Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis

PubMed Central

2013-01-01

Background In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Results Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. Conclusion mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry

Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Chowdhury, Debabani Roy; Bhadra, Utpal; Pal-Bhadra, Manika

2013-01-24

In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof¹/+; mnkp⁶/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF

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.

The mRNA-bound proteome of the early fly embryo

PubMed Central

Wessels, Hans-Hermann; Imami, Koshi; Baltz, Alexander G.; Kolinski, Marcin; Beldovskaya, Anastasia; Selbach, Matthias; Small, Stephen; Ohler, Uwe; Landthaler, Markus

2016-01-01

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation. PMID:27197210

Biochemical defects of mutant nudel alleles causing early developmental arrest or dorsalization of the Drosophila embryo.

PubMed Central

LeMosy, E K; Leclerc, C L; Hashimoto, C

2000-01-01

The nudel gene of Drosophila is maternally required both for structural integrity of the egg and for dorsoventral patterning of the embryo. It encodes a structurally modular protein that is secreted by ovarian follicle cells. Genetic and molecular studies have suggested that the Nudel protein is also functionally modular, with a serine protease domain that is specifically required for ventral development. Here we describe biochemical and immunolocalization studies that provide insight into the molecular basis for the distinct phenotypes produced by nudel mutations and for the interactions between these alleles. Mutations causing loss of embryonic dorsoventral polarity result in a failure to activate the protease domain of Nudel. Our analyses support previous findings that catalytic activity of the protease domain is required for dorsoventral patterning and that the Nudel protease is auto-activated and reveal an important role for a region adjacent to the protease domain in Nudel protease function. Mutations causing egg fragility and early embryonic arrest result in a significant decrease in extracellular Nudel protein, due to defects in post-translational processing, stability, or secretion. On the basis of these and other studies of serine proteases, we suggest potential mechanisms for the complementary and antagonistic interactions between the nudel alleles. PMID:10628985

The Development of Motor Coordination in Drosophila Embryos

PubMed Central

Crisp, Sarah; Evers, Jan Felix; Fiala, André; Bate, Michael

2012-01-01

We use non-invasive muscle imaging to study onset of motor activity and emergence of coordinated movement in Drosophila embryos. Earliest movements are myogenic and neurally controlled muscle contractions first appear with the onset of bursting activity 17 hours after egg laying. Initial episodes of activity are poorly organised and coordinated crawling sequences only begin to appear after a further hour of bursting. Thus network performance improves during this first period of activity. The embryo continues to exhibit bursts of crawling like sequences until shortly before hatching, while other reflexes also mature. Bursting does not begin as a reflex response to sensory input but appears to reflect the onset of spontaneous activity in the motor network. It does not require GABA-ergic transmission, and using a light activated channel to excite the network we demonstrate activity dependent depression that may cause burst termination. PMID:18927150

Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size.

PubMed

Puah, Wee Choo; Chinta, Rambabu; Wasser, Martin

2017-03-15

Time-lapse microscopy is a powerful tool to investigate cellular and developmental dynamics. In Drosophila melanogaster , it can be used to study division cycles in embryogenesis. To obtain quantitative information from 3D time-lapse data and track proliferating nuclei from the syncytial stage until gastrulation, we developed an image analysis pipeline consisting of nuclear segmentation, tracking, annotation and quantification. Image analysis of maternal-haploid ( mh ) embryos revealed that a fraction of haploid syncytial nuclei fused to give rise to nuclei of higher ploidy (2n, 3n, 4n). Moreover, nuclear densities in mh embryos at the mid-blastula transition varied over threefold. By tracking synchronized nuclei of different karyotypes side-by-side, we show that DNA content determines nuclear growth rate and size in early interphase, while the nuclear to cytoplasmic ratio constrains nuclear growth during late interphase. mh encodes the Drosophila ortholog of human Spartan, a protein involved in DNA damage tolerance. To explore the link between mh and chromosome instability, we fluorescently tagged Mh protein to study its subcellular localization. We show Mh-mKO2 localizes to nuclear speckles that increase in numbers as nuclei expand in interphase. In summary, quantitative microscopy can provide new insights into well-studied genes and biological processes. © 2017. Published by The Company of Biologists Ltd.

Multiscale diffusion in the mitotic Drosophila melanogaster syncytial blastoderm

PubMed Central

Daniels, Brian R.; Rikhy, Richa; Renz, Malte; Dobrowsky, Terrence M.; Lippincott-Schwartz, Jennifer

2012-01-01

Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional “compartmentalization” has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins. Our results demonstrate that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Using a combination of live-cell experiments and computational modeling, we characterize these effects and relate effective bulk diffusion rates to instantaneous diffusion coefficients throughout the syncytial blastoderm nuclear cycle phase of the early embryo. This multiscale effect may be related to the effect of interphase nuclei on effective diffusion, and thus we propose that an as-yet-unidentified role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients. PMID:22592793

Convergence of topological domain boundaries, insulators, and polytene interbands revealed by high-resolution mapping of chromatin contacts in the early Drosophila melanogaster embryo

PubMed Central

Stadler, Michael R; Haines, Jenna E

2017-01-01

High-throughput assays of three-dimensional interactions of chromosomes have shed considerable light on the structure of animal chromatin. Despite this progress, the precise physical nature of observed structures and the forces that govern their establishment remain poorly understood. Here we present high resolution Hi-C data from early Drosophila embryos. We demonstrate that boundaries between topological domains of various sizes map to DNA elements that resemble classical insulator elements: short genomic regions sensitive to DNase digestion that are strongly bound by known insulator proteins and are frequently located between divergent promoters. Further, we show a striking correspondence between these elements and the locations of mapped polytene interband regions. We believe it is likely this relationship between insulators, topological boundaries, and polytene interbands extends across the genome, and we therefore propose a model in which decompaction of boundary-insulator-interband regions drives the organization of interphase chromosomes by creating stable physical separation between adjacent domains. PMID:29148971

Methanol exposure interferes with morphological cell movements in the Drosophila embryo and causes increased apoptosis in the CNS.

PubMed

Mellerick, Dervla M; Liu, Heather

2004-09-05

Despite the significant contributions of tissue culture and bacterial models to toxicology, whole animal models for developmental neurotoxins are limited in availability and ease of experimentation. Because Drosophila is a well understood model for embryonic development that is highly accessible, we asked whether it could be used to study methanol developmental neurotoxicity. In the presence of 4% methanol, approximately 35% of embryos die and methanol exposure leads to severe CNS defects in about half those embryos, where the longitudinal connectives are dorsally displaced and commissure formation is severely reduced. In addition, a range of morphological defects in other germ layers is seen, and cell movement is adversely affected by methanol exposure. Although we did not find any evidence to suggest that methanol exposure affects the capacity of neuroblasts to divide or induces inappropriate apoptosis in these cells, in the CNS of germ band retracted embryos, the number of apoptotic nuclei is significantly increased in methanol-exposed embryos in comparison to controls, particularly in and adjacent to the ventral midline. Apoptosis contributes significantly to methanol neurotoxicity because embryos lacking the cell death genes grim, hid, and reaper have milder CNS defects resulting from methanol exposure than wild-type embryos. Our data suggest that when neurons and glia are severely adversely affected by methanol exposure, the damaged cells are cleared by apoptosis, leading to embryonic death. Thus, the Drosophila embryo may prove useful in identifying and unraveling mechanistic aspects of developmental neurotoxicity, specifically in relation to methanol toxicity.

The use of centrifugation to study early Drosophila embryogenesis

NASA Technical Reports Server (NTRS)

Abbott, M. K.; Spooner, B. S. (Principal Investigator)

1993-01-01

By the end of 10th nuclear cycle, the somatic nuclei of the Drosophila embryo have migrated to the periphery of the egg. Centrifugation of embryos did not result in the displacement of these nuclei, since cytoskeletal elements anchor them to the cortex. But, mild centrifugal forces displace the centrally located, nascent yolk nuclei. If this increased sensitivity to hypergravity occurs before the beginning of nuclear differentiation during cycle 8, when the nascent yolk and somatic nuclei physically separate, then it would mark the earliest functional difference between these two lineages.

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay.

PubMed

Hudry, Bruno; Viala, Séverine; Graba, Yacine; Merabet, Samir

2011-01-28

Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.

Metabolome analysis of Drosophila melanogaster during embryogenesis.

PubMed

An, Phan Nguyen Thuy; Yamaguchi, Masamitsu; Bamba, Takeshi; Fukusaki, Eiichiro

2014-01-01

The Drosophila melanogaster embryo has been widely utilized as a model for genetics and developmental biology due to its small size, short generation time, and large brood size. Information on embryonic metabolism during developmental progression is important for further understanding the mechanisms of Drosophila embryogenesis. Therefore, the aim of this study is to assess the changes in embryos' metabolome that occur at different stages of the Drosophila embryonic development. Time course samples of Drosophila embryos were subjected to GC/MS-based metabolome analysis for profiling of low molecular weight hydrophilic metabolites, including sugars, amino acids, and organic acids. The results showed that the metabolic profiles of Drosophila embryo varied during the course of development and there was a strong correlation between the metabolome and different embryonic stages. Using the metabolome information, we were able to establish a prediction model for developmental stages of embryos starting from their high-resolution quantitative metabolite composition. Among the important metabolites revealed from our model, we suggest that different amino acids appear to play distinct roles in different developmental stages and an appropriate balance in trehalose-glucose ratio is crucial to supply the carbohydrate source for the development of Drosophila embryo.

Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos.

PubMed

Deneke, Victoria E; Melbinger, Anna; Vergassola, Massimo; Di Talia, Stefano

2016-08-22

Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. Copyright © 2016 Elsevier Inc. All rights reserved.

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay

PubMed Central

2011-01-01

Background Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Results Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Conclusion Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development. PMID:21276241

Metabolome Analysis of Drosophila melanogaster during Embryogenesis

PubMed Central

An, Phan Nguyen Thuy; Yamaguchi, Masamitsu; Bamba, Takeshi; Fukusaki, Eiichiro

2014-01-01

The Drosophila melanogaster embryo has been widely utilized as a model for genetics and developmental biology due to its small size, short generation time, and large brood size. Information on embryonic metabolism during developmental progression is important for further understanding the mechanisms of Drosophila embryogenesis. Therefore, the aim of this study is to assess the changes in embryos’ metabolome that occur at different stages of the Drosophila embryonic development. Time course samples of Drosophila embryos were subjected to GC/MS-based metabolome analysis for profiling of low molecular weight hydrophilic metabolites, including sugars, amino acids, and organic acids. The results showed that the metabolic profiles of Drosophila embryo varied during the course of development and there was a strong correlation between the metabolome and different embryonic stages. Using the metabolome information, we were able to establish a prediction model for developmental stages of embryos starting from their high-resolution quantitative metabolite composition. Among the important metabolites revealed from our model, we suggest that different amino acids appear to play distinct roles in different developmental stages and an appropriate balance in trehalose-glucose ratio is crucial to supply the carbohydrate source for the development of Drosophila embryo. PMID:25121768

Spatial Reorganization of the Endoplasmic Reticulum during Mitosis Relies on Mitotic Kinase Cyclin A in the Early Drosophila Embryo

PubMed Central

Bergman, Zane J.; Mclaurin, Justin D.; Eritano, Anthony S.; Johnson, Brittany M.; Sims, Amanda Q.; Riggs, Blake

2015-01-01

Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope. PMID:25689737

Protein O-Mannosyltransferases Affect Sensory Axon Wiring and Dynamic Chirality of Body Posture in the Drosophila Embryo.

PubMed

Baker, Ryan; Nakamura, Naosuke; Chandel, Ishita; Howell, Brooke; Lyalin, Dmitry; Panin, Vladislav M

2018-02-14

Genetic defects in protein O-mannosyltransferase 1 (POMT1) and POMT2 underlie severe muscular dystrophies. POMT genes are evolutionarily conserved in metazoan organisms. In Drosophila , both male and female POMT mutants show a clockwise rotation of adult abdominal segments, suggesting a chirality of underlying pathogenic mechanisms. Here we described and analyzed a similar phenotype in POMT mutant embryos that shows left-handed body torsion. Our experiments demonstrated that coordinated muscle contraction waves are associated with asymmetric embryo rolling, unveiling a new chirality marker in Drosophila development. Using genetic and live-imaging approaches, we revealed that the torsion phenotype results from differential rolling and aberrant patterning of peristaltic waves of muscle contractions. Our results demonstrated that peripheral sensory neurons are required for normal contractions that prevent the accumulation of torsion. We found that POMT mutants show abnormal axonal connections of sensory neurons. POMT transgenic expression limited to sensory neurons significantly rescued the torsion phenotype, axonal connectivity defects, and abnormal contractions in POMT mutant embryos. Together, our data suggested that protein O-mannosylation is required for normal sensory feedback to control coordinated muscle contractions and body posture. This mechanism may shed light on analogous functions of POMT genes in mammals and help to elucidate the etiology of neurological defects in muscular dystrophies. SIGNIFICANCE STATEMENT Protein O-mannosyltransferases (POMTs) are evolutionarily conserved in metazoans. Mutations in POMTs cause severe muscular dystrophies associated with pronounced neurological defects. However, neurological functions of POMTs remain poorly understood. We demonstrated that POMT mutations in Drosophila result in abnormal muscle contractions and cause embryo torsion. Our experiments uncovered a chirality of embryo movements and a unique POMT -dependent

Evidence for Functional Differentiation among Drosophila Septins in Cytokinesis and Cellularization

PubMed Central

Adam, Jennifer C.; Pringle, John R.; Peifer, Mark

2000-01-01

The septins are a conserved family of proteins that are involved in cytokinesis and other aspects of cell-surface organization. In Drosophila melanogaster, null mutations in the pnut septin gene are recessive lethal, but homozygous pnut mutants complete embryogenesis and survive until the pupal stage. Because the completion of cellularization and other aspects of early development seemed likely to be due to maternally contributed Pnut product, we attempted to generate embryos lacking the maternal contribution in order to explore the roles of Pnut in these processes. We used two methods, the production of germline clones homozygous for a pnut mutation and the rescue of pnut homozygous mutant flies by a pnut+ transgene under control of the hsp70 promoter. Remarkably, the pnut germline-clone females produced eggs, indicating that stem-cell and cystoblast divisions in the female germline do not require Pnut. Moreover, the Pnut-deficient embryos obtained by either method completed early syncytial development and began cellularization of the embryo normally. However, during the later stages of cellularization, the organization of the actin cytoskeleton at the leading edge of the invaginating furrows became progressively more abnormal, and the embryos displayed widespread defects in cell and embryo morphology beginning at gastrulation. Examination of two other septins showed that Sep1 was not detectable at the cellularization front in the Pnut-deficient embryos, whereas Sep2 was still present in normal levels. Thus, it is possible that Sep2 (perhaps in conjunction with other septins such as Sep4 and Sep5) fulfills an essential septin role during the organization and initial ingression of the cellularization furrow even in the absence of Pnut and Sep1. Together, the results suggest that some cell-division events in Drosophila do not require septin function, that there is functional differentiation among the Drosophila septins, or both. PMID:10982405

Loss of PTB or Negative Regulation of Notch mRNA Reveals Distinct Zones of Notch and Actin Protein Accumulation in Drosophila Embryo

PubMed Central

Wesley, Cedric S.; Guo, Heng; Chaudhry, Kanita A.; Thali, Markus J.; Yin, Jerry C.; Clason, Todd; Wesley, Umadevi V.

2011-01-01

Polypyrimidine Tract Binding (PTB) protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1) the Notch mRNA is a potential target of PTB, (2) PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3) the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions. PMID:21750738

Origin and specification of type II neuroblasts in the Drosophila embryo.

PubMed

Álvarez, José-Andrés; Díaz-Benjumea, Fernando J

2018-04-05

In Drosophila , neural stem cells or neuroblasts (NBs) acquire different identities according to their site of origin in the embryonic neuroectoderm. Their identity determines the number of times they will divide and the types of daughter cells they will generate. All NBs divide asymmetrically, with type I NBs undergoing self-renewal and generating another cell that will divide only once more. By contrast, a small set of NBs in the larval brain, type II NBs, divides differently, undergoing self-renewal and generating an intermediate neural progenitor (INP) that continues to divide asymmetrically several more times, generating larger lineages. In this study, we have analysed the origin of type II NBs and how they are specified. Our results indicate that these cells originate in three distinct clusters in the dorsal protocerebrum during stage 12 of embryonic development. Moreover, it appears that their specification requires the combined action of EGFR signalling and the activity of the related genes buttonhead and Drosophila Sp1 In addition, we also show that the INPs generated in the embryo enter quiescence at the end of embryogenesis, resuming proliferation during the larval stage. © 2018. Published by The Company of Biologists Ltd.

Ultra-deep sequencing of ribosome-associated poly-adenylated RNA in early Drosophila embryos reveals hundreds of conserved translated sORFs.

PubMed

Li, Hongmei; Hu, Chuansheng; Bai, Ling; Li, Hua; Li, Mingfa; Zhao, Xiaodong; Czajkowsky, Daniel M; Shao, Zhifeng

2016-12-01

There is growing recognition that small open reading frames (sORFs) encoding peptides shorter than 100 amino acids are an important class of functional elements in the eukaryotic genome, with several already identified to play critical roles in growth, development, and disease. However, our understanding of their biological importance has been hindered owing to the significant technical challenges limiting their annotation. Here we combined ultra-deep sequencing of ribosome-associated poly-adenylated RNAs with rigorous conservation analysis to identify a comprehensive population of translated sORFs during early Drosophila embryogenesis. In total, we identify 399 sORFs, including those previously annotated but without evidence of translational capacity, those found within transcripts previously classified as non-coding, and those not previously known to be transcribed. Further, we find, for the first time, evidence for translation of many sORFs with different isoforms, suggesting their regulation is as complex as longer ORFs. Furthermore, many sORFs are found not associated with ribosomes in late-stage Drosophila S2 cells, suggesting that many of the translated sORFs may have stage-specific functions during embryogenesis. These results thus provide the first comprehensive annotation of the sORFs present during early Drosophila embryogenesis, a necessary basis for a detailed delineation of their function in embryogenesis and other biological processes. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Glassfrog embryos hatch early after parental desertion.

PubMed

Delia, Jesse R J; Ramírez-Bautista, Aurelio; Summers, Kyle

2014-06-22

Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations.

Glassfrog embryos hatch early after parental desertion

PubMed Central

Delia, Jesse R. J.; Ramírez-Bautista, Aurelio; Summers, Kyle

2014-01-01

Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations. PMID:24789892

Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids

PubMed Central

Doubrovinski, Konstantin; Swan, Michael; Polyakov, Oleg; Wieschaus, Eric F.

2017-01-01

Many models of morphogenesis are forced to assume specific mechanical properties of cells, because the actual mechanical properties of living tissues are largely unknown. Here, we measure the rheology of epithelial cells in the cellularizing Drosophila embryo by injecting magnetic particles and studying their response to external actuation. We establish that, on timescales relevant to epithelial morphogenesis, the cytoplasm is predominantly viscous, whereas the cellular cortex is elastic. The timescale of elastic stress relaxation has a lower bound of 4 min, which is comparable to the time required for internalization of the ventral furrow during gastrulation. The cytoplasm was measured to be ∼103-fold as viscous as water. We show that elasticity depends on the actin cytoskeleton and conclude by discussing how these results relate to existing mechanical models of morphogenesis. PMID:28096360

Embryo sac formation and early embryo development in Agave tequilana (Asparagaceae).

PubMed

González-Gutiérrez, Alejandra G; Gutiérrez-Mora, Antonia; Rodríguez-Garay, Benjamín

2014-01-01

Agave tequilana is an angiosperm species that belongs to the family Asparagaceae (formerly Agavaceae). Even though there is information regarding to some aspects related to the megagametogenesis of A. tequilana, this is the first report describing the complete process of megasporogenesis, megagametogenesis, the early embryo and endosperm development process in detail. The objective of this work was to study and characterize all the above processes and the distinctive morphological changes of the micropylar and chalazal extremes after fertilization in this species. The agave plant material for the present study was collected from commercial plantations in the state of Jalisco, Mexico. Ovules and immature seeds, previously fixed in FAA and kept in ethanol 70%, were stained based on a tissue clarification technique by using a Mayer's-Hematoxylin solution. The tissue clarification technique was successfully used for the characterization of the megasporogenesis, megagametogenesis, mature embryo sac formation, the early embryo and endosperm development processes by studying intact cells. The embryo sac of A. tequilana was confirmed to be of the monosporic Polygonum-type and an helobial endosperm formation. Also, the time-lapse of the developmental processes studied was recorded.

Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

PubMed

Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

2014-10-13

Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1 hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

Efficient harvesting methods for early-stage snake and turtle embryos.

PubMed

Matsubara, Yoshiyuki; Kuroiwa, Atsushi; Suzuki, Takayuki

2016-04-01

Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle. © 2016 Japanese Society of Developmental Biologists.

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

Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila

PubMed Central

Sysoev, Vasiliy O.; Fischer, Bernd; Frese, Christian K.; Gupta, Ishaan; Krijgsveld, Jeroen; Hentze, Matthias W.; Castello, Alfredo; Ephrussi, Anne

2016-01-01

The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development. PMID:27378189

Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage

PubMed Central

Pires, Camilla Valente; Freitas, Flávia Cristina de Paula; Cristino, Alexandre S.; Dearden, Peter K.; Simões, Zilá Luz Paulino

2016-01-01

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development. PMID:26751956

Sulfation of Eggshell Proteins by Pipe Defines Dorsal-Ventral Polarity in the Drosophila embryo

PubMed Central

Zhang, Zhenyu; Stevens, Leslie M.; Stein, David

2009-01-01

Summary Drosophila embryonic dorsal-ventral (DV) polarity is controlled by a group of sequentially acting serine proteases located in the fluid-filled perivitelline space between the embryonic membrane and the eggshell, which generate the ligand for the Toll receptor on the ventral side of the embryo [1, 2, 3]. Spatial control of the protease cascade relies on the Pipe sulfotransferase, a fly homologue of vertebrate glycosaminoglycan modifying enzymes [4, 5, 6], which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte. The identification of the Pipe enzymatic target has remained a major gap in our understanding of the mechanism controlling the perivitelline protease cascade, and hence embryonic DV patterning. Here we show that the protein Vitelline Membrane-Like (VML) [7] undergoes Pipe-dependent sulfation and, consistent with a role in conveying positional information from the egg chamber to the embryo, becomes incorporated into the eggshell at a position corresponding to the location of the follicle cells from which it was secreted. Although VML influences embryonic DV pattern in a sensitized genetic background, VML is not essential for DV axis formation, suggesting that there is redundancy in the composition of the Pipe enzymatic target. Correspondingly, we find that additional structural components of the vitelline membrane undergo Pipe-dependent sulfation. In identifying the elusive targets of Pipe, this ork points to the vitelline membrane as the source of signals that generate the Drosophila DV axis and provides a framework for understanding the mechanism controlling spatially-specific activation of serine protease activity during embryonic pattern formation. PMID:19540119

Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos

PubMed Central

Lye, Claire M.; Naylor, Huw W.; Sanson, Bénédicte

2014-01-01

A key challenge in the post-genomic area is to identify the function of the genes discovered, with many still uncharacterised in all metazoans. A first step is transcription pattern characterisation, for which we now have near whole-genome coverage in Drosophila. However, we have much more limited information about the expression and subcellular localisation of the corresponding proteins. The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. We have systematically analysed subcellular localisations at cellularisation (stage 5) and recorded expression patterns at stage 5, at mid-embryogenesis (stage 11) and at late embryogenesis (stages 15-17). At stage 5, 31% of the nuclear lines (41) and 26% of the cytoplasmic lines (67) show discrete localisations that provide clues on the function of the protein and markers for organelles or regions, including nucleoli, the nuclear envelope, nuclear speckles, centrosomes, mitochondria, the endoplasmic reticulum, Golgi, lysosomes and peroxisomes. We characterised the membranous/cortical lines (102) throughout stage 5 to 10 during epithelial morphogenesis, documenting their apico-basal position and identifying those secreted in the extracellular space. We identified the tricellular vertices as a specialized membrane domain marked by the integral membrane protein Sidekick. Finally, we categorised the localisation of the membranous/cortical proteins during cytokinesis. PMID:25294944

Dynamic Hsp83 RNA localization during Drosophila oogenesis and embryogenesis.

PubMed Central

Ding, D; Parkhurst, S M; Halsell, S R; Lipshitz, H D

1993-01-01

Hsp83 is the Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. We show that maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. This protection of Hsp83 RNA occurs in wild-type embryos and embryos produced by females carrying the maternal effect mutations nanos and pumilio, which eliminate components of the posterior polar plasm without disrupting polar granule integrity. In contrast, Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules--cappuccino, oskar, spire, staufen, tudor, valois, and vasa. Mislocalization of oskar RNA to the anterior pole, which has been shown to result in induction of germ cells at the anterior, leads to anterior protection of maternal Hsp83 RNA. These results suggest that Hsp83 RNA is a component of the posterior polar plasm that might be associated with polar granules. In addition, we show that zygotic expression of Hsp83 commences in the anterior third of the embryo at the syncytial blastoderm stage and is regulated by the anterior morphogen, bicoid. We consider the possible developmental significance of this complex control of Hsp83 transcript distribution. Images PMID:7684502

[Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

PubMed

Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

2014-06-01

To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos.

PubMed

Onischenko, Evgeny A; Gubanova, Natalia V; Kiseleva, Elena V; Hallberg, Einar

2005-11-01

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

Expression of renin–angiotensin system components in the early bovine embryo

PubMed Central

Pijacka, Wioletta; Hunter, Morag G; Broughton Pipkin, Fiona; Luck, Martin R

2012-01-01

The renin–angiotensin system (RAS), mainly associated with the regulation of blood pressure, has been recently investigated in female reproductive organs and the developing foetus. Angiotensin II (Ang II) influences oviductal gamete movements and foetal development, but there is no information about RAS in the early embryo. The aim of this study was to determine whether RAS components are present in the pre-implantation embryo, to determine how early they are expressed and to investigate their putative role at this stage of development. Bovine embryos produced in vitro were used for analysis of RAS transcripts (RT-PCR) and localisation of the receptors AGTR1 and AGTR2 (immunofluorescent labelling). We also investigated the effects of Ang II, Olmesartan (AGTR1 antagonist) and PD123319 (AGTR2 antagonist) on oocyte cleavage, embryo expansion and hatching. Pre-implanted embryos possessed AGTR1 and AGTR2 but not the other RAS components. Both receptors were present in the trophectoderm and in the inner cell mass of the blastocyst. AGTR1 was mainly localised in granular-like structures in the cytoplasm, suggesting its internalisation into clathrin-coated vesicles, and AGTR2 was found mainly in the nuclear membrane and in the mitotic spindle of dividing trophoblastic cells. Treating embryos with PD123319 increased the proportion of hatched embryos compared with the control. These results, the first on RAS in the early embryo, suggest that the pre-implanted embryo responds to Ang II from the mother rather than from the embryo itself. This may be a route by which the maternal RAS influences blastocyst hatching and early embryonic development. PMID:23781300

Tissue morphodynamics shaping the early mouse embryo.

PubMed

Sutherland, Ann E

2016-07-01

Generation of the elongated vertebrate body plan from the initially radially symmetrical embryo requires comprehensive changes to tissue form. These shape changes are generated by specific underlying cell behaviors, coordinated in time and space. Major principles and also specifics are emerging, from studies in many model systems, of the cell and physical biology of how region-specific cell behaviors produce regional tissue morphogenesis, and how these, in turn, are integrated at the level of the embryo. New technical approaches have made it possible more recently, to examine the morphogenesis of the mouse embryo in depth, and to elucidate the underlying cellular mechanisms. This review focuses on recent advances in understanding the cellular basis for the early fundamental events that establish the basic form of the embryo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Impact of PCOS on early embryo cleavage kinetics.

PubMed

Wissing, M L; Bjerge, M R; Olesen, A I G; Hoest, T; Mikkelsen, A L

2014-04-01

This study investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage and cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t₂, t₃, t₄, t₅, t₆, t₇, t₈, t(M), t(B)) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. Compared with controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown, t₂, t₃, t₄ and t₇ but not at t₅, t₆, t₈, t(M) or t(B). Embryos from hyperandrogenic PCOS women had developed slower from fertilization to the 8-cell stage compared with embryos from controls. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Male Killing Spiroplasma Preferentially Disrupts Neural Development in the Drosophila melanogaster Embryo

PubMed Central

Martin, Jennifer; Chong, Trisha; Ferree, Patrick M.

2013-01-01

Male killing bacteria such as Spiroplasma are widespread pathogens of numerous arthropods including Drosophila melanogaster. These maternally transmitted bacteria can bias host sex ratios toward the female sex in order to ‘selfishly’ enhance bacterial transmission. However, little is known about the specific means by which these pathogens disrupt host development in order to kill males. Here we show that a male-killing Spiroplasma strain severely disrupts nervous tissue development in male but not female D. melanogaster embryos. The neuroblasts, or neuron progenitors, form properly and their daughter cells differentiate into neurons of the ventral nerve chord. However, the neurons fail to pack together properly and they produce highly abnormal axons. In contrast, non-neural tissue, such as mesoderm, and body segmentation appear normal during this time, although the entire male embryo becomes highly abnormal during later stages. Finally, we found that Spiroplasma is altogether absent from the neural tissue but localizes within the gut and the epithelium immediately surrounding the neural tissue, suggesting that the bacterium secretes a toxin that affects neural tissue development across tissue boundaries. Together these findings demonstrate the unique ability of this insect pathogen to preferentially affect development of a specific embryonic tissue to induce male killing. PMID:24236124

­Back-to-back mechanisms drive actomyosin ring closure during Drosophila embryo cleavage

PubMed Central

2016-01-01

Contraction of actomyosin rings during cytokinesis is typically attributed to actin filaments sliding toward each other via Myosin-2 motor activity. However, rings constrict in some cells in the absence of Myosin-2 activity. Thus, ring closure uses Myosin-2–dependent and –independent mechanisms. But what the Myosin-2–independent mechanisms are, and to what extent they are sufficient to drive closure, remains unclear. During cleavage in Drosophila melanogaster embryos, actomyosin rings constrict in two sequential and mechanistically distinct phases. We show that these phases differ in constriction speed and are genetically and pharmacologically separable. Further, Myosin-2 activity is required for slow constriction in “phase 1” but is largely dispensable for fast constriction in “phase 2,” and F-actin disassembly is only required for fast constriction in phase 2. Switching from phase 1 to phase 2 seemingly relies on the spatial organization of F-actin as controlled by Cofilin, Anillin, and Septin. Our work shows that fly embryos present a singular opportunity to compare separable ring constriction mechanisms, with varying Myosin-2 dependencies, in one cell type and in vivo. PMID:27799369

Kinesin-5–dependent Poleward Flux and Spindle Length Control in Drosophila Embryo Mitosis

PubMed Central

Brust-Mascher, Ingrid; Sommi, Patrizia; Cheerambathur, Dhanya K.

2009-01-01

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system. PMID:19158379

Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy.

PubMed

Watanabe, Tomoko; Thayil, Anisha; Jesacher, Alexander; Grieve, Kate; Debarre, Delphine; Wilson, Tony; Booth, Martin; Srinivas, Shankar

2010-06-03

Lipid droplets (LD) are organelles with an important role in normal metabolism and disease. The lipid content of embryos has a major impact on viability and development. LD in Drosophila embryos and cultured cell lines have been shown to move and fuse in a microtubule dependent manner. Due to limitations in current imaging technology, little is known about the behaviour of LD in the mammalian embryo. Harmonic generation microscopy (HGM) allows one to image LD without the use of exogenous labels. Adaptive optics can be used to correct aberrations that would otherwise degrade the quality and information content of images. We have built a harmonic generation microscope with adaptive optics to characterise early mouse embryogenesis. At fertilization, LD are small and uniformly distributed, but in the implanting blastocyst, LD are larger and enriched in the invading giant cells of the trophectoderm. Time-lapse studies reveal that LD move continuously and collide but do not fuse, instead forming aggregates that subsequently behave as single units. Using specific inhibitors, we show that the velocity and dynamic behaviour of LD is dependent not only on microtubules as in other systems, but also on microfilaments. We explore the limits within which HGM can be used to study living embryos without compromising viability and make the counterintuitive finding that 16 J of energy delivered continuously over a period of minutes can be less deleterious than an order of magnitude lower energy delivered dis-continuously over a period of hours. LD in pre-implantation mouse embryos show a previously unappreciated complexity of behaviour that is dependent not only on microtubules, but also microfilaments. Unlike LD in other systems, LD in the mouse embryo do not fuse but form aggregates. This study establishes HGM with adaptive optics as a powerful tool for the study of LD biology and provides insights into the photo-toxic effects of imaging embryos.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions.

PubMed

Koyle, Melinda L; Veloz, Madeline; Judd, Alec M; Wong, Adam C-N; Newell, Peter D; Douglas, Angela E; Chaston, John M

2016-07-30

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos.

PubMed

Hiraoka, Y; Agard, D A; Sedat, J W

1990-12-01

The spatial and temporal dynamics of diploid chromosome organization, microtubule arrangement, and the state of the nuclear envelope have been analyzed in syncytial blastoderm embryos of Drosophila melanogaster during the transition from prophase to metaphase, by three-dimensional optical sectioning microscopy. Time-lapse, three-dimensional data recorded in living embryos revealed that congression of chromosomes (the process whereby chromosomes move to form the metaphase plate) at prometaphase occurs as a wave, starting at the top of the nucleus near the embryo surface and proceeding through the nucleus to the bottom. The time-lapse analysis was augmented by a high-resolution analysis of fixed embryos where it was possible to unambiguously trace the three-dimensional paths of individual chromosomes. In prophase, the centromeres were found to be clustered at the top of the nucleus while the telomeres were situated at the bottom of the nucleus or towards the embryo interior. This polarized centromere-telomere orientation, perpendicular to the embryo surface, was preserved during the process of prometaphase chromosome congression. Correspondingly, breakdown of the nuclear envelope started at the top of the nucleus with the mitotic spindle being formed at the positions of the partial breakdown of the nuclear envelope. Our observation provide an example in which nuclear structures are spatially organized and their functions are locally and coordinately controlled in three dimensions.

Extra-embryonic tissue spreading directs early embryo morphogenesis in killifish

PubMed Central

Reig, Germán; Cerda, Mauricio; Sepúlveda, Néstor; Flores, Daniela; Castañeda, Victor; Tada, Masazumi; Härtel, Steffen; Concha, Miguel L.

2017-01-01

The spreading of mesenchymal-like cell layers is critical for embryo morphogenesis and tissue repair, yet we know little of this process in vivo. Here we take advantage of unique developmental features of the non-conventional annual killifish embryo to study the principles underlying tissue spreading in a simple cellular environment, devoid of patterning signals and major morphogenetic cell movements. Using in vivo experimentation and physical modelling we reveal that the extra-embryonic epithelial enveloping cell layer, thought mainly to provide protection to the embryo, directs cell migration and the spreading of embryonic tissue during early development. This function relies on the ability of embryonic cells to couple their autonomous random motility to non-autonomous signals arising from the expansion of the extra-embryonic epithelium, mediated by cell membrane adhesion and tension. Thus, we present a mechanism of extra-embryonic control of embryo morphogenesis that couples the mechanical properties of adjacent tissues in the early killifish embryo. PMID:28580937

Notch and PKC are involved in formation of the lateral region of the dorso-ventral axis in Drosophila embryos.

PubMed

Tremmel, Daniel M; Resad, Sedat; Little, Christopher J; Wesley, Cedric S

2013-01-01

The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD), an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis.

Notch and PKC Are Involved in Formation of the Lateral Region of the Dorso-Ventral Axis in Drosophila Embryos

PubMed Central

Tremmel, Daniel M.; Resad, Sedat; Little, Christopher J.; Wesley, Cedric S.

2013-01-01

The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD), an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis. PMID:23861806

Exceptional preservation of tiny embryos documents seed dormancy in early angiosperms.

PubMed

Friis, Else Marie; Crane, Peter R; Pedersen, Kaj Raunsgaard; Stampanoni, Marco; Marone, Federica

2015-12-24

The rapid diversification of angiosperms through the Early Cretaceous period, between about 130-100 million years ago, initiated fundamental changes in the composition of terrestrial vegetation and is increasingly well understood on the basis of a wealth of palaeobotanical discoveries over the past four decades and their integration with improved knowledge of living angiosperms. Prevailing hypotheses, based on evidence both from living and from fossil plants, emphasize that the earliest angiosperms were plants of small stature with rapid life cycles that exploited disturbed habitats in open, or perhaps understorey, conditions. However, direct palaeontogical data relevant to understanding the seed biology and germination ecology of Early Cretaceous angiosperms are sparse. Here we report the discovery of embryos and their associated nutrient storage tissues in exceptionally well-preserved angiosperm seeds from the Early Cretaceous. Synchrotron radiation X-ray tomographic microscopy of the fossil embryos from many taxa reveals that all were tiny at the time of dispersal. These results support hypotheses based on extant plants that tiny embryos and seed dormancy are basic for angiosperms as a whole. The minute size of the fossil embryos, and the modest nutrient storage tissues dictated by the overall small seed size, is also consistent with the interpretation that many early angiosperms were opportunistic, early successional colonizers of disturbance-prone habitats.

The L1-type cell adhesion molecule neuroglian influences the stability of neural ankyrin in the Drosophila embryo but not its axonal localization.

PubMed

Bouley, M; Tian, M Z; Paisley, K; Shen, Y C; Malhotra, J D; Hortsch, M

2000-06-15

Ankyrins are linker proteins, which connect various membrane proteins, including members of the L1 family of neural cell adhesion molecules, with the submembranous actin-spectrin skeleton. Here we report the cloning and characterization of a second, novel Drosophila ankyrin gene (Dank2) that appears to be the result of a gene duplication event during arthropod evolution. The Drosophila L1-type protein neuroglian interacts with products from both Drosophila ankyrin genes. Whereas the previously described ankyrin gene is ubiquitously expressed during embryogenesis, the expression of Dank2 is restricted to the nervous system in the Drosophila embryo. The absence of neuroglian protein in a neuroglian null mutant line causes decreased levels of Dank2 protein in most neuronal cells. This suggests that neuroglian is important for the stability of Dank2 protein. However, neuroglian is not required for Dank2 axonal localization. In temperature-sensitive neuroglian mutants in which neuroglian protein is mislocated at the restrictive temperature to an intracellular location in the neuronal soma, Dank2 protein can still be detected along embryonic nerve tracts.

Chronology of early embryonic development and embryo uterine migration in alpacas.

PubMed

Picha, Y; Tibary, A; Memon, M; Kasimanickam, R; Sumar, J

2013-03-01

The objectives were to: (1) describe the chronology of early embryonic development from ovulation to entry into the uterus; and (2) to determine the timing of embryo migration to the left uterine horn when ovulation occurred from the right ovary. The experiment was conducted in Peru. Females (n = 132) were randomly assigned to 15 experimental groups. All females were mated to an intact male, given 50 μg GnRH im (Cystorelin) and ovulation time determined by transrectal ultrasonography, conducted every 6 hours, starting 24 hours postmating. Animals were slaughtered at a specific intervals postovulation and reproductive tracts were recovered and subjected to oviductal and uterine flushing for females slaughtered between 1 and 6 days postovulation (dpo; Day 0 = ovulation) and uterine flushing for females slaughtered from 7 to 15 dpo for recovery of oocytes/embryos. Season of mating did not influence the interval from mating to ovulation (winter: 29 ± 6 hours vs. summer: 30 ± 6 hours; P = 0.49). Ovulation rates for females mated during winter and summer were 92% versus 100%, respectively (P = 0.05). Fertilization rates for winter and summer mated females were 72% and 82% (P = 0.29). Unfertilized ova were not retained in the uterine tube. All embryos collected were in the uterine tube ipsilateral to the side of ovulation between 1 and 5 dpo. Embryos reached the uterus on 6 dpo. Embryos began to elongate on 9 dpo; at this time, 83% of embryos derived from right-ovary ovulations were collected from the left uterine horn. Embryos occupied the entire uterine cavity by 10 dpo. In conclusion, we characterized early embryo development and location of embryo during its early developmental stages in alpaca. This was apparently the first report regarding chronology of embryo development and migration to the left horn in alpaca which merits further investigation regarding its role in maternal recognition of pregnancy. Copyright © 2013 Elsevier Inc. All rights reserved.

Reduced cell number in the hindgut epithelium disrupts hindgut left-right asymmetry in a mutant of pebble, encoding a RhoGEF, in Drosophila embryos.

PubMed

Nakamura, Mitsutoshi; Matsumoto, Kenjiroo; Iwamoto, Yuta; Muguruma, Takeshi; Nakazawa, Naotaka; Hatori, Ryo; Taniguchi, Kiichiro; Maeda, Reo; Matsuno, Kenji

2013-02-01

Animals often show left-right (LR) asymmetry in their body structures. In some vertebrates, the mechanisms underlying LR symmetry breaking and the subsequent signals responsible for LR asymmetric development are well understood. However, in invertebrates, the molecular bases of these processes are largely unknown. Therefore, we have been studying the genetic pathway of LR asymmetric development in Drosophila. The embryonic gut is the first organ that shows directional LR asymmetry during Drosophila development. We performed a genetic screen to identify mutations affecting LR asymmetric development of the embryonic gut. From this screen, we isolated pebble (pbl), which encodes a homolog of a mammalian RhoGEF, Ect2. The laterality of the hindgut was randomized in embryos homozygous for a null mutant of pbl. Pbl is a multi-functional protein required for cytokinesis and the epithelial-to-mesenchymal transition in Drosophila. Consistent with Pbl's role in cytokinesis, we found reduced numbers of cells in the hindgut epithelium in pbl homozygous embryos. The specific expression of pbl in the hindgut epithelium, but not in other tissues, rescued the LR defects and reduced cell number in embryonic pbl homozygotes. Embryos homozygous for string (stg), a mutant that reduces cell number through a different mechanism, also showed LR defects of the hindgut. However, the reduction in cell number in the pbl mutants was not accompanied by defects in the specification of hindgut epithelial tissues or their integrity. Based on these results, we speculate that the reduction in cell number may be one reason for the LR asymmetry defect of the pbl hindgut, although we cannot exclude contributions from other functions of Pbl, including regulation of the actin cytoskeleton through its RhoGEF activity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Evolution and inheritance of early embryonic patterning in Drosophila simulans and D. sechellia.

PubMed

Lott, Susan E; Ludwig, Michael Z; Kreitman, Martin

2011-05-01

Pattern formation in Drosophila is a widely studied example of a robust developmental system. Such robust systems pose a challenge to adaptive evolution, as they mask variation that selection may otherwise act upon. Yet we find variation in the localization of expression domains (henceforth "stripe allometry") in the pattern formation pathway. Specifically, we characterize differences in the gap genes giant and Kruppel, and the pair-rule gene even-skipped, which differ between the sibling species Drosophila simulans and D. sechellia. In a double-backcross experiment, stripe allometry is consistent with maternal inheritance of stripe positioning and multiple genetic factors, with a distinct genetic basis from embryo length. Embryos produced by F1 and F2 backcross mothers exhibit novel spatial patterns of gene expression relative to the parental species, with no measurable increase in positional variance among individuals. Buffering of novel spatial patterns in the backcross genotypes suggests that robustness need not be disrupted in order for the trait to evolve, and perhaps the system is incapable of evolving to prevent the expression of all genetic variation. This limitation, and the ability of natural selection to act on minute genetic differences that are within the "margin of error" for the buffering mechanism, indicates that developmentally buffered traits can evolve without disruption of robustness. © 2010 The Author(s). Evolution© 2010 The Society for the Study of Evolution.

Argonaute-1 functions as a mitotic regulator by controlling Cyclin B during Drosophila early embryogenesis.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Bag, Indira; Hunt, Clayton R; Ramaiah, M Janaki; Pandita, Tej K; Bhadra, Utpal; Pal-Bhadra, Manika

2014-02-01

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. In this study, we established evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. We identified involvement of 2 novel embryonic miRNAs--miR-981 and miR--317-for spatiotemporal regulation of cyclin B. In summary, our results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (mei-41 and grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, our results have established a novel role of Ago-1 as a regulator of the cell cycle.

Multiple transcription factor codes activate epidermal wound–response genes in Drosophila

PubMed Central

Pearson, Joseph C.; Juarez, Michelle T.; Kim, Myungjin; Drivenes, Øyvind; McGinnis, William

2009-01-01

Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. We characterize the transcriptional regulatory enhancers of 4 genes—Ddc, ple, msn, and kkv—that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarily conserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. We propose that the combination of GRH and FOS is part of an ancient wound–response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. Our results indicate that this is an overly simplistic view. PMID:19168633

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.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

PubMed Central

Raissig, Michael T.; Gagliardini, Valeria; Jaenisch, Johan; Grossniklaus, Ueli; Baroux, Célia

2013-01-01

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays. PMID:23770918

Mitotic waves in the early embryogenesis of Drosophila: Bistability traded for speed.

PubMed

Vergassola, Massimo; Deneke, Victoria E; Di Talia, Stefano

2018-03-06

Early embryogenesis of most metazoans is characterized by rapid and synchronous cleavage divisions. Chemical waves of Cdk1 activity were previously shown to spread across Drosophila embryos, and the underlying molecular processes were dissected. Here, we present the theory of the physical mechanisms that control Cdk1 waves in Drosophila The in vivo dynamics of Cdk1 are captured by a transiently bistable reaction-diffusion model, where time-dependent reaction terms account for the growing level of cyclins and Cdk1 activation across the cell cycle. We identify two distinct regimes. The first one is observed in mutants of the mitotic switch. There, waves are triggered by the classical mechanism of a stable state invading a metastable one. Conversely, waves in wild type reflect a transient phase that preserves the Cdk1 spatial gradients while the overall level of Cdk1 activity is swept upward by the time-dependent reaction terms. This unique mechanism generates a wave-like spreading that differs from bistable waves for its dependence on dynamic parameters and its faster speed. Namely, the speed of "sweep" waves strikingly decreases as the strength of the reaction terms increases and scales as the powers 3/4, -1/2, and 7/12 of Cdk1 molecular diffusivity, noise amplitude, and rate of increase of Cdk1 activity in the cell-cycle S phase, respectively. Theoretical predictions are supported by numerical simulations and experiments that couple quantitative measurements of Cdk1 activity and genetic perturbations of the accumulation rate of cyclins. Finally, our analysis bears upon the inhibition required to suppress Cdk1 waves at the cell-cycle pause for the maternal-to-zygotic transition.

Autophagy can promote but is not required for epithelial cell extrusion in the amnioserosa of the Drosophila embryo.

PubMed

Cormier, Olga; Mohseni, Nilufar; Voytyuk, Iryna; Reed, Bruce H

2012-02-01

During Drosophila embryogenesis the majority of the extra-embryonic epithelium known as the amnioserosa (AS) undergoes programmed cell death (PCD) following the completion of the morphogenetic process of dorsal closure. Approximately ten percent of AS cells, however, are eliminated during dorsal closure by extrusion from the epithelium. Using biosensors that report autophagy and caspase activity in vivo, we demonstrate that AS cell extrusion occurs in the context of elevated autophagy and caspase activation. Furthermore, we evaluate AS extrusion rates, autophagy, and caspase activation in embryos in which caspase activity or autophagy are altered by genetic manipulation. This includes using the GAL4/UAS system to drive expression of p35, reaper, dINR (ACT) and Atg1 in the AS; we also analyze embryos lacking both maternal and zygotic expression of Atg1. Based on our results we suggest that autophagy can promote, but is not required for, epithelial extrusion and caspase activation in the amnioserosa.

Early Activation of MAPK and Apoptosis in Nutritive Embryos of Calyptraeid Gastropods.

PubMed

Lesoway, Maryna P; Collin, Rachel; Abouheif, Ehab

2017-07-01

Investigation of alternative phenotypes, different morphologies produced by a single genome, has contributed novel insights into development and evolution. Yet, the mechanisms underlying developmental switch points between alternative phenotypes remain poorly understood. The calyptraeid snails Crepidula navicella and Calyptraea lichen produce two phenotypes: viable and nutritive embryos, where nutritive embryos arrest their development after gastrulation and are ingested by their viable siblings as a form of intracapsular nutrition. Here, we investigate the activity of mitogen-activated protein kinase (MAPK, ERK1/2) and apoptosis during early cleavage. MAPK and apoptosis, found in a previous transcriptomic study, are known to be involved in organization of other spiralian embryos and nutritive embryo development, respectively. In the model Crepidula fornicata, MAPK activation begins at the 16-cell stage. In contrast, we discovered in C. navicella and C. lichen that many embryos begin MAPK activation at the one-cell stage. A subset of embryos shows a similar pattern of MAPK activation to C. fornicata at later stages. In all stages where MAPK is detected, the activation pattern is highly variable, frequently occurring in all quadrants or in multiple tiers of cells. We also detected apoptosis in cleaving embryos, while C. fornicata and Crepidula lessoni, which do not produce nutritive embryos, show no signs of apoptosis during cleavage. Our results show that MAPK and apoptosis are expressed during early development in species with nutritive embryos, and raises the possibility that these processes may play a role and even interact with one another in producing the nutritive embryo phenotype. © 2017 Wiley Periodicals, Inc.

Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

PubMed Central

Tamada, Masako; Zallen, Jennifer A.

2015-01-01

Summary Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. Here we identify a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand, Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation. PMID:26506305

Maternal control of the Drosophila dorsal–ventral body axis

PubMed Central

Stein, David S.; Stevens, Leslie M.

2016-01-01

The pathway that generates the dorsal–ventral (DV) axis of the Drosophila embryo has been the subject of intense investigation over the previous three decades. The initial asymmetric signal originates during oogenesis by the movement of the oocyte nucleus to an anterior corner of the oocyte, which establishes DV polarity within the follicle through signaling between Gurken, the Drosophila Transforming Growth Factor (TGF)-α homologue secreted from the oocyte, and the Drosophila Epidermal Growth Factor Receptor (EGFR) that is expressed by the follicular epithelium cells that envelop the oocyte. Follicle cells that are not exposed to Gurken follow a ventral fate and express Pipe, a sulfotransferase that enzymatically modifies components of the inner vitelline membrane layer of the eggshell, thereby transferring DV spatial information from the follicle to the egg. These ventrally sulfated eggshell proteins comprise a localized cue that directs the ventrally restricted formation of the active Spätzle ligand within the perivitelline space between the eggshell and the embryonic membrane. Spätzle activates Toll, a transmembrane receptor in the embryonic membrane. Transmission of the Toll signal into the embryo leads to the formation of a ventral-to-dorsal gradient of the transcription factor Dorsal within the nuclei of the syncytial blastoderm stage embryo. Dorsal controls the spatially specific expression of a large constellation of zygotic target genes, the Dorsal gene regulatory network, along the embryonic DV circumference. This article reviews classic studies and integrates them with the details of more recent work that has advanced our understanding of the complex pathway that establishes Drosophila embryo DV polarity. PMID:25124754

Embryo density and medium volume effects on early murine embryo development.

PubMed

Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

1992-10-01

One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

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.

Noncoding RNAs of the Ultrabithorax Domain of the Drosophila Bithorax Complex

PubMed Central

Pease, Benjamin; Borges, Ana C.; Bender, Welcome

2013-01-01

RNA transcripts without obvious coding potential are widespread in many creatures, including the fruit fly, Drosophila melanogaster. Several noncoding RNAs have been identified within the Drosophila bithorax complex. These first appear in blastoderm stage embryos, and their expression patterns indicate that they are transcribed only from active domains of the bithorax complex. It has been suggested that these noncoding RNAs have a role in establishing active domains, perhaps by setting the state of Polycomb Response Elements A comprehensive survey across the proximal half of the bithorax complex has now revealed nine distinct noncoding RNA transcripts, including four within the Ultrabithorax transcription unit. At the blastoderm stage, the noncoding transcripts collectively span ∼75% of the 135 kb surveyed. Recombination-mediated cassette exchange was used to invert the promoter of one of the noncoding RNAs, a 23-kb transcript from the bxd domain of the bithorax complex. The resulting animals fail to make the normal bxd noncoding RNA and show no transcription across the bxd Polycomb Response Element in early embryos. The mutant flies look normal; the regulation of the bxd domain appears unaffected. Thus, the bxd noncoding RNA has no apparent function. PMID:24077301

The Drosophila Pericentrin-like-protein (PLP) cooperates with Cnn to maintain the integrity of the outer PCM

PubMed Central

Richens, Jennifer H.; Barros, Teresa P.; Lucas, Eliana P.; Peel, Nina; Pinto, David Miguel Susano; Wainman, Alan; Raff, Jordan W.

2015-01-01

ABSTRACT Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs). As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM. PMID:26157019

The Drosophila Pericentrin-like-protein (PLP) cooperates with Cnn to maintain the integrity of the outer PCM.

PubMed

Richens, Jennifer H; Barros, Teresa P; Lucas, Eliana P; Peel, Nina; Pinto, David Miguel Susano; Wainman, Alan; Raff, Jordan W

2015-07-08

Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. Here we investigate the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, we find that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal microtubules (MTs). As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM. © 2015. Published by The Company of Biologists Ltd.

Early events in speciation: polymorphism for hybrid male sterility in Drosophila.

PubMed

Reed, Laura K; Markow, Therese A

2004-06-15

Capturing the process of speciation early enough to determine the initial genetic causes of reproductive isolation remains a major challenge in evolutionary biology. We have found, to our knowledge, the first example of substantial intraspecific polymorphism for genetic factors contributing to hybrid male sterility. Specifically, we show that the occurrence of hybrid male sterility in crosses between Drosophila mojavensis and its sister species, Drosophila arizonae, is controlled by factors present at different frequencies in different populations of D. mojavensis. In addition, we show that hybrid male sterility is a complex phenotype; some hybrid males with motile sperm still cannot sire offspring. Because male sterility factors in hybrids between these species are not yet fixed within D. mojavensis, this system provides an invaluable opportunity to characterize the genetics of reproductive isolation at an early stage.

Expression of voltage-activated calcium channels in the early zebrafish embryo.

PubMed

Sanhueza, Dayán; Montoya, Andro; Sierralta, Jimena; Kukuljan, Manuel

2009-05-01

Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

Real-Time Three-Dimensional Cell Segmentation in Large-Scale Microscopy Data of Developing Embryos.

PubMed

Stegmaier, Johannes; Amat, Fernando; Lemon, William C; McDole, Katie; Wan, Yinan; Teodoro, George; Mikut, Ralf; Keller, Philipp J

2016-01-25

We present the Real-time Accurate Cell-shape Extractor (RACE), a high-throughput image analysis framework for automated three-dimensional cell segmentation in large-scale images. RACE is 55-330 times faster and 2-5 times more accurate than state-of-the-art methods. We demonstrate the generality of RACE by extracting cell-shape information from entire Drosophila, zebrafish, and mouse embryos imaged with confocal and light-sheet microscopes. Using RACE, we automatically reconstructed cellular-resolution tissue anisotropy maps across developing Drosophila embryos and quantified differences in cell-shape dynamics in wild-type and mutant embryos. We furthermore integrated RACE with our framework for automated cell lineaging and performed joint segmentation and cell tracking in entire Drosophila embryos. RACE processed these terabyte-sized datasets on a single computer within 1.4 days. RACE is easy to use, as it requires adjustment of only three parameters, takes full advantage of state-of-the-art multi-core processors and graphics cards, and is available as open-source software for Windows, Linux, and Mac OS. Copyright © 2016 Elsevier Inc. All rights reserved.

Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention.

PubMed

Mitchell, Megan; Schulz, Samantha L; Armstrong, David T; Lane, Michelle

2009-04-01

Dietary supply of nutrients, both periconception and during pregnancy, influence the growth and development of the fetus and offspring and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. The present study examined embryo development following periconception exposure of females to a high-protein diet (HPD) or a low-protein diet (LPD) relative to a medium-protein diet (MPD; control), and we hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and LPD reduced the number of inner cell mass cells in the blastocyst-stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from HPD females had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and, potentially, the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus, suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo, and the nature of the perturbation differs between HPD and LPD exposure.

Regulation of the protein kinase activity of Shaggy(Zeste-white3) by components of the wingless pathway in Drosophila cells and embryos.

PubMed

Ruel, L; Stambolic, V; Ali, A; Manoukian, A S; Woodgett, J R

1999-07-30

The protein-serine kinase Shaggy(Zeste-white3) (Sgg(Zw3)) is the Drosophila homolog of mammalian glycogen synthase kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. Sgg(Zw3) is a putative component of the Wingless (Wg) pathway, and epistasis analyses suggest that Sgg(Zw3) function is repressed by Wg signaling. Here, we have investigated the biochemical consequences of Wg signaling with respect to the Sgg(Zw3) protein kinase in two types of Drosophila cell lines and in embryos. Our results demonstrate that Sgg(Zw3) activity is inhibited following exposure of cells to Wg protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of Sgg(Zw3) is accompanied by serine phosphorylation. We also show that the level of Sgg(Zw3) activity regulates the stability of Armadillo protein and modulates the level of phosphorylation of D-Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling proteins.

Sensitivity of early mouse embryos to (/sup 3/H)thymidine

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

Spindle, A.; Wu, K.; Pedersen, R.A.

1982-12-01

Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to (/sup 3/H)thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of (/sup 3/H)thymidine, concentrations as low as 0.2 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs were reduced, and at 10 nCi/ml the number of embryos developing to all threemore » post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highst concentration (/sup 3/H)thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to (/sup 3/H)thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages. The 24-h exposure of immunosurgically isolated ICMS to (/sup 3/H)thymidine revealed that the high sensitivity of the ICM to (/sup 3/H)thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate (/sup 3/H)thymidine into the DNA.« less

Early events in speciation: Polymorphism for hybrid male sterility in Drosophila

PubMed Central

Reed, Laura K.; Markow, Therese A.

2004-01-01

Capturing the process of speciation early enough to determine the initial genetic causes of reproductive isolation remains a major challenge in evolutionary biology. We have found, to our knowledge, the first example of substantial intraspecific polymorphism for genetic factors contributing to hybrid male sterility. Specifically, we show that the occurrence of hybrid male sterility in crosses between Drosophila mojavensis and its sister species, Drosophila arizonae, is controlled by factors present at different frequencies in different populations of D. mojavensis. In addition, we show that hybrid male sterility is a complex phenotype; some hybrid males with motile sperm still cannot sire offspring. Because male sterility factors in hybrids between these species are not yet fixed within D. mojavensis, this system provides an invaluable opportunity to characterize the genetics of reproductive isolation at an early stage. PMID:15184657

Innexin 3, a New Gene Required for Dorsal Closure in Drosophila Embryo

PubMed Central

Giuliani, Fabrizio; Giuliani, Giuliano; Bauer, Reinhard; Rabouille, Catherine

2013-01-01

Background Dorsal closure is a morphogenetic event that occurs during mid-embryogenesis in many insects including Drosophila, during which the ectoderm migrates on the extraembryonic amnioserosa to seal the embryo dorsally. The contribution of the ectoderm in this event has been known for a long time. However, amnioserosa tension and contractibility have recently been shown also to be instrumental to the closure. A critical pre-requisite for dorsal closure is integrity of these tissues that in part is mediated by cell-cell junctions and cell adhesion. In this regard, mutations impairing junction formation and/or adhesion lead to dorsal closure. However, no role for the gap junction proteins Innexins has so far been described. Results and Discussion Here, we show that Innexin 1, 2 and 3, are present in the ectoderm but also in the amnioserosa in plaques consistent with gap junctions. However, only the loss of Inx3 leads to dorsal closure defects that are completely rescued by overexpression of inx3::GFP in the whole embryo. Loss of Inx3 leads to the destabilisation of Inx1, Inx2 and DE-cadherin at the plasma membrane, suggesting that these four proteins form a complex. Accordingly, in addition to the known interaction of Inx2 with DE-cadherin, we show that Inx3 can bind to DE-cadherin. Furthermore, Inx3-GFP overexpression recruits DE-cadherin from its wildtype plasma membrane domain to typical Innexin plaques, strengthening the notion that they form a complex. Finally, we show that Inx3 stability is directly dependent on tissue tension. Taken together, we propose that Inx3 is a critical factor for dorsal closure and that it mediates the stability of Inx1, 2 and DE-cadherin by forming a complex. PMID:23894431

DYZ1 copy number variation, Y chromosome polymorphism and early recurrent spontaneous abortion/early embryo growth arrest.

PubMed

Yan, Junhao; Fan, Lingling; Zhao, Yueran; You, Li; Wang, Laicheng; Zhao, Han; Li, Yuan; Chen, Zi-Jiang

2011-12-01

To find the association between recurrent spontaneous abortion (RSA)/early embryo growth arrest and Y chromosome polymorphism. Peripheral blood samples of the male patients of big Y chromosome, small Y chromosome and other male patients whose partners suffered from unexplained RSA/early embryo growth arrest were collected. PCR and real-time fluorescent quantitative PCR were used to test the deletion and the copy number variation of DYZ1 region in Y chromosome of the patients. A total of 79 big Y chromosome patients (48 of whose partners suffered from RSA or early embryo growth arrest), 7 small Y chromosome patients, 106 other male patients whose partners had suffered from unexplained RSA or early embryo growth arrest, and 100 normal male controls were enrolled. There was no fraction deletion of DYZ1 detected both in big Y patients and in normal men. Of RSA patients, 1 case showed deletion of 266bp from the gene locus 25-290bp, and 2 cases showed deletion of 773bp from 1347 to 2119bp. Of only 7 small Y chromosome patients, 2 cases showed deletion of 266bp from 25 to 290bp, and 4 cases showed deletion of 773bp from 1347 to 2119bp and 275bp from 3128 to 3420bp. The mean of DYZ1 copies was 3900 in normal control men; the mean in big Y patients was 5571, in RSA patients was 2655, and in small Y patients was 1059. All of the others were significantly different (P<0.01) compared with normal control men, which meant that DYZ1 copy number in normal control men was less than that of big Y chromosome patients, and was more than that of unexplained early RSA patients and small Y patients. The integrity and copy number variation of DYZ1 are closely related to the Y chromosome length under microscope. The cause of RSA/early embryo growth arrest in some couples may be the increase (big Y patients) or decrease of DYZ1 copy number in the husbands' Y chromosome. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The influence of early embryo traits on human embryonic stem cell derivation efficiency.

PubMed

O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

2011-05-01

Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

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

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

PubMed

Liu, C; Liu, Y; Larsen, K; Hou, Y P; Callesen, H

2018-01-01

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila.

PubMed

Lőrincz, Péter; Lakatos, Zsolt; Varga, Ágnes; Maruzs, Tamás; Simon-Vecsei, Zsófia; Darula, Zsuzsanna; Benkő, Péter; Csordás, Gábor; Lippai, Mónika; Andó, István; Hegedűs, Krisztina; Medzihradszky, Katalin F; Takáts, Szabolcs; Juhász, Gábor

2016-06-02

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila.

Myoblast fusion in Drosophila

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

Haralalka, Shruti; Abmayr, Susan M., E-mail: sma@stowers.org; Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, MO 66160

2010-11-01

The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral sidemore » of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.« less

DRhoGEF2 and Diaphanous Regulate Contractile Force during Segmental Groove Morphogenesis in the Drosophila Embryo

PubMed Central

Mulinari, Shai; Barmchi, Mojgan Padash

2008-01-01

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. Here, we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. We further show that the Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis. PMID:18287521

Microtubule Flux and Sliding in Mitotic Spindles of Drosophila EmbryosV⃞

PubMed Central

Brust-Mascher, Ingrid; Scholey, Jonathan M.

2002-01-01

We proposed that spindle morphogenesis in Drosophila embryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 μm/s, too slow to drive chromatid-to-pole motion at 0.11 μm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart. PMID:12429839

The early-stage diagnosis of albinic embryos by applying optical coherence tomography

NASA Astrophysics Data System (ADS)

Yang, Bor-Wen; Wang, Shih-Yuan; Wang, Yu-Yen; Cai, Jyun-Jhang; Chang, Chung-Hao

2013-09-01

Albinism is a kind of congenital disease of abnormal metabolism. Poecilia reticulata (guppy fish) is chosen as the model to study the development of albinic embryos as it is albinic, ovoviviparous and with short life period. This study proposed an imaging method for penetrative embryo investigation using optical coherence tomography. By imaging through guppy mother’s reproduction purse, we found the embryo’s eyes were the early-developed albinism features. As human’s ocular albinism typically appear at about four weeks old, it is the time to determine if an embryo will grow into an albino.

A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate.

PubMed

de los Santos, Maria José; Arroyo, Gemma; Busquet, Ana; Calderón, Gloria; Cuadros, Jorge; Hurtado de Mendoza, Maria Victoria; Moragas, Marta; Herrer, Raquel; Ortiz, Agueda; Pons, Carme; Ten, Jorge; Vilches, Miguel Angel; Figueroa, Maria José

2014-04-01

To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. Prospective cross-sectional study. Multicenter study. Seven hundred embryo transfers and 1,028 early-stage human embryos. None. Implantation according to the presence of EC and embryo quality. The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

CLOCK expression identifies developing circadian oscillator neurons in the brains of Drosophila embryos.

PubMed

Houl, Jerry H; Ng, Fanny; Taylor, Pete; Hardin, Paul E

2008-12-18

The Drosophila circadian oscillator is composed of transcriptional feedback loops in which CLOCK-CYCLE (CLK-CYC) heterodimers activate their feedback regulators period (per) and timeless (tim) via E-box mediated transcription. These feedback loop oscillators are present in distinct clusters of dorsal and lateral neurons in the adult brain, but how this pattern of expression is established during development is not known. Since CLK is required to initiate feedback loop function, defining the pattern of CLK expression in embryos and larvae will shed light on oscillator neuron development. A novel CLK antiserum is used to show that CLK expression in the larval CNS and adult brain is limited to circadian oscillator cells. CLK is initially expressed in presumptive small ventral lateral neurons (s-LNvs), dorsal neurons 2 s (DN2s), and dorsal neuron 1 s (DN1s) at embryonic stage (ES) 16, and this CLK expression pattern persists through larval development. PER then accumulates in all CLK-expressing cells except presumptive DN2s during late ES 16 and ES 17, consistent with the delayed accumulation of PER in adult oscillator neurons and antiphase cycling of PER in larval DN2s. PER is also expressed in non-CLK-expressing cells in the embryonic CNS starting at ES 12. Although PER expression in CLK-negative cells continues in ClkJrk embryos, PER expression in cells that co-express PER and CLK is eliminated. These data demonstrate that brain oscillator neurons begin development during embryogenesis, that PER expression in non-oscillator cells is CLK-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons. These results define the temporal and spatial coordinates of factors that initiate Clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that PER functions in a different capacity before oscillator cell development is initiated.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila

PubMed Central

Lőrincz, Péter; Lakatos, Zsolt; Varga, Ágnes; Maruzs, Tamás; Simon-Vecsei, Zsófia; Darula, Zsuzsanna; Benkő, Péter; Csordás, Gábor; Lippai, Mónika; Andó, István; Hegedűs, Krisztina; Medzihradszky, Katalin F; Takáts, Szabolcs; Juhász, Gábor

2016-01-01

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.14226.001 PMID:27253064

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.

Macrophages and cellular immunity in Drosophila melanogaster

PubMed Central

Gold, Katrina S.; Brückner, Katja

2016-01-01

The invertebrate Drosophila melanogaster has been a powerful model for understanding blood cell development and immunity. Drosophila is a holometabolous insect, which transitions through a series of life stages from embryo, larva and pupa to adulthood. In spite of this, remarkable parallels exist between Drosophila and vertebrate macrophages, both in terms of development and function. More than 90% of Drosophila blood cells (hemocytes) are macrophages (plasmatocytes), making this highly tractable genetic system attractive for studying a variety of questions in macrophage biology. In vertebrates, recent findings revealed that macrophages have two independent origins: self-renewing macrophages, which reside and proliferate in local microenvironments in a variety of tissues, and macrophages of the monocyte lineage, which derive from hematopoietic stem or progenitor cells. Like vertebrates, Drosophila possesses two macrophage lineages with a conserved dual ontogeny. These parallels allow us to take advantage of the Drosophila model when investigating macrophage lineage specification, maintenance and amplification, and the induction of macrophages and their progenitors by local microenvironments and systemic cues. Beyond macrophage development, Drosophila further serves as a paradigm for understanding the mechanisms underlying macrophage function and cellular immunity in infection, tissue homeostasis and cancer, throughout development and adult life. PMID:27117654

Central cell-derived peptides regulate early embryo patterning in flowering plants.

PubMed

Costa, Liliana M; Marshall, Eleanor; Tesfaye, Mesfin; Silverstein, Kevin A T; Mori, Masashi; Umetsu, Yoshitaka; Otterbach, Sophie L; Papareddy, Ranjith; Dickinson, Hugh G; Boutiller, Kim; VandenBosch, Kathryn A; Ohki, Shinya; Gutierrez-Marcos, José F

2014-04-11

Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non-cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

Coordinated Development of Muscles and Tendon-Like Structures: Early Interactions in the Drosophila Leg.

PubMed

Soler, Cedric; Laddada, Lilia; Jagla, Krzysztof

2016-01-01

The formation of the musculoskeletal system is a remarkable example of tissue assembly. In both vertebrates and invertebrates, precise connectivity between muscles and skeleton (or exoskeleton) via tendons or equivalent structures is fundamental for movement and stability of the body. The molecular and cellular processes underpinning muscle formation are well-established and significant advances have been made in understanding tendon development. However, the mechanisms contributing to proper connection between these two tissues have received less attention. Observations of coordinated development of tendons and muscles suggest these tissues may interact during the different steps in their development. There is growing evidence that, depending on animal model and muscle type, these interactions can take place from progenitor induction to the final step of the formation of the musculoskeletal system. Here, we briefly review and compare the mechanisms behind muscle and tendon interaction throughout the development of vertebrates and Drosophila before going on to discuss our recent findings on the coordinated development of muscles and tendon-like structures in Drosophila leg. By altering apodeme formation (the functional Drosophila equivalent of tendons in vertebrates) during the early steps of leg development, we affect the spatial localization of subsequent myoblasts. These findings provide the first evidence of the developmental impact of early interactions between muscle and tendon-like precursors, and confirm the appendicular Drosophila muscle system as a valuable model for studying these processes.

Coordinated Development of Muscles and Tendon-Like Structures: Early Interactions in the Drosophila Leg

PubMed Central

Soler, Cedric; Laddada, Lilia; Jagla, Krzysztof

2016-01-01

The formation of the musculoskeletal system is a remarkable example of tissue assembly. In both vertebrates and invertebrates, precise connectivity between muscles and skeleton (or exoskeleton) via tendons or equivalent structures is fundamental for movement and stability of the body. The molecular and cellular processes underpinning muscle formation are well-established and significant advances have been made in understanding tendon development. However, the mechanisms contributing to proper connection between these two tissues have received less attention. Observations of coordinated development of tendons and muscles suggest these tissues may interact during the different steps in their development. There is growing evidence that, depending on animal model and muscle type, these interactions can take place from progenitor induction to the final step of the formation of the musculoskeletal system. Here, we briefly review and compare the mechanisms behind muscle and tendon interaction throughout the development of vertebrates and Drosophila before going on to discuss our recent findings on the coordinated development of muscles and tendon-like structures in Drosophila leg. By altering apodeme formation (the functional Drosophila equivalent of tendons in vertebrates) during the early steps of leg development, we affect the spatial localization of subsequent myoblasts. These findings provide the first evidence of the developmental impact of early interactions between muscle and tendon-like precursors, and confirm the appendicular Drosophila muscle system as a valuable model for studying these processes. PMID:26869938

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

PubMed Central

Thomson, Travis; Liu, Niankun; Arkov, Alexey; Lehmann, Ruth; Lasko, Paul

2008-01-01

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components we isolated proteins that associate in early embryos with Vasa (VAS) and Tudor (TUD), two known polar granule associated molecules. We identified Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) as components of both VAS and TUD complexes and confirmed their localization to polar granules by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. Our recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly. PMID:18590813

Early embryonic survival and embryo development in two lines of rabbits divergently selected for uterine capacity.

PubMed

Peiró, R; Santacreu, M A; Climent, A; Blasco, A

2007-07-01

The aim of this work is to study early embryo survival and development in 2 lines divergently selected for high and low uterine capacity throughout 10 generations. A total of 162 female rabbits from the high line and 133 from the low line were slaughtered at 25, 48, or 62 h of gestation. There were no differences in ovulation rate and fertilization rate between lines in any of the 3 stages of gestation. Embryo survival, estimated as the number of normal embryos recovered at a constant ovulation rate, was similar in both lines at 25 and 48 h. Embryo survival was greater in the high line [D (posterior mean of the difference between the high and low lines) = 0.57 embryos] at 62 h of gestation. There was no difference in embryonic stage of development at 25 h, but at 48 and 62 h of gestation, the high line, compared with the low line, had a greater percentage of early morulae (83 vs. 72%) and compacted morulae (55 vs. 38%). Divergent selection for uterine capacity appeared to modify embryo development, at least from 48 h of gestation, and embryo survival from 62 h.

[Specification of cell destiny in early Caenorhabditis elegans embryo].

PubMed

Schierenberg, E

1997-02-01

Embryogenesis of the nematode Caenorhabditis elegans has been described completely on a cell-by-cell basis and found to be essentially invariant. With this knowledge in hands, micromanipulated embryos and mutants have been analyzed for cell lineage defects and the distribution of specific gene products. The results challenge the classical view of cell-autonomous development in nematodes and indicate that the early embryo of C. elegans is a highly dynamic system. A network of inductive events between neighboring cells is being revealed, which is necessary to assign different developmental programs to blastomeres. In those cases where molecules involved in these cell-cell interactions have been identified, homologies to cell surface receptors, ligands and transcription factors found in other systems have become obvious.

Developmental analysis of the dopamine-containing neurons of the Drosophila brain

PubMed Central

Hartenstein, Volker; Cruz, Louie; Lovick, Jennifer K.; Guo, Ming

2016-01-01

The Drosophila dopaminergic (DA) system consists of a relatively small number of neurons clustered throughout the brain and ventral nerve cord. Previous work shows that clusters of DA neurons innervate different brain compartments, which in part accounts for functional diversity of the DA system. In this paper, we analyzed the association between DA neuron clusters and specific brain lineages, developmental and structural units of the Drosophila brain which provide a framework of connections that can be followed throughout development. The hatching larval brain contains six groups of primary DA neurons (born in the embryo), which we assign to six distinct lineages. We can show that all larval DA clusters persist into the adult brain. Some clusters increase in cell number during late larval stages while others do not become DA-positive until early pupa. Ablating neuroblasts with hydroxyurea (HU) prior to onset of larval proliferation (generates secondary neurons) confirms these added DA clusters are primary neurons born in the embryo, rather than secondary neurons. A single cluster that becomes DA-positive in the late pupa, PAM1/lineage DALcm1/2, forms part of a secondary lineage which can be ablated by larval HU application. By supplying lineage information for each DA cluster, our analysis promotes further developmental and functional analyses of this important system of neurons. PMID:27350102

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.

Sildenafil citrate (Viagra) impairs fertilization and early embryo development in mice.

PubMed

Glenn, David R J; McClure, Neil; Cosby, S Louise; Stevenson, Michael; Lewis, Sheena E M

2009-03-01

To determine the effects of sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor known to affect sperm function, on fertilization and early embryo cleavage. This acute mammal study included male and female mice assigned randomly, the females sacrificed after mating and their oocytes/embryos evaluated at four time periods after treatment. Academic research environment. Male and female CBAB(6) mice. Female mice were injected intraperitoneally with 5 IU gonadotropin (hCG) to stimulate follicular growth and induce ovulation. They were each caged with a male that had been gavaged with sildenafil citrate (0.06 mg/0.05 mL) and allowed to mate. After 12, 36, 60, and 84 h, females were killed, their oviducts were dissected out, and retrieved embryos were assessed for blastomere number and quality. Fertilization rates and numbers of embryos were evaluated after treatment. Fertilization rates (day 1) were markedly reduced (-33%) in matings where the male had taken sildenafil citrate. Over days 2-4, the numbers of embryos developing in the treated group were significantly fewer than in the control group. There was also a trend for impaired cleavage rates within those embryos, although this did not reach significance. The impairments to fertility caused by sildenafil citrate have important implications for infertility centers and for couples who are using this drug precoitally while attempting to conceive.

Early embryo mortality in natural human reproduction: What the data say

PubMed Central

Jarvis, Gavin E.

2017-01-01

How many human embryos die between fertilisation and birth under natural conditions? It is widely accepted that natural human embryo mortality is high, particularly during the first weeks after fertilisation, with total prenatal losses of 70% and higher frequently claimed. However, the first external sign of pregnancy occurs two weeks after fertilisation with a missed menstrual period, and establishing the fate of embryos before this is challenging. Calculations are additionally hampered by a lack of data on the efficiency of fertilisation under natural conditions. Four distinct sources are used to justify quantitative claims regarding embryo loss: (i) a hypothesis published by Roberts & Lowe in The Lancet  is widely cited but has no practical quantitative value; (ii) life table analyses give consistent assessments of clinical pregnancy loss, but cannot illuminate losses at earlier stages of development; (iii) studies that measure human chorionic gonadotrophin (hCG) reveal losses in the second week of development and beyond, but not before; and (iv) the classic studies of Hertig and Rock offer the only direct insight into the fate of human embryos from fertilisation under natural conditions. Re-examination of Hertig’s data demonstrates that his estimates for fertilisation rate and early embryo loss are highly imprecise and casts doubt on the validity of his numerical analysis. A recent re-analysis of hCG study data concluded that approximately 40-60% of embryos may be lost between fertilisation and birth, although this will vary substantially between individual women. In conclusion, natural human embryo mortality is lower than often claimed and widely accepted. Estimates for total prenatal mortality of 70% or higher are exaggerated and not supported by the available data. PMID:28580126

Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos.

PubMed

Hrubik, Jelena; Glisic, Branka; Samardzija, Dragana; Stanic, Bojana; Pogrmic-Majkic, Kristina; Fa, Svetlana; Andric, Nebojsa

2016-12-01

Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200μg/L) at different time points after fertilization. Results showed that 200μgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Embryo selection using time-lapse analysis (Early Embryo Viability Assessment) in conjunction with standard morphology: a prospective two-center pilot study.

PubMed

Kieslinger, Dorit C; De Gheselle, Stefanie; Lambalk, Cornelis B; De Sutter, Petra; Kostelijk, E Hanna; Twisk, Jos W R; van Rijswijk, Joukje; Van den Abbeel, Etienne; Vergouw, Carlijn G

2016-11-01

Does prospective embryo selection using the results from the Eava Test (Early Embryo Viability Assessment) in combination with standard morphology increase the pregnancy rate of IVF and ICSI patients compared to embryo selection based on morphology only? Embryo selection using the Eeva Test plus standard morphology on Day 3 results in comparable pregnancy rates as conventional morphological embryo selection. Time-lapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. The Eeva Test records the development of each embryo with a cell-tracking system and predicts the likelihood (High, Medium or Low) that an embryo will form a blastocyst based on an automated analysis of early cell division timings. This trial was designed as a prospective, observational, two-center pilot study with a propensity matched control group. The analysis involved 280 of 302 enrolled patients who were included in the Eeva Test group in 2013 and 560 control patients who were treated in the years 2011-2013. The majority of transfers (98%) were single embryo transfers. Two academic hospitals (VUmc Amsterdam and UZ Gent) enrolled patients <41 years old, with <3 previous attempts and ≥5 normally fertilized eggs. Propensity matching was used to identify a propensity matched control group from a cohort of 1777 patients based on age, cycle number, oocyte number and number of fertilized oocytes. There was no difference in patient baseline characteristics between the two groups. The ongoing pregnancy rate (OPR) of patients enrolled in the Eeva Test group (34.3%; 96/280) did not differ significantly from the OPR in the propensity matched control group (34.6%, 194/560; P = 0.92). However, significantly less top quality embryos (eight-cell embryos with ≤25% fragmentation) were transferred in the Eeva Test group compared to the propensity matched control group (70.4% vs. 82.3%; P < 0.001). The transfer of Eeva High and Medium embryos resulted in a

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.

Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis

PubMed Central

Yan, Dong; Lin, Xinhua

2007-01-01

Summary Previous studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. Here we show that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, we analyzed the role of Dlp in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, our results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila. PMID:17959166

Transfer of Dorsoventral and Terminal Information from the Ovary to the Embryo by a Common Group of Eggshell Proteins in Drosophila.

PubMed

Mineo, Alessandro; Furriols, Marc; Casanova, Jordi

2017-04-01

The Drosophila eggshell is an extracellular matrix that confers protection to the egg and also plays a role in transferring positional information from the ovary to pattern the embryo. Among the constituents of the Drosophila eggshell, Nasrat, Polehole, and Closca form a group of proteins related by sequence, secreted by the oocyte, and mutually required for their incorporation into the eggshell. Besides their role in eggshell integrity, Nasrat, Polehole, and Closca are also required for embryonic terminal patterning by anchoring or stabilizing Torso-like at the eggshell. Here, we show that they are also required for dorsoventral patterning, thereby unveiling that the dorsoventral and terminal systems, hitherto considered independent, share a common extracellular step. Furthermore, we show that Nasrat, Polehole, and Closca are required for proper Nudel activity, a protease acting both in embryonic dorsoventral patterning and eggshell integrity, thus providing a means to account for the role of Nasrat, Polehole, and Closca. We propose that a Nasrat/Polehole/Closca complex acts as a multifunctional hub to anchor various proteins synthesized at oogenesis, ensuring their spatial and temporal restricted function. Copyright © 2017 by the Genetics Society of America.

Macrophages and cellular immunity in Drosophila melanogaster.

PubMed

Gold, Katrina S; Brückner, Katja

2015-12-01

The invertebrate Drosophila melanogaster has been a powerful model for understanding blood cell development and immunity. Drosophila is a holometabolous insect, which transitions through a series of life stages from embryo, larva and pupa to adulthood. In spite of this, remarkable parallels exist between Drosophila and vertebrate macrophages, both in terms of development and function. More than 90% of Drosophila blood cells (hemocytes) are macrophages (plasmatocytes), making this highly tractable genetic system attractive for studying a variety of questions in macrophage biology. In vertebrates, recent findings revealed that macrophages have two independent origins: self-renewing macrophages, which reside and proliferate in local microenvironments in a variety of tissues, and macrophages of the monocyte lineage, which derive from hematopoietic stem or progenitor cells. Like vertebrates, Drosophila possesses two macrophage lineages with a conserved dual ontogeny. These parallels allow us to take advantage of the Drosophila model when investigating macrophage lineage specification, maintenance and amplification, and the induction of macrophages and their progenitors by local microenvironments and systemic cues. Beyond macrophage development, Drosophila further serves as a paradigm for understanding the mechanisms underlying macrophage function and cellular immunity in infection, tissue homeostasis and cancer, throughout development and adult life. Copyright © 2016. Published by Elsevier Ltd.

Maternal Nanos-Dependent RNA Stabilization in the Primordial Germ Cells of Drosophila Embryos.

PubMed

Sugimori, Seiko; Kumata, Yuji; Kobayashi, Satoru

2018-01-01

Nanos (Nos) is an evolutionary conserved protein expressed in the germline of various animal species. In Drosophila, maternal Nos protein is essential for germline development. In the germline progenitors, or the primordial germ cells (PGCs), Nos binds to the 3' UTR of target mRNAs to repress their translation. In contrast to this prevailing role of Nos, here we report that the 3' UTR of CG32425 mRNA mediates Nos-dependent RNA stabilization in PGCs. We found that the level of mRNA expressed from a reporter gene fused to the CG32425 3' UTR was significantly reduced in PGCs lacking maternal Nos (nos PGCs) as compared with normal PGCs. By deleting the CG32425 3' UTR, we identified the region required for mRNA stabilization, which includes Nos-binding sites. In normal embryos, CG32425 mRNA was maternally supplied into PGCs and remained in this cell type during embryogenesis. However, as expected from our reporter assay, the levels of CG32425 mRNA and its protein product expressed in nos PGCs were lower than in normal PGCs. Thus, we propose that Nos protein has dual functions in translational repression and stabilization of specific RNAs to ensure proper germline development. © 2017 Japanese Society of Developmental Biologists.

Paternal Diet-Induced Obesity Retards Early Mouse Embryo Development, Mitochondrial Activity and Pregnancy Health

PubMed Central

Binder, Natalie K.; Hannan, Natalie J.; Gardner, David K.

2012-01-01

Worldwide, 48% of adult males are overweight or obese. An association between infertility and excessive body weight is now accepted, although focus remains primarily on females. It has been shown that parental obesity results in compromised embryo development, disproportionate changes in embryo metabolism and reduced blastocyst cell number. The aim of this study was to determine whether paternal obesity has negative effects on the resultant embryo. Specifically, using in vitro fertilisation (IVF), we wanted to isolate the functional effects of obesity on sperm by examining the subsequent embryo both pre- and post-implantation. Epididymal sperm was collected from age matched normal and obese C57BL/6 mice and cryopreserved for subsequent IVF with oocytes collected from Swiss females (normal diet/weight). Obesity was induced in male mice by feeding a high fat diet of 22% fat for 10 weeks. Resultant embryos were cultured individually and development monitored using time-lapse microscopy. Paternal obesity resulted in a significant delay in preimplantation embryo development as early as syngamy (P<0.05). Metabolic parameters were measured across key developmental stages, demonstrating significant reduction in mitochondrial membrane potential (P<0.01). Blastocysts were stained to determine trophectoderm (TE) and inner cell mass (ICM) cell numbers, revealing significant differences in the ratio of cell allocation to TE and ICM lineages (P<0.01). Functional studies examining blastocyst attachment, growth and implantation demonstrated that blastocysts derived from sperm of obese males displayed significantly reduced outgrowth on fibronectin in vitro (P<0.05) and retarded fetal development in vivo following embryo transfer (P<0.05). Taken together, these data clearly demonstrate that paternal obesity has significant negative effects on the embryo at a variety of key early developmental stages, resulting in delayed development, reduced placental size and smaller offspring

Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos

PubMed Central

Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

2014-01-01

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development. PMID:24523664

The effect of flurbiprofen on the development of anencephaly in early stage chicken embryos.

PubMed

Özeren, Ersin; Er, Uygur; Güvenç, Yahya; Demirci, Adnan; Arıkök, Ata Türker; Şenveli, Engin; Ergün, Rüçhan Behzat

2015-04-01

The study investigated the effect of flurbiprofen on the development of anencephaly in early stage chicken embryos. We looked at four groups with a total of 36 embryos. There was a control group, a normal saline group, a normal-dose group and a high-dose group with ten, ten, eight and eight eggs with embryo respectively. Two embryos in the control group, studied with light microscopy at 48 h, were consistent with 28-29 hours' incubation in the Hamburger-Hamilton System. They had open neural tubes. The other embryos in this group were considered normal. One embryo in the normal saline group was on the occlusion stage at 48 h. One embryo showed an open neural tube. They were compatible with 28-29 hours' incubation in the Hamburger-Hamilton system. The remaining eight embryos showed normal development. In the normal dose group, one embryo showed underdevelopment of the embryonic disc and the embryo was dead. In four embryos, the neural tubes were open. One cranial malformation was found that was complicated with anencephaly in one embryo. In two embryos the neural tubes were closed, as they showed normal development, and they reached their expected stages according to the Hamburger-Hamilton classification. There was no malformation or growth retardation. Four experimental embryos were anencephalic in the high dose group, and three embryos had open neural tubes. One embryo exhibited both anencephaly and a neural tube closure defect. None of the embryos in this group showed normal development. Even the usual therapeutic doses of flurbiprofen increased the risk of neural tube defect. Flurbiprofen was found to significantly increase the risk of anencephaly. The provision of improved technical materials and studies with larger sample sizes will reveal the stage of morphological disruption during the development of embryos.

Drosophila I-R hybrid dysgenesis is associated with catastrophic meiosis and abnormal zygote formation.

PubMed

Orsi, Guillermo A; Joyce, Eric F; Couble, Pierre; McKim, Kim S; Loppin, Benjamin

2010-10-15

The Drosophila I-R type of hybrid dysgenesis is a sterility syndrome (SF sterility) associated with the mobilization of the I retrotransposon in female germ cells. SF sterility results from a maternal-effect embryonic lethality whose origin has remained unclear since its discovery about 40 years ago. Here, we show that meiotic divisions in SF oocytes are catastrophic and systematically fail to produce a functional female pronucleus at fertilization. As a consequence, most embryos from SF females rapidly arrest their development with aneuploid or damaged nuclei, whereas others develop as non-viable, androgenetic haploid embryos. Finally, we show that, in contrast to mutants affecting the biogenesis of piRNAs, SF egg chambers do not accumulate persistent DNA double-strand breaks, suggesting that I-element activity might perturb the functional organization of meiotic chromosomes without triggering an early DNA damage response.

Uncovering a Dynamic Feature of the Transcriptional Regulatory Network for Anterior-Posterior Patterning in the Drosophila Embryo

PubMed Central

Liu, Junbo; Ma, Jun

2013-01-01

Anterior-posterior (AP) patterning in the Drosophila embryo is dependent on the Bicoid (Bcd) morphogen gradient. However, most target genes of Bcd also require additional inputs to establish their expression domains, reflective of the operation of a cross-regulatory network and contributions of other maternal signals. This is in contrast to hunchback (hb), which has an anterior expression domain driven by an enhancer that appears to respond primarily to the Bcd input. To gain a better understanding of the regulatory logic of the AP patterning network, we perform quantitative studies that specifically investigate the dynamics of hb transcription during development. We show that Bcd-dependent hb transcription, monitored by the intron-containing nascent transcripts near the P2 promoter, is turned off quickly–on the order of a few minutes–upon entering the interphase of nuclear cycle 14A. This shutdown contrasts with earlier cycles during which active hb transcription can persist until the moment when the nucleus enters mitosis. The shutdown takes place at a time when the nuclear Bcd gradient profile in the embryo remains largely intact, suggesting that this is a process likely subject to control of a currently unknown regulatory mechanism. We suggest that this dynamic feature offers a window of opportunity for hb to faithfully interpret, and directly benefit from, Bcd gradient properties, including its scaling properties, to help craft a robust AP patterning outcome. PMID:23646132

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors.

PubMed

Kimber, S J; Sneddon, S F; Bloor, D J; El-Bareg, A M; Hawkhead, J A; Metcalfe, A D; Houghton, F D; Leese, H J; Rutherford, A; Lieberman, B A; Brison, D R

2008-05-01

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

A novel function for the IκB inhibitor Cactus in promoting Dorsal nuclear localization and activity in the Drosophila embryo.

PubMed

Cardoso, Maira Arruda; Fontenele, Marcio; Lim, Bomyi; Bisch, Paulo Mascarello; Shvartsman, Stanislav Y; Araujo, Helena Marcolla

2017-08-15

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals. © 2017. Published by The Company of Biologists Ltd.

Perturbed glial scaffold formation precedes axon tract malformation in Drosophila mutants.

PubMed

Jacobs, J R

1993-05-01

The longitudinal glia (LG), progeny of a single glioblast, form a scaffold that presages the formation of longitudinal tracts in the ventral nerve cord (VNC) of the Drosophila embryo. The LG are used as a substrate during the extension of the first axons of the longitudinal tract. I have examined the differentiation of the LG in six mutations in which the longitudinal tracts were absent, displaced, or interrupted to determine whether the axon tract malformations may be attributable to disruptions in the LG scaffold. Embryos mutant for the gene prospero had no longitudinal tracts, and glial differentiation remained arrested at a preaxonogenic state. Two mutants of the Polycomb group also lacked longitudinal tracts; here the glia failed to form an oriented scaffold, but cytological differentiation of the LG was unperturbed. The longitudinal tracts in embryos mutant for slit fused at the VNC midline and scaffold formation was normal, except that it was medially displaced. Longitudinal tracts had intersegmental interruptions in embryos mutant for hindsight and midline. In hindsight, there were intersegmental gaps in the glial scaffold. In midline, the glial scaffold retracted after initial extension. LG morphogenesis during axonogenesis was abnormal in midline. Commitment to glial identity and glial differentiation also occurred before scaffold formation. In all mutants examined, the early distribution of the glycoprotein neuroglian was perturbed. This was indicative of early alterations in VNC pattern present before LG scaffold formation began. Therefore, some changes in scaffold formation may have reflected changes in the placement and differentiation of other cells of the VNC. In all mutants, alterations in scaffold formation preceded longitudinal axon tract formation.

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models

PubMed Central

2011-01-01

Background PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. Methods Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. Results PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control). Conclusions PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF

P element excision in drosophila melanogaster and related drosophilids

USDA-ARS?s Scientific Manuscript database

The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species, Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlik...

Method of Electroporation for the Early Chick Embryo

NASA Astrophysics Data System (ADS)

Hatakeyama, Jun; Shimamura, Kenji

Chick embryos have long been one of the favored model systems in the field of embryology and developmental biology. Recent advances in the gene manipulation technologies (Muramatsu et al., 1997; Nakamura et al., 2004) make this model system even more attractive for the developmental biologists (see review by Stern, 2005). Thanks to its two dimensional geometry, easiness in accessibility and observation, and well-established fate maps (e.g. Couly and Le Douarin, 1988; Garcia-Martinez et al., 1993; Hatada and Stern, 1994; Psychoyos and Stern, 1996; Sawada and Aoyama, 1999; Cobos et al., 2001; Lopez-Sanchez et al., 2001; Redkar et al., 2001; Fernandez-Garre et al., 2002; Kimura et al., 2006; Matsushita et al., 2008), it has great advantages especially for studies at the early embryonic stages, such as the processes of gastrulation, neural induction, left-right patterning, etc. For such purposes, a whole embryo culture system, originally invented by Dennis A. T. New (New, 1955), and its derivatives (Flamme, 1987; Sundin and Eichele, 1992; Stern, 1993; Chapman et al., 2001) have been widely used.

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

PubMed Central

Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

2017-01-01

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers.

PubMed

Lopes-da-Costa, L; Chagas e Silva, J; Deloche, M C; Jeanguyot, N; Humblot, P; Horta, A E M

2011-08-01

The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size at transfer (whole or demi embryo) on Day 7 of the estrous cycle (Day 0 = estrus) and P4 supplementation between Days 7 to 19 through an intravaginal device (yes or no) on plasma P4 and PSPB concentrations and on embryo measurements. Plasma P4 concentrations were measured by RIA on Days 0, 7, 14, 19, 21, 25, 35, 42, 49, 56 and 63 of pregnancy and, PSPB concentrations were measured by ELISA on Days 7, 21, 25, 35, 42, 49, 56 and 63. The presence of an embryonic vesicle was detected on Day 25, embryonic/fetal movements and heartbeat were evaluated on Days 42 and 63 and embryo measurements [crown-rump length (CRL) and width at mid body] were obtained on Day 42 through ultrasonography. In non-supplemented pregnancies, Day 42 whole embryos had higher (P < 0.05) CRL and width than demi embryos, but the difference averaged only 1 to 2 mm. In P4 supplemented pregnancies, whole and demi embryos attained a similar size on Day 42 of pregnancy. Embryo size at transfer, early exogenous P4 supplementation and their interactions had no effects (P > 0.05) on plasma P4 concentrations. However, the post-hoc LSD evaluation showed that plasma P4 concentrations on Day 25 were higher (P < 0.001) in whole than in demi embryo derived pregnancies and, that exogenous P4 supplementation increased (P < 0.05) plasma P4 concentrations on Day 19 of pregnancy. The plasma PSPB detection rate on Days 7 to 63 of pregnancy was similar in pregnancies resulting from the transfer of whole and demi embryos. From a total of 93 recipients remaining pregnant until Day 63, plasma PSPB was constantly undetectable on Day 7, was detected in 4% of

The Effect of Levetiracetam on Closure of the Midline in Early Chicken Embryos.

PubMed

Ozgural, Onur; Armagan, Ercan; Bozkurt, Melih; Eroglu, Umit; Kahilogullari, Gokmen; Unlu, Agahan

2015-01-01

Genetic predisposition and some environmental factors play an important role in the development of neural tube defects. Levetiracetam is a new drug that has been approved in the treatment of partial seizures. We aimed in this study to determine the effect of levetiracetam on chick embryos. One hundred and sixty fertile non-pathogenic Super Nick eggs were incubated for 24 hours and were divided into four groups of 40 eggs each. Levetiracetam was administered via the sub-blastodermic route. The eggs were incubated for another 24 hours. All eggs were opened at the 48th hour, and the embryos were evaluated morphologically and histopathologically. The effects of levetiracetam on the embryo were correlated with the dose of levetiracetam. In the light of the results, it was determined that the use of increasing doses of levetiracetam led to defects of midline closure in early chicken embryos. Levetiracetam, a new antiepileptic drug that is effective especially on calcium ion concentration, leads to defects in midline closure in embryos in a dose-dependent manner. Further studies are needed to show the mechanism of embryonic damage and the mechanisms of its teratogenous effects associated with genetic and environmental factors.

Bovine oocytes and early embryos express Staufen and ELAVL RNA-binding proteins.

PubMed

Calder, M D; Madan, P; Watson, A J

2008-05-01

RNA-binding proteins (RBP) influence RNA editing, localization, stability and translation and may contribute to oocyte developmental competence by regulating the stability and turnover of oogenetic mRNAs. The expression of Staufen 1 and 2 and ELAVL1, ELAVL2 RNA-binding proteins during cow early development was characterized. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, matured, inseminated and subjected to embryo culture in vitro. Oocyte or preimplantation embryo pools were processed for RT-PCR and whole-mount immunofluorescence analysis of mRNA expression and protein distribution. STAU1 and STAU2 and ELAVL1 mRNAs and proteins were detected throughout cow preimplantation development from the germinal vesicle (GV) oocyte to the blastocyst stage. ELAVL2 mRNAs were detectable from the GV to the morula stage, whereas ELAVL2 protein was in all stages examined and localized to both cytoplasm and nuclei. The findings provide a foundation for investigating the role of RBPs during mammalian oocyte maturation and early embryogenesis.

Drosophila non-muscle myosin II motor activity determines the rate of tissue folding

PubMed Central

Vasquez, Claudia G; Heissler, Sarah M; Billington, Neil; Sellers, James R; Martin, Adam C

2016-01-01

Non-muscle cell contractility is critical for tissues to adopt shape changes. Although, the non-muscle myosin II holoenzyme (myosin) is a molecular motor that powers contraction of actin cytoskeleton networks, recent studies have questioned the importance of myosin motor activity cell and tissue shape changes. Here, combining the biochemical analysis of enzymatic and motile properties for purified myosin mutants with in vivo measurements of apical constriction for the same mutants, we show that in vivo constriction rate scales with myosin motor activity. We show that so-called phosphomimetic mutants of the Drosophila regulatory light chain (RLC) do not mimic the phosphorylated RLC state in vitro. The defect in the myosin motor activity in these mutants is evident in developing Drosophila embryos where tissue recoil following laser ablation is decreased compared to wild-type tissue. Overall, our data highlights that myosin activity is required for rapid cell contraction and tissue folding in developing Drosophila embryos. DOI: http://dx.doi.org/10.7554/eLife.20828.001 PMID:28035903

Supplementation with CTGF, SDF1, NGF, and HGF promotes ovine in vitro oocyte maturation and early embryo development.

PubMed

Wang, D H; Ren, J; Zhou, C J; Han, Z; Wang, L; Liang, C G

2018-05-17

The strategies for improving the in vitro maturation (IVM) of domestic animal oocytes focus on promoting nuclear and cytoplasmic maturation. The identification of paracrine factors and their supplementation in the culture medium represent effective approaches for oocyte maturation and embryo development. This study investigated the effects of paracrine factor supplementation including connective tissue growth factor (CTGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and stromal derived factor 1 (SDF1) on ovine oocytes and early parthenogenetic embryos using an in vitro culture system. First, we identified the optimal concentrations of CTGF (30 ng/mL), SDF1 (10 ng/mL), NGF (3 ng/mL), and HGF (100 ng/mL) for promoting oocyte maturation, which combined, induced nuclear maturation in 94.19% of oocytes. This combination also promoted cumulus cell expansion and inhibited oocyte/cumulus apoptosis, while enabling a larger proportion (33.04%) of embryos to develop into blastocysts than in the controls and prevented embryo apoptosis. These novel findings demonstrate that the paracrine factors CTGF, SDF1, NGF, and HGF facilitate ovine oocyte and early parthenogenetic embryo development in vitro. Thus, supplementation with these factors may help optimize the IVM of ovine oocytes and early parthenogenetic embryo development strategies. Copyright © 2018 Elsevier Inc. All rights reserved.

Mouse Embryo Compaction.

PubMed

White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

2016-01-01

Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. © 2016 Elsevier Inc. All rights reserved.

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

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.

PubMed

Flores, Luis E; Hildebrandt, Thomas B; Kühl, Anja A; Drews, Barbara

2014-05-10

Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9-11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert's membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy

PubMed Central

2014-01-01

Background Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. Methods In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. Results The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9–11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert’s membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and

Moral uncertainty in bioethical argumentation: a new understanding of the pro-life view on early human embryos.

PubMed

Żuradzki, Tomasz

2014-12-01

In this article, I present a new interpretation of the pro-life view on the status of early human embryos. In my understanding, this position is based not on presumptions about the ontological status of embryos and their developmental capabilities but on the specific criteria of rational decisions under uncertainty and on a cautious response to the ambiguous status of embryos. This view, which uses the decision theory model of moral reasoning, promises to reconcile the uncertainty about the ontological status of embryos with the certainty about normative obligations. I will demonstrate that my interpretation of the pro-life view, although seeming to be stronger than the standard one, has limited scope and cannot be used to limit destructive research on human embryos.

Pathway to a Phenocopy: Heat Stress Effects in Early Embryogenesis

PubMed Central

Crews, Sarah M.; McCleery, W. Tyler; Hutson, M. Shane

2015-01-01

Background Heat shocks applied at the onset of gastrulation in early Drosophila embryos frequently lead to phenocopies of U-shaped mutants – having characteristic failures in the late morphogenetic processes of germband retraction and dorsal closure. The pathway from non-specific heat stress to phenocopied abnormalities is unknown. Results Drosophila embryos subjected to 30-min, 38-°C heat shocks at gastrulation appear to recover and restart morphogenesis. Post-heat-shock development appears normal, albeit slower, until a large fraction of embryos develop amnioserosa holes (diameters > 100 μm). These holes are positively correlated with terminal U-shaped phenocopies. They initiate between amnioserosa cells and open over tens of minutes by evading normal wound healing responses. They are not caused by tissue-wide increases in mechanical stress or decreases in cell-cell adhesion, but instead appear to initiate from isolated apoptosis of amnioserosa cells. Conclusions The pathway from heat shock to U-shaped phenocopies involves the opening of one or more large holes in the amnioserosa that compromise its structural integrity and lead to failures in morphogenetic processes that rely on amnioserosa-generated tensile forces. The proposed mechanism by which heat shock leads to hole initiation and expansion is heterochonicity – i.e., disruption of morphogenetic coordination between embryonic and extra-embryonic cell types. PMID:26498920

From egg to gastrula: How the cell cycle is remodeled during the Drosophila mid-blastula transition

PubMed Central

Farrell, Jeffrey A.; O’Farrell, Patrick H.

2015-01-01

Many, if not most, embryos begin development with extremely short cell cycles that exhibit unusually rapid DNA replication and no gap phases. The commitment to the cell cycle in the early embryo appears to preclude many other cellular processes which only emerge as the cell cycle slows, at a major embryonic transition known as the mid-blastula transition (MBT) just prior to gastrulation. As reviewed here, genetic and molecular studies in Drosophila have identified changes that extend S phase and introduce a post-replicative gap phase, G2, to slow the cell cycle. While many mysteries remain about the upstream regulators of these changes, we review the core mechanisms of the change in cell cycle regulation and discuss advances in our understanding of how these might be timed and triggered. Finally, we consider how the elements of this program may be conserved or changed in other organisms. PMID:25195504

Stochastic model for gene transcription on Drosophila melanogaster embryos

NASA Astrophysics Data System (ADS)

Prata, Guilherme N.; Hornos, José Eduardo M.; Ramos, Alexandre F.

2016-02-01

We examine immunostaining experimental data for the formation of stripe 2 of even-skipped (eve) transcripts on D. melanogaster embryos. An estimate of the factor converting immunofluorescence intensity units into molecular numbers is given. The analysis of the eve dynamics at the region of stripe 2 suggests that the promoter site of the gene has two distinct regimes: an earlier phase when it is predominantly activated until a critical time when it becomes mainly repressed. That suggests proposing a stochastic binary model for gene transcription on D. melanogaster embryos. Our model has two random variables: the transcripts number and the state of the source of mRNAs given as active or repressed. We are able to reproduce available experimental data for the average number of transcripts. An analysis of the random fluctuations on the number of eves and their consequences on the spatial precision of stripe 2 is presented. We show that the position of the anterior or posterior borders fluctuate around their average position by ˜1 % of the embryo length, which is similar to what is found experimentally. The fitting of data by such a simple model suggests that it can be useful to understand the functions of randomness during developmental processes.

Characterization of a Drosophila homologue of the human myelodysplasia/myeloid leukemia factor (MLF).

PubMed

Ohno, K; Takahashi, Y; Hirose, F; Inoue, Y H; Taguchi, O; Nishida, Y; Matsukage, A; Yamaguchi, M

2000-12-30

The transcription factor DREF regulates proliferation-related genes in Drosophila. With two-hybrid screening using DREF as a bait, we have obtained a clone encoding a protein homologous to human myelodysplasia/myeloid leukemia factor 1 (hMLF1). We termed the protein Drosophila MLF (dMLF); it consists of a polypeptide of 309 amino acid residues, whose sequence shares 23.1% identity with hMLF1. High conservation of 54.2% identity over 107 amino acids was found in the central region. The dMLF gene was mapped to 52D on the second chromosome by in situ hybridization. Interaction between dMLF and DREF in vitro could be confirmed by glutathione S-transferase pull-down assay, with the conserved central region appearing to play an important role in this. Northern blot hybridization analysis revealed dMLF mRNA levels to be high in unfertilized eggs, early embryos, pupae and adult males, and relatively low in adult females and larvae. This fluctuation of mRNA during Drosophila development is similar to that observed for DREF mRNA, except in the pupa and adult male. Using a specific antibody against the dMLF, we performed immunofluorescent staining of Drosophila Kc cells and showed a primarily cytoplasmic staining, whereas DREF localizes in the nucleus. However, dMLF protein contains a putative 14-3-3 binding motif involved in the subcellular localization of various regulatory molecules, and interaction with DREF could be regulated through this motif. The transgenic fly data suggesting the genetic interaction between DREF and dMLF support this possibility. Characterization of dMLF in the present study provides the molecular basis for analysis of its significance in Drosophila.

Maternal hCG concentrations in early IVF pregnancies: associations with number of cells in the Day 2 embryo and oocytes retrieved.

PubMed

Tanbo, T G; Eskild, A

2015-12-01

Do number of cells in the transferred cleavage stage embryo and number of oocytes retrieved for IVF influence maternal hCG concentrations in early pregnancies? Compared with transfer of a 2-cell embryo, transfer of a 4-cell embryo results in higher hCG concentrations on Day 12 after transfer, and more than 20 oocytes retrieved were associated with low hCG concentrations. Maternal hCG concentration in very early pregnancy varies considerably among women, but is likely to be an indicator of time since implantation of the embryo into the endometrium, in addition to number and function of trophoblast cells. We followed 1047 pregnancies after IVF/ICSI from oocyte retrieval until Day 12 after embryo transfer. Women were recruited in Norway during the years 2005-2013. Successful pregnancies after transfer of one single embryo that had been cultured for 2 days were included. Maternal hCG was quantified on Day 12 after embryo transfer by chemiluminescence immunoassay, which measures intact hCG and the free β-hCG chain. Information on a successful pregnancy, defined as birth after >16 weeks, was obtained by linkage to the Medical Birth Registry of Norway. Transfer of a 4-cell embryo resulted in higher maternal hCG concentrations compared with transfer of a 2-cell embryo (134.8 versus 87.8 IU/l, P < 0.05). A high number of oocytes retrieved (>20) was associated with low hCG concentrations (P < 0.05). The factors studied explain a limited part of the total variation of hCG concentrations in early pregnancy. Although embryo transfer was performed at the same time after fertilization, we do not know the exact time of implantation. A further limitation to our study is that the number of pregnancies after transfer of a 2-cell embryo was small (27 cases). Number of cells in the transferred embryo and number of oocytes retrieved may influence the conditions and timing for embryo implantation in different ways and thereby influence maternal hCG concentrations. Such knowledge may be

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

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

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

Early maternal serum ß-human chorionic gonadotropin (ß-hCG) levels and sex-related growth difference of IVF embryos.

PubMed

Esh-Broder, Efrat; Oron, Galia; Son, Weon-Young; Holzer, Hananel; Tulandi, Togas

2015-10-01

Maternal serum ß-human chorionic gonadotropin (ß-hCG) represents the trophoblastic cell mass and is an indirect measurement of embryo development at early implantation stage. Studies in animals and human embryos detected sex-related growth differences (SRGD) in favour of male embryos during the pre-implantation period. The purpose of our study was to correlate SRGD and maternal serum ß-hCG at 16 days after embryo transfer. We retrospectively analysed all (fresh and frozen) non-donor, single embryo transfers (SET), elective and not elective, that were performed between December 2008 and December 2013. We included ß-hCG values from day 16 after oocyte collection of pregnancies resulting in live birth. Neonatal gender was retrieved from patient files. Male and female embryos were further grouped to cleavage and blastocyst stage transfers. Regression analysis for confounding variables included maternal age, maternal body mass index (BMI), use of micromanipulation (ICSI), embryo quality (grade), assisted hatching, day of transfer and fresh or frozen embryo transfer. Seven hundred eighty-six non-donor SETs resulted in live birth. After including only day 16 serum ß-hCG results, 525 SETs were analysed. Neonatal gender was available for 522 cases. Mean maternal serum ß-hCG levels were similar, 347 ± 191 IU/L in the male newborn group and 371 ± 200 IU/L in the female group. The difference between ß-hCG levels remained insignificant after adjusting for confounding variables. Early maternal ß-hCG levels after embryo transfers did not represent SRGD in our study.

Genetic control of cuticle formation during embryonic development of Drosophila melanogaster.

PubMed Central

Ostrowski, Stephen; Dierick, Herman A; Bejsovec, Amy

2002-01-01

The embryonic cuticle of Drosophila melanogaster is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. We have characterized mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion reversed within the eggshell (the "retroactive" phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the "blimp" phenotype). We find that one of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and that a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. We also demonstrate that two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and we describe a new mutation, called zeppelin (zep), that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do. PMID:12019232

Chicken Embryos as a Potential New Model for Early Onset Type I Diabetes

PubMed Central

Shi, Liheng; Ko, Michael L.; Huang, Cathy Chia-Yu; Park, So-Young; Hong, Min-Pyo; Ko, Gladys Y.-P.

2014-01-01

Diabetic retinopathy (DR) is the leading cause of blindness among the American working population. The purpose of this study is to establish a new diabetic animal model using a cone-dominant avian species to address the distorted color vision and altered cone pathway responses in prediabetic and early diabetic patients. Chicken embryos were injected with either streptozotocin (STZ), high concentration of glucose (high-glucose), or vehicle at embryonic day 11. Cataracts occurred in varying degrees in both STZ- and high glucose-induced diabetic chick embryos at E18. Streptozotocin-diabetic chicken embryos had decreased levels of blood insulin, glucose transporter 4 (Glut4), and phosphorylated protein kinase B (pAKT). In STZ-injected E20 embryos, the ERG amplitudes of both a- and b-waves were significantly decreased, the implicit time of the a-wave was delayed, while that of the b-wave was significantly increased. Photoreceptors cultured from STZ-injected E18 embryos had a significant decrease in L-type voltage-gated calcium channel (L-VGCC) currents, which was reflected in the decreased level of L-VGCCα1D subunit in the STZ-diabetic retinas. Through these independent lines of evidence, STZ-injection was able to induce pathological conditions in the chicken embryonic retina, and it is promising to use chickens as a potential new animal model for type I diabetes. PMID:25133191

The developmental basis for germline mosaicism in mouse and Drosophila melanogaster.

PubMed

Drost, J B; Lee, W R

1998-01-01

Data involving germline mosaics in Drosophila melanogaster and mouse are reconciled with developmental observations. Mutations that become fixed in the early embryo before separation of soma from the germline may, by the sampling process of development, continue as part of germline and/or differentiate into any somatic tissue. The cuticle of adult D. melanogaster, because of segmental development, can be used to estimate the proportion of mutant nuclei in the early embryo, but most somatic tissues and the germlines of both species continue from samples too small to be representative of the early embryo. Because of the small sample of cells/nuclei that remain in the germline after separation of soma in both species, mosaic germlines have percentages of mutant cells that vary widely, with a mean of 50% and an unusual platykurtic, flat-topped distribution. While the sampling process leads to similar statistical results for both species, their patterns of development are very different. In D. melanogaster the first differentiation is the separation of soma from germline with the germline continuing from a sample of only two to four nuclei, whereas the adult cuticle is a representative sample of cleavage nuclei. The presence of mosaicism in D. melanogaster germline is independent of mosaicism in the eye, head, and thorax. This independence was used to determine that mutations can occur at any of the early embryonic cell divisions and still average 50% mutant germ cells when the germline is mosaic; however, the later the mutation occurs, the higher the proportion of completely nonmutant germlines. In contrast to D. melanogaster, the first differentiation in the mouse does not separate soma from germline but produces the inner cell mass that is representative of the cleavage nuclei. Following formation of the primitive streak, the primordial germ cells develop at the base of the allantois and among a clonally related sample of cells, providing the same statistical

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

PubMed Central

Murray, Jessica R.; Stanciauskas, Monika E.; Aralere, Tejas S.; Saha, Margaret S.

2014-01-01

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access. PMID:24999108

Embryonic multipotent progenitors remodel the Drosophila airways during metamorphosis

PubMed Central

Pitsouli, Chrysoula; Perrimon, Norbert

2010-01-01

Adult structures in holometabolous insects such as Drosophila are generated by groups of imaginal cells dedicated to the formation of different organs. Imaginal cells are specified in the embryo and remain quiescent until the larval stages, when they proliferate and differentiate to form organs. The Drosophila tracheal system is extensively remodeled during metamorphosis by a small number of airway progenitors. Among these, the spiracular branch tracheoblasts are responsible for the generation of the pupal and adult abdominal airways. To understand the coordination of proliferation and differentiation during organogenesis of tubular organs, we analyzed the remodeling of Drosophila airways during metamorphosis. We show that the embryonic spiracular branch tracheoblasts are multipotent cells that express the homeobox transcription factor Cut, which is necessary for their survival and normal development. They give rise to three distinct cell populations at the end of larval development, which generate the adult tracheal tubes, the spiracle and the epidermis surrounding the spiracle. Our study establishes the series of events that lead to the formation of an adult tubular structure in Drosophila. PMID:20940225

From Embryo to Adult: Hematopoiesis along the Drosophila Life Cycle.

PubMed

Ramond, Elodie; Meister, Marie; Lemaitre, Bruno

2015-05-26

Studies on Drosophila hematopoiesis have thus far focused on the embryonic and larval origin of hemocytes, the fly blood cells. In this issue of Developmental Cell, Ghosh et al. (2015) identify adult hematopoietic hubs containing progenitors that can differentiate into different blood cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

Disparities in reproductive outcomes according to the endometrial preparation protocol in frozen embryo transfer : The risk of early pregnancy loss in frozen embryo transfer cycles.

PubMed

Hatoum, I; Bellon, L; Swierkowski, N; Ouazana, M; Bouba, S; Fathallah, K; Paillusson, B; Bailly, M; Boitrelle, F; Alter, L; Bergère, M; Selva, J; Wainer, R

2018-03-01

The purpose of this study was to determine the effect of stimulated and artificial endometrial preparation protocols on reproductive outcomes in frozen embryo transfer (FET) cycles. We performed a retrospective study of 1926 FET cycles over a 3.5-year period in the Fertility Unit at a University Hospital. Stimulated and artificial protocols were used for endometrial preparation. The embryos for FET were obtained from either in vitro fertilization or intracytoplasmic sperm injection cycles. Live birth rate and early pregnancy loss rates were retrospectively compared. In artificial protocols, oral or vaginal administration of oestradiol 2 mg two or three times a day was followed by vaginal supplementation with progesterone 200 mg two or three times a day. In stimulated protocols, recombinant follicle-stimulating hormone was administered from day 4 onward. Vaginal ultrasound was used for endometrial and ovarian monitoring. A pregnancy test was performed 14 days after FET. If it was positive, oestradiol and progesterone were administered up until the 12th week of gestation in artificial cycles. We defined early pregnancy losses as biochemical pregnancies (preclinical losses) and miscarriages. Data on 865 artificial cycles (45% of the total) and 1061 stimulated cycles (55%) were collected. Early pregnancy loss rate was significantly lower for stimulated cycles (34.2%) than for artificial cycles (56.9%), and the live birth rate was significantly higher for stimulated cycles (59.7%) than for artificial cycles (29.1%). In frozen embryo transfer, artificial cycles were associated with more early pregnancy loss and lower live birth rate than stimulated cycles.

Stochastic phase of ventral furrow formation in the Drosophila embryo: cellular constriction chains, mechanical feedback, and robustness

NASA Astrophysics Data System (ADS)

Blawzdziewicz, Jerzy; Gao, Guo-Jie J.; Holcomb, Michael C.; Thomas, Jeffrey H.

The key process giving rise to ventral furrow formation (VFF) in Drosophila embryo is apical constriction of cells in the ventral region. The constriction produces negative spontaneous curvature of the cell layer. During the initial slower phase of VFF approximately 40% of cells constrict in a seemingly random order. We show that this initial phase of VFF does not depend on random uncorrelated events. Instead, constricted cell apices form well-defined correlated structures, i.e., cellular constriction chains (CCCs), indicative of strong spatial and directional correlations between the constriction events. We argue that this chain formation is a signature of mechanical signaling that coordinates apical constrictions through tensile stress. To gain insights into the mechanisms involved in this correlated constriction process, we propose an active granular fluid (AGF) model which considers a tissue as a collection of mechanically active, stress-responsive objects. Our AGF molecular dynamics simulations show that cell constriction sensitivity to tensile stress results in formation of CCCs whereas compressive-stress sensitivity leads to compact constricted cell clusters; the CCCs, which can penetrate less-active regions, increase the robustness of the VFF process.

The early Cambrian fossil embryo Pseudooides is a direct-developing cnidarian, not an early ecdysozoan.

PubMed

Duan, Baichuan; Dong, Xi-Ping; Porras, Luis; Vargas, Kelly; Cunningham, John A; Donoghue, Philip C J

2017-12-20

Early Cambrian Pseudooides prima has been described from embryonic and post-embryonic stages of development, exhibiting long germ-band development. There has been some debate about the pattern of segmentation, but this interpretation, as among the earliest records of ecdysozoans, has been generally accepted. Here, we show that the 'germ band' of P. prima embryos separates along its mid axis during development, with the transverse furrows between the 'somites' unfolding into the polar aperture of the ten-sided theca of Hexaconularia sichuanensis , conventionally interpreted as a scyphozoan cnidarian; co-occurring post-embryonic remains of ecdysozoans are unrelated. We recognize H. sichuanensis as a junior synonym of P. prima as a consequence of identifying these two form-taxa as distinct developmental stages of the same organism. Direct development in P. prima parallels the co-occuring olivooids Olivooides, and Quadrapyrgites and Bayesian phylogenetic analysis of a novel phenotype dataset indicates that, despite differences in their tetra-, penta- and pseudo-hexa-radial symmetry, these hexangulaconulariids comprise a clade of scyphozoan medusozoans, with Arthrochites and conulariids, that all exhibit direct development from embryo to thecate polyp. The affinity of hexangulaconulariids and olivooids to extant scyphozoan medusozoans indicates that the prevalence of tetraradial symmetry and indirect development are a vestige of a broader spectrum of body-plan symmetries and developmental modes that was manifest in their early Phanerozoic counterparts. © 2017 The Authors.

The early Cambrian fossil embryo Pseudooides is a direct-developing cnidarian, not an early ecdysozoan

PubMed Central

2017-01-01

Early Cambrian Pseudooides prima has been described from embryonic and post-embryonic stages of development, exhibiting long germ-band development. There has been some debate about the pattern of segmentation, but this interpretation, as among the earliest records of ecdysozoans, has been generally accepted. Here, we show that the ‘germ band’ of P. prima embryos separates along its mid axis during development, with the transverse furrows between the ‘somites’ unfolding into the polar aperture of the ten-sided theca of Hexaconularia sichuanensis, conventionally interpreted as a scyphozoan cnidarian; co-occurring post-embryonic remains of ecdysozoans are unrelated. We recognize H. sichuanensis as a junior synonym of P. prima as a consequence of identifying these two form-taxa as distinct developmental stages of the same organism. Direct development in P. prima parallels the co-occuring olivooids Olivooides, and Quadrapyrgites and Bayesian phylogenetic analysis of a novel phenotype dataset indicates that, despite differences in their tetra-, penta- and pseudo-hexa-radial symmetry, these hexangulaconulariids comprise a clade of scyphozoan medusozoans, with Arthrochites and conulariids, that all exhibit direct development from embryo to thecate polyp. The affinity of hexangulaconulariids and olivooids to extant scyphozoan medusozoans indicates that the prevalence of tetraradial symmetry and indirect development are a vestige of a broader spectrum of body-plan symmetries and developmental modes that was manifest in their early Phanerozoic counterparts. PMID:29237861

Early developmental gene regulation in Strongylocentrotus purpuratus embryos in response to elevated CO₂ seawater conditions.

PubMed

Hammond, LaTisha M; Hofmann, Gretchen E

2012-07-15

Ocean acidification, or the increased uptake of CO(2) by the ocean due to elevated atmospheric CO(2) concentrations, may variably impact marine early life history stages, as they may be especially susceptible to changes in ocean chemistry. Investigating the regulatory mechanisms of early development in an environmental context, or ecological development, will contribute to increased understanding of potential organismal responses to such rapid, large-scale environmental changes. We examined transcript-level responses to elevated seawater CO(2) during gastrulation and the initiation of spiculogenesis, two crucial developmental processes in the purple sea urchin, Strongylocentrotus purpuratus. Embryos were reared at the current, accepted oceanic CO(2) concentration of 380 microatmospheres (μatm), and at the elevated levels of 1000 and 1350 μatm, simulating predictions for oceans and upwelling regions, respectively. The seven genes of interest comprised a subset of pathways in the primary mesenchyme cell gene regulatory network (PMC GRN) shown to be necessary for the regulation and execution of gastrulation and spiculogenesis. Of the seven genes, qPCR analysis indicated that elevated CO(2) concentrations only had a significant but subtle effect on two genes, one important for early embryo patterning, Wnt8, and the other an integral component in spiculogenesis and biomineralization, SM30b. Protein levels of another spicule matrix component, SM50, demonstrated significant variable responses to elevated CO(2). These data link the regulation of crucial early developmental processes with the environment that these embryos would be developing within, situating the study of organismal responses to ocean acidification in a developmental context.

Studies on trypsin-like enzymes in sperm and early embryos

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

Penn, A.

1975-12-09

Results are reported from a study of acrosomal proteinase, a trypsin-like enzyme (TLE), found in the acrosome of all eutherian mammals studied to date. It has been implicated in the dissolution of a passage for the sperm through the zona pellucida of the egg, a step necessary for in vivo fertilization. A cytochemical procedure employing autoradiographic film as a gelatin substrate is described for in situ detection and localization of acrosomal proteolytic activity. A role for TLE in the early development of embryos is suggested. (CH)

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

Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1.

PubMed

Bieber, A J; Snow, P M; Hortsch, M; Patel, N H; Jacobs, J R; Traquina, Z R; Schilling, J; Goodman, C S

1989-11-03

Drosophila neuroglian is an integral membrane glycoprotein that is expressed on a variety of cell types in the Drosophila embryo, including expression on a large subset of glial and neuronal cell bodies in the central and peripheral nervous systems and on the fasciculating axons that extend along them. Neuroglian cDNA clones were isolated by expression cloning. cDNA sequence analysis reveals that neuroglian is a member of the immunoglobulin superfamily. The extracellular portion of the protein consists of six immunoglobulin C2-type domains followed by five fibronectin type III domains. Neuroglian is closely related to the immunoglobulin-like vertebrate neural adhesion molecules and, among them, shows most extensive homology to mouse L1. Its homology to L1 and its embryonic localization suggest that neuroglian may play a role in neural and glial cell adhesion in the developing Drosophila embryo. We report here on the identification of a lethal mutation in the neuroglian gene.

The First Human Cloned Embryo.

ERIC Educational Resources Information Center

Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

2002-01-01

Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

Supramolecular delivery of photoactivatable fluorophores in developing embryos

NASA Astrophysics Data System (ADS)

Zhang, Yang; Tang, Sicheng; Sansalone, Lorenzo; Thapaliya, Ek Raj; Baker, James D.; Raymo, Françisco M.

2017-02-01

The identification of noninvasive strategies to monitor dynamics within living organisms in real time is essential to elucidate the fundamental factors governing a diversity of biological processes. This study demonstrates that the supramolecular delivery of photoactivatable fluorophores in Drosophila melanogaster embryos allows the real-time tracking of translocating molecules. The designed photoactivatable fluorophores switch from an emissive reactant to an emissive product with spectrally-resolved fluorescence, under moderate blue-light irradiation conditions. These hydrophobic fluorescent probes can be encapsulated within supramolecular hosts and delivered to the cellular blastoderm of the embryos. Thus, the combination of supramolecular delivery and fluorescence photoactivation translates into a noninvasive method to monitor dynamics in vivo and can evolve into a general chemical tool to track motion in biological specimens.

From stem cell to embryo without centrioles.

PubMed

Stevens, Naomi R; Raposo, Alexandre A S F; Basto, Renata; St Johnston, Daniel; Raff, Jordan W

2007-09-04

Centrosome asymmetry plays a key role in ensuring the asymmetric division of Drosophila neural stem cells (neuroblasts [NBs]) and male germline stem cells (GSCs) [1-3]. In both cases, one centrosome is anchored close to a specific cortical region during interphase, thus defining the orientation of the spindle during the ensuing mitosis. To test whether asymmetric centrosome behavior is a general feature of stem cells, we have studied female GSCs, which divide asymmetrically, producing another GSC and a cystoblast. The cystoblast then divides and matures into an oocyte, a process in which centrosomes exhibit a series of complex behaviors proposed to play a crucial role in oogenesis [4-6]. We show that the interphase centrosome does not define spindle orientation in female GSCs and that DSas-4 mutant GSCs [7], lacking centrioles and centrosomes, invariably divide asymmetrically to produce cystoblasts that proceed normally through oogenesis-remarkably, oocyte specification, microtubule organization, and mRNA localization are all unperturbed. Mature oocytes can be fertilized, but embryos that cannot support centriole replication arrest very early in development. Thus, centrosomes are dispensable for oogenesis but essential for early embryogenesis. These results reveal that asymmetric centrosome behavior is not an essential feature of stem cell divisions.

Early embryo development in Fucus distichus is auxin sensitive

NASA Technical Reports Server (NTRS)

Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

2002-01-01

Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development.

Redundant roles of Sox17 and Sox18 in early cardiovascular development of mouse embryos

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

Sakamoto, Youhei; Hara, Kenshiro; Kanai-Azuma, Masami

Sox7, -17 and -18 constitute the Sox subgroup F (SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posteriormore » dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.« less

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development.

PubMed

Xu, Peng; Johnson, Tamara L; Stoller-Conrad, Jessica R; Schulz, Robert A

2012-01-01

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.

Brain tumor is a sequence-specific RNA-binding protein that directs maternal mRNA clearance during the Drosophila maternal-to-zygotic transition.

PubMed

Laver, John D; Li, Xiao; Ray, Debashish; Cook, Kate B; Hahn, Noah A; Nabeel-Shah, Syed; Kekis, Mariana; Luo, Hua; Marsolais, Alexander J; Fung, Karen Yy; Hughes, Timothy R; Westwood, J Timothy; Sidhu, Sachdev S; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

2015-05-12

Brain tumor (BRAT) is a Drosophila member of the TRIM-NHL protein family. This family is conserved among metazoans and its members function as post-transcriptional regulators. BRAT was thought to be recruited to mRNAs indirectly through interaction with the RNA-binding protein Pumilio (PUM). However, it has recently been demonstrated that BRAT directly binds to RNA. The precise sequence recognized by BRAT, the extent of BRAT-mediated regulation, and the exact roles of PUM and BRAT in post-transcriptional regulation are unknown. Genome-wide identification of transcripts associated with BRAT or with PUM in Drosophila embryos shows that they bind largely non-overlapping sets of mRNAs. BRAT binds mRNAs that encode proteins associated with a variety of functions, many of which are distinct from those implemented by PUM-associated transcripts. Computational analysis of in vitro and in vivo data identified a novel RNA motif recognized by BRAT that confers BRAT-mediated regulation in tissue culture cells. The regulatory status of BRAT-associated mRNAs suggests a prominent role for BRAT in post-transcriptional regulation, including a previously unidentified role in transcript degradation. Transcriptomic analysis of embryos lacking functional BRAT reveals an important role in mediating the decay of hundreds of maternal mRNAs during the maternal-to-zygotic transition. Our results represent the first genome-wide analysis of the mRNAs associated with a TRIM-NHL protein and the first identification of an RNA motif bound by this protein family. BRAT is a prominent post-transcriptional regulator in the early embryo through mechanisms that are largely independent of PUM.

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

Quantitative 4D analyses of epithelial folding during Drosophila gastrulation.

PubMed

Khan, Zia; Wang, Yu-Chiun; Wieschaus, Eric F; Kaschube, Matthias

2014-07-01

Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts. © 2014. Published by The Company of Biologists Ltd.

The developmental transcriptome of Drosophila melanogaster

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

University of Connecticut; Graveley, Brenton R.; Brooks, Angela N.

of genes. Whereas, 20% of Drosophila genes are annotated as encoding alternatively spliced premRNAs, splice-junction microarray experiments indicate that this number is at least 40% (ref. 7). Determining the diversity of mRNAs generated by alternative promoters, alternative splicing and RNA editing will substantially increase the inferred protein repertoire. Non-coding RNA genes (ncRNAs) including short interfering RNAs (siRNAs) and microRNAS (miRNAs) (reviewed in ref. 10), and longer ncRNAs such as bxd (ref. 11) and rox (ref. 12), have important roles in gene regulation, whereas others such as small nucleolar RNAs (snoRNAs)and small nuclear RNAs (snRNAs) are important components of macromolecular machines such as the ribosome and spliceosome. The transcription and processing of these ncRNAs must also be fully documented and mapped. As part of the modENCODE project to annotate the functional elements of the D. melanogaster and Caenorhabditis elegans genomes, we used RNA-Seq and tiling microarrays to sample the Drosophila transcriptome at unprecedented depth throughout development from early embryo to ageing male and female adults. We report on a high-resolution view of the discovery, structure and dynamic expression of the D. melanogaster transcriptome.« less

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer

PubMed Central

Struffi, Paolo; Corado, Maria; Kaplan, Leah; Yu, Danyang; Rushlow, Christine; Small, Stephen

2011-01-01

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo. PMID:21865322

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

PubMed Central

Mayer-Jaekel, R E; Baumgartner, S; Bilbe, G; Ohkura, H; Glover, D M; Hemmings, B A

1992-01-01

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner. Images PMID:1320961

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.

Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development.

PubMed

Wang, Chao-Jie; Wang, Guang; Wang, Xiao-Yu; Liu, Meng; Chuai, Manli; Lee, Kenneth Ka Ho; He, Xiao-Song; Lu, Da-Xiang; Yang, Xuesong

2016-06-15

Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

γ-BUTYROBETAINE AS A SPECIFIC ANTAGONIST FOR CARNITINE IN THE DEVELOPMENT OF THE EARLY CHICK EMBRYO

PubMed Central

Ito, Toshio; Fraenkel, G.

1957-01-01

The effect of γ-butyrobetaine alone and with the addition of carnitine on the development of the early excised chick embryo has been studied. γ-Butyrobetaine in appropriate amounts exerts an inhibitory effect which can be relieved or annulled by the inclusion of appropriate amounts of carnitine. This has been interpreted as a metabolite-antimetabolite relationship, in which the normal metabolite, carnitine, is antagonized by the structurally closely related γ-butyrobetaine, and is regarded as evidence of an important role of carnitine in the metabolism of the developing chick embryo. PMID:13475691

Embryo apoptosis identification: Oocyte grade or cleavage stage?

PubMed Central

Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

2015-01-01

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

Laser ablation of Drosophila embryonic motoneurons causes ectopic innervation of target muscle fibers

NASA Technical Reports Server (NTRS)

Chang, T. N.; Keshishian, H.

1996-01-01

We have tested the effects of neuromuscular denervation in Drosophila by laser-ablating the RP motoneurons in intact embryos before synaptogenesis. We examined the consequences of this ablation on local synaptic connectivity in both 1st and 3rd instar larvae. We find that the partial or complete loss of native innervation correlates with the appearance of alternate inputs from neighboring motor endings and axons. These collateral inputs are found at ectopic sites on the denervated target muscle fibers. The foreign motor endings are electrophysiologically functional and are observed on the denervated muscle fibers by the 1st instar larval stage. Our data are consistent with the existence of a local signal from the target environment, which is regulated by innervation and influences synaptic connectivity. Our results show that, despite the stereotypy of Drosophila neuromuscular connections, denervation can induce local changes in connectivity in wild-type Drosophila, suggesting that mechanisms of synaptic plasticity may also be involved in normal Drosophila neuromuscular development.

Refrigeration of rainbow trout gametes and embryos.

PubMed

Babiak, Igor; Dabrowski, Konrad

2003-12-01

Prolonged access to early embryos composed of undifferentiated, totipotent blastomeres is desirable in situations when multiple collections of gametes are not possible. The objective of the present study is to examine whether the refrigeration of rainbow trout Oncorhynchus mykiss gametes and early embryos would be a suitable, reliable, and efficient tool for prolonging the availability of early developmental stages up to the advanced blastula stage. The study was conducted continuously during fall, winter, and spring spawning seasons. In all, more than 500 experimental variants were performed involving individual samples from 26 females and 33 males derived from three strains. These strains represented three possible circumstances. In optimal one, gametes from good quality donors were obtained soon after ovulation. In the two non-optimal sources, either donors were of poor genetic quality or gametes were collected from a distant location and transported as unfertilized gametes. A highly significant effect of variability of individual sample quality on efficiency of gamete and embryo refrigeration was revealed. The source of gametes significantly affected viability of refrigerated oocytes and embryos, but not spermatozoa. On average, oocytes from optimal source retained full fertilization viability for seven days of chilled storage, significantly longer than from non-optimal sources. Spermatozoa, regardless of storage method, retained full fertilization ability for the first week of storage. Refrigeration of embryos at 1.4+/-0.4 degrees C significantly slowed the development. Two- week-old embryos were still in blastula stage. Average survival rate of embryos refrigerated for 10 days and then transferred to regular incubation temperatures of 9-14 degrees C was 92% in optimal and 51 and 71% in non-optimal source variants. No effect of gamete and embryo refrigeration on the occurrence of developmental abnormalities was observed. Cumulative refrigeration of oocytes and

Drosophila as a genetic and cellular model for studies on axonal growth

PubMed Central

Sánchez-Soriano, Natalia; Tear, Guy; Whitington, Paul; Prokop, Andreas

2007-01-01

One of the most fascinating processes during nervous system development is the establishment of stereotypic neuronal networks. An essential step in this process is the outgrowth and precise navigation (pathfinding) of axons and dendrites towards their synaptic partner cells. This phenomenon was first described more than a century ago and, over the past decades, increasing insights have been gained into the cellular and molecular mechanisms regulating neuronal growth and navigation. Progress in this area has been greatly assisted by the use of simple and genetically tractable invertebrate model systems, such as the fruit fly Drosophila melanogaster. This review is dedicated to Drosophila as a genetic and cellular model to study axonal growth and demonstrates how it can and has been used for this research. We describe the various cellular systems of Drosophila used for such studies, insights into axonal growth cones and their cytoskeletal dynamics, and summarise identified molecular signalling pathways required for growth cone navigation, with particular focus on pathfinding decisions in the ventral nerve cord of Drosophila embryos. These Drosophila-specific aspects are viewed in the general context of our current knowledge about neuronal growth. PMID:17475018

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

PubMed

Shiokawa, K; Kai, M; Higo, T; Kaito, C; Yokoska, J; Yasuhiko, Y; Kajita, E; Nagano, M; Yamada, Y; Shibata, M; Muto, T; Shinga, J; Hara, H; Takayama, E; Fukamachi, H; Yaoita, Y; Igarashi, K

2000-06-01

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in early Xenopus embryos induces cell dissociation and inhibits transition from the blastula to gastrula stage.

PubMed

Shibata, M; Shinga, J; Yasuhiko, Y; Kai, M; Miura, K; Shimogori, T; Kashiwagi, K; Igarashi, K; Shiokawa, K

1998-07-01

Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.

The effect of repeated light-dark shifts on uterine receptivity and early gestation in mice undergoing embryo transfer.

PubMed

Goldstein, Cathy A; O'Brien, Louise M; Bergin, Ingrid L; Saunders, Thomas L

2018-04-01

Female shift workers are at increased risk for negative reproductive outcomes, and animal evidence suggests that manipulation of the light-dark cycle is detrimental to early gestation in female mice. Specifically, failure of implantation may be responsible for these findings. The objective of this study was to better delineate which reproductive processes are vulnerable to detrimental effects of maternal circadian disturbance. We exposed mice undergoing embryo transfer to repetitive phase advances of the photoperiod. Embryos were derived from donor sperm and eggs from mice living in normal light-dark conditions to isolate the effects of photoperiod disruption on uterine receptivity and early gestation. Twenty-eight mice receiving embryo transfer underwent an experimental light-dark condition (advance of lights on and lights off by 6 hours every 4 days). Twenty-eight mice remained in a normal light-dark condition. Animals lived in their assigned light-dark condition beginning 2 weeks prior to embryo transfer and ending the day of uterine necropsy (post-coitus day 14.5). Wilcoxon-Mann-Whitney test demonstrated no significant differences between control and experimental light-dark conditions in pups (Z=0.10, p=.92), resorptions (Z=0.20, p=.84), or implantations (Z=-0.34, p=.73). Pup and placental weights were similar between groups. In this investigation, uterine receptivity and maintenance of early gestation were preserved despite recurrent phase advances in photoperiod. This finding, in the context of the current literature, suggests that the negative effects of circadian disruption are mediated by reproductive processes upstream of implantation.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm.

PubMed

Elwell, Jennifer A; Lovato, TyAnna L; Adams, Melanie M; Baca, Erica M; Lee, Thai; Cripps, Richard M

2015-04-15

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 arises 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 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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Spatial harmonics and pattern specification in early Drosophila development. Part II. The four colour wheels model.

PubMed

Kauffman, S A; Goodwin, B C

1990-06-07

We review the evidence presented in Part I showing that transcripts and protein products of maternal, gap, pair-rule, and segment polarity genes exhibit increasingly complex, multipeaked longitudinal waveforms in the early Drosophila embryo. The central problem we address in Part II is the use the embryo makes of these wave forms to specify longitudinal pattern. Based on the fact that mutants of many of these genes generate deletions and mirror symmetrical duplications of pattern elements on length scales ranging from about half the egg to within segments, we propose that position is specified by measuring a "phase angle" by use of the ratios of two or more variables. Pictorially, such a phase angle can be thought of as a colour on a colour wheel. Any such model contains a phaseless singularity where all or many phases, or colours, come together. We suppose as well that positional values sufficiently close to the singularity are meaningless, hence a "dead zone". Duplications and deletions are accounted for by deformation of the cycle of morphogen values occurring along the antero-posterior axis. If the cycle of values surrounds the singularity and lies outside the dead zone, pattern is normal. If the curve transects the dead zone, pattern elements are deleted. If the curve lies entirely on one side of the singularity, pattern elements are deleted and others are duplicated with mirror symmetry. The existence of different wavelength transcript patterns in maternal, gap, pair-rule, and segment polarity genes and the roles of those same genes in generating deletions and mirror symmetrical duplications on a variety of length scales lead us to propose that position is measured simultaneously on at least four colour wheels, which cycle different numbers of times along the anterior-posterior axis. These yield progressively finer grained positional information. Normal pattern specification requires a unique angle, outside of the dead zone, from each of the four wheels

Localization and activation of the Drosophila protease easter require the ER-resident saposin-like protein seele.

PubMed

Stein, David; Charatsi, Iphigenie; Cho, Yong Suk; Zhang, Zhenyu; Nguyen, Jesse; DeLotto, Robert; Luschnig, Stefan; Moussian, Bernard

2010-11-09

Drosophila embryonic dorsal-ventral polarity is generated by a series of serine protease processing events in the egg perivitelline space. Gastrulation Defective processes Snake, which then cleaves Easter, which then processes Spätzle into the activating ligand for the Toll receptor. seele was identified in a screen for mutations that, when homozygous in ovarian germline clones, lead to the formation of progeny embryos with altered embryonic patterning; maternal loss of seele function leads to the production of moderately dorsalized embryos. By combining constitutively active versions of Gastrulation Defective, Snake, Easter, and Spätzle with loss-of-function alleles of seele, we find that Seele activity is dispensable for Spätzle-mediated activation of Toll but is required for Easter, Snake, and Gastrulation Defective to exert their effects on dorsal-ventral patterning. Moreover, Seele function is required specifically for secretion of Easter from the developing embryo into the perivitelline space and for Easter processing. Seele protein resides in the endoplasmic reticulum of blastoderm embryos, suggesting a role in the trafficking of Easter to the perivitelline space, prerequisite to its processing and function. Easter transport to the perivitelline space represents a previously unappreciated control point in the signal transduction pathway that controls Drosophila embryonic dorsal-ventral polarity. Copyright © 2010 Elsevier Ltd. All rights reserved.

Rapid and highly accurate detection of Drosophila suzukii, spotted wing Drosophila (Diptera: Drosophilidae) by loop-mediated isothermal amplification assays

USDA-ARS?s Scientific Manuscript database

Drosophila suzukii, the spotted wing drosophila (SWD), is currently a major pest that causes severe economic losses to thin-skinned, small fruit growers in North America and Europe. The monitoring and early detection of SWD in the field is of the utmost importance for its proper management. Althou...

[Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

PubMed

Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

2007-09-01

To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

Nucleolus Precursor Bodies and Ribosome Biogenesis in Early Mammalian Embryos: Old Theories and New Discoveries.

PubMed

Fulka, Helena; Aoki, Fugaku

2016-06-01

In mammals, mature oocytes and early preimplantation embryos contain transcriptionally inactive structures termed nucleolus precursor bodies instead of the typical fibrillo-granular nucleoli. These nuclear organelles are essential and strictly of maternal origin. If they are removed from oocytes, the resulting embryos are unable to replace them and consequently fail to develop. Historically, nucleolus precursor bodies have been perceived as a passive repository site of nucleolar proteins that are required for embryos to form fully functional nucleoli. Recent results, however, contradict this long-standing dogma and show that these organelles are dispensable for nucleologenesis and ribosome biogenesis. In this article, we discuss the possible roles of nucleolus precursor bodies and propose how they might be involved in embryogenesis. Furthermore, we argue that these organelles are essential only shortly after fertilization and suggest that they might actively participate in centromeric chromatin establishment. © 2016 by the Society for the Study of Reproduction, Inc.

Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice.

PubMed

Wu, Yibo; Lv, Zhuo; Yang, Yang; Dong, Guoying; Yu, Yang; Cui, Yiqiang; Tong, Man; Wang, Liu; Zhou, Zuomin; Zhu, Hui; Zhou, Qi; Sha, Jiahao

2014-05-01

Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.

Selective Loss of Sperm Bearing a Compound Chromosome in the Drosophila Female

PubMed Central

Dernburg, A. F.; Daily, D. R.; Yook, K. J.; Corbin, J. A.; Sedat, J. W.; Sullivan, W.

1996-01-01

The Drosophila compound entire second chromosome, C(2)EN, displays paternal transmission well below Mendelian expectations (NOVITSKI et al. 1981). Because C(2)EN stocks also show higher-than-expected rates of zygotic lethality, it was proposed that this reduced paternal inheritance might be wholly or partially due to postfertilization events. Efforts to investigate this phenomenon have been hampered because the progeny of crosses between C(2)EN-bearing individuals and those with normal karyotypes die during embryogenesis. We have circumvented this obstacle by employing fluorescence in situ hybridization to directly karyotype early embryos from crosses involving C(2)EN-bearing individuals. This analysis reveals that the distortion in paternal transmission is established before fertilization. Moreover, measurement of the sperm ratios within both the male and female reproductive organs demonstrates that C(2)EN-bearing sperm are selectively lost after sperm transfer to the female and before storage of sperm in the seminal receptacles and spermathecae. Our results are consistent with a model of meiotic drive in which aberrations occuring early in meiosis lead ultimately to sperm dysfunction. PMID:8844151

Selective loss of sperm bearing a compound chromosome in the Drosophila female.

PubMed

Dernburg, A F; Daily, D R; Yook, K J; Corbin, J A; Sedat, J W; Sullivan, W

1996-08-01

The Drosophila compound entire second chromosome, C(2)EN, displays paternal transmission well below Mendelian expectations (NOVITSKI et al. 1981). Because C(2)EN stocks also show higher-than-expected rates of zygotic lethality, it was proposed that this reduced paternal inheritance might be wholly or partially due to postfertilization events. Efforts to investigate this phenomenon have been hampered because the progeny of crosses between C(2)EN-bearing individuals and those with normal karyotypes die during embryogenesis. We have circumvented this obstacle by employing fluorescence in situ hybridization to directly karyotype early embryos from crosses involving C(2)EN-bearing individuals. This analysis reveals that the distortion in paternal transmission is established before fertilization. Moreover, measurement of the sperm ratios within both the male and female reproductive organs demonstrates that C(2)EN-bearing sperm are selectively lost after sperm transfer to the female and before storage of sperm in the seminal receptacles and spermathecae. Our results are consistent with a model of meiotic drive in which aberrations occurring early in meiosis lead ultimately to sperm dysfunction.

Pipe-dependent ventral processing of Easter by Snake is the defining step in Drosophila embryo DV axis formation.

PubMed

Cho, Yong Suk; Stevens, Leslie M; Stein, David

2010-06-22

The establishment of Drosophila embryonic dorsal-ventral (DV) polarity relies on serine proteolytic activity in the perivitelline space between the embryonic membrane and the eggshell. Gastrulation Defective cleaves and activates Snake, which processes and activates Easter, which cleaves Spätzle to form the activating ligand for the Toll receptor. Ventral restriction of ligand formation depends on the Pipe sulfotransferase, which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte. Pipe modifies components of the developing eggshell to produce a ventral cue embedded in the vitelline membrane. This ventral cue is believed to promote one or more of the proteolysis steps in the perivitelline space. By examining the processing of transgenic, tagged versions of the perivitelline proteins during DV patterning, we find that the proteolysis of Easter by Snake is the first Pipe-dependent step and therefore the key ventrally restricted event in the protease cascade. We also find that Snake and Easter associate together in a complex in both wild-type and pipe mutant-derived embryos. This observation suggests a mechanism in which the sulfated target of Pipe promotes a productive interaction between Snake and Easter, perhaps by facilitating conformational changes in a complex containing the two proteins. Copyright 2010 Elsevier Ltd. All rights reserved.

Early Embryo Development in Fucus distichus Is Auxin Sensitive1

PubMed Central

Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

2002-01-01

Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [3H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development. PMID:12226509

The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo.

PubMed

Martin, Veronica; Mrkusich, Eli; Steinel, Martin C; Rice, Jason; Merritt, David J; Whitington, Paul M

2008-04-08

Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth. We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, we suggest that Neuroglian

The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo

PubMed Central

Martin, Veronica; Mrkusich, Eli; Steinel, Martin C; Rice, Jason; Merritt, David J; Whitington, Paul M

2008-01-01

Background Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth. Results We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. Conclusion We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, we

Sleep Deprivation During Early-Adult Development Results in Long-Lasting Learning Deficits in Adult Drosophila

PubMed Central

Seugnet, Laurent; Suzuki, Yasuko; Donlea, Jeff M.; Gottschalk, Laura; Shaw, Paul J.

2011-01-01

Study Objectives: Multiple lines of evidence indicate that sleep is important for the developing brain, although little is known about which cellular and molecular pathways are affected. Thus, the aim of this study was to determine whether the early adult life of Drosophila, which is associated with high amounts of sleep and critical periods of brain plasticity, could be used as a model to identify developmental processes that require sleep. Subjects: Wild type Canton-S Drosophila melanogaster. Design; Intervention: Flies were sleep deprived on their first full day of adult life and allowed to recover undisturbed for at least 3 days. The animals were then tested for short-term memory and response-inhibition using aversive phototaxis suppression (APS). Components of dopamine signaling were further evaluated using mRNA profiling, immunohistochemistry, and pharmacological treatments. Measurements and Results: Flies exposed to acute sleep deprivation on their first day of life showed impairments in short-term memory and response inhibition that persisted for at least 6 days. These impairments in adult performance were reversed by dopamine agonists, suggesting that the deficits were a consequence of reduced dopamine signaling. However, sleep deprivation did not impact dopaminergic neurons as measured by their number or by the levels of dopamine, pale (tyrosine hydroxylase), dopadecarboxylase, and the Dopamine transporter. However, dopamine pathways were impacted as measured by increased transcript levels of the dopamine receptors D2R and dDA1. Importantly, blocking signaling through the dDA1 receptor in animals that were sleep deprived during their critical developmental window prevented subsequent adult learning impairments. Conclusions: These data indicate that sleep plays an important and phylogenetically conserved role in the developing brain. Citation: Seugnet L; Suzuki Y; Donlea JM; Gottschalk L; Shaw PJ. Sleep deprivation during early-adult development results in

Rap1 and Canoe/afadin are essential for establishment of apical–basal polarity in the Drosophila embryo

PubMed Central

Choi, Wangsun; Harris, Nathan J.; Sumigray, Kaelyn D.; Peifer, Mark

2013-01-01

The establishment and maintenance of apical–basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. We found that the small GTPase Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. We next tested the current linear model for polarity establishment. Both Bazooka and aPKC regulate Canoe localization despite being “downstream” of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data reshape our view, suggesting that polarity establishment is regulated by a protein network rather than a linear pathway. PMID:23363604

The onset of homologous chromosome pairing during Drosophila melanogaster embryogenesis.

PubMed

Hiraoka, Y; Dernburg, A F; Parmelee, S J; Rykowski, M C; Agard, D A; Sedat, J W

1993-02-01

We have determined the position within the nucleus of homologous sites of the histone gene cluster in Drosophila melanogaster using in situ hybridization and high-resolution, three-dimensional wide field fluorescence microscopy. A 4.8-kb biotinylated probe for the histone gene repeat, located approximately midway along the short arm of chromosome 2, was hybridized to whole-mount embryos in late syncytial and early cellular blastoderm stages. Our results show that the two homologous histone loci are distinct and separate through all stages of the cell cycle up to nuclear cycle 13. By dramatic contrast, the two homologous clusters were found to colocalize with high frequency during interphase of cycle 14. Concomitant with homolog pairing at cycle 14, both histone loci were also found to move from their position near the midline of the nucleus toward the apical side. This result suggests that coincident with the initiation of zygotic transcription, there is dramatic chromosome and nuclear reorganization between nuclear cycles 13 and 14.

The Deadbeat Paternal Effect of Uncapped Sperm Telomeres on Cell Cycle Progression and Chromosome Behavior in Drosophila melanogaster

PubMed Central

Yamaki, Takuo; Yasuda, Glenn K.; Wakimoto, Barbara T.

2016-01-01

Telomere-capping complexes (TCCs) protect the ends of linear chromosomes from illegitimate repair and end-to-end fusions and are required for genome stability. The identity and assembly of TCC components have been extensively studied, but whether TCCs require active maintenance in nondividing cells remains an open question. Here we show that Drosophila melanogaster requires Deadbeat (Ddbt), a sperm nuclear basic protein (SNBP) that is recruited to the telomere by the TCC and is required for TCC maintenance during genome-wide chromatin remodeling, which transforms spermatids to mature sperm. Ddbt-deficient males produce sperm lacking TCCs. Their offspring delay the initiation of anaphase as early as cycle 1 but progress through the first two cycles. Persistence of uncapped paternal chromosomes induces arrest at or around cycle 3. This early arrest can be rescued by selective elimination of paternal chromosomes and production of gynogenetic haploid or haploid mosaics. Progression past cycle 3 can also occur if embryos have reduced levels of the maternally provided checkpoint kinase Chk2. The findings provide insights into how telomere integrity affects the regulation of the earliest embryonic cell cycles. They also suggest that other SNBPs, including those in humans, may have analogous roles and manifest as paternal effects on embryo quality. PMID:27029731

Collective synchronization of divisions in Drosophila development

NASA Astrophysics Data System (ADS)

Vergassola, Massimo

Mitoses in the early development of most metazoans are rapid and synchronized across the entire embryo. While diffusion is too slow, in vitro experiments have shown that waves of the cell-cycle regulator Cdk1 can transfer information rapidly across hundreds of microns. However, the signaling dynamics and the physical properties of chemical waves during embryonic development remain unclear. We develop FRET biosensors for the activity of Cdk1 and the checkpoint kinase Chk1 in Drosophila embryos and exploit them to measure waves in vivo. We demonstrate that Cdk1 chemical waves control mitotic waves and that their speed is regulated by the activity of Cdk1 during the S-phase (and not mitosis). We quantify the progressive slowdown of the waves with developmental cycles and identify its underlying control mechanism by the DNA replication checkpoint through the Chk1/Wee1 pathway. The global dynamics of the mitotic signaling network illustrates a novel control principle: the S-phase activity of Cdk1 regulates the speed of the mitotic wave, while the Cdk1 positive feedback ensures an invariantly rapid onset of mitosis. Mathematical modeling captures the speed of the waves and predicts a fundamental distinction between the S-phase Cdk1 trigger waves and the mitotic phase waves, which is illustrated by embryonic ablation experiments. In collaboration with Victoria Deneke1, Anna Melbinger2, and Stefano Di Talia1 1 Department of Cell Biology, Duke University Medical Center 2 Department of Physics, University of California San Diego.

Drosophila embryogenesis scales uniformly across temperature in developmentally diverse species.

PubMed

Kuntz, Steven G; Eisen, Michael B

2014-04-01

Temperature affects both the timing and outcome of animal development, but the detailed effects of temperature on the progress of early development have been poorly characterized. To determine the impact of temperature on the order and timing of events during Drosophila melanogaster embryogenesis, we used time-lapse imaging to track the progress of embryos from shortly after egg laying through hatching at seven precisely maintained temperatures between 17.5 °C and 32.5 °C. We employed a combination of automated and manual annotation to determine when 36 milestones occurred in each embryo. D. melanogaster embryogenesis takes [Formula: see text]33 hours at 17.5 °C, and accelerates with increasing temperature to a low of 16 hours at 27.5 °C, above which embryogenesis slows slightly. Remarkably, while the total time of embryogenesis varies over two fold, the relative timing of events from cellularization through hatching is constant across temperatures. To further explore the relationship between temperature and embryogenesis, we expanded our analysis to cover ten additional Drosophila species of varying climatic origins. Six of these species, like D. melanogaster, are of tropical origin, and embryogenesis time at different temperatures was similar for them all. D. mojavensis, a sub-tropical fly, develops slower than the tropical species at lower temperatures, while D. virilis, a temperate fly, exhibits slower development at all temperatures. The alpine sister species D. persimilis and D. pseudoobscura develop as rapidly as tropical flies at cooler temperatures, but exhibit diminished acceleration above 22.5 °C and have drastically slowed development by 30 °C. Despite ranging from 13 hours for D. erecta at 30 °C to 46 hours for D. virilis at 17.5 °C, the relative timing of events from cellularization through hatching is constant across all species and temperatures examined here, suggesting the existence of a previously unrecognized timer controlling the

Cytoskeletal changes in oocytes and early embryos during in vitro fertilization process in mice.

PubMed

Gumus, E; Bulut, H E; Kaloglu, C

2010-02-01

The cytoskeleton plays crucial roles in the development and fertilization of germ cells and in the early embryo development. The growth, maturation and fertilization of oocytes require an active movement and a correct localization of cellular organelles. This is performed by the re-organization of microtubules and actin filaments. Therefore, the aim of the present study was to determine the changes in cytoskeleton during in vitro fertilization process using appropriate immunofluorescence techniques. While the chromatin content was found to be scattered throughout the nucleus during the oocyte maturation period, it was seen only around nucleolus following the completion of the maturation. Microtubules, during oocyte maturation, were regularly distributed throughout the ooplasm which was then localized in the subcortical region of oocytes. Similarly microfilaments were scattered throughout the ooplasm during the oocyte maturation period whereas they were seen in the subcortical region around the polar body and above the meiotic spindle throughout the late developmental stages. In conclusion, those changes occurred in microtubules and microfilaments might be closely related to the re-organization of the genetic material during the oocyte maturation and early embryo development.

Zonda is a novel early component of the autophagy pathway in Drosophila

PubMed Central

Melani, Mariana; Valko, Ayelén; Romero, Nuria M.; Aguilera, Milton O.; Acevedo, Julieta M.; Bhujabal, Zambarlal; Perez-Perri, Joel; de la Riva-Carrasco, Rocío V.; Katz, Maximiliano J.; Sorianello, Eleonora; D’Alessio, Cecilia; Juhász, Gabor; Johansen, Terje; Colombo, María I.; Wappner, Pablo

2017-01-01

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum–associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation. PMID:28904211

Development of the embryonic heat shock response and the impact of repeated thermal stress in early stage lake whitefish (Coregonus clupeaformis) embryos.

PubMed

Whitehouse, Lindy M; McDougall, Chance S; Stefanovic, Daniel I; Boreham, Douglas R; Somers, Christopher M; Wilson, Joanna Y; Manzon, Richard G

2017-10-01

Lake whitefish (Coregonus clupeaformis) embryos were exposed to thermal stress (TS) at different developmental stages to determine when the heat shock response (HSR) can be initiated and if it is altered by exposure to repeated TS. First, embryos were subject to one of three different TS temperatures (6, 9, or 12°C above control) at 4 points in development (21, 38, 60 and 70 days post-fertilisation (dpf)) for 2h followed by a 2h recovery to understand the ontogeny of the HSR. A second experiment explored the effects of repeated TS on the HSR in embryos from 15 to 75 dpf. Embryos were subjected to one of two TS regimes; +6°C TS for 1h every 6 days or +9°C TS for 1h every 6 days. Following a 2h recovery, a subset of embryos was sampled. Our results show that embryos could initiate a HSR via upregulation of heat shock protein 70 (hsp70) mRNA at all developmental ages studied, but that this response varied with age and was only observed with a TS of +9 or +12°C. In comparison, when embryos received multiple TS treatments, hsp70 was not induced in response to the 1h TS and 2h recovery, and a downregulation was observed at 39 dpf. Downregulation of hsp47 and hsp90α mRNA was also observed in early age embryos. Collectively, these data suggest that embryos are capable of initiating a HSR at early age and throughout embryogenesis, but that repeated TS can alter the HSR, and may result in either reduced responsiveness or a downregulation of inducible hsps. Our findings warrant further investigation into both the short- and long-term effects of repeated TS on lake whitefish development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Forces directing germ-band extension in Drosophila embryos.

PubMed

Kong, Deqing; Wolf, Fred; Großhans, Jörg

2017-04-01

Body axis elongation by convergent extension is a conserved developmental process found in all metazoans. Drosophila embryonic germ-band extension is an important morphogenetic process during embryogenesis, by which the length of the germ-band is more than doubled along the anterior-posterior axis. This lengthening is achieved by typical convergent extension, i.e. narrowing the lateral epidermis along the dorsal-ventral axis and simultaneous extension along the anterior-posterior axis. Germ-band extension is largely driven by cell intercalation, whose directionality is determined by the planar polarity of the tissue and ultimately by the anterior-posterior patterning system. In addition, extrinsic tensile forces originating from the invaginating endoderm induce cell shape changes, which transiently contribute to germ-band extension. Here, we review recent progress in understanding of the role of mechanical forces in germ-band extension. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

DOE PAGES

Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz; ...

2017-03-22

Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

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

Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz

Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

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

Ju, Yan; Liu, Chunying; Lu, Wenwen

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysismore » revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.« less

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.

PubMed

Gandhi, Shashank; Piacentino, Michael L; Vieceli, Felipe M; Bronner, Marianne E

2017-12-01

The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Radiation induced abnormalities in early in vitro mouse embryos

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

Kirkpatrick, J.F.

1973-08-01

Female mice were superovulated and mated, and the two-cell embryos were collected and cultured in vitro. The embryos were exposed to x-irradiation (0 to 491 rads) during the two-cell stage before the appearance of the next cleavage plate, placed in new unirradiated culture medium and observed during subsequent development. Morphological abnormalities, which occurred as a result of irradiation, included fragmentation, disintegration, granlation, incomplete cleavage, cleavage cessation, nuclear degeneration and pycnosis and cytoplasmic vacuolization. There was no damage to the zona pellucida. The types of abnormalities indicate an agreement with the results of previous in vivo studies. A distinct correlation existedmore » between morphological abnormalities and embryo death. The greatest number of abnormalities resulted within five hours following irradiation, but increased through 20 hours post-exposure. At doses above 300 rads, the magnitude of damage was greater in the in vitro embryos than that shown in previous in vivo studies. (auth)« less

Shorter Exposures to Harder X-Rays Trigger Early Apoptotic Events in Xenopus laevis Embryos

PubMed Central

Dong, JiaJia; Mury, Sean P.; Drahos, Karen E.; Moscovitch, Marko

2010-01-01

Background A long-standing conventional view of radiation-induced apoptosis is that increased exposure results in augmented apoptosis in a biological system, with a threshold below which radiation doses do not cause any significant increase in cell death. The consequences of this belief impact the extent to which malignant diseases and non-malignant conditions are therapeutically treated and how radiation is used in combination with other therapies. Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems. Methodology/Principal Findings We explored how the energy of individual X-ray photons and exposure time, both factors that determine the total dose, influence the occurrence of cell death in early Xenopus embryo. Three different experimental scenarios were analyzed and morphological and biochemical hallmarks of apoptosis were evaluated. Initially, we examined cell death events in embryos exposed to increasing incident energies when the exposure time was preset. Then, we evaluated the embryo's response when the exposure time was augmented while the energy value remained constant. Lastly, we studied the incidence of apoptosis in embryos exposed to an equal total dose of radiation that resulted from increasing the incoming energy while lowering the exposure time. Conclusions/Significance Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system. In particular, for embryos exposed under identical conditions and delivered the same absorbed dose of radiation, the response is significantly increased when shorter bursts of more energetic photons are used. These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and utilized for therapeutic

Role of nucleation-promoting factors in mouse early embryo development.

PubMed

Wang, Qiao-Chu; Liu, Jun; Wang, Fei; Duan, Xing; Dai, Xiao-Xin; Wang, Teng; Liu, Hong-Lin; Cui, Xiang-Shun; Sun, Shao-Chen; Kim, Nam-Hyung

2013-06-01

During mitosis nucleation-promoting factors (NPFs) bind to the Arp2/3 complex and activate actin assembly. JMY and WAVE2 are two critical members of the NPFs. Previous studies have demonstrated that NPFs promote multiple processes such as cell migration and cytokinesis. However, the role of NPFs in development of mammalian embryos is still unknown. Results of the present study show that the NPFs JMY and WAVE2 are critical for cytokinesis during development of mouse embryos. Both JMY and WAVE2 are expressed in mouse embryos. After injection of JMY or WAVE2 siRNA, all embryos failed to develop to the morula or blastocyst stages. Moreover, using fluorescence intensity analysis, we found that the expression of actin decreased, and multiple nuclei were observed within a single cell indicating that NPFs-induced actin reduction caused the failure of cell division. In addition, injection of JMY and WAVE2 siRNA also caused ARP2 degradation, indicating that involvement of NPFs in development of mouse embryos is mainly through regulation of ARP2/3-induced actin assembly. Taken together, these data suggested that WAVE2 and JMY are involved in development of mouse embryos, and their regulation may be through a NPFs-Arp2/3-actin pathway.

Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos.

PubMed

Strobl, Frederic; Stelzer, Ernst H K

2014-06-01

Insect development has contributed significantly to our understanding of metazoan development. However, most information has been obtained by analyzing a single species, the fruit fly Drosophila melanogaster. Embryonic development of the red flour beetle Tribolium castaneum differs fundamentally from that of Drosophila in aspects such as short-germ development, embryonic leg development, extensive extra-embryonic membrane formation and non-involuted head development. Although Tribolium has become the second most important insect model organism, previous live imaging attempts have addressed only specific questions and no long-term live imaging data of Tribolium embryogenesis have been available. By combining light sheet-based fluorescence microscopy with a novel mounting method, we achieved complete, continuous and non-invasive fluorescence live imaging of Tribolium embryogenesis at high spatiotemporal resolution. The embryos survived the 2-day or longer imaging process, developed into adults and produced fertile progeny. Our data document all morphogenetic processes from the rearrangement of the uniform blastoderm to the onset of regular muscular movement in the same embryo and in four orientations, contributing significantly to the understanding of Tribolium development. Furthermore, we created a comprehensive chronological table of Tribolium embryogenesis, integrating most previous work and providing a reference for future studies. Based on our observations, we provide evidence that serosa window closure and serosa opening, although deferred by more than 1 day, are linked. All our long-term imaging datasets are available as a resource for the community. Tribolium is only the second insect species, after Drosophila, for which non-invasive long-term fluorescence live imaging has been achieved. © 2014. Published by The Company of Biologists Ltd.

The role of Mixer in patterning the early Xenopus embryo.

PubMed

Kofron, Matt; Wylie, Chris; Heasman, Janet

2004-05-01

The transcription factor VegT, is required in early Xenopus embryos for the formation of both the mesoderm and endoderm germ layers. Inherited as a maternal mRNA localized only in vegetal cells, VegT activates the transcription of a large number of transcription factors, as well as signaling ligands that induce cells in the vegetal mass to form endoderm, and the marginal zone to form mesoderm. It is important now to understand the extent to which transcription factors downstream of VegT play individual, or overlapping, roles in the specification and patterning of the endoderm and mesoderm. In addition, it is important to understand the mechanism that specifies the boundary between endoderm and mesoderm. One of the downstream targets of VegT, the homeodomain protein Mixer, is expressed at high levels at the mesoderm/endoderm boundary at the late blastula stage. We therefore examined its functions by blocking its translation using morpholino oligos. In Mixer-depleted embryos, the expression of many signaling ligands and transcription factors was affected. In particular, we found that the expression of several genes, including several normally expressed in mesoderm, was upregulated. Functional assays of Mixer-depleted vegetal cells showed that they have increased mesoderm-inducing activity. This demonstrates that Mixer plays an essential role in controlling the amount of mesoderm induction by the vegetal cells.

The flipflop orphan genes are required for limb bud eversion in the Tribolium embryo.

PubMed

Thümecke, Susanne; Beermann, Anke; Klingler, Martin; Schröder, Reinhard

2017-01-01

Unlike Drosophila but similar to other arthropod and vertebrate embryos, the flour beetle Tribolium castaneum develops everted limb buds during embryogenesis. However, the molecular processes directing the evagination of epithelia are only poorly understood. Here we show that the newly discovered genes Tc-flipflop1 and Tc-flipflop2 are involved in regulating the directional budding of appendages. RNAi-knockdown of Tc-flipflop results in a variety of phenotypic traits. Most prominently, embryonic limb buds frequently grow inwards rather than out, leading to the development of inverted appendages inside the larval body. Moreover, affected embryos display dorsal closure defects. The Tc-flipflop genes are evolutionarily non-conserved, and their molecular function is not evident. We further found that Tc-RhoGEF2 , a highly-conserved gene known to be involved in actomyosin-dependent cell movement and cell shape changes, shows a Tc-flipflop -like RNAi-phenotype. The similarity of the inverted appendage phenotype in both the flipflop - and the RhoGEF2 RNAi gene knockdown led us to conclude that the Tc-flipflop orphan genes act in a Rho-dependent pathway that is essential for the early morphogenesis of polarised epithelial movements. Our work describes one of the few examples of an orphan gene playing a crucial role in an important developmental process.

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 �

Negative regulation of P element excision by the somatic product and terminal sequences of P in drosophila melanogaster

USDA-ARS?s Scientific Manuscript database

A transient in vivo P element excision assay was used to test the regulatory properties of putative repressor-encoding plasmids in Drosophila melanogaster embryos. The somatic expression of an unmodified transposase transcription unit under the control of a heat shock gene promoter (phsn) effectivel...

Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

PubMed Central

Petrek, Jiri; Zitka, Ondrej; Adam, Vojtech; Bartusek, Karel; Anjum, Naser A.; Pereira, Eduarda; Havel, Ladislav; Kizek, Rene

2015-01-01

Background Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. Methodology/Principal Findings The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we

Early Cambrian Pentamerous Cubozoan Embryos from South China

PubMed Central

Han, Jian; Kubota, Shin; Li, Guoxiang; Yao, Xiaoyong; Yang, Xiaoguang; Shu, Degan; Li, Yong; Kinoshita, Shunichi; Sasaki, Osamu; Komiya, Tsuyoshi; Yan, Gang

2013-01-01

Background Extant cubozoans are voracious predators characterized by their square shape, four evenly spaced outstretched tentacles and well-developed eyes. A few cubozoan fossils are known from the Middle Cambrian Marjum Formation of Utah and the well-known Carboniferous Mazon Creek Formation of Illinois. Undisputed cubozoan fossils were previously unknown from the early Cambrian; by that time probably all representatives of the living marine phyla, especially those of basal animals, should have evolved. Methods Microscopic fossils were recovered from a phosphatic limestone in the Lower Cambrian Kuanchuanpu Formation of South China using traditional acetic-acid maceration. Seven of the pre-hatched pentamerous cubozoan embryos, each of which bears five pairs of subumbrellar tentacle buds, were analyzed in detail through computed microtomography (Micro-CT) and scanning electron microscopy (SEM) without coating. Results The figured microscopic fossils are unequivocal pre-hatching embryos based on their spherical fertilization envelope and the enclosed soft-tissue that has preserved key anatomical features arranged in perfect pentaradial symmetry, allowing detailed comparison with modern cnidarians, especially medusozoans. A combination of features, such as the claustrum, gonad-lamella, suspensorium and velarium suspended by the frenula, occur exclusively in the gastrovascular system of extant cubozoans, indicating a cubozoan affinity for these fossils. Additionally, the interior anatomy of these embryonic cubozoan fossils unprecedentedly exhibits the development of many new septum-derived lamellae and well-partitioned gastric pockets unknown in living cubozoans, implying that ancestral cubozoans had already evolved highly specialized structures displaying unexpected complexity at the dawn of the Cambrian. The well-developed endodermic lamellae and gastric pockets developed in the late embryonic stages of these cubozoan fossils are comparable with extant pelagic

Lineage-associated tracts defining the anatomy of the Drosophila first instar larval brain

PubMed Central

Hartenstein, Volker; Younossi-Hartenstein, Amelia; Lovick, Jennifer; Kong, Angel; Omoto, Jaison; Ngo, Kathy; Viktorin, Gudrun

2015-01-01

Fixed lineages derived from unique, genetically specified neuroblasts form the anatomical building blocks of the Drosophila brain. Neurons belonging to the same lineage project their axons in a common tract, which is labeled by neuronal markers. In this paper, we present a detailed atlas of the lineage-associated tracts forming the brain of the early Drosophila larva, based on the use of global markers (anti-Neuroglian, anti-Neurotactin, Inscuteable-Gal4>UAS-chRFP-Tub) and lineage-specific reporters. We describe 68 discrete fiber bundles that contain axons of one lineage or pairs/small sets of adjacent lineages. Bundles enter the neuropil at invariant locations, the lineage tract entry portals. Within the neuropil, these fiber bundles form larger fascicles that can be classified, by their main orientation, into longitudinal, transverse, and vertical (ascending/descending) fascicles. We present 3D digital models of lineage tract entry portals and neuropil fascicles, set into relationship to commonly used, easily recognizable reference structures such as the mushroom body, the antennal lobe, the optic lobe, and the Fasciclin II-positive fiber bundles that connect the brain and ventral nerve cord. Correspondences and differences between early larval tract anatomy and the previously described late larval and adult lineage patterns are highlighted. Our L1 neuro-anatomical atlas of lineages constitutes an essential step towards following morphologically defined lineages to the neuroblasts of the early embryo, which will ultimately make it possible to link the structure and connectivity of a lineage to the expression of genes in the particular neuroblast that gives rise to that lineage. Furthermore, the L1 atlas will be important for a host of ongoing work that attempts to reconstruct neuronal connectivity at the level of resolution of single neurons and their synapses. PMID:26141956

Lineage-associated tracts defining the anatomy of the Drosophila first instar larval brain.

PubMed

Hartenstein, Volker; Younossi-Hartenstein, Amelia; Lovick, Jennifer K; Kong, Angel; Omoto, Jaison J; Ngo, Kathy T; Viktorin, Gudrun

2015-10-01

Fixed lineages derived from unique, genetically specified neuroblasts form the anatomical building blocks of the Drosophila brain. Neurons belonging to the same lineage project their axons in a common tract, which is labeled by neuronal markers. In this paper, we present a detailed atlas of the lineage-associated tracts forming the brain of the early Drosophila larva, based on the use of global markers (anti-Neuroglian, anti-Neurotactin, inscuteable-Gal4>UAS-chRFP-Tub) and lineage-specific reporters. We describe 68 discrete fiber bundles that contain axons of one lineage or pairs/small sets of adjacent lineages. Bundles enter the neuropil at invariant locations, the lineage tract entry portals. Within the neuropil, these fiber bundles form larger fascicles that can be classified, by their main orientation, into longitudinal, transverse, and vertical (ascending/descending) fascicles. We present 3D digital models of lineage tract entry portals and neuropil fascicles, set into relationship to commonly used, easily recognizable reference structures such as the mushroom body, the antennal lobe, the optic lobe, and the Fasciclin II-positive fiber bundles that connect the brain and ventral nerve cord. Correspondences and differences between early larval tract anatomy and the previously described late larval and adult lineage patterns are highlighted. Our L1 neuro-anatomical atlas of lineages constitutes an essential step towards following morphologically defined lineages to the neuroblasts of the early embryo, which will ultimately make it possible to link the structure and connectivity of a lineage to the expression of genes in the particular neuroblast that gives rise to that lineage. Furthermore, the L1 atlas will be important for a host of ongoing work that attempts to reconstruct neuronal connectivity at the level of resolution of single neurons and their synapses. Copyright © 2015 Elsevier Inc. All rights reserved.

Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

PubMed

Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S; Gao, Guanjun

2014-03-21

Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor. Copyright © 2014 Xue et al.

A technique for sexing fully developed embryos and early-instar larvae of the gypsy moth

Treesearch

Gilbert Levesque

1963-01-01

Because variation in sex ratio is an important factor in the population dynamics of the gypsy moth (Porthetria dispar), it is necessary to have some means of determining the ratio of males to females in a population at the beginning of the larval period as well as in the later stages. For determining the sex of fully developed embryos and early-...

Induction of the early-late Ddc gene during Drosophila metamorphosis by the ecdysone receptor.

PubMed

Chen, Li; Reece, Christian; O'Keefe, Sandra L; Hawryluk, Gregory W L; Engstrom, Monica M; Hodgetts, Ross B

2002-06-01

During Drosophila metamorphosis, the 'early-late' genes constitute a unique class regulated by the steroid hormone 20-hydroxyecdysone. Their induction is comprised of both a primary and a secondary response to ecdysone. Previous work has suggested that the epidermal expression of the dopa decarboxylase gene (Ddc) is likely that of a typical early-late gene. Accumulation of the Ddc transcript is rapidly initiated in the absence of protein synthesis, which implies that the ecdysone receptor plays a direct role in induction. However, full Ddc expression requires the participation of one of the transcription factors encoded by the Broad-Complex. In this paper, we characterize an ecdysone response element (EcRE) that contributes to the primary response. Using gel mobility shift assays and transgenic assays, we identified a single functional EcRE, located at position -97 to -83 bp relative to the transcription initiation site. This is the first report of an EcRE associated with an early-late gene in Drosophila. Competition experiments indicated that the affinity of the Ddc EcRE for the ecdysone receptor complex was at least four-fold less than that of the canonical EcRE of the hsp27 gene. Using in vitro mutagenesis, we determined that the reduced affinity of the EcRE resided at two positions where the nucleotides differed from those found in the canonical sequence. The ecdysone receptor, acting through this EcRE, releases Ddc from a silencing mechanism, whose cis-acting domain we have mapped to the 5'-upstream region between -2067 and -1427 bp. Deletion of this repressive element resulted in precocious expression of Ddc in both epidermis and imaginal discs. Thus, epidermal Ddc induction at pupariation is under the control of an extended genomic region that contains both positive and negative regulatory elements. Copyright 2002 Elsevier Science Ireland Ltd.

Rapid DNA Synthesis During Early Drosophila Embryogenesis Is Sensitive to Maternal Humpty Dumpty Protein Function.

PubMed

Lesly, Shera; Bandura, Jennifer L; Calvi, Brian R

2017-11-01

Problems with DNA replication cause cancer and developmental malformations. It is not fully understood how DNA replication is coordinated with development and perturbed in disease. We had previously identified the Drosophila gene humpty dumpty ( hd ), and showed that null alleles cause incomplete DNA replication, tissue undergrowth, and lethality. Animals homozygous for the missense allele, hd 272-9 , were viable, but adult females had impaired amplification of eggshell protein genes in the ovary, resulting in the maternal effects of thin eggshells and embryonic lethality. Here, we show that expression of an hd transgene in somatic cells of the ovary rescues amplification and eggshell synthesis but not embryo viability. The germline of these mothers remain mutant for the hd 272-9 allele, resulting in reduced maternal Hd protein and embryonic arrest during mitosis of the first few S/M nuclear cleavage cycles with chromosome instability and chromosome bridges. Epistasis analysis of hd with the rereplication mutation plutonium indicates that the chromosome bridges of hd embryos are the result of a failed attempt to segregate incompletely replicated sister chromatids. This study reveals that maternally encoded Humpty dumpty protein is essential for DNA replication and genome integrity during the little-understood embryonic S/M cycles. Moreover, the two hd 272-9 maternal-effect phenotypes suggest that ovarian gene amplification and embryonic cleavage are two time periods in development that are particularly sensitive to mild deficits in DNA replication function. This last observation has broader relevance for interpreting why mild mutations in the human ortholog of humpty dumpty and other DNA replication genes cause tissue-specific malformations of microcephalic dwarfisms. Copyright © 2017 by the Genetics Society of America.

Development of the Drosophila entero-endocrine lineage and its specification by the Notch signaling pathway

PubMed Central

Takashima, Shigeo; Adams, Katrina L.; Ortiz, Paola A.; Ying, Chong T.; Moridzadeh, Rameen; Younossi-Hartenstein, Amelia; Hartenstein, Volker

2013-01-01

In this paper we have investigated the developmental-genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors. As in the embryo, activation of the Notch pathway triggers enterocyte differentiation, and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells. PMID:21382366

Superresolution imaging of Drosophila tissues using expansion microscopy.

PubMed

Jiang, Nan; Kim, Hyeon-Jin; Chozinski, Tyler J; Azpurua, Jorge E; Eaton, Benjamin A; Vaughan, Joshua C; Parrish, Jay Z

2018-06-15

The limited resolving power of conventional diffraction-limited microscopy hinders analysis of small, densely packed structural elements in cells. Expansion microscopy (ExM) provides an elegant solution to this problem, allowing for increased resolution with standard microscopes via physical expansion of the specimen in a swellable polymer hydrogel. Here, we apply, validate, and optimize ExM protocols that enable the study of Drosophila embryos, larval brains, and larval and adult body walls. We achieve a lateral resolution of ∼70 nm in Drosophila tissues using a standard confocal microscope, and we use ExM to analyze fine intracellular structures and intercellular interactions. First, we find that ExM reveals features of presynaptic active zone (AZ) structure that are observable with other superresolution imaging techniques but not with standard confocal microscopy. We further show that synapses known to exhibit age-dependent changes in activity also exhibit age-dependent changes in AZ structure. Finally, we use the significantly improved axial resolution of ExM to show that dendrites of somatosensory neurons are inserted into epithelial cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to Drosophila tissues and underscores the importance of tissue-specific optimization of ExM procedures.

Drosophila TNF Modulates Tissue Tension in the Embryo to Facilitate Macrophage Invasive Migration.

PubMed

Ratheesh, Aparna; Biebl, Julia; Vesela, Jana; Smutny, Michael; Papusheva, Ekaterina; Krens, S F Gabriel; Kaufmann, Walter; Gyoergy, Attila; Casano, Alessandra Maria; Siekhaus, Daria E

2018-05-07

Migrating cells penetrate tissue barriers during development, inflammatory responses, and tumor metastasis. We study if migration in vivo in such three-dimensionally confined environments requires changes in the mechanical properties of the surrounding cells using embryonic Drosophila melanogaster hemocytes, also called macrophages, as a model. We find that macrophage invasion into the germband through transient separation of the apposing ectoderm and mesoderm requires cell deformations and reductions in apical tension in the ectoderm. Interestingly, the genetic pathway governing these mechanical shifts acts downstream of the only known tumor necrosis factor superfamily member in Drosophila, Eiger, and its receptor, Grindelwald. Eiger-Grindelwald signaling reduces levels of active Myosin in the germband ectodermal cortex through the localization of a Crumbs complex component, Patj (Pals-1-associated tight junction protein). We therefore elucidate a distinct molecular pathway that controls tissue tension and demonstrate the importance of such regulation for invasive migration in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

Use of Early Ripening Cultivars to Avoid Infestation and Mass Trapping to Manage Drosophila suzukii (Diptera: Drosophilidae) in Vaccinium corymbosum (Ericales: Ericaceae).

PubMed

Hampton, Emily; Koski, Carissa; Barsoian, Olivia; Faubert, Heather; Cowles, Richard S; Alm, Steven R

2014-10-01

Use of early ripening highbush blueberry cultivars to avoid infestation and mass trapping were evaluated for managing spotted wing drosophila, Drosophila suzukii (Matsumura). Fourteen highbush blueberry cultivars were sampled for spotted wing drosophila infestation. Most 'Earliblue', 'Bluetta', and 'Collins' fruit were harvested before spotted wing drosophila oviposition commenced, and so escaped injury. Most fruit from 'Bluejay', 'Blueray', and 'Bluehaven' were also harvested before the first week of August, after which spotted wing drosophila activity led to high levels of blueberry infestation. In a separate experiment, damage to cultivars was related to the week in which fruit were harvested, with greater damage to fruit observed as the season progressed. Attractant traps placed within blueberry bushes increased nearby berry infestation by 5%, irrespective of cultivar and harvest date. The significant linear reduction in infestation with increasing distance from the attractant trap suggests that traps are influencing fly behavior to at least 5.5 m. Insecticides applied to the exterior of traps, compared with untreated traps, revealed that only 10-30% of flies visiting traps enter the traps and drown. Low trap efficiency may jeopardize surrounding fruits by increasing local spotted wing drosophila activity. To protect crops, traps for mass trapping should be placed in a perimeter outside fruit fields and insecticides need to be applied to the surface of traps or on nearby fruit to function as an attract-and-kill strategy. © 2014 Entomological Society of America.

Analysis of the effects of early nutritional environment on inbreeding depression in Drosophila melanogaster.

PubMed

Valtonen, T M; Roff, D A; Rantala, M J

2011-01-01

The impact of nutritional deficiencies early in life in determining life-history variation in organisms is well recognized. The negative effects of inbreeding on fitness are also well known. Contrary to studies on vertebrates, studies on invertebrates are not consistent with the observation that inbreeding compromises resistance to parasites and pathogens. In this study, we investigated the effect of early nutrition on the magnitude of inbreeding depression in development time, adult body size and adult resistance to the bacterium Serratia marcescens in Drosophila melanogaster. We found that early nutritional environment had no effect on the magnitude of inbreeding depression in development time or adult body size but may have played a small role in adult resistance to the bacterial infection. Estimates of heritabilities for development time under the poor nutritional environment were larger than those measured under the standard nutritional conditions. © 2010 The Authors. Journal of Evolutionary Biology © 2010 European Society For Evolutionary Biology.

Essential Role of Chromatin Remodeling Protein Bptf in Early Mouse Embryos and Embryonic Stem Cells

PubMed Central

Landry, Joseph; Sharov, Alexei A.; Piao, Yulan; Sharova, Lioudmila V.; Xiao, Hua; Southon, Eileen; Matta, Jennifer; Tessarollo, Lino; Zhang, Ying E.; Ko, Minoru S. H.; Kuehn, Michael R.; Yamaguchi, Terry P.; Wu, Carl

2008-01-01

We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf−/− embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf−/− embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo. PMID:18974875

Fish embryos on land: terrestrial embryo deposition lowers oxygen uptake without altering growth or survival in the amphibious fish Kryptolebias marmoratus.

PubMed

Wells, Michael W; Turko, Andy J; Wright, Patricia A

2015-10-01

Few teleost fishes incubate embryos out of water, but the oxygen-rich terrestrial environment could provide advantages for early growth and development. We tested the hypothesis that embryonic oxygen uptake is limited in aquatic environments relative to air using the self-fertilizing amphibious mangrove rivulus, Kryptolebias marmoratus, which typically inhabits hypoxic, water-filled crab burrows. We found that adult mangrove rivulus released twice as many embryos in terrestrial versus aquatic environments and that air-reared embryos had accelerated developmental rates. Surprisingly, air-reared embryos consumed 44% less oxygen and possessed larger yolk reserves, but attained the same mass, length and chorion thickness. Water-reared embryos moved their opercula ∼2.5 more times per minute compared with air-reared embryos at 7 days post-release, which probably contributed to the higher rates of oxygen uptake and yolk utilization we observed. Genetically identical air- and water-reared embryos from the same parent were raised to maturity, but the embryonic environment did not affect growth, reproduction or emersion ability in adults. Therefore, although aspects of early development were plastic, these early differences were not sustained into adulthood. Kryptolebias marmoratus embryos hatched out of water when exposed to aerial hypoxia. We conclude that exposure to a terrestrial environment reduces the energetic costs of development partly by reducing the necessity of embryonic movements to dispel stagnant boundary layers. Terrestrial incubation of young would be especially beneficial to amphibious fishes that occupy aquatic habitats of poor water quality, assuming low terrestrial predation and desiccation risks. © 2015. Published by The Company of Biologists Ltd.

The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

PubMed Central

Zhang, Gang; Beati, Hamze; Nilsson, Jakob; Wodarz, Andreas

2013-01-01

Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs) are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs) have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs. PMID:23593258

Endogenous Nod-Factor-Like Signal Molecules Promote Early Somatic Embryo Development in Norway Spruce1

PubMed Central

Dyachok, Julia V.; Wiweger, Malgorzata; Kenne, Lennart; von Arnold, Sara

2002-01-01

Embryogenic cultures of Norway spruce (Picea abies) are composed of pro-embryogenic masses (PEMs) and somatic embryos of various developmental stages. Auxin is important for PEM formation and proliferation. In this report we show that depletion of auxin blocks PEM development and causes large-scale cell death. Extracts of the media conditioned by embryogenic cultures stimulate development of PEM aggregates in auxin-deficient cultures. Partial characterization of the conditioning factor shows that it is a lipophilic, low-molecular-weight molecule, which is sensitive to chitinase and contains GlcNAc residues. On the basis of this information, we propose that the factor is a lipophilic chitin oligosaccharide (LCO). The amount of LCO correlates to the developmental stages of PEMs and embryos, with the highest level in the media conditioned by developmentally blocked cultures. LCO is not present in nonembryogenic cultures. Cell death, induced by withdrawal of auxin, is suppressed by extra supply of endogenous LCO or Nod factor from Rhizobium sp. NGR234. The effect can be mimicked by a chitotetraose or chitinase from Streptomyces griseus. Taken together, our data suggest that endogenous LCO acts as a signal molecule stimulating PEM and early embryo development in Norway spruce. PMID:11842156

Time-lapse culture with morphokinetic embryo selection improves pregnancy and live birth chances and reduces early pregnancy loss: a meta-analysis.

PubMed

Pribenszky, Csaba; Nilselid, Anna-Maria; Montag, Markus

2017-11-01

Embryo evaluation and selection is fundamental in clinical IVF. Time-lapse follow-up of embryo development comprises undisturbed culture and the application of the visual information to support embryo evaluation. A meta-analysis of randomized controlled trials was carried out to study whether time-lapse monitoring with the prospective use of a morphokinetic algorithm for selection of embryos improves overall clinical outcome (pregnancy, early pregnancy loss, stillbirth and live birth rate) compared with embryo selection based on single time-point morphology in IVF cycles. The meta-analysis of five randomized controlled trials (n = 1637) showed that the application of time-lapse monitoring was associated with a significantly higher ongoing clinical pregnancy rate (51.0% versus 39.9%), with a pooled odds ratio of 1.542 (P < 0.001), significantly lower early pregnancy loss (15.3% versus 21.3%; OR: 0.662; P = 0.019) and a significantly increased live birth rate (44.2% versus 31.3%; OR 1.668; P = 0.009). Difference in stillbirth was not significant between groups (4.7% versus 2.4%). Quality of the evidence was moderate to low owing to inconsistencies across the studies. Selective application and variability were also limitations. Although time-lapse is shown to significantly improve overall clinical outcome, further high-quality evidence is needed before universal conclusions can be drawn. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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.

A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila.

PubMed

Verd, Berta; Clark, Erik; Wotton, Karl R; Janssens, Hilde; Jiménez-Guri, Eva; Crombach, Anton; Jaeger, Johannes

2018-02-01

Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects.

A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila

PubMed Central

Verd, Berta; Clark, Erik; Wotton, Karl R.; Janssens, Hilde; Jiménez-Guri, Eva; Crombach, Anton

2018-01-01

Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects. PMID:29451884

Selection and dynamics of embryonic stem cell integration into early mouse embryos

PubMed Central

Alexandrova, Stoyana; Kalkan, Tuzer; Humphreys, Peter; Riddell, Andrew; Scognamiglio, Roberta; Trumpp, Andreas; Nichols, Jennifer

2016-01-01

The process by which pluripotent cells incorporate into host embryos is of interest to investigate cell potency and cell fate decisions. Previous studies suggest that only a minority of the embryonic stem cell (ESC) inoculum contributes to the adult chimaera. How incoming cells are chosen for integration or elimination remains unclear. By comparing a heterogeneous mix of undifferentiated and differentiating ESCs (serum/LIF) with more homogeneous undifferentiated culture (2i/LIF), we examine the role of cellular heterogeneity in this process. Time-lapse ex vivo imaging revealed a drastic elimination of serum/LIF ESCs during early development in comparison with 2i/LIF ESCs. Using a fluorescent reporter for naive pluripotency (Rex1-GFP), we established that the acutely eliminated serum/LIF ESCs had started to differentiate. The rejected cells were apparently killed by apoptosis. We conclude that a selection process exists by which unwanted differentiating cells are eliminated from the embryo. However, occasional Rex1− cells were able to integrate. Upregulation of Rex1 occurred in a proportion of these cells, reflecting the potential of the embryonic environment to expedite diversion from differentiation priming to enhance the developing embryonic epiblast. PMID:26586221

Nuclear localization signal-dependent and -independent movements of Drosophila melanogaster dUTPase isoforms during nuclear cleavage

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

Muha, Villo; Zagyva, Imre; Venkei, Zsolt

2009-04-03

Two dUTPase isoforms (23 kDa and 21 kDa) are present in the fruitfly with the sole difference of an N-terminal extension. In Drosophila embryo, both isoforms are detected inside the nucleus. Here, we investigated the function of the N-terminal segment using eYFP-dUTPase constructs. In Schneider 2 cells, only the 23 kDa construct showed nuclear localization arguing that it may contain a nuclear localization signal (NLS). Sequence comparisons identified a lysine-rich nonapeptide with similarity to the human c-myc NLS. In Drosophila embryos during nuclear cleavages, the 23 kDa isoform showed the expected localization shifts. Contrariwise, although the 21 kDa isoform wasmore » excluded from the nuclei during interphase, it was shifted to the nucleus during prophase and forthcoming mitotic steps. The observed dynamic localization character showed strict timing to the nuclear cleavage phases and explained how both isoforms can be present within the nuclear microenvironment, although at different stages of cell cycle.« less

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.

Genome-wide analysis of promoter architecture in Drosophila melanogaster

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

Hoskins, Roger A.; Landolin, Jane M.; Brown, James B.

2010-10-20

Core promoters are critical regions for gene regulation in higher eukaryotes. However, the boundaries of promoter regions, the relative rates of initiation at the transcription start sites (TSSs) distributed within them, and the functional significance of promoter architecture remain poorly understood. We produced a high-resolution map of promoters active in the Drosophila melanogaster embryo by integrating data from three independent and complementary methods: 21 million cap analysis of gene expression (CAGE) tags, 1.2 million RNA ligase mediated rapid amplification of cDNA ends (RLMRACE) reads, and 50,000 cap-trapped expressed sequence tags (ESTs). We defined 12,454 promoters of 8037 genes. Our analysismore » indicates that, due to non-promoter-associated RNA background signal, previous studies have likely overestimated the number of promoter-associated CAGE clusters by fivefold. We show that TSS distributions form a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% of cases. This suggests that these distributions are generally determined by static elements such as local DNA sequence and are not modulated by dynamic signals such as histone modifications. Transcription factor binding motifs are differentially enriched as a function of promoter shape, and peaked promoter shape is correlated with both temporal and spatial regulation of gene expression. Our results contribute to the emerging view that core promoters are functionally diverse and control patterning of gene expression in Drosophila and mammals.« less

Endometrium as an early sensor of in vitro embryo manipulation technologies

PubMed Central

Mansouri-Attia, Nadéra; Sandra, Olivier; Aubert, Julie; Degrelle, Séverine; Everts, Robin E.; Giraud-Delville, Corinne; Heyman, Yvan; Galio, Laurent; Hue, Isabelle; Yang, Xiangzhong; Tian, X. Cindy; Lewin, Harris A.; Renard, Jean-Paul

2009-01-01

Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy. PMID:19297625

Identification and functional analysis of long non-coding RNAs in human and mouse early embryos based on single-cell transcriptome data

PubMed Central

Qiu, Jia-jun; Ren, Zhao-rui; Yan, Jing-bin

2016-01-01

Epigenetics regulations have an important role in fertilization and proper embryonic development, and several human diseases are associated with epigenetic modification disorders, such as Rett syndrome, Beckwith-Wiedemann syndrome and Angelman syndrome. However, the dynamics and functions of long non-coding RNAs (lncRNAs), one type of epigenetic regulators, in human pre-implantation development have not yet been demonstrated. In this study, a comprehensive analysis of human and mouse early-stage embryonic lncRNAs was performed based on public single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs are expressed in a developmental stage–specific manner during human early-stage embryonic development, whereas a more temporal-specific expression pattern was identified in mouse embryos. Weighted gene co-expression network analysis suggested that lncRNAs involved in human early-stage embryonic development are associated with several important functions and processes, such as oocyte maturation, zygotic genome activation and mitochondrial functions. We also found that the network of lncRNAs involved in zygotic genome activation was highly preservative between human and mouse embryos, whereas in other stages no strong correlation between human and mouse embryo was observed. This study provides insight into the molecular mechanism underlying lncRNA involvement in human pre-implantation embryonic development. PMID:27542205

Single-cell transcriptome of early embryos and cultured embryonic stem cells of cynomolgus monkeys

PubMed Central

Nakamura, Tomonori; Yabuta, Yukihiro; Okamoto, Ikuhiro; Sasaki, Kotaro; Iwatani, Chizuru; Tsuchiya, Hideaki; Saitou, Mitinori

2017-01-01

In mammals, the development of pluripotency and specification of primordial germ cells (PGCs) have been studied predominantly using mice as a model organism. However, divergences among mammalian species for such processes have begun to be recognized. Between humans and mice, pre-implantation development appears relatively similar, but the manner and morphology of post-implantation development are significantly different. Nevertheless, the embryogenesis just after implantation in primates, including the specification of PGCs, has been unexplored due to the difficulties in analyzing the embryos at relevant developmental stages. Here, we present a comprehensive single-cell transcriptome dataset of pre- and early post-implantation embryo cells, PGCs and embryonic stem cells (ESCs) of cynomolgus monkeys as a model of higher primates. The identities of each transcriptome were also validated rigorously by other way such as immunofluorescent analysis. The information reported here will serve as a foundation for our understanding of a wide range of processes in the developmental biology of primates, including humans. PMID:28649393

Genes encoding cuticular proteins are components of the Nimrod gene cluster in Drosophila.

PubMed

Cinege, Gyöngyi; Zsámboki, János; Vidal-Quadras, Maite; Uv, Anne; Csordás, Gábor; Honti, Viktor; Gábor, Erika; Hegedűs, Zoltán; Varga, Gergely I B; Kovács, Attila L; Juhász, Gábor; Williams, Michael J; Andó, István; Kurucz, Éva

2017-08-01

The Nimrod gene cluster, located on the second chromosome of Drosophila melanogaster, is the largest synthenic unit of the Drosophila genome. Nimrod genes show blood cell specific expression and code for phagocytosis receptors that play a major role in fruit fly innate immune functions. We previously identified three homologous genes (vajk-1, vajk-2 and vajk-3) located within the Nimrod cluster, which are unrelated to the Nimrod genes, but are homologous to a fourth gene (vajk-4) located outside the cluster. Here we show that, unlike the Nimrod candidates, the Vajk proteins are expressed in cuticular structures of the late embryo and the late pupa, indicating that they contribute to cuticular barrier functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sodium Butyrate Improves Locomotor Impairment and Early Mortality in a Rotenone-Induced Drosophila Model of Parkinson’s Disease

PubMed Central

St. Laurent, Robyn; O’Brien, Liam M.; Ahmad, S. Tariq

2013-01-01

Parkinson’s disease (PD) is a neurodegenerative disorder primarily affecting the dopaminergic neurons in the nigrastriatal pathway resulting in debilitating motor impairment in both familial and sporadic cases. Histone deacetylase (HDAC) inhibitors have been recently implicated as a therapeutic candidate because of their ability to correct the disrupted HDAC activity in PD and other neurodegenerative diseases. Sodium butyrate (SB), an HDAC inhibitor, reduces degeneration of dopaminergic neurons in a mutant alpha-synuclein Drosophila transgenic model of familial PD. Chronic exposure to the pesticide rotenone also causes selective degeneration of dopaminergic neurons and causes locomotor impairment and early mortality in a Drosophila model of chemically-induced PD. This study investigated the effects of sodium butyrate on locomotor impairment and early mortality in a rotenone-induced PD model. We show that treatment with 10 mM SB-supplemented food rescued the rotenone-induced locomotor impairment and early mortality in flies. Additionally, flies with the genetic knockdown of HDAC activity through Sin3A loss-of-function mutation (Sin3Alof) were resistant to rotenone-induced locomotor impairment and early mortality. Furthermore, SB-supplemented Sin3Alof flies had a modest additive effect for improving locomotor impairment. We also show SB-mediated improvement of rotenone-induced locomotor impairment was associated with elevated dopamine levels in the brain. However, the possibility of SB-mediated protective role through mechanisms independent from dopamine system is also discussed. These findings demonstrate that HDAC inhibitors like SB can ameliorate locomotor impairment in a rotenone-induced PD model. PMID:23623990

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.

Molecular cloning, developmental expression, and cellular localization of the 70-kDa RPA-1 subunit of Drosophila melanogaster.

PubMed

Perdigão, J; Logarinho, E; Avides, M C; Sunkel, C E

1999-12-01

Replication protein A (RPA) is a highly conserved multifunctional heterotrimeric complex, involved in DNA replication, repair, recombination, and possibly transcription. Here, we report the cloning of the gene that codes for the largest subunit of the Drosophila melanogaster RPA homolog, dmRPA70. In situ hybridization showed that dmRPA70 RNA is present in developing embryos during the first 16 cycles. After this point, dm-RPA70 expression is downregulated in cells that enter a G1 phase and exit the mitotic cycle, becoming restricted to brief bursts of accumulation from late G1 to S phase. This pattern of regulated expression is also observed in the developing eye imaginal disc. In addition, we have shown that the presence of cyclin E is necessary and sufficient to drive the expression of dmRPA70 in embryonic cells arrested in G1 but is not required in tissues undergoing endoreduplication. Immunolocalization showed that in early developing embryos, the dmRPA70 protein associates with chromatin from the end of mitosis until the beginning of the next prophase in a dynamic speckled pattern that is strongly suggestive of its association with replication foci.

IN VITRO CULTURE OF POSTIMPLANTATION HAMSTER EMBRYOS

EPA Science Inventory

In vitro culture of intact rat and mouse embryos has been described extensively, but information on the culture of other species is sparse. The present study examined some culture requirements of early somite stage hamster embryos and assessed the embryotoxic effects of sodium sa...

Patterns of protein synthesis in oocytes and early embryos of Rana esculenta complex.

PubMed

Chen, P S; Stumm-Zollinger, E

1986-01-01

We have used isotopic labelling and both one-and two-dimensional electrophoretic procedures to analyse the protien synthesis patterns in oocytes and early embryos of three phenotypes of the European green frogs. The results demonstrated that protein patterns of Rana ridibunda and R. esculenta are identical, but that they differ from those of R. lessonae. Progeny of the lethal cross R. esculenta × R. esculenta showed a distinct delay in the appearance of stage-specific proteins during early embryogenesis. The heat-shock response of R. ridibunda and R. esculenta oocytes was found to be identical, but different from that of Xenopus laevis. The implications of these findings, with respect to hybridogenesis in R. esculenta complex and variations in the regulations of heat shock genes in different amphibian species, are discussed.

Drosophila Growth Cones Advance by Forward Translocation of the Neuronal Cytoskeletal Meshwork In Vivo

PubMed Central

Roossien, Douglas H.; Lamoureux, Phillip; Van Vactor, David; Miller, Kyle E.

2013-01-01

In vitro studies conducted in Aplysia and chick sensory neurons indicate that in addition to microtubule assembly, long microtubules in the C-domain of the growth cone move forward as a coherent bundle during axonal elongation. Nonetheless, whether this mode of microtubule translocation contributes to growth cone motility in vivo is unknown. To address this question, we turned to the model system Drosophila. Using docked mitochondria as fiduciary markers for the translocation of long microtubules, we first examined motion along the axon to test if the pattern of axonal elongation is conserved between Drosophila and other species in vitro. When Drosophila neurons were cultured on Drosophila extracellular matrix proteins collected from the Drosophila Kc167 cell line, docked mitochondria moved in a pattern indicative of bulk microtubule translocation, similar to that observed in chick sensory neurons grown on laminin. To investigate whether the C-domain is stationary or advances in vivo, we tracked the movement of mitochondria during elongation of the aCC motor neuron in stage 16 Drosophila embryos. We found docked mitochondria moved forward along the axon shaft and in the growth cone C-domain. This work confirms that the physical mechanism of growth cone advance is similar between Drosophila and vertebrate neurons and suggests forward translocation of the microtubule meshwork in the axon underlies the advance of the growth cone C-domain in vivo. These results highlight the need for incorporating en masse microtubule translocation, in addition to assembly, into models of axonal elongation. PMID:24244629

Structure of interphase chromosomes in the nuclei of Drosophila cells.

PubMed

Banfalvi, Gaspar

2006-10-01

Fluorescent images of interphase chromatin structures and chromosome structures isolated from reversibly permeable Drosophila cells were analyzed. Decondensed chromatin in early S phase (2.0-2.5 C-value) consisted of a veil-like fibrillary network. Fibrillar chromatin formed rodlets later in the early S phase (2.5-2.75 C). Drosophila chromosomes contain several smaller subunits called rodlets. Fibrillar chromatin turned to chromatin ribbon and the early mid-S-phase globular chromosomes (2.75-3.0 C), then to opened fibrous globular forms later in the mid-S-phase (3.0-3.25 C), to late-S-phase supercoiled ribbons (3.25-3.5 C), end-S-phase elongated prechromosomes (3.5-3.75 C), bent and linear chromosomes (3.75-4.0 C). Early-S phase chromatin fibrils in the nuclei of Drosophila cells are thinner than the veil-like structures in mammalian cells. The connectivity of chromosomes shows linear arrangement (3, 1, 2, 4), with larger chromosomes (1 and 2) inside and smaller chromosomes (3, 4) at the two ends in the chromosomal chain.

The glutamate dehydrogenase GENE of Drosophila melanogaster: molecular analysis and expression.

PubMed

Papadopoulou, D; Louis, C

2000-09-01

Glutamate dehydrogenase is an enzyme that, in addition to its role in the energy metabolism in mitochondria, is involved in neuromuscular transmission. Here we present the structure and sequence of the Gdh gene of Drosophila melanogaster, as well as the analysis of its spatial and temporal pattern of expression. Unlike all other organisms analyzed so far, two forms of the enzyme, differing by the inclusion of 13 extra amino acids, are found in the fruitfly. We show the presence of Gdh mRNA in several tissues of the developing embryo, including the central nervous system, muscles and the alimentary tract. Moreover, we detect the localization of the Gdh protein in specific areas of the muscles, a fact that is consistent both with an involvement in energy metabolism and the role of glutamate as the major neuromuscular transmitter in Drosophila.

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.

HNK-1 immunoreactivity during early morphogenesis of the head region in a nonmodel vertebrate, crocodile embryo

NASA Astrophysics Data System (ADS)

Kundrát, Martin

2008-11-01

The present study examines HNK-1 immunoidentification of a population of the neural crest (NC) during early head morphogenesis in the nonmodel vertebrate, the crocodile ( Crocodylus niloticus) embryos. Although HNK-1 is not an exclusive NC marker among vertebrates, temporospatial immunoreactive patterns found in the crocodile are almost consistent with NC patterns derived from gene expression studies known in birds (the closest living relatives of crocodiles) and mammals. In contrast to birds, the HNK-1 epitope is immunoreactive in NC cells at the neural fold level in crocodile embryos and therefore provides sufficient base to assess early migratory events of the cephalic NC. I found that crocodile NC forms three classic migratory pathways in the head: mandibular, hyoid, and branchial. Further, I demonstrate that, besides this classic phenotype, there is also a forebrain-derived migratory population, which consolidates into a premandibular stream in the crocodile. In contrast to the closely related chick model, crocodilian premandibular and mandibular NC cells arise from the open neural tube suggesting that species-specific heterochronic behavior of NC may be involved in the formation of different vertebrate facial phenotypes.

Dynamic Properties of Electrotonic Coupling between Cells of Early Xenopus Embryos

PubMed Central

DiCaprio, R. A.; French, A. S.; Sanders, E. J.

1974-01-01

Frequency response functions were measured between the cells of Xenopus laevis embryos during the first two cleavage stages. Linear systems theory was then used to produce electronic models which account for the electrical behavior of the systems. Coupling between the cells may be explained by models which have simple resistive elements joining each cell to its neighbors. The vitelline, or fertilization, membrane which surrounds the embryos has no detectable resistance to the passage of electric current. The electrical properties of the four-cell embryo can only be explained by the existence of individual junctions linking each pair of cells. This arrangement suggests that electrotonic coupling is important in the development of the embryos, at least until the four-cell stage. ImagesFIGURE 5FIGURE 14FIGURE 15 PMID:19431351

Effects of hypo-O-GlcNAcylation on Drosophila development.

PubMed

Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

2018-05-11

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc ( O -GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila , null mutants of the Polycomb gene O -GlcNAc transferase ( OGT ; also known as super sex combs ( sxc )) display homeotic phenotypes. To dissect the requirement for O -GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxc K872M , was recessive lethal, whereas a second mutant, the hypomorphic sxc H537A , was homozygous viable. We observed that reduced total protein O -GlcNAcylation in the sxc H537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc H537A and a null allele of Drosophila host cell factor ( dHcf ), encoding an extensively O -GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxc H537A flies lacking a copy of skuld ( skd ), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxc H537A enabled us to identify pleiotropic effects of globally reduced protein O -GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development. © 2018 Mariappa et al.

Drosophila caspases involved in developmentally regulated programmed cell death of peptidergic neurons during early metamorphosis.

PubMed

Lee, Gyunghee; Wang, Zixing; Sehgal, Ritika; Chen, Chun-Hong; Kikuno, Keiko; Hay, Bruce; Park, Jae H

2011-01-01

A great number of obsolete larval neurons in the Drosophila central nervous system are eliminated by developmentally programmed cell death (PCD) during early metamorphosis. To elucidate the mechanisms of neuronal PCD occurring during this period, we undertook genetic dissection of seven currently known Drosophila caspases in the PCD of a group of interneurons (vCrz) that produce corazonin (Crz) neuropeptide in the ventral nerve cord. The molecular death program in the vCrz neurons initiates within 1 hour after pupariation, as demonstrated by the cytological signs of cell death and caspase activation. PCD was significantly suppressed in dronc-null mutants, but not in null mutants of either dredd or strica. A double mutation lacking both dronc and strica impaired PCD phenotype more severely than did a dronc mutation alone, but comparably to a triple dredd/strica/dronc mutation, indicating that dronc is a main initiator caspase, while strica plays a minor role that overlaps with dronc's. As for effector caspases, vCrz PCD requires both ice and dcp-1 functions, as they work cooperatively for a timely removal of the vCrz neurons. Interestingly, the activation of the Ice and Dcp-1 is not solely dependent on Dronc and Strica, implying an alternative pathway to activate the effectors. Two remaining effector caspase genes, decay and damm, found no apparent functions in the neuronal PCD, at least during early metamorphosis. Overall, our work revealed that vCrz PCD utilizes dronc, strica, dcp-1, and ice wherein the activation of Ice and Dcp-1 requires a novel pathway in addition to the initiator caspases.

Novel synthetic Medea selfish genetic elements drive population replacement in Drosophila; a theoretical exploration of Medea-dependent population suppression.

PubMed

Akbari, Omar S; Chen, Chun-Hong; Marshall, John M; Huang, Haixia; Antoshechkin, Igor; Hay, Bruce A

2014-12-19

Insects act as vectors for diseases of plants, animals, and humans. Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a potentially self-perpetuating method of disease prevention. Population replacement requires a gene drive mechanism in order to spread linked genes mediating disease refractoriness through wild populations. We previously reported the creation of synthetic Medea selfish genetic elements able to drive population replacement in Drosophila. These elements use microRNA-mediated silencing of myd88, a maternally expressed gene required for embryonic dorso-ventral pattern formation, coupled with early zygotic expression of a rescuing transgene, to bring about gene drive. Medea elements that work through additional mechanisms are needed in order to be able to carry out cycles of population replacement and/or remove existing transgenes from the population, using second-generation elements that spread while driving first-generation elements out of the population. Here we report the synthesis and population genetic behavior of two new synthetic Medea elements that drive population replacement through manipulation of signaling pathways involved in cellular blastoderm formation or Notch signaling, demonstrating that in Drosophila Medea elements can be generated through manipulation of diverse signaling pathways. We also describe the mRNA and small RNA changes in ovaries and early embryos associated from Medea-bearing females. Finally, we use modeling to illustrate how Medea elements carrying genes that result in diapause-dependent female lethality could be used to bring about population suppression.

The Potential Role of As-sumo-1 in the Embryonic Diapause Process and Early Embryo Development of Artemia sinica

PubMed Central

Chu, Bing; Yao, Feng; Cheng, Cheng; Wu, Yang; Mei, Yanli; Li, Xuejie; Liu, Yan; Wang, Peisheng; Hou, Lin; Zou, Xiangyang

2014-01-01

During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica. PMID:24404204

Intracellular pH in early Xenopus embryos: its effect on current flow between blastomeres.

PubMed Central

Turin, L; Warner, A E

1980-01-01

1. Electrophysiological techniques were used to monitor the flow of electric current from one cell to the next in Xenopus laevis embryos between the 4-cell and early blastula stages of development. Intracellular pH and blastocoel pH were determined using pH-sensitive micro-electrodes. 2. The resting intracellular pH was 7.74+/-0.02 (S.E. of mean, n = 29); there were no systematic differences between developmental stages. Blastocoel cavity pH was 8.4+/-0.06 (S.E. of mean, n = 10). The intracellular buffer value was 18 m-equiv. H+/pH unit per litre. 3. In embryos treated with bicarbonate buffered Holtfreter solution equilibrated with 100% CO2 the intracellular pH fell to 6.3+/-0.17 (S.D., n = 8). The membrane potential fell and the input resistance increased. The size of the effect on membrane potential and input resistance varied. 4. From the 32-cell stage onwards current flow from one cell to the next was abolished when the intracellular pH fell to below 6.5; the effect was rapid in onset and completely reversible. At cleavage stages of development lowering intracellular pH with CO2 had no effect on current flow from cell to cell. 5. The relationship between intracellular pH and current flow from cell to cell was sigmoid and covered between 0.2 and 0.4 pH units. The pH at which current flow was completely abolished ranged from 6.85 to 6.4. 6. Alterations in extraembryonic pH over the range 5.8-7.5 had no effect on any parameter measured. 7. We conclude that lowering the intracellular pH increases the resistance of both non-junctional junctional membranes. The data do not allow us to extract the pH junctional conductance relationship. 8. Variations in intracellular pH may provide a useful tool for the study of the functional role of direct cell to cell communication in both adult organs and early embryos. PMID:6770084

Primary Sex Determination in Drosophila melanogaster Does Not Rely on the Male-Specific Lethal Complex.

PubMed

Erickson, James W

2016-02-01

It has been proposed that the Male Specific Lethal (MSL) complex is active in Drosophila melanogaster embryos of both sexes prior to the maternal-to-zygotic transition. Elevated gene expression from the two X chromosomes of female embryos is proposed to facilitate the stable establishment of Sex-lethal (Sxl) expression, which determines sex and represses further activity of the MSL complex, leaving it active only in males. Important supporting data included female-lethal genetic interactions between the seven msl genes and either Sxl or scute and sisterlessA, two of the X-signal elements (XSE) that regulate early Sxl expression. Here I report contrary findings that there are no female-lethal genetic interactions between the msl genes and Sxl or its XSE regulators. Fly stocks containing the msl3(1) allele were found to exhibit a maternal-effect interaction with Sxl, scute, and sisterlessA mutations, but genetic complementation experiments showed that msl3 is neither necessary nor sufficient for the female-lethal interactions, which appear to be due to an unidentified maternal regulator of Sxl. Published data cited as evidence for an early function of the MSL complex in females, including a maternal effect of msl2, have been reevaluated and found not to support a maternal, or other effect, of the MSL complex in sex determination. These findings suggest that the MSL complex is not involved in primary sex determination or in X chromosome dosage compensation prior to the maternal-to-zygotic transition. Copyright © 2016 by the Genetics Society of America.

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

Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos.

PubMed

Tao, T; Reichelt, B; Niemann, H

1995-07-01

Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Spiroplasma infection causes either early or late male killing in Drosophila, depending on maternal host age

NASA Astrophysics Data System (ADS)

Kageyama, Daisuke; Anbutsu, Hisashi; Shimada, Masakazu; Fukatsu, Takema

2007-04-01

Symbiont-induced male-killing phenotypes have been found in a variety of insects. Conventionally, these phenotypes have been divided into two categories according to the timing of action: early male killing at embryonic stages and late male killing at late larval stages. In Drosophila species, endosymbiotic bacteria of the genus Spiroplasma have been known to cause early male killing. Here, we report that a spiroplasma strain normally causing early male killing also induces late male killing depending on the maternal host age: male-specific mortality of larvae and pupae was more frequently observed in the offspring of young females. As the lowest spiroplasma density and occasional male production were also associated with newly emerged females, we proposed the density-dependent hypothesis for the expression of early and late male-killing phenotypes. Our finding suggested that (1) early and late male-killing phenotypes can be caused by the same symbiont and probably by the same mechanism; (2) late male killing may occur as an attenuated expression of early male killing; (3) expression of early and late male-killing phenotypes may be dependent on the symbiont density, and thus, could potentially be affected by the host immunity and regulation; and (4) early male killing and late male killing could be alternative strategies adopted by microbial reproductive manipulators.

Optogenetic pacing in Drosophila models (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wu, Penghe; Li, Airong; Men, Jing; Tans, Rudolph E.; Zhou, Chao

2017-02-01

The Drosophila melanogaster shares many similarities with vertebrates in heart development. Comparison of heart structural and functional characteristic between male and female Drosophila melanogaster at different developmental stages is helpful to understand heart morphogenesis and function for different genders. And also, it opens up the possibility to uncover the role of sex-related genes in heart development. In this longitudinal study, we cultured and tracked dozens of individually labeled flies throughout their lifecycle. The heart characteristic was measured at different developmental stages during culturing. The gender of each individual fly was determined by adult stage so that the collected data of early stages could be classified to male or female group. We adapted a high-speed optical coherence microscopy (OCM) system with axial and transverse resolution of 2um and 4um, respectively, to perform non-invasive M-mode imaging at a frame rate of 132Hz in Drosophila heart at third instar larva, early pupa and adult stage. Based on those GPU processed M-mode OCM images, we segmented the fly heart region and then quantified the cardiac structural and functional parameters such as heart rate, heart chamber size and so on. Despite large variances of wild type Drosophila in terms of some cardiac characteristic, our results suggest that the heart rate is lower for male flies than for female flies, especially at third instar larva stage. The end diastolic area (EDA) and end systolic area (ESA) of the heart are both slightly larger in female flies than in male flies at larva and adult stage. In summary, we showed gender differences of wild type drosophila in heart functional and structural characteristic.

Ethanol impedes embryo transport and impairs oviduct epithelium.

PubMed

Xu, Tonghui; Yang, Qiuhong; Liu, Ruoxi; Wang, Wenfu; Wang, Shuanglian; Liu, Chuanyong; Li, Jingxin

2016-05-16

Most studies have demonstrated that alcohol consumption is associated with decreased fertility. The aim of this study was to investigate the effects of alcohol on pre-implantation embryo transport and/or early embryo development in the oviduct. We reported here that ethanol concentration-dependently suppressed the spontaneous motility of isolated human oviduct strips (EC50 50±6mM), which was largely attenuated in the present of L-NAME, a classical nitric oxide synthase(NOS) competitive inhibitor. Notably, either acute or chronic alcohol intake delayed egg transport and retarded early development of the embryo in the mouse oviduct, which was largely rescued by co-administration of L-NAME in a acute alcohol intake group but not in chronic alcohol intake group. It is worth mentioning that the oviductal epithelium destruction was verified by scanning electron microscope (SEM) observations in chronic alcohol intake group. In conclusion, alcohol intake delayed egg transport and retarded early development of the embryo in the oviduct by suppressing the spontaneous motility of oviduct and/or impairing oviductal epithelium. These findings suggested that alcohol abuse increases the incident of ectopic pregnancy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

Novel Synthetic Medea selfish genetic elements drive population replacement in Drosophila, and a theoretical exploration of Medea-dependent population suppression

PubMed Central

Akbari, Omar S.; Chen, Chun-Hong; Marshall, John M.; Huang, Haixia; Antoshechkin, Igor; Hay, Bruce A.

2013-01-01

Insects act as vectors for diseases of plants, animals and humans. Replacement of wild insect populations with genetically modified individuals unable to transmit disease provides a potentially self-perpetuating method of disease prevention. Population replacement requires a gene drive mechanism in order to spread linked genes mediating disease refractoriness through wild populations. We previously reported the creation of synthetic Medea selfish genetic elements able to drive population replacement in Drosophila. These elements use microRNA-mediated silencing of myd88, a maternally expressed gene required for embryonic dorso-ventral pattern formation, coupled with early zygotic expression of a rescuing transgene, to bring about gene drive. Medea elements that work through additional mechanisms are needed in order to be able to carry out cycles of population replacement and/or remove existing transgenes from the population, using second-generation elements that spread while driving first-generation elements out of the population. Here we report the synthesis and population genetic behavior of two new synthetic Medea elements that drive population replacement through manipulation of signaling pathways involved in cellular blastoderm formation or Notch signaling, demonstrating that in Drosophila Medea elements can be generated through manipulation of diverse signaling pathways. We also describe the mRNA and small RNA changes in ovaries and early embryos associated from Medea-bearing females. Finally, we use modeling to illustrate how Medea elements carrying genes that result in diapause-dependent female lethality could be used to bring about population suppression. PMID:23654248

The effects of the early uterine environment on the subsequent development of embryo and fetus.

PubMed

Barnes, F L

2000-01-15

Synchrony between the embryo and the uterine endometrium is essential for the establishment of pregnancy and birth in people and livestock. When asynchronous conditions occur a variety of complication result that include failure of the embryo to implant, early embryonic mortality, retarded development and growth, and accelerated development and growth. These complications all appear to be induced within the first week of embryo development and not withstanding the immediate endpoint of large or small size at birth, may alter the course of development throughout the life of the animal. Progesterone appears to play a causative role in establishing the abnormal growth of the fetus by decelerating or accelerating embryonic development. This may act through increasing the transport of blood born growth factors into the uterine lumen or by stimulating the release of growth factors from the endometrium directly. It can not be ruled out that progesterone mediated abundance of, or absence of, appropriate nutrition may bring about the same lifelong outcome. In vitro culture situations that include serum and/or co-culture can also bring about these abnormalities of growth. It is hypothesized that exposure to growth factors "out of phase" may result in an irreversible induction of abnormal development. The described abnormalities that occur in sheep and cattle have not yet been described for children resulting from IVF.

Stress Induces AMP-Dependent Loss of Potency Factors Id2 and Cdx2 in Early Embryos and Stem Cells

PubMed Central

Xie, Yufen; Awonuga, Awoniyi; Liu, Jian; Rings, Edmond; Puscheck, Elizabeth Ella

2013-01-01

The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id)2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the blastocyst stage. The caudal-related homeodomain protein (Cdx)2 is a regulatory gene for determining TSC, the earliest placental lineage in the preimplantation mouse embryo, but is expressed in the oocyte and in early cleavage stage embryos before TSC arise. We assayed the expression of putative potency-maintaining phosphorylated Cdx2 ser60 in the oocyte, two-cell stage embryo, blastocyst, and in TSC. We studied the loss of Cdx2 phospho ser60 expression induced by hyperosmolar stress and its underlying mechanisms. Hyperosmolar stress caused rapid loss of nuclear Cdx2 phospho ser60 and Id2 in the two-cell stage embryo by 0.5 h. Stress-induced Cdx2 phospho ser60 and Id2 loss is reversed by the AMPK inhibitor compound C and is induced by the AMPK agonist 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide in the absence of stress. In the two-cell stage embryo and TSC hyperosmolar, stress caused AMPK-mediated loss of Cdx2 phospho ser60 as detected by immunofluorescence and immunoblot. We propose that AMPK may be the master regulatory enzyme for mediating stress-induced loss of potency as AMPK is also required for stress-induced loss of Id2 in blastocysts and TSC. Since AMPK mediates potency loss in embryos and stem cells it will be important to measure, test mechanisms for, and manage the AMPK function to optimize the stem cell and embryo quality in vitro and in vivo. PMID:23316940

Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin.

PubMed

Lucas, Eliana P; Raff, Jordan W

2007-08-27

Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. We show that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles "rocket" around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and we observe only subtle defects in the asymmetric divisions of mutant neuroblasts. We conclude that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts.

The peripheral nervous system supports blood cell homing and survival in the Drosophila larva

PubMed Central

Makhijani, Kalpana; Alexander, Brandy; Tanaka, Tsubasa; Rulifson, Eric; Brückner, Katja

2011-01-01

Interactions of hematopoietic cells with their microenvironment control blood cell colonization, homing and hematopoiesis. Here, we introduce larval hematopoiesis as the first Drosophila model for hematopoietic colonization and the role of the peripheral nervous system (PNS) as a microenvironment in hematopoiesis. The Drosophila larval hematopoietic system is founded by differentiated hemocytes of the embryo, which colonize segmentally repeated epidermal-muscular pockets and proliferate in these locations. Importantly, we show that these resident hemocytes tightly colocalize with peripheral neurons and we demonstrate that larval hemocytes depend on the PNS as an attractive and trophic microenvironment. atonal (ato) mutant or genetically ablated larvae, which are deficient for subsets of peripheral neurons, show a progressive apoptotic decline in hemocytes and an incomplete resident hemocyte pattern, whereas supernumerary peripheral neurons induced by ectopic expression of the proneural gene scute (sc) misdirect hemocytes to these ectopic locations. This PNS-hematopoietic connection in Drosophila parallels the emerging role of the PNS in hematopoiesis and immune functions in vertebrates, and provides the basis for the systematic genetic dissection of the PNS-hematopoietic axis in the future. PMID:22071105

[Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

PubMed

Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

2008-10-14

To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

The immunologic and antioxidant effects of L-phenylalanine on the uterine implantation of mice embryos during early pregnancy.

PubMed

Dong, Yulan; Bai, Yongping; Liu, Guanhui; Wang, Zixu; Cao, Jing; Chen, Yaoxing; Yang, Hongliang

2014-10-01

L-phenylalanine (L-PHE) is a synthetic precursor of catecholamines. Because it cannot be synthesised by an organism, it must be absorbed from the environment. Despite the wide use of L-PHE, whether L-PHE has a negative impact on embryo implantation and development is poorly understood. This study attempted to determine the roles of L-PHE in embryo implantation and development and in the immune response and antioxidant status of the uterus in early pregnancy mice injected intraperitoneally with 320 mg/kg L-PHE. The embryo number of treated mice decreased by 57.6%, and the size of their embryos was reduced by 2.8% (P⟩0.05) along the long diameter and 11.9% (P⟨0.05) along the short diameter at E9 compared with control mice. In addition, L-PHE significantly suppressed B lymphocyte proliferation. L-PHE increased IL-2 secretion but decreased the IL-4 concentration, thereby up-regulating the ratio of IL-2/IL-4 to 1.37-8.45. An analysis of the oxidant and antioxidant status showed that, compared with the control mice, the level of superoxide dismutase activity decreased by 21.54-39.94% and the glutathione peroxidase activity decreased by 15.27-18.96% among the L-PHE-treated mice at E1-E9. However, the malonaldehyde content increased by 14.29%-90.11% among the L-PHE-treated mice. Therefore, L-PHE impaired embryo implantation by disrupting cytokine-based immunity and oxidative stress in the uterus.

Transcriptome analysis of PCOS arrested 2-cell embryos.

PubMed

Lu, Cuiling; Chi, Hongbin; Wang, Yapeng; Feng, Xue; Wang, Lina; Huang, Shuo; Yan, Liying; Lin, Shengli; Liu, Ping; Qiao, Jie

2018-06-18

In an attempt to explore the early developmental arrest in embryos from polycystic ovarian syndrome (PCOS) patients, we sequenced the transcriptome profiles of PCOS arrested 2-cell embryos, non-PCOS arrested 2-cell embryos and non-arrested 2-cell embryos using single-cell RNA-Seq technique. Differential expression analysis was performed using the DEGSeq R package. Gene Ontology (GO) enrichment was analyzed using the GOseq R package. Data revealed 62 differentially expressed genes between non-PCOS arrested and PCOS arrested embryos and 2217 differentially expressed genes between PCOS arrested and non-arrested 2-cell embryos. A total of 49 differently expressed genes (DEGs) were annotated with GO terms in the up-regulated genes between PCOS arrested and non-PCOS arrested embryos after GO enrichment. A total of 29 DEGs were annotated with GO terms in the down-regulated genes between PCOS arrested and non-arrested 2-cell embryos after GO enrichment. These data can provide a reference for screening specific genes involved in the arrest of PCOS embryos.

Spatial distribution of the capacity to initiate a secondary embryo in the 32-cell embryo of Xenopus laevis.

PubMed

Kageura, H

1990-12-01

To examine the spatial distribution of dorsal determinants in the early embryos of Xenopus laevis, individual cells from the 32-cell embryo were transplanted into the same tier of the ventral side of a synchronous recipient. Their abilities to initiate a secondary embryo were measured by the incidence of secondary embryos and by the length of the secondary axis relative to the primary embryo. The ability was found to be localized in all cells (A1, B1, C1, and D1) of the dorsal most column and in the vegetal cells (C2 and D2) of the dorsolateral column. Transplanted C1 (subequatorial) cells caused the highest incidence of a secondary embryo and the average relative length of the secondary embryo was also greatest. Effectiveness decreased in the order: D1, B1, D2, C2, and A1. When these results were compared with Dale and Slack's fate map of the 32-cell embryo, it was concluded that the distribution of dorsal determinants is unique and does not coincide with the prospective regions for any tissues, though it is somewhat similar to the prospective region of dorsal endoderm or notochord. From these results it seems that dorsal determinants do not determine a particular tissue in an embryo but rather the "dorsal" region of an embryo.

Color Processing in the Early Visual System of Drosophila.

PubMed

Schnaitmann, Christopher; Haikala, Väinö; Abraham, Eva; Oberhauser, Vitus; Thestrup, Thomas; Griesbeck, Oliver; Reiff, Dierk F

2018-01-11

Color vision extracts spectral information by comparing signals from photoreceptors with different visual pigments. Such comparisons are encoded by color-opponent neurons that are excited at one wavelength and inhibited at another. Here, we examine the circuit implementation of color-opponent processing in the Drosophila visual system by combining two-photon calcium imaging with genetic dissection of visual circuits. We report that color-opponent processing of UV short /blue and UV long /green is already implemented in R7/R8 inner photoreceptor terminals of "pale" and "yellow" ommatidia, respectively. R7 and R8 photoreceptors of the same type of ommatidia mutually inhibit each other directly via HisCl1 histamine receptors and receive additional feedback inhibition that requires the second histamine receptor Ort. Color-opponent processing at the first visual synapse represents an unexpected commonality between Drosophila and vertebrates; however, the differences in the molecular and cellular implementation suggest that the same principles evolved independently. Copyright © 2017 Elsevier Inc. All rights reserved.

A distinct class of homeodomain proteins is encoded by two sequentially expressed Drosophila genes from the 93D/E cluster.

PubMed Central

Jagla, K; Stanceva, I; Dretzen, G; Bellard, F; Bellard, M

1994-01-01

Homeodomains appear to be one of the most frequently employed DNA-binding domains in a superfamily of transacting factors. It is likely that during evolution several sub-types of homeodomain have evolved from a common ancestral domain, resulting in distinct but closely related DNA-binding preferences. Here we describe the conservation of a distinct type of homeodomain encoded by the Drosophila lady-bird-late (lbl) gene, previously named nkch4 (1). Using degenerate PCR primers corresponding to the most divergent regions of the first and third helix of the Lbl homeodomain we have amplified, from genomic DNA of the fly, a lady-bird-like homeobox fragment. The Drosophila PCR products contained both the lbl (1) and a highly related homeobox sequence, which we named lady-bird-early (lbe). This new Drosophila gene resides directly upstream to lbl and together with tinman/NK4 (2, 3, 4, 5), bagpipe/NK3 (2, 4) S59/NK1 (4, 6) and 93Bal (7) compose the 93D/E homeobox gene cluster. Ibe and lbl are transcribed from the same strand and in a temporal order corresponding to their 5'-3' chromosomal location. Transcripts of both genes are found in the epiderm of Drosophila embryos, in cells known to express a segment polarity gene wingless (8), and their spatial and temporal colinearity of expression strongly suggests that they cooperate during segmentation. The amino-acid composition of both Lady-bird homeodomains differ from that of Antp-type at several positions involved in DNA recognition. These substitutions appear to modify DNA-binding preferences since Lbl homeodomain is unable to recognize the most common homeodomain binding TAAT motif in gel retardation experiments. Images PMID:7909370

A Sponge-like Structure Involved in the Association and Transport of Maternal Products during Drosophila Oogenesis

PubMed Central

Wilsch-Bräuninger, Michaela; Schwarz, Heinz; Nüsslein-Volhard, Christiane

1997-01-01

Localization of maternally provided RNAs during oogenesis is required for formation of the antero–posterior axis of the Drosophila embryo. Here we describe a subcellular structure in nurse cells and oocytes which may function as an intracellular compartment for assembly and transport of maternal products involved in RNA localization. This structure, which we have termed “sponge body,” consists of ER-like cisternae, embedded in an amorphous electron-dense mass. It lacks a surrounding membrane and is frequently associated with mitochondria. The sponge bodies are not identical to the Golgi complexes. We suggest that the sponge bodies are homologous to the mitochondrial cloud in Xenopus oocytes, a granulo-fibrillar structure that contains RNAs involved in patterning of the embryo. Exuperantia protein, the earliest factor known to be required for the localization of bicoid mRNA to the anterior pole of the Drosophila oocyte, is highly enriched in the sponge bodies but not an essential structural component of these. RNA staining indicates that sponge bodies contain RNA. However, neither the intensity of this staining nor the accumulation of Exuperantia in the sponge bodies is dependent on the amount of bicoid mRNA present in the ovaries. Sponge bodies surround nuage, a possible polar granule precursor. Microtubules and microfilaments are not present in sponge bodies, although transport of the sponge bodies through the cells is implied by their presence in cytoplasmic bridges. We propose that the sponge bodies are structures that, by assembly and transport of included molecules or associated structures, are involved in localization of mRNAs in Drosophila oocytes. PMID:9348297

Early life sensory ability-ventilatory responses of thornback ray embryos (Raja clavata) to predator-type electric fields.

PubMed

Ball, Rachel Emma; Oliver, Matthew Kenneth; Gill, Andrew Bruce

2016-07-01

Predator avoidance is fundamental for survival and it can be particularly challenging for prey animals if physical movement away from a predatory threat is restricted. Many sharks and rays begin life within an egg capsule that is attached to the sea bed. The vulnerability of this sedentary life stage is exacerbated in skates (Rajidae) as the compulsory ventilatory activity of embryos makes them conspicuous to potential predators. Embryos can reduce this risk by mediating ventilatory activity if they detect the presence of a predator using an acute electrosense. To determine how early in embryonic life predator elicited behavioral responses can occur, the reactions of three different age groups (1/3 developed, 2/3 developed, and near hatching) of embryonic thornback rays Raja clavata were tested using predator-type electric field stimuli. Egg capsules were exposed to continuous or intermittent stimuli in order to assess varying predator-type encounter scenarios on the ventilatory behavior of different developmental stages. All embryos reacted with a "freeze response" following initial electric field (E-field) exposure, ceasing ventilatory behavior in response to predator presence, demonstrating electroreceptive functionality for the first time at the earliest possible stage in ontogeny. This ability coincided with the onset of egg ventilatory behavior and may represent an effective means to enhance survival. A continuous application of stimuli over time revealed that embryos can adapt their behavior and resume normal activity, whereas when presented intermittently, the E-field resulted in a significant reduction in overall ventilatory activity across all ages. Recovery from stimuli was significantly quicker in older embryos, potentially indicative of the trade-off between avoiding predation and adequate respiration. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 721-729, 2016. © 2015 Wiley Periodicals, Inc.

Drosophila hemocyte migration: an in vivo assay for directional cell migration.

PubMed

Moreira, Carolina G A; Regan, Jennifer C; Zaidman-Rémy, Anna; Jacinto, Antonio; Prag, Soren

2011-01-01

This protocol describes an in vivo assay for random and directed hemocyte migration in Drosophila. Drosophila is becoming an increasingly powerful model system for in vivo cell migration analysis, combining unique genetic tools with translucency of the embryo and pupa, which allows direct imaging and traceability of different cell types. In the assay we present here, we make use of the hemocyte response to epithelium wounding to experimentally induce a transition from random to directed migration. Time-lapse confocal microscopy of hemocyte migration in untreated conditions provides a random cell migration assay that allows identification of molecular mechanisms involved in this complex process. Upon laser-induced wounding of the thorax epithelium, a rapid chemotactic response changes hemocyte migratory behavior into a directed migration toward the wound site. This protocol provides a direct comparison of cells during both types of migration in vivo, and combined with recently developed resources such as transgenic RNAi, is ideal for forward genetic screens.

Deep cytoplasmic rearrangements in ventralized Xenopus embryos

NASA Technical Reports Server (NTRS)

Brown, E. E.; Denegre, J. M.; Danilchik, M. V.

1993-01-01

Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos

Gene–environment interplay in Drosophila melanogaster: Chronic food deprivation in early life affects adult exploratory and fitness traits

PubMed Central

Burns, James Geoffrey; Svetec, Nicolas; Rowe, Locke; Mery, Frederic; Dolan, Michael J.; Boyce, W. Thomas; Sokolowski, Marla B.

2012-01-01

Early life adversity has known impacts on adult health and behavior, yet little is known about the gene–environment interactions (GEIs) that underlie these consequences. We used the fruit fly Drosophila melanogaster to show that chronic early nutritional adversity interacts with rover and sitter allelic variants of foraging (for) to affect adult exploratory behavior, a phenotype that is critical for foraging, and reproductive fitness. Chronic nutritional adversity during adulthood did not affect rover or sitter adult exploratory behavior; however, early nutritional adversity in the larval period increased sitter but not rover adult exploratory behavior. Increasing for gene expression in the mushroom bodies, an important center of integration in the fly brain, changed the amount of exploratory behavior exhibited by sitter adults when they did not experience early nutritional adversity but had no effect in sitters that experienced early nutritional adversity. Manipulation of the larval nutritional environment also affected adult reproductive output of sitters but not rovers, indicating GEIs on fitness itself. The natural for variants are an excellent model to examine how GEIs underlie the biological embedding of early experience. PMID:23045644

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.

Climatic factors affecting quantity and quality grade of in vivo derived embryos of cattle.

PubMed

Chinchilla-Vargas, Josué; Jahnke, Marianna M; Dohlman, Tyler M; Rothschild, Max F; Gunn, Patrick J

2018-05-01

The present study investigated the effects of climatic variables on the quality grade and quantity of in vivo derived cattle embryos in the Midwestern United States. Climatic information included greatest and least daily temperature, average daily wind speed and average temperature-humidity index for each of the 765 records. The response variables included the number of ovarian structures, viable embryos, quality grade 1 embryos, quality grade 2 embryos, quality grade 3 embryos, freezable embryos (sum of quality grade 1 and quality grade 2 embryos), transferable embryos (sum of quality grade 1-3 embryos), degenerate embryos and unfertilized ova. Measures for variables among the breeds of donors and sires grouped by geographical origin were compared. A negative effect of greater temperatures during the early embryonic development stage tended (P < 0.10) to be associated with a decrease in the quality of embryos recovered. Interestingly, the greater the Temperature-Humidity Index (THI) during the early ovarian antral follicular development stage 40-45 days prior to ovulation was associated with a tendency for greater numbers of total number of freezable and transferable embryos recovered per uterine flushing (P < 0.10). Increased wind speed at the early antral follicular phase 40-45 days prior to ovulation was associated with an increase in the percentage of quality grade 1 embryos recovered (P < 0.05). Wind speed during the estrous synchronization period was also associated with a lesser number of embryos recovered (P < 0.05). This retrospective study confirms that climatic variables have significant effects on the in vivo production of cattle embryos and that wind speed should be considered in future analyses of factors affecting embryo quality. Copyright © 2018 Elsevier B.V. All rights reserved.

Enhance beef cattle improvement by embryo biotechnologies.

PubMed

Wu, B; Zan, L

2012-10-01

Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer (in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions. © 2011 Blackwell Verlag GmbH.

Recognition of the CDEI motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo.

PubMed Central

Blangy, A; Léopold, P; Vidal, F; Rassoulzadegan, M; Cuzin, F

1991-01-01

We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development. Images PMID:1766880

Toxicity and cardiac effects of carbaryl in early developing zebrafish (Danio rerio) embryos

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

Lin, C.C.; Hui, Michelle N.Y.; Cheng, S.H. E-mail: bhcheng@cityu.edu.hk

2007-07-15

Carbaryl, an acetylcholinesterase inhibitor, is known to be moderately toxic to adult zebrafish and has been reported to cause heart malformations and irregular heartbeat in medaka. We performed experiments to study the toxicity of carbaryl, specifically its effects on the heart, in early developing zebrafish embryos. LC50 and EC50 values for carbaryl at 28 h post-fertilization were 44.66 {mu}g/ml and 7.52 {mu}g/ml, respectively, and 10 {mu}g/ml carbaryl was used in subsequent experiments. After confirming acetylcholinesterase inhibition by carbaryl using an enzymatic method, we observed red blood cell accumulation, delayed hatching and pericardial edema, but not heart malformation as described inmore » some previous reports. Our chronic exposure data also demonstrated carbaryl-induced bradycardia, which is a common effect of acetylcholinesterase inhibitors due to the accumulation of acetylcholine, in embryos from 1 day post-fertilization (dpf) to 5 dpf. The distance between the sinus venosus, the point where blood enters the atrium, and the bulbus arteriosus, the point where blood leaves the ventricle, indicated normal looping of the heart tube. Immunostaining of myosin heavy chains with the ventricle-specific antibody MF20 and the atrium-specific antibody S46 showed normal development of heart chambers. At the same time, acute exposure resulted in carbaryl-induced bradycardia. Heart rate dropped significantly after a 10-min exposure to 100 {mu}g/ml carbaryl but recovered when carbaryl was removed. The novel observation of carbaryl-induced bradycardia in 1- and 2-dpf embryos suggested that carbaryl affected cardiac function possibly through an alternative mechanism other than acetylcholinesterase inhibition such as inhibition of calcium ion channels, since acetylcholine receptors in zebrafish are not functional until 3 dpf. However, the exact nature of this mechanism is currently unknown, and thus further studies are required.« less

Excess Imidacloprid Exposure Causes the Heart Tube Malformation of Chick Embryos.

PubMed

Gao, Lin-Rui; Li, Shuai; Zhang, Jing; Liang, Chang; Chen, En-Ni; Zhang, Shi-Yao; Chuai, Manli; Bao, Yong-Ping; Wang, Guang; Yang, Xuesong

2016-11-30

As a neonicotinoid pesticide, imidacloprid is widely used to control sucking insects on agricultural planting and fleas on domestic animals. However, the extent to which imidacloprid exposure has an influence on cardiogensis in early embryogenesis is still poorly understood. In vertebrates, the heart is the first organ to be formed. In this study, to address whether imidacloprid exposure affects early heart development, the early chick embryo has been used as an experimental model because of its accessibility at its early developmental stage. The results demonstrate that exposure of the early chick embryo to imidacloprid caused malformation of heart tube. Furthermore, the data reveal that down-regulation of GATA4, NKX2.5, and BMP4 and up-regulation of Wnt3a led to aberrant cardiomyocyte differentiation. In addition, imidacloprid exposure interfered with basement membrane breakdown, E-cadherin/laminin expression, and mesoderm formation during the epithelial-mesenchymal transition (EMT) in gastrula chick embryos. Finally, the DiI-labeled cell migration trajectory indicated that imidacloprid restricted the cell migration of cardiac progenitors to primary heart field in gastrula chick embryos. A similar observation was also obtained from the cell migration assay of scratch wounds in vitro. Additionally, imidacloprid exposure negatively affected the cytoskeleton structure and expression of corresponding adhesion molecules. Taken together, these results reveal that the improper EMT, cardiac progenitor migration, and differentiation are responsible for imidacloprid exposure-induced malformation of heart tube during chick embryo development.

Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos

PubMed Central

Gupta, Prabuddha; Martin, René; Knölker, Hans-Joachim; Nihalani, Deepak; Kumar Sinha, Deepak

2017-01-01

Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis. PMID:28678859

Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function.

PubMed

Faivre-Sarrailh, Catherine; Banerjee, Swati; Li, Jingjun; Hortsch, Michael; Laval, Monique; Bhat, Manzoor A

2004-10-01

Septate junctions (SJs) in epithelial and neuronal cells play an important role in the formation and maintenance of charge and size selective barriers. They form the basis for the ensheathment of nerve fibers in Drosophila and for the attachment of myelin loops to axonal surface in vertebrates. The cell-adhesion molecules NRX IV/Caspr/Paranodin (NCP1), contactin and Neurofascin-155 (NF-155) are all present at the vertebrate axo-glial SJs. Mutational analyses have shown that vertebrate NCP1 and its Drosophila homolog, Neurexin IV (NRX IV) are required for the formation of SJs. In this study, we report the genetic, molecular and biochemical characterization of the Drosophila homolog of vertebrate contactin, CONT. Ultrastructural and dye-exclusion analyses of Cont mutant embryos show that CONT is required for organization of SJs and paracellular barrier function. We show that CONT, Neuroglian (NRG) (Drosophila homolog of NF-155) and NRX IV are interdependent for their SJ localization and these proteins form a tripartite complex. Hence, our data provide evidence that the organization of SJs is dependent on the interactions between these highly conserved cell-adhesion molecules.

Expression of the Drosophila homeobox gene, Distal-less supports an ancestral role in neural development

PubMed Central

Plavicki, Jessica S.; Squirrell, Jayne M.; Eliceiri, Kevin W.; Boekhoff-Falk, Grace

2015-01-01

Background Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. Results Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos we show that Dll is expressed in the embryonic, larval and adult CNS and PNS in embryonic and larval neurons, brain and ventral nerve cord (VNC) glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes. Conclusions Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts. PMID:26472170

A novel muscle LIM-only protein is generated from the paxillin gene locus in Drosophila.

PubMed

Yagi, R; Ishimaru, S; Yano, H; Gaul, U; Hanafusa, H; Sabe, H

2001-09-01

Paxillin is a protein containing four LIM domains, and functions in integrin signaling. We report here that two transcripts are generated from the paxillin gene locus in Drosophila; one encodes a protein homolog of the vertebrate Paxillin (DPxn37), and the other a protein with only three LIM domains, partly encoded by its own specific exon (PDLP). At the myotendinous junctions of Drosophila embryos where integrins play important roles, both DPxn37 and PDLP are highly expressed with different patterns; DPxn37 is predominantly concentrated at the center of the junctions, whereas PDLP is highly enriched at neighboring sides of the junction centers, primarily expressed in the mesodermal myotubes. Northern blot analysis revealed that DPxn37 is ubiquitously expressed throughout the life cycle, whereas PDLP expression exhibits a biphasic pattern during development, largely concomitant with muscle generation and remodeling. Our results collectively reveal that a unique system exists in Drosophila for the generation of a novel type of LIM-only protein, highly expressed in the embryonic musculature, largely utilizing the Paxillin LIM domains.

Myosin II Dynamics during Embryo Morphogenesis

NASA Astrophysics Data System (ADS)

Kasza, Karen

2013-03-01

During embryonic morphogenesis, the myosin II motor protein generates forces that help to shape tissues, organs, and the overall body form. In one dramatic example in the Drosophila melanogaster embryo, the epithelial tissue that will give rise to the body of the adult animal elongates more than two-fold along the head-to-tail axis in less than an hour. This elongation is accomplished primarily through directional rearrangements of cells within the plane of the tissue. Just prior to elongation, polarized assemblies of myosin II accumulate perpendicular to the elongation axis. The contractile forces generated by myosin activity orient cell movements along a common axis, promoting local cell rearrangements that contribute to global tissue elongation. The molecular and mechanical mechanisms by which myosin drives this massive change in embryo shape are poorly understood. To investigate these mechanisms, we generated a collection of transgenic flies expressing variants of myosin II with altered motor function and regulation. We found that variants that are predicted to have increased myosin activity cause defects in tissue elongation. Using biophysical approaches, we found that these myosin variants also have decreased turnover dynamics within cells. To explore the mechanisms by which molecular-level myosin dynamics are translated into tissue-level elongation, we are using time-lapse confocal imaging to observe cell movements in embryos with altered myosin activity. We are utilizing computational approaches to quantify the dynamics and directionality of myosin localization and cell rearrangements. These studies will help elucidate how myosin-generated forces control cell movements within tissues. This work is in collaboration with J. Zallen at the Sloan-Kettering Institute.

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 �

Luna, a Drosophila KLF6/KLF7, Is Maternally Required for Synchronized Nuclear and Centrosome Cycles in the Preblastoderm Embryo

PubMed Central

Weber, Ursula; Rodriguez, Estefania; Martignetti, John; Mlodzik, Marek

2014-01-01

Krüppel like factors (KLFs) are conserved transcription factors that have been implicated in many developmental processes including differentiation, organ patterning, or regulation of stem cell pluripotency. We report the generation and analysis of loss-of-function mutants of Drosophila Klf6/7, the luna gene. We demonstrate that luna mutants are associated with very early embryonic defects prior to cellularization at the syncytial stage and cause DNA separation defects during the rapid mitotic cycles resulting in un-coupled DNA and centrosome cycles. These defects manifest themselves, both in animals that are maternally homozygous and heterozygous mutant. Surprisingly, luna is only required during the syncytial stages and not later in development, suggesting that the DNA segregation defect is linked to centrosomes, since centrosomes are dispensable for later cell divisions. PMID:24915236

Zonda is a novel early component of the autophagy pathway in Drosophila.

PubMed

Melani, Mariana; Valko, Ayelén; Romero, Nuria M; Aguilera, Milton O; Acevedo, Julieta M; Bhujabal, Zambarlal; Perez-Perri, Joel; de la Riva-Carrasco, Rocío V; Katz, Maximiliano J; Sorianello, Eleonora; D'Alessio, Cecilia; Juhász, Gabor; Johansen, Terje; Colombo, María I; Wappner, Pablo

2017-11-01

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation. © 2017 Melani, Valko, Romero, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A new level of plasticity: Drosophila smooth-like testes muscles compensate failure of myoblast fusion

PubMed Central

Kuckwa, Jessica; Fritzen, Katharina; Buttgereit, Detlev; Rothenbusch-Fender, Silke; Renkawitz-Pohl, Renate

2016-01-01

The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. Here, we show that adhesion molecules are distinctly localized on the seminal vesicles; founder cell (FC)-like myoblasts express Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast (FCM)-like cells mainly express Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst probably act independently in this context. Despite reduced fusion in all of these RNAi-treated animals, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments were arranged as in the wild type and spermatogenesis proceeded normally. Hence, the testes muscles compensate for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild type and male fertility is guaranteed. PMID:26657767

Conservation of regulatory sequences and gene expression patterns in the disintegrating Drosophila Hox gene complex

PubMed Central

Negre, Bárbara; Casillas, Sònia; Suzanne, Magali; Sánchez-Herrero, Ernesto; Akam, Michael; Nefedov, Michael; Barbadilla, Antonio; de Jong, Pieter; Ruiz, Alfredo

2005-01-01

Homeotic (Hox) genes are usually clustered and arranged in the same order as they are expressed along the anteroposterior body axis of metazoans. The mechanistic explanation for this colinearity has been elusive, and it may well be that a single and universal cause does not exist. The Hox-gene complex (HOM-C) has been rearranged differently in several Drosophila species, producing a striking diversity of Hox gene organizations. We investigated the genomic and functional consequences of the two HOM-C splits present in Drosophila buzzatii. Firstly, we sequenced two regions of the D. buzzatii genome, one containing the genes labial and abdominal A, and another one including proboscipedia, and compared their organization with that of D. melanogaster and D. pseudoobscura in order to map precisely the two splits. Then, a plethora of conserved noncoding sequences, which are putative enhancers, were identified around the three Hox genes closer to the splits. The position and order of these enhancers are conserved, with minor exceptions, between the three Drosophila species. Finally, we analyzed the expression patterns of the same three genes in embryos and imaginal discs of four Drosophila species with different Hox-gene organizations. The results show that their expression patterns are conserved despite the HOM-C splits. We conclude that, in Drosophila, Hox-gene clustering is not an absolute requirement for proper function. Rather, the organization of Hox genes is modular, and their clustering seems the result of phylogenetic inertia more than functional necessity. PMID:15867430

Cooling strategies for brazilian flounder Paralichthys orbignyanus embryos.

PubMed

Varela, A S; Cardoso, T F; Fernandes E Silva, E; Goularte, K L; Okamoto, M H; Sampaio, L A; Jardim, R D; Corcini, C D

Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). Control embryos were maintained at 23 degree C and treated embryos were cooled to 15 degree C, 10 degree C and 5 degree C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5 degree C for 30 min are recommended.

Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos

PubMed Central

Famiglietti, Amber L.; Wei, Zheng; Beres, Thomas M.; Milac, Adina L.; Tran, Duy T.; Patel, Divya; Angerer, Robert C.; Angerer, Lynne M.

2017-01-01

Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered

Reduced heart rate and cardiac output differentially affect angiogenesis, growth, and development in early chicken embryos (Gallus domesticus).

PubMed

Branum, Sylvia R; Yamada-Fisher, Miho; Burggren, Warren

2013-01-01

An increase in both vascular circumferential tension and shear stress in the developing vasculature of the chicken embryo has been hypothesized to stimulate angiogenesis in the developing peripheral circulation chorioallantoic membrane (CAM). To test this hypothesis, angiogenesis in the CAM, development, and growth were measured in the early chicken embryo, following acute and chronic topical application of the purely bradycardic drug ZD7288. At hour 56, ZD7288 reduced heart rate (f(H)) by ~30% but had no significant effect on stroke volume (~0.19 ± 0.2 μL), collectively resulting in a significant fall in cardiac output (CO) from ~27 ± 3 to 18 ± 2 μL min(-1). Mean f(H) at 72 h of development was similarly significantly lowered by acute ZD7288 treatment (250 μM) to 128 ± 0.3 beats min(-1), compared with 174.5 ± 0.3 and 174.7 ± 0.8 beats min(-1) in control and Pannett-Compton (P-C) saline-treated embryos, respectively. Chronic dosing with ZD7288-and the attendant decreases in f(H) and CO-did not change eye diameter or cervical flexion (key indicators of development rate) at 120 h but significantly reduced overall growth (wet and dry body mass decreased by 20%). CAM vessel density index (reflecting angiogenesis) measured 200-400 μm from the umbilical stalk was not altered, but ZD7288 reduced vessel numbers-and therefore vessel density-by 13%-16% more distally (500-600 μm from umbilical stalk) in the CAM. In the ZD7288-treated embryos, a decrease in vessel length was found within the second branch order (~300-400 μm from the umbilical stock), while a decrease in vessel diameter was found closer to the umbilical stock, beginning in the first branch order (~200-300 μm). Paradoxically, chronic application of P-C saline also reduced peripheral CAM vessel density index at 500 and 600 μm by 13% and 7%, respectively, likely from washout of local angiogenic factors. In summary, decreased f(H) with reduced CO did not slow development rate but reduced embryonic

Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

PubMed

Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

2013-10-01

approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence. This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest. ClinicalTrials.gov Identifier: NCT01397136.

Types of neural cells in the spinal ganglia of human embryos and early fetuses.

PubMed

Olszewska, B; Woźniak, W; Gardner, E; O'Rahilly, R

1979-01-01

Spinal ganglial of human embryos and fetuses ranging in C.-R. length from 15 to 74 mm and in age from 6 1/2 to 11 postovulatory weeks were studied by light and electron microscopy. A sequence of events in differentiation and maturation enabled five types of cells to be distinguished: 1. apolar, undifferentiated neuroblasts are the main cells at 6 1/2 to 7 1/2 weeks; 2. early bipolar neuroblasts (strictly speaking, types 2 to 5 are immature neurons) predominate at the end of the embryonic period proper (8 postovulatory weeks); 3. intermediate bipolar neuroblasts are characteristic of the early fetal period; 4. late bipolar neuroblasts, in which two proceses arise separately from one pole of the cell, appear at about 10 postovulatory weeks; 5. unipolar neuroblasts are found within another week and, by that time, cells of types 1 and 2 are no longer present.

Apical targeting of the formin Diaphanous in Drosophila tubular epithelia

PubMed Central

Rousso, Tal; Shewan, Annette M; Mostov, Keith E; Schejter, Eyal D; Shilo, Ben-Zion

2013-01-01

Apical secretion from epithelial tubes of the Drosophila embryo is mediated by apical F-actin cables generated by the formin-family protein Diaphanous (Dia). Apical localization and activity of Dia are at the core of restricting F-actin formation to the correct membrane domain. Here we identify the mechanisms that target Dia to the apical surface. PI(4,5)P2 levels at the apical membrane regulate Dia localization in both the MDCK cyst model and in Drosophila tubular epithelia. An N-terminal basic domain of Dia is crucial for apical localization, implying direct binding to PI(4,5)P2. Dia apical targeting also depends on binding to Rho1, which is critical for activation-induced conformational change, as well as physically anchoring Dia to the apical membrane. We demonstrate that binding to Rho1 facilitates interaction with PI(4,5)P2 at the plane of the membrane. Together these cues ensure efficient and distinct restriction of Dia to the apical membrane. DOI: http://dx.doi.org/10.7554/eLife.00666.001 PMID:23853710

Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

PubMed Central

Belacortu, Yaiza; Weiss, Ron; Kadener, Sebastian; Paricio, Nuria

2012-01-01

Background Cabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. Methodology/Principal Findings To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. Conclusions/Significance Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of

Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila.

PubMed

Shen, Baohe; Manley, James L

2002-04-01

The Drosophila Pelle kinase plays a key role in the evolutionarily conserved Toll signaling pathway, but the mechanism responsible for its activation has been unknown. We present in vivo and in vitro evidence establishing an important role for concentration-dependent autophosphorylation in the signaling process. We first show that Pelle phosphorylation can be detected transiently in early embryos, concomitant with activation of signaling. Importantly, Pelle phosphorylation is enhanced in a gain-of-function Toll mutant (Toll(10b)), but decreased by loss-of-function Toll alleles. Next we found that Pelle is phosphorylated in transfected Schneider L2 cells in a concentration-dependent manner such that significant modification is observed only at high Pelle concentrations, which coincide with levels required for phosphorylation and activation of the downstream target, Dorsal. Pelle phosphorylation is also enhanced in L2 cells co-expressing Toll(10b), and is dependent on Pelle kinase activity. In vitro kinase assays revealed that recombinant, autophosphorylated Pelle is far more active than unphosphorylated Pelle. Importantly, unphosphorylated Pelle becomes autophosphorylated, and activated, by incubation at high concentrations. We discuss these results in the context of Toll-like receptor mediated signaling in both flies and mammals.

The Origin of the Second Centriole in the Zygote of Drosophila melanogaster

PubMed Central

Blachon, Stephanie; Khire, Atul; Avidor-Reiss, Tomer

2014-01-01

Centrosomes are composed of two centrioles surrounded by pericentriolar material (PCM). However, the sperm and the oocyte modify or lose their centrosomes. Consequently, how the zygote establishes its first centrosome, and in particular, the origin of the second zygotic centriole, is uncertain. Drosophila melanogaster spermatids contain a single centriole called the Giant Centriole (GC) and a Proximal centriole-like (PCL) structure whose function is unknown. We found that, like the centriole, the PCL loses its protein markers at the end of spermiogenesis. After fertilization, the first two centrioles are observed via the recruitment of the zygotic PCM proteins and are seen in asterless mutant embryos that cannot form centrioles. The zygote’s centriolar proteins label only the daughter centrioles of the first two centrioles. These observations demonstrate that the PCL is the origin for the second centriole in the Drosophila zygote and that a paternal centriole precursor, without centriolar proteins, is transmitted to the egg during fertilization. PMID:24532732

Extensive Use of RNA-Binding Proteins in Drosophila Sensory Neuron Dendrite Morphogenesis

PubMed Central

Olesnicky, Eugenia C.; Killian, Darrell J.; Garcia, Evelyn; Morton, Mary C.; Rathjen, Alan R.; Sola, Ismail E.; Gavis, Elizabeth R.

2013-01-01

The large number of RNA-binding proteins and translation factors encoded in the Drosophila and other metazoan genomes predicts widespread use of post-transcriptional regulation in cellular and developmental processes. Previous studies identified roles for several RNA-binding proteins in dendrite branching morphogenesis of Drosophila larval sensory neurons. To determine the larger contribution of post-transcriptional gene regulation to neuronal morphogenesis, we conducted an RNA interference screen to identify additional Drosophila proteins annotated as either RNA-binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 genes encoding such proteins whose knockdown resulted in aberrant dendritic morphology, including alterations in dendritic branch number, branch length, field size, and patterning of the dendritic tree. In particular, splicing and translation initiation factors were associated with distinct and characteristic phenotypes, suggesting that different morphogenetic events are best controlled at specific steps in post-transcriptional messenger RNA metabolism. Many of the factors identified in the screen have been implicated in controlling the subcellular distributions and translation of maternal messenger RNAs; thus, common post-transcriptional regulatory strategies may be used in neurogenesis and in the generation of asymmetry in the female germline and embryo. PMID:24347626

Automated cell tracking identifies mechanically oriented cell divisions during Drosophila axis elongation.

PubMed

Wang, Michael F Z; Hunter, Miranda V; Wang, Gang; McFaul, Christopher; Yip, Christopher M; Fernandez-Gonzalez, Rodrigo

2017-04-01

Embryos extend their anterior-posterior (AP) axis in a conserved process known as axis elongation. Drosophila axis elongation occurs in an epithelial monolayer, the germband, and is driven by cell intercalation, cell shape changes, and oriented cell divisions at the posterior germband. Anterior germband cells also divide during axis elongation. We developed image analysis and pattern-recognition methods to track dividing cells from confocal microscopy movies in a generally applicable approach. Mesectoderm cells, forming the ventral midline, divided parallel to the AP axis, while lateral cells displayed a uniform distribution of division orientations. Mesectoderm cells did not intercalate and sustained increased AP strain before cell division. After division, mesectoderm cell density increased along the AP axis, thus relieving strain. We used laser ablation to isolate mesectoderm cells from the influence of other tissues. Uncoupling the mesectoderm from intercalating cells did not affect cell division orientation. Conversely, separating the mesectoderm from the anterior and posterior poles of the embryo resulted in uniformly oriented divisions. Our data suggest that mesectoderm cells align their division angle to reduce strain caused by mechanical forces along the AP axis of the embryo. © 2017. Published by The Company of Biologists Ltd.

The evolution of porcine embryo in vitro production.

PubMed

Grupen, Christopher G

2014-01-01

The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Programmed cell death acts at different stages of Drosophila neurodevelopment to shape the central nervous system

PubMed Central

Desplan, Claude

2016-01-01

Nervous system development is a process that integrates cell proliferation, differentiation and programmed cell death (PCD). PCD is an evolutionary conserved mechanism and a fundamental developmental process by which the final cell number in a nervous system is established. In vertebrates and invertebrates, PCD can be determined intrinsically by cell lineage and age, as well as extrinsically by nutritional, metabolic and hormonal states. Drosophila has been an instrumental model for understanding how this mechanism is regulated. We review the role of PCD in Drosophila central nervous system development from neural progenitors to neurons, its molecular mechanism and function, how it is regulated and implemented, and how it ultimately shapes the fly central nervous system from the embryo to the adult. Finally, we discuss ideas that emerge while integrating this information. PMID:27404003

The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila.

PubMed

Laval, Monique; Bel, Christophe; Faivre-Sarrailh, Catherine

2008-07-18

A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions. We conducted photobleaching experiments on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. We show that GFP-tagged Nrg and Nrx IV molecules exhibit very stable association with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP becomes distributed to the baso-lateral membrane in null mutants of vari and cora, and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the null mutation of nrx IV on the lateral mobility of Nrg-GFP. The strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility of CAMs is strongly limited when they take part to the multimolecular complex forming septate junctions. This restricted lateral diffusion of CAMs depends on both adhesive interactions and clustering by scaffolding molecules. The lateral mobility of CAMs is strongly increased in embryos presenting alteration of septate junctions. The stronger effect of vari by comparison with nrx IV null mutation supports

Progesterone is critical for the development of mouse embryos.

PubMed

Zhang, Cong; Murphy, Bruce D

2014-08-01

Infertility affects approximately 10-15 % of reproductive-aged couples, and embryo loss due to preimplantation death is common to many mammals. Previous studies showed that a complex series of interactive molecular events are associated with this process, especially hormones (progesterone and estrogens) and growth factors, and are important for the cleavage and differentiation of the blastocysts. Yet, the mechanism of preimplantation embryo development is unclear. Using conditional knockout mice (CKO), we showed the development of blastocyst is tightly controlled by the level of progesterone (P4); furthermore, we found that the time when P4 should increase is also crucial for the formation of blastocysts. In CKO mice whose Lrh1 (liver receptor homolog 1) is deleted under the expression of Cre recombinase driven by progesterone receptor promoter, which reduced P4 synthesis, few of their embryos can reach blastocyst stage. When these CKO mice were supplied with P4 in the afternoon of dpc 1 (day post copulation), most of the embryos can form blastocysts; when CKO mice were supplied with P4 from the morning of dpc1, one-third of the embryos can reach blastocyst stage; however, the supplement of P4 in the morning of dpc 2 made very few of the embryos become blastocysts. We conclude that early exposure to P4 is essential for timely progression of early embryogenesis in the mouse.

Starch Turnover and Metabolism during Flower and Early Embryo Development1[CC-BY

PubMed Central

Pazmino, Diana; Gagliardini, Valeria

2016-01-01

The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed. PMID:27794100

Sex determination of duck embryos: observations on syrinx development

USGS Publications Warehouse

Wilson, Robert E.; Sonsthagen, Sarah A.; Franson, J. Christian

2013-01-01

Ducks exhibit sexual dimorphism in vocal anatomy. Asymmetrical ossification of the syrinx (bulla syringealis) is discernable at about 10 days of age in male Pekin duck (Anas platyrhynchos domestica) embryos, but information is lacking on the early development of the bulla in wild ducks. To evaluate the reliability of this characteristic for sexing developing embryos, we examined the syrinx of dead embryos and compared results with molecular sexing techniques in high arctic nesting Common Eiders (Somateria mollissima). Embryos 8 days or older were accurately (100%) sexed based on the presence/absence of a bulla, 2 days earlier than Pekin duck. The use of the tracheal bulla can be a valuable technique when sex identification of embryos or young ducklings is required.

5Alpha-Reduced Steroids Are Major Metabolites in the Early Equine Embryo Proper and Its Membranes.

PubMed

Raeside, James I; Christie, Heather L; Betteridge, Keith J

2015-09-01

Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast, allantois) were incubated with tritium-labeled substrates. 5Alpha-reduced metabolites (5alpha-dihydro- and 3beta,5alpha-tetrahydro- steroids) as radiolabeled products were identified from a series of chromatographic steps using four solvent systems for high-performance liquid chromatography. Use of a 5alpha-reductase inhibitor confirmed the metabolites were indeed 5alpha-reduced steroids. For the embryo, the only products from androstenedione were 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione, with no evidence of more polar metabolites; there was some 3beta,5alpha-tetrahydrotestosterone but no 5alpha-dihydrotestosterone from testosterone, and formation of androstenedione was followed by the production of 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione. The major 5alpha-reduced product from progesterone was 3beta,5alpha-tetrahydroprogesterone, with lesser amounts of 5alpha-dihydroprogesterone. For the membranes, reductions to tetrahydro, 5alpha-reduced steroids were prominent in most instances, but also present were considerable amounts of products more polar than the substrates. The well-recognized activity of some 5alpha-reduced steroids--for example, 5alpha-dihydrotestosterone in male sexual differentiation--provokes interest in their even earlier appearance, as seen in this study, and suggests a possible role for them in

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.

Early events in speciation: Cryptic species of Drosophila aldrichi.

PubMed

Castro Vargas, Cynthia; Richmond, Maxi Polihronakis; Ramirez Loustalot Laclette, Mariana; Markow, Therese Ann

2017-06-01

Understanding the earliest events in speciation remains a major challenge in evolutionary biology. Thus identifying species whose populations are beginning to diverge can provide useful systems to study the process of speciation. Drosophila aldrichi , a cactophilic fruit fly species with a broad distribution in North America, has long been assumed to be a single species owing to its morphological uniformity. While previous reports either of genetic divergence or reproductive isolation among different D. aldrichi strains have hinted at the existence of cryptic species, the evolutionary relationships of this species across its range have not been thoroughly investigated. Here we show that D. aldrichi actually is paraphyletic with respect to its closest relative, Drosophila wheeleri , and that divergent D. aldrichi lineages show complete hybrid male sterility when crossed. Our data support the interpretation that there are at least two species of D. aldrichi, making these flies particularly attractive for studies of speciation in an ecological and geographical context.

Hypoxia transiently sequesters mps1 and polo to collagenase-sensitive filaments in Drosophila prometaphase oocytes.

PubMed

Gilliland, William D; Vietti, Dana L; Schweppe, Nicole M; Guo, Fengli; Johnson, Teri J; Hawley, R Scott

2009-10-22

The protein kinases Mps1 and Polo, which are required for proper cell cycle regulation in meiosis and mitosis, localize to numerous ooplasmic filaments during prometaphase in Drosophila oocytes. These filaments first appear throughout the oocyte at the end of prophase and are disassembled after egg activation. We showed here that Mps1 and Polo proteins undergo dynamic and reversible localization to static ooplasmic filaments as part of an oocyte-specific response to hypoxia. The observation that Mps1- and Polo-associated filaments reappear in the same locations through multiple cycles of oxygen deprivation demonstrates that underlying structural components of the filaments must still be present during normoxic conditions. Using immuno-electron microscopy, we observed triple-helical binding of Mps1 to numerous electron-dense filaments, with the gold label wrapped around the outside of the filaments like a garland. In addition, we showed that in live oocytes the relocalization of Mps1 and Polo to filaments is sensitive to injection of collagenase, suggesting that the structural components of the filaments are composed of collagen-like fibrils. However, the collagen-like genes we have been able to test so far (vkg and CG42453) did not appear to be associated with the filaments, demonstrating that the collagenase-sensitive component of the filaments is one of a number of other Drosophila proteins bearing a collagenase cleavage site. Finally, as hypoxia is known to cause Mps1 protein to accumulate at kinetochores in syncytial embryos, we also show that GFP-Polo accumulates at both kinetochores and centrosomes in hypoxic syncytial embryos. These findings identify both a novel cellular structure (the ooplasmic filaments) as well as a new localization pattern for Mps1 and Polo and demonstrate that hypoxia affects Polo localization in Drosophila.

Sec61α is required for dorsal closure during Drosophila embryogenesis through its regulation of Dpp signaling

PubMed Central

Wang, Xiaochen; Ward, Robert E.

2010-01-01

During dorsal closure in Drosophila, signaling events in the dorsalmost row of epidermal cells (DME cells) direct the migration of lateral epidermal sheets towards the dorsal midline where they fuse to enclose the embryo. A Jun amino-terminal kinase (JNK) cascade in the DME cells induces the expression of Decapentaplegic (Dpp). Dpp signaling then regulates the cytoskeleton in the DME cells and amnioserosa to affect the cell shape changes necessary to complete dorsal closure. We identified a mutation in Sec61α that specifically perturbs dorsal closure. Sec61α encodes the main subunit of the translocon complex for co-translational import of proteins into the ER. JNK signaling is normal in Sec61α mutant embryos, but Dpp signaling is attenuated and the DME cells fail to maintain an actinomyosin cable as epithelial migration fails. Consistent with this model, dorsal closure is rescued in Sec61α mutant embryos by an activated form of the Dpp receptor Thick veins. PMID:20112345

Identification of early zygotic genes in the yellow fever mosquito Aedes aegypti and discovery of a motif involved in early zygotic genome activation.

PubMed

Biedler, James K; Hu, Wanqi; Tae, Hongseok; Tu, Zhijian

2012-01-01

During early embryogenesis the zygotic genome is transcriptionally silent and all mRNAs present are of maternal origin. The maternal-zygotic transition marks the time over which embryogenesis changes its dependence from maternal RNAs to zygotically transcribed RNAs. Here we present the first systematic investigation of early zygotic genes (EZGs) in a mosquito species and focus on genes involved in the onset of transcription during 2-4 hr. We used transcriptome sequencing to identify the "pure" (without maternal expression) EZGs by analyzing transcripts from four embryonic time ranges of 0-2, 2-4, 4-8, and 8-12 hr, which includes the time of cellular blastoderm formation and up to the start of gastrulation. Blast of 16,789 annotated transcripts vs. the transcriptome reads revealed evidence for 63 (P<0.001) and 143 (P<0.05) nonmaternally derived transcripts having a significant increase in expression at 2-4 hr. One third of the 63 EZG transcripts do not have predicted introns compared to 10% of all Ae. aegypti genes. We have confirmed by RT-PCR that zygotic transcription starts as early as 2-3 hours. A degenerate motif VBRGGTA was found to be overrepresented in the upstream sequences of the identified EZGs using a motif identification software called SCOPE. We find evidence for homology between this motif and the TAGteam motif found in Drosophila that has been implicated in EZG activation. A 38 bp sequence in the proximal upstream sequence of a kinesin light chain EZG (KLC2.1) contains two copies of the mosquito motif. This sequence was shown to support EZG transcription by luciferase reporter assays performed on injected early embryos, and confers early zygotic activity to a heterologous promoter from a divergent mosquito species. The results of these studies are consistent with the model of early zygotic genome activation via transcriptional activators, similar to what has been found recently in Drosophila.

Identification of ER proteins involved in the functional organisation of the early secretory pathway in Drosophila cells by a targeted RNAi screen.

PubMed

Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

2011-02-23

In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for "more and smaller Golgi") upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation.

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila.

PubMed

Ji, Yingbiao; Tulin, Alexei V

2016-10-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3' untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3' UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3' UTR, increasing the translation in vivo and in vitro hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila

PubMed Central

Ji, Yingbiao

2016-01-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3′ untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3′ UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3′ UTR, increasing the translation in vivo and in vitro. hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. PMID:27402862

Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos.

PubMed

Karnowski, Karol; Ajduk, Anna; Wieloch, Bartosz; Tamborski, Szymon; Krawiec, Krzysztof; Wojtkowski, Maciej; Szkulmowski, Maciej

2017-06-23

Imaging of living cells based on traditional fluorescence and confocal laser scanning microscopy has delivered an enormous amount of information critical for understanding biological processes in single cells. However, the requirement for a high numerical aperture and fluorescent markers still limits researchers' ability to visualize the cellular architecture without causing short- and long-term photodamage. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of fluorescence imaging techniques and provides unique access to fundamental aspects of early embryonic development, without the requirement for sample pre-processing or labeling. In the present paper, we utilized the internal motion of cytoplasm, as well as custom scanning and signal processing protocols, to effectively reduce the speckle noise typical for standard OCM and enable high-resolution intracellular time-lapse imaging. To test our imaging system we used mouse and pig oocytes and embryos and visualized them through fertilization and the first embryonic division, as well as at selected stages of oogenesis and preimplantation development. Because all morphological and morphokinetic properties recorded by OCM are believed to be biomarkers of oocyte/embryo quality, OCM may represent a new chapter in imaging-based preimplantation embryo diagnostics.

Glucocorticoid teratogenesis in mouse whole embryo culture.

PubMed

Pratt, R M; Perry, E L; Chapman, L M; Goulding, E H

1984-08-01

Glucocorticoids, such as triamcinolone acetonide (TAC-A) and triamcinolone hexacetonide (TAC-HA), are potent inducers of cleft palate in vivo in various mouse strains when administered on day 11 of gestation, whereas they are poor or ineffective inducers of cleft lip when given on day 7. The purpose of the present study was to determine whether glucocorticoids are capable of interfering with early embryonic development in culture. CD-1 mouse embryos were cultured for 48 hours starting either on day 8 (plug day 0) with the embryo inside the yolk sac, or on day 10 with the embryo exteriorized from its functional yolk sac. At the end of the culture period, embryos were examined grossly for malformations and biochemically for altered DNA and protein levels. With the day 8 cultures, TAC-A produced a dose-dependent inhibition of growth along with malformations consisting of cardiac irregularities, abnormal rotation, and irregular neural tube closure. With the day 10 cultures, these malformations were not observed, presumably due to the advanced stage of development when the embryos were exposed to TAC-A; however, TAC-A did produce growth inhibition along with cleft lip. When TAC-HA was administered in vivo to pregnant donor females on day 7, in combination with TAC-A added on day 10 to the culture medium, there was a dramatic increase in the frequency of cleft lip along with other alterations in craniofacial appearance. Our results demonstrate that glucocorticoids are capable of directly affecting embryonic growth and development during the early stages of organogenesis.

Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program.

PubMed

Herrera, C; Morikawa, M I; Bello, M B; von Meyeren, M; Centeno, J Eusebio; Dufourq, P; Martinez, M M; Llorente, J

2014-03-15

Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300-1000, and >1000 μm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7-10 hours and 63% for 1-2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the

Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

NASA Astrophysics Data System (ADS)

Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

2015-11-01

Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

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

Drosophila Polypyrimidine Tract-Binding Protein (DmPTB) Regulates Dorso-Ventral Patterning Genes in Embryos

PubMed Central

Huntley, Jim; Wesley, Cedric S.; Singh, Ravinder

2014-01-01

The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during embryogenesis. A loss of function mutation, heph03429, results in varied defects in embryonic developmental processes, leading to embryonic lethality. However, the suite of molecular functions that are disrupted in the mutant remains unknown. We have used an unbiased high throughput sequencing approach to identify transcripts that are misregulated in this mutant. Misregulated transcripts show evidence of significantly altered patterns of splicing (exon skipping, 5′ and 3′ splice site switching), alternative 5′ ends, and mRNA level changes (up and down regulation). These findings are independently supported by reverse-transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridization. We show that a group of genes, such as Zerknüllt, z600 and screw are among the most upregulated in the mutant and have been functionally linked to dorso-ventral patterning and/or dorsal closure processes. Thus, loss of dmPTB function results in specific misregulated transcripts, including those that provide the missing link between the loss of dmPTB function and observed developmental defects in embryogenesis. This study provides the first comprehensive repertoire of genes affected in vivo in the heph mutant in Drosophila and offers insight into the role of dmPTB during embryonic development. PMID:25014769

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

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

A Vertex Model of Drosophila Ventral Furrow Formation

PubMed Central

Spahn, Philipp; Reuter, Rolf

2013-01-01

Ventral furrow formation in Drosophila is an outstanding model system to study the mechanisms involved in large-scale tissue rearrangements. Ventral cells accumulate myosin at their apical sides and, while being tightly coupled to each other via apical adherens junctions, execute actomyosin contractions that lead to reduction of their apical cell surface. Thereby, a band of constricted cells along the ventral epithelium emerges which will form a tissue indentation along the ventral midline (the ventral furrow). Here we adopt a 2D vertex model to simulate ventral furrow formation in a surface view allowing easy comparison with confocal live-recordings. We show that in order to reproduce furrow morphology seen in vivo, a gradient of contractility must be assumed in the ventral epithelium which renders cells more contractile the closer they lie to the ventral midline. The model predicts previous experimental findings, such as the gain of eccentric morphology of constricting cells and an incremental fashion of apical cell area reduction. Analysis of the model suggests that this incremental area reduction is caused by the dynamical interplay of cell elasticity and stochastic contractility as well as by the opposing forces from contracting neighbour cells. We underpin results from the model through in vivo analysis of ventral furrow formation in wildtype and twi mutant embryos. Our results show that ventral furrow formation can be accomplished as a “tug-of-war” between stochastically contracting, mechanically coupled cells and may require less rigorous regulation than previously thought. Summary For the developmental biologist it is a fascinating question how cells can coordinate major tissue movements during embryonic development. The so-called ventral furrow of the Drosophila embryo is a well-studied example of such a process when cells from a ventral band, spanning nearly the entire length of the embryo, undergo dramatic shape change by contracting their tips and

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

PubMed

Morel, Alexandre; Teyssier, Caroline; Trontin, Jean-François; Eliášová, Kateřina; Pešek, Bedřich; Beaufour, Martine; Morabito, Domenico; Boizot, Nathalie; Le Metté, Claire; Belal-Bessai, Leila; Reymond, Isabelle; Harvengt, Luc; Cadene, Martine; Corbineau, Françoise; Vágner, Martin; Label, Philippe; Lelu-Walter, Marie-Anne

2014-09-01

Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets. © 2014 Scandinavian Plant Physiology Society.

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.

Undernutrition affects embryo quality of superovulated ewes.

PubMed

Abecia, J A; Forcada, F; Palacín, I; Sánchez-Prieto, L; Sosa, C; Fernández-Foren, A; Meikle, A

2015-02-01

To determine the effect of undernutrition on embryo production and quality in superovulated sheep, 45 ewes were allocated into two groups to be fed diets that provided 1.5 (control, C; n = 20) or 0.5 (low nutrition, L; n = 25) times daily requirements for maintenance, from oestrous synchronization with intravaginal sponges to embryo collection. Embryos were collected 7 days after the onset of oestrus (day 0). Low nutrition resulted in lower live weight and body condition at embryo collection (P < 0.05). Diet (P < 0.01) and day of sampling (P < 0.001) significantly affected plasma non-esterified fatty acid (NEFA) and insulin concentrations. Plasma leptin concentrations decreased on day 7 only in L ewes. A significant effect of dietary treatment (P < 0.05) and day (P < 0.0001) was observed on plasma insulin-like growth factor (IGF)-I concentrations. The number of recovered oocytes and embryos did not differ between the groups (L: 15.4 ± 0.4; C: 12.4 ± 0.4). Recovery rate was lower (P < 0.05) in the L (60%) than in the C group (73%). The total number of embryos and number of viable-transferable embryos (5.0 ± 0.3 and 3.4 ± 0.3 embryos, respectively) of the L group were lower (P < 0.1) when compared with controls (8.4 ± 0.4 and 6.2 ± 0.4 embryos, respectively). Undernutrition during the period of superovulation and early embryonic development reduced total and viable number of embryos. These effects might be mediated by disruption of endocrine homeostasis, oviduct environment and/or oocyte quality.

Experimenting with embryos: can philosophy help?

PubMed

Heyd, David

1996-10-01

Beyond the well-known ethical issues involved in medical experimentation on human subjects, experimenting with embryos raises unique and particularly hard problems. Besides the psychological obstacles connected with the fear of "playing God" and the awe with which we hold the process of the creation of human beings, there are three philosophical problems which are the main subject of the article: 1. The logical problem of circularity: the morality of experimenting on embryos is dependent on the status of the embryo, which in turn is partly decided by experimentation; 2. The metaphysical problem: experiments are justified by the benefits they bring to human subjects; but it is doubtful whether an early embryo is a "subject" and whether coming into being is a "benefit"; 3. The moral problem: the standard constraint on medical experiments is that they benefit either the individual subject or at least members of a relevantly defined group of patients suffering from the same syndrome. But embryo experimentation is often associated with potential cure to people of a completely different category (like geriatric patients). Finally, the article discusses the limits of the force of philosophical arguments in the formation of actual policies for regulating such practices as experimenting with embryos. The widely-shared fourteen-day limit is shown to be a sound practical compromise despite the difficulties in justifying it philosophically.

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.

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells.

PubMed

Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

2017-08-01

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells

PubMed Central

Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

2017-01-01

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA

Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

PubMed

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2012-11-01

Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

A method of microinjection: delivering monoclonal antibody 1223 into sea urchin embryos.

PubMed

Cho, J W

1999-08-31

In this paper, a simpler method of microinjecting sea urchin embryos without using the conventional microinjection chamber designed by Kiehart is reported. A trough was made on a surface of 0.6% agarose gel dissolved in artificial sea water. Approximately fifty hatched embryos could be loaded in the trough and, consequently, swimming embryos were trapped in the trough. Monoclonal antibody (mAB) 1223 which blocks spiculogenesis in vitro was delivered into the blastocoels of sea urchin embryos to test whether this antibody inhibits spiculogenesis in vivo and also, whether this new technique is effective for the microinjection of the sea urchin embryos. The embryos were injected with mAB1223 at the hatched blastula, early mesenchyme blastula and early gastrula stages, and 63%, 90% and 97% of the embryos did not form spicules at the late gastrula stage, respectively. Therefore, mAB1223 was shown to also block spiculogenesis in vivo. From the fact that spiculogenesis occurred at a lower rate when mAB1223 was injected at the hatched blastula stage than at later stages, it may be speculated that endogenous proteases degraded the injected antibodies. Using this technique, extracellular events in the blastocoel or the function of certain molecules expressed in blastocoel can be easily investigated in vivo.

A Drosophila receptor tyrosine phosphatase expressed in the embryonic CNS and larval optic lobes is a member of the set of proteins bearing the "HRP" carbohydrate epitope.

PubMed

Desai, C J; Popova, E; Zinn, K

1994-12-01

Recent studies have defined several cell surface glycoproteins expressed in the developing nervous system of insect embryos that may be involved in axon outgrowth and guidance processes. These glycoproteins include the fasciclins and a group of receptor-linked protein tyrosine phosphatases (R-PTPs). In embryos, the fasciclins are localized to axonal subsets, while the R-PTPs appear to be expressed on most or all CNS axons. To identify other neuronal cell surface glycoproteins in the Drosophila embryo, we have taken a biochemical approach. This is based on the observation that antisera against horseradish peroxidase (HRP) recognize a carbohydrate epitope that is selectively expressed in the insect nervous system. A large number of neuronal glycoproteins (denoted "HRP proteins") apparently bear the HRP carbohydrate epitope. We have used polyclonal anti-HRP antibodies to purify these proteins from Drosophila embryos, and have obtained protein sequences from seven HRP protein bands. These data define three major HRP proteins as neurotactin, fasciclin I, and an R-PTP, DPTP69D. Western blotting data suggest that fasciclin II, neuroglian, DPTP10D, and DPTP99A are also HRP proteins. We show that DPTP69D, like the previously characterized R-PTPs, is localized to CNS axons in the embryo. In third instar larvae, DPTP69D expression is restricted to subsets of neuronal processes in the brain, ventral nerve cord, and eye disk. In the optic lobes, DPTP69D is localized to the neuropils of the lamina and medulla, and to an array of parallel thick bundles that may be the transmedullary fibers of the developing lobula complex.

Hydroxyurea-mediated neuroblast ablation establishes birthdates of secondary lineages and addresses neuronal interactions in the developing Drosophila brain

PubMed Central

Lovick, Jennifer K.; Hartenstein, Volker

2015-01-01

The Drosophila brain is comprised of neurons formed by approximately 100 lineages, each of which is derived from a stereotyped, asymmetrically dividing neuroblast. Lineages serve as structural and developmental units of Drosophila brain anatomy and reconstruction of lineage projection patterns represents a suitable map of Drosophila brain circuitry at the level of neuron populations (“macro-circuitry”). Two phases of neuroblast proliferation, the first in the embryo and the second during the larval phase (following a period of mitotic quiescence), produce primary and secondary lineages, respectively. Using temporally controlled pulses of hydroxyurea (HU) to ablate neuroblasts and their corresponding secondary lineages during the larval phase, we analyzed the effect on development of primary and secondary lineages in the late larval and adult brain. Our findings indicate that timing of neuroblast re-activation is highly stereotyped, allowing us to establish “birth dates” for all secondary lineages. Furthermore, our results demonstrate that, whereas the trajectory and projection pattern of primary and secondary lineages is established in a largely independent manner, the final branching pattern of secondary neurons is dependent upon the presence of appropriate neuronal targets. Taken together, our data provide new insights into the degree of neuronal plasticity during Drosophila brain development. PMID:25773365

ME31B globally represses maternal mRNAs by two distinct mechanisms during the Drosophila maternal-to-zygotic transition.

PubMed

Wang, Miranda; Ly, Michael; Lugowski, Andrew; Laver, John D; Lipshitz, Howard D; Smibert, Craig A; Rissland, Olivia S

2017-09-06

In animal embryos, control of development is passed from exclusively maternal gene products to those encoded by the embryonic genome in a process referred to as the maternal-to-zygotic transition (MZT). We show that the RNA-binding protein, ME31B, binds to and represses the expression of thousands of maternal mRNAs during the Drosophila MZT. However, ME31B carries out repression in different ways during different phases of the MZT. Early, it represses translation while, later, its binding leads to mRNA destruction, most likely as a consequence of translational repression in the context of robust mRNA decay. In a process dependent on the PNG kinase, levels of ME31B and its partners, Cup and Trailer Hitch (TRAL), decrease by over 10-fold during the MZT, leading to a change in the composition of mRNA-protein complexes. We propose that ME31B is a global repressor whose regulatory impact changes based on its biological context.

The sex of specific neurons controls female body growth in Drosophila.

PubMed

Sawala, Annick; Gould, Alex P

2017-10-01

Sexual dimorphisms in body size are widespread throughout the animal kingdom but their underlying mechanisms are not well characterized. Most models for how sex chromosome genes specify size dimorphism have emphasized the importance of gonadal hormones and cell-autonomous influences in mammals versus strictly cell-autonomous mechanisms in Drosophila melanogaster. Here, we use tissue-specific genetics to investigate how sexual size dimorphism (SSD) is established in Drosophila. We find that the larger body size characteristic of Drosophila females is established very early in larval development via an increase in the growth rate per unit of body mass. We demonstrate that the female sex determination gene, Sex-lethal (Sxl), functions in central nervous system (CNS) neurons as part of a relay that specifies the early sex-specific growth trajectories of larval but not imaginal tissues. Neuronal Sxl acts additively in 2 neuronal subpopulations, one of which corresponds to 7 median neurosecretory cells: the insulin-producing cells (IPCs). Surprisingly, however, male-female differences in the production of insulin-like peptides (Ilps) from the IPCs do not appear to be involved in establishing SSD in early larvae, although they may play a later role. These findings support a relay model in which Sxl in neurons and Sxl in local tissues act together to specify the female-specific growth of the larval body. They also reveal that, even though the sex determination pathways in Drosophila and mammals are different, they both modulate body growth via a combination of tissue-autonomous and nonautonomous inputs.

The sex of specific neurons controls female body growth in Drosophila

PubMed Central

Sawala, Annick

2017-01-01

Sexual dimorphisms in body size are widespread throughout the animal kingdom but their underlying mechanisms are not well characterized. Most models for how sex chromosome genes specify size dimorphism have emphasized the importance of gonadal hormones and cell-autonomous influences in mammals versus strictly cell-autonomous mechanisms in Drosophila melanogaster. Here, we use tissue-specific genetics to investigate how sexual size dimorphism (SSD) is established in Drosophila. We find that the larger body size characteristic of Drosophila females is established very early in larval development via an increase in the growth rate per unit of body mass. We demonstrate that the female sex determination gene, Sex-lethal (Sxl), functions in central nervous system (CNS) neurons as part of a relay that specifies the early sex-specific growth trajectories of larval but not imaginal tissues. Neuronal Sxl acts additively in 2 neuronal subpopulations, one of which corresponds to 7 median neurosecretory cells: the insulin-producing cells (IPCs). Surprisingly, however, male-female differences in the production of insulin-like peptides (Ilps) from the IPCs do not appear to be involved in establishing SSD in early larvae, although they may play a later role. These findings support a relay model in which Sxl in neurons and Sxl in local tissues act together to specify the female-specific growth of the larval body. They also reveal that, even though the sex determination pathways in Drosophila and mammals are different, they both modulate body growth via a combination of tissue-autonomous and nonautonomous inputs. PMID:28976974

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.

Drosophila segmentation: supercomputer simulation of prepattern hierarchy.

PubMed

Hunding, A; Kauffman, S A; Goodwin, B C

1990-08-09

Spontaneous prepattern formation in a two level hierarchy of reaction-diffusion systems is simulated in three space co-ordinates and time, mimicking gap gene and primary pair-rule gene expression. The model rests on the idea of Turing systems of the second kind, in which one prepattern generates position dependent rate constants for a subsequent reaction-diffusion system. Maternal genes are assumed responsible for setting up gradients from the anterior and posterior ends, one of which is needed to stabilize a double period prepattern suggested to underly the read out of the gap genes. The resulting double period pattern in turn stabilizes the next prepattern in the hierarchy, which has a short wavelength with many characteristics of the stripes seen in actual primary pair-rule gene expression. Without such hierarchical stabilization, reaction-diffusion mechanisms yield highly patchy short wave length patterns, and thus unreliable stripes. The model yields seven stable stripes located in the middle of the embryo, with the potential for additional expression near the poles, as observed experimentally. The model does not rely on specific chemical reaction kinetics, rather the effect is general to many such kinetic schemes. This makes it robust to parameter changes, and it has good potential for adapting to size and shape changes as well. The study thus suggests that the crucial organizing principle in early Drosophila embryogenesis is based on global field mechanisms, not on particular local interactions.

In vitro and in vivo effects of ulipristal acetate on fertilization and early embryo development in mice.

PubMed

Gómez-Elías, Matías D; Munuce, María J; Bahamondes, Luis; Cuasnicú, Patricia S; Cohen, Débora J

2016-01-01

Does ulipristal acetate (UPA), a selective progesterone receptor modulator used for emergency contraception (EC), interfere with fertilization or early embryo development in vitro and in vivo? At doses similar to those used for EC, UPA does not affect mouse gamete transport, fertilization or embryo development. UPA acts as an emergency contraceptive mainly by inhibiting or delaying ovulation. However, there is little information regarding its effects on post-ovulatory events preceding implantation. This was an in vitro and in vivo experimental study involving the use of mouse gametes and embryos from at least three animals in each set of experiments. For in vitro fertilization experiments, mouse epididymal spermatozoa capacitated in the presence of different concentrations of UPA (0-1000 ng/ml) were used to inseminate cumulus-intact or cumulus-free eggs in the presence or absence of UPA during gamete co-incubation, and the percentage of fertilized eggs was determined. For in vivo fertilization experiments, superovulated females caged with proven fertile males were injected with UPA (40 mg/kg) or vehicle just before or just after mating and the percentage of fertilized eggs recovered from the ampulla was determined. To investigate the effect of UPA on embryo development, zygotes were recovered from mated females, cultured in the presence of UPA (1000 ng/ml) for 4 days and the progression of embryo development was monitored daily. In vitro studies revealed that the presence of UPA during capacitation and/or gamete co-incubation does not affect fertilization. Whereas the in vivo administration of UPA at the same time as hCG injection produced a decrease in the number of eggs ovulated compared with controls (vehicle injected animals, P < 0.05), no effects on fertilization were observed when UPA was administered shortly before or after mating. No differences were observed in either the percentage of cleaved embryos or the cleavage speed when UPA was present during in

Drosophila melanogaster, a genetic model system for alcohol research.

PubMed

Guarnieri, Douglas J; Heberlein, Ulrike

2003-01-01

In its natural environment, which consists of fermenting plant materials, the fruit fly Drosophila melanogaster encounters high levels of ethanol. Flies are well equipped to deal with the toxic effects of ethanol; they use it as an energy source and for lipid biosynthesis. The primary ethanol-metabolizing pathway in flies involves the enzymes alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH); their role in adaptation to ethanol-rich environments has been studied extensively. The similarity between Drosophila and mammals is not restricted to the manner in which they metabolize ethanol; behaviors elicited by ethanol exposure are also remarkably similar in these organisms. Flies show signs of acute intoxication, which range from locomotor stimulation at low doses to complete sedation at higher doses, they develop tolerance upon intermittent ethanol exposure, and they appear to like ethanol, showing preference for ethanol-containing media. Molecular genetic analysis of ethanol-induced behaviors in Drosophila, while still in its early stages, has already revealed some surprising parallels with mammals. The availability of powerful tools for genetic manipulation in Drosophila, together with the high degree of conservation at the genomic level, make Drosophila a promising model organism to study the mechanism by which ethanol regulates behavior and the mechanisms underlying the organism's adaptation to long-term ethanol exposure.

Effect of progesterone supplementation in the first week post conception on embryo survival in beef heifers.

PubMed

Beltman, M E; Lonergan, P; Diskin, M G; Roche, J F; Crowe, M A

2009-04-15

Progesterone is essential for establishment and maintenance of pregnancy in mammals. The objective of this study was to examine the effect of elevating progesterone during the different physiological stages of early embryo development on embryo survival. Estrus was synchronized in cross-bred beef heifers (n=197, approximately 2-years old) and they were inseminated 12-18h after estrus onset (=Day 0). Inseminated heifers were randomly assigned to 1 of 3 treatments: (1) Control, n=69; (2) progesterone supplementation using a Controlled Internal Drug Release Device (CIDR) from Day 3 to 6.5, n=64; or (3) progesterone supplementation using a CIDR from Day 4.5 to 8, n=64. Body condition (BCS) and locomotion scores (scale of 1-5) were recorded for all animals. Animals with a locomotion score >/=4 (very lame) were excluded. Embryo survival rate was determined at slaughter on Day 25. Conceptus length and weight were recorded and the corpus luteum (CL) of all pregnant animals was dissected and weighed. Supplementation with exogenous progesterone increased (P<0.05) peripheral progesterone concentrations, but did not affect embryo survival rate compared with controls. Mean CL weight, conceptus length and conceptus weight were not different between treatments. There was a positive relationship (P<0.04) between the increase in progesterone concentrations from Days 3 to 6.5 and embryo survival rate in treated heifers and a similar trend existed between the increase from Days 4.5 to 8 (P<0.06). There was also a positive relationship (P<0.05) between the progesterone concentration on Day 6.5 and the embryo survival rate in treated heifers. A direct correlation was seen between locomotion score and embryo survival rate, with higher (P<0.05) early embryo survival rates in heifers with a lower locomotion score. In conclusion, supplementation with progesterone at different stages of early embryo development increased peripheral progesterone concentration and resulted in a positive

Analysis of global gene expression profiles to identify differentially expressed genes critical for embryo development in Brassica rapa.

PubMed

Zhang, Yu; Peng, Lifang; Wu, Ya; Shen, Yanyue; Wu, Xiaoming; Wang, Jianbo

2014-11-01

Embryo development represents a crucial developmental period in the life cycle of flowering plants. To gain insights into the genetic programs that control embryo development in Brassica rapa L., RNA sequencing technology was used to perform transcriptome profiling analysis of B. rapa developing embryos. The results generated 42,906,229 sequence reads aligned with 32,941 genes. In total, 27,760, 28,871, 28,384, and 25,653 genes were identified from embryos at globular, heart, early cotyledon, and mature developmental stages, respectively, and analysis between stages revealed a subset of stage-specific genes. We next investigated 9,884 differentially expressed genes with more than fivefold changes in expression and false discovery rate ≤ 0.001 from three adjacent-stage comparisons; 1,514, 3,831, and 6,633 genes were detected between globular and heart stage embryo libraries, heart stage and early cotyledon stage, and early cotyledon and mature stage, respectively. Large numbers of genes related to cellular process, metabolism process, response to stimulus, and biological process were expressed during the early and middle stages of embryo development. Fatty acid biosynthesis, biosynthesis of secondary metabolites, and photosynthesis-related genes were expressed predominantly in embryos at the middle stage. Genes for lipid metabolism and storage proteins were highly expressed in the middle and late stages of embryo development. We also identified 911 transcription factor genes that show differential expression across embryo developmental stages. These results increase our understanding of the complex molecular and cellular events during embryo development in B. rapa and provide a foundation for future studies on other oilseed crops.

The current status and future of commercial embryo transfer in cattle.

PubMed

Hasler, John F

2003-12-15

A commercially viable cattle embryo transfer (ET) industry was established in North America during the early 1970s, approximately 80 years after the first successful embryo transfer was reported in a mammal. Initially, techniques for recovering and transferring cattle embryos were exclusively surgical. However, by the late 1970s, most embryos were recovered and transferred nonsurgically. Successful cryopreservation of embryos was widespread by the early 1980s, followed by the introduction of embryo splitting, in vitro procedures, direct transfer of frozen embryos and sexing of embryos. The wide spread adoption of ethylene glycol as a cryoprotectant has simplified the thaw-transfer procedures for frozen embryos. The number of embryos recovered annually has not grown appreciably over the last 10 years in North America and Europe; however, there has been significant growth of commercial ET in South America. Within North America, ET activity has been relatively constant in Holstein cattle, whereas there has been a large ET increase in the Angus breed and a concomitant ET decrease in some other beef breeds. Although a number of new technologies have been adopted within the ET industry in the last decade, the basic procedure of superovulation of donor cattle has undergone little improvement over the last 20 years. The export-import of frozen cattle embryos has become a well-established industry, governed by specific health regulations. The international movement of embryos is subject to sudden and dramatic disturbances, as exemplified by the 2001 outbreak of foot and mouth disease in Great Britain. It is probable that there will be an increased influence of animal rights issues on the ET industry in the future. Several companies in North America are currently commercially producing cloned cattle. The sexing of bovine semen with the use of flow cytometry is extremely accurate and moderate pregnancy rates in heifers have been achieved in field trials, but sexed semen

Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

PubMed Central

Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

2011-01-01

Background In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. PMID:21383842

Identification and characterization of pyrokinin and CAPA peptides, and corresponding GPCRs from spotted wing drosophila, Drosophila suzukii.

PubMed

Choi, Man-Yeon; Ahn, Seung-Joon; Kim, A Young; Koh, Youngho

2017-05-15

The family of FXPRLamide peptides serves as a major insect hormone. It is characterized by a core active amino acid sequence conserved at the C-terminal ends, and provides various physiological roles across the Insecta. In this study we identified and characterized pyrokinin (PK) and CAPA cDNAs encoding two FXPRLamide peptides, pyrokinin and CAPA-DH (diapause hormone), and two corresponding G protein-coupled receptors (GPCRs) from spotted wing drosophila (SWD), Drosophila suzukii. Expressions of PK and CAPA mRNAs were differentially observed during all life stages except the embryo, and the detection of CAPA transcription was relatively strong compared with the PK gene in SWD. Both D. suzukii pyrokinin receptor (DrosuPKr) and CAPA-DH receptor (DrosuCAPA-DHr) were functionally expressed and confirmed through binding to PK and DH peptides. Differential expression of two GPCRs occurred during all life stages; a strong transcription of DrosuPKr was observed in the 3rd instar. DrosuCAPA-DHr was clearly expressed from the embryo to the larva, but not detected in the adult. Gene regulation during the life stages was not synchronized between ligand and receptor. For example, SWD CAPA mRNA has been up-regulated in the adult while CAPA-DHr was down-regulated. The difference could be from the CAPA mRNA translating multiple peptides including CAPA-DH and two CAPA-PVK (periviscerokinin) peptides to act on different receptors. Comparing the genes of SWD PK, CAPA, PKr and CAPA-DHr to four corresponding genes of D. melanogaster, SWD CAPA and the receptor are more similar to D. melanogaster than PK and the receptor. These data suggest that the CAPA gene could be evolutionally more conserved to have a common biological role in insects. In addition, the effect of Kozak sequences was investigated by the expression of the GPCRs with or without Kozak sequences in Sf9 insect cells. The Kozak sequenced PK receptor was significantly less active than the original (= no Kozak sequenced

Fossilized embryos are widespread but the record is temporally and taxonomically biased

USGS Publications Warehouse

Donoghue, P.C.J.; Kouchinsky, A.; Waloszek, Dieter; Bengtson, S.; Dong, X.-P.; Val'Kov, A.K.; Cunningham, J.A.; Repetski, J.E.

2006-01-01

We report new discoveries of embryos and egg capsules from the Lower Cambrian of Siberia, Middle Cambrian of Australia and Lower Ordovician of North America. Together with existing records, embryos have now been recorded from four of the seven continents. However, the new discoveries highlight secular and systematic biases in the fossil record of embryonic stages. The temporal window within which the embryos and egg capsules are found is of relatively short duration; it ends in the Early Ordovician and is roughly coincident with that of typical "Orsten"-type faunas. The reduced occurrence of such fossils has been attributed to reducing levels of phosphate in marine waters during the early Paleozoic, but may also be owing to the increasing depth of sediment mixing by infaunal metazoans. Furthermore, most records younger than the earliest Cambrian are of a single kind - large eggs and embryos of the priapulid-like scalidophoran Markuelia. We explore alternative explanations for the low taxonomic diversity of embryos recovered thus far, including sampling, size, anatomy, ecology, and environment, concluding that the preponderance of Markuelia embryos is due to its precocious development of cuticle at an embryonic stage, predisposing it to preservation through action as a substrate on which microbially mediated precipitation of authigenic calcium phosphate may occur. The fossil record of embryos may be limited to a late Neoproterozoic to early Ordovician snapshot that is subject to dramatic systematic bias. Together, these biases must be considered seriously in attempts to use the fossil record to arbitrate between hypotheses of developmental and life history evolution implicated in the origin of metazoan clades. ?? 2006 Blackwell Publishing Ltd.

Cell death during Drosophila melanogaster early oogenesis is mediated through autophagy.

PubMed

Nezis, Ioannis P; Lamark, Trond; Velentzas, Athanassios D; Rusten, Tor Erik; Bjørkøy, Geir; Johansen, Terje; Papassideri, Issidora S; Stravopodis, Dimitrios J; Margaritis, Lukas H; Stenmark, Harald; Brech, Andreas

2009-04-01

Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. Herein, we report that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila melanogaster. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atg1 or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development.

A potential determinant role of adiponectin and receptors for the early embryo development in PCOS patients with obesity hinted by quantitative profiling.

PubMed

Zhang, Ning; Hao, Cuifang; Liu, Xiaoyan; Zhang, Shouxin; Zhang, Fengrong; Zhuang, Lili; Zhao, Dongmei

2017-02-01

To identify the quantitative profiling of adiponectin and its receptors (AdipoR1, AdipoR2, and T-cadherin) in cumulus cells (CCs) and to evaluate their roles in the early embryo development of polycystic ovary syndrome (PCOS) patients, in part, with obesity. Fifty-five subjects were divided into two groups according to the body mass index. Oocytes were further inseminated and only mature and normal fertilized oocytes (2PN) were included in this research. Real-time PCR and western blot were performed to identify adiponectin and its receptors in CCs. Adiponectin and receptors were ubiquitously expressed in CCs of PCOS and non-PCOS patients. The level of AdipoR2 in CCs from the oocytes yielding blastocyst after 5/6 days in vitro culture was markedly higher than in those from oocytes could not develop to blastocyst stage after Day 6, for non-obese or obese PCOS patients (0.1647 ± 0.0161 versus 0.0783 ± 0.0385, 0.1948 ± 0.0307 versus 0.1057 ± 0.0236, respectively, p < 0.05). In addition, only in patients with PCOS and concurrent obesity the AdipoR1 in CCs was considerably increased in CC-B + compared with CC-B - subgroup (0.5162 ± 0.0371 versus 0.2448 ± 0.0333, p < 0.01). The development of early embryo was associated with the up-regulation of AdipoR1 and AdipoR2 in PCOS patients. Our results suggested that adiponectin could positively modulate embryo development in humans. Further investigations should be carried out to unlock the crucial role that adiponectin plays in embryo development.

Nucleus positioning within Drosophila egg chamber.

PubMed

Bernard, Fred; Lepesant, Jean-Antoine; Guichet, Antoine

2017-10-19

Both types of Drosophila egg chamber germ cells, i.e. oocyte and nurse cells, have to control their nucleus positions in order to produce a viable gamete. Interestingly, while actin microfilaments are crucial to position the nuclei in nurse cells, these are the microtubules that are important for oocyte nucleus to migrate and adopt the correct position. In this review, we discuss the mechanisms underlying these positioning processes in the two cell types with respect to the organization and dynamics of the actin and microtubule skeleton. In the nurse cells it is essential to keep firmly the nuclei in a central position to prevent them from obstructing the ring canals when the cytoplasmic content of the cells is dumped into the oocyte cells toward the end of oogenesis. This is achieved by the assembly of thick filopodia-like actin cables anchored to the plasma membrane, which grow inwardly and eventually encase tightly the nuclei in a cage-like structure. In the oocyte, the migration at an early stage of oogenesis of the nucleus from a posterior location to an anchorage site at an asymmetric anterior position, is an essential step in the setting up of the dorsoventral polarity axis of the future embryo. This process is controlled by an interplay between MT networks that just start to be untangled. Although both mechanisms have evolved to fulfill cell-type specific cell processes in the context of fly oogenesis, interesting parallels can be drawn with other nuclear positioning mechanisms in the mouse oocyte and the developing muscle respectively. Copyright © 2017. Published by Elsevier Ltd.

Early somatic embryo induction events in alfalfa callus cultures. [Medicago sativa

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

El-Bakry, A.A.; Hildebrand, D.F.

1987-04-01

High and low regenerating alfalfa Medicago sativa L. cv Regen S full sibs were isolated from a callus culture screen on modified Blaydes medium. The average number of embryos per ovary were thirty and zero for the high and low genotypes respectively after six weeks in culture. Proembryonic cell masses (4-8 celled) were observed after 4-5 days in culture and maximum meristematic activity was at 6-7 days in culture, for the high regenerating genotypes. Well formed globular embryos, both epidermal and subepidermal in origin, were observed after 2 weeks is culture. Samples in culture for 3, 6 and 14 daysmore » from the high and low regenerating genotypes were radiolabeled in vivo with /sup 35/S-methionine and run both on one and two dimension gels. The results will be discussed in relation to differences in proteins between the high and low regenerating genotypes at the stage of maximum meristematic activity (day 6) and differences occurring relative to the appearance of globular stage embryos (day 14) will be presented.« less

Properties of Barley Seed Chitinases and Release of Embryo-Associated Isoforms during Early Stages of Imbibition 1

PubMed Central

Swegle, Mark; Kramer, Karl J.; Muthukrishnan, Subbaratnam

1992-01-01

Barley (Hordeum vulgare L.) seeds contain at least five proteins with chitinase (CH) activity. Two of these (CH1 and CH2) are found primarily in the aleurone and endosperm tissues, and the other three (CH3, CH4, and CH5) are enriched in the embryo. From the bran fraction, three of these CHs (CH1, CH2, and CH3) were purified to apparent homogeneity. These three CHs have apparent molecular masses of 27, 34, and 35 kilodaltons and isoelectric points of 9.3, 9.2, and 8.7, respectively. CH2 and CH3 have amino terminal sequences resembling a portion of the chitin-binding domain of lectins and other plant defense proteins. CH1 lacks this domain. All three CHs exhibit antifungal activity and inhibit the mycelial growth of some species of trichoderma and Fusarium in vitro. During the early period of imbibition by seeds, two of the embryo-associated CHs are selectively released into the surrounding aqueous medium. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:16668964

Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene.

PubMed

Schneider, Mary D; Bains, Anupinder K; Rajendra, T K; Dominski, Zbigniew; Matera, A Gregory; Simmonds, Andrew J

2010-11-01

MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.

A Baculovirus Immediate-Early Gene, ie1, Promoter Drives Efficient Expression of a Transgene in Both Drosophila melanogaster and Bombyx mori

PubMed Central

Masumoto, Mika; Ohde, Takahiro; Shiomi, Kunihiro; Yaginuma, Toshinobu; Niimi, Teruyuki

2012-01-01

Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, ie1, from the Bombyx mori nucleopolyhedrovirus (BmNPV) could be used to drive efficient transgene expression in a wide variety of insects. We used a piggyBac-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV ie1 promoter; we performed a detailed investigation of the promoter in transgene expression pattern in Drosophila melanogaster and in B. mori. Drosophila and Bombyx belong to different insect orders (Diptera and Lepidoptera, respectively); however, and to our surprise, ie1 promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole) in both insects. Furthermore, in both species, the ie1 promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous ie1 promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the ie1 promoter can be used as an efficient expression driver in a diverse range of insect species. PMID:23152896

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

Lethal effect of dehydroleucodine (DhL) on amphibian Bufo arenarum embryos.

PubMed

Moreno, Liliana Elizabeth; Juárez, Américo Osvaldo; Pelzer, Lilian Eugenia

2012-03-01

The dehydroleucodine is a sesquiterpene lactone isolated from Artemisia douglasiana Besser which is used in popular medicine. Toxicity tests using embryos of amphibian have been widely used in order to predict toxic effects of different compounds. However, to our knowledge, there are not studies focussed on the toxic effects of dehydroleucodine on Bufo arenarum, which is an anuran widely distributed in South America. The effect of dehydroleucodine on the survival of embryos was evaluated in an acute test during the early life stage of B. arenarum embryos. Lethality and the degree of adverse effects were dehydroleucodine dose-dependent. Overall, amphibian early life stages appeared to be more susceptible to the embryotoxicity associated with exposure to dehydroleucodine, especially at concentration greater that 3mM. This increased susceptibility may result from the relatively high rate of cellular differentiation and morphogenesis that occurs at this early stage of development. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo

NASA Technical Reports Server (NTRS)

Halfon, M. S.; Keshishian, H.

1998-01-01

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

Three-Dimensional Genome Organization and Function in Drosophila

PubMed Central

Schwartz, Yuri B.; Cavalli, Giacomo

2017-01-01

Understanding how the metazoan genome is used during development and cell differentiation is one of the major challenges in the postgenomic era. Early studies in Drosophila suggested that three-dimensional (3D) chromosome organization plays important regulatory roles in this process and recent technological advances started to reveal connections at the molecular level. Here we will consider general features of the architectural organization of the Drosophila genome, providing historical perspective and insights from recent work. We will compare the linear and spatial segmentation of the fly genome and focus on the two key regulators of genome architecture: insulator components and Polycomb group proteins. With its unique set of genetic tools and a compact, well annotated genome, Drosophila is poised to remain a model system of choice for rapid progress in understanding principles of genome organization and to serve as a proving ground for development of 3D genome-engineering techniques. PMID:28049701

Reversal of an ancient sex chromosome to an autosome in Drosophila.

PubMed

Vicoso, Beatriz; Bachtrog, Doris

2013-07-18

Although transitions of sex-determination mechanisms are frequent in species with homomorphic sex chromosomes, heteromorphic sex chromosomes are thought to represent a terminal evolutionary stage owing to chromosome-specific adaptations such as dosage compensation or an accumulation of sex-specific mutations. Here we show that an autosome of Drosophila, the dot chromosome, was ancestrally a differentiated X chromosome. We analyse the whole genome of true fruitflies (Tephritidae), flesh flies (Sarcophagidae) and soldier flies (Stratiomyidae) to show that genes located on the dot chromosome of Drosophila are X-linked in outgroup species, whereas Drosophila X-linked genes are autosomal. We date this chromosomal transition to early drosophilid evolution by sequencing the genome of other Drosophilidae. Our results reveal several puzzling aspects of Drosophila dot chromosome biology to be possible remnants of its former life as a sex chromosome, such as its minor feminizing role in sex determination or its targeting by a chromosome-specific regulatory mechanism. We also show that patterns of biased gene expression of the dot chromosome during early embryogenesis, oogenesis and spermatogenesis resemble that of the current X chromosome. Thus, although sex chromosomes are not necessarily evolutionary end points and can revert back to an autosomal inheritance, the highly specialized genome architecture of this former X chromosome suggests that severe fitness costs must be overcome for such a turnover to occur.

Developmental consequences of cryopreservation of mammalian oocytes and embryos.

PubMed

Smith, Gary D; Silva E Silva, Cristine Ane

2004-08-01

During the last three decades, significant advances have been made in successful cryopreservation of mammalian preimplantation embryos, and more recently oocytes. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte/embryo cryopreservation success has increased over time, there is still room for improvement. Oocytes and embryos are susceptible to cryo-damage, which collectively entails cellular damage caused by mechanical, chemical, or thermal forces during the freeze-thaw process. Basic studies focused on understanding cellular structures, their composition, and more importantly their functions, in normal cell developments will continue to be critical in assessing, understanding, and correcting oocyte/embryo cryo-damage. This review will delineate many of the oocyte/embryo intracellular and extracellular structures that are or may be compromised during cryopreservation. A global theme presented throughout this review is that many structural components of the oocyte/embryo also have essential functional roles in development. Compromising these cellular structures, and thus their cellular homeostatic functions, can deleteriously influence initial cryo-survival or compromise subsequent normal development through effects on the oocyte and/or early embryo.

Viability of bovine demi- and quarter-embryos after transfer.

PubMed

Bredbacka, P; Huhtinen, M; Aalto, J; Rainio, V

1992-07-01

The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6/13) for heifers and 33.3% (4/12) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8/13) for heifers and 8.3% (1/12) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo.

PubMed

Haillot, Emmanuel; Molina, Maria Dolores; Lapraz, François; Lepage, Thierry

2015-01-01

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo

PubMed Central

Haillot, Emmanuel; Molina, Maria Dolores; Lapraz, François; Lepage, Thierry

2015-01-01

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1

Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos.

PubMed

Takai, Y; Tsutsumi, O; Ikezuki, Y; Hiroi, H; Osuga, Y; Momoeda, M; Yano, T; Taketani, Y

2000-04-21

The effects of bisphenol A, a xenoestrogen widely used in industry and dentistry, were studied in early preimplantation mouse embryos. Two-cell mouse embryos were cultured with 100 pM to 100 microM bisphenol A with or without 100 nM tamoxifen and evaluated at 24-h intervals for their development to eight-cell and blastocyst stages. At 72 h, blastocysts were cultured for another 48 h without bisphenol A, and surface areas of trophoblast spread were measured. At 24 h, more embryos exposed to 3 nM bisphenol A than to controls had reached the eight-cell stage. At 48 h, more embryos exposed to 1 nM and 3 nM bisphenol A than to controls had become blastocysts. At 100 microM, bisphenol A decreased frequency of development to blastocysts. Tamoxifen counteracted both stimulatory and inhibitory effects of bisphenol A on blastocyst formation. Although bisphenol A did not alter blastocyst morphology or cell number, early exposure to 100 microM bisphenol A increased subsequent trophoblast areas. These findings suggest that bisphenol A may not only effect early embryonic development via estrogen receptors even at low, environmentally relevant doses, but also exert some late effects on subsequent development of these embryos. Copyright 2000 Academic Press.

Early miscarriage rate in lean polycystic ovary syndrome women after euploid embryo transfer - a matched-pair study.

PubMed

Luo, Lu; Gu, Fang; Jie, Huying; Ding, Chenhui; Zhao, Qiang; Wang, Qiong; Zhou, Canquan

2017-11-01

The early miscarriage rate is reported to be higher in patients with polycystic ovary syndrome (PCOS) compared with non-PCOS patients. However, whether PCOS is an independent risk factor for early miscarriage is still controversial; to what extent embryonic aneuploidy accounts for miscarriages of PCOS is still unknown. In this 1:3 matched-pair study, 67 lean PCOS patients and 201 controls matched for age, body mass index (BMI) and embryo scores undergoing a single euploid blastocyst transfer in vitrified-warmed cycles were analysed. Clinical pregnancy, early miscarriage and live birth rates were compared. Logistic regression analysis was performed to further evaluate the factors associated with early miscarriage and live birth. Clinical pregnancy rates were 50.7% in PCOS and 55.2% in control groups. Early miscarriage rate was significantly (P = 0.029) increased in the PCOS group compared with controls; non-PCOS patients had a significantly higher live birth rate than PCOS patients, P < 0.001. Further regression analyses showed that PCOS was significantly associated with a higher risk of early miscarriage and decreased chance of live birth. In conclusion, PCOS in women undergoing pre-implantation genetic diagnosis may, independently from BMI and karyotype, increase the risk of miscarriage. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Effect of Culture Conditions on Viability of Mouse and Rat Embryos Developed in Vitro

PubMed Central

Popova, Elena; Bader, Michael; Krivokharchenko, Alexander

2011-01-01

Currently in vitro culture of mouse preimplantation embryos has become a very important technique to investigate different mechanisms of early embryogenesis. However, there is a big difference in the preimplantation development between mammalian species. Despite close relatedness to mice, in vitro cultivation of rat preimplantation embryos is still delicate and needs further investigation and optimizations. In this study we have compared the in vitro developmental potential of mouse and rat embryos cultured at different culture conditions in parallel experiments. Interestingly, mouse zygotes developed in vitro until blastocyst stage even in inadequate medium without any phosphates and with low osmolarity which was formulated especially for cultivation of rat embryos. Rat parthenotes and zygotes developed in M16 medium formulated for mouse embryos only till 2-cell stage and further development is blocked completely at this stage. Moreover, developmental ability of rat embryos in vitro was significantly lower in comparison with mouse even in special rat mR1ECM medium. Mouse and rat embryos at 2-cell stage obtained in vivo developed until blastocyst stages significantly more efficiently compared to zygotes. Culture of mouse zygotes in glass capillaries resulted in a significantly higher rate of morula and blastocyst development compared with dishes. The Well-of-the-Well system resulted in a significant improvement when compared with dishes for the culture of rat zygotes only until morula stage. Reduced oxygen tension increased the developmental rate of rat but not mouse zygotes until blastocyst stage. This study demonstrates that development of early preimplantation embryos is altered by different culture conditions and show strong differences even between two related species such as mice and rats. Therefore, for understanding the fundamental mechanisms of early mammalian development it is very important to use embryos of various species. PMID:24710194

Drosophila melanogaster Hedgehog cooperates with Frazzled to guide axons through a non-canonical signalling pathway.

PubMed

Ricolo, Delia; Butí, Elisenda; Araújo, Sofia J

2015-08-01

We report that the morphogen Hedgehog (Hh) is an axonal chemoattractant in the midline of Drosophila melanogaster embryos. Hh is present in the ventral nerve cord during axonal guidance and overexpression of hh in the midline causes ectopic midline crossing of FasII-positive axonal tracts. In addition, we show that Hh influences axonal guidance via a non-canonical signalling pathway dependent on Ptc. Our results reveal that the Hh pathway cooperates with the Netrin/Frazzled pathway to guide axons through the midline in invertebrates. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

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.

Characterization of a Smad motif similar to Drosophila mad in the mouse Msx 1 promoter.

PubMed

Alvarez Martinez, Cristina E; Binato, Renata; Gonzalez, Sayonara; Pereira, Monica; Robert, Benoit; Abdelhay, Eliana

2002-03-01

Mouse Msx 1 gene, orthologous of the Drosophila msh, is involved in several developmental processes. BMP family members are major proteins in the regulation of Msx 1 expression. BMP signaling activates Smad 1/5/8 proteins, which associate to Smad 4 before translocating to the nucleus. Analysis of Msx 1 promoter revealed the presence of three elements similar to the consensus established for Mad, the Smad 1 Drosophila counterpart. Notably, such an element was identified in an enhancer important for Msx 1 regulation. Gel shift analysis demonstrated that proteins from 13.5 dpc embryo associate to this enhancer. Remarkably, supershift assays showed that Smad proteins are present in the complex. Purified Smad 1 and 4 also bind to this fragment. We demonstrate that functional binding sites in this enhancer are confined to the Mad motif and flanking region. Our data suggest that this Mad motif may be functional in response to BMP signaling. ©2002 Elsevier Science (USA).

Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

PubMed

Kurosawa, Hiroki; Utsunomiya, Hiroki; Shiga, Naomi; Takahashi, Aiko; Ihara, Motomasa; Ishibashi, Masumi; Nishimoto, Mitsuo; Watanabe, Zen; Abe, Hiroyuki; Kumagai, Jin; Terada, Yukihiro; Igarashi, Hideki; Takahashi, Toshifumi; Fukui, Atsushi; Suganuma, Ryota; Tachibana, Masahito; Yaegashi, Nobuo

2016-10-01

. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality

Equine cloning: in vitro and in vivo development of aggregated embryos.

PubMed

Gambini, Andrés; Jarazo, Javier; Olivera, Ramiro; Salamone, Daniel F

2012-07-01

The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.

Drosophila homologues of adenomatous polyposis coli (APC) and the formin diaphanous collaborate by a conserved mechanism to stimulate actin filament assembly.

PubMed

Jaiswal, Richa; Stepanik, Vince; Rankova, Aneliya; Molinar, Olivia; Goode, Bruce L; McCartney, Brooke M

2013-05-10

Vertebrate APC collaborates with Dia through its Basic domain to assemble actin filaments. Despite limited sequence homology between the vertebrate and Drosophila APC Basic domains, Drosophila APC1 collaborates with Dia to stimulate actin assembly in vitro. The mechanism of actin assembly is highly conserved over evolution. APC-Dia collaborations may be crucial in a wide range of animal cells. Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad

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.

Characterization of the intergenic RNA profile at abdominal-A and Abdominal-B in the Drosophila bithorax complex

PubMed Central

Bae, Esther; Calhoun, Vincent C.; Levine, Michael; Lewis, Edward B.; Drewell, Robert A.

2002-01-01

The correct spatial expression of two Drosophila bithorax complex (BX-C) genes, abdominal-A (abdA) and Abdominal-B (AbdB), is dependent on the 100-kb intergenic infraabdominal (iab) region. The iab region is known to contain a number of different domains (iab2 through iab8) that harbor cis-regulatory elements responsible for directing expression of abdA and AbdB in the second through eighth abdominal segments. Here, we use in situ hybridization to perform high-resolution mapping of the transcriptional activity in the iab control regions. We show that transcription of the control regions themselves is abundant and precedes activation of the abdA and AbdB genes. As with the homeotic genes of the BX-C, the transcription patterns of the RNAs from the iab control regions demonstrate colinearity with the sequence of the iab regions along the chromosome and the domains in the embryo under the control of the specific iab regions. These observations suggest that the intergenic RNAs may play a role in initiating cis regulation at the BX-C early in development. PMID:12481037

Maternal organism and embryo biosensoring: insights from ruminants.

PubMed

Sandra, Olivier; Constant, Fabienne; Vitorino Carvalho, Anais; Eozénou, Caroline; Valour, Damien; Mauffré, Vincent; Hue, Isabelle; Charpigny, Gilles

2015-04-01

In terms of contribution to pregnancy, the mother not only produces gametes, but also hosts gestation, whose progression in the uterus is conditioned by early events during implantation. In ruminants, this period is associated with elongation of the extra-embryonic tissues, gastrulation of the embryonic disk and cross-talk with the endometrium. Recent data have prompted the need for accurate staging of the bovine conceptus and shown that asynchrony between elongation and gastrulation processes may account for pregnancy failure. Data mining of endometrial gene signatures has allowed the identification of molecular pathways and new factors regulated by the conceptus (e.g. FOXL2, SOCS6). Interferon-tau has been recognised to be the major signal of pregnancy recognition, but prostaglandins and lysophospholipids have also been demonstrated to be critical players at the conceptus-endometrium interface. Interestingly, up-regulation of interferon-regulated gene expression has been identified in circulating immune cells during implantation, making these factors a potential source of non-invasive biomarkers for early pregnancy. Distinct endometrial responses have been shown to be elicited by embryos produced by artificial insemination, in vitro fertilisation or somatic cell nuclear transfer. These findings have led to the concept that endometrium is an early biosensor of embryo quality. This biological property first demonstrated in cattle has been recently extended and associated with embryo selection in humans. Hence, compromised or suboptimal endometrial quality can subtly or deeply affect embryo development, with visible and sometimes severe consequences for placentation, foetal development, pregnancy outcome and the long-term health of the offspring. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Protein Phosphorylation during Coconut Zygotic Embryo Development1

PubMed Central

Islas-Flores, Ignacio; Oropeza, Carlos; Hernández-Sotomayor, S.M. Teresa

1998-01-01

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species. PMID:9733545

Culture of Cells from Amphibian Embryos.

ERIC Educational Resources Information Center

Stanisstreet, Martin

1983-01-01

Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

Telomere lengthening early in development.

PubMed

Liu, Lin; Bailey, Susan M; Okuka, Maja; Muñoz, Purificación; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L

2007-12-01

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

Drosophila increase exploration after visually detecting predators.

PubMed

de la Flor, Miguel; Chen, Lijian; Manson-Bishop, Claire; Chu, Tzu-Chun; Zamora, Kathya; Robbins, Danielle; Gunaratne, Gemunu; Roman, Gregg

2017-01-01

Novel stimuli elicit behaviors that are collectively known as specific exploration. These behaviors allow the animal to become more familiar with the novel objects within its environment. Specific exploration is frequently suppressed by defensive reactions to predator cues. Herein, we examine if this suppression occurs in Drosophila melanogaster by measuring the response of these flies to wild harvested predators. The flies used in our experiments have been cultured and had not lived under predator threat for multiple decades. In a circular arena with centrally-caged predators, wild type Drosophila actively avoided the pantropical jumping spider, Plexippus paykulli, and the Texas unicorn mantis, Phyllovates chlorophaena, indicating an innate defensive reaction to these predators. Interestingly, wild type Drosophila males also avoided a centrally-caged mock spider, and the avoidance of the mock spider became exaggerated when it was made to move within the cage. Visually impaired Drosophila failed to detect and avoid the Plexippus paykulli and the moving mock spider, while the broadly anosmic orco2 mutants were fully capable of detecting and avoiding Plexippus paykulli, indicating that these flies principally relied upon vison to perceive the predator stimuli. During early exploration of the arena, exploratory activity increased in the presence of Plexippus paykulli and the moving mock spider. The elevated activity induced by Plexippus paykulli disappeared after the fly had finished exploring, suggesting the flies were capable of habituating the predator cues. Taken together, these results indicate that despite being isolated from predators for decades Drosophila will visually detect these predators, retain innate defensive behaviors, respond by increasing exploratory activity in the arena rather than suppressing activity, and may habituate to normal predator cues.

Drosophila increase exploration after visually detecting predators

PubMed Central

Manson-Bishop, Claire; Chu, Tzu-Chun; Zamora, Kathya; Robbins, Danielle; Gunaratne, Gemunu

2017-01-01

Novel stimuli elicit behaviors that are collectively known as specific exploration. These behaviors allow the animal to become more familiar with the novel objects within its environment. Specific exploration is frequently suppressed by defensive reactions to predator cues. Herein, we examine if this suppression occurs in Drosophila melanogaster by measuring the response of these flies to wild harvested predators. The flies used in our experiments have been cultured and had not lived under predator threat for multiple decades. In a circular arena with centrally-caged predators, wild type Drosophila actively avoided the pantropical jumping spider, Plexippus paykulli, and the Texas unicorn mantis, Phyllovates chlorophaena, indicating an innate defensive reaction to these predators. Interestingly, wild type Drosophila males also avoided a centrally-caged mock spider, and the avoidance of the mock spider became exaggerated when it was made to move within the cage. Visually impaired Drosophila failed to detect and avoid the Plexippus paykulli and the moving mock spider, while the broadly anosmic orco2 mutants were fully capable of detecting and avoiding Plexippus paykulli, indicating that these flies principally relied upon vison to perceive the predator stimuli. During early exploration of the arena, exploratory activity increased in the presence of Plexippus paykulli and the moving mock spider. The elevated activity induced by Plexippus paykulli disappeared after the fly had finished exploring, suggesting the flies were capable of habituating the predator cues. Taken together, these results indicate that despite being isolated from predators for decades Drosophila will visually detect these predators, retain innate defensive behaviors, respond by increasing exploratory activity in the arena rather than suppressing activity, and may habituate to normal predator cues. PMID:28746346

Transgenerational propagation and quantitative maintenance of paternal centromeres depends on Cid/Cenp-A presence in Drosophila sperm.

PubMed

Raychaudhuri, Nitika; Dubruille, Raphaelle; Orsi, Guillermo A; Bagheri, Homayoun C; Loppin, Benjamin; Lehner, Christian F

2012-01-01

In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.

Transgenerational Propagation and Quantitative Maintenance of Paternal Centromeres Depends on Cid/Cenp-A Presence in Drosophila Sperm

PubMed Central

Raychaudhuri, Nitika; Dubruille, Raphaelle; Orsi, Guillermo A.; Bagheri, Homayoun C.; Loppin, Benjamin; Lehner, Christian F.

2012-01-01

In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses. PMID:23300376

The first cell-fate decisions in the mouse embryo: destiny is a matter of both chance and choice.

PubMed

Zernicka-Goetz, Magdalena

2006-08-01

Development of the early mouse embryo has always been classified as regulative, meaning that when parts or blastomeres of the embryo are isolated they change their developmental fate and can even reconstruct the whole. However, regulative development does not mean that, in situ, these parts or blastomeres are equivalent; it does not mean that the early mammalian embryo is a ball of identical cells without any bias. Regulative development simply means that whatever bias the regions of the embryo might have they still remain flexible and can respond to experimental interference by changes of fate. This realization -- that regulative development and patterning can co-exist -- has led to a renaissance of interest in the first days of development of the mouse embryo, and several laboratories have provided evidence for some early bias. Now the challenge is to gain some understanding of the molecular basis of this bias.

Live imaging of rat embryos with Doppler swept-source optical coherence tomography

NASA Astrophysics Data System (ADS)

Larina, Irina V.; Furushima, Kenryo; Dickinson, Mary E.; Behringer, Richard R.; Larin, Kirill V.

2009-09-01

The rat has long been considered an excellent system to study mammalian embryonic cardiovascular physiology, but has lacked the extensive genetic tools available in the mouse to be able to create single gene mutations. However, the recent establishment of rat embryonic stem cell lines facilitates the generation of new models in the rat embryo to link changes in physiology with altered gene function to define the underlying mechanisms behind congenital cardiovascular birth defects. Along with the ability to create new rat genotypes there is a strong need for tools to analyze phenotypes with high spatial and temporal resolution. Doppler OCT has been previously used for 3-D structural analysis and blood flow imaging in other model species. We use Doppler swept-source OCT for live imaging of early postimplantation rat embryos. Structural imaging is used for 3-D reconstruction of embryo morphology and dynamic imaging of the beating heart and vessels, while Doppler-mode imaging is used to visualize blood flow. We demonstrate that Doppler swept-source OCT can provide essential information about the dynamics of early rat embryos and serve as a basis for a wide range of studies on functional evaluation of rat embryo physiology.

Live imaging of rat embryos with Doppler swept-source optical coherence tomography

PubMed Central

Larina, Irina V.; Furushima, Kenryo; Dickinson, Mary E.; Behringer, Richard R.; Larin, Kirill V.

2009-01-01

The rat has long been considered an excellent system to study mammalian embryonic cardiovascular physiology, but has lacked the extensive genetic tools available in the mouse to be able to create single gene mutations. However, the recent establishment of rat embryonic stem cell lines facilitates the generation of new models in the rat embryo to link changes in physiology with altered gene function to define the underlying mechanisms behind congenital cardiovascular birth defects. Along with the ability to create new rat genotypes there is a strong need for tools to analyze phenotypes with high spatial and temporal resolution. Doppler OCT has been previously used for 3-D structural analysis and blood flow imaging in other model species. We use Doppler swept-source OCT for live imaging of early postimplantation rat embryos. Structural imaging is used for 3-D reconstruction of embryo morphology and dynamic imaging of the beating heart and vessels, while Doppler-mode imaging is used to visualize blood flow. We demonstrate that Doppler swept-source OCT can provide essential information about the dynamics of early rat embryos and serve as a basis for a wide range of studies on functional evaluation of rat embryo physiology. PMID:19895102

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

Use of the Coelomic Grafting Technique for Prolonged ex utero Cultivation of Late Preprimitive Streak-Stage Rabbit Embryos.

PubMed

Püschel, Bernd; Männer, Jörg

2016-01-01

Due to its morphological similarity with the early human embryo, the pregastrulation-stage rabbit may represent an appropriate mammalian model for studying processes involved in early human development. The usability of mammalian embryos for experimental studies depends on the availability of whole embryo culture methods facilitating prolonged ex utero development. While currently used culture methods yield high success rates for embryos from primitive streak stages onward, the success rate of extended cultivation of preprimitive streak-stage mammalian embryos is low for all previously established methods and for all studied species. This limits the usability of preprimitive streak-stage rabbit embryos in experimental embryology. We have tested whether the extraembryonic coelom of 4-day-old chick embryos may be used for prolonged ex utero culture of preprimitive streak-stage rabbit embryos (stage 2, 6.2 days post coitum). We found that, within this environment, stage 2 rabbit blastocysts can be cultured at decreasing success rates (55% after 1 day, 35% after 2 days, 15% after 3 days) up to a maximum of 72 h. Grafted blastocysts can continue development from the onset of gastrulation to early organogenesis and thereby form all structures characterizing age-matched controls (e.g. neural tube, somites, beating heart). Compared to normal controls, successfully cultured embryos developed at a slower rate and finally showed some structural and gross morphological anomalies. The method presented here was originally developed for whole embryo culture of mouse embryos by Gluecksohn-Schoenheimer in 1941. It is a simple and inexpensive method that may represent a useful extension to presently available ex utero culture systems for rabbit embryos. © 2016 S. Karger AG, Basel.

Neurotactin functions in concert with other identified CAMs in growth cone guidance in Drosophila.

PubMed

Speicher, S; García-Alonso, L; Carmena, A; Martín-Bermudo, M D; de la Escalera, S; Jiménez, F

1998-02-01

We have isolated and characterized mutations in Drosophila neurotactin, a gene that encodes a cell adhesion protein widely expressed during neural development. Analysis of both loss and gain of gene function conditions during embryonic and postembryonic development revealed specific requirements for neurotactin during axon outgrowth, fasciculation, and guidance. Furthermore, embryos of some double mutant combinations of neurotactin and other genes encoding adhesion/signaling molecules, including neuroglian, derailed, and kekkon1, displayed phenotypic synergy. This result provides evidence for functional cooperativity in vivo between the adhesion and signaling pathways controlled by neurotactin and the other three genes.

Effects of growth hormone on the ultrastructure of bovine preimplantation embryos.

PubMed

Kölle, Sabine; Stojkovic, Miodrag; Reese, Sven; Reichenbach, Horst-Dieter; Wolf, Eckhard; Sinowatz, Fred

2004-07-01

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.

Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos

PubMed Central

D’Souza, Fiona; Pudakalakatti, Shivanand M.; Uppangala, Shubhashree; Honguntikar, Sachin; Salian, Sujith Raj; Kalthur, Guruprasad; Pasricha, Renu; Appajigowda, Divya; Atreya, Hanudatta S.; Adiga, Satish Kumar

2016-01-01

Early development of certain mammalian embryos is protected by complex checkpoint systems to maintain the genomic integrity. Several metabolic pathways are modulated in response to genetic insults in mammalian cells. The present study investigated the relationship between the genetic integrity, embryo metabolites and developmental competence in preimplantation stage mouse embryos with the aim to identify early biomarkers which can predict embryonic genetic integrity using spent medium profiling by NMR spectroscopy. Embryos carrying induced DNA lesions (IDL) developed normally for the first 2.5 days, but began to exhibit a developmental delay at embryonic day 3.5(E3.5) though they were morphologically indistinguishable from control embryos. Analysis of metabolites in the spent medium on E3.5 revealed a significant association between pyruvate, lactate, glucose, proline, lysine, alanine, valine, isoleucine and thymine and the extent of genetic instability observed in the embryos on E4.5. Further analysis revealed an association of apoptosis and micronuclei frequency with P53 and Bax transcripts in IDL embryos on the E4.5 owing to delayed induction of chromosome instability. We conclude that estimation of metabolites on E3.5 in spent medium may serve as a biomarker to predict the genetic integrity in pre-implantation stage embryos which opens up new avenues to improve outcomes in clinical IVF programs. PMID:27853269

Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Mitotic Cycles

PubMed Central

Su, Tin Tin; O'Farrell, Patrick H.

1997-01-01

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM–chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM–chromatin association. PMID:9314525