silencing by methylation of specific gene promoters, defense of the host genome from retrovirus, and transcriptional suppression of transgenes. In addition, genomic imprinting, by which certain genes are expressed in a parent-of-origin-specific manner, has been observed in a wide range of plants and animals and has been associated with differential methylation. However, imprinting phenomena of DNA methylation effects have not been revealed in chickens. To analyze whether genomic imprinting occurs in chickens, methyl-DNA immunoprecipitation array analysis was applied across the entire genome of germ cells in early chick embryos. A differentially methylated region (DMR) was detected in the eighth intron of the l-arginine:glycine amidinotransferase (GATM) gene. When the DMR in GATM was analyzed by bisulfite sequencing, the methylation in male primordial germ cells (PGC) of 6-d-old embryos was higher than that in female PGC (57.5 vs. 35.0%). At 8 d, the DMR methylation of GATM in male PGC was 3.7-fold higher than that in female PGC (65.0 vs. 17.5%). Subsequently, to investigate mono- or biallelic expression of the GATM gene during embryo development, we found 2 indel sequences (GTTTAATGC and CAAAAA) within the GATM 3'-untranslated region in Korean Oge (KO) and White Leghorn (WL) chickens. When individual WL and KO chickens were genotyped for indel sequences, 3 allele combinations (homozygous insertion, homozygous deletion, and heterozygotes) were detected in both breeds using a gel shift assay and high-resolution melt assay. The deletion allele was predominant in KO, whereas the insertion allele was predominant in WL. Heterozygous animals were evenly distributed in both breeds (P < 0.01). Despite the different methylation status between male and female PGC, the GATM gene conclusively displayed biallelic expression in PGC as well as somatic embryonic, extraembryonic, and adult chicken tissues.

Gene Expression in Pre-MBT Embryos and Activation of Maternally-Inherited Program of Apoptosis to be Executed at around MBT as a Fail-Safe Mechanism in Xenopus Early Embryogenesis

PubMed Central

Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Uchiyama, Hiroaki; Kuroyanagi, Shinsaku; Takai, Jun-Ichi; Takahashi, Senji; Kajitani, Masayuki; Kaito, Chikara; Sekimizu, Kazuhisa; Takayama, Eiji; Igarashi, Kazuei; Hara, Hiroshi

2008-01-01

S-adenosylmethionine decarboxylase (SAMDC) is an enzyme which converts S-adenosylmethione (SAM), a methyl donor, to decarboxylated SAM (dcSAM), an aminopropyl donor for polyamine biosynthesis. In our studies on gene expression control in Xenopus early embryogenesis, we cloned the mRNA for Xenopus SAMDC, and overexpressed the enzyme by microinjecting its mRNA into Xenopus fertilized eggs. In the mRNA-injected embryos, the level of SAMDC was enormously increased, the SAM was exhausted, and protein synthesis was greatly inhibited, but cellular polyamine content did not change appreciably. SAMDC-overexpressed embryos cleaved and developed normally up to the early blastula stage, but at the midblastula stage, or the stage of midblastula transition (MBT), all the embryos were dissociated into cells, and destroyed due to execution of apoptosis. During cleavage SAMDC-overexpressed embryos transcribed caspase-8 gene, and this was followed by activation of caspase-9. When we overexpressed p53 mRNA in fertilized eggs, similar apoptosis took place at MBT, but in this case, transcription of caspase-8 did not occur, however activation of caspase-9 took place. Apoptosis induced by SAMDC-overexpression was completely suppressed by Bcl-2, whereas apoptosis induced by p53 overexpression or treatments with other toxic agents was only partially rescued. When we injected SAMDC mRNA into only one blastomere of 8- to 32-celled embryos, descendant cells of the mRNA-injected blastomere were segregated into the blastocoel and underwent apoptosis within the blastocoel, although such embryos continued to develop and became tadpoles with various extents of anomaly, reflecting the developmental fate of the eliminated cells. Thus, embryonic cells appear to check themselves at MBT and if physiologically severely-damaged cells occur, they are eliminated from the embryo by activation and execution of the maternally-inherited program of apoptosis. We assume that the apoptosis executed at MBT is a “fail-safe” mechanism of early development to save the embryo from accidental damages that take place during cleavage. PMID:19787085

Transgenesis in fish.

PubMed

Houdebine, L M; Chourrout, D

1991-09-15

Gene transfer into fish embryo is being performed in several species (trout, salmon, carps, tilapia, medaka, goldfish, zebrafish, loach, catfish, etc.). In most cases, pronuclei are not visible and microinjection must be done into the cytoplasm of early embryos. Several million copies of the gene are generally injected. In medaka, transgenesis was attempted by injection of the foreign gene into the nucleus of oocyte. Several reports indicate that the injected DNA was rapidly replicated in the early phase of embryo development, regardless of the origin and the sequence of the foreign DNA. The survival of the injected embryos was reasonably good and a large number reached maturity. The proportion of transgenic animals ranged from 1 to 50% or more, according to species and to experimentators. The reasons for this discrepancy have not been elucidated. In all species, the transgenic animals were mosaic. The copy number of the foreign DNA was different in the various tissues of an animal and a proportion lower than 50% of F1 offsprings received the gene from their parents. This suggests that the foreign DNA was integrated into the fish genome at the two cells stage or later. An examination of the integrated DNA in different cell types of an animal revealed that integration occurred mainly during early development. The transgene was found essentially unrearranged in the fish genome of the founders and offsprings. The transgenes were therefore stably transmitted to progeny in a Mendelian fashion. Southern blot analysis revealed the presence of possible junction fragments and also of minor bands which may result from a rearrangement of the injected DNA. In all species, the integrated DNA appeared mainly as random end-to-end concatemers. In adult trout blood cells, a small proportion of the foreign DNA was maintained in the form of non-integrated concatemers, as judged by the existence of end fragments. The transgenes were generally only poorly expressed. The majority of the injected gene constructs contained essentially mammalian or higher vertebrates sequences. The comparison of the expression efficiency of these constructs in transfected fish and mammalian cells indicates that some of the mammalian DNA sequences are most efficiently understood by the fish cell machinery. Chloramphenicol acetyl transferase gene under the control of promoters from Rous sarcoma virus, and human cytomegalovirus, was expressed in several tissues of transgenic fish. Chicken delta-crystallin gene was expressed in several tissues of transgenic fish.(ABSTRACT TRUNCATED AT 400 WORDS)

Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes.

PubMed

Biedler, James K; Tu, Zhijian

2010-07-08

The maternal zygotic transition marks the time at which transcription from the zygotic genome is initiated and a subset of maternal RNAs are progressively degraded in the developing embryo. A number of early zygotic genes have been identified in Drosophila melanogaster and comparisons to sequenced mosquito genomes suggest that some of these early zygotic genes such as bottleneck are fast-evolving or subject to turnover in dipteran insects. One objective of this study is to identify early zygotic genes from the yellow fever mosquito Aedes aegypti to study their evolution. We are also interested in obtaining early zygotic promoters that will direct transgene expression in the early embryo as part of a Medea gene drive system. Two novel early zygotic kinesin light chain genes we call AaKLC2.1 and AaKLC2.2 were identified by transcriptome sequencing of Aedes aegypti embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These AaKLC2 genes have a paralogous gene (AaKLC1) in Ae. aegypti. Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus. In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae. There is only one KLC gene in D. melanogaster and other sequenced holometabolous insects that appears to be similar to AaKLC1. Unlike AaKLC2, AaKLC1 is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the An. gambiae and D. melanogaster KLC genes. Phylogenetic inference also suggests that AaKLC2 genes and their likely C. quinquefasciatus ortholog are fast-evolving genes relative to the highly conserved AaKLC1-like paralogs. Embryonic injection of a luciferase reporter under the control of a 1 kb fragment upstream of the AaKLC2.1 start codon shows promoter activity at least as early as 3 hours in the developing Ae. aegypti embryo. The AaKLC2.1 promoter activity reached ~1600 fold over the negative control at 5 hr after egg deposition. Transcriptome profiling by use of high throughput sequencing technologies has proven to be a valuable method for the identification and discovery of early and transient zygotic genes. The evolutionary investigation of the KLC gene family reveals that duplication is a source for the evolution of new genes that play a role in the dynamic process of early embryonic development. AaKLC2.1 may provide a promoter for early zygotic-specific transgene expression, which is a key component of the Medea gene drive system.

Embryonal carcinoma antigen and the T/t locus of the mouse.

PubMed Central

Kemler, R; Babinet, C; Condamine, H; Gachelin, G; Guenet, J L; Jacob, F

1976-01-01

The presence of the F9 antigen and of four other antigens related to the T/t locus of the mouse was investigated by immunofluorescence on preimplantation embryos. In morulae heterozygous for any of these t haplotypes, both the appropriate t antigen and the F9 antigen are expressed. The F9 antigen segregates among the progeny of crosses producing embryos homozygous for some (tw32 and tw5) but not for other haplotypes. It is concluded that (i) whatever the time of action of a t haplotype, its corresponding antigen is expressed during cleavage and (ii) the F9 antigen is specified by a gene(s) in the region of the T/t locus. Images PMID:1069295

Quantitative assessment of Hox complex expression in the indirect development of the polychaete annelid Chaetopterus sp

NASA Technical Reports Server (NTRS)

Peterson, K. J.; Irvine, S. Q.; Cameron, R. A.; Davidson, E. H.

2000-01-01

A prediction from the set-aside theory of bilaterian origins is that pattern formation processes such as those controlled by the Hox cluster genes are required specifically for adult body plan formation. This prediction can be tested in animals that use maximal indirect development, in which the embryonic formation of the larva and the postembryonic formation of the adult body plan are temporally and spatially distinct. To this end, we quantitatively measured the amount of transcripts for five Hox genes in embryos of a lophotrochozoan, the polychaete annelid Chaetopterus sp. The polychaete Hox complex is shown not to be expressed during embryogenesis, but transcripts of all measured Hox complex genes are detected at significant levels during the initial stages of adult body plan formation. Temporal colinearity in the sequence of their activation is observed, so that activation follows the 3'-5' arrangement of the genes. Moreover, Hox gene expression is spatially localized to the region of teloblastic set-aside cells of the later-stage embryos. This study shows that an indirectly developing lophotrochozoan shares with an indirectly developing deuterostome, the sea urchin, a common mode of Hox complex utilization: construction of the larva, whether a trochophore or dipleurula, does not involve Hox cluster expression, but in both forms the complex is expressed in the set-aside cells from which the adult body plan derives.

The Chromatin Regulator Brpf1 Regulates Embryo Development and Cell Proliferation*

PubMed Central

You, Linya; Yan, Kezhi; Zou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

2015-01-01

With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs. PMID:25773539

The role of Sox6 in zebrafish muscle fiber type specification.

PubMed

Jackson, Harriet E; Ono, Yosuke; Wang, Xingang; Elworthy, Stone; Cunliffe, Vincent T; Ingham, Philip W

2015-01-01

The transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification. The GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres. Ectopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine. The Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation in the zebrafish, a role similar to that ascribed to its murine ortholog.

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos.

PubMed

Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei

2010-02-01

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

PubMed

You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

2010-02-01

The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P < 0.05) level in SCNT oocytes that were treated post-fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos. (c) 2009 Wiley-Liss, Inc.

Differential expression of Toll-like receptor pathway genes in chicken embryo fibroblasts from chickens resistant and susceptible to Marek’s disease

USDA-ARS?s Scientific Manuscript database

The Toll-like receptor (TLR) signaling pathway is one of the innate immune defense mechanisms against pathogens in vertebrates and invertebrates. However, the role of TLR in non-MHC genetic resistance or susceptibility to Marek’s disease (MD) in the chicken is yet to be elucidated. Chicken embryo fi...

PITX2 and NODAL expression during axis formation in the early rabbit embryo.

PubMed

Plöger, Ruben; Viebahn, Christoph

2018-04-26

Attaining molecular and morphological axial polarity during gastrulation is a fundamental early requirement for normal development of the embryo. In mammals, the first morphological sign of the anterior-posterior axis appears anteriorly in the form of the anterior marginal crescent (or anterior visceral endoderm) while in the avian the first such sign is the Koller's sickle at the posterior pole of the embryonic disc. Despite this inverse mode of axis formation many genes and molecular pathways involved in various steps of this process seem to be evolutionary conserved amongst amniotes, the nodal gene being a well-known example with its functional involvement prior and during gastrulation. The pitx2 gene, however, is a new candidate described in the chick as an early marker for anterior-posterior polarity and as regulator of axis formation including twinning. To find out whether pitx2 has retained its inductive and early marker function during the evolution of mammals, this study analyzes pitx2 and nodal expression at parallel stages during formation of the anterior-posterior polarity in the early rabbit embryo using whole-mount in situ hybridization and serial light-microscopical sections. At a late pre-gastrulation stage a localized reduction of nodal expression presages the position of the anterior pole of the embryonic disc and thus serves as the earliest molecular marker of anterior-posterior polarity known so far. pitx2 is expressed in a polarized manner in the anterior marginal crescent and in the posterior half of the embryonic disc during further development only while nodal expression in the anterior segment of the posterior pitx2 expression domain helps to define the so-called anterior streak domain (ASD), a novel progenitor region of the anterior half of the primitive streak. The expression patterns of both genes thus serve as signs of a conserved involvement in early axis formation in amniotes and, possibly, in twinning in mammals as well. Copyright © 2018 Elsevier GmbH. All rights reserved.

Heat shock during early somitogenesis induces caudal vertebral column defects in Atlantic salmon (Salmo salar).

PubMed

Wargelius, Anna; Fjelldal, Per Gunnar; Hansen, Tom

2005-07-01

In several terrestrial vertebrates, heat shock (HS) during somitogenesis causes vertebral deformities. To determine if vertebral deformities can occur due to sudden temperature changes during early development in fish, Atlantic salmon embryos were HS treated during somitogenesis. Ten months later these individuals displayed a high prevalence of caudal vertebral column condensations (27-34%). The defects were located caudally of the abdominal cavity, displaying an even distribution in this region independent of time of HS. To determine if HS disturbed vertebral development during somitogenesis, two genes coding for markers of skeletal development were identified, namely, the secreted protein Shh (Sashh) and the transcription factor Twist (Satwist). These proteins are involved in the proliferation and specification of presumptive skeletal cells (sclerotome) in vertebrates. The spatial expression pattern of sashh and satwist in salmon indicated a functional conservation of these proteins. Furthermore, HS embryos displayed expressional disturbance in both sashh and satwist, indicating an effect of HS on sclerotomal cell patterning. However, the HS-protecting ability in embryos seems to be individually regulated because reduction in gene expression was not detected at all stages; in addition, HS did not induce somitic disturbance and vertebral deformity in all embryos.

Cloning, expression pattern, and potential role of apoptosis inhibitor 5 in the termination of embryonic diapause and early embryo development of Artemia sinica.

PubMed

Zhang, Shuang; Yao, Feng; Jing, Ting; Zhang, Mengchen; Zhao, Wei; Zou, Xiangyang; Sui, Linlin; Hou, Lin

2017-09-10

During the embryonic development of Artemia sinica, the diapause phenomenon can be induced by high salinity or low temperature conditions. The diapause embryo at the gastrula stage is maintained under the threat of apoptosis to guarantee the embryo's normal development. In this process, apoptosis inhibitor proteins play vital roles in protecting embryos against apoptosis. Apoptosis inhibitor5 (API5) plays a pivotal role in regulating the cell cycle and preventing programmed cell death after growth factor starvation. In the present study, we cloned the full-length cDNA representing the api5 gene from A. sinica (As-api5), which encodes a 372-amino acid protein. In situ hybridization experiments revealed that As-api5 expression is not tissue or organ specific. Quantitative real-time PCR analyses of the developmental expression of As-api5 showed that it reached its highest level at 10h, after which its expression decreased. High salinity and low temperature treatments increased the expression of As-api5. Western blotting was used to assess the abundance of As-API5 and related proteins (As-CyclinA, As-CyclinE, As-E2F1, As-CDK2, As-APAF1, and As-Caspase9). Downregulation of As-api5 expression using a short interfering RNA resulted in increased mortality and embryo malformation of A. sinica. Taken together, the results indicated that API5 plays a crucial role in embryonic diapause termination and early embryo development of A. sinica. Copyright © 2017. Published by Elsevier B.V.

Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species.

PubMed

Lagutina, Irina; Zakhartchenko, Valeri; Fulka, Helena; Colleoni, Silvia; Wolf, Eckhard; Fulka, Josef; Lazzari, Giovanna; Galli, Cesare

2011-04-01

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

Arsenic Exposure to Killifish During Embryogenesis Alters Muscle Development

PubMed Central

Gaworecki, Kristen M.; Chapman, Robert W.; Neely, Marion G.; D’Amico, Angela R.; Bain, Lisa J.

2012-01-01

Epidemiological studies have correlated arsenic exposure in drinking water with adverse developmental outcomes such as stillbirths, spontaneous abortions, neonatal mortality, low birth weight, delays in the use of musculature, and altered locomotor activity. Killifish (Fundulus heteroclitus) were used as a model to help to determine the mechanisms by which arsenic could impact development. Killifish embryos were exposed to three different sodium arsenite concentrations and were collected at 32 h post-fertilization (hpf), 42 hpf, 168 hpf, or < 24 h post-hatch. A killifish oligo microarray was developed and used to examine gene expression changes between control and 25-ppm arsenic-exposed hatchlings. With artificial neural network analysis of the transcriptomic data, accurate prediction of each group (control vs. arsenic-exposed embryos) was obtained using a small subset of only 332 genes. The genes differentially expressed include those involved in cell cycle, development, ubiquitination, and the musculature. Several of the genes involved in cell cycle regulation and muscle formation, such as fetuin B, cyclin D–binding protein 1, and CapZ, were differentially expressed in the embryos in a time- and dose-dependent manner. Examining muscle structure in the hatchlings showed that arsenic exposure during embryogenesis significantly reduces the average muscle fiber size, which is coupled with a significant 2.1- and 1.6-fold upregulation of skeletal myosin light and heavy chains, respectively. These findings collectively indicate that arsenic exposure during embryogenesis can initiate molecular changes that appear to lead to aberrant muscle formation. PMID:22058191

Cornelia de Lange Syndrome: NIPBL haploinsufficiency downregulates canonical Wnt pathway in zebrafish embryos and patients fibroblasts.

PubMed

Pistocchi, A; Fazio, G; Cereda, A; Ferrari, L; Bettini, L R; Messina, G; Cotelli, F; Biondi, A; Selicorni, A; Massa, V

2013-10-17

Cornelia de Lange Syndrome is a severe genetic disorder characterized by malformations affecting multiple systems, with a common feature of severe mental retardation. Genetic variants within four genes (NIPBL (Nipped-B-like), SMC1A, SMC3, and HDAC8) are believed to be responsible for the majority of cases; all these genes encode proteins that are part of the 'cohesin complex'. Cohesins exhibit two temporally separated major roles in cells: one controlling the cell cycle and the other involved in regulating the gene expression. The present study focuses on the role of the zebrafish nipblb paralog during neural development, examining its expression in the central nervous system, and analyzing the consequences of nipblb loss of function. Neural development was impaired by the knockdown of nipblb in zebrafish. nipblb-loss-of-function embryos presented with increased apoptosis in the developing neural tissues, downregulation of canonical Wnt pathway genes, and subsequent decreased Cyclin D1 (Ccnd1) levels. Importantly, the same pattern of canonical WNT pathway and CCND1 downregulation was observed in NIPBL-mutated patient-specific fibroblasts. Finally, chemical activation of the pathway in nipblb-loss-of-function embryos rescued the adverse phenotype and restored the physiological levels of cell death.

Auxin Import and Local Auxin Biosynthesis Are Required for Mitotic Divisions, Cell Expansion and Cell Specification during Female Gametophyte Development in Arabidopsis thaliana

PubMed Central

Panoli, Aneesh; Martin, Maria Victoria; Alandete-Saez, Monica; Simon, Marissa; Neff, Christina; Swarup, Ranjan; Bellido, Andrés; Yuan, Li; Pagnussat, Gabriela C.; Sundaresan, Venkatesan

2015-01-01

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development. PMID:25970627

Auxin Import and Local Auxin Biosynthesis Are Required for Mitotic Divisions, Cell Expansion and Cell Specification during Female Gametophyte Development in Arabidopsis thaliana.

PubMed

Panoli, Aneesh; Martin, Maria Victoria; Alandete-Saez, Monica; Simon, Marissa; Neff, Christina; Swarup, Ranjan; Bellido, Andrés; Yuan, Li; Pagnussat, Gabriela C; Sundaresan, Venkatesan

2015-01-01

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development.

Do rice suspension-cultured cells treated with abscisic acid mimic developing seeds?

PubMed

Matsuno, Koya; Fujimura, Tatsuhito

2015-08-01

Starch synthesis is activated in the endosperm during seed development and also in rice suspension cells cultured with abscisic acid. In the anticipation that the mechanisms of starch synthesis are similar between the endosperm and the suspension cells cultured with abscisic acid, expression of genes involved in starch synthesis was evaluated in the suspension cells after abscisic acid treatment. However, it was found that the regulatory mechanism of starch synthesis in the suspension cells cultured with abscisic acid was different from that in developing seeds. Expression analyses of genes involved in oil bodies, which accumulate in the embryo and aleurone layer, and seed storage proteins, which accumulate mainly in the endosperm, showed that the former were activated in the suspension cells cultured with abscisic acid, but the latter were not. Master regulators for embryogenesis, OsVP1 (homologue of AtABI3) and OsLFL1 (homologue of AtFUS3 or AtLFL2), were expressed in the suspension cells at levels comparable to those in the embryo. From these results, it is suggested that interactions between regulators and abscisic acid control the synthesis of phytic acid and oil bodies in the cultured cells and embryo. We suggest that the system of suspension cells cultured with abscisic acid helps to reveal the mechanisms of phytic acid and oil body synthesis in embryo.

Nano-nutrition of chicken embryos. The effect of in ovo administration of diamond nanoparticles and L-glutamine on molecular responses in chicken embryo pectoral muscles.

PubMed

Grodzik, Marta; Sawosz, Filip; Sawosz, Ewa; Hotowy, Anna; Wierzbicki, Mateusz; Kutwin, Marta; Jaworski, Sławomir; Chwalibog, André

2013-11-20

It has been demonstrated that the content of certain amino acids in eggs is not sufficient to fully support embryonic development. One possibility to supply the embryo with extra nutrients and energy is in ovo administration of nutrients. Nanoparticles of diamond are highly biocompatible non-toxic carbonic structures, and we hypothesized that bio-complexes of diamond nanoparticles with L-glutamine may affect molecular responses in breast muscle. The objective of the investigation was to evaluate the effect of diamond nanoparticle (ND) and L-glutamine (Gln) on expression of growth and differentiation factors of chicken embryo pectoral muscles. ND, Gln, and Gln/ND solutions (50 mg/L) were injected into fertilized broiler chicken eggs at the beginning of embryogenesis. Muscle tissue was dissected at day 20 of incubation and analysed for gene expression of FGF2, VEGF-A, and MyoD1. ND and especially Gln/ND up-regulated expression of genes related to muscle cell proliferation (FGF2) and differentiation (MyoD1). Furthermore, the ratio between FGF2 and MyoD1 was highest in the Gln/ND group. At the end of embryogenesis, Gln/ND enhanced both proliferation and differentiation of pectoral muscle cells and differentiation dominated over proliferation. These preliminary results suggest that the bio-complex of glutamine and diamond nanoparticles may accelerate growth and maturation of muscle cells.

Zebrafish aussicht mutant embryos exhibit widespread overexpression of ace (fgf8) and coincident defects in CNS development.

PubMed

Heisenberg, C P; Brennan, C; Wilson, S W

1999-05-01

During the development of the zebrafish nervous system both noi, a zebrafish pax2 homolog, and ace, a zebrafish fgf8 homolog, are required for development of the midbrain and cerebellum. Here we describe a dominant mutation, aussicht (aus), in which the expression of noi and ace is upregulated. In aus mutant embryos, ace is upregulated at many sites in the embryo, while noi expression is only upregulated in regions of the forebrain and midbrain which also express ace. Subsequent to the alterations in noi and ace expression, aus mutants exhibit defects in the differentiation of the forebrain, midbrain and eyes. Within the forebrain, the formation of the anterior and postoptic commissures is delayed and the expression of markers within the pretectal area is reduced. Within the midbrain, En and wnt1 expression is expanded. In heterozygous aus embryos, there is ectopic outgrowth of neural retina in the temporal half of the eyes, whereas in putative homozygous aus embryos, the ventral retina is reduced and the pigmented retinal epithelium is expanded towards the midline. The observation that aus mutant embryos exhibit widespread upregulation of ace raised the possibility that aus might represent an allele of the ace gene itself. However, by crossing carriers for both aus and ace, we were able to generate homozygous ace mutant embryos that also exhibited the aus phenotype. This indicated that aus is not tightly linked to ace and is unlikely to be a mutation directly affecting the ace locus. However, increased Ace activity may underly many aspects of the aus phenotype and we show that the upregulation of noi in the forebrain of aus mutants is partially dependent upon functional Ace activity. Conversely, increased ace expression in the forebrain of aus mutants is not dependent upon functional Noi activity. We conclude that aus represents a mutation involving a locus normally required for the regulation of ace expression during embryogenesis.

Gene regulation in amphioxus: An insight from transgenic studies in amphioxus and vertebrates.

PubMed

Kozmikova, Iryna; Kozmik, Zbynek

2015-12-01

Cephalochordates, commonly known as amphioxus or lancelets, are the most basal subphylum of chordates. Cephalochordates are thus key to understanding the origin of vertebrates and molecular mechanisms underlying vertebrate evolution. The evolution of developmental control mechanisms during invertebrate-to-vertebrate transition involved not only gene duplication events, but also specific changes in spatial and temporal expression of many genes. To get insight into the spatiotemporal regulation of gene expression during invertebrate-to-vertebrate transition, functional studies of amphioxus gene regulatory elements are highly warranted. Here, we review transgenic studies performed in amphioxus and vertebrates using promoters and enhancers derived from the genome of Branchiostoma floridae. We describe the current methods of transgenesis in amphioxus, provide evidence of Tol2 transposon-generated transgenic embryos of Branchiostoma lanceolatum and discuss possible future directions. We envision that comparative transgenic analysis of gene regulatory sequences in the context of amphioxus and vertebrate embryos will likely provide an important mechanistic insight into the evolution of vertebrate body plan. Copyright © 2015 Elsevier B.V. All rights reserved.

Coordinated activation of the secretory pathway during notochord formation in the Xenopus embryo.

PubMed

Tanegashima, Kosuke; Zhao, Hui; Rebbert, Martha L; Dawid, Igor B

2009-11-01

We compared the transcriptome in the developing notochord of Xenopus laevis embryos with that of other embryonic regions. A coordinated and intense activation of a large set of secretory pathway genes was observed in the notochord, but not in notochord precursors in the axial mesoderm at early gastrula stage. The genes encoding Xbp1 and Creb3l2 were also activated in the notochord. These two transcription factors are implicated in the activation of secretory pathway genes during the unfolded protein response, where cells react to the stress of a build-up of unfolded proteins in their endoplasmic reticulum. Xbp1 and Creb3l2 are differentially expressed but not differentially activated in the notochord. Reduction of expression of Xbp1 or Creb3l2 by injection of antisense morpholinos led to strong deficits in notochord but not somitic muscle development. In addition, the expression of some, but not all, genes encoding secretory proteins was inhibited by injection of xbp1 morpholinos. Furthermore, expression of activated forms of Xbp1 or Creb3l2 in animal explants could activate a similar subset of secretory pathway genes. We conclude that coordinated activation of a battery of secretory pathway genes mediated by Xbp1 and Creb/ATF factors is a characteristic and necessary feature of notochord formation.

Cloning and expression patterns of two Smad genes during embryonic development and shell formation of the Pacific oyster Crassostrea gigas

NASA Astrophysics Data System (ADS)

Liu, Gang; Huan, Pin; Liu, Baozhong

2014-11-01

Increasing evidence indicates that transforming growth factor β (TGF-β) signaling pathways play many important roles in the early development of mollusks. However, limited information is known concerning their detailed mechanisms. Here, we describe the identification, cloning and characterization of two Smad genes, the key components of TGF-β signaling pathways, from the Pacific oyster Crassostrea gigas. Sequence analysis of the two genes, designated as cgi-smad1/ 5/ 8 and cgi-smad4, revealed conserved functional characteristics. The two genes were widely expressed in embryos and larvae, suggesting multiple roles in the early development of C. gigas. The mRNA of the two genes aggregated in the D quadrant and cgi-smad4 was highly expressed on the dorsal side of the gastrula, indicating that TGF-β signaling pathways may be involved in dorsoventral patterning in C. gigas. Furthermore, high expression levels of the two genes in the shell fields of embryos at different stages suggested important roles for TGF-β signaling pathways in particular phases of shell development, including the formation of the initial shell field and the biomineralization of larval shells. The results of this study provide fundamental support for elucidating how TGF-β signaling pathways participate in the early development of bivalve mollusks, and suggest that further work is warranted to this end.

MRG-1, an autosome-associated protein, silences X-linked genes and protects germline immortality in Caenorhabditis elegans

PubMed Central

Takasaki, Teruaki; Liu, Zheng; Habara, Yasuaki; Nishiwaki, Kiyoji; Nakayama, Jun-ichi; Inoue, Kunio; Sakamoto, Hiroshi; Strome, Susan

2008-01-01

MRG15, a mammalian protein related to the mortality factor MORF4, is required for cell proliferation and embryo survival. Our genetic analysis has revealed that the Caenorhabditis elegans ortholog MRG-1 serves similar roles. Maternal MRG-1 promotes embryo survival and is required for proliferation and immortality of the primordial germ cells (PGCs). As expected of a chromodomain protein, MRG-1 associates with chromatin. Unexpectedly, it is concentrated on the autosomes and not detectable on the X chromosomes. This association is not dependent on the autosome-enriched protein MES-4. Focusing on possible roles of MRG-1 in regulating gene expression, we determined that MRG-1 is required to maintain repression in the maternal germ line of transgenes on extrachromosomal arrays, and of several X-linked genes previously shown to depend on MES-4 for repression. MRG-1 is not required for PGCs to acquire transcriptional competence or for the turn-on of expression of several PGC-expressed genes (pgl-1, glh-1, glh-4 and nos-1). By contrast to this result in PGCs, MRG-1 is required for ectopic expression of those germline genes in somatic cells lacking the NuRD complex component MEP-1. We discuss how an autosome-enriched protein might repress genes on the X chromosome, promote PGC proliferation and survival, and influence the germ versus soma distinction. PMID:17215300

Progesterone and norgestrel alter transcriptional expression of genes along the hypothalamic-pituitary-thyroid axis in zebrafish embryos-larvae.

PubMed

Liang, Yan-Qiu; Huang, Guo-Yong; Ying, Guang-Guo; Liu, Shuang-Shuang; Jiang, Yu-Xia; Liu, Shan

2015-01-01

The aim of this study was to investigate the effects of progestins on the hypothalamic-pituitary-thyroid (HPT) axis in the early stage of zebrafish. Zebrafish embryos were exposed to progesterone (P4) or norgestrel (NGT) at 5, 50 and 100 ng L(-1) for 144 h post fertilization (hpf), and the transcriptional levels of target genes along the hypothalamic-pituitary-thyroid axis were determined daily. The results showed that P4 had only minor effects on the mRNA expression of thyroglobulin (Tg), iodothyronine deiodinase type Ι (Dio1) and thyroid hormone receptor β (Thrb) genes. Similarly, the effects of NGT on transcripts of thyrotropin-releasing hormone (Trh), Dio1, iodothyronine deiodinase type II (Dio2) and thyroid hormone receptor α (Thra) genes were generally low. In addition, NGT resulted in some alterations of Tg and Thrb transcripts at different time points. However, a strong induction of Nis mRNA by P4 and NGT was observed in zebrafish embryos-larvae. The overall results showed that besides Nis no effects on the hypothalamic-pituitary-thyroid (HPT) axis are observed following exposure to P4 and NGT, which imply that both P4 and NGT have potential effects on the thyroid endocrine system by inducing transcript of Nis gene during the early stage of zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

PubMed

Han, Chengquan; Deng, Ruizhi; Mao, Tingchao; Luo, Yan; Wei, Biao; Meng, Peng; Zhao, Lu; Zhang, Qing; Quan, Fusheng; Liu, Jun; Zhang, Yong

2018-05-23

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency. © 2018 Federation of European Biochemical Societies.

Equivalency of Buffalo (Bubalus Bubalis) Embryonic Stem Cells Derived From Fertilized, Parthenogenetic, and Hand-Made Cloned Embryos

PubMed Central

Muzaffar, Musharifa; Selokar, Naresh L.; Singh, Karn P.; Zandi, Mohammad; Singh, Manoj K.; Shah, Riaz A.; Chauhan, Manmohan S.; Singla, Suresh K.; Palta, Prabhat

2012-01-01

Abstract This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production. PMID:22582863

MOLECULAR AND MORPHOLOGICAL CHANGES IN ZEBRAFISH FOLLOWING TRANSIENT ETHANOL EXPOSURE DURING DEFINED DEVELOPMENTAL STAGES

PubMed Central

Zhang, Chengjin; Frazier, Jared M.; Chen, Hao; Liu, Yao; Lee, Ju-Ahng; Cole, Gregory J.

2014-01-01

Alcohol is a teratogen that has diverse effects on brain and craniofacial development, leading to a constellation of developmental disorders referred to as fetal alcohol spectrum disorder (FASD). The molecular basis of ethanol insult remains poorly understood, as does the relationship between molecular and behavioral changes as a consequence of prenatal ethanol exposure. Zebrafish embryos were exposed to a range of ethanol concentrations (0.5–5.0%) during defined developmental stages, and examined for morphological phenotypes characteristic of FASD. Embryos were also analyzed by in situ hybridization for changes in expression of defined cell markers for neural cell types that are sonic hedgehog-dependent. We show that transient binge-like ethanol exposures during defined developmental stages, such as early gastrulation and early neurulation, result in a range of phenotypes and changes in expression of Shh-dependent genes. The severity of fetal alcohol syndrome (FAS) morphological phenotypes, such as microphthalmia, depends on the embryonic stage and concentration of alcohol exposure, as does diminution of retinal Pax6a or forebrain and hindbrain GAD1 gene expression. We also show that changes in eye and brain morphology correlate with changes in Pax6a and GAD1 gene expression. Our results therefore show that transient binge-like ethanol exposures in zebrafish embryos produce the stereotypical morphological phenotypes of FAS, with the severity of phenotypes depending on the developmental stage and alcohol concentration of exposure. PMID:24929233

Equivalency of buffalo (Bubalus bubalis) embryonic stem cells derived from fertilized, parthenogenetic, and hand-made cloned embryos.

PubMed

Muzaffar, Musharifa; Selokar, Naresh L; Singh, Karn P; Zandi, Mohammad; Singh, Manoj K; Shah, Riaz A; Chauhan, Manmohan S; Singla, Suresh K; Palta, Prabhat; Manik, Radheysham

2012-06-01

This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production.

Interferon-induced TRAIL-independent cell death in DNase II-/- embryos.

PubMed

Kitahara, Yusuke; Kawane, Kohki; Nagata, Shigekazu

2010-09-01

The chromosomal DNA of apoptotic cells and the nuclear DNA expelled from erythroid precursors is cleaved by DNase II in lysosomes after the cells or nuclei are engulfed by macrophages. DNase II(-/-) embryos suffer from lethal anemia due to IFN-beta produced in the macrophages carrying undigested DNA. Here, we show that Type I IFN induced a caspase-dependent cell death in human epithelial cells that were transformed to express a high level of IFN type I receptor. During this death process, a set of genes was strongly activated, one of which encoded TRAIL, a death ligand. A high level of TRAIL mRNA was also found in the fetal liver of the lethally anemic DNase II(-/-) embryos, and a lack of IFN type I receptor in the DNase II(-/-) IFN-IR(-/-) embryos blocked the expression of TRAIL mRNA. However, a null mutation in TRAIL did not rescue the lethal anemia of the DNase II(-/-) embryos, indicating that TRAIL is dispensable for inducing the apoptosis of erythroid cells in DNase II(-/-) embryos, and therefore, that there is a TRAIL-independent mechanism for the IFN-induced apoptosis.

Transcriptome analysis and gene expression profiling of abortive and developing ovules during fruit development in hazelnut.

PubMed

Cheng, Yunqing; Liu, Jianfeng; Zhang, Huidi; Wang, Ju; Zhao, Yixin; Geng, Wanting

2015-01-01

A high ratio of blank fruit in hazelnut (Corylus heterophylla Fisch) is a very common phenomenon that causes serious yield losses in northeast China. The development of blank fruit in the Corylus genus is known to be associated with embryo abortion. However, little is known about the molecular mechanisms responsible for embryo abortion during the nut development stage. Genomic information for C. heterophylla Fisch is not available; therefore, data related to transcriptome and gene expression profiling of developing and abortive ovules are needed. In this study, de novo transcriptome sequencing and RNA-seq analysis were conducted using short-read sequencing technology (Illumina HiSeq 2000). The results of the transcriptome assembly analysis revealed genetic information that was associated with the fruit development stage. Two digital gene expression libraries were constructed, one for a full (normally developing) ovule and one for an empty (abortive) ovule. Transcriptome sequencing and assembly results revealed 55,353 unigenes, including 18,751 clusters and 36,602 singletons. These results were annotated using the public databases NR, NT, Swiss-Prot, KEGG, COG, and GO. Using digital gene expression profiling, gene expression differences in developing and abortive ovules were identified. A total of 1,637 and 715 unigenes were significantly upregulated and downregulated, respectively, in abortive ovules, compared with developing ovules. Quantitative real-time polymerase chain reaction analysis was used in order to verify the differential expression of some genes. The transcriptome and digital gene expression profiling data of normally developing and abortive ovules in hazelnut provide exhaustive information that will improve our understanding of the molecular mechanisms of abortive ovule formation in hazelnut.

Uterine Gene Expression in the Live-Bearing Lizard, Chalcides ocellatus, Reveals Convergence of Squamate Reptile and Mammalian Pregnancy Mechanisms

PubMed Central

Brandley, Matthew C.; Young, Rebecca L.; Warren, Dan L.; Thompson, Michael B.; Wagner, Günter P.

2012-01-01

Although the morphological and physiological changes involved in pregnancy in live-bearing reptiles are well studied, the genetic mechanisms that underlie these changes are not known. We used the viviparous African Ocellated Skink, Chalcides ocellatus, as a model to identify a near complete gene expression profile associated with pregnancy using RNA-Seq analyses of uterine transcriptomes. Pregnancy in C. ocellatus is associated with upregulation of uterine genes involved with metabolism, cell proliferation and death, and cellular transport. Moreover, there are clear parallels between the genetic processes associated with pregnancy in mammals and Chalcides in expression of genes related to tissue remodeling, angiogenesis, immune system regulation, and nutrient provisioning to the embryo. In particular, the pregnant uterine transcriptome is dominated by expression of proteolytic enzymes that we speculate are involved both with remodeling the chorioallantoic placenta and histotrophy in the omphaloplacenta. Elements of the maternal innate immune system are downregulated in the pregnant uterus, indicating a potential mechanism to avoid rejection of the embryo. We found a downregulation of major histocompatability complex loci and estrogen and progesterone receptors in the pregnant uterus. This pattern is similar to mammals but cannot be explained by the mammalian model. The latter finding provides evidence that pregnancy is controlled by different endocrinological mechanisms in mammals and reptiles. Finally, 88% of the identified genes are expressed in both the pregnant and the nonpregnant uterus, and thus, morphological and physiological changes associated with C. ocellatus pregnancy are likely a result of regulation of genes continually expressed in the uterus rather than the initiation of expression of unique genes. PMID:22333490

A Nodal-independent and tissue-intrinsic mechanism controls heart-looping chirality

NASA Astrophysics Data System (ADS)

Noël, Emily S.; Verhoeven, Manon; Lagendijk, Anne Karine; Tessadori, Federico; Smith, Kelly; Choorapoikayil, Suma; den Hertog, Jeroen; Bakkers, Jeroen

2013-11-01

Breaking left-right symmetry in bilateria is a major event during embryo development that is required for asymmetric organ position, directional organ looping and lateralized organ function in the adult. Asymmetric expression of Nodal-related genes is hypothesized to be the driving force behind regulation of organ laterality. Here we identify a Nodal-independent mechanism that drives asymmetric heart looping in zebrafish embryos. In a unique mutant defective for the Nodal-related southpaw gene, preferential dextral looping in the heart is maintained, whereas gut and brain asymmetries are randomized. As genetic and pharmacological inhibition of Nodal signalling does not abolish heart asymmetry, a yet undiscovered mechanism controls heart chirality. This mechanism is tissue intrinsic, as explanted hearts maintain ex vivo retain chiral looping behaviour and require actin polymerization and myosin II activity. We find that Nodal signalling regulates actin gene expression, supporting a model in which Nodal signalling amplifies this tissue-intrinsic mechanism of heart looping.

ACR4, a putative receptor kinase gene of Arabidopsis thaliana, that is expressed in the outer cell layers of embryos and plants, is involved in proper embryogenesis.

PubMed

Tanaka, Hirokazu; Watanabe, Masaru; Watanabe, Daisuke; Tanaka, Toshihiro; Machida, Chiyoko; Machida, Yasunori

2002-04-01

The surfaces of higher plants are characterized by epidermis, which usually consists of a single layer of cells. The epidermis is derived from the outer cell layer of the embryo or protoderm, which arises as a result of periclinal cell division. After seed germination, most of the epidermal cells of the aerial parts of plants are derived from the outer cell layer of the shoot apical meristem (the L1 layer). Thus, knowledge of how the protoderm and/or L1 layer is established is fundamental to understanding the morphogenesis of higher plants. Here, we report the isolation of a gene encoding an Arabidopsis homologue (ACR4) of the maize putative receptor kinase CRINKLY4 (CR4), which is involved in epidermal differentiation. The domain organization of the predicted amino acid sequence of ACR4 is essentially identical to that of CR4. ACR4-GFP fusion protein localized to the cell surface when expressed in tobacco cell (BY-2) culture. ACR4 transcripts were detected in all the organs of the Arabidopsis plant. In developing embryos and shoot apices, ACR4 transcripts accumulated in protoderm and epidermis at relatively higher levels than in the inner tissues. Over-expression of antisense ACR4 in Arabidopsis plants resulted in malformation of embryos to varying degrees. These results suggest that ACR4 is, at a minimum, involved in the normal morphogenesis of embryos, most likely through properly differentiating protoderm cells.

The first whole transcriptomic exploration of pre-oviposited early chicken embryos using single and bulked embryonic RNA-sequencing.

PubMed

Hwang, Young Sun; Seo, Minseok; Choi, Hee Jung; Kim, Sang Kyung; Kim, Heebal; Han, Jae Yong

2018-04-01

The chicken is a valuable model organism, especially in evolutionary and embryology research because its embryonic development occurs in the egg. However, despite its scientific importance, no transcriptome data have been generated for deciphering the early developmental stages of the chicken because of practical and technical constraints in accessing pre-oviposited embryos. Here, we determine the entire transcriptome of pre-oviposited avian embryos, including oocyte, zygote, and intrauterine embryos from Eyal-giladi and Kochav stage I (EGK.I) to EGK.X collected using a noninvasive approach for the first time. We also compare RNA-sequencing data obtained using a bulked embryo sequencing and single embryo/cell sequencing technique. The raw sequencing data were preprocessed with two genome builds, Galgal4 and Galgal5, and the expression of 17,108 and 26,102 genes was quantified in the respective builds. There were some differences between the two techniques, as well as between the two genome builds, and these were affected by the emergence of long intergenic noncoding RNA annotations. The first transcriptome datasets of pre-oviposited early chicken embryos based on bulked and single embryo sequencing techniques will serve as a valuable resource for investigating early avian embryogenesis, for comparative studies among vertebrates, and for novel gene annotation in the chicken genome.

Transient Shifts of Incubation Temperature Reveal Immediate and Long-Term Transcriptional Response in Chicken Breast Muscle Underpinning Resilience and Phenotypic Plasticity.

PubMed

Naraballobh, Watcharapong; Trakooljul, Nares; Murani, Eduard; Brunner, Ronald; Krischek, Carsten; Janisch, Sabine; Wicke, Michael; Ponsuksili, Siriluck; Wimmers, Klaus

2016-01-01

Variations in egg incubation temperatures can have acute or long-term effects on gene transcription in avian species. Altered gene expression may, in turn, affect muscle traits in poultry and indirectly influence commercial production. To determine how changes in eggshell temperature affect gene expression, incubation temperatures were varied [36.8°C (low), 37.8°C (control), 38.8°C (high)] at specific time periods reflecting two stages of myogenesis [embryonic days (ED) 7-10 and 10-13]. Gene expression was compared between interventions and matching controls by microarrays in broiler breast muscle at ED10 or ED13 and post-hatch at day 35. Early (ED7-10) high incubation temperature (H10ΔC) resulted in 1370 differentially expressed genes (DEGs) in embryos. Ingenuity pathway analysis revealed temporary activation of cell maintenance, organismal development, and survival ability genes, but these effects were not maintained in adults. Late high incubation temperature (ED10-13) (H13ΔC) had slightly negative impacts on development of cellular components in embryos, but a cumulative effect was observed in adults, in which tissue development and nutrition metabolism were affected. Early low incubation temperature (L10ΔC) produced 368 DEGs, most of which were down-regulated and involved in differentiation and formation of muscle cells. In adults, this treatment down-regulated pathways of transcriptional processes, but up-regulated cell proliferation. Late low temperature incubation (L13ΔC) produced 795 DEGs in embryos, and activated organismal survival and post-transcriptional regulation pathways. In adults this treatment activated cellular and organ development, nutrition and small molecule activity, and survival rate, but deactivated size of body and muscle cells. Thermal interventions during incubation initiate immediate and delayed transcriptional responses that are specific for timing and direction of treatment. Interestingly, the transcriptional response to transiently decreased incubation temperature, which did not affect the phenotypes, prompts compensatory effects reflecting resilience. In contrast, higher incubation temperature triggers gene expression and has long-term effects on the phenotype. These mechanisms of considerable phenotypic plasticity contribute to the biodiversity and broaden the basis for managing poultry populations.

UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity.

PubMed

Wang, Chaochen; Lee, Ji-Eun; Cho, Young-Wook; Xiao, Ying; Jin, Qihuang; Liu, Chengyu; Ge, Kai

2012-09-18

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.

[Influence of the activator of transcription GAL4 on growth and development of embryos and embryonic cells in primary cultures of sand dollar].

PubMed

Odintsova, N A; Kiselev, K V; Bulgakov, V P; Kol'tsova, E A; Iakovlev, K V

2003-01-01

In order to solve many tasks of biotechnology, constant lines of the cells of marine invertebrates with a high growth potential are required, which are absent at present. We used the universal activator of transcription gal4 to change the degree of expression of genes of growth factors in embryonic sea urchin cells and, thereby, increase their proliferative activity. The fertilized sea urchin eggs and dissociated embryonic cells at the blastula stage were treated with plasmids containing both the functional gene gal4 and the gene devoid of the regions encoding the activator domain. The transfection of embryonic sea urchin eggs with the functional gene led to cell dedifferentiation and formation of tumor-like structures in the embryos or increased number of embryonic cells in culture. In the cells obtained from the transfected embryos, the pigments were found within two months of cultivation, whose absorption spectrum coincided with that of echinochrome.

The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean.

PubMed

Barraza, Aarón; Cabrera-Ponce, José L; Gamboa-Becerra, Roberto; Luna-Martínez, Francisco; Winkler, Robert; Álvarez-Venegas, Raúl

2015-01-01

Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.

Cell wall invertase as a regulator in determining sequential development of endosperm and embryo through glucose signaling early in seed development.

PubMed

Wang, Lu; Liao, Shengjin; Ruan, Yong-Ling

2013-01-01

Seed development depends on coordination among embryo, endosperm and seed coat. Endosperm undergoes nuclear division soon after fertilization, whereas embryo remains quiescent for a while. Such a developmental sequence is of great importance for proper seed development. However, the underlying mechanism remains unclear. Recent results on the cellular domain- and stage-specific expression of invertase genes in cotton and Arabidopsis revealed that cell wall invertase may positively and specifically regulate nuclear division of endosperm after fertilization, thereby playing a role in determining the sequential development of endosperm and embryo, probably through glucose signaling.

Chicken HOXA3 Gene: Its Expression Pattern and Role in Branchial Nerve Precursor Cell Migration

PubMed Central

Watari-Goshima, Natsuko; Chisaka, Osamu

2011-01-01

In vertebrates, the proximal and distal sensory ganglia of the branchial nerves are derived from neural crest cells (NCCs) and placodes, respectively. We previously reported that in Hoxa3 knockout mouse embryos, NCCs and placode-derived cells of the glossopharyngeal nerve were defective in their migration. In this report, to determine the cell-type origin for this Hoxa3 knockout phenotype, we blocked the expression of the gene with antisense morpholino oligonucleotides (MO) specifically in either NCCs/neural tube or placodal cells of chicken embryos. Our results showed that HOXA3 function was required for the migration of the epibranchial placode-derived cells and that HOXA3 regulated this cell migration in both NCCs/neural tube and placodal cells. We also report that the expression pattern of chicken HOXA3 was slightly different from that of mouse Hoxa3. PMID:21278919

Early embryonic expression of FGF4/6/9 gene and its role in the induction of mesenchyme and notochord in Ciona savignyi embryos.

PubMed

Imai, Kaoru S; Satoh, Nori; Satou, Yutaka

2002-04-01

In early Ciona savignyi embryos, nuclear localization of beta-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of beta-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 was transient and the transcript was seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene was then expressed again in cells of the nervous system after neurulation. When the gene function was suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells was completely blocked, and the fate of presumptive mesenchyme cells appeared to change into that of muscle cells. The inhibition of mesenchyme differentiation was abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of beta-catenin nuclear localization resulted in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in beta-catenin-downregulated embryos evoked mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. On the other hand, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction was inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes were expressed later to form a partial notochord.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

PubMed

Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

2016-01-01

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

Identification of vimentin- and elastin-like transcripts specifically expressed in developing notochord of Atlantic salmon (Salmo salar L.).

PubMed

Sagstad, Anita; Grotmol, Sindre; Kryvi, Harald; Krossøy, Christel; Totland, Geir K; Malde, Ketil; Wang, Shou; Hansen, Tom; Wargelius, Anna

2011-11-01

The notochord functions as the midline structural element of all vertebrate embryos, and allows movement and growth at early developmental stages. Moreover, during embryonic development, notochord cells produce secreted factors that provide positional and fate information to a broad variety of cells within adjacent tissues, for instance those of the vertebrae, central nervous system and somites. Due to the large size of the embryo, the salmon notochord is useful to study as a model for exploring notochord development. To investigate factors that might be involved in notochord development, a normalized cDNA library was constructed from a mix of notochords from ∼500 to ∼800 day°. From the 1968 Sanger-sequenced transcripts, 22 genes were identified to be predominantly expressed in the notochord compared to other organs of salmon. Twelve of these genes were found to show expressional regulation around mineralization of the notochord sheath; 11 genes were up-regulated and one gene was down-regulated. Two genes were found to be specifically expressed in the notochord; these genes showed similarity to vimentin (acc. no GT297094) and elastin (acc. no GT297478). In-situ results showed that the vimentin- like transcript was expressed in both chordocytes and chordoblasts, whereas the elastin- like transcript was uniquely expressed in the chordoblasts lining the notochordal sheath. In salmon aquaculture, vertebral deformities are a common problem, and some malformations have been linked to the notochord. The expression of identified transcripts provides further insight into processes taking place in the developing notochord, prior to and during the early mineralization period.

EYA1 mutations associated with the branchio-oto-renal syndrome result in defective otic development in Xenopus laevis

PubMed Central

Li, Youe; Manaligod, Jose M.; Weeks, Daniel L.

2009-01-01

Background information. The BOR (branchio-oto-renal) syndrome is a dominant disorder most commonly caused by mutations in the EYA1 (Eyes Absent 1) gene. Symptoms commonly include deafness and renal anomalies. Results. We have used the embryos of the frog Xenopus laevis as an animal model for early ear development to examine the effects of different EYA1 mutations. Four eya1 mRNAs encoding proteins correlated with congenital anomalies in human were injected into early stage embryos. We show that the expression of mutations associated with BOR, even in the presence of normal levels of endogenous eya1 mRNA, leads to morphologically abnormal ear development as measured by overall otic vesicle size, establishment of sensory tissue and otic innervation. The molecular consequences of mutant eya1 expression were assessed by QPCR (quantitative PCR) analysis and in situ hybridization. Embryos expressing mutant eya1 showed altered levels of multiple genes (six1, dach, neuroD, ngnr-1 and nt3) important for normal ear development. Conclusions. These studies lend support to the hypothesis that dominant-negative effects of EYA1 mutations may have a role in the pathogenesis of BOR. PMID:19951260

The role of the embryo and ethylene in avocado fruit mesocarp discoloration

PubMed Central

Hershkovitz, Vera; Friedman, Haya; Goldschmidt, Eliezer E.; Pesis, Edna

2009-01-01

Chilling injury (CI) symptoms in avocado (Persea americana Mill.) fruit, expressed as mesocarp discoloration, were found to be associated with embryo growth and ethylene production during cold storage. In cvs Ettinger and Arad most mesocarp discoloration was located close to the base of the seed and was induced by ethylene treatment in seeded avocado fruit. However, ethylene did not increase mesocarp discoloration in seedless fruit stored at 5 °C. Application of ethylene to whole fruit induced embryo development inside the seed. It also induced seedling elongation when seeds were imbibed separately. Persea americana ethylene receptor (PaETR) gene expression and polyphenol oxidase activity were highest close to the base of the seed and decreased gradually toward the blossom end. By contrast, expressions of PaETR transcript and polyphenol oxidase activity in seedless avocado fruit were similar throughout the pulp at the base of the fruit. Application of the ethylene inhibitor, 1-methylcyclopropene, decreased mesocarp browning, embryo development, seedling growth, and ion leakage, and down-regulated polyphenol oxidase activity. The results demonstrate that ethylene-mediated embryo growth in whole fruit is involved in the mesocarp response to ethylene perception and the development of CI disorders. PMID:19196750

The role of the embryo and ethylene in avocado fruit mesocarp discoloration.

PubMed

Hershkovitz, Vera; Friedman, Haya; Goldschmidt, Eliezer E; Pesis, Edna

2009-01-01

Chilling injury (CI) symptoms in avocado (Persea americana Mill.) fruit, expressed as mesocarp discoloration, were found to be associated with embryo growth and ethylene production during cold storage. In cvs Ettinger and Arad most mesocarp discoloration was located close to the base of the seed and was induced by ethylene treatment in seeded avocado fruit. However, ethylene did not increase mesocarp discoloration in seedless fruit stored at 5 degrees C. Application of ethylene to whole fruit induced embryo development inside the seed. It also induced seedling elongation when seeds were imbibed separately. Persea americana ethylene receptor (PaETR) gene expression and polyphenol oxidase activity were highest close to the base of the seed and decreased gradually toward the blossom end. By contrast, expressions of PaETR transcript and polyphenol oxidase activity in seedless avocado fruit were similar throughout the pulp at the base of the fruit. Application of the ethylene inhibitor, 1-methylcyclopropene, decreased mesocarp browning, embryo development, seedling growth, and ion leakage, and down-regulated polyphenol oxidase activity. The results demonstrate that ethylene-mediated embryo growth in whole fruit is involved in the mesocarp response to ethylene perception and the development of CI disorders.

An amphioxus Msx gene expressed predominantly in the dorsal neural tube.

PubMed

Sharman, A C; Shimeld, S M; Holland, P W

1999-04-01

Genomic and cDNA clones of an Msx class homeobox gene were isolated from amphioxus (Branchiostoma floridae). The gene, AmphiMsx, is expressed in the neural plate from late gastrulation; in later embryos it is expressed in dorsal cells of the neural tube, excluding anterior and posterior regions, in an irregular reiterated pattern. There is transient expression in dorsal cells within somites, reminiscent of migrating neural crest cells of vertebrates. In larvae, mRNA is detected in two patches of anterior ectoderm proposed to be placodes. Evolutionary analyses show there is little phylogenetic information in Msx protein sequences; however, it is likely that duplication of Msx genes occurred in the vertebrate lineage.

The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation.

PubMed

Liao, W; Bisgrove, B W; Sawyer, H; Hug, B; Bell, B; Peters, K; Grunwald, D J; Stainier, D Y

1997-01-01

The zebrafish cloche mutation affects both the endothelial and hematopoietic lineages at a very early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W., Zon, L. I. and Fishman, M. C. (1995). Development 121, 3141-3150). The most striking vascular phenotype is the absence of endocardial cells from the heart. Microscopic examination of mutant embryos reveals the presence of endothelial-like cells in the lower trunk and tail regions while head vessels appear to be missing, indicating a molecular diversification of the endothelial lineage. Cell transplantation experiments show that cloche acts cell-autonomously within the endothelial lineage. To analyze further the role of cloche in regulating endothelial cell differentiation, we have examined the expression of flk-1 and tie, two receptor tyrosine kinase genes expressed early and sequentially in the endothelial lineage. In wild-type fish, flk-1-positive cells are found throughout the embryo and differentiate to form the nascent vasculature. In cloche mutants, flk-1-positive cells are found only in the lower trunk and tail regions, and this expression is delayed as compared to wild-type. Unlike the flk-1-positive cells in wild-type embryos, those in cloche mutants do not go on to express tie, suggesting that their differentiation is halted at an early stage. We also find that the cloche mutation is not linked to flk-1. These data indicate that cloche affects the differentiation of all endothelial cells and that it acts at a very early stage, either by directly regulating flk-1 expression or by controlling the differentiation of cells that normally develop to express flk-1. cloche mutants also have a blood deficit and their hematopoietic tissues show no expression of the hematopoietic transcription factor genes GATA-1 or GATA-2 at early stages. Because the appearance of distinct levels of flk-1 expression is delayed in cloche mutants, we examined GATA-1 expression at late embryonic stages and found some blood cell differentiation that appears to be limited to the region lined by the flk-1-expressing cells. The spatial restriction of blood in the ventroposterior-most region of cloche mutant embryos may be indicative of a ventral source of signal(s) controlling hematopoietic differentiation. In addition, the restricted colocalization of blood and endothelium in cloche mutants suggests that important interactions occur between these two lineages during normal development.

Wnt6 activates endoderm in the sea urchin gene regulatory network

PubMed Central

Croce, Jenifer; Range, Ryan; Wu, Shu-Yu; Miranda, Esther; Lhomond, Guy; Peng, Jeff Chieh-fu; Lepage, Thierry; McClay, David R.

2011-01-01

In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network. PMID:21750039

Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron.

PubMed

Hardy, Melissa E; Ross, Louis V; Chien, Chi-Bin

2007-11-01

Misexpression of genes in a temporally and spatially controlled fashion is an important tool for assessing gene function during development. Because few tissue-specific promoters have been identified in zebrafish, inducible systems such as the Cre/LoxP and Tet repressor systems are of limited utility. Here we describe a new method of misexpression: local heat shock using a modified soldering iron. Zebrafish carrying transgenes under the control of a heat shock promoter (hsp70) are focally heated with the soldering iron to induce gene expression in a small area of the embryo. We have validated this method in three stable transgenic lines and at three developmental timepoints. Local heat shock is a fast, easy, and inexpensive method for gene misexpression. Copyright 2007 Wiley-Liss, Inc.

Effects of varying Notch1 signal strength on embryogenesis and vasculogenesis in compound mutant heterozygotes

PubMed Central

2010-01-01

Background Identifying developmental processes regulated by Notch1 can be addressed in part by characterizing mice with graded levels of Notch1 signaling strength. Here we examine development in embryos expressing various combinations of Notch1 mutant alleles. Mice homozygous for the hypomorphic Notch112f allele, which removes the single O-fucose glycan in epidermal growth factor-like repeat 12 (EGF12) of the Notch1 ligand binding domain (lbd), exhibit reduced growth after weaning and defective T cell development. Mice homozygous for the inactive Notch1lbd allele express Notch1 missing an ~20 kDa internal segment including the canonical Notch1 ligand binding domain, and die at embryonic day ~E9.5. The embryonic and vascular phenotypes of compound heterozygous Notch112f/lbd embryos were compared with Notch1+/12f, Notch112f/12f, and Notch1lbd/lbd embryos. Embryonic stem (ES) cells derived from these embryos were also examined in Notch signaling assays. While Notch1 signaling was stronger in Notch112f/lbd compound heterozygotes compared to Notch1lbd/lbd embryos and ES cells, Notch1 signaling was even stronger in embryos carrying Notch112f and a null Notch1 allele. Results Mouse embryos expressing the hypomorphic Notch112f allele, in combination with the inactive Notch1lbd allele which lacks the Notch1 ligand binding domain, died at ~E11.5-12.5. Notch112f/lbd ES cells signaled less well than Notch112f/12f ES cells but more strongly than Notch1lbd/lbd ES cells. However, vascular defects in Notch112f/lbd yolk sac were severe and similar to Notch1lbd/lbd yolk sac. By contrast, vascular disorganization was milder in Notch112f/lbd compared to Notch1lbd/lbd embryos. The expression of Notch1 target genes was low in Notch112f/lbd yolk sac and embryo head, whereas Vegf and Vegfr2 transcripts were increased. The severity of the compound heterozygous Notch112f/lbd yolk sac phenotype suggested that the allelic products may functionally interact. By contrast, compound heterozygotes with Notch112f in combination with a Notch1 null allele (Notch1tm1Con) were capable of surviving to birth. Conclusions Notch1 signaling in Notch112f/lbd compound heterozygous embryos is more defective than in compound heterozygotes expressing a hypomorphic Notch112f allele and a Notch1 null allele. The data suggest that the gene products Notch1lbd and Notch112f interact to reduce the activity of Notch112f. PMID:20346184

The Tribolium homeotic gene Abdominal is homologous to abdominal-A of the Drosophila bithorax complex

NASA Technical Reports Server (NTRS)

Stuart, J. J.; Brown, S. J.; Beeman, R. W.; Denell, R. E.; Spooner, B. S. (Principal Investigator)

1993-01-01

The Abdominal gene is a member of the single homeotic complex of the beetle, Tribolium castaneum. An integrated developmental genetic and molecular analysis shows that Abdominal is homologous to the abdominal-A gene of the bithorax complex of Drosophila. abdominal-A mutant embryos display strong homeotic transformations of the anterior abdomen (parasegments 7-9) to PS6, whereas developmental commitments in the posterior abdomen depend primarily on Abdominal-B. In beetle embryos lacking Abdominal function, parasegments throughout the abdomen are transformed to PS6. This observation demonstrates the general functional significance of parasegmental expression among insects and shows that the control of determinative decisions in the posterior abdomen by homeotic selector genes has undergone considerable evolutionary modification.

The Tribolium homeotic gene Abdominal is homologous to abdominal-A of the Drosophila bithorax complex.

PubMed

Stuart, J J; Brown, S J; Beeman, R W; Denell, R E

1993-01-01

The Abdominal gene is a member of the single homeotic complex of the beetle, Tribolium castaneum. An integrated developmental genetic and molecular analysis shows that Abdominal is homologous to the abdominal-A gene of the bithorax complex of Drosophila. abdominal-A mutant embryos display strong homeotic transformations of the anterior abdomen (parasegments 7-9) to PS6, whereas developmental commitments in the posterior abdomen depend primarily on Abdominal-B. In beetle embryos lacking Abdominal function, parasegments throughout the abdomen are transformed to PS6. This observation demonstrates the general functional significance of parasegmental expression among insects and shows that the control of determinative decisions in the posterior abdomen by homeotic selector genes has undergone considerable evolutionary modification.

The oxidized state of the nanocomposite Carbo-Iron® causes no adverse effects on growth, survival and differential gene expression in zebrafish.

PubMed

Weil, Mirco; Meißner, Tobias; Busch, Wibke; Springer, Armin; Kühnel, Dana; Schulz, Ralf; Duis, Karen

2015-10-15

For degradation of halogenated chemicals in groundwater Carbo-Iron®, a composite of activated carbon and nano-sized Fe(0), was developed (Mackenzie et al., 2012). Potential effects of this nanocomposite on fish were assessed. Beyond the contaminated zone Fe(0) can be expected to have oxidized and Carbo-Iron was used in its oxidized form in ecotoxicological tests. Potential effects of Carbo Iron in zebrafish (Danio rerio) were investigated using a 48 h embryo toxicity test under static conditions, a 96 h acute test with adult fish under semi-static conditions and a 34 d fish early life stage test (FELST) in a flow-through system. Particle diameters in test suspensions were determined via dynamic light scattering (DLS) and ranged from 266 to 497 nm. Particle concentrations were measured weekly in samples from the FELST using a method based on the count rate in DLS. Additionally, uptake of particles into test organisms was investigated using microscopic methods. Furthermore, effects of Carbo-Iron on gene expression were investigated by microarray analysis in zebrafish embryos. In all tests performed, no significant lethal effects were observed. Furthermore, Carbo-Iron had no significant influence on weight and length of fish as determined in the FELST. In the embryo test and the early life stage test, growth of fungi on the chorion was observed at Carbo-Iron concentrations between 6.3 and 25mg/L. Fungal growth did not affect survival, hatching success and growth. In the embryo test, no passage of Carbo-Iron particles into the perivitelline space or the embryo was observed. In juvenile and adult fish, Carbo-Iron was detected in the gut at the end of exposure. In juvenile fish exposed to Carbo-Iron for 29 d and subsequently kept for 5d in control water, Carbo-Iron was no longer detectable in the gut. Global gene expression in zebrafish embryos was not significantly influenced by Carbo-Iron. Copyright © 2015 Elsevier B.V. All rights reserved.

Microcystin-LR affects the hypothalamic-pituitary-inter-renal (HPI) axis in early life stages (embryos and larvae) of zebrafish.

PubMed

Chen, Liang; Wang, Yeke; Giesy, John P; Chen, Feng; Shi, Ting; Chen, Jun; Xie, Ping

2018-05-22

Frequencies and durations of blooms of cyanobacteria are increasing. Some cyanobacteria can produce cyanotoxins including microcystins (MCs). MCs are the most common toxic products of hazardous algal blooms (HABs), with the greatest potential for exposure and to cause toxicity. Recently, MCs have been shown to disrupt endocrine functions. In this study, for the first time, effects of MC-LR on the hypothalamic-pituitary-inter-renal (HPI) axis during early embryonic development (embryos/larvae) of zebrafish (Danio rerio), were investigated. Embryos/larvae of zebrafish were exposed to 1, 10, 100, or 300 μg MC-LR/L during the period of 4-168 h post-fertilization (hpf). Exposure to 300 μg MC-LR/L resulted in significantly greater concentrations of whole-body cortisol than those in controls. Expressions of genes along the HPI axis and mineralocorticoid receptor (MR-) and glucocorticoid receptor (GR-) centered gene networks were evaluated by use of quantitative real-time PCR. Expression of mRNA for crh was significantly down-regulated by exposure to 300 μg MC-LR/L, while expressions of crhbp, crhr1, and crhr2 were significantly up-regulated, relative to controls. MC-LR caused significantly lesser levels of mRNA for steroidogenic genes including hmgra, star, and cyp17, but expression of mRNA for hsd20b was significantly greater than that of controls. Treatment with MC-LR also altered profiles of transcription of MR- and GR-centered gene networks, which might result in multiple responses. Taken together, these results demonstrated that MC-LR affected the corticosteroid-endocrine system of larvae of zebrafish. This study provided valuable insights into molecular mechanisms behind potential toxicity and endocrine disruption of MCs. Copyright © 2018 Elsevier Ltd. All rights reserved.

Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

PubMed Central

Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

2016-01-01

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis. PMID:26493868

Expression of Wise in chick embryos.

PubMed

Shigetani, Y; Itasaki, N

2007-08-01

We have performed in situ hybridization to study the expression of Wise in early chick embryos. Wise expression is first detectable in the ectoderm at posterior levels of late neurula. As development proceeds, Wise expression is seen in specific patterns in the ectoderm of the trunk region, pharyngeal arches, limb buds, and feather buds. In addition to these areas, particular cartilages such as the ones in the maxillary process and limbs start to express Wise at the late pharyngula stage, and the expression in these cartilages becomes stronger than that in epidermal components at later stages. Importantly, Wise is expressed in regions where other signaling molecules such as Wnt, Bmp, and Shh are known to function in morphogenesis and differentiation. Direct comparisons of the expression of Wise and these genes are also demonstrated. (c) 2007 Wiley-Liss, Inc.

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

PubMed

Ono, Yukiko; Kono, Tomohiro

2006-08-01

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus × H. tuberosus

PubMed Central

Chiappetta, A.; Fambrini, M.; Petrarulo, M.; Rapparini, F.; Michelotti, V.; Bruno, L.; Greco, M.; Baraldi, R.; Salvini, M.; Pugliesi, C.; Bitonti, M. B.

2009-01-01

Background and Aims The clone EMB-2 of the interspecific hybrid Helianthus annuus × H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. Methods Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography–mass spectrometry–selected ion monitoring method and an immuno-cytochemical approach. Key Results Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. Conclusions In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone. PMID:19151043

Modulation by Cocaine of Dopamine Receptors through miRNA-133b in Zebrafish Embryos

PubMed Central

Barreto-Valer, Katherine; López-Bellido, Roger; Macho Sánchez-Simón, Fátima; Rodríguez, Raquel E.

2012-01-01

The use of cocaine during pregnancy can affect the mother and indirectly might alter the development of the embryo/foetus. Accordingly, in the present work our aim was to study in vivo (in zebrafish embryos) the effects of cocaine on the expression of dopamine receptors and on miR-133b. These embryos were exposed to cocaine hydrochloride (HCl) at 5 hours post-fertilization (hpf) and were then collected at 8, 16, 24, 48 and 72 hpf to study the expression of dopamine receptors, drd1, drd2a, drd2b and drd3, by quantitative real time PCR (qPCR) and in situ hybridization (ISH, only at 24 hpf). Our results indicate that cocaine alters the expression of the genes studied, depending on the stage of the developing embryo and the type of dopamine receptor. We found that cocaine reduced the expression of miR-133b at 24 and 48 hpf in the central nervous system (CNS) and at the periphery by qPCR and also that the spatial distribution of miR-133b was mainly seen in somites, a finding that suggests the involvement of miR-133b in the development of the skeletal muscle. In contrast, at the level of the CNS miR-133b had a weak and moderate expression at 24 and 48 hpf. We also analysed the interaction of miR-133b with the Pitx3 and Pitx3 target genes drd2a and drd2b, tyrosine hydroxylase (th) and dopamine transporter (dat) by microinjection of the Pitx3-3'UTR sequence. Microinjection of Pitx3-3'UTR affected the expression of pitx3, drd2a, drd2b, th and dat. In conclusion, in the present work we describe a possible mechanism to account for cocaine activity by controlling miR-133b transcription in zebrafish. Via miR-133b cocaine would modulate the expression of pitx3 and subsequently of dopamine receptors, dat and th. These results indicate that miRNAs can play an important role during embryogenesis and in drug addiction. PMID:23285158

Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes.

PubMed

Salvemini, Marco; D'Amato, Rocco; Petrella, Valeria; Ippolito, Domenica; Ventre, Giuseppe; Zhang, Ying; Saccone, Giuseppe

2014-01-01

The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal. We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) anddifferential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome. We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.

Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes

PubMed Central

2014-01-01

The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal. We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) and differential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome. We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function. PMID:25472628

Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos.

PubMed

Zhang, Qisen; Zhang, Xiaoqi; Pettolino, Filomena; Zhou, Gaofeng; Li, Chengdao

2016-02-01

Barley (Hordeum vulgare L.) seed germination initiates many important biological processes such as DNA, membrane and mitochondrial repairs. However, little is known on cell wall modifications in germinating embryos. We have investigated cell wall polysaccharide composition change, gene transcription and alternative splicing events in four barley varieties at 24h and 48 h germination. Cell wall components in germinating barley embryos changed rapidly, with increases in cellulose and (1,3)(1,4)-β-D-glucan (20-100%) within 24h, but decreases in heteroxylan and arabinan (3-50%). There were also significant changes in the levels of type I arabinogalactans and heteromannans. Alternative splicing played very important roles in cell wall modifications. At least 22 cell wall transcripts were detected to undergo either alternative 3' splicing, alternative 5' splicing or intron retention type of alternative splicing. These genes coded enzymes catalyzing synthesis and degradation of cellulose, heteroxylan, (1,3)(1,4)-β-D-glucan and other cell wall polymers. Furthermore, transcriptional regulation also played very important roles in cell wall modifications. Transcript levels of primary wall cellulase synthase, heteroxylan synthesizing and nucleotide sugar inter-conversion genes were very high in germinating embryos. At least 50 cell wall genes changed transcript levels significantly. Expression patterns of many cell wall genes coincided with changes in polysaccharide composition. Our data showed that cell wall polysaccharide metabolism was very active in germinating barley embryos, which was regulated at both transcriptional and post-transcriptional levels. Copyright © 2015 Elsevier GmbH. All rights reserved.

Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters DNA methyltransferase (dnmt) expression in zebrafish (Danio rerio)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aluru, Neelakanteswar, E-mail: naluru@whoi.edu; Kuo, Elaine; Stanford University, 450 Serra Mall, Stanford, CA 94305

2015-04-15

DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5 nM TCDD for 1 h from 4 to 5 h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNAmore » methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns. - Highlights: • TCDD altered the dnmt expression in a gene and developmental time-specific manner. • TCDD hypermethylated ahrra and hypomethylated c-fos proximal promoter regions. • Functional analysis suggests that ahr2 can regulate dnmt3a1, 3a2, and 3b2 expression. • Dnmt3b genes are expressed early whereas dnmt3a are abundant later in development.« less

Effects of the histone deacetylase inhibitor 'Scriptaid' on the developmental competence of mouse embryos generated through round spermatid injection.

PubMed

Kong, Pengcheng; Yin, Mingru; Chen, Dongbao; Li, Shangang; Li, Yao; Xing, Fengying; Jiang, Manxi; Fang, Zhenfu; Lyu, Qifeng; Chen, Xuejin

2017-01-01

Can the histone deacetylase inhibitor Scriptaid improve the efficiency of the development of round spermatid injection (ROSI)-fertilized embryos in a mouse model? Treatment of ROSI mouse zygotes with Scriptaid increased the expression levels of several development-related genes at the blastocyst stage, resulting in more efficient in vitro development of the blastocyst and an increased birth rate of ROSI-derived embryos. The full-term development of embryos derived through ROSI is significantly lower than that following ICSI in humans and other species. Oocytes, spermatozoa and round spermatids were collected from BDF1 (C57BL/6 × DBA/2) mice. For in vitro development experiments, mouse ROSI-derived zygotes were treated with Scriptaid at different concentrations (0, 125, 250, 500 and 1000 nM) and for different exposure times (0, 6, 10, 16 or 24 h). Next, blastocysts of the optimal Scriptaid-treated group and the non-treated ROSI group were separately transferred into surrogate ICR mice to compare in vivo development with the ICSI group (control). Each experiment was repeated at least three times. Metaphase II (MII) oocytes, spermatozoa and round spermatids were obtained from sexually mature BDF1 female or male mice. The developmental potential of embryos among the three groups (the ICSI, ROSI and optimal Scriptaid-treated ROSI groups) was assessed based on the rates of obtaining zygotes, two-cell stage embryos, four-cell stage embryos, blastocysts and full-term offspring. In addition, the expression levels of development-related genes (Oct4, Nanog, Klf4 and Sox2) were analysed using real-time PCR, and the methylation states of imprinted genes (H19 and Snrpn) in these three groups were detected using methylation-specific PCR (MS-PCR) sequencing following bisulfite treatment. The in vitro experiments revealed that treating ROSI-derived zygotes with 250 nM Scriptaid for 10 h significantly improved the blastocyst formation rate (59%) compared with the non-treated group (38%) and further increased the birth rates of ROSI-derived embryos from 21% to 40% in vivo. Moreover, in ROSI-derived embryos, the expression of the Oct4, Nanog and Sox2 genes at the blastocyst stage was decreased, but the optimal Scriptaid treatment restored expression to a level similar to their ICSI counterparts. In addition, Scriptaid treatment moderately repaired the abnormal DNA methylation pattern in the imprinting control regions (ICRs) of H19 and Snrpn. N/A LIMITATIONS, REASONS FOR CAUTION: Because of the ethics regarding the use of human gametes for ROSI studies, the mouse model was used as an approach to explore the effects of Scriptaid on the developmental potential of ROSI-derived embryos. However, to determine whether these findings can be applied to humans, further investigation will be required. Scriptaid treatment provides a new means of improving the efficiency and safety of clinical human ROSI. The study was financially supported through grants from the National Key Research Program of China (No. 2016YFC1304800); the National Natural Science Foundation of China (Nos: 81170756, 81571486); the Natural Science Foundation of Shanghai (Nos: 15140901700, 15ZR1424900) and the Programme for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. There are no conflicts of interest to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli

NASA Astrophysics Data System (ADS)

Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert E.; Zemp, Roger

2015-10-01

To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9× dilution sample was 55, suggesting that ˜20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between <1 and 20 mm apart from each other, and imaged with the appropriate imaging modality. Photoacoustic imaging could resolve the two tubes of melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene for visualizing bacteria, and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

Enantioselectivity in Developmental Toxicity of rac-metalaxyl and R-metalaxyl in Zebrafish (Danio rerio) Embryo.

PubMed

Zhang, Yinjun; Zhang, Yi; Chen, An; Zhang, Wei; Chen, Hao; Zhang, Quan

2016-06-01

Enantioselectivity of chiral pesticides in environmental safety has attracted more and more attention. In this study, we evaluated the enantioselective toxicity of rac-metalaxyl and R-metalaxyl to zebrafish (Danio rerio) embryos through various malformations including pericardial edema, yolk sac edema, crooked body, and short tails. The results showed that there were significant differences in toxicity to zebrafish embryos caused by rac-metalaxyl and R-metalaxyl, and the LC50 s at 96 h are 416.41 (353.91, 499.29) mg · L(-1) and 320.650 (279.80, 363.46) mg · L(-1) , respectively. In order to explore the possible mechanism of the development defects, the genes involved in the hypothalamic-pituitary-gonadal axis (vtg1, vtg2, cyp17, cyp19a, cyp19b) and hypothalamic-pituitary-thyroid axis (dio1, dio2, nis, tg, tpo) were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed that there were no significant differences in the expression of vtg1, vtg2, cyp17, cyp19a, and cyp19b after exposure to rac-metalaxyl. However, the expression of vtg1, cyp19a, and cyp19b decreased significantly after exposure to R-metalaxyl. And likewise, rac-metalaxyl only caused the upregulation of dio2, while R-metalaxyl suppressed the expression of dio1 and tpo and induced the expression of dio2 and nis. The change of gene expression may cause the enantioselectivity in developmental toxicity in zebrafish embryo. The data provided here will be helpful for us to comprehensively understand the potential ecological risks of the currently used chiral fungicides. Chirality 28:489-494, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The CSR-1 endogenous RNAi pathway ensures accurate transcriptional reprogramming during the oocyte-to-embryo transition in Caenorhabditis elegans.

PubMed

Fassnacht, Christina; Tocchini, Cristina; Kumari, Pooja; Gaidatzis, Dimos; Stadler, Michael B; Ciosk, Rafal

2018-03-01

Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition.

The CSR-1 endogenous RNAi pathway ensures accurate transcriptional reprogramming during the oocyte-to-embryo transition in Caenorhabditis elegans

PubMed Central

Tocchini, Cristina; Kumari, Pooja; Gaidatzis, Dimos

2018-01-01

Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition. PMID:29579041

Laminarin improves developmental competence of porcine early stage embryos by inhibiting oxidative stress.

PubMed

Jiang, Hao; Liang, Shuang; Yao, Xue-Rui; Jin, Yong-Xun; Shen, Xing-Hui; Yuan, Bao; Zhang, Jia-Bao; Kim, Nam-Hyung

2018-04-23

Laminarin (LMA), a β-glucan mixture with good biocompatibility, improves the growth performance and immune response when used as food additives and nutraceuticals. The aim of the present research was to explore the effects of LMA on porcine early stage embryo development, as well as the underlying mechanisms. The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LMA supplementation during the in vitro culture period. The presence of 20 μg/mL LMA during the in vitro culture period significantly improved cleavage rate, blastocyst formation rates, hatching rate, and total cell number in the blastocyst compared to that in the control group. Notably, LMA attenuated the intracellular reactive oxygen species generation induced by H 2 O 2 . Furthermore, LMA not only increased intracellular glutathione levels, but also ameliorated mitochondrial membrane potential. In addition, the expression of a zygotic genome activation related gene (YAP1), pluripotency-related genes (OCT4, NANOG, and SOX2), and hatching-related genes (COX2, GATA4, and ITGA5) were up-regulated following LMA supplementation during porcine early stage embryo development. These results demonstrate that LMA has beneficial effects on the development of porcine early stage embryos via regulation of oxidative stress. This evidence provides a novel method for embryo development improvement associated with exposure to LMA. Copyright © 2018 Elsevier Inc. All rights reserved.

De Novo Assembly of the Manila Clam Ruditapes philippinarum Transcriptome Provides New Insights into Expression Bias, Mitochondrial Doubly Uniparental Inheritance and Sex Determination

PubMed Central

Ghiselli, Fabrizio; Milani, Liliana; Chang, Peter L.; Hedgecock, Dennis; Davis, Jonathan P.; Nuzhdin, Sergey V.; Passamonti, Marco

2012-01-01

Males and females share the same genome, thus, phenotypic divergence requires differential gene expression and sex-specific regulation. Accordingly, the analysis of expression patterns is pivotal to the understanding of sex determination mechanisms. Many bivalves are stable gonochoric species, but the mechanism of gonad sexualization and the genes involved are still unknown. Moreover, during the period of sexual rest, a gonad is not present and sex cannot be determined. A mechanism associated with germ line differentiation in some bivalves, including the Manila clam Ruditapes philippinarum, is the doubly uniparental inheritance (DUI) of mitochondria, a variation of strict maternal inheritance. Two mitochondrial lineages are present, one transmitted through eggs and the other through sperm, as well as a mother-dependent sex bias of the progeny. We produced a de novo annotation of 17,186 transcripts from R. philippinarum and compared the transcriptomes of males and females and identified 1,575 genes with strong sex-specific expression and 166 sex-specific single nucleotide polymorphisms, obtaining preliminary information about genes that could be involved in sex determination. Then we compared the transcriptomes between a family producing predominantly females and a family producing predominantly males to identify candidate genes involved in regulation of sex-specific aspects of DUI system, finding a relationship between sex bias and differential expression of several ubiquitination genes. In mammalian embryos, sperm mitochondria are degraded by ubiquitination. A modification of this mechanism is hypothesized to be responsible for the retention of sperm mitochondria in male embryos of DUI species. Ubiquitination can additionally regulate gene expression, playing a role in sex determination of several animals. These data enable us to develop a model that incorporates both the DUI literature and our new findings. PMID:21976711

Impacts of oxidative stress on acetylcholinesterase transcription, and activity in embryos of zebrafish (Danio rerio) following Chlorpyrifos exposure.

PubMed

Rodríguez-Fuentes, Gabriela; Rubio-Escalante, Fernando J; Noreña-Barroso, Elsa; Escalante-Herrera, Karla S; Schlenk, Daniel

2015-01-01

Organophosphate pesticides cause irreversible inhibition of AChE which leads to neuronal overstimulation and death. Thus, dogma indicates that the target of OP pesticides is AChE, but many authors postulate that these compounds also disturb cellular redox processes, and change the activities of antioxidant enzymes. Interestingly, it has also been reported that oxidative stress plays also a role in the regulation and activity of AChE. The aims of this study were to determine the effects of the antioxidant, vitamin C (VC), the oxidant, t-butyl hydroperoxide (tBOOH) and the organophosphate Chlorpyrifos (CPF), on AChE gene transcription and activity in zebrafish embryos after 72h exposure. In addition, oxidative stress was evaluated by measuring antioxidant enzymes activities and transcription, and quantification of total glutathione. Apical effects on the development of zebrafish embryos were also measured. With the exception of AChE inhibition and enhanced gene expression, limited effects of CPF on oxidative stress and apical endpoints were found at this developmental stage. Addition of VC had little effect on oxidative stress or AChE, but increased pericardial area and heartbeat rate through an unknown mechanism. TBOOH diminished AChE gene expression and activity, and caused oxidative stress when administered alone. However, in combination with CPF, only reductions in AChE activity were observed with no significant changes in oxidative stress suggesting the adverse apical endpoints in the embryos may have been due to AChE inhibition by CPF rather than oxidative stress. These results give additional evidence to support the role of prooxidants in AChE activity and expression. Copyright © 2015 Elsevier Inc. All rights reserved.

Network analysis reveals stage-specific changes in zebrafish embryo development using time course whole transcriptome profiling and prior biological knowledge.

PubMed

Zhang, Yuji

2015-01-01

Molecular networks act as the backbone of molecular activities within cells, offering a unique opportunity to better understand the mechanism of diseases. While network data usually constitute only static network maps, integrating them with time course gene expression information can provide clues to the dynamic features of these networks and unravel the mechanistic driver genes characterizing cellular responses. Time course gene expression data allow us to broadly "watch" the dynamics of the system. However, one challenge in the analysis of such data is to establish and characterize the interplay among genes that are altered at different time points in the context of a biological process or functional category. Integrative analysis of these data sources will lead us a more complete understanding of how biological entities (e.g., genes and proteins) coordinately perform their biological functions in biological systems. In this paper, we introduced a novel network-based approach to extract functional knowledge from time-dependent biological processes at a system level using time course mRNA sequencing data in zebrafish embryo development. The proposed method was applied to investigate 1α, 25(OH)2D3-altered mechanisms in zebrafish embryo development. We applied the proposed method to a public zebrafish time course mRNA-Seq dataset, containing two different treatments along four time points. We constructed networks between gene ontology biological process categories, which were enriched in differential expressed genes between consecutive time points and different conditions. The temporal propagation of 1α, 25-Dihydroxyvitamin D3-altered transcriptional changes started from a few genes that were altered initially at earlier stage, to large groups of biological coherent genes at later stages. The most notable biological processes included neuronal and retinal development and generalized stress response. In addition, we also investigated the relationship among biological processes enriched in co-expressed genes under different conditions. The enriched biological processes include translation elongation, nucleosome assembly, and retina development. These network dynamics provide new insights into the impact of 1α, 25-Dihydroxyvitamin D3 treatment in bone and cartilage development. We developed a network-based approach to analyzing the DEGs at different time points by integrating molecular interactions and gene ontology information. These results demonstrate that the proposed approach can provide insight on the molecular mechanisms taking place in vertebrate embryo development upon treatment with 1α, 25(OH)2D3. Our approach enables the monitoring of biological processes that can serve as a basis for generating new testable hypotheses. Such network-based integration approach can be easily extended to any temporal- or condition-dependent genomic data analyses.

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of themore » TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.« less

Habenular commissure formation in zebrafish is regulated by the pineal gland-specific gene unc119c.

PubMed

Toyama, Reiko; Kim, Mi Ha; Rebbert, Martha L; Gonzales, John; Burgess, Harold; Dawid, Igor B

2013-09-01

The zebrafish pineal gland (epiphysis) is a site of melatonin production, contains photoreceptor cells, and functions as a circadian clock pacemaker. Since it is located on the surface of the forebrain, it is accessible for manipulation and, therefore, is a useful model system to analyze pineal gland function and development. We previously analyzed the pineal transcriptome during development and showed that many genes exhibit a highly dynamic expression pattern in the pineal gland. Among genes preferentially expressed in the zebrafish pineal gland, we identified a tissue-specific form of the unc119 gene family, unc119c, which is highly preferentially expressed in the pineal gland during day and night at all stages examined from embryo to adult. When expression of unc119c was inhibited, the formation of the habenular commissure (HC) was specifically compromised. The Unc119c interacting factors Arl3l1 and Arl3l2 as well as Wnt4a also proved indispensible for HC formation. We suggest that Unc119c, together with Arl3l1/2, plays an important role in modulating Wnt4a production and secretion during HC formation in the forebrain of the zebrafish embryo. Copyright © 2013 Wiley Periodicals, Inc.

Habenular commissure formation in zebrafish is regulated by the pineal gland specific gene unc119c

PubMed Central

Toyama, Reiko; Kim, Mi Ha; Rebbert, Martha L.; Gonzales, John; Burgess, Harold; Dawid, Igor B.

2013-01-01

Background The zebrafish pineal gland (epiphysis) is a site of melatonin production, contains photoreceptor cells, and functions as a circadian clock pacemaker. Since it is located on the surface of the forebrain, it is accessible for manipulation and therefore is a useful model system to analyze pineal gland function and development. We previously analyzed the pineal transcriptome during development and showed that many genes exhibit a highly dynamic expression pattern in the pineal gland. Results Among genes preferentially expressed in the zebrafish pineal gland, we identified a tissue-specific form of the unc119 gene family, unc119c, which is highly preferentially expressed in the pineal gland during day and night at all stages examined from embryo to adult. When expression of unc119c was inhibited, the formation of the habenular commissure (HC) was specifically compromised. The Unc119c interacting factors Arl3l1 and Arl3l2 as well as Wnt4a also proved indispensible for HC formation. Conclusions We suggest that Unc119c, together with Arl3l1/2, plays an important role in modulating Wnt4a production and secretion during HC formation in the forebrain of the zebrafish embryo. PMID:23749482

Genome editing reveals a role for OCT4 in human embryogenesis.

PubMed

Fogarty, Norah M E; McCarthy, Afshan; Snijders, Kirsten E; Powell, Benjamin E; Kubikova, Nada; Blakeley, Paul; Lea, Rebecca; Elder, Kay; Wamaitha, Sissy E; Kim, Daesik; Maciulyte, Valdone; Kleinjung, Jens; Kim, Jin-Soo; Wells, Dagan; Vallier, Ludovic; Bertero, Alessandro; Turner, James M A; Niakan, Kathy K

2017-10-05

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

PubMed Central

Balbach, Sebastian Thomas; Esteves, Telma Cristina; Houghton, Franchesca Dawn; Siatkowski, Marcin; Pfeiffer, Martin Johannes; Tsurumi, Chizuko; Kanzler, Benoit; Fuellen, Georg; Boiani, Michele

2012-01-01

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development. PMID:22530006

Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

PubMed

Cooper, Caitlin A; Challagulla, Arjun; Jenkins, Kristie A; Wise, Terry G; O'Neil, Terri E; Morris, Kirsten R; Tizard, Mark L; Doran, Timothy J

2017-06-01

Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

The expression dynamics of mechanosensitive genes in extra-embryonic vasculature after heart starts to beat in chick embryo.

PubMed

Rajendran, Saranya; Sundaresan, Lakshmikirupa; Rajendran, Krithika; Selvaraj, Monica; Gupta, Ravi; Chatterjee, Suvro

2016-02-11

Fluid flow plays an important role in vascular development. However, the detailed mechanisms, particularly the link between flow and modulation of gene expression during vascular development, remain unexplored. In chick embryo, the key events of vascular development from initiation of heart beat to establishment of effective blood flow occur between the stages HH10 and HH13. Therefore, we propose a novel in vivo model to study the flow experienced by developing endothelium. Using this model, we aimed to capture the transcriptome dynamics of the pre- and post-flow conditions. RNA was isolated from extra embryonic area vasculosa (EE-AV) pooled from three chick embryos between HH10-HH13 and RNA sequencing was performed. The whole transcriptome sequencing of chick identified up-regulation of some of the previously well-known mechanosensitive genes including NFR2, HAND1, CTGF and KDR. GO analyses of the up-regulated genes revealed enrichment of several biological processes including heart development, extracellular matrix organization, cell-matrix adhesion, cell migration, blood vessel development, patterning of blood vessels, collagen fibril organization. Genes encoding for gap junctions proteins which are involved in vascular remodeling and arterial-venous differentiation, and genes involved in cell-cell adhesion, and ECM interactions were significantly up-regulated. Validation of selected genes through semi quantitative PCR was performed. The study indicates that shear stress plays a major role in development. Through appropriate validation, this platform can serve as an in vivo model to study conditions of disturbed flow in pathology as well as normal flow during development.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popp, R.A.; Marsh, C.L.; Skow, L.C.

Hemoglobins of mouse embryos at 11.5 through 16.5 days of gestation were separated by electrophoresis on cellulose acetate and quantitated by a scanning densitometer to study the effects of two radiation-induced mutations on the expression of embryonic hemoglobin genes in mice. Normal mice produce three kinds of embryonic hemoglobins. In heterozygous ..cap alpha..-thalassemic embryos, expression of EI (x/sub 2/y/sub 2/) and EII (..cap alpha../sub 2/y/sub 2/) is deficient because the x- and ..cap alpha..-globin genes of one of the allelic pairs of Hba on chromosome 11 was deleted or otherwise inactivated by X irradiation. Simultaneous inactivation of the x- andmore » ..cap alpha..-globin genes indicates that these genes must be closely linked. Reduced x- and ..cap alpha..-chain synthesis results in an excess of y chains that associate as homotetramers. This unique y/sub 4/ hemoglobin also appears in ..beta..-duplication embryos where excess y chains are produced by the presence of three rather than two functional alleles of y- and ..beta..-globin genes. In double heterozygotes, which have a single functional allele of x- and ..cap alpha..-globin genes and three functional alleles of y- and ..beta..-globin genes, synthesis of ..cap alpha.. and non-..cap alpha.. chains is severely imbalanced and half of the total hemoglobin is y/sub 4/. Mouse y/sub 4/ has a high affinity for oxygen, P/sub 50/ of less than 10 mm Hg, but it lacks cooperativity so is inefficient for oxygen transport. The death of double heterozygotes in late fetal or neonatal life may be in large part to oxygen deprivation to the tissues.« less

Segmental expression of Pax3/7 and engrailed homologs in tardigrade development.

PubMed

Gabriel, Willow N; Goldstein, Bob

2007-06-01

How morphological diversity arises through evolution of gene sequence is a major question in biology. In Drosophila, the genetic basis for body patterning and morphological segmentation has been studied intensively. It is clear that some of the genes in the Drosophila segmentation program are functioning similarly in certain other taxa, although many questions remain about when these gene functions arose and which taxa use these genes similarly to establish diverse body plans. Tardigrades are an outgroup to arthropods in the Ecdysozoa and, as such, can provide insight into how gene functions have evolved among the arthropods and their close relatives. We developed immunostaining methods for tardigrade embryos, and we used cross-reactive antibodies to investigate the expression of homologs of the pair-rule gene paired (Pax3/7) and the segment polarity gene engrailed in the tardigrade Hypsibius dujardini. We find that in H. dujardini embryos, Pax3/7 protein localizes not in a pair-rule pattern but in a segmentally iterated pattern, after the segments are established, in regions of the embryo where neurons later arise. Engrailed protein localizes in the posterior ectoderm of each segment before ectodermal segmentation is apparent. Together with previous results from others, our data support the conclusions that the pair-rule function of Pax3/7 is specific to the arthropods, that some of the ancient functions of Pax3/7 and Engrailed in ancestral bilaterians may have been in neurogenesis, and that Engrailed may have a function in establishing morphological boundaries between segments that is conserved at least among the Panarthropoda.

Analyses of interactions among pair-rule genes and the gap gene Krüppel in Bombyx segmentation.

PubMed

Nakao, Hajime

2015-09-01

In the short-germ insect Tribolium, a pair-rule gene circuit consisting of the Tribolium homologs of even-skipped, runt, and odd-skipped (Tc-eve, Tc-run and Tc-odd, respectively) has been implicated in segment formation. To examine the application of the model to other taxa, I studied the expression and function of pair-rule genes in Bombyx mori, together with a Bombyx homolog of Krüppel (Bm-Kr), a known gap gene. Knockdown embryos of Bombyx homologs of eve, run and odd (Bm-eve, Bm-run and Bm-odd) exhibited asegmental phenotypes similar to those of Tribolium knockdowns. However, pair-rule gene interactions were similar to those of both Tribolium and Drosophila, which, different from Tribolium, shows a hierarchical segmentation mode. Additionally, the Bm-odd expression pattern shares characteristics with those of Drosophila pair-rule genes that receive upstream regulatory input. On the other hand, Bm-Kr knockdowns exhibited a large posterior segment deletion as observed in short-germ insects. However, a detailed analysis of these embryos indicated that Bm-Kr modulates expression of pair-rule genes like in Drosophila, although the mechanisms appear to be different. This suggested hierarchical interactions between Bm-Kr and pair-rule genes. Based on these results, I concluded that the pair-rule gene circuit model that describes Tribolium development is not applicable to Bombyx. Copyright © 2015 Elsevier Inc. All rights reserved.

R-spondin 3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling in zebrafish embryos.

PubMed

Rong, Xiaozhi; Chen, Chen; Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

2014-01-01

The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos.

R-Spondin 3 Regulates Dorsoventral and Anteroposterior Patterning by Antagonizing Wnt/β-Catenin Signaling in Zebrafish Embryos

PubMed Central

Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

2014-01-01

The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos. PMID:24918770

Ectoderm gene activation in sea urchin embryos mediated by the CCAAT-binding factor.

PubMed

Li, Xiaotao; Bhattacharya, Chitralekha; Dayal, Sandeep; Maity, Sankar; Klein, William H

2002-05-01

Transcriptional enhancers are short stretches of DNA that function to achieve highly specific patterns of gene expression. To identify the mechanisms by which enhancers achieve their specificity, we made use of an enhancer from the aboral ectoderm-specific spec2a gene of the sea urchin Strongylocentrotus purpuratus. The spec2a enhancer contains five cis-regulatory elements within 78 base pairs that interact with five distinct DNA-binding proteins to confer aboral ectoderm expression. Here, we present an analysis of the sea urchin CCAAT binding factor (CBF), which binds to a CCAAT motif within the spec2a enhancer. S. purpuratus CBF and SpOtx, a ubiquitously expressed factor, act together at closely placed cis-regulatory elements to mediate spec2a transcription in the ectoderm. SpCBF was the sole factor that bound to the spec2a CCAAT element, and two of the three subunits that make up the CBF holoprotein were cloned and shown to have high sequence conservation with their vertebrate orthologs. Based on its involvement in the regulation of several other sea urchin genes, SpCBF appears to be a major transcription factor in the sea urchin embryo for positive regulation of ectoderm gene expression. In addition to its role in vertebrate cell growth and proliferation, our results indicate that CBF also functions at the early stages of germ layer formation, namely ectoderm differentiation.

Genomic structure, promoter identification, and chromosomal mapping of a mouse nuclear orphan receptor expressed in embryos and adult testes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, C.H.; Wei, Li-Na; Copeland, N.G.

We have isolated and characterized overlapping genomic clones containing the complete transcribed region of a newly isolated mouse cDNA encoding an orphan receptor expressed specifically in midgestation embryos and adult testis. This gene spans a distance of more than 50 kb and is organized into 13 exons. The transcription initiation site is located at the 158th nucleotide upstream from the translation initiation codon. All the exon/intron junction sequences follow the GT/AG rule. Based upon Northern blot analysis and the size of the transcribed region of the gene, its transcript was determined to be approximately 2.5 kb. Within approximately 500 hpmore » upstream from the transcription initiation site, several immune response regulatory elements were identified but no TATA box was located. This gene was mapped to the distal region of mouse chromosome 10 and its locus has been designated Tr2-11. Immunohistochemical studies show that the Tr2-11 protein is present mainly in advanced germ cell populations of mature testes and that Tr2-11 gene expression is dramatically decreased in vitamin A-depleted animals. 23 refs., 7 figs.« less

Elk3 is essential for the progression from progenitor to definitive neural crest cell

PubMed Central

Rogers, Crystal D.; Phillips, Jacquelyn L.; Bronner, Marianne E.

2013-01-01

Elk3/Net/Sap2 (here referred to as Elk3) is an Ets ternary complex transcriptional repressor known for its involvement in angiogenesis during embryonic development. Although Elk3 is expressed in various tissues, additional roles for the protein outside of vasculature development have yet to be reported. Here, we characterize the early spatiotemporal expression pattern of Elk3 in the avian embryo using whole mount in situ hybridization and quantitative RT-PCR and examine the effects of its loss of function on neural crest development. At early stages, Elk3 is expressed in the head folds, head mesenchyme, intersomitic vessels, and migratory cranial neural crest (NC) cells. Loss of the Elk3 protein results in the retention of Pax7+ precursors in the dorsal neural tube that fail to upregulate neural crest specifier genes, FoxD3, Sox10 and Snail2, resulting in embryos with severe migration defects. The results putatively place Elk3 downstream of neural plate border genes, but upstream of neural crest specifier genes in the neural crest gene regulatory network (NC-GRN), suggesting that it is critical for the progression from progenitor to definitive neural crest cell. PMID:23266330

Large clusters of co-expressed genes in the Drosophila genome.

PubMed

Boutanaev, Alexander M; Kalmykova, Alla I; Shevelyov, Yuri Y; Nurminsky, Dmitry I

2002-12-12

Clustering of co-expressed, non-homologous genes on chromosomes implies their co-regulation. In lower eukaryotes, co-expressed genes are often found in pairs. Clustering of genes that share aspects of transcriptional regulation has also been reported in higher eukaryotes. To advance our understanding of the mode of coordinated gene regulation in multicellular organisms, we performed a genome-wide analysis of the chromosomal distribution of co-expressed genes in Drosophila. We identified a total of 1,661 testes-specific genes, one-third of which are clustered on chromosomes. The number of clusters of three or more genes is much higher than expected by chance. We observed a similar trend for genes upregulated in the embryo and in the adult head, although the expression pattern of individual genes cannot be predicted on the basis of chromosomal position alone. Our data suggest that the prevalent mechanism of transcriptional co-regulation in higher eukaryotes operates with extensive chromatin domains that comprise multiple genes.

Effects of High Hydrostatic Pressure on Expression Profiles of In Vitro Produced Vitrified Bovine Blastocysts

PubMed Central

Jiang, Zongliang; Harrington, Patrick; Zhang, Ming; Marjani, Sadie L.; Park, Joonghoon; Kuo, Lynn; Pribenszky, Csaba; Tian, Xiuchun (Cindy)

2016-01-01

High hydrostatic pressure (HHP) has been used to pre-condition embryos before essential, yet potentially detrimental procedures such as cryopreservation. However, the mechanisms for HHP are poorly understood. We treated bovine blastocysts with three different HHP (40, 60 and 80 MPa) in combination with three recovery periods (0, 1 h, 2 h post HHP). Re-expansion rates were significantly higher at 40 and 60 but lower at 80 MPa after vitrification-warming in the treated groups than controls. Microarray analysis revealed 399 differentially expressed transcripts, representing 254 unique genes, among different groups. Gene ontology analysis indicated that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes in cell death and apoptosis, and up-regulation of genes in RNA processing, cellular growth and proliferation. In contrast, 80 MPa up-regulated genes in apoptosis, and down-regulated protein folding and cell cycle-related genes. Moreover, gene expression was also influenced by the length of the recovery time after HHP. The significantly over-represented categories were apoptosis and cell death in the 1 h group, and protein folding, response to unfolded protein and cell cycle in the 2 h group compared to 0 h. Taken together, HHP promotes competence of vitrified bovine blastocysts through modest transcriptional changes. PMID:26883277

Nuclear Photosynthetic Gene Expression Is Synergistically Modulated by Rates of Protein Synthesis in Chloroplasts and Mitochondria[W

PubMed Central

Pesaresi, Paolo; Masiero, Simona; Eubel, Holger; Braun, Hans-Peter; Bhushan, Shashi; Glaser, Elzbieta; Salamini, Francesco; Leister, Dario

2006-01-01

Arabidopsis thaliana mutants prors1-1 and -2 were identified on the basis of a decrease in effective photosystem II quantum yield. Mutations were localized to the 5′-untranslated region of the nuclear gene PROLYL-tRNA SYNTHETASE1 (PRORS1), which acts in both plastids and mitochondria. In prors1-1 and -2, PRORS1 expression is reduced, along with protein synthesis in both organelles. PRORS1 null alleles (prors1-3 and -4) result in embryo sac and embryo development arrest. In mutants with the leaky prors1-1 and -2 alleles, transcription of nuclear genes for proteins involved in photosynthetic light reactions is downregulated, whereas genes for other chloroplast proteins are upregulated. Downregulation of nuclear photosynthetic genes is not associated with a marked increase in the level of reactive oxygen species in leaves and persists in the dark, suggesting that the transcriptional response is light and photooxidative stress independent. The mrpl11 and prpl11 mutants are impaired in the mitochondrial and plastid ribosomal L11 proteins, respectively. The prpl11 mrpl11 double mutant, but neither of the single mutants, resulted in strong downregulation of nuclear photosynthetic genes, like that seen in leaky mutants for PRORS1, implying that, when organellar translation is perturbed, signals derived from both types of organelles cooperate in the regulation of nuclear photosynthetic gene expression. PMID:16517761

Gene discovery in an invasive tephritid model pest species, the Mediterranean fruit fly, Ceratitis capitata

PubMed Central

Gomulski, Ludvik M; Dimopoulos, George; Xi, Zhiyong; Soares, Marcelo B; Bonaldo, Maria F; Malacrida, Anna R; Gasperi, Giuliano

2008-01-01

Background The medfly, Ceratitis capitata, is a highly invasive agricultural pest that has become a model insect for the development of biological control programs. Despite research into the behavior and classical and population genetics of this organism, the quantity of sequence data available is limited. We have utilized an expressed sequence tag (EST) approach to obtain detailed information on transcriptome signatures that relate to a variety of physiological systems in the medfly; this information emphasizes on reproduction, sex determination, and chemosensory perception, since the study was based on normalized cDNA libraries from embryos and adult heads. Results A total of 21,253 high-quality ESTs were obtained from the embryo and head libraries. Clustering analyses performed separately for each library resulted in 5201 embryo and 6684 head transcripts. Considering an estimated 19% overlap in the transcriptomes of the two libraries, they represent about 9614 unique transcripts involved in a wide range of biological processes and molecular functions. Of particular interest are the sequences that share homology with Drosophila genes involved in sex determination, olfaction, and reproductive behavior. The medfly transformer2 (tra2) homolog was identified among the embryonic sequences, and its genomic organization and expression were characterized. Conclusion The sequences obtained in this study represent the first major dataset of expressed genes in a tephritid species of agricultural importance. This resource provides essential information to support the investigation of numerous questions regarding the biology of the medfly and other related species and also constitutes an invaluable tool for the annotation of complete genome sequences. Our study has revealed intriguing findings regarding the transcript regulation of tra2 and other sex determination genes, as well as insights into the comparative genomics of genes implicated in chemosensory reception and reproduction. PMID:18500975

The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene.

PubMed

Lynn, K; Fernandez, A; Aida, M; Sedbrook, J; Tasaka, M; Masson, P; Barton, M K

1999-02-01

Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to form shoot apical meristems. Factors required for this competence are expected to cause a defect in shoot apical meristem formation when inactivated and to be expressed or active preferentially in the adaxial leaf domain. PINHEAD, a member of a family of proteins that includes the translation factor eIF2C, is required for reliable formation of primary and axillary shoot apical meristems. In addition to high-level expression in the vasculature, we find that low-level PINHEAD expression defines a novel domain of positional identity in the plant. This domain consists of adaxial leaf primordia and the meristem. These findings suggest that the PINHEAD gene product may be a component of a hypothetical meristem forming competence factor. We also describe defects in floral organ number and shape, as well as aberrant embryo and ovule development associated with pinhead mutants, thus elaborating on the role of PINHEAD in Arabidopsis development. In addition, we find that embryos doubly mutant for PINHEAD and ARGONAUTE1, a related, ubiquitously expressed family member, fail to progress to bilateral symmetry and do not accumulate the SHOOT MERISTEMLESS protein. Therefore PINHEAD and ARGONAUTE1 together act to allow wild-type growth and gene expression patterns during embryogenesis.

Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

PubMed Central

Timme-Laragy, Alicia R.; Goldstone, Jared V.; Imhoff, Barry R.; Stegeman, John J.; Hahn, Mark E.; Hansen, Jason M.

2013-01-01

Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous non-protein antioxidant defense molecule is the tri-peptide glutathione (γ-glutamyl-cysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0–5 days post-fertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione (GSH, GSSG) using HPLC, and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0–120 hours of zebrafish development (including mature oocytes, fertilization, mid-blastula transition, gastrulation, somitogenesis, pharyngula, pre-hatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12 hours post fertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12 h, and then oscillated around −190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (−220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis. PMID:23770340

Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes

PubMed Central

dos Santos Neto, Pedro C.; Cuadro, Federico; Bosolasco, Diego; Mulet, Ana P.; Crispo, Martina

2018-01-01

High lipid content of oocytes and embryos in domestic animals is one of the well-known factors associated with poor cryosurvival. Herein, we wanted to determine whether the use of delipidated estrous sheep serum during in vitro maturation (IVM) of ovine oocytes reduces the cytoplasmic lipid droplets content and improves embryo development and cryotolerance after vitrification. Cumulus oocytes complexes (COCs) were matured in vitro for 24 h in medium supplemented with whole or delipidated estrous sheep serum prior to vitrification. Neutral lipid present in lipid droplets of COCs, cleavage rate, embryo development rate on Day 6 and Day 8, and hatching rate on Day 8, were compared among experimental groups. Endoplasmic reticulum stress genes were evaluated in in vitro matured COCs under different lipid conditions prior to vitrification. The lipid droplets’ content (mean fluorescence intensity) of oocytes cultured with IVM media supplemented with delipidated serum was lower than COCs matured with whole serum (7.6 ± 1.7 vs. 22.8 ± 5.0 arbitrary units, respectively; P< 0.05). Despite IVM treatment, oocytes subjected to vitrification showed impaired competence compared with the non-vitrified groups (P<0.05). No significant differences in embryo production were observed in non-vitrified COCs after maturation in delipidated or whole serum (33.4±4.9 vs 31.9 ±4.2). COCs matured in delipidated serum and subjected to vitrification showed increased expression of ATF4, ATF6, GRP78, and CHOP10 genes (ER stress markers). Collectively, our results demonstrate that although supplementation of IVM medium with delipidated estrous sheep serum reduces the presence of cytoplasmic lipid droplets in oocytes after maturation, oocyte cryotolerance is not improved. Notably, the expression of genes associated with the unfolded protein response (UPR) was increased in COCs, with fewer lipid droplets subjected to vitrification, suggesting that oocyte cryopreservation is associated with ER stress and activation of adaptive responses. PMID:29912910

A mutation in the tuft mouse disrupts TET1 activity and alters the expression of genes that are crucial for neural tube closure.

PubMed

Fong, Keith S K; Hufnagel, Robert B; Khadka, Vedbar S; Corley, Michael J; Maunakea, Alika K; Fogelgren, Ben; Ahmed, Zubair M; Lozanoff, Scott

2016-05-01

Genetic variations affecting neural tube closure along the head result in malformations of the face and brain. Neural tube defects (NTDs) are among the most common birth defects in humans. We previously reported a mouse mutant called tuft that arose spontaneously in our wild-type 3H1 colony. Adult tuft mice present midline craniofacial malformations with or without an anterior cephalocele. In addition, affected embryos presented neural tube closure defects resulting in insufficient closure of the anterior neuropore or exencephaly. Here, through whole-genome sequencing, we identified a nonsense mutation in the Tet1 gene, which encodes a methylcytosine dioxygenase (TET1), co-segregating with the tuft phenotype. This mutation resulted in premature termination that disrupts the catalytic domain that is involved in the demethylation of cytosine. We detected a significant loss of TET enzyme activity in the heads of tuft embryos that were homozygous for the mutation and had NTDs. RNA-Seq transcriptome analysis indicated that multiple gene pathways associated with neural tube closure were dysregulated in tuft embryo heads. Among them, the expressions of Cecr2, Epha7 and Grhl2 were significantly reduced in some embryos presenting neural tube closure defects, whereas one or more components of the non-canonical WNT signaling pathway mediating planar cell polarity and convergent extension were affected in others. We further show that the recombinant mutant TET1 protein was capable of entering the nucleus and affected the expression of endogenous Grhl2 in IMCD-3 (inner medullary collecting duct) cells. These results indicate that TET1 is an epigenetic determinant for regulating genes that are crucial to closure of the anterior neural tube and its mutation has implications to craniofacial development, as presented by the tuft mouse. © 2016. Published by The Company of Biologists Ltd.

Expression of neuronal antigens and related ventral and dorsal proteins in the normal spinal cord and a surgically induced open neural tube defect of the spine in chick embryos: an immunohistochemical study.

PubMed

Lee, Do-Hun; Phi, Ji Hoon; Chung, You-Nam; Lee, Yun-Jin; Kim, Seung-Ki; Cho, Byung-Kyu; Kim, Dong Won; Park, Moon-Sik; Wang, Kyu-Chang

2010-05-01

The aims of this study were to elucidate the processes of neuronal differentiation and ventrodorsal patterning in the spinal cord of the chick embryo from embryonic day (E) 3 to E17 and to study the effect of a prenatal spinal open neural tube defect (ONTD) on these processes. Expression patterns of neuronal antigens (neuronal nuclear antigen, neurofilament-associated protein (NAP), and synaptophysin) and related ventral markers [sonic hedgehog, paired box gene (PAX)6, and islet-1], and dorsal markers (bone morphogenetic protein, Notch homolog 1, and PAX7) were investigated in the normal spinal cord and in a surgically induced spinal ONTD in chick embryos. Four normal and ONTD chick embryos were used for each antigen group. There were no differences in the expression of neuronal and ventrodorsal markers between the control and ONTD groups. NAP and synaptophysin were useful for identifying dorsal structures in the distorted anatomy of the ONTD chicks.

Cloning and expression of sheep DNA methyltransferase 1 and its development-specific isoform.

PubMed

Taylor, Jane; Moore, Hannah; Beaujean, Nathalie; Gardner, John; Wilmut, Ian; Meehan, Richard; Young, Lorraine

2009-05-01

Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8-16 cell stage. Attempts to clone oocyte-specific 5' regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi-mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation. (c) 2008 Wiley-Liss, Inc.

A transcriptional blueprint for a spiral-cleaving embryo.

PubMed

Chou, Hsien-Chao; Pruitt, Margaret M; Bastin, Benjamin R; Schneider, Stephan Q

2016-08-05

The spiral cleavage mode of early development is utilized in over one-third of all animal phyla and generates embryonic cells of different size, position, and fate through a conserved set of stereotypic and invariant asymmetric cell divisions. Despite the widespread use of spiral cleavage, regulatory and molecular features for any spiral-cleaving embryo are largely uncharted. To address this gap we use RNA-sequencing on the spiralian model Platynereis dumerilii to capture and quantify the first complete genome-wide transcriptional landscape of early spiral cleavage. RNA-sequencing datasets from seven stages in early Platynereis development, from the zygote to the protrochophore, are described here including the de novo assembly and annotation of ~17,200 Platynereis genes. Depth and quality of the RNA-sequencing datasets allow the identification of the temporal onset and level of transcription for each annotated gene, even if the expression is restricted to a single cell. Over 4000 transcripts are maternally contributed and cleared by the end of the early spiral cleavage phase. Small early waves of zygotic expression are followed by major waves of thousands of genes, demarcating the maternal to zygotic transition shortly after the completion of spiral cleavages in this annelid species. Our comprehensive stage-specific transcriptional analysis of early embryonic stages in Platynereis elucidates the regulatory genome during early spiral embryogenesis and defines the maternal to zygotic transition in Platynereis embryos. This transcriptome assembly provides the first systems-level view of the transcriptional and regulatory landscape for a spiral-cleaving embryo.

The role of arginine metabolic pathway during embryogenesis and germination in maritime pine (Pinus pinaster Ait.).

PubMed

Llebrés, María-Teresa; Pascual, María-Belén; Debille, Sandrine; Trontin, Jean-François; Harvengt, Luc; Avila, Concepción; Cánovas, Francisco M

2018-03-01

Vegetative propagation through somatic embryogenesis is critical in conifer biotechnology towards multivarietal forestry that uses elite varieties to cope with environmental and socio-economic issues. An important and still sub-optimal process during in vitro maturation of somatic embryos (SE) is the biosynthesis and deposition of storage proteins, which are rich in amino acids with high nitrogen (N) content, such as arginine. Mobilization of these N-rich proteins is essential for the germination and production of vigorous somatic seedlings. Somatic embryos accumulate lower levels of N reserves than zygotic embryos (ZE) at a similar stage of development. To understand the molecular basis for this difference, the arginine metabolic pathway has been characterized in maritime pine (Pinus pinaster Ait.). The genes involved in arginine metabolism have been identified and GFP-fusion constructs were used to locate the enzymes in different cellular compartments and clarify their metabolic roles during embryogenesis and germination. Analysis of gene expression during somatic embryo maturation revealed high levels of transcripts for genes involved in the biosynthesis and metabolic utilization of arginine. By contrast, enhanced expression levels were only observed during the last stages of maturation and germination of ZE, consistent with the adequate accumulation and mobilization of protein reserves. These results suggest that arginine metabolism is unbalanced in SE (simultaneous biosynthesis and degradation of arginine) and could explain the lower accumulation of storage proteins observed during the late stages of somatic embryogenesis.

Spontaneous neural tube defects in splotch mice supplemented with selected micronutrients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wlodarczyk, Bogdan J.; Tang, Louisa S.; Triplett, Aleata

Splotch (Sp/Sp) mice homozygous for a mutation in the Pax3 gene inevitably present with neural tube defects (NTDs), along with other associated congenital anomalies. The affected mutant embryos usually die by gestation days (E) 12-13. In the present study, the effect of modifier genes from a new genetic background (CXL-Sp) and periconceptional supplementation with selected micronutrients (folic acid, 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, methionine, myoinositol, thiamine, thymidine, and {alpha}-tocopherol) was determined with respect to the incidence of NTDs. In order to explore how different exposure parameters (time, dose, and route of compound administration) modulate the beneficial effects of micronutrient supplementation, female mice receivedmore » either short- or long-term nutrient supplements via enteral or parenteral routes. Embryos were collected on E12.5 and examined for the presence of anterior or posterior NTDs. Additionally, whole mount in situ hybridization studies were conducted in order to reveal/confirm normal expression patterns of the Pax3 gene during neurulation in the wild-type and Sp/Sp homozygous mutant mouse embryos utilized in this study. A strong Pax3 signal was demonstrated in CXL-Sp embryos during neural tube closure (E9.5 to E10.5). The intensity and spatial pattern of expression were similar to other Splotch mutant mice. Of all the micronutrients tested, only supplementation with folic acid or 5-methyltetrahydrofolate rescued the normal phenotype in Sp/Sp embryos. When the folate supplementation dose was increased to 200 mg/kg in the diet, the incidence of rescued splotch homozygotes reached 30%; however, this was accompanied by six-fold increased resorption rate.« less

Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent

PubMed Central

2009-01-01

Background In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin. Results When tested against different alternate selection agents our studies show that 0.16 μg/mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl)-L-cysteine) (AEC) and the acetolactate synthase (ALS) inhibitors Exceed® and Synchrony® both at 150 μg/mL inhibited soybean somatic embryo growth. Even at the concentration of 2 mg/mL, lysine+threonine (LT) were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike the plant enzyme, dihydrodipicolinate synthase (DHPS) from E. coli is not feed-back inhibited by physiological concentrations of lysine. The dapA gene which codes for E. coli DHPS was expressed in soybean somatic embryos under the control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed. Conclusion Genetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate) (Roundup®), AEC and the ALS inhibitors Exceed® and Synchrony® against different tissues of soybean PMID:19922622

Epigenomic Landscape of Human Fetal Brain, Heart, and Liver.

PubMed

Yan, Liying; Guo, Hongshan; Hu, Boqiang; Li, Rong; Yong, Jun; Zhao, Yangyu; Zhi, Xu; Fan, Xiaoying; Guo, Fan; Wang, Xiaoye; Wang, Wei; Wei, Yuan; Wang, Yan; Wen, Lu; Qiao, Jie; Tang, Fuchou

2016-02-26

The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression analysis of fertilization/early embryogenesis-associated genes in Phalaenopsis orchids.

PubMed

Chen, Jhun-Chen; Wei, Miao-Ju; Fang, Su-Chiung

2016-10-02

One of the distinct reproductive programs in orchid species is pollination-triggered ovule development and megasporogenesis. During sexual reproduction, fertilization occurs days to months after pollination. The molecular mechanisms evolved to carry out this strategic reproductive program remain unclear. In the August issue of Plant Physiology 1 , we report comprehensive studies of comparative genome-wide gene expression in various reproductive tissues and the molecular events associated with developmental transitions unique to sexual reproduction of Phalaenopsis aphrodite. Transcriptional factors and signaling components whose expression is specifically enriched in interior ovary tissues when fertilization occurs and embryos start to develop have been identified. Here, we report verification of additional fertilization-associated genes, DOMAINS REARRANGED METHYLTRANSFERASE 1 (PaDRM1), CHROMOMETHYLTRANSFERASE 1 (PaCMT1), SU(VAR)3-9 RELATED PROTEIN 1 (PaSUVR1), INDOLE-3-ACETIC ACID inducible 30-like 1 (PaIAA30L1), and ETHYLENE INSENSITIVE 3-like 1 (PaEIN3L1), and discuss their potential roles in gametophyte development, epigenetic reprogramming, and hormone regulation during fertilization and establishment of embryo development in Phalaenopsis orchids.