Tension (re)builds: Biophysical mechanisms of embryonic wound repair.

PubMed

Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo

2017-04-01

Embryonic tissues display an outstanding ability to rapidly repair wounds. Epithelia, in particular, serve as protective layers that line internal organs and form the skin. Thus, maintenance of epithelial integrity is of utmost importance for animal survival, particularly at embryonic stages, when an immune system has not yet fully developed. Rapid embryonic repair of epithelial tissues is conserved across species, and involves the collective migration of the cells around the wound. The migratory cell behaviours associated with wound repair require the generation and transmission of mechanical forces, not only for the cells to move, but also to coordinate their movements. Here, we review the forces involved in embryonic wound repair. We discuss how different force-generating structures are assembled at the molecular level, and the mechanisms that maintain the balance between force-generating structures as wounds close. Finally, we describe the mechanisms that cells use to coordinate the generation of mechanical forces around the wound. Collective cell movements and their misregulation have been associated with defective tissue repair, developmental abnormalities and cancer metastasis. Thus, we propose that understanding the role of mechanical forces during embryonic wound closure will be crucial to develop therapeutic interventions that promote or prevent collective cell movements under pathological conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Diversity and Complexity in Chromatin Recognition by TFII-I Transcription Factors in Pluripotent Embryonic Stem Cells and Embryonic Tissues

PubMed Central

Makeyev, Aleksandr V.; Enkhmandakh, Badam; Hong, Seung-Hyun; Joshi, Pujan; Shin, Dong-Guk; Bayarsaihan, Dashzeveg

2012-01-01

GTF2I and GTF2IRD1 encode a family of closely related transcription factors TFII-I and BEN critical in embryonic development. Both genes are deleted in Williams-Beuren syndrome, a complex genetic disorder associated with neurocognitive, craniofacial, dental and skeletal abnormalities. Although genome-wide promoter analysis has revealed the existence of multiple TFII-I binding sites in embryonic stem cells (ESCs), there was no correlation between TFII-I occupancy and gene expression. Surprisingly, TFII-I recognizes the promoter sequences enriched for H3K4me3/K27me3 bivalent domain, an epigenetic signature of developmentally important genes. Moreover, we discovered significant differences in the association between TFII-I and BEN with the cis-regulatory elements in ESCs and embryonic craniofacial tissues. Our data indicate that in embryonic tissues BEN, but not the highly homologous TFII-I, is primarily recruited to target gene promoters. We propose a “feed-forward model” of gene regulation to explain the specificity of promoter recognition by TFII-I factors in eukaryotic cells. PMID:22970219

Diversity and complexity in chromatin recognition by TFII-I transcription factors in pluripotent embryonic stem cells and embryonic tissues.

PubMed

Makeyev, Aleksandr V; Enkhmandakh, Badam; Hong, Seung-Hyun; Joshi, Pujan; Shin, Dong-Guk; Bayarsaihan, Dashzeveg

2012-01-01

GTF2I and GTF2IRD1 encode a family of closely related transcription factors TFII-I and BEN critical in embryonic development. Both genes are deleted in Williams-Beuren syndrome, a complex genetic disorder associated with neurocognitive, craniofacial, dental and skeletal abnormalities. Although genome-wide promoter analysis has revealed the existence of multiple TFII-I binding sites in embryonic stem cells (ESCs), there was no correlation between TFII-I occupancy and gene expression. Surprisingly, TFII-I recognizes the promoter sequences enriched for H3K4me3/K27me3 bivalent domain, an epigenetic signature of developmentally important genes. Moreover, we discovered significant differences in the association between TFII-I and BEN with the cis-regulatory elements in ESCs and embryonic craniofacial tissues. Our data indicate that in embryonic tissues BEN, but not the highly homologous TFII-I, is primarily recruited to target gene promoters. We propose a "feed-forward model" of gene regulation to explain the specificity of promoter recognition by TFII-I factors in eukaryotic cells.

Engineering human cell spheroids to model embryonic tissue fusion in vitro.

EPA Science Inventory

Epithelial-mesenchymal interactions drive embryonic fusion events during development and upon perturbation can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known abo...

The role of nanotechnology in induced pluripotent and embryonic stem cells research.

PubMed

Chen, Lukui; Qiu, Rong; Li, Lushen

2014-12-01

This paper reviews the recent studies on development of nanotechnology in the field of induced pluripotent and embryonic stem cells. Stem cell therapy is a promising therapy that can improve the quality of life for patients with refractory diseases. However, this option is limited by the scarcity of tissues, ethical problem, and tumorigenicity. Nanotechnology is another promising therapy that can be used to mimic the extracellular matrix, label the implanted cells, and also can be applied in the tissue engineering. In this review, we briefly introduce implementation of nanotechnology in induced pluripotent and embryonic stem cells research. Finally, the potential application of nanotechnology in tissue engineering and regenerative medicine is also discussed.

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

PubMed Central

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

2011-01-01

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

A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

PubMed

Ratajczak, Mariusz Z; Ratajczak, Janina; Suszynska, Malwina; Miller, Donald M; Kucia, Magda; Shin, Dong-Myung

2017-01-06

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine. © 2017 American Heart Association, Inc.

Conserved developmental alternative splicing of muscleblind-like (MBNL) transcripts regulates MBNL localization and activity.

PubMed

Terenzi, Fulvia; Ladd, Andrea N

2010-01-01

Muscleblind-like (MBNL) proteins have been shown to regulate pre-mRNA alternative splicing, and MBNL1 has been implicated in regulating fetal-to-adult transitions in alternative splicing in the heart. MBNL1 is highly conserved, exhibiting more than 95% identity at the amino acid level between birds and mammals. To investigate MBNL1 expression during embryonic heart development, we examined MBNL1 transcript and protein expression in the embryonic chicken heart from the formation of the primitive heart tube through cardiac morphogenesis (embryonic days 1.5 through 8). MBNL1 transcript levels remained steady throughout these stages, whereas MBNL1 protein levels increased and exhibited a shift in isoforms. MBNL1 has several alternatively spliced exons. Using RT-PCR, we determined that the inclusion of one of these, exon 5, decreases dramatically during cardiac morphogenesis. This developmental transition is conserved in mice. Functional analyses of MBNL1 isoforms containing or lacking exon 5-encoded sequences revealed that exon 5 is important for the regulation of the subcellular localization, RNA binding affinity, and alternative splicing activity of MBNL1 proteins. A second MBNL protein, MBNL2, is also expressed in the embryonic heart. We found that MBNL2 exon 5, which is paralogous to MBNL1 exon 5, is similarly regulated during embryonic heart development. Analysis of MBNL1 and MBNL2 transcripts in several embryonic tissues in chicken and mouse indicate that exon 5 alternative splicing is highly conserved and tissue-specific. Thus, we propose that conserved developmental stage- and tissue-specific alternative splicing of MBNL transcripts is an important mechanism by which MBNL activity is regulated during embryonic development.

Generation of stomach tissue from mouse embryonic stem cells.

PubMed

Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

2015-08-01

Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

Structure and function of gap junction proteins: role of gap junction proteins in embryonic heart development.

PubMed

Ahir, Bhavesh K; Pratten, Margaret K

2014-01-01

Intercellular (cell-to-cell) communication is a crucial and complex mechanism during embryonic heart development. In the cardiovascular system, the beating of the heart is a dynamic and key regulatory process, which is functionally regulated by the coordinated spread of electrical activity through heart muscle cells. Heart tissues are composed of individual cells, each bearing specialized cell surface membrane structures called gap junctions that permit the intercellular exchange of ions and low molecular weight molecules. Gap junction channels are essential in normal heart function and they assist in the mediated spread of electrical impulses that stimulate synchronized contraction (via an electrical syncytium) of cardiac tissues. This present review describes the current knowledge of gap junction biology. In the first part, we summarise some relevant biochemical and physiological properties of gap junction proteins, including their structure and function. In the second part, we review the current evidence demonstrating the role of gap junction proteins in embryonic development with particular reference to those involved in embryonic heart development. Genetics and transgenic animal studies of gap junction protein function in embryonic heart development are considered and the alteration/disruption of gap junction intercellular communication which may lead to abnormal heart development is also discussed.

Tenascin-C and mechanotransduction in the development and diseases of cardiovascular system

PubMed Central

Imanaka-Yoshida, Kyoko; Aoki, Hiroki

2014-01-01

Living tissue is composed of cells and extracellular matrix (ECM). In the heart and blood vessels, which are constantly subjected to mechanical stress, ECM molecules form well-developed fibrous frameworks to maintain tissue structure. ECM is also important for biological signaling, which influences various cellular functions in embryonic development, and physiological/pathological responses to extrinsic stimuli. Among ECM molecules, increased attention has been focused on matricellular proteins. Matricellular proteins are a growing group of non-structural ECM proteins highly up-regulated at active tissue remodeling, serving as biological mediators. Tenascin-C (TNC) is a typical matricellular protein, which is highly expressed during embryonic development, wound healing, inflammation, and cancer invasion. The expression is tightly regulated, dependent on the microenvironment, including various growth factors, cytokines, and mechanical stress. In the heart, TNC appears in a spatiotemporal-restricted manner during early stages of development, sparsely detected in normal adults, but transiently re-expressed at restricted sites associated with tissue injury and inflammation. Similarly, in the vascular system, TNC is strongly up-regulated during embryonic development and under pathological conditions with an increase in hemodynamic stress. Despite its intriguing expression pattern, cardiovascular system develops normally in TNC knockout mice. However, deletion of TNC causes acute aortic dissection (AAD) under strong mechanical and humoral stress. Accumulating reports suggest that TNC may modulate the inflammatory response and contribute to elasticity of the tissue, so that it may protect cardiovascular tissue from destructive stress responses. TNC may be a key molecule to control cellular activity during development, adaptation, or pathological tissue remodeling. PMID:25120494

Mechanical control of notochord morphogenesis by extra-embryonic tissues in mouse embryos.

PubMed

Imuta, Yu; Koyama, Hiroshi; Shi, Dongbo; Eiraku, Mototsugu; Fujimori, Toshihiko; Sasaki, Hiroshi

2014-05-01

Mammalian embryos develop in coordination with extraembryonic tissues, which support embryonic development by implanting embryos into the uterus, supplying nutrition, providing a confined niche, and also providing patterning signals to embryos. Here, we show that in mouse embryos, the expansion of the amniotic cavity (AC), which is formed between embryonic and extraembryonic tissues, provides the mechanical forces required for a type of morphogenetic movement of the notochord known as convergent extension (CE) in which the cells converge to the midline and the tissue elongates along the antero-posterior (AP) axis. The notochord is stretched along the AP axis, and the expansion of the AC is required for CE. Both mathematical modeling and physical simulation showed that a rectangular morphology of the early notochord caused the application of anisotropic force along the AP axis to the notochord through the isotropic expansion of the AC. AC expansion acts upstream of planar cell polarity (PCP) signaling, which regulates CE movement. Our results highlight the importance of extraembryonic tissues as a source of the forces that control the morphogenesis of embryos. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Development of a 3D co-culture model using human stem cells for studying embryonic palatal fusion.

EPA Science Inventory

Morphogenetic tissue fusion is a critical and complex event in embryonic development and failure of this event leads to birth defects, such as cleft palate. Palatal fusion requires adhesion and subsequent dissolution of the medial epithelial layer of the mesenchymal palatal shelv...

Adiposity associated changes in serum glucose and adiponectin levels modulate ovarian steroidogenesis during delayed embryonic development in the fruit bat, Cynopterus sphinx.

PubMed

Anuradha; Krishna, Amitabh

2018-06-01

The aim of the present study was to evaluate the mechanism by which embryonic development in Cynopterus sphinx is impaired during the period of increased accumulation of white adipose tissue during winter scarcity of food. The change in the mass of white adipose tissue during adipogenesis showed significant positive correlation with the circulating glucose level. But increase in circulating glucose level during the adipogenesis showed negative correlation with circulating progesterone and adiponectin levels. The in vivo study showed increased glucose uptake by the adipose tissue during adipogenesis due to increased expression of insulin receptor (IR) and glucose transporter (GLUT) 4 proteins. This study showed decline in the adiponectin level during fat accumulation. In the in vitro study, ovary treated with high doses of glucose showed impaired progesterone synthesis. This is due to decreased glucose uptake mediated decrease in the expression of luteinizing hormone-receptor, steroidogenic acute regulatory protein, IR, GLUT4 and AdipoR1 proteins. But the ovary treated with adiponectin either alone or with higher concentration of glucose showed improvement in progesterone synthesis due to increased expression of IR, GLUT4 and AdipoR1 mediated increased glucose uptake. In conclusion, increased circulating glucose level prior to winter dormancy preferably transported to white adipose tissue for fat accumulation diverting glucose away from the ovary. Consequently the decreased availability of adiponectin and glucose to the ovary and utero-embryonic unit may be responsible for impaired progesterone synthesis and delayed embryonic development. The delayed embryonic development in Cynopterus sphinx may have evolved, in part, as a mechanism to prevent pregnancy loss during the period of decreased energy availability. Copyright © 2018 Elsevier Inc. All rights reserved.

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation.

PubMed

Inman, Kimberly E; Downs, Karen M

2006-10-01

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.

Scaffolding for Three-Dimensional Embryonic Vasculogenesis

NASA Astrophysics Data System (ADS)

Kraehenbuehl, Thomas P.; Aday, Sezin; Ferreira, Lino S.

Biomaterial scaffolds have great potential to support efficient vascular differentiation of embryonic stem cells. Vascular cell fate-specific biochemical and biophysical cues have been identified and incorporated into three-dimensional (3D) biomaterials to efficiently direct embryonic vasculogenesis. The resulting vascular-like tissue can be used for regenerative medicine applications, further elucidation of biophysical and biochemical cues governing vasculogenesis, and drug discovery. In this chapter, we give an overview on the following: (1) developmental cues for directed differentiation of human embryonic stem cells (hESCs) into vascular cells, (2) 3D vascular differentiation in embryoid bodies (EBs), (3) preparation of 3D scaffolds for the vascular differentiation of hESCs, and (4) the most significant studies combining scaffolding and hESCs for development of vascular-like tissue.

Stem cells: sources and applications.

PubMed

Vats, A; Tolley, N S; Polak, J M; Buttery, L D K

2002-08-01

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of function of organs through implantation of cells/tissues grown outside the body, or stimulating cells to grow into implanted matrix. In this short review, some of the most recent developments in the use of stem cells for tissue repair and regeneration will be discussed. There is no doubt that stem cells derived from adult and embryonic sources hold great therapeutic potential but it is clear that there is still much research required before their use is commonplace. There is much debate over adult versus embryonic stem cells and whether both are required. It is probably too early to disregard one or other of these cell sources. With regard to embryonic stem cells, the major concern relates to the ethics of their creation and the proposed practice of therapeutic cloning.

Expression analysis of the insulin-like growth factors I and II during embryonic and early larval development of turbot ( Scophthalmus maximus)

NASA Astrophysics Data System (ADS)

Wen, Haishen; Qi, Qian; Hu, Jian; Si, Yufeng; He, Feng; Li, Jifang

2015-04-01

The insulin-like growth factors I and II (IGF-I and IGF-II) are important proteins involved in fish growth and development. Here, we report the isolation of IGF-II and expression analysis of IGFs in turbot Scophthalmus maximus, aiming to clarify their function in embryonic and larval development of fish. The deduced IGF-II gene is 808 bp in full length, which encodes a protein of 219 amino acids and is 93% similar with that of Paralichthys olicaceus in amino acid sequence. The tissue abundance and the expression pattern of IGFs in a turbot at early development stages were investigated via reverse transcription-polymer chain reaction. Result showed that the IGF-I and IGF-II genes were widely expressed in tissues of S. maximus. IGF-I was detected in all tissues except intestines with the highest level in liver, while IGF-II transcript presented in all tissues except muscle. At the stages of embryonic and larval development, the mRNA levels of IGFs sharply increased from the stage of unfertilized egg to post larva, followed by a decrease with larval development. However, there was an increase in IGF-I at the embryonic stage and IGF-II at the gastrula stage, respectively. These results suggested that IGFs play important roles in cell growth and division of the turbot. Our study provides reference data for further investigation of growth regulation in turbot, which can guarantee better understanding of the physiological role that IGFs play in fish.

Embryonic rather than extraembryonic tissues have more impact on the development of placental hyperplasia in cloned mice.

PubMed

Miki, H; Wakisaka, N; Inoue, K; Ogonuki, N; Mori, M; Kim, J-M; Ohta, A; Ogura, A

2009-06-01

Somatic cell cloning by nuclear transfer (NT) in mice is associated with hyperplastic placentas at term. To dissect the effects of embryonic and extraembryonic tissues on this clone-associated phenotype, we constructed diploid (2n) fused with (<-->) tetraploid (4n) chimeras from NT- and fertilization-derived (FD) embryos. Generally, the 4n cells contributed efficiently to all the extraembryonic tissues but not to the embryo itself. Embryos constructed by 2n NT<-->4n FD aggregation developed hyperplastic placentas (0.33+/-0.22 g) with a predominant contribution by NT-derived cells. Even when the population of FD-derived cells in placentas was increased using multiple FD embryos (up to four) for aggregation, most placentas remained hyperplastic (0.36+/-0.13 g). By contrast, placentas of the reciprocal combination, 2n FD<-->4n NT, were less hyperplastic (0.15+/-0.02 g). These nearly normal-looking placentas had a large proportion of NT-derived cells. Thus, embryonic rather than extraembryonic tissues had more impact on the onset of placental hyperplasia, and that the abnormal placentation in clones occurs in a noncell-autonomous manner. These findings suggest that for improvement of cloning efficiency we should understand the mechanisms regulating placentation, especially those of embryonic origin that might control the proliferation of trophoblastic lineage cells.

Derivation of Multipotent Mesenchymal Precursors from Human Embryonic Stem Cells

PubMed Central

Barberi, Tiziano; Willis, Lucy M; Socci, Nicholas D; Studer, Lorenz

2005-01-01

Background Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. Methods and Findings Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. Conclusion Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications. PMID:15971941

[The legal and ethical aspects of nerve tissue transplantation].

PubMed

Sramka, M; Rattaj, M

1992-01-01

The authors have specified the following criteria for the withdrawal of embryonal tissue at their department: 1) only tissue from dead fetus is allowed to be used in neurotransplantation; 2) embryonal tissue is to be obtained after spontaneous abortions from volunteers or from women asking for artificial abortion; 3) the women should be informed about the curative purposes of embryonal tissue voluntary donorship and they must give a written consent; 4) decision on abortion should be separated from the use of embryonal tissue; 5) women should not know recipients; no payments should be made for tissue; 6) the donor is not permitted to impregnate in order to use embryos for research or clinical purposes; 7) sampling of BWR, HBsAG, anti-HIV, cytomegalovirus, herpes I and II is to be made for serologic examinations and that from the cervix for cultivation and sensitivity, as well as ultrasound verification of a germinal age is done in potential donors; 8) consent should be signed to embryonal brain transplantation by recipient or his legitimate deputy if the recipient is certifiable. The above criteria should protect both the donor and the recipient. The use of embryonal tissue cultures seems to be promising. In addition to legal and ethic problems, immunological problems and problems concerning the aseptic withdrawal of embryonal tissue are falling off.

Comparison of NMDA and AMPA Channel Expression and Function between Embryonic and Adult Neurons Utilizing Microelectrode Array Systems.

PubMed

Edwards, Darin; Sommerhage, Frank; Berry, Bonnie; Nummer, Hanna; Raquet, Martina; Clymer, Brad; Stancescu, Maria; Hickman, James J

2017-12-11

Microelectrode arrays (MEAs) are innovative tools used to perform electrophysiological experiments for the study of electrical activity and connectivity in populations of neurons from dissociated cultures. Reliance upon neurons derived from embryonic tissue is a common limitation of neuronal/MEA hybrid systems and perhaps of neuroscience research in general, and the use of adult neurons could model fully functional in vivo parameters more closely. Spontaneous network activity was concurrently recorded from both embryonic and adult rat neurons cultured on MEAs for up to 10 weeks in vitro to characterize the synaptic connections between cell types. The cultures were exposed to synaptic transmission antagonists against NMDA and AMPA channels, which revealed significantly different receptor profiles of adult and embryonic networks in vitro. In addition, both embryonic and adult neurons were evaluated for NMDA and AMPA channel subunit expression over five weeks in vitro. The results established that neurons derived from embryonic tissue did not express mature synaptic channels for several weeks in vitro under defined conditions. Consequently, the embryonic response to synaptic antagonists was significantly different than that of neurons derived from adult tissue sources. These results are especially significant because most studies reported with embryonic hippocampal neurons do not begin at two to four weeks in culture. In addition, the utilization of MEAs in lieu of patch-clamp electrophysiology avoided a large-scale, labor-intensive study. These results establish the utility of this unique hybrid system derived from adult hippocampal tissue in combination with MEAs and offer a more appropriate representation of in vivo function for drug discovery. It has application for neuronal development and regeneration as well as for investigations into neurodegenerative disease, traumatic brain injury, and stroke.

A novel approach for studying the temporal modulation of embryonic skeletal development using organotypic bone cultures and microcomputed tomography.

PubMed

Kanczler, Janos M; Smith, Emma L; Roberts, Carol A; Oreffo, Richard O C

2012-10-01

Understanding the structural development of embryonic bone in a three dimensional framework is fundamental to developing new strategies for the recapitulation of bone tissue in latter life. We present an innovative combined approach of an organotypic embryonic femur culture model, microcomputed tomography (μCT) and immunohistochemistry to examine the development and modulation of the three dimensional structures of the developing embryonic femur. Isolated embryonic chick femurs were organotypic (air/liquid interface) cultured for 10 days in either basal, chondrogenic, or osteogenic supplemented culture conditions. The growth development and modulating effects of basal, chondrogenic, or osteogenic culture media of the embryonic chick femurs was investigated using μCT, immunohistochemistry, and histology. The growth and development of noncultured embryonic chick femur stages E10, E11, E12, E13, E15, and E17 were very closely correlated with increased morphometric indices of bone formation as determined by μCT. After 10 days in the organotpyic culture set up, the early aged femurs (E10 and E11) demonstrated a dramatic response to the chondrogenic or osteogenic culture conditions compared to the basal cultured femurs as determined by a change in μCT morphometric indices and modified expression of chondrogenic and osteogenic markers. Although the later aged femurs (E12 and E13) increased in size and structure after 10 days organotpypic culture, the effects of the osteogenic and chondrogenic organotypic cultures on these femurs were not significantly altered compared to basal conditions. We have demonstrated that the embryonic chick femur organotpyic culture model combined with the μCT and immunohistochemical analysis can provide an integral methodology for investigating the modulation of bone development in an ex vivo culture setting. Hence, these interdisciplinary techniques of μCT and whole organ bone cultures will enable us to delineate some of the temporal, structural developmental paradigms and modulation of bone tissue formation to underpin innovative skeletal regenerative technology for clinical therapeutic strategies in musculoskeletal trauma and diseases.

Changes in WNT signaling-related gene expression associated with development and cloning in bovine extra-embryonic and endometrial tissues during the peri-implantation period.

PubMed

Biase, Fernando H; Rabel, Chanaka; Guillomot, Michel; Sandra, Olivier; Andropolis, Kalista; Olmstead, Colleen; Oliveira, Rosane; Wallace, Richard; Le Bourhis, Daniel; Richard, Christophe; Campion, Evelyne; Chaulot-Talmon, Aurélie; Giraud-Delville, Corinne; Taghouti, Géraldine; Jammes, Hélène; Hue, Isabelle; Renard, Jean Paul; Lewin, Harris A

2013-12-01

We determined if somatic cell nuclear transfer (SCNT) cloning is associated with WNT-related gene expression in cattle development, and if the expression of genes in the WNT pathway changes during the peri-implantation period. Extra-embryonic and endometrial tissues were collected at gestation days 18 and 34 (d18, d34). WNT5A, FZD4, FZD5, LRP5, CTNNB1, GNAI2, KDM1A, BCL2L1, and SFRP1 transcripts were localized in extra-embryonic tissue, whereas SFRP1 and DKK1 were localized in the endometrium. There were no differences in the localization of these transcripts in extra-embryonic tissue or endometrium from SCNT or artificial insemination (AI) pregnancies. Expression levels of WNT5A were 11-fold greater in the allantois of SCNT than AI samples. In the trophoblast, expression of WNT5A, FZD5, CTNNB1, and DKK1 increased significantly from d18 to d34, whereas expression of KDM1A and SFRP1 decreased, indicating that implantation is associated with major changes in WNT signaling. SCNT was associated with altered WNT5A expression in trophoblasts, with levels increasing 2.3-fold more in AI than SCNT conceptuses from d18 to d34. In the allantois, expression of WNT5A increased 6.3-fold more in SCNT than AI conceptuses from d18 to d34. Endometrial tissue expression levels of the genes tested did not differ between AI or SCNT pregnancies, although expression of individual genes showed variation across developmental stages. Our results demonstrate that SCNT is associated with altered expression of specific WNT-related genes in extra-embryonic tissue in a time- and tissue-specific manner. The pattern of gene expression in the WNT pathway suggests that noncanonical WNT signal transduction is important for implantation of cattle conceptuses. © 2013 Wiley Periodicals, Inc.

Engineering epithelial-stromal interactions in vitro for toxicology assessment.

PubMed

Belair, David G; Abbott, Barbara D

2017-05-01

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues. Published by Elsevier B.V.

Engineering epithelial-stromal interactions in vitro for toxicology assessment

PubMed Central

Belair, David G.; Abbott, Barbara D.

2018-01-01

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues. PMID:28285100

Mechanical preconditioning enables electrophysiologic coupling of skeletal myoblast cells to myocardium

PubMed Central

Treskes, Philipp; Cowan, Douglas B.; Stamm, Christof; Rubach, Martin; Adelmann, Roland; Wittwer, Thorsten; Wahlers, Thorsten

2015-01-01

Objective The effect of mechanical preconditioning on skeletal myoblasts in engineered tissue constructs was investigated to resolve issues associated with conduction block between skeletal myoblast cells and cardiomyocytes. Methods Murine skeletal myoblasts were used to generate engineered tissue constructs with or without application of mechanical strain. After in vitro myotube formation, engineered tissue constructs were co-cultured for 6 days with viable embryonic heart slices. With the use of sharp electrodes, electrical coupling between engineered tissue constructs and embryonic heart slices was assessed in the presence or absence of pharmacologic agents. Results The isolation and expansion procedure for skeletal myoblasts resulted in high yields of homogeneously desmin-positive (97.1% ± 0.1%) cells. Mechanical strain was exerted on myotubes within engineered tissue constructs during gelation of the matrix, generating preconditioned engineered tissue constructs. Electrical coupling between preconditioned engineered tissue constructs and embryonic heart slices was observed; however, no coupling was apparent when engineered tissue constructs were not subjected to mechanical strain. Coupling of cells from engineered tissue constructs to cells in embryonic heart slices showed slower conduction velocities than myocardial cells with the embryonic heart slices (preconditioned engineered tissue constructs vs embryonic heart slices: 0.04 ± 0.02 ms vs 0.10 ± 0.05 ms, P = .011), lower stimulation frequencies (preconditioned engineered tissue constructs vs maximum embryonic heart slices: 4.82 ± 1.42 Hz vs 10.58 ± 1.56 Hz; P = .0009), and higher sensitivities to the gap junction inhibitor (preconditioned engineered tissue constructs vs embryonic heart slices: 0.22 ± 0.07 mmol/L vs 0.93 ± 0.15 mmol/L; P = .0004). Conclusions We have generated skeletal myoblast–based transplantable grafts that electrically couple to myocardium. PMID:22980065

Identification and Characterization of Long Non-Coding RNAs Related to Mouse Embryonic Brain Development from Available Transcriptomic Data

PubMed Central

He, Hongjuan; Xiu, Youcheng; Guo, Jing; Liu, Hui; Liu, Qi; Zeng, Tiebo; Chen, Yan; Zhang, Yan; Wu, Qiong

2013-01-01

Long non-coding RNAs (lncRNAs) as a key group of non-coding RNAs have gained widely attention. Though lncRNAs have been functionally annotated and systematic explored in higher mammals, few are under systematical identification and annotation. Owing to the expression specificity, known lncRNAs expressed in embryonic brain tissues remain still limited. Considering a large number of lncRNAs are only transcribed in brain tissues, studies of lncRNAs in developmental brain are therefore of special interest. Here, publicly available RNA-sequencing (RNA-seq) data in embryonic brain are integrated to identify thousands of embryonic brain lncRNAs by a customized pipeline. A significant proportion of novel transcripts have not been annotated by available genomic resources. The putative embryonic brain lncRNAs are shorter in length, less spliced and show less conservation than known genes. The expression of putative lncRNAs is in one tenth on average of known coding genes, while comparable with known lncRNAs. From chromatin data, putative embryonic brain lncRNAs are associated with active chromatin marks, comparable with known lncRNAs. Embryonic brain expressed lncRNAs are also indicated to have expression though not evident in adult brain. Gene Ontology analysis of putative embryonic brain lncRNAs suggests that they are associated with brain development. The putative lncRNAs are shown to be related to possible cis-regulatory roles in imprinting even themselves are deemed to be imprinted lncRNAs. Re-analysis of one knockdown data suggests that four regulators are associated with lncRNAs. Taken together, the identification and systematic analysis of putative lncRNAs would provide novel insights into uncharacterized mouse non-coding regions and the relationships with mammalian embryonic brain development. PMID:23967161

Blastocyst-like structures generated solely from stem cells.

PubMed

Rivron, Nicolas C; Frias-Aldeguer, Javier; Vrij, Erik J; Boisset, Jean-Charles; Korving, Jeroen; Vivié, Judith; Truckenmüller, Roman K; van Oudenaarden, Alexander; van Blitterswijk, Clemens A; Geijsen, Niels

2018-05-01

The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells 1 . From mouse blastocysts, it is possible to derive both trophoblast 2 and embryonic stem-cell lines 3 , which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal-KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation.

Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: relating tissue engineering using adult mesenchymal stem cells to embryonic development.

PubMed

Hellingman, Catharine A; Koevoet, Wendy; Kops, Nicole; Farrell, Eric; Jahr, Holger; Liu, Wei; Baatenburg de Jong, Robert J; Frenz, Dorothy A; van Osch, Gerjo J V M

2010-02-01

Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

In utero mouse embryonic imaging with OCT for ophthalmologic research

NASA Astrophysics Data System (ADS)

Syed, Saba H.; Larina, Irina V.; Dickinson, Mary E.; Larin, Kirill V.

2011-03-01

Live imaging of an eye during embryonic development in mammalian model is important for understanding dynamic aspects of normal and abnormal eye morphogenesis. In this study, we used Swept Source Optical Coherence Tomography (SS-OCT) for live structural imaging of mouse embryonic eye through the uterine wall. The eye structure was reconstructed in mouse embryos at 13.5 to 17.5 days post coitus (dpc). Despite the limited imaging depth of OCT in turbid tissues, we were able to visualize the whole eye globe at these stages. These results suggest that live in utero OCT imaging is a useful tool to study embryonic eye development in the mouse model.

Engineering cellular fibers for musculoskeletal soft tissues using directed self-assembly.

PubMed

Schiele, Nathan R; Koppes, Ryan A; Chrisey, Douglas B; Corr, David T

2013-05-01

Engineering strategies guided by developmental biology may enhance and accelerate in vitro tissue formation for tissue engineering and regenerative medicine applications. In this study, we looked toward embryonic tendon development as a model system to guide our soft tissue engineering approach. To direct cellular self-assembly, we utilized laser micromachined, differentially adherent growth channels lined with fibronectin. The micromachined growth channels directed human dermal fibroblast cells to form single cellular fibers, without the need for a provisional three-dimensional extracellular matrix or scaffold to establish a fiber structure. Therefore, the resulting tissue structure and mechanical characteristics were determined solely by the cells. Due to the self-assembly nature of this approach, the growing fibers exhibit some key aspects of embryonic tendon development, such as high cellularity, the rapid formation (within 24 h) of a highly organized and aligned cellular structure, and the expression of cadherin-11 (indicating direct cell-to-cell adhesions). To provide a dynamic mechanical environment, we have also developed and characterized a method to apply precise cyclic tensile strain to the cellular fibers as they develop. After an initial period of cellular fiber formation (24 h postseeding), cyclic strain was applied for 48 h, in 8-h intervals, with tensile strain increasing from 0.7% to 1.0%, and at a frequency of 0.5 Hz. Dynamic loading dramatically increased cellular fiber mechanical properties with a nearly twofold increase in both the linear region stiffness and maximum load at failure, thereby demonstrating a mechanism for enhancing cellular fiber formation and mechanical properties. Tissue engineering strategies, designed to capture key aspects of embryonic development, may provide unique insight into accelerated maturation of engineered replacement tissue, and offer significant advances for regenerative medicine applications in tendon, ligament, and other fibrous soft tissues.

[Embryonic stem cells and therapeutic cloning].

PubMed

Sunde, A; Eftedal, I

2001-08-30

Increased interest in the therapeutic use of human stem cells has emerged following significant progress in ongoing research. The cloning of a sheep, the isolation of human embryonic stem cells, and the discovery that adult stem cells may be reprogrammed taken together give substance to hopes that novel principles of treatment may be developed for a variety of serious conditions. Embryonic stem cells are derived from pre-embryos at the blastocyst stage and may give rise to all bodily tissues and cells. Animal models have demonstrated that embryonic stem cells when transplanted into adult hosts may differentiate and develop into cells and tissues applicable for treatment of a variety of conditions, including Parkinson's disease, multiple sclerosis, spinal injuries, cardiac stroke and cancer. Transplanted embryonic stem cells are exposed to immune reactions similar to those acting on organ transplants, hence immunosuppression of the recipient is generally required. It is, however, possible to obtain embryonic stem cells that are genetically identical to the patient's own cells by means of therapeutic cloning techniques. The nucleus from a somatic cell is transferred into an egg after removal of the egg's own genetic material. Under specific condition the egg will use genetic information from the somatic cell in organising the formation of a blastocyst which in turn generates embryonic stem cells. These cells have a genetic composition identical to that of the patient and are suitable for stem cell therapy.

In vitro developmental model of the gastrointestinal tract from mouse embryonic stem cells.

PubMed

Torihashi, Shigeko; Kuwahara, Masaki; Kurahashi, Masaaki

2007-10-01

Mouse embryonic stem (ES) cells are pluripotent and retain their potential to form cells, tissues and organs originated from three embryonic germ layers. Recently, we developed in vitro organ--gut-like structures--from mouse ES cells. They had basically similar morphological features to a mouse gastrointestinal tract in vivo composed of three distinct layers (i.e., epithelium, connective tissue and musculature). Gut-like structures showed spontaneous contractions derived from pacemaker cells (interstitial cells of Cajal) in the musculature. We also examined their formation process and expression pattern of transcription factors crucial for gut organogenesis such as Id2, Sox17, HNF3beta/Foxa2 and GATA4. We found that they mimic the development of embryonic gut in vivo and showed a similar expression pattern of common transcription factors. They also maintain their developmental potential after transplantation to a renal capsule. Therefore, gut-like structures are suitable for in vitro models of gastrointestinal tracts and their development. In addition, we pointed out several unique features different from gut in vivo that provide useful and advantageous tools to investigate the developmental mechanism of the gastrointestinal tract.

The promise of human embryonic stem cells in aging-associated diseases

PubMed Central

Yabut, Odessa; Bernstein, Harold S.

2011-01-01

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications. PMID:21566262

Advanced technique for long term culture of epithelia in a continuous luminal-basal medium gradient.

PubMed

Schumacher, Karl; Strehl, Raimund; de, Vries Uwe; Minuth, Will W

2002-02-01

The majority of epithelia in our organism perform barrier functions on being exposed to different fluids at the luminal and basal sides. To simulate this natural situation under in vitro conditions for biomaterial testing and tissue engineering the epithelia have to withstand mechanical and fluid stress over a prolonged period of time. Leakage, edge damage and pressure differences in the culture system have to be avoided so that the epithelial barrier function is maintained. Besides, the environmental influences on important cell biological features such as, sealing or transport functions, have to remain upregulated and a loss of characteristics by dedifferentiation is prevented. Our aim is to expose embryonic renal collecting duct (CD) epithelia as model tissue for 14 days to fluid gradients and to monitor the development of tissue-specific features. For these experiments, cultured embryonic epithelia are placed in tissue carriers and in gradient containers, where different media are superfused at the luminal and basal sides. Epithelia growing on the tissue carriers act as a physiological barrier during the whole culture period. To avoid mechanical damage of the tissue and to suppress fluid pressure differences between the luminal and basal compartments improved transport of the medium and an elimination of unilaterally accumulated gas bubbles in the gradient container compartments by newly developed gas expander modules is introduced. By the application of these tools the yield of embryonic renal collecting duct epithelia with intact barrier function on a fragile natural support material could be increased significantly as compared to earlier experiments. Epithelia treated with a luminal NaCl load ranging from 3 to 24 mmol l were analyzed by immunohistochemical methods to determine the degree of differentiation. The tissue showed an upregulation of individual CD cell features as compared to embryonic epithelia in the neonatal kidney.

Direct measurement of local material properties within living embryonic tissues

NASA Astrophysics Data System (ADS)

Serwane, Friedhelm; Mongera, Alessandro; Rowghanian, Payam; Kealhofer, David; Lucio, Adam; Hockenbery, Zachary; Campàs, Otger

The shaping of biological matter requires the control of its mechanical properties across multiple scales, ranging from single molecules to cells and tissues. Despite their relevance, measurements of the mechanical properties of sub-cellular, cellular and supra-cellular structures within living embryos pose severe challenges to existing techniques. We have developed a technique that uses magnetic droplets to measure the mechanical properties of complex fluids, including in situ and in vivo measurements within living embryos ,across multiple length and time scales. By actuating the droplets with magnetic fields and recording their deformation we probe the local mechanical properties, at any length scale we choose by varying the droplets' diameter. We use the technique to determine the subcellular mechanics of individual blastomeres of zebrafish embryos, and bridge the gap to the tissue scale by measuring the local viscosity and elasticity of zebrafish embryonic tissues. Using this technique, we show that embryonic zebrafish tissues are viscoelastic with a fluid-like behavior at long time scales. This technique will enable mechanobiology and mechano-transduction studies in vivo, including the study of diseases correlated with tissue stiffness, such as cancer.

Studies of teratomas in mice: possibilities for the future production of animal models.

PubMed Central

Lehman, J. M.

1980-01-01

The murine teratoma-teratocarcinoma has become an interesting model for the study of neoplastic transformation, developmental biology, and possibly a useful system for genetic studies. These tumors arise spontaneously in 129 strain mice and can be induced in other strains by transplanting early embryos or portions of embryos into extrauterine sites. The majority of these tumors are benign, but some are capable of transplantation due to the presence of the stem cell, embryonal carcinoma, which is a multipotential cell able to proliferate and also differentiate into tissues and cell types representative of all the embryonic germ layers. It has been elegantly shown by transplantation of embryonal carcinoma cells into blastocysts which are then placed into a pseudopregnant mouse that a normal mouse is obtained composed of cells from the host blastocyst and also cells from the malignant embryonal carcinoma. Therefore, under this set of circumstances, embryonal carcinoma cells are induced to functionally differentiate into multiple cell and tissue types which are benign and able to contribute to the development of a mouse. The adaptation of the embryonal carcinoma cell to tissue culture has allowed the manipulation of these cells with subsequent selection of mutant cells which can be further transplanted into blastocysts to obtain a mouse which contains these mutant cells. If the mutant cells have populated the germ line, it may be possible to obtain a stock of mice with the lesion present in all cells. This system may be exploitable for studies in neoplasia, developmental biology, and with proper selection procedures, allow the development of new genetic strains of mice. PMID:7457573

Concentration dependent survival and neural differentiation of murine embryonic stem cells cultured on polyethylene glycol dimethacrylate hydrogels possessing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-Asp-Lle.

PubMed

Lim, Hyun Ju; Mosley, Matthew C; Kurosu, Yuki; Smith Callahan, Laura A

2017-07-01

N-cadherin cell-cell signaling plays a key role in the structure and function of the nervous system. However, few studies have incorporated bioactive signaling from n-cadherin into tissue engineering matrices. The present study uses a continuous gradient approach in polyethylene glycol dimethacrylate hydrogels to identify concentration dependent effects of n-cadherin peptide, His-Ala-Val-Asp-Lle (HAVDI), on murine embryonic stem cell survival and neural differentiation. The n-cadherin peptide was found to affect the expression of pluripotency marker, alkaline phosphatase, in murine embryonic stem cells cultured on n-cadherin peptide containing hydrogels in a concentration dependent manner. Increasing n-cadherin peptide concentrations in the hydrogels elicited a biphasic response in neurite extension length and mRNA expression of neural differentiation marker, neuron-specific class III β-tubulin, in murine embryonic stem cells cultured on the hydrogels. High concentrations of n-cadherin peptide in the hydrogels were found to increase the expression of apoptotic marker, caspase 3/7, in murine embryonic stem cells compared to that of murine embryonic stem cell cultures on hydrogels containing lower concentrations of n-cadherin peptide. Increasing the n-cadherin peptide concentration in the hydrogels facilitated greater survival of murine embryonic stem cells exposed to increasing oxidative stress caused by hydrogen peroxide exposure. The combinatorial approach presented in this work demonstrates concentration dependent effects of n-cadherin signaling on mouse embryonic stem cell behavior, underscoring the need for the greater use of systematic approaches in tissue engineering matrix design in order to understand and optimize bioactive signaling in the matrix for tissue formation. Single cell encapsulation is common in tissue engineering matrices. This eliminates cellular access to cell-cell signaling. N-cadherin, a cell-cell signaling molecule, plays a vital role in the development of neural tissues, but has not been well studied as a bioactive signaling element in neural tissue engineering matrices. The present study uses a systematic continuous gradient approach to identify concentration dependent effects of n-cadherin derived peptide, HAVDI, on the survival and neural differentiation of murine embryonic stem cells. This work underscores the need for greater use to combinatorial strategies to understand the effect complex bioactive signaling, such as n-cadherin, and the need to optimize the concentration of such bioactive signaling within tissue engineering matrices for maximal cellular response. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

[Chromosome aberration frequency in workers in tire and industrial ruber manufacture].

PubMed

Aleksandrov, S E

1982-01-01

A cytogenetic study was carried out with the view to analyse blood and embryonic tissue cultures taken from female workers of various shops of tyre and rubber industry. In workers of preparation shops the level of chromosome aberrations in blood was equal to 2.63%, while in embryonic tissues the value was 6.33%. The number of aberrations on blood and embryonic tissue exhibited by workers of chemical shops was equal to 1.34 and 2.79%, respectively. No specific differences were observed in the sub-group of women having been in frequent contacts with gasoline or curing gases, as compared with the group on the whole. Curing accelerators which are ingredients of toxic dust in preparation shops of tyre and rubber industry cause a sharp increase in the number of chromosome aberrations both in blood and in the embryonic tissues of women. Data on induced abortions may be used for evaluation of the influence of chemicals on the developing fetus and can serve as a test models of mutagenic and embryotoxic effect. They also may be regarded as a part of the general system of the evaluation of mutagenic effects of chemicals in humans.

A Novel Approach for Studying the Temporal Modulation of Embryonic Skeletal Development Using Organotypic Bone Cultures and Microcomputed Tomography

PubMed Central

Smith, Emma L.; Roberts, Carol A.

2012-01-01

Understanding the structural development of embryonic bone in a three dimensional framework is fundamental to developing new strategies for the recapitulation of bone tissue in latter life. We present an innovative combined approach of an organotypic embryonic femur culture model, microcomputed tomography (μCT) and immunohistochemistry to examine the development and modulation of the three dimensional structures of the developing embryonic femur. Isolated embryonic chick femurs were organotypic (air/liquid interface) cultured for 10 days in either basal, chondrogenic, or osteogenic supplemented culture conditions. The growth development and modulating effects of basal, chondrogenic, or osteogenic culture media of the embryonic chick femurs was investigated using μCT, immunohistochemistry, and histology. The growth and development of noncultured embryonic chick femur stages E10, E11, E12, E13, E15, and E17 were very closely correlated with increased morphometric indices of bone formation as determined by μCT. After 10 days in the organotpyic culture set up, the early aged femurs (E10 and E11) demonstrated a dramatic response to the chondrogenic or osteogenic culture conditions compared to the basal cultured femurs as determined by a change in μCT morphometric indices and modified expression of chondrogenic and osteogenic markers. Although the later aged femurs (E12 and E13) increased in size and structure after 10 days organotpypic culture, the effects of the osteogenic and chondrogenic organotypic cultures on these femurs were not significantly altered compared to basal conditions. We have demonstrated that the embryonic chick femur organotpyic culture model combined with the μCT and immunohistochemical analysis can provide an integral methodology for investigating the modulation of bone development in an ex vivo culture setting. Hence, these interdisciplinary techniques of μCT and whole organ bone cultures will enable us to delineate some of the temporal, structural developmental paradigms and modulation of bone tissue formation to underpin innovative skeletal regenerative technology for clinical therapeutic strategies in musculoskeletal trauma and diseases. PMID:22472170

Generation of Functional Thyroid Tissue Using 3D-Based Culture of Embryonic Stem Cells.

PubMed

Antonica, Francesco; Kasprzyk, Dominika Figini; Schiavo, Andrea Alex; Romitti, Mírian; Costagliola, Sabine

2017-01-01

During the last decade three-dimensional (3D) cultures of pluripotent stem cells have been intensively used to understand morphogenesis and molecular signaling important for the embryonic development of many tissues. In addition, pluripotent stem cells have been shown to be a valid tool for the in vitro modeling of several congenital or chronic human diseases, opening new possibilities to study their physiopathology without using animal models. Even more interestingly, 3D culture has proved to be a powerful and versatile tool to successfully generate functional tissues ex vivo. Using similar approaches, we here describe a protocol for the generation of functional thyroid tissue using mouse embryonic stem cells and give all the details and references for its characterization and analysis both in vitro and in vivo. This model is a valid approach to study the expression and the function of genes involved in the correct morphogenesis of thyroid gland, to elucidate the mechanisms of production and secretion of thyroid hormones and to test anti-thyroid drugs.

Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

PubMed

Qian, Chen; Wong, Carol Wing Yan; Wu, Zhongluan; He, Qiuming; Xia, Huimin; Tam, Paul Kwong Hang; Wong, Kenneth Kak Yuen; Lui, Vincent Chi Hang

2017-01-01

Platelet-derived growth factor receptor alpha (PDGFRα) is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures. To address the temporal requirement of Pdgfra in embryonic development. We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies. Current study showed that (i) conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5) resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii) the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives. Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a) the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b) if mutations / sequence variations of these regulatory elements cause these anomalies.

Case Study: Organotypic human in vitro models of embryonic ...

EPA Pesticide Factsheets

Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell-matrix interactions that drive proliferation, differentiation, and morphogenesis. Chemical low-dose exposures can disrupt morphogenesis across space and time by interfering with key embryonic fusion events. The Morphogenetic Fusion Task uses computer and in vitro models to elucidate consequences of developmental exposures. The Morphogenetic Fusion Task integrates multiple approaches to model responses to chemicals that leaad to birth defects, including integrative mining on ToxCast DB, ToxRefDB, and chemical structures, advanced computer agent-based models, and human cell-based cultures that model disruption of cellular and molecular behaviors including mechanisms predicted from integrative data mining and agent-based models. The purpose of the poster is to indicate progress on the CSS 17.02 Virtual Tissue Models Morphogenesis Task 1 products for the Board of Scientific Counselors meeting on Nov 16-17.

Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight

NASA Technical Reports Server (NTRS)

Spooner, B. S.; Hardman, P.; Paulsen, A.

1994-01-01

Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

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.

Correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced apoptotic cell death in the embryonic vasculature with embryotoxicity

USGS Publications Warehouse

Cantrell, Susannah M.; Joy-Schlezinger, Jennifer; Stegeman, John J.; Tillitt, Donald E.; Hannington, Mark D.

1998-01-01

Vertebrate embryos are particularly sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Identification of tissues that are susceptible to the adverse effects of TCDD is requisite for understanding the embryo toxic effects of TCDD. The objective of the present study was to quantitate the temporal appearance of and dose dependence of apoptosis in TCDD-exposed medaka embryos (Oryzias latipes). A fluorescent-based DNA end-labeling assay provided a sensitive method for detection of TCDD-induced apoptosis in tissue sections of medaka embryos. Apoptotic cells were readily apparent in the medial yolk vein at all observed embryonic stages in TCDD-exposed embryos. Slope-comparison analysis indicated that TCDD-induced programmed cell death in the embryonic medial yolk vein was mechanistically linked to embryo mortality. These data are consistent with the hypothesis that vascular damage contributes to the acute embryo toxic effects of TCDD. However, as sublethal concentrations of dioxin-like compounds are more typical of environmental exposures, tissue damage was also assessed in medaka fry that were exposed to low doses of TCDD during embryonic development. Cell death was detected in gill and digestive tissues in visibly healthy medaka fry that had been exposed to low doses of TCDD during embryonic development. Increased expression of cytochrome P450 1A is a major biochemical consequence of TCDD exposure and is often used as a biomarker for exposure to dioxin-like compounds. Therefore, we compared the tissue distribution of TCDD-induced P450 1A expression and TCDD-induced programmed cell death. TCDD-induced programmed cell death co-localized with TCDD-induced P450 1A expression in both embryos and in visibly healthy post-hatch fry. Our results suggest that aberrant programmed cell death may be a suitable marker for exposure of feral organisms to dioxin-like compounds.

Stem cell maintenance by manipulating signaling pathways: past, current and future

PubMed Central

Chen, Xi; Ye, Shoudong; Ying, Qi-Long

2015-01-01

Pluripotent stem cells only exist in a narrow window during early embryonic development, whereas multipotent stem cells are abundant throughout embryonic development and are retainedin various adult tissues and organs. While pluripotent stem cell lines have been established from several species, including mouse, rat, and human, it is still challenging to establish stable multipotent stem cell lines from embryonic or adult tissues. Based on current knowledge, we anticipate that by manipulating extrinsic and intrinsic signaling pathways, most if not all types of stem cells can be maintained in a long-term culture. In this article, we summarize current culture conditions established for the long-term maintenance of authentic pluripotent and multipotent stem cells and the signaling pathways involved. We also discuss the general principles of stem cell maintenance and propose several strategies on the establishment of novel stem cell lines through manipulation of signaling pathways. [BMB Reports 2015; 48(12): 668-676] PMID:26497581

Effects of chemicals and pathway inhibitors on a human in vitro model of secondary palatal fusion.

EPA Science Inventory

The mechanisms of tissue and organ formation during embryonic development are unique, but many tissues like the iris, urethra, heart, neural tube, and palate rely upon common cellular and tissue events including tissue fusion. Few human in vitro assays exist to study human embryo...

Embryonic Explant Culture: Studying Effects of Regulatory Molecules on Gene Expression in Craniofacial Tissues.

PubMed

Närhi, Katja

2017-01-01

The ex vivo culture of embryonic tissue explants permits the continuous monitoring of growth and morphogenesis at specific embryonic stages. The functions of soluble regulatory molecules can be analyzed by introducing them into culture medium or locally with beads to the tissue. Gene expression in the manipulated tissue explants can be analyzed using in situ hybridization, quantitative PCR, and reporter constructs combined to organ culture to examine the functions of the signaling molecules.

The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: potential role in temperature-dependent sex determination and testis development.

PubMed

Rhen, Turk; Jangula, Adam; Schroeder, Anthony; Woodward-Bosh, Rikki

2009-05-01

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-alpha and PdgfR-beta in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-alpha mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-beta mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-alpha and PdgfR-beta in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle.

The platelet-derived growth factor signaling system in snapping turtle embryos, Chelydra serpentina: potential role in temperature-dependent sex determination and testis development

PubMed Central

Rhen, Turk; Jangula, Adam; Schroeder, Anthony; Woodward-Bosh, Rikki

2009-01-01

The platelet-derived growth factor (Pdgf) signaling system is known to play a significant role during embryonic and postnatal development of testes in mammals and birds. In contrast, genes that comprise the Pdgf system in reptiles have never been cloned or studied in any tissue, let alone developing gonads. To explore the potential role of PDGF ligands and their receptors during embryogenesis, we cloned cDNA fragments of Pdgf-A, Pdgf-B, and receptors PdgfR-α and PdgfR-β in the snapping turtle, a reptile with temperature-dependent sex determination (TSD). We then compared gene expression profiles in gonads from embryos incubated at a male-producing temperature to those from embryos at a female-producing temperature, as well as between hatchling testes and ovaries. Expression of Pdgf-B mRNA in embryonic gonads was significantly higher at a male temperature than at a female temperature, but there was no difference between hatchling testes and ovaries. This developmental pattern was reversed for Pdgf-A and PdgfR-α mRNA: expression of these genes did not differ in embryos, but diverged in hatchling testes and ovaries. Levels of PdgfR-β mRNA in embryonic gonads were not affected by temperature and did not differ between testes and ovaries. However, expression of both receptors increased at least an order of magnitude from the embryonic to the post-hatching period. Finally, we characterized expression of these genes in several other embryonic tissues. The brain, heart, and liver displayed unique expression patterns that distinguished these tissues from each other and from intestine, lung, and muscle. Incubation temperature had a significant effect on expression of PdgfR-α and PdgfR-β in the heart but not other tissues. Together, these findings demonstrate that temperature has tissue specific effects on the Pdgf system and suggest that Pdgf signaling is involved in sex determination and the ensuing differentiation of testes in the snapping turtle. PMID:19523392

Transport of organic anions and cations in murine embryonic kidney development and in serially-reaggregated engineered kidneys

PubMed Central

Lawrence, Melanie L.; Chang, C-Hong; Davies, Jamie A.

2015-01-01

Recent advances in renal tissue engineering have shown that dissociated, early renogenic tissue from the developing embryo can self-assemble into morphologically accurate kidney-like organs arranged around a central collecting duct tree. In order for such self-assembled kidneys to be useful therapeutically or as models for drug screening, it is necessary to demonstrate that they are functional. One of the main functional characteristics of mature kidneys is transport of organic anions and cations into and out of the proximal tubule. Here, we show that the transport function of embryonic kidneys allowed to develop in culture follows a developmental time-course that is comparable to embryonic kidney development in vivo. We also demonstrate that serially-reaggregated engineered kidneys can transport organic anions and cations through specific uptake and efflux channels. These results support the physiological relevance of kidneys grown in culture, a commonly used model for kidney development and research, and suggest that serially-reaggregated kidneys self-assembled from separated cells have some functional characteristics of intact kidneys. PMID:25766625

[Cell therapy for Parkinson's disease: III. Neonatal, fetal and embryonic stem cell-based applications].

PubMed

Anisimov, S V

2009-01-01

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

Gene and protein expressions of vimentin and desmin during embryonic development of the mylohyoid muscle.

PubMed

Kishi, Asuka; Yamamoto, Masahito; Kikuchi, Akihito; Iwanuma, Osamu; Watanabe, Yutaka; Ide, Yoshinobu; Abe, Shinichi

2012-09-01

Meckel's cartilage is known to be involved in formation of the prenatal mandible. However, the relationship between Meckel's cartilage and the embryonic mylohyoid muscle during growth and development has been investigated only rarely. This study examined the expression of intermediate filaments in Meckel's cartilage and the embryonic mylohyoid muscle in fetal mice during morphological development. Specimens of E12-16 ICR mice sectioned in the frontal direction were subjected to immunohistochemistry for vimentin and desmin. Hematoxylin and eosin sections showed that the immature mylohyoid muscle began to grow along Meckel's cartilage during fetal development. Weak vimentin expression was detected in the mylohyoid muscle and surrounding tissues at E12. Desmin expression was detected specifically in the mylohyoid, and strong expression was evident after E13, and increased with age. It was inferred that the mylohyoid muscle is one the tissues developing from Meckel's cartilage, the latter exerting a continuous influence on the growth of the former. In the early stage, the surrounding mesenchymal tissues expressing vimentin formed a scaffold for the developing mylohyoid muscle. Muscle attachment at E13 showed steady desmin expression, which continued until maturity. This study suggested the possibility that Meckel's cartilage has an influence not only on the mandibular bone, but also on the development of the mylohyoid muscle attached to the mandibular bone. Furthermore, it revealed a stage of the developmental process of the mylohyoid muscle in which the expression of vimentin, which is a common protein in the surrounding tissue such as muscle and bone, induces the morphological formation of the mylohyoid muscle, cooperating with the surrounding structures.

Functional optical coherence tomography for live dynamic analysis of mouse embryonic cardiogenesis

NASA Astrophysics Data System (ADS)

Wang, Shang; Lopez, Andrew L.; Larina, Irina V.

2018-02-01

Blood flow, heart contraction, and tissue stiffness are important regulators of cardiac morphogenesis and function during embryonic development. Defining how these factors are integrated is critically important to advance prevention, diagnostics, and treatment of congenital heart defects. Mammalian embryonic development is taking place deep within the female body, which makes cardiodynamic imaging and analysis during early developmental stages in humans inaccessible. With thousands of mutant lines available and well-established genetic manipulation tools, mouse is a great model to understand how biomechanical factors are integrated with molecular pathways to regulate cardiac function and development. Dynamic imaging and quantitative analysis of the biomechanics of live mouse embryos have become increasingly important, which demands continuous advancements in imaging techniques and live assessment approaches. This has been one of the major drives to keep pushing the frontier of embryonic imaging for better resolution, higher speed, deeper penetration, and more diverse and effective contrasts. Optical coherence tomography (OCT) has played a significant role in addressing such demands, and its features in non-labeling imaging, 3D capability, a large working distance, and various functional derivatives allow OCT to cover a number of specific applications in embryonic imaging. Recently, our group has made several technical improvements in using OCT to probe the biomechanical aspects of live developing mouse embryos at early stages. These include the direct volumetric structural and functional imaging of the cardiodynamics, four-dimensional quantitative Doppler imaging and analysis of the cardiac blood flow, and fourdimensional blood flow separation from the cardiac wall tissue in the beating embryonic heart. Here, we present a short review of these studies together with brief descriptions of the previous work that demonstrate OCT as a valuable and useful imaging tool for the research in developmental cardiology.

Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues.

PubMed

Tremblay, Kimberly D; Zaret, Kenneth S

2005-04-01

The location and movement of mammalian gut tissue progenitors, prior to the expression of tissue-specific genes, has been unknown, but this knowledge is essential to identify transitions that lead to cell type specification. To address this, we used vital dyes to label exposed anterior endoderm cells of early somite stage mouse embryos, cultured the embryos into the tissue bud phase of development, and determined the tissue fate of the dye labeled cells. This approach was performed at three embryonic stages that are prior to, or coincident with, foregut tissue patterning (1-3 somites, 4-6 somites, and 7-10 somites). Short-term labeling experiments tracked the movement of tissue progenitor cells during foregut closure. Surprisingly, we found that two distinct types of endoderm-progenitor cells, lateral and medial, arising from three spatially separated embryonic domains, converge to generate the epithelial cells of the liver bud. Whereas the lateral endoderm-progenitors give rise to descendants that are constrained in tissue fate and position along the anterior-posterior axis of the gut, the medial gut endoderm-progenitors give rise to descendants that stream along the anterior-posterior axis at the ventral midline and contribute to multiple gut tissues. The fate map reveals extensive morphogenetic movement of progenitors prior to tissue specification, it permits a detailed analysis of endoderm tissue patterning, and it illustrates that diverse progenitor domains can give rise to individual tissue cell types.

Cell counting in whole mount tissue volumes using expansion OCT (Conference Presentation)

NASA Astrophysics Data System (ADS)

Liu, Yehe; Gu, Shi; Watanabe, Michiko; Rollins, Andrew M.; Jenkins, Michael W.

2017-02-01

Abnormal cell proliferation and migration during heart development can lead to severe congenital heart defects (CHDs). Studying the spatial distribution of cells during embryonic development helps our understanding of how the heart develops and the etiology of certain CHDs. However, imaging large groups of single cells in intact tissue volumes is challenging. No current technique can accomplish this task in both a time-efficient and cost-effective manner. OCT has potential with its large field of view and micron-scale resolution, but even the highest resolution OCT systems have poor contrast for counting cells and have a small field of view compared to conventional OCT. We propose using a conventional OCT system and processing the sample to enhance cellular contrast. Inspired by the recently developed Expansion Microscopy, we permeated whole-mount embryonic tissue with a superabsorbent monomer solution and polymerized into a hydrogel. When hydrated in DI water, the tissue-hydrogel complex was uniformly enlarged ( 5X in all dimensions) without distorting the microscopic structure. This had a twofold effect: it increased the resolution by a factor of 5 and decreased scattering, which allowed us to resolve cellular level features deep in the tissue with high contrast using conventional OCT. We noted that cell nuclei caused significantly more backscattering than the other subcellular structures after expansion. Based on this property, we were able to distinguish individual cell nuclei, and thus count cells, in expanded OCT images with simple intensity thresholding. We demonstrate the technique with embryonic quail hearts at various developmental stages.

Influences of Reduced Expression of Maternal Bone Morphogenetic Protein 2 on Embryonic Development

PubMed Central

Singh, Ajeet P.; Castranio, Trisha; Scott, Greg; Guo, Dayong; Harris, Marie A.; Ray, Manas; Harris, Stephan E.; Mishina, Yuji

2009-01-01

Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. During a course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeting events in embryonic stem cells, in the 3’ untranslated region of exon 3 of Bmp2, reduced the expression levels of Bmp2 both in embryonic and maternal tissues. Some of the embryos that were genotyped as transheterozygous for the floxed allele with the neo cassette over the conventional null allele (fn/−) showed a lethal phenotype including defects in cephalic neural tube closure and ventral abdominal wall closure. Embryos exhibiting these abnormalities were increased when genotypes of the pregnant females were different; when expression levels of Bmp2 in maternal tissues were lower, a larger proportion of fn/− embryos exhibit these abnormalities. These results suggest that the expression levels of Bmp2 together in both in embryonic and maternal tissues influence the normal neural tube closure and body wall closure with different thresholds. PMID:18769073

Localization of trefoil factor family peptide 3 (TFF3) in epithelial tissues originating from the three germ layers of developing mouse embryo.

PubMed

Bijelić, Nikola; Belovari, Tatjana; Tolušić Levak, Maja; Baus Lončar, Mirela

2017-08-20

Trefoil factor family (TFF) peptides are involved in the maintenance of epithelial integrity and epithelial restitution. Mature epithelial tissues originate from different embryonic germ layers. The objective of this research was to explore the presence and localization of TFF3 peptide in mouse embryonic epithelia and to examine if the occurrence of TFF3 peptide is germ layer-dependent. Mouse embryos (14-18 days old) were fixed in 4% paraformaldehyde and embedded in paraffin. Immunohistochemistry was performed with affinity purified rabbit anti-TFF3 antibody, goat anti-rabbit biotinylated secondary antibody and streptavidin-horseradish peroxidase, followed by 3,3'-diaminobenzidine. TFF3 peptide was present in the gastric and intestinal mucosa, respiratory mucosa in the upper and lower airways, pancreas, kidney tubules, epidermis, and oral cavity. The presence and localization of TFF3 peptide was associated with the embryonic stage and tissue differentiation. TFF3 peptide distribution specific to the germ layers was not observed. The role of TFF3 peptide in cell migration and differentiation, immune response, and apoptosis might be associated with specific embryonic epithelial cells. TFF3 peptide may also be considered as a marker for mucosal maturation.

Origin and Functions of Tissue Macrophages

PubMed Central

Epelman, Slava; Lavine, Kory J.; Randolph, Gwendalyn J.

2015-01-01

Macrophages are distributed in tissues throughout the body and contribute to both homeostasis and disease. Recently, it has become evident that most adult tissue macrophages originate during embryonic development and not from circulating monocytes. Each tissue has its own composition of embryonically derived and adult-derived macrophages, but it is unclear whether macrophages of distinct origins are functionally interchangeable or have unique roles at steady state. This new understanding also prompts reconsideration of the function of circulating monocytes. Classical Ly6chi monocytes patrol the extravascular space in resting organs, and Ly6clo nonclassical monocytes patrol the vasculature. Inflammation triggers monocytes to differentiate into macrophages, but whether resident and newly recruited macrophages possess similar functions during inflammation is unclear. Here, we define the tools used for identifying the complex origin of tissue macrophages and discuss the relative contributions of tissue niche versus ontological origin to the regulation of macrophage functions during steady state and inflammation. PMID:25035951

Combination Chemotherapy in Treating Patients With Previously Untreated Rhabdomyosarcoma

ClinicalTrials.gov

2013-06-13

Adult Malignant Mesenchymoma; Adult Rhabdomyosarcoma; Alveolar Childhood Rhabdomyosarcoma; Childhood Malignant Mesenchymoma; Embryonal Childhood Rhabdomyosarcoma; Embryonal-botryoid Childhood Rhabdomyosarcoma; Nonmetastatic Childhood Soft Tissue Sarcoma; Previously Untreated Childhood Rhabdomyosarcoma; Stage I Adult Soft Tissue Sarcoma; Stage II Adult Soft Tissue Sarcoma; Stage III Adult Soft Tissue Sarcoma

Isolation and expression of homeobox genes from the embryonic chicken eye.

PubMed

Dhawan, R R; Schoen, T J; Beebe, D C

1997-06-11

To identify homeobox-containing genes that may play a role in the differentiation of ocular tissues. Total RNA was isolated from microdissected chicken embryo eye tissues at 3.5 days of development (embryonic day 3.5; E3.5). An "anchor-oligo-dT primer" was used for the synthesis of cDNA. Degenerate oligonucleotides designed from highly-conserved sequences in the third helix of the homeobox and the "anchor-primer" were used to amplify cDNAs by polymerase chain reaction (PCR). PCR products were cloned and sequenced. The spatial and temporal expression of selected transcripts was mapped by whole-mount in situ hybridization and northern blot analysis. After sequencing eighteen clones we identified a member of the distal-less family (dlx-3) in cDNA from presumptive neural retina and three chicken homologs of the Xenopus "anterior neural fold" (Xanf-1) in cDNA from anterior eye tissue. Dlx transcripts were mapped by in situ hybridization. Expression began at Hamburger and Hamilton stage 14 (E2.5) and was widely distributed in embryonic mesenchyme on E3 and E4. Expression increased in the retina during early development and persisted until after hatching. The one anf clone selected for further study was not detected by in situ or northern blot analysis. It is feasible to isolate homeobox cDNAs directly from microdissected embryonic tissues. Chicken dlx-3 mRNA has a wider distribution in the embryo than expected, based on the expression of the mouse homolog. Dlx-3 may play a role in establishing or maintaining the differentiation of the retina.

Can physics help to explain embryonic development? An overview.

PubMed

Fleury, V

2013-10-01

Recent technical advances including digital imaging and particle image velocimetry can be used to extract the full range of embryonic movements that constitute the instantaneous 'morphogenetic fields' of a developing animal. The final shape of the animal results from the sum over time (integral) of the movements that make up the velocity fields of all the tissue constituents. In vivo microscopy can be used to capture the details of vertebrate development at the earliest embryonic stages. The movements thus observed can be quantitatively compared to physical models that provide velocity fields based on simple hypotheses about the nature of living matter (a visco-elastic gel). This approach has cast new light on the interpretation of embryonic movement, folding, and organisation. It has established that several major discontinuities in development are simple physical changes in boundary conditions. In other words, with no change in biology, the physical consequences of collisions between folds largely explain the morphogenesis of the major structures (such as the head). Other discontinuities result from changes in physical conditions, such as bifurcations (changes in physical behaviour beyond specific yield points). For instance, beyond a certain level of stress, a tissue folds, without any new gene being involved. An understanding of the physical features of movement provides insights into the levers that drive evolution; the origin of animals is seen more clearly when viewed under the light of the fundamental physical laws (Newton's principle, action-reaction law, changes in symmetry breaking scale). This article describes the genesis of a vertebrate embryo from the shapeless stage (round mass of tissue) to the development of a small, elongated, bilaterally symmetric structure containing vertebral precursors, hip and shoulder enlarges, and a head. Copyright © 2013. Published by Elsevier Masson SAS.

Impaired intervertebral disc development and premature disc degeneration in mice with notochord-specific deletion of CCN2.

PubMed

Bedore, Jake; Sha, Wei; McCann, Matthew R; Liu, Shangxi; Leask, Andrew; Séguin, Cheryle A

2013-10-01

Currently, our ability to treat intervertebral disc (IVD) degeneration is hampered by an incomplete understanding of disc development and aging. The specific function of matricellular proteins, including CCN2, during these processes remains an enigma. The aim of this study was to determine the tissue-specific localization of CCN proteins and to characterize their role in IVD tissues during embryonic development and age-related degeneration by using a mouse model of notochord-specific CCN2 deletion. Expression of CCN proteins was assessed in IVD tissues from wild-type mice beginning on embryonic day 15.5 to 17 months of age. Given the enrichment of CCN2 in notochord-derived tissues, we generated notochord-specific CCN2-null mice to assess the impact on the IVD structure and extracellular matrix composition. Using a combination of histologic evaluation and magnetic resonance imaging (MRI), IVD health was assessed. Loss of the CCN2 gene in notochord-derived cells disrupted the formation of IVDs in embryonic and newborn mice, resulting in decreased levels of aggrecan and type II collagen and concomitantly increased levels of type I collagen within the nucleus pulposus. CCN2-knockout mice also had altered expression of CCN1 (Cyr61) and CCN3 (Nov). Mirroring its role during early development, notochord-specific CCN2 deletion accelerated age-associated degeneration of IVDs. Using a notochord-specific gene targeting strategy, this study demonstrates that CCN2 expression by nucleus pulposus cells is essential to the regulation of IVD development and age-associated tissue maintenance. The ability of CCN2 to regulate the composition of the intervertebral disc suggests that it may represent an intriguing clinical target for the treatment of disc degeneration. Copyright © 2013 by the American College of Rheumatology.

Combination Chemotherapy and Radiation Therapy in Treating Patients With Newly Diagnosed Rhabdomyosarcoma

ClinicalTrials.gov

2017-06-27

Adult Malignant Mesenchymoma; Adult Rhabdomyosarcoma; Childhood Alveolar Rhabdomyosarcoma; Childhood Botryoid-Type Embryonal Rhabdomyosarcoma; Childhood Embryonal Rhabdomyosarcoma; Childhood Malignant Mesenchymoma; Non-Metastatic Childhood Soft Tissue Sarcoma; Stage I Adult Soft Tissue Sarcoma; Stage II Adult Soft Tissue Sarcoma; Stage III Adult Soft Tissue Sarcoma; Untreated Childhood Rhabdomyosarcoma

Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

PubMed

Preynat-Seauve, Olivier; Suter, David M; Tirefort, Diderik; Turchi, Laurent; Virolle, Thierry; Chneiweiss, Herve; Foti, Michelangelo; Lobrinus, Johannes-Alexander; Stoppini, Luc; Feki, Anis; Dubois-Dauphin, Michel; Krause, Karl Heinz

2009-03-01

Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic tissue-inducing culture.

PubMed

Sakakura, Eriko; Eiraku, Mototsugu; Takata, Nozomu

2016-08-01

The eyes are subdivided from the rostral diencephalon in early development. How the neuroectoderm regulates this subdivision, however, is largely unknown. Taking advantage of embryonic stem cell (ESC) culture using a Rax reporter line to monitor rostral diencephalon formation, we found that ESC-derived tissues at day 7 grown in Glasgow Minimum Expression Media (GMEM) containing knockout serum replacement (KSR) exhibited higher levels of expression of axin2, a Wnt target gene, than those grown in chemically defined medium (CDM). Surprisingly, Wnt agonist facilitated eye field-like tissue specification in CDM. In contrast, the addition of Wnt antagonist diminished eye field tissue formation in GMEM+KSR. Furthermore, the morphological formation of the eye tissue anlage, including the optic vesicle, was accompanied by Wnt signaling activation. Additionally, using CDM culture, we developed an efficient method for generating Rax+/Chx10+ retinal progenitors, which could become fully stratified retina. Here we provide a new avenue for exploring the mechanisms of eye field specification in vitro. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Osteopontin is a Novel Marker of Pancreatic Ductal Tissues and of Undifferentiated Pancreatic Precursors in Mice

PubMed Central

Kilic, Gamze; Wang, Junfeng; Sosa-Pineda, Beatriz

2008-01-01

Matricellular proteins mediate both tissue morphogenesis and tissue homeostasis in important ways because they modulate cell-matrix and cell-cell interactions. In this study, we found that the matricellular protein osteopontin (Opn) is a novel marker of undifferentiated pancreatic precursors and pancreatic ductal tissues in mice. Our analysis also underscored a specific, dynamic profile of Opn expression in embryonic pancreatic tissues that suggests the participation of this protein’s function in processes involving cell migration, cell-cell interactions, or both. Surprisingly, our analysis of Opn-deficient pancreata did not reveal obvious alterations in the morphology or differentiation of these tissues. Therefore, in embryonic pancreatic tissues, it is possible that other proteins act redundantly to Opn or that this protein’s function is dispensable for pancreas development. Finally, the maintenance of Opn expression in pancreatic tissues of adults argues for a possible function of this protein in injury and pathologic responses. PMID:16518820

DNA methylation analysis of the gene CDKN2B in Gallus gallus (chicken).

PubMed

Gryzińska, Magdalena; Andraszek, Katarzyna; Jocek, Grzegorz

2013-01-01

Methylation is an epigenetic modification of DNA affecting gene expression without changing the structure of nucleotides. It plays a crucial role in the embryonic and post-embryonic development of living organisms. Methylation level is tissue and species-specific and changes with age. The study was aimed at identifying the methylation of the CDKN2B gene situated at locus bar in Polbar chickens on the 6th and 18th day of embryonic development using the MSP (methylation-specific PCR) method. Methylation was not detected in the promoter region of gene CDKN2B on the 6th and 18th day of embryonic development. As one of the five genes responsible for melanine activity in melanocytes and highly active, it can contribute to the production of this pigment. The present research broadens the current knowledge of the chicken epigenome and the mechanism of autosexing in birds.

Redeployment of germ layers related TFs shows regionalized expression during two non-embryonic developments.

PubMed

Ricci, Lorenzo; Cabrera, Fabien; Lotito, Sonia; Tiozzo, Stefano

2016-08-01

In all non-vertebrate metazoan phyla, species that evolved non-embryonic developmental pathways as means of propagation or regeneration can be found. In this context, new bodies arise through asexual reproduction processes (such as budding) or whole body regeneration, that lack the familiar temporal and spatial cues classically associated with embryogenesis, like maternal determinants, or gastrulation. The molecular mechanisms underlying those non-embryonic developments (i.e., regeneration and asexual reproduction), and their relationship to those deployed during embryogenesis are poorly understood. We have addressed this question in the colonial ascidian Botryllus schlosseri, which undergoes an asexual reproductive process via palleal budding (PB), as well as a whole body regeneration by vascular budding (VB). We identified early regenerative structures during VB and then followed the fate of differentiating tissues during both non-embryonic developments (PB and VB) by monitoring the expression of genes known to play key functions in germ layer specification with well conserved expression patterns in solitary ascidian embryogenesis. The expression patterns of FoxA1, GATAa, GATAb, Otx, Bra, Gsc and Tbx2/3 were analysed during both PB and VB. We found that the majority of these transcription factors were expressed during both non-embryonic developmental processes, revealing a regionalization of the palleal and vascular buds. Knockdown of GATAa by siRNA in palleal buds confirmed that preventing the correct development of one of these regions blocks further tissue specification. Our results indicate that during both normal and injury-induced budding, a similar alternative developmental program operates via early commitment of epithelial regions. Copyright © 2016. Published by Elsevier Inc.

Surgical inflammatory stress: the embryo takes hold of the reins again

PubMed Central

2013-01-01

The surgical inflammatory response can be a type of high-grade acute stress response associated with an increasingly complex trophic functional system for using oxygen. This systemic neuro-immune-endocrine response seems to induce the re-expression of 2 extraembryonic-like functional axes, i.e. coelomic-amniotic and trophoblastic-yolk-sac-related, within injured tissues and organs, thus favoring their re-development. Accordingly, through the up-regulation of two systemic inflammatory phenotypes, i.e. neurogenic and immune-related, a gestational-like response using embryonic functions would be induced in the patient’s injured tissues and organs, which would therefore result in their repair. Here we establish a comparison between the pathophysiological mechanisms that are produced during the inflammatory response and the physiological mechanisms that are expressed during early embryonic development. In this way, surgical inflammation could be a high-grade stress response whose pathophysiological mechanisms would be based on the recapitulation of ontogenic and phylogenetic-related functions. Thus, the ultimate objective of surgical inflammation, as a gestational process, is creating new tissues/organs for repairing the injured ones. Since surgical inflammation and early embryonic development share common production mechanisms, the factors that hamper the wound healing reaction in surgical patients could be similar to those that impair the gestational process. PMID:23374964

A 3D magnetic tissue stretcher for remote mechanical control of embryonic stem cell differentiation.

PubMed

Du, Vicard; Luciani, Nathalie; Richard, Sophie; Mary, Gaëtan; Gay, Cyprien; Mazuel, François; Reffay, Myriam; Menasché, Philippe; Agbulut, Onnik; Wilhelm, Claire

2017-09-12

The ability to create a 3D tissue structure from individual cells and then to stimulate it at will is a major goal for both the biophysics and regenerative medicine communities. Here we show an integrated set of magnetic techniques that meet this challenge using embryonic stem cells (ESCs). We assessed the impact of magnetic nanoparticles internalization on ESCs viability, proliferation, pluripotency and differentiation profiles. We developed magnetic attractors capable of aggregating the cells remotely into a 3D embryoid body. This magnetic approach to embryoid body formation has no discernible impact on ESC differentiation pathways, as compared to the hanging drop method. It is also the base of the final magnetic device, composed of opposing magnetic attractors in order to form embryoid bodies in situ, then stretch them, and mechanically stimulate them at will. These stretched and cyclic purely mechanical stimulations were sufficient to drive ESCs differentiation towards the mesodermal cardiac pathway.The development of embryoid bodies that are responsive to external stimuli is of great interest in tissue engineering. Here, the authors culture embryonic stem cells with magnetic nanoparticles and show that the presence of magnetic fields could affect their aggregation and differentiation.

Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice.

PubMed

Nakagawa, Kimie; Sawada, Natsumi; Hirota, Yoshihisa; Uchino, Yuri; Suhara, Yoshitomo; Hasegawa, Tomoka; Amizuka, Norio; Okamoto, Tadashi; Tsugawa, Naoko; Kamao, Maya; Funahashi, Nobuaki; Okano, Toshio

2014-01-01

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

Proinsulin in development: New roles for an ancient prohormone.

PubMed

Hernández-Sánchez, C; Mansilla, A; de la Rosa, E J; de Pablo, F

2006-06-01

In postnatal organisms, insulin is well known as an essential anabolic hormone responsible for maintaining glucose homeostasis. Its biosynthesis by the pancreatic beta cell has been considered a model of tissue-specific gene expression. However, proinsulin mRNA and protein have been found in embryonic stages before the formation of the pancreatic primordium, and later, in extrapancreatic tissues including the nervous system. Phylogenetic studies have also confirmed that production of insulin-like peptides antecedes the morphogenesis of a pancreas, and that these peptides contribute to normal development. In recent years, other roles for insulin distinct from its metabolic function have emerged also in vertebrates. During embryonic development, insulin acts as a survival factor and is involved in early morphogenesis. These findings are consistent with the observation that, at these stages, the proinsulin gene product remains as the precursor form, proinsulin. Independent of its low metabolic activity, proinsulin stimulates proliferation in developing neuroretina, as well as cell survival and cardiogenesis in early embryos. Insulin/proinsulin levels are finely regulated during development, since an excess of the protein interferes with correct morphogenesis and is deleterious for the embryo. This fine-tuned regulation is achieved by the expression of alternative embryonic proinsulin transcripts that have diminished translational activity.

Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi.

PubMed

Takata, Nozomu; Sakakura, Eriko; Sakuma, Tetsushi; Yamamoto, Takashi

2017-01-01

Approaches to investigate gene functions in experimental biology are becoming more diverse and reliable. Furthermore, several kinds of tissues and organs that possess their original identities can be generated in petri dishes from stem cells including embryonic, adult and induced pluripotent stem cells. Researchers now have several choices of experimental methods and their combinations to analyze gene functions in various biological systems. Here, as an example we describe one of the better protocols, which combines three-dimensional embryonic stem cell culture with small regulatory RNA-mediated technologies, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), and inducible RNA interference (RNAi). This protocol allows investigation of genes of interest to better understand gene functions in target tissues (or organs) during in vitro development.

ChIP-seq Identification of Weakly Conserved Heart Enhancers

PubMed Central

Blow, Matthew J.; McCulley, David J.; Li, Zirong; Zhang, Tao; Akiyama, Jennifer A.; Holt, Amy; Plajzer-Frick, Ingrid; Shoukry, Malak; Wright, Crystal; Chen, Feng; Afzal, Veena; Bristow, James; Ren, Bing; Black, Brian L.; Rubin, Edward M.; Visel, Axel; Pennacchio, Len A.

2011-01-01

Accurate control of tissue-specific gene expression plays a pivotal role in heart development, but few cardiac transcriptional enhancers have thus far been identified. Extreme non-coding sequence conservation successfully predicts enhancers active in many tissues, but fails to identify substantial numbers of heart enhancers. Here we used ChIP-seq with the enhancer-associated protein p300 from mouse embryonic day 11.5 heart tissue to identify over three thousand candidate heart enhancers genome-wide. Compared to other tissues studied at this time-point, most candidate heart enhancers are less deeply conserved in vertebrate evolution. Nevertheless, the testing of 130 candidate regions in a transgenic mouse assay revealed that most of them reproducibly function as enhancers active in the heart, irrespective of their degree of evolutionary constraint. These results provide evidence for a large population of poorly conserved heart enhancers and suggest that the evolutionary constraint of embryonic enhancers can vary depending on tissue type. PMID:20729851

Spatial distribution of endogenous retinoids in the murine embryonic mandible.

PubMed

Kronmiller, J E; Beeman, C S

1994-12-01

Retinoids play an important part in pattern formation during embryonic development. Exogenous retinoids alter the pattern of skeletal, neural and odontogenic tissues. Endogenous retinoids have been demonstrated previously in the murine embryonic mandible, reaching a concentration peak during the initiation of odontogenesis. It was now found that endogenous retinoids are present in a concentration gradient in the embryonic mouse mandible at the time of the initiation of the dental lamina. All-trans-retinoic acid was more concentrated in the incisor region and retinol in the molar region. These results, and the fact that exogenous retinoids produce supernumerary incisors and missing molars, suggest that all-trans-retinoic acid may instruct incisor morphology.

Effect of allo- and xenotransplantation of embryonic nervous tissue and umbilical cord blood-derived stem cells on structural and functional state of cerebral cortex of albino rats in posttraumatic period.

PubMed

Ereniev, S I; Semchenko, V V; Sysheva, E V; Bogdashin, I V; Shapovalova, V V; Khizhnyak, A S; Gasanenko, L N

2005-11-01

Comparative study of the structural and functional state of cerebral cortex of adult albino rats after intracerebral allo- and xenotransplantation of embryonic nervous tissue and intravenous injection of umbilical cord blood-derived stem cells at different terms after diffuse-focal cerebral trauma revealed the best cerebroprotective effect on day 7 of posttraumatic period in animals receiving embryonic nervous tissue.

Skeletal tissue engineering using mesenchymal or embryonic stem cells: clinical and experimental data.

PubMed

Gamie, Zakareya; MacFarlane, Robert J; Tomkinson, Alicia; Moniakis, Alexandros; Tran, Gui Tong; Gamie, Yehya; Mantalaris, Athanasios; Tsiridis, Eleftherios

2014-11-01

Mesenchymal stem cells (MSCs) can be obtained from a wide variety of tissues for bone tissue engineering such as bone marrow, adipose, birth-associated, peripheral blood, periosteum, dental and muscle. MSCs from human fetal bone marrow and embryonic stem cells (ESCs) are also promising cell sources. In vitro, in vivo and clinical evidence was collected using MEDLINE® (1950 to January 2014), EMBASE (1980 to January 2014) and Google Scholar (1980 to January 2014) databases. Enhanced results have been found when combining bone marrow-derived mesenchymal stem cells (BMMSCs) with recently developed scaffolds such as glass ceramics and starch-based polymeric scaffolds. Preclinical studies investigating adipose tissue-derived stem cells and umbilical cord tissue-derived stem cells suggest that they are likely to become promising alternatives. Stem cells derived from periosteum and dental tissues such as the periodontal ligament have an osteogenic potential similar to BMMSCs. Stem cells from human fetal bone marrow have demonstrated superior proliferation and osteogenic differentiation than perinatal and postnatal tissues. Despite ethical concerns and potential for teratoma formation, developments have also been made for the use of ESCs in terms of culture and ideal scaffold.

A novel perfused rotary bioreactor for cardiomyogenesis of embryonic stem cells.

PubMed

Teo, Ailing; Mantalaris, Athanasios; Song, Kedong; Lim, Mayasari

2014-05-01

Developments in bioprocessing technology play an important role for overcoming challenges in cardiac tissue engineering. To this end, our laboratory has developed a novel rotary perfused bioreactor for supporting three-dimensional cardiac tissue engineering. The dynamic culture environments provided by our novel perfused rotary bioreactor and/or the high-aspect rotating vessel produced constructs with higher viability and significantly higher cell numbers (up to 4 × 10(5) cells/bead) than static tissue culture flasks. Furthermore, cells in the perfused rotary bioreactor showed earlier gene expressions of cardiac troponin-T, α- and β-myosin heavy chains with higher percentages of cardiac troponin-I-positive cells and better uniformity of sacromeric α-actinin expression. A dynamic and perfused environment, as provided by this bioreactor, provides a superior culture performance in cardiac differentiation for embryonic stem cells particularly for larger 3D constructs.

Long-range ordered vorticity patterns in living tissue induced by cell division

NASA Astrophysics Data System (ADS)

Rossen, Ninna S.; Tarp, Jens M.; Mathiesen, Joachim; Jensen, Mogens H.; Oddershede, Lene B.

2014-12-01

In healthy blood vessels with a laminar blood flow, the endothelial cell division rate is low, only sufficient to replace apoptotic cells. The division rate significantly increases during embryonic development and under halted or turbulent flow. Cells in barrier tissue are connected and their motility is highly correlated. Here we investigate the long-range dynamics induced by cell division in an endothelial monolayer under non-flow conditions, mimicking the conditions during vessel formation or around blood clots. Cell divisions induce long-range, well-ordered vortex patterns extending several cell diameters away from the division site, in spite of the system’s low Reynolds number. Our experimental results are reproduced by a hydrodynamic continuum model simulating division as a local pressure increase corresponding to a local tension decrease. Such long-range physical communication may be crucial for embryonic development and for healing tissue, for instance around blood clots.

Vertebrate homologues of Frodo are dynamically expressed during embryonic development in tissues undergoing extensive morphogenetic movements.

PubMed

Hunter, Nina L; Hikasa, Hiroki; Dymecki, Susan M; Sokol, Sergei Y

2006-01-01

Frodo has been identified as a protein interacting with Dishevelled, an essential mediator of the Wnt signaling pathway, critical for the determination of cell fate and polarity in embryonic development. In this study, we use specific gene probes to characterize stage- and tissue-specific expression patterns of the mouse Frodo homologue and compare them with Frodo expression patterns in Xenopus embryos. In situ hybridization analysis of mouse Frodo transcripts demonstrates that, similar to Xenopus Frodo, mouse Frodo is expressed in primitive streak mesoderm, neuroectoderm, neural crest, presomitic mesoderm, and somites. In many cases, Frodo expression is confined to tissues undergoing extensive morphogenesis, suggesting that Frodo may be involved in the regulation of cell shape and motility. Highly conserved dynamic expression patterns of Frodo homologues indicate a similar function for these proteins in different vertebrates. 2005 Wiley-Liss, Inc.

Adult Human Gingival Epithelial Cells as a Source for Whole-tooth Bioengineering

PubMed Central

Angelova Volponi, A.; Kawasaki, M.; Sharpe, P.T.

2013-01-01

Teeth develop from interactions between embryonic oral epithelium and neural-crest-derived mesenchyme. These cells can be separated into single-cell populations and recombined to form normal teeth, providing a basis for bioengineering new teeth if suitable, non-embryonic cell sources can be identified. We show here that cells can be isolated from adult human gingival tissue that can be expanded in vitro and, when combined with mouse embryonic tooth mesenchyme cells, form teeth. Teeth with developing roots can be produced from this cell combination following transplantation into renal capsules. These bioengineered teeth contain dentin and enamel with ameloblast-like cells and rests of Malassez of human origin. PMID:23458883

[Stem cells--cloning, plasticity, bioethic].

PubMed

Pflegerl, Pamina; Keller, Thomas; Hantusch, Brigitte; Hoffmann, Thomas Sören; Kenner, Lukas

2008-01-01

Stem cells with certain characteristics have become promising tools for molecular medicine. They have the potential to self-regenerate and to differentiate into specific tissues. Besides their great potential, embryonic stem cells (ESC) run the risk of enhanced tumorigenesis. The use of human embryonic stem cells (hESC) is ethically problematic because their isolation involves the destruction of human embryos. Recently developed methods generate are able to pluripotent stem cells from fibroblasts. Alternatives for ESC are adult stem cells (ASC) derived from bone marrow, cord blood, amniotic fluid and other tissues. The following article is on the basis of testimony of Lukas Kenner for the German Bundestag about the use of ESC for research, therapy and drug development. Ethical aspects are taken into consideration.

Initiating head development in mouse embryos: integrating signalling and transcriptional activity.

PubMed

Arkell, Ruth M; Tam, Patrick P L

2012-03-01

The generation of an embryonic body plan is the outcome of inductive interactions between the progenitor tissues that underpin their specification, regionalization and morphogenesis. The intercellular signalling activity driving these processes is deployed in a time- and site-specific manner, and the signal strength must be precisely controlled. Receptor and ligand functions are modulated by secreted antagonists to impose a dynamic pattern of globally controlled and locally graded signals onto the tissues of early post-implantation mouse embryo. In response to the WNT, Nodal and Bone Morphogenetic Protein (BMP) signalling cascades, the embryo acquires its body plan, which manifests as differences in the developmental fate of cells located at different positions in the anterior-posterior body axis. The initial formation of the anterior (head) structures in the mouse embryo is critically dependent on the morphogenetic activity emanating from two signalling centres that are juxtaposed with the progenitor tissues of the head. A common property of these centres is that they are the source of antagonistic factors and the hub of transcriptional activities that negatively modulate the function of WNT, Nodal and BMP signalling cascades. These events generate the scaffold of the embryonic head by the early-somite stage of development. Beyond this, additional tissue interactions continue to support the growth, regionalization, differentiation and morphogenesis required for the elaboration of the structure recognizable as the embryonic head.

Transcriptional Profiling of Ectoderm Specification to Keratinocyte Fate in Human Embryonic Stem Cells

PubMed Central

Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

2015-01-01

In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ–secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions. PMID:25849374

Embryonic Stem Cells Contribute to Mouse Chimeras in the Absence of Detectable Cell Fusion

PubMed Central

Kidder, Benjamin L.; Oseth, Leann; Miller, Shanna; Hirsch, Betsy; Verfaillie, Catherine

2008-01-01

Abstract Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras. PMID:18338954

Relationship between delayed embryonic development and metabolic factors and fat deposition in fruit bat Cynopterus sphinx.

PubMed

Banerjee, Arnab; Meenakumari, K J; Krishna, Amitabh

2007-01-01

The present study was undertaken in the fruit bat Cynopterus sphinx, which breeds twice in quick succession at Varanasi, India. Its gestation period varies significantly in the two successive pregnancies of the year owing to delayed embryonic development during the first (winter) pregnancy. The primary aim of the present study was to determine the role of metabolic factors in delayed embryonic development in the fruit bat C. sphinx. Variation in bodyweight, fat deposition, oxygen (O(2)) consumption rate, basal metabolic rate (BMR), body temperature (Tb) and hepatic succinate dehydrogenase (SDH) activity, along with circulating levels of thyroid hormones (tri-iodothyronine and thyroxine), were examined as metabolic factors during the two successive pregnancies in C. sphinx. The increase in bodyweight observed in November was due to accumulation of white adipose tissue in the posterior abdominal region. A significant decline in O(2) consumption rate, BMR, Tb and SDH activity was found in early winter in November-December, which coincides closely with the period of fat accumulation and with the period of delayed embryonic development in C. sphinx. A significantly higher O(2) consumption rate, BMR, Tb and SDH activity was noted during the second pregnancy in, when embryonic development was relatively faster. Thyroid hormone levels were high during the period of embryonic delay compared with levels during the remaining months. The results of the present study suggest that the delayed embryonic development in C. sphinx during early winter may be due to a low O(2) consumption rate, BMR, Tb and SDH activity in November-December. The energy saved by suppressing embryonic development in this species may be advantageous for fat accumulation. Increased thyroid hormone levels during the early winter period might facilitate fat accumulation in C. sphinx.

A New Wnt1-CRE TomatoRosa Embryonic Stem Cell Line: A Tool for Studying Neural Crest Cell Integration Capacity.

PubMed

Acuna-Mendoza, Soledad; Martin, Sabrina; Kuchler-Bopp, Sabine; Ribes, Sandy; Thalgott, Jérémy; Chaussain, Catherine; Creuzet, Sophie; Lesot, Hervé; Lebrin, Franck; Poliard, Anne

2017-12-01

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 Rosa TomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.

Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice.

PubMed

Rosen, Chava; Shezen, Elias; Aronovich, Anna; Klionsky, Yael Zlotnikov; Yaakov, Yasmin; Assayag, Miri; Biton, Inbal Eti; Tal, Orna; Shakhar, Guy; Ben-Hur, Herzel; Shneider, David; Vaknin, Zvi; Sadan, Oscar; Evron, Shmuel; Freud, Enrique; Shoseyov, David; Wilschanski, Michael; Berkman, Neville; Fibbe, Willem E; Hagin, David; Hillel-Karniel, Carmit; Krentsis, Irit Milman; Bachar-Lustig, Esther; Reisner, Yair

2015-08-01

Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

Very small embryonic-like (VSEL) stem cells in adult organs and their potential role in rejuvenation of tissues and longevity

PubMed Central

Ratajczak, Mariusz Z.; Zuba-Surma, Ewa K.; Shin, Dong-Myung; Ratajczak, Janina; Kucia, Magda

2011-01-01

Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4+) small stem cells (SCs) from the murine bone marrow (BM) that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs (VSELs). Our data indicate that VSELs are also present in many other organs in mice and that they may differentiate into cells from all three germ layers. Similar SCs were also isolated from human cord blood (CB) and mobilized peripheral blood (mPB). We hypothesize that VSELs are deposited during gastrulation and organogenesis in developing organs/tissues of mammals as a population of pluripotent stem cells (PSCs) that give rise to tissue committed monopotent SCs and that their number decreases with age. Therefore VSELs could play a pivotal role in normal rejuvenation of adult tissues as well as involvement in regeneration of damaged organs. Thus, these cells are potential SCs candidates for regenerative medicine and we envision that the regenerative potential of these cells could be harnessed to decelerate the aging processes. PMID:18601995

Genetic deletion of the EGFR ligand epigen does not affect mouse embryonic development and tissue homeostasis.

PubMed

Dahlhoff, Maik; Schäfer, Matthias; Wolf, Eckhard; Schneider, Marlon R

2013-02-15

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor with manifold functions during development, tissue homeostasis and disease. EGFR activation, the formation of homodimers or heterodimers (with the related ERBB2-4 receptors) and downstream signaling is initiated by the binding of a family of structurally related growth factors, the EGFR ligands. Genetic deletion experiments clarified the biological function of all family members except for the last characterized ligand, epigen. We employed gene targeting in mouse embryonic stem cells to generate mice lacking epigen expression. Loss of epigen did not affect mouse development, fertility, or organ physiology. Quantitative RT-PCR analysis revealed increased expression of betacellulin and EGF in a few organs of epigen-deficient mice, suggesting a functional compensation by these ligands. In conclusion, we completed the genetic analysis of EGFR ligands and show that epigen has non-essential functions or functions that can be compensated by other EGFR ligands during growth and tissue homeostasis. Copyright © 2012 Elsevier Inc. All rights reserved.

Early zebrafish development: It’s in the maternal genes

PubMed Central

Abrams, Elliott W.; Mullins, Mary C.

2009-01-01

Summary The earliest stages of embryonic development in all animals examined rely on maternal gene products that are generated during oogenesis and supplied to the egg. The period of maternal control of embryonic development varies among animals according to the onset of zygotic transcription and the persistence of maternal gene products. This maternal regulation has been little studied in vertebrates, due to the difficulty in manipulating maternal gene function and lack of basic molecular information. However, recent maternal-effect screens in the zebrafish have generated more than 40 unique mutants that are providing new molecular entry points to the maternal control of early vertebrate development. Here we discuss recent studies of 12 zebrafish mutant genes that illuminate the maternal molecular controls on embryonic development, including advances in the regulation of animal-vegetal polarity, egg activation, cleavage development, body plan formation, tissue morphogenesis, microRNA function and germ cell development. PMID:19608405

Ferritin Is Required in Multiple Tissues during Drosophila melanogaster Development.

PubMed

González-Morales, Nicanor; Mendoza-Ortíz, Miguel Ángel; Blowes, Liisa M; Missirlis, Fanis; Riesgo-Escovar, Juan R

2015-01-01

In Drosophila melanogaster, iron is stored in the cellular endomembrane system inside a protein cage formed by 24 ferritin subunits of two types (Fer1HCH and Fer2LCH) in a 1:1 stoichiometry. In larvae, ferritin accumulates in the midgut, hemolymph, garland, pericardial cells and in the nervous system. Here we present analyses of embryonic phenotypes for mutations in Fer1HCH, Fer2LCH and in both genes simultaneously. Mutations in either gene or deletion of both genes results in a similar set of cuticular embryonic phenotypes, ranging from non-deposition of cuticle to defects associated with germ band retraction, dorsal closure and head involution. A fraction of ferritin mutants have embryonic nervous systems with ventral nerve cord disruptions, misguided axonal projections and brain malformations. Ferritin mutants die with ectopic apoptotic events. Furthermore, we show that ferritin maternal contribution, which varies reflecting the mother's iron stores, is used in early development. We also evaluated phenotypes arising from the blockage of COPII transport from the endoplasmic reticulum to the Golgi apparatus, feeding the secretory pathway, plus analysis of ectopically expressed and fluorescently marked Fer1HCH and Fer2LCH. Overall, our results are consistent with insect ferritin combining three functions: iron storage, intercellular iron transport, and protection from iron-induced oxidative stress. These functions are required in multiple tissues during Drosophila embryonic development.

[Cell therapy for Parkinson's disease: IV. Risks and future trends].

PubMed

Anisimov, S V

2009-01-01

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

Expression of ribosomopathy genes during Xenopus tropicalis embryogenesis.

PubMed

Robson, Andrew; Owens, Nick D L; Baserga, Susan J; Khokha, Mustafa K; Griffin, John N

2016-10-26

Because ribosomes are ubiquitously required for protein production, it was long assumed that any inherited defect in ribosome manufacture would be embryonically lethal. However, several human congenital diseases have been found to be associated with mutations in ribosome biogenesis factors. Surprisingly, despite the global requirement for ribosomes, these "ribosomopathies" are characterized by distinct and tissue specific phenotypes. The reasons for such tissue proclivity in ribosomopathies remain mysterious but may include differential expression of ribosome biogenesis factors in distinct tissues. Here we use in situ hybridization of labeled antisense mRNA probes and ultra high temporal resolution RNA-Seq data to examine and compare expression of 13 disease associated ribosome biogenesis factors at six key stages in Xenopus tropicalis development. Rather than being ubiquitously expressed during development, mRNAs of all examined ribosome biogenesis factors were highly enriched in specific tissues, including the cranial neural crest and ventral blood islands. Interestingly, expression of ribosome biogenesis factors demonstrates clear differences in timing, transcript number and tissue localization. Ribosome biogenesis factor expression is more spatiotemporally regulated during embryonic development than previously expected and correlates closely with many of the common ribosomopathy phenotypes. Our findings provide information on the dynamic use of ribosome production machinery components during development and advance our understanding of their roles in disease.

Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis.

PubMed

Duncan, S A

2005-12-01

Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

The Development of the Ciliary Epithelium in the Embryonic Chicken Eye

DTIC Science & Technology

1989-08-04

aqueous humor, which nourishes the avascular tissues in the anterior segment and generates intraocular pressure (IOP). IOP is the pressure contained...lightly stained. At higher magnification, tissue necrosis is evident in the central region. (60x). j 168 Figure 44. Graph of the labelling index

VISUALIZATION OF TISSUE DISTRIBUTION AND METABOLISM OF BENZO[A]PYRENE IN EARLY EMBRYONIC MEDAKA (ORYZIAS LATIPES)

EPA Science Inventory

Fish early life stages are highly sensitive to exposure to persistent bioaccumulative toxicants (PBTs). The factors that contribute to this are unknown, but may include the distribution of PBTs to sensitive tissues during critical stages of development. Multiphoton laser scannin...

Regulation of Embryonic and Postnatal Development by the CSF-1 Receptor

PubMed Central

Chitu, Violeta; Stanley, E. Richard

2017-01-01

Macrophages are found in all tissues and regulate tissue morphogenesis during development through trophic and scavenger functions. The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) is the major regulator of tissue macrophage development and maintenance. In combination with receptor activator of nuclear factor κB (RANK), the CSF-1R also regulates the differentiation of the bone-resorbing osteoclast and controls bone remodeling during embryonic and early postnatal development. CSF-1R-regulated macrophages play trophic and remodeling roles in development. Outside the mononuclear phagocytic system, the CSF-1R directly regulates neuronal survival and differentiation, the development of intestinal Paneth cells and of preimplantation embryos, as well as trophoblast innate immune function. Consistent with the pleiotropic roles of the receptor during development, CSF-1R deficiency in most mouse strains causes embryonic or perinatal death and the surviving mice exhibit multiple developmental and functional deficits. The CSF-1R is activated by two dimeric glycoprotein ligands, CSF-1, and interleukin-34 (IL-34). Homozygous Csf1-null mutations phenocopy most of the deficits of Csf1r-null mice. In contrast, Il34-null mice have no gross phenotype, except for decreased numbers of Langerhans cells and microglia, indicating that CSF-1 plays the major developmental role. Homozygous inactivating mutations of the Csf1r or its ligands have not been reported in man. However, heterozygous inactivating mutations in the Csf1r lead to a dominantly inherited adult-onset progressive dementia, highlighting the importance of CSF-1R signaling in the brain. PMID:28236968

Regulation of Embryonic and Postnatal Development by the CSF-1 Receptor.

PubMed

Chitu, Violeta; Stanley, E Richard

2017-01-01

Macrophages are found in all tissues and regulate tissue morphogenesis during development through trophic and scavenger functions. The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) is the major regulator of tissue macrophage development and maintenance. In combination with receptor activator of nuclear factor κB (RANK), the CSF-1R also regulates the differentiation of the bone-resorbing osteoclast and controls bone remodeling during embryonic and early postnatal development. CSF-1R-regulated macrophages play trophic and remodeling roles in development. Outside the mononuclear phagocytic system, the CSF-1R directly regulates neuronal survival and differentiation, the development of intestinal Paneth cells and of preimplantation embryos, as well as trophoblast innate immune function. Consistent with the pleiotropic roles of the receptor during development, CSF-1R deficiency in most mouse strains causes embryonic or perinatal death and the surviving mice exhibit multiple developmental and functional deficits. The CSF-1R is activated by two dimeric glycoprotein ligands, CSF-1, and interleukin-34 (IL-34). Homozygous Csf1-null mutations phenocopy most of the deficits of Csf1r-null mice. In contrast, Il34-null mice have no gross phenotype, except for decreased numbers of Langerhans cells and microglia, indicating that CSF-1 plays the major developmental role. Homozygous inactivating mutations of the Csf1r or its ligands have not been reported in man. However, heterozygous inactivating mutations in the Csf1r lead to a dominantly inherited adult-onset progressive dementia, highlighting the importance of CSF-1R signaling in the brain. © 2017 Elsevier Inc. All rights reserved.

Expression and activity of the 5'-adenosine monophosphate-activated protein kinase pathway in selected tissues during chicken embryonic development.

PubMed

Proszkowiec-Weglarz, M; Richards, M P

2009-01-01

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine-threonine protein kinase and a key part of a kinase-signaling cascade that senses cellular energy status (adenosine monophosphate:adenosine triphosphate ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating metabolic pathways. The objective of this study was to investigate aspects of the AMPK pathway in the liver, brain, breast muscle, and heart from d 12 of incubation through hatch in chickens. We first determined mRNA and protein expression profiles for a major upstream AMPK kinase, LKB1, which is known to activate (phosphorylate) AMPK in response to increases in the adenosine monophosphate:adenosine triphosphate ratio. Expression of LKB1 protein was greatest in the brain, which demonstrated tissue-specific patterns for phosphorylation. Next, AMPK subunit mRNA and protein expression profiles were determined. Significant changes in AMPK subunit mRNA expression occurred in all tissues from d 12 of incubation to hatch. Differences in the levels of active (phosphorylated) AMPK as well as alpha and beta subunit proteins were observed in all 4 tissues during embryonic development. Finally, we determined the protein level and phosphorylation status of an important downstream target for AMPK, acetyl-coenzyme A carboxylase. The expression of acetyl-co-enzyme A carboxylase and phosphorylated acetyl-coenzyme A was greater in the brain than the liver, but was undetectable by Western blotting in the breast muscle and heart throughout the period of study. Together, our results are the first to demonstrate the expression and activity of the AMPK pathway in key tissues during the transition from embryonic to posthatch development in chickens.

In vitro organogenesis of gut-like structures from mouse embryonic stem cells.

PubMed

Kuwahara, M; Ogaeri, T; Matsuura, R; Kogo, H; Fujimoto, T; Torihashi, S

2004-04-01

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues, including representatives of all three germ layers in the embryo. We have reported previously that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e. epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neurone and ICC development.

3D bio-etching of a complex composite-like embryonic tissue.

PubMed

Hazar, Melis; Kim, Yong Tae; Song, Jiho; LeDuc, Philip R; Davidson, Lance A; Messner, William C

2015-08-21

Morphogenesis involves a complex series of cell signaling, migration and differentiation events that are coordinated as tissues self-assemble during embryonic development. Collective cell movements such as those that occur during morphogenesis have typically been studied in 2D with single layers of cultured cells adhering to rigid substrates such as glass or plastic. In vivo, the intricacies of the 3D microenvironment and complex 3D responses are pivotal in the formation of functional tissues. To study such processes as collective cell movements within 3D multilayered tissues, we developed a microfluidic technique capable of producing complex 3D laminar multicellular structures. We call this technique "3D tissue-etching" because it is analogous to techniques used in the microelectromechanics (MEMS) field where complex 3D structures are built by successively removing material from a monolithic solid through subtractive manufacturing. We use a custom-designed microfluidic control system to deliver a range of tissue etching reagents (detergents, chelators, proteases, etc.) to specific regions of multilayered tissues. These tissues were previously isolated by microsurgical excision from embryos of the African claw-toed frog, Xenopus laevis. The ability to shape the 3D form of multicellular tissues and to control 3D stimulation will have a high impact on tissue engineering and regeneration applications in bioengineering and medicine as well as provide significant improvements in the synthesis of highly complex 3D integrated multicellular biosystems.

Embryonic development during chronic acceleration

NASA Technical Reports Server (NTRS)

Smith, A. H.; Abbott, U. K.

1982-01-01

Experiments carried out on chicken eggs indicate that the embryo is affected during very early development, especially over the first four days, and during hatching. In the first four days, the brain develops as well as the anlage for all other organs. In addition, the heart commences to function and the extraembryonic membranes that compartmentalize the egg contents form. The latter require an appreciable extension and folding of tissue which may be disrupted by the mechanical load. Observations of embryonic abnormalities that occur during chronic acceleration suggest an inhibition of development of the axial skeleton, which is rarely seen otherwise, a general retardation of embryonic growth, and circulatory problems. The final stages of development (after 18 days) involve the uptake of fluids, the transition to aerial respiration, and the reorientation of the embryo into a normal hatching position. At 4 G mortality is very high during this period, with a majority of embryos failing to reorient into the normal hatching position.

The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-β3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

PubMed

Smith, Emma L; Rashidi, Hassan; Kanczler, Janos M; Shakesheff, Kevin M; Oreffo, Richard O C

2015-01-01

Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

Thymosin β4 overexpression regulates neuron production and spatial distribution in the developing avian optic tectum.

PubMed

Lever, Mael; Theiss, Carsten; Morosan-Puopolo, Gabriela; Brand-Saberi, Beate

2017-05-01

Thymosin β4 (Tβ4), the principal G-actin regulating entity in eukaryotic cells, has also multiple intra- and extracellular functions related to tissue regeneration and healing. While its effect in adult organs is being widely investigated, currently, little is known about its influence on embryonic tissues, i.e., in the developing nervous system. The importance of Tβ4 for neural stem cell proliferation in the embryonic chicken optic tectum (OT) has previously been shown by us for the first time. In the present study, using in ovo electroporation, we carried out a quantification of the effects of the Tβ4-overexpression on the developing chicken OT between E4 and E6 at the hemisphere as well as cellular level. We precisely examined tissue growth and characterized cells arising from the elevated mitotic activity of progenitor cells. By using spinning-disk confocal laser scanning microscopy, we were able to visualize these effects across whole OT sections. Our experiments now demonstrate more clearly that the overexpression of Tβ4 leads to a tangential expansion of the treated OT-hemisphere and that, under these circumstances, overall density of tectal and in particular of postmitotic neuronal cells is increased. Thanks to this new quantitative approach, the present results extend our previous findings that Tβ4 is important for the proliferation of progenitor cells, neurogenesis, tangential expansion, and tissue growth in the young embryonic chicken optic tectum. Taken together, our results further illustrate and support the current idea that Tβ4 is widely implicated in shaping and maintenance of the nervous system.

Cloning and expression of sheep DNA methyltransferase 1 and its development-specific isoform.

PubMed

Taylor, Jane; Moore, Hannah; Beaujean, Nathalie; Gardner, John; Wilmut, Ian; Meehan, Richard; Young, Lorraine

2009-05-01

Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8-16 cell stage. Attempts to clone oocyte-specific 5' regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi-mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation. (c) 2008 Wiley-Liss, Inc.

Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate

PubMed Central

Ratajczak, M Z; Zuba-Surma, E; Wojakowski, W; Suszynska, M; Mierzejewska, K; Liu, R; Ratajczak, J; Shin, D M; Kucia, M

2014-01-01

The concept that adult tissue, including bone marrow (BM), contains early-development cells with broader differentiation potential has again been recently challenged. In response, we would like to review the accumulated evidence from several independent laboratories that adult tissues, including BM, harbor a population of very rare stem cells that may cross germ layers in their differentiation potential. Thus, the BM stem cell compartment hierarchy needs to be revisited. These dormant, early-development cells that our group described as very small embryonic-like stem cells (VSELs) most likely overlap with similar populations of stem cells that have been identified in adult tissues by other investigators as the result of various experimental strategies and have been given various names. As reported, murine VSELs have some pluripotent stem cell characteristics. Moreover, they display several epiblast/germline markers that suggest their embryonic origin and developmental deposition in adult BM. Moreover, at the molecular level, changes in expression of parentally imprinted genes (for example, Igf2–H19) and resistance to insulin/insulin-like growth factor signaling (IIS) regulates their quiescent state in adult tissues. In several emergency situations related to organ damage, VSELs can be activated and mobilized into peripheral blood, and in appropriate animal models they contribute to tissue organ/regeneration. Interestingly, their number correlates with lifespan in mice, and they may also be involved in some malignancies. VSELs have been successfully isolated in several laboratories; however, some investigators experience problems with their isolation. PMID:24018851

Correlation of Versican Expression, Accumulation, and Degradation during Embryonic Development by Quantitative Immunohistochemistry

PubMed Central

Snyder, Jessica M.; Washington, Ida M.; Birkland, Timothy; Chang, Mary Y.; Frevert, Charles W.

2015-01-01

Versican, a chondroitin sulfate proteoglycan, is important in embryonic development, and disruption of the versican gene is embryonically lethal in the mouse. Although several studies show that versican is increased in various organs during development, a focused quantitative study on versican expression and distribution during lung and central nervous system development in the mouse has not previously been performed. We tracked changes in versican (Vcan) gene expression and in the accumulation and degradation of versican. Vcan expression and quantitative immunohistochemistry performed from embryonic day (E) 11.5 to E15.5 showed peak Vcan expression at E13.5 in the lungs and brain. Quantitative mRNA analysis and versican immunohistochemistry showed differences in the expression of the versican isoforms in the embryonic lung and head. The expression of Vcan mRNA and accumulation of versican in tissues was complementary. Immunohistochemistry demonstrated co-localization of versican accumulation and degradation, suggesting distinct roles of versican deposition and degradation in embryogenesis. Very little versican mRNA or protein was found in the lungs of 12- to 16-week-old mice but versican accumulation was significantly increased in mice with Pseudomonas aeruginosa lung infection. These data suggest that versican plays an important role in fundamental, overlapping cellular processes in lung development and infection. PMID:26385570

Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues.

PubMed

Cohen, Shahar; Leshansky, Lucy; Zussman, Eyal; Burman, Michael; Srouji, Samer; Livne, Erella; Abramov, Natalie; Itskovitz-Eldor, Joseph

2010-10-01

The use of stem cells for tissue engineering (TE) encourages scientists to design new platforms in the field of regenerative and reconstructive medicine. Human embryonic stem cells (hESC) have been proposed to be an important cell source for cell-based TE applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESC lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. We describe a simple method for engineering tendon grafts that can successfully repair injured Achilles tendons and restore the ankle joint extension movement in mice. We also show the CTP's ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. This study offers evidence for the possibility of using stem cell-derived engineered grafts to replace missing tissues, and sets a basic platform for future cell-based TE applications in the fields of orthopedics and reconstructive surgery.

PTBP1 Is Required for Embryonic Development before Gastrulation

PubMed Central

Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A. Francis; Solimena, Michele

2011-01-01

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures. PMID:21423341

PTBP1 is required for embryonic development before gastrulation.

PubMed

Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A Francis; Solimena, Michele

2011-02-17

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

DEVELOPMENT OF AN AMPHIBIAN METAMORPHOSIS MODEL FOR DETECTING THYROID AXIS DISRUPTION

EPA Science Inventory

Metamorphosis in Xenopus laevis represents an elaborate process of post-embryonic development which is thyroid hormone (TH) dependent. The development of a functional thyroid axis and the responses of tissues to different TH concentrations are well defined in this species, provid...

Preattachment Embryos of Domestic Animals: Insights into Development and Paracrine Secretions.

PubMed

Sandra, Olivier; Charpigny, Gilles; Galio, Laurent; Hue, Isabelle

2017-02-08

In mammalian species, endometrial receptivity is driven by maternal factors independently of embryo signals. When pregnancy initiates, paracrine secretions of the preattachment embryo are essential both for maternal recognition and endometrium preparation for implantation and for coordinating development of embryonic and extraembryonic tissues of the conceptus. This review mainly focuses on domestic large animal species. We first illustrate the major steps of preattachment embryo development, including elongation in bovine, ovine, porcine, and equine species. We next highlight conceptus secretions that are involved in the communication between extraembryonic and embryonic tissues, as well as between the conceptus and the endometrium. Finally, we introduce experimental data demonstrating the intimate connection between conceptus secretions and endometrial activity and how adverse events perturbing this interplay may affect the progression of implantation that will subsequently impact pregnancy outcome, postnatal health, and expression of production traits in livestock offspring.

Characterizing the distribution of steroid sulfatase during embryonic development: when and where might metabolites of maternal steroids be reactivated?

PubMed

Paitz, Ryan T; Duffield, Kristin R; Bowden, Rachel M

2017-12-15

All vertebrate embryos are exposed to maternally derived steroids during development. In placental vertebrates, metabolism of maternal steroids by the placenta modulates embryonic exposure, but how exposure is regulated in oviparous vertebrates is less clear. Recent work in oviparous vertebrates has demonstrated that steroids are not static molecules, as they can be converted to more polar steroid sulfates by sulfotransferase enzymes. Importantly, these steroid sulfates can be converted back to the parent compound by the enzyme steroid sulfatase (STS). We investigated when and where STS was present during embryonic development in the red-eared slider turtle, Trachemys scripta We report that STS is present during all stages of development and in all tissues we examined. We conclude that STS activity may be particularly important for regulating maternal steroid exposure in oviparous vertebrates. © 2017. Published by The Company of Biologists Ltd.

Iodine-enhanced micro-CT imaging: methodological refinements for the study of the soft-tissue anatomy of post-embryonic vertebrates.

PubMed

Gignac, Paul M; Kley, Nathan J

2014-05-01

The now widespread use of non-destructive X-ray computed tomography (CT) and micro-CT (µCT) has greatly augmented our ability to comprehensively detail and quantify the internal hard-tissue anatomy of vertebrates. However, the utility of X-ray imaging for gaining similar insights into vertebrate soft-tissue anatomy has yet to be fully realized due to the naturally low X-ray absorption of non-mineralized tissues. In this study, we show how a wide diversity of soft-tissue structures within the vertebrate head-including muscles, glands, fat deposits, perichondria, dural venous sinuses, white and gray matter of the brain, as well as cranial nerves and associated ganglia-can be rapidly visualized in their natural relationships with extraordinary levels of detail using iodine-enhanced (i-e) µCT imaging. To date, Lugol's iodine solution (I2 KI) has been used as a contrast agent for µCT imaging of small invertebrates, vertebrate embryos, and certain isolated parts of larger, post-embryonic vertebrates. These previous studies have all yielded promising results, but visualization of soft tissues in smaller invertebrate and embryonic vertebrate specimens has generally been more complete than that for larger, post-embryonic vertebrates. Our research builds on these previous studies by using high-energy µCT together with more highly concentrated I2 KI solutions and longer staining times to optimize the imaging and differentiation of soft tissues within the heads of post-embryonic archosaurs (Alligator mississippiensis and Dromaius novaehollandiae). We systematically quantify the intensities of tissue staining, demonstrate the range of anatomical structures that can be visualized, and generate a partial three-dimensional reconstruction of alligator cephalic soft-tissue anatomy. © 2014 Wiley Periodicals, Inc.

Engineered three-dimensional multicellular culture model to recapitulate morphogenetic fusion using human cells

EPA Science Inventory

Tissue fusion during early mammalian development requires crosstalk between multiple cell types. For example, paracrine signaling between palatal epithelial cells and palatal mesenchyme mediates the fusion of opposing palatal shelves during embryonic development. Fusion events in...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta.

PubMed

Misra, Ashish; Feng, Zhonghui; Zhang, Jiasheng; Lou, Zhi-Yin; Greif, Daniel M

2017-09-12

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration.

PubMed

Mort, Richard L; Keighren, Margaret; Hay, Leonard; Jackson, Ian J

2014-05-19

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles(1,2). Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture(3-6). Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue(3,6). High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture(6). By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

PubMed

Seiler, Christoph; Gebhart, Nichole; Zhang, Yong; Shinton, Susan A; Li, Yue-sheng; Ross, Nicola L; Liu, Xingjun; Li, Qin; Bilbee, Alison N; Varshney, Gaurav K; LaFave, Matthew C; Burgess, Shawn M; Balciuniene, Jorune; Balciunas, Darius; Hardy, Richard R; Kappes, Dietmar J; Wiest, David L; Rhodes, Jennifer

2015-01-01

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

Effects of breed, parity, and folic Acid supplement on the expression of folate metabolism genes in endometrial and embryonic tissues from sows in early pregnancy.

PubMed

Vallée, Maud; Guay, Frédéric; Beaudry, Danièle; Matte, Jacques; Blouin, Richard; Laforest, Jean-Paul; Lessard, Martin; Palin, Marie-France

2002-10-01

Folic acid and glycine are factors of great importance in early gestation. In sows, folic acid supplement can increase litter size through a decrease in embryonic mortality, while glycine, the most abundant amino acid in the sow oviduct, uterine, and allantoic fluids, is reported to act as an organic osmoregulator. In this study, we report the characterization of cytoplasmic serine hydroxymethyltransferase (cSHMT), T-protein, and vT-protein (variant T-protein) mRNA expression levels in endometrial and embryonic tissues in gestating sows on Day 25 of gestation according to the breed, parity, and folic acid + glycine supplementation. Expression levels of cSHMT, T-protein, and vT-protein mRNA in endometrial and embryonic tissues were performed using semiquantitative reverse transcription-polymerase chain reaction. We also report, for the first time, an alternative splicing event in the porcine T-protein gene. Results showed that a T-protein splice variant, vT-protein, is present in all the tested sow populations. Further characterizations revealed that this T-protein splice variant contains a coding intron that can adopt a secondary structure. Results demonstrated that cSHMT mRNA expression levels were significantly higher in sows receiving the folic acid + glycine supplementation, independently of the breed or parity and in both endometrial and embryonic tissues. Upon receiving the same treatment, the vT-protein and T-protein mRNA expression levels were significantly reduced in the endometrial tissue of Yorkshire-Landrace sows only. These results indicate that modulation of specific gene expression levels in endometrial and embryonic tissues of sows in early gestation could be one of the mechanism involved with the role of folic acid on improving swine reproduction traits.

The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development.

PubMed Central

Hardtke, C S; Berleth, T

1998-01-01

The vascular tissues of flowering plants form networks of interconnected cells throughout the plant body. The molecular mechanisms directing the routes of vascular strands and ensuring tissue continuity within the vascular system are not known, but are likely to depend on general cues directing plant cell orientation along the apical-basal axis. Mutations in the Arabidopsis gene MONOPTEROS (MP) interfere with the formation of vascular strands at all stages and also with the initiation of the body axis in the early embryo. Here we report the isolation of the MP gene by positional cloning. The predicted protein product contains functional nuclear localization sequences and a DNA binding domain highly similar to a domain shown to bind to control elements of auxin inducible promoters. During embryogenesis, as well as organ development, MP is initially expressed in broad domains that become gradually confined towards the vascular tissues. These observations suggest that the MP gene has an early function in the establishment of vascular and body patterns in embryonic and post-embryonic development. PMID:9482737

Transcriptome architecture across tissues in the pig

PubMed Central

Ferraz, André LJ; Ojeda, Ana; López-Béjar, Manel; Fernandes, Lana T; Castelló, Anna; Folch, Josep M; Pérez-Enciso, Miguel

2008-01-01

Background Artificial selection has resulted in animal breeds with extreme phenotypes. As an organism is made up of many different tissues and organs, each with its own genetic programme, it is pertinent to ask: How relevant is tissue in terms of total transcriptome variability? Which are the genes most distinctly expressed between tissues? Does breed or sex equally affect the transcriptome across tissues? Results In order to gain insight on these issues, we conducted microarray expression profiling of 16 different tissues from four animals of two extreme pig breeds, Large White and Iberian, two males and two females. Mixed model analysis and neighbor – joining trees showed that tissues with similar developmental origin clustered closer than those with different embryonic origins. Often a sound biological interpretation was possible for overrepresented gene ontology categories within differentially expressed genes between groups of tissues. For instance, an excess of nervous system or muscle development genes were found among tissues of ectoderm or mesoderm origins, respectively. Tissue accounted for ~11 times more variability than sex or breed. Nevertheless, we were able to confidently identify genes with differential expression across tissues between breeds (33 genes) and between sexes (19 genes). The genes primarily affected by sex were overall different than those affected by breed or tissue. Interaction with tissue can be important for differentially expressed genes between breeds but not so much for genes whose expression differ between sexes. Conclusion Embryonic development leaves an enduring footprint on the transcriptome. The interaction in gene × tissue for differentially expressed genes between breeds suggests that animal breeding has targeted differentially each tissue's transcriptome. PMID:18416811

Endothelin-1 signalling controls early embryonic heart rate in vitro and in vivo.

PubMed

Karppinen, S; Rapila, R; Mäkikallio, K; Hänninen, S L; Rysä, J; Vuolteenaho, O; Tavi, P

2014-02-01

Spontaneous activity of embryonic cardiomyocytes originates from sarcoplasmic reticulum (SR) Ca(2+) release during early cardiogenesis. However, the regulation of heart rate during embryonic development is still not clear. The aim of this study was to determine how endothelin-1 (ET-1) affects the heart rate of embryonic mice, as well as the pathway through which it exerts its effects. The effects of ET-1 and ET-1 receptor inhibition on cardiac contraction were studied using confocal Ca(2+) imaging of isolated mouse embryonic ventricular cardiomyocytes and ultrasonographic examination of embryonic cardiac contractions in utero. In addition, the amount of ET-1 peptide and ET receptor a (ETa) and b (ETb) mRNA levels were measured during different stages of development of the cardiac muscle. High ET-1 concentration and expression of both ETa and ETb receptors was observed in early cardiac tissue. ET-1 was found to increase the frequency of spontaneous Ca(2+) oscillations in E10.5 embryonic cardiomyocytes in vitro. Non-specific inhibition of ET receptors with tezosentan caused arrhythmia and bradycardia in isolated embryonic cardiomyocytes and in whole embryonic hearts both in vitro (E10.5) and in utero (E12.5). ET-1-mediated stimulation of early heart rate was found to occur via ETb receptors and subsequent inositol trisphosphate receptor activation and increased SR Ca(2+) leak. Endothelin-1 is required to maintain a sufficient heart rate, as well as to prevent arrhythmia during early development of the mouse heart. This is achieved through ETb receptor, which stimulates Ca(2+) leak through IP3 receptors. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Motility-Driven Glass and Jamming Transitions in Biological Tissues

NASA Astrophysics Data System (ADS)

Bi, Dapeng; Yang, Xingbo; Marchetti, M. Cristina; Manning, M. Lisa

2016-04-01

Cell motion inside dense tissues governs many biological processes, including embryonic development and cancer metastasis, and recent experiments suggest that these tissues exhibit collective glassy behavior. To make quantitative predictions about glass transitions in tissues, we study a self-propelled Voronoi model that simultaneously captures polarized cell motility and multibody cell-cell interactions in a confluent tissue, where there are no gaps between cells. We demonstrate that the model exhibits a jamming transition from a solidlike state to a fluidlike state that is controlled by three parameters: the single-cell motile speed, the persistence time of single-cell tracks, and a target shape index that characterizes the competition between cell-cell adhesion and cortical tension. In contrast to traditional particulate glasses, we are able to identify an experimentally accessible structural order parameter that specifies the entire jamming surface as a function of model parameters. We demonstrate that a continuum soft glassy rheology model precisely captures this transition in the limit of small persistence times and explain how it fails in the limit of large persistence times. These results provide a framework for understanding the collective solid-to-liquid transitions that have been observed in embryonic development and cancer progression, which may be associated with epithelial-to-mesenchymal transition in these tissues.

Tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras

PubMed Central

Karagenç, Levent; Sandikci, Mustafa

2010-01-01

The objective of the current study was to determine the tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras. Quail-chick chimeras were constructed by transferring dissociated cells from the area opaca of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in embryonic as well as extra-embryonic tissues of the recipient embryo were examined using the QCPN monoclonal antibody after 6 days of incubation in serial sections taken at 100-μm intervals. Data gathered in the present study demonstrated that, when introduced into the subgerminal cavity of a recipient embryo, cells of the area opaca are able to populate not only extra-embryonic structures such as the amnion and the yolk sac, but also various embryonic tissues derived from the ectoderm and less frequently the mesoderm. Ectodermal chimerism was confined mainly to the head region and was observed in tissues derived from the neural ectoderm and the surface ectoderm, including the optic cup, diencephalon and lens. Although the possibility of random incorporation of transplanted cells into these embryonic structures cannot be excluded, these results would suggest that area opaca, a peripheral ring of cells in the avian embryo destined to form the extra-embryonic ectoderm and endoderm of the yolk sac, might harbor cells that have the potential to give rise to various cell types in the recipient chick embryo, including those derived from the surface ectoderm and neural ectoderm. PMID:19900180

Mesenchymal-epithelial interaction techniques

PubMed Central

Baskin, Lawrence

2016-01-01

This paper reviews the importance of mesenchymal-epithelial interactions in development and gives detailed technical protocols for investigating these interactions. Successful analysis of mesenchymal-epithelial interactions requires knowing the ages in which embryonic, neonatal and adult organs can be separated into mesenchymal and epithelial tissues. Methods for separation of mesenchymal and epithelial and preparation of tissue recombinants are described. PMID:26610327

Zebrafish Caudal Haematopoietic Embryonic Stromal Tissue (CHEST) Cells Support Haematopoiesis.

PubMed

Wolf, Anja; Aggio, Julian; Campbell, Clyde; Wright, Francis; Marquez, Gabriel; Traver, David; Stachura, David L

2017-03-16

Haematopoiesis is an essential process in early vertebrate development that occurs in different distinct spatial locations in the embryo that shift over time. These different sites have distinct functions: in some anatomical locations specific hematopoietic stem and progenitor cells (HSPCs) are generated de novo. In others, HSPCs expand. HSPCs differentiate and renew in other locations, ensuring homeostatic maintenance. These niches primarily control haematopoiesis through a combination of cell-to-cell signalling and cytokine secretion that elicit unique biological effects in progenitors. To understand the molecular signals generated by these niches, we report the generation of caudal hematopoietic embryonic stromal tissue (CHEST) cells from 72-hours post fertilization (hpf) caudal hematopoietic tissue (CHT), the site of embryonic HSPC expansion in fish. CHEST cells are a primary cell line with perivascular endothelial properties that expand hematopoietic cells in vitro. Morphological and transcript analysis of these cultures indicates lymphoid, myeloid, and erythroid differentiation, indicating that CHEST cells are a useful tool for identifying molecular signals critical for HSPC proliferation and differentiation in the zebrafish. These findings permit comparison with other temporally and spatially distinct haematopoietic-supportive zebrafish niches, as well as with mammalian haematopoietic-supportive cells to further the understanding of the evolution of the vertebrate hematopoietic system.

Generation of erythroid cells from polyploid giant cancer cells: re-thinking about tumor blood supply.

PubMed

Yang, Zhigang; Yao, Hong; Fei, Fei; Li, Yuwei; Qu, Jie; Li, Chunyuan; Zhang, Shiwu

2018-04-01

During development and tumor progression, cells need a sufficient blood supply to maintain development and rapid growth. It is reported that there are three patterns of blood supply for tumor growth: endothelium-dependent vessels, mosaic vessels, and vasculogenic mimicry (VM). VM was first reported in highly aggressive uveal melanomas, with tumor cells mimicking the presence and function of endothelial cells forming the walls of VM vessels. The walls of mosaic vessels are randomly lined with both endothelial cells and tumor cells. We previously proposed a three-stage process, beginning with VM, progressing to mosaic vessels, and eventually leading to endothelium-dependent vessels. However, many phenomena unique to VM channel formation remain to be elucidated, such as the origin of erythrocytes before VM vessels connect with endothelium-dependent vessels. In adults, erythroid cells are generally believed to be generated from hematopoietic stem cells in the bone marrow. In contrast, embryonic tissue obtains oxygen through formation of blood islands, which are largely composed of embryonic hemoglobin with a higher affinity with oxygen, in the absence of mature erythrocytes. Recent data from our laboratory suggest that embryonic blood-forming mechanisms also exist in cancer tissue, particularly when these tissues are under environmental stress such as hypoxia. We review the evidence from induced pluripotent stem cells in vitro and in vivo to support this previously underappreciated cell functionality in normal and cancer cells, including the ability to generate erythroid cells. We will also summarize the current understanding of tumor angiogenesis, VM, and our recent work on polyploid giant cancer cells, with emphasis on their ability to generate erythroid cells and their association with tumor growth under hypoxia. An alternative embryonic pathway to obtain oxygen in cancer cells exists, particularly when they are under hypoxic conditions.

Electron microscopy of the amphibian model systems Xenopus laevis and Ambystoma mexicanum.

PubMed

Kurth, Thomas; Berger, Jürgen; Wilsch-Bräuninger, Michaela; Kretschmar, Susanne; Cerny, Robert; Schwarz, Heinz; Löfberg, Jan; Piendl, Thomas; Epperlein, Hans H

2010-01-01

In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy. Copyright © 2010 Elsevier Inc. All rights reserved.

Tissue engineering strategies to study cartilage development, degeneration and regeneration.

PubMed

Bhattacharjee, Maumita; Coburn, Jeannine; Centola, Matteo; Murab, Sumit; Barbero, Andrea; Kaplan, David L; Martin, Ivan; Ghosh, Sourabh

2015-04-01

Cartilage tissue engineering has primarily focused on the generation of grafts to repair cartilage defects due to traumatic injury and disease. However engineered cartilage tissues have also a strong scientific value as advanced 3D culture models. Here we first describe key aspects of embryonic chondrogenesis and possible cell sources/culture systems for in vitro cartilage generation. We then review how a tissue engineering approach has been and could be further exploited to investigate different aspects of cartilage development and degeneration. The generated knowledge is expected to inform new cartilage regeneration strategies, beyond a classical tissue engineering paradigm. Copyright © 2014 Elsevier B.V. All rights reserved.

A multiplexed quantitative proteomics approach for investigating protein expression in the developing central nervous system.

PubMed

Orme, Rowan P; Gates, Monte A; Fricker-Gates, Rosemary A

2010-08-15

Cell transplantation using stem cell-derived neurons is commonly viewed as a candidate therapy for neurodegenerative diseases. However, methods for differentiating stem cells into homogenous populations of neurons suitable for transplant remain elusive. This suggests that there are as yet unknown signalling factors working in vivo to specify neuronal cell fate during development. These factors could be manipulated to better differentiate stem cells into neural populations useful for therapeutic transplantation. Here a quantitative proteomics approach is described for investigating cell signalling in the developing central nervous system (CNS), using the embryonic ventral mesencephalon as a model. Briefly, total protein was extracted from embryonic ventral midbrain tissue before, during and after the birth of dopaminergic neurons, and digested using trypsin. Two-dimensional liquid chromatography, coupled with tandem mass spectrometry, was then used to identify proteins from the tryptic peptides. Isobaric tagging for relative and absolute quantification (iTRAQ) reagents were used to label the tryptic peptides and facilitate relative quantitative analysis. The success of the experiment was confirmed by the identification of proteins known to be expressed in the developing ventral midbrain, as well as by Western blotting, and immunolabelling of embryonic tissue sections. This method of protein discovery improves upon previous attempts to identify novel signalling factors through microarray analysis. Importantly, the methods described here could be applied to virtually any aspect of development. (c) 2010 Elsevier B.V. All rights reserved.

Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways.

PubMed

Liu, Jing; Zhao, Yong; Ge, Wei; Zhang, Pengfei; Liu, Xinqi; Zhang, Weidong; Hao, Yanan; Yu, Shuai; Li, Lan; Chu, Meiqiang; Min, Lingjiang; Zhang, Hongfu; Shen, Wei

2017-06-27

The impacts of zinc oxide nanoparticles on embryonic development following oocyte stage exposure are unknown and the underlying mechanisms are sparsely understood. In the current investigation, intact nanoparticles were detected in ovarian tissue in vivo and cultured cells in vitro under zinc oxide nanoparticles treatment. Zinc oxide nanoparticles exposure during the oocyte stage inhibited embryonic development. Notably, in vitro culture data closely matched in vivo embryonic data, in that the impairments caused by Zinc oxide nanoparticles treatment passed through cell generations; and both gamma-H2AX and NF-kappaB pathways were involved in zinc oxide nanoparticles caused embryo-toxicity. Copper oxide and silicon dioxide nanoparticles have been used to confirm that particles are important for the toxicity of zinc oxide nanoparticles. The toxic effects of zinc oxide nanoparticles emanate from both intact nanoparticles and Zn2+. Our investigation along with others suggests that zinc oxide nanoparticles are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic and that precaution should be taken regarding human exposure to their everyday use.

Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways

PubMed Central

Liu, Jing; Zhao, Yong; Ge, Wei; Zhang, Pengfei; Liu, Xinqi; Zhang, Weidong; Hao, Yanan; Yu, Shuai; Li, Lan; Chu, Meiqiang; Min, Lingjiang; Zhang, Hongfu; Shen, Wei

2017-01-01

The impacts of zinc oxide nanoparticles on embryonic development following oocyte stage exposure are unknown and the underlying mechanisms are sparsely understood. In the current investigation, intact nanoparticles were detected in ovarian tissue in vivo and cultured cells in vitro under zinc oxide nanoparticles treatment. Zinc oxide nanoparticles exposure during the oocyte stage inhibited embryonic development. Notably, in vitro culture data closely matched in vivo embryonic data, in that the impairments caused by Zinc oxide nanoparticles treatment passed through cell generations; and both gamma-H2AX and NF-kappaB pathways were involved in zinc oxide nanoparticles caused embryo-toxicity. Copper oxide and silicon dioxide nanoparticles have been used to confirm that particles are important for the toxicity of zinc oxide nanoparticles. The toxic effects of zinc oxide nanoparticles emanate from both intact nanoparticles and Zn2+. Our investigation along with others suggests that zinc oxide nanoparticles are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic and that precaution should be taken regarding human exposure to their everyday use. PMID:28487501

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

PubMed

Sambi, Manpreet; Chow, Theresa; Whiteley, Jennifer; Li, Mira; Chua, Shawn; Raileanu, Vanessa; Rogers, Ian M

2017-08-01

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

Temporal distribution of endogenous retinoids in the embryonic mouse mandible.

PubMed

Beeman, C S; Kronmiller, J E

1994-09-01

Retinoids play an important part in embryonic pattern formation. They are necessary for normal differentiation of odontogenic tissues and, in excess, disrupt the pattern of tooth formation. Excess retinoids produce supernumerary buds of the dental lamina in the diastema region of the mouse embryonic mandible where teeth do not normally form. This effect is coincident with an increase in epithelial proliferation and an alteration in epidermal growth factor mRNA expression (a gene product necessary for tooth formation). It was found by high-performance liquid chromatography that endogenous retinoids are present in the developing murine mandible and that concentrations of some retinoids reach a peak at the time of the initiation of odontogenesis (dental lamina formation).

SpSM30 gene family expression patterns in embryonic and adult biomineralized tissues of the sea urchin, Strongylocentrotus purpuratus.

PubMed

Killian, Christopher E; Croker, Lindsay; Wilt, Fred H

2010-01-01

The SpSM30 gene family of the sea urchin, Strongylocentrotus purpuratus, is comprised of six members, designated SpSM30A through SpSM30F (Livingston et al., 2006). The SpSM30 proteins are found uniquely in embryonic and adult mineralized tissues of the sea urchin. Previous studies have revealed that SpSM30 proteins are occluded within the embryonic endoskeleton and adult mineralized tissues (Killian and Wilt, 1996; Mann et al., 2008a,b; Urry et al., 2000). Furthermore, some of the SpSM30 proteins are among the most abundant of the approximately four-dozen integral matrix proteins of the larval spicule (Killian and Wilt, 1996). The amino acid sequence, protein domain architecture, and contiguity within the genome strongly support the supposition that the six genes constitute a gene family. Reverse transcription-polymerase chain reaction (RT-PCR) is used in the present study to describe the time course of expression of the family members during embryonic development, and their expression in adult tissues. SpSM30A, B, C and E are expressed, albeit at different levels, during overt spicule deposition in the embryo with some differences in the precise timing of expression. SpSM30D is not expressed in the embryo, and SpSM30F is expressed transiently and at low levels just prior to overt spicule formation. Whole mount in situ hybridization studies show that SpSM30A, B, C, and E are expressed exclusively in primary mesenchyme (PMC) cells and their descendants. In addition, tissue fractionation studies indicate that SpSM30F expression is highly enriched in PMCs. Each adult tissue examined expresses a different cohort of the SpSM30 family members at varying levels: SpSM30A mRNA is not expressed in adult tissues. Its expression is limited to the embryo. Conversely, SpSM30D mRNA is not expressed in the embryo, but is expressed in adult spines and teeth. SpSM30B and SpSM30C are expressed at modest levels in all mineralized adult tissues; SpSM30E is expressed highly in tooth and test; and SpSM30F is expressed in spine and at low levels in the other adult tissues except the test. Relative levels of expression of the several family members in these different tissues vary widely. It is likely SpSM30 proteins play a vital, but still unknown, role in biomineralization of these tissues during development. Copyright 2010 Elsevier B.V. All rights reserved.

Stage-dependent and locus-specific role of histone demethylase Jumonji D3 (JMJD3) in the embryonic stages of lung development.

PubMed

Li, Qingtian; Wang, Helen Y; Chepelev, Iouri; Zhu, Qingyuan; Wei, Gang; Zhao, Keji; Wang, Rong-Fu

2014-07-01

Histone demethylases have emerged as important players in developmental processes. Jumonji domain containing-3 (Jmjd3) has been identified as a key histone demethylase that plays a critical role in the regulation of gene expression; however, the in vivo function of Jmjd3 in embryonic development remains largely unknown. To this end, we generated Jmjd3 global and conditional knockout mice. Global deletion of Jmjd3 induces perinatal lethality associated with defective lung development. Tissue and stage-specific deletion revealed that Jmjd3 is dispensable in the later stage of embryonic lung development. Jmjd3 ablation downregulates the expression of genes critical for lung development and function, including AQP-5 and SP-B. Jmjd3-mediated alterations in gene expression are associated with locus-specific changes in the methylation status of H3K27 and H3K4. Furthermore, Jmjd3 is recruited to the SP-B promoter through interactions with the transcription factor Nkx2.1 and the epigenetic protein Brg1. Taken together, these findings demonstrate that Jmjd3 plays a stage-dependent and locus-specific role in the mouse lung development. Our study provides molecular insights into the mechanisms by which Jmjd3 regulates target gene expression in the embryonic stages of lung development.

Enzymatic Metabolism of Vitamin A in Developing Vertebrate Embryos

PubMed Central

Metzler, Melissa A.; Sandell, Lisa L.

2016-01-01

Embryonic development is orchestrated by a small number of signaling pathways, one of which is the retinoic acid (RA) signaling pathway. Vitamin A is essential for vertebrate embryonic development because it is the molecular precursor of the essential signaling molecule RA. The level and distribution of RA signaling within a developing embryo must be tightly regulated; too much, or too little, or abnormal distribution, all disrupt embryonic development. Precise regulation of RA signaling during embryogenesis is achieved by proteins involved in vitamin A metabolism, retinoid transport, nuclear signaling, and RA catabolism. The reversible first step in conversion of the precursor vitamin A to the active retinoid RA is mediated by retinol dehydrogenase 10 (RDH10) and dehydrogenase/reductase (SDR family) member 3 (DHRS3), two related membrane-bound proteins that functionally activate each other to mediate the interconversion of retinol and retinal. Alcohol dehydrogenase (ADH) enzymes do not contribute to RA production under normal conditions during embryogenesis. Genes involved in vitamin A metabolism and RA catabolism are expressed in tissue-specific patterns and are subject to feedback regulation. Mutations in genes encoding these proteins disrupt morphogenesis of many systems in a developing embryo. Together these observations demonstrate the importance of vitamin A metabolism in regulating RA signaling during embryonic development in vertebrates. PMID:27983671

Ca2+ signalling and early embryonic patterning during zebrafish development.

PubMed

Webb, Sarah E; Miller, Andrew L

2007-09-01

1. It has been proposed that Ca2+ signalling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern-forming events during early vertebrate development. 2. With reference to the embryo of the zebrafish (Danio rerio), herein we review the Ca2+ transients reported from the cleavage to segmentation periods. This time-window includes most of the major pattern-forming events of early development, which transform a single-cell zygote into a complex multicellular embryo with established primary germ layers and body axes. 3. Data are presented to support our proposal that intracellular Ca2+ waves are an essential feature of embryonic cytokinesis and that propagating intercellular Ca2+ waves (both long and short range) may play a crucial role in: (i) the establishment of the embryonic periderm and the coordination of cell movements during epiboly, convergence and extension; (ii) the establishment of the basic embryonic axes and germ layers; and (iii) definition of the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen. 4. The potential downstream targets of these Ca2+ transients are also discussed, as well as how they may integrate with other pattern-forming signalling pathways known to modulate early developmental events.

The Evolutionary Economics of Embryonic-Sac Fluids in Squamate Reptiles.

PubMed

Bonnet, Xavier; Naulleau, Guy; Shine, Richard

2017-03-01

The parchment-shelled eggs of squamate reptiles take up substantial water from the nest environment, enabling the conversion of yolk into neonatal tissue and buffering the embryo against the possibility of subsequent dry weather. During development, increasing amounts of water are stored in the embryonic sacs (i.e., membranes around the embryo: amnion, allantois, and chorion). The evolution of viviparity (prolonged uterine retention of developing embryos) means that embryonic-sac fluid storage now imposes a cost (increased maternal burdening), confers less benefit (because the mother buffers fetal water balance), and introduces a potential conflict among uterine siblings (for access to finite water supplies). Our data on nine species of squamate reptiles and published information on three species show that the embryonic-sac fluids comprise around 33% of neonatal mass in viviparous species versus 94% in full-term eggs of oviparous squamates. Data on parturition in 149 vipers (Vipera aspis, a viviparous species) show that larger offspring store more fluids in their fetal sacs and that an increase in litter size is associated with a decrease in fluid-sac mass per offspring. Overall, the evolutionary transition from oviparity to viviparity may have substantially altered selective forces on offspring packaging and created competition among offspring for access to water reserves during embryonic development.

The miR-290-295 cluster as multi-faceted players in mouse embryonic stem cells.

PubMed

Yuan, Kai; Ai, Wen-Bing; Wan, Lin-Yan; Tan, Xiao; Wu, Jiang-Feng

2017-01-01

Increasing evidence indicates that embryonic stem cell specific microRNAs (miRNAs) play an essential role in the early development of embryo. Among them, the miR-290-295 cluster is the most highly expressed in the mouse embryonic stem cells and involved in various biological processes. In this paper, we reviewed the research progress of the function of the miR-290-295 cluster in embryonic stem cells. The miR-290-295 cluster is involved in regulating embryonic stem cell pluripotency maintenance, self-renewal, and reprogramming somatic cells to an embryonic stem cell-like state. Moreover, the miR-290-295 cluster has a latent pro-survival function in embryonic stem cells and involved in tumourigenesis and senescence with a great significance. Elucidating the interaction between the miR-290-295 cluster and other modes of gene regulation will provide us new ideas on the biology of pluripotent stem cells. In the near future, the broad prospects of the miRNA cluster will be shown in the stem cell field, such as altering cell identities with high efficiency through the transient introduction of tissue-specific miRNA cluster.

RESISTANCE TO X-IRRADIATION BY EMBRYONIC CELLS OF THE LIMB-BUDS OF TADPOLES

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

Allen, B.M.; Ewell, L.M.

1959-01-01

Both total-body irradiation and shielding of the trunk were used to study the effects of x irradiation from 1000 to 30000 r upon the limb-buds of Bufo boreas and Hyla regilla tadpoles. The object was to test the view that the younger the cells the more sensitive they are to irradiation. The answer is negative. If there is any special susceptibility of these undifferentiated cells it should appear at levels far below the 30000 r maximum employed. A sharp distinction is made between the very susceptible mitotic cells and the resistant non-dividing embryonic cells that have been accumulated in suchmore » numbers that they may rapidly differentiate into the characteristic limb tissues under the stimulus of the thyroid hormone. Many irradiated ectoderm cells were changed to form bizarre excrescences but were not destroyed. Unicellular cuthneous gland cells continued to arise even after the heaviest irradiation. Irradiated tadpoles with hind limb-buds from 0.6 mm down to 0.2 mm length were unable to develop normal limbs. This capacity was propontional to the number of non-dividing embryonic cells stored at the time of irradiation. Irradiation of 5000, equal degree but the rapidity was greatest in the cases of higher dosage. Not only did these levels of irradiation fail to destroy the non-dividing embryonic cells but they did not effect their pre-deterrmined specificity nor modify their capacity for subsequent differentiation and growth. Exposure to a thyroxin solution caused the hind limb-buds without visible differentiation of cells to grow from a length of 0.8 or 0.9 mm or 1.0 mm at the time of irradiation to a length of as much as 5.0 mm in the course of 7 days. Development of thigh, shank, ankle, and toes was complete. Microscopic studies showed characteristic tissues such as cartilage, connective tissue, and muscle, developed to a comparable degree in control and irradiated specimens. (auth)« less

Rate of Dehydration and Cumulative Desiccation Stress Interacted to Modulate Desiccation Tolerance of Recalcitrant Cocoa and Ginkgo Embryonic Tissues1

PubMed Central

Liang, Yongheng; Sun, Wendell Q.

2002-01-01

Rate of dehydration greatly affects desiccation tolerance of recalcitrant seeds. This effect is presumably related to two different stress vectors: direct mechanical or physical stress because of the loss of water and physicochemical damage of tissues as a result of metabolic alterations during drying. The present study proposed a new theoretic approach to represent these two types of stresses and investigated how seed tissues responded differently to two stress vectors, using the models of isolated cocoa (Theobroma cacao) and ginkgo (Ginkgo biloba) embryonic tissues dehydrated under various drying conditions. This approach used the differential change in axis water potential (ΔΨ/Δt) to quantify rate of dehydration and the intensity of direct physical stress experienced by embryonic tissues during desiccation. Physicochemical effect of drying was expressed by cumulative desiccation stress [∫\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\pagestyle{empty} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{o}^{t}}}\\end{equation*}\\end{document}f(ψ,t)], a function of both the rate and time of dehydration. Rapid dehydration increased the sensitivity of embryonic tissues to desiccation as indicated by high critical water contents, below which desiccation damage occurred. Cumulative desiccation stress increased sharply under slow drying conditions, which was also detrimental to embryonic tissues. This quantitative analysis of the stress-time-response relationship helps to understand the physiological basis for the existence of an optimal dehydration rate, with which maximum desiccation tolerance could be achieved. The established numerical analysis model will prove valuable for the design of experiments that aim to elucidate biochemical and physiological mechanisms of desiccation tolerance. PMID:11950981

Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

PubMed

Uchida, Keiko; Nakazawa, Maki; Yamagishi, Chihiro; Mikoshiba, Katsuhiko; Yamagishi, Hiroyuki

2016-10-01

The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface. Copyright © 2016 Elsevier Inc. All rights reserved.

A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury.

PubMed

Guenther, Catherine A; Wang, Zhen; Li, Emma; Tran, Misha C; Logan, Catriona Y; Nusse, Roel; Pantalena-Filho, Luiz; Yang, George P; Kingsley, David M

2015-08-01

Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an "injury response" control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals. Copyright © 2015 Elsevier Inc. All rights reserved.

FGF signalling controls anterior extraembryonic and embryonic fate in the beetle Tribolium.

PubMed

Sharma, Rahul; Beermann, Anke; Schröder, Reinhard

2013-09-01

Fibroblast growth factor (FGF) signalling plays a key role in early embryonic development and cell migration in vertebrates and in invertebrates. To gain novel insights into FGF signalling in an arthropod, we characterized the fgf1b ortholog in the beetle Tribolium that is not represented in the Drosophila genome. We found that FGF1b dependent signalling organizes the anterior to posterior axis of the early embryo. The loss of Tc-fgf1b function in Tribolium by RNA interference resulted in the reduction of the anteriormost extraembryonic fate, in an anterior shift of embryonic fate and in the loss or malformation of anterior embryonic structures. Without intact extraembryonic membranes the serosa and the amnion, Tc-fgf1b(RNAi) embryos did not undergo morphogenetic movements and remained posteriorly localized throughout embryogenesis. Only weakly affected embryos developed into a cuticle that show dorsally curved bodies with head defects and a dorsal opening. Except for the posterior dorsal amnion, the overall topology of the dorsal-ventral axis seemed unaffected. Moreover, FGF signalling was not required for the onset of mesoderm formation but for fine-tuning this tissue during later development. We also show that in affected embryos the dorsal epidermis was expanded and expressed Tc-dpp at a higher level. We conclude that in the Tribolium blastoderm embryo, FGF1-signalling organizes patterning along the AP-axis and also balances the expression level of Dpp in the dorsal epidermis, a tissue critically involved in dorsal closure. Copyright © 2013 Elsevier Inc. All rights reserved.

TORC2 signaling antagonizes SKN-1 to induce C. elegans mesendodermal embryonic development

PubMed Central

Ruf, Vanessa; Holzem, Christina; Peyman, Tobias; Walz, Gerd; Blackwell, T. Keith; Neumann-Haefelin, Elke

2013-01-01

The evolutionarily conserved target of rapamycin (TOR) kinase controls fundamental metabolic processes to support cell and tissue growth. TOR functions within the context of two distinct complexes, TORC1 and TORC2. TORC2, with its specific component Rictor, has been recently implicated in aging and regulation of growth and metabolism. Here, we identify rict-1/Rictor as a regulator of embryonic development in C. elegans. The transcription factor skn-1 establishes development of the mesendoderm in embryos, and is required for cellular homeostasis and longevity in adults. Loss of maternal skn-1 function leads to misspecification of the mesendodermal precursor and failure to form intestine and pharynx. We found that genetic inactivation of rict-1 suppressed skn-1-associated lethality by restoring mesendodermal specification in skn-1 deficient embryos. Inactivation of other TORC2 but not TORC1 components also partially rescued skn-1 embryonic lethality. The SGK-1 kinase mediated these functions downstream of rict-1/TORC2, as a sgk-1 gain-of-function mutant suppressed the rict-1 mutant phenotype. These data indicate that TORC2 and SGK-1 antagonize SKN-1 during embryonic development. PMID:23973804

Growth factor involvement in tension-induced skeletal muscle growth

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman W.

1987-01-01

New muscle tissue culture techniques were developed to grow embryonic skeletal myofibers which are able to differentiate into more adultlike myofibers. Studies on mechanical simulation of cultured muscle cell growth will now be more directly applicable to mechanically-induced growth in adult muscle, and lead to better models for understanding muscle tissue atrophy caused by disuse in the microgravity of space.

A cell-autonomous requirement for neutral sphingomyelinase 2 in bone mineralization

PubMed Central

Khavandgar, Zohreh; Poirier, Christophe; Clarke, Christopher J.; Li, Jingjing; Wang, Nicholas; McKee, Marc D.; Hannun, Yusuf A.

2011-01-01

A deletion mutation called fro (fragilitas ossium) in the murine Smpd3 (sphingomyelin phosphodiesterase 3) gene leads to a severe skeletal dysplasia. Smpd3 encodes a neutral sphingomyelinase (nSMase2), which cleaves sphingomyelin to generate bioactive lipid metabolites. We examined endochondral ossification in embryonic day 15.5 fro/fro mouse embryos and observed impaired apoptosis of hypertrophic chondrocytes and severely undermineralized cortical bones in the developing skeleton. In a recent study, it was suggested that nSMase2 activity in the brain regulates skeletal development through endocrine factors. However, we detected Smpd3 expression in both embryonic and postnatal skeletal tissues in wild-type mice. To investigate whether nSMase2 plays a cell-autonomous role in these tissues, we examined the in vitro mineralization properties of fro/fro osteoblast cultures. fro/fro cultures mineralized less than the control osteoblast cultures. We next generated fro/fro;Col1a1-Smpd3 mice, in which osteoblast-specific expression of Smpd3 corrected the bone abnormalities observed in fro/fro embryos without affecting the cartilage phenotype. Our data suggest tissue-specific roles for nSMase2 in skeletal tissues. PMID:21788370

Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

PubMed

Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

2015-02-25

Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

Neural Stem Cells Derived Directly from Adipose Tissue.

PubMed

Petersen, Eric D; Zenchak, Jessica R; Lossia, Olivia V; Hochgeschwender, Ute

2018-05-01

Neural stem cells (NSCs) are characterized as self-renewing cell populations with the ability to differentiate into the multiple tissue types of the central nervous system. These cells can differentiate into mature neurons, astrocytes, and oligodendrocytes. This category of stem cells has been shown to be a promisingly effective treatment for neurodegenerative diseases and neuronal injury. Most treatment studies with NSCs in animal models use embryonic brain-derived NSCs. This approach presents both ethical and feasibility issues for translation to human patients. Adult tissue is a more practical source of stem cells for transplantation therapies in humans. Some adult tissues such as adipose tissue and bone marrow contain a wide variety of stem cell populations, some of which have been shown to be similar to embryonic stem cells, possessing many pluripotent properties. Of these stem cell populations, some are able to respond to neuronal growth factors and can be expanded in vitro, forming neurospheres analogous to cells harvested from embryonic brain tissue. In this study, we describe a method for the collection and culture of cells from adipose tissue that directly, without going through intermediates such as mesenchymal stem cells, results in a population of NSCs that are able to be expanded in vitro and be differentiated into functional neuronal cells. These adipose-derived NSCs display a similar phenotype to those directly derived from embryonic brain. When differentiated into neurons, cells derived from adipose tissue have spontaneous spiking activity with network characteristics similar to that of neuronal cultures.

MSX-1 gene expression and regulation in embryonic palatal tissue.

PubMed

Nugent, P; Greene, R M

1998-01-01

The palatal cleft seen in Msx-1 knock-out mice suggests a role for this gene in normal palate development. The cleft is presumed secondary to tooth and jaw malformations, since in situ hybridization suggests that Msx-1 mRNA is not highly expressed in developing palatal tissue. In this study we demonstrate, by Northern blot analysis, the expression of Msx-1, but not Msx-2, in the developing palate and in primary cultures of murine embryonic palate mesenchymal cells. Furthermore, we propose a role for Msx-1 in retinoic acid-induced cleft palate, since retinoic acid inhibits Msx-1 mRNA expression in palate mesenchymal cells. We also demonstrate that transforming growth factor beta inhibits Msx-1 mRNA expression in palate mesenchymal cells, with retinoic acid and transforming growth factor beta acting synergistically when added simultaneously to these cells. These data suggest a mechanistic interaction between retinoic acid, transforming growth factor beta, and Msx-1 in the etiology of retinoic acid-induced cleft palate.

UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity.

PubMed

Wang, Chaochen; Lee, Ji-Eun; Cho, Young-Wook; Xiao, Ying; Jin, Qihuang; Liu, Chengyu; Ge, Kai

2012-09-18

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.

Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro.

PubMed

Harrison, Sarah Ellys; Sozen, Berna; Christodoulou, Neophytos; Kyprianou, Christos; Zernicka-Goetz, Magdalena

2017-04-14

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos. Copyright © 2017, American Association for the Advancement of Science.

Anterograde Tracing Method using DiI to Label Vagal Innervation of the Embryonic and Early Postnatal Mouse Gastrointestinal Tract

PubMed Central

Murphy, Michelle C.; Fox, Edward A.

2007-01-01

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations, or pharmacological manipulations. Therefore, a method using 1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development. PMID:17418900

The effect of matrix composition of 3D constructs on embryonic stem cell differentiation.

PubMed

Battista, Sabrina; Guarnieri, Daniela; Borselli, Cristina; Zeppetelli, Stefania; Borzacchiello, Assunta; Mayol, Laura; Gerbasio, Diego; Keene, Douglas R; Ambrosio, Luigi; Netti, Paolo A

2005-11-01

The use of embryonic stem (ES) cells as unlimited cell source in tissue engineering has ignited the hope of regenerating any kind of tissue in vitro. However, the role of the material in control and guidance of their development and commitment into complex and viable three-dimensional (3D) tissues is still poorly understood. In this work, we investigate the role of material composition and structure on promoting ES cells growth and differentiation, by culturing mouse ES cell-derived embryoid bodies (EBs) in various semi-interpenetrating polymer networks (SIPNs), made of collagen, fibronectin (FN) and laminin (LM). We show that both composition and strength of the supportive matrix play an important role in EBs development. High collagen concentrations inhibit EBs cavitation and hence the following EBs differentiation, by inhibiting apoptosis. The presence of FN in 3D collagen constructs strongly stimulates endothelial cell differentiation and vascularization. Conversely, LM increases the ability of ES cells to differentiate into beating cardiomyocytes. Our data suggest that matrix composition has an important role in EBs development and that it is possible to influence stem cell differentiation toward preferential pattern, by modulating the physical and biochemical properties of the scaffold.

Fibroblast growth factor receptor 2 (FGFR2) is required for corneal epithelial cell proliferation and differentiation during embryonic development.

PubMed

Zhang, Jinglin; Upadhya, Dinesh; Lu, Lin; Reneker, Lixing W

2015-01-01

Fibroblast growth factors (FGFs) play important roles in many aspects of embryonic development. During eye development, the lens and corneal epithelium are derived from the same surface ectodermal tissue. FGF receptor (FGFR)-signaling is essential for lens cell differentiation and survival, but its role in corneal development has not been fully investigated. In this study, we examined the corneal defects in Fgfr2 conditional knockout mice in which Cre expression is activated at lens induction stage by Pax6 P0 promoter. The cornea in LeCre, Fgfr2(loxP/loxP) mice (referred as Fgfr2(CKO)) was analyzed to assess changes in cell proliferation, differentiation and survival. We found that Fgfr2(CKO) cornea was much thinner in epithelial and stromal layer when compared to WT cornea. At embryonic day 12.5-13.5 (E12.5-13.5) shortly after the lens vesicle detaches from the overlying surface ectoderm, cell proliferation (judged by labeling indices of Ki-67, BrdU and phospho-histone H3) was significantly reduced in corneal epithelium in Fgfr2(CKO) mice. At later stage, cell differentiation markers for corneal epithelium and underlying stromal mesenchyme, keratin-12 and keratocan respectively, were not expressed in Fgfr2(CKO) cornea. Furthermore, Pax6, a transcription factor essential for eye development, was not present in the Fgfr2(CKO) mutant corneal epithelial at E16.5 but was expressed normally at E12.5, suggesting that FGFR2-signaling is required for maintaining Pax6 expression in this tissue. Interestingly, the role of FGFR2 in corneal epithelial development is independent of ERK1/2-signaling. In contrast to the lens, FGFR2 is not required for cell survival in cornea. This study demonstrates for the first time that FGFR2 plays an essential role in controlling cell proliferation and differentiation, and maintaining Pax6 levels in corneal epithelium via ERK-independent pathways during embryonic development.

Preliminary observations on the effects of selenate on the development of the embryonic skate, Raja eglanteria

NASA Technical Reports Server (NTRS)

Conrad, G. W.; Luer, C. A.; Paulsen, A. Q.; Funderburgh, J. L.; Spooner, B. S. (Principal Investigator)

1993-01-01

Morphogenesis of the clearnose skate, Raja eglanteria, was not significantly inhibited as a result of 7 days of exposure to 1-2 mM selenate in the sea water during Days 59-69 of embryonic development (hatching would normally have occurred at 82 +/- 4 days of incubation). Although corneal transparency appeared normal in the eye, preliminary measurements of the thickness of Bowman's layer of the cornea suggested that it was significantly thinner in the corneas of embryos exposed to 1-2 mM selenate. Selenate is an ion reported to inhibit sulfation of glycosaminoglycans in connective tissue.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds.

PubMed

Zhou, Jin; Zhang, Ye; Lin, Qiuxia; Liu, Zhiqiang; Wang, Haibin; Duan, Cuimi; Wang, Yanmeng; Hao, Tong; Wu, Kuiwu; Wang, Changyong

2010-07-01

Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture, they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment. Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

The mechanics of development: models and methods for tissue morphogenesis

PubMed Central

Gjorevski, Nikolce; Nelson, Celeste M.

2011-01-01

Embryonic development is a physical process during which masses of cells are sculpted into functional organs. The mechanical properties of tissues and the forces exerted on them serve as epigenetic regulators of morphogenesis. Understanding these mechanobiological effects in the embryo requires new experimental approaches. Here we focus on branching of the lung airways and bending of the heart tube to describe examples of mechanical and physical cues that guide cell fate decisions and organogenesis. We highlight recent technological advances to measure tissue elasticity and endogenous mechanical stresses in real time during organ development. We also discuss recent progress in manipulating forces in intact embryos. PMID:20860059

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

PubMed

Russ, Holger A; Landsman, Limor; Moss, Christopher L; Higdon, Roger; Greer, Renee L; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

PubMed Central

Russ, Holger A.; Landsman, Limor; Moss, Christopher L.; Higdon, Roger; Greer, Renee L.; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos.

PubMed

Masaki, Hideki; Kato-Itoh, Megumi; Takahashi, Yusuke; Umino, Ayumi; Sato, Hideyuki; Ito, Keiichi; Yanagida, Ayaka; Nishimura, Toshinobu; Yamaguchi, Tomoyuki; Hirabayashi, Masumi; Era, Takumi; Loh, Kyle M; Wu, Sean M; Weissman, Irving L; Nakauchi, Hiromitsu

2016-11-03

Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17 + endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17 + endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine. Copyright © 2016 Elsevier Inc. All rights reserved.

Deciphering the Epigenetic Code in Embryonic and Dental Pulp Stem Cells

PubMed Central

Bayarsaihan, Dashzeveg

2016-01-01

A close cooperation between chromatin states, transcriptional modulation, and epigenetic modifications is required for establishing appropriate regulatory circuits underlying self-renewal and differentiation of adult and embryonic stem cells. A growing body of research has established that the epigenome topology provides a structural framework for engaging genes in the non-random chromosomal interactions to orchestrate complex processes such as cell-matrix interactions, cell adhesion and cell migration during lineage commitment. Over the past few years, the functional dissection of the epigenetic landscape has become increasingly important for understanding gene expression dynamics in stem cells naturally found in most tissues. Adult stem cells of the human dental pulp hold great promise for tissue engineering, particularly in the skeletal and tooth regenerative medicine. It is therefore likely that progress towards pulp regeneration will have a substantial impact on the clinical research. This review summarizes the current state of knowledge regarding epigenetic cues that have evolved to regulate the pluripotent differentiation potential of embryonic stem cells and the lineage determination of developing dental pulp progenitors. PMID:28018144

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

PubMed

Eiraku, Mototsugu; Sasai, Yoshiki

2011-12-15

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

Histological image data of limb skeletal tissue from larval and adult Ambystoma mexicanum.

PubMed

McCusker, Catherine D; Diaz-Castillo, Carlos; Sosnik, Julian; Phan, Anne; Gardiner, David M

2016-09-01

The data presented in this article are related to the article entitled "Cartilage and bone cells do not participate in skeletal regeneration in Ambystoma mexicanum limbs" [1]. Here we present image data of the post-embryonic development of the forelimb skeletal tissue of Ambystoma Mexicanum. Histological staining was performed on sections from the intact limbs of young (6.5 cm) and old (25 cm) animals, and on dissected skeletal tissues (cartilage, bone, and periosteum) from these animals.

Impaired Embryonic Development in Mice Overexpressing the RNA-Binding Protein TIAR

PubMed Central

Kharraz, Yacine; Salmand, Pierre-Adrien; Camus, Anne; Auriol, Jacques; Gueydan, Cyril; Kruys, Véronique; Morello, Dominique

2010-01-01

Background TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. Methodology/Principal Findings To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR) allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2α that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. Conclusions/Significance This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming. PMID:20596534

Plant development. Integration of growth and patterning during vascular tissue formation in Arabidopsis.

PubMed

De Rybel, Bert; Adibi, Milad; Breda, Alice S; Wendrich, Jos R; Smit, Margot E; Novák, Ondřej; Yamaguchi, Nobutoshi; Yoshida, Saiko; Van Isterdael, Gert; Palovaara, Joakim; Nijsse, Bart; Boekschoten, Mark V; Hooiveld, Guido; Beeckman, Tom; Wagner, Doris; Ljung, Karin; Fleck, Christian; Weijers, Dolf

2014-08-08

Coordination of cell division and pattern formation is central to tissue and organ development, particularly in plants where walls prevent cell migration. Auxin and cytokinin are both critical for division and patterning, but it is unknown how these hormones converge upon tissue development. We identify a genetic network that reinforces an early embryonic bias in auxin distribution to create a local, nonresponding cytokinin source within the root vascular tissue. Experimental and theoretical evidence shows that these cells act as a tissue organizer by positioning the domain of oriented cell divisions. We further demonstrate that the auxin-cytokinin interaction acts as a spatial incoherent feed-forward loop, which is essential to generate distinct hormonal response zones, thus establishing a stable pattern within a growing vascular tissue. Copyright © 2014, American Association for the Advancement of Science.

The energy cost of embryonic development in fishes and amphibians, with emphasis on new data from the Australian lungfish, Neoceratodus forsteri.

PubMed

Mueller, Casey A; Joss, Jean M P; Seymour, Roger S

2011-01-01

The rate of oxygen consumption throughout embryonic development is used to indirectly determine the 'cost' of development, which includes both differentiation and growth. This cost is affected by temperature and the duration of incubation in anamniote fish and amphibian embryos. The influences of temperature on embryonic development rate, respiration rate and energetics were investigated in the Australian lungfish, Neoceratodus forsteri, and compared with published data. Developmental stage and oxygen consumption rate were measured until hatching, upon which wet and dry gut-free masses were determined. A measure of the cost of development, the total oxygen required to produce 1 mg of embryonic dry tissue, increased as temperature decreased. The relationship between the oxygen cost of development (C, ml mg(-1)) and dry hatchling mass (M, mg) in fishes and amphibians is described by C = 0.30 M(0.22 0.13 (95% CI)), r (2) = 0.52. The scaling exponent indicates that the cost of embryonic development increases disproportionally with increasing hatchling mass. At 15 and 20°C, N. forsteri cost of development is significantly lower than the regression mean for all species, and at 25°C is lower than the allometrically scaled data set. Unexpectedly, incubation of N. forsteri is long, despite natural development under relatively warm conditions, and may be related to a large genome size. The low cost of development may be associated with construction of a rather sluggish fish with a low capacity for aerobic metabolism. The metabolic rate is lower in N. forsteri hatchlings than in any other fishes or amphibians at the same temperature, which matches the extremely low aerobic metabolic scope of the juveniles.

Optical coherence tomography for embryonic imaging: a review

PubMed Central

Raghunathan, Raksha; Singh, Manmohan; Dickinson, Mary E.; Larin, Kirill V.

2016-01-01

Abstract. Embryogenesis is a highly complex and dynamic process, and its visualization is crucial for understanding basic physiological processes during development and for identifying and assessing possible defects, malformations, and diseases. While traditional imaging modalities, such as ultrasound biomicroscopy, micro-magnetic resonance imaging, and micro-computed tomography, have long been adapted for embryonic imaging, these techniques generally have limitations in their speed, spatial resolution, and contrast to capture processes such as cardiodynamics during embryogenesis. Optical coherence tomography (OCT) is a noninvasive imaging modality with micrometer-scale spatial resolution and imaging depth up to a few millimeters in tissue. OCT has bridged the gap between ultrahigh resolution imaging techniques with limited imaging depth like confocal microscopy and modalities, such as ultrasound sonography, which have deeper penetration but poorer spatial resolution. Moreover, the noninvasive nature of OCT has enabled live imaging of embryos without any external contrast agents. We review how OCT has been utilized to study developing embryos and also discuss advances in techniques used in conjunction with OCT to understand embryonic development. PMID:27228503

Contribution of Caudal Müllerian Duct Mesenchyme to Prostate Development

PubMed Central

Brechka, Hannah; McAuley, Erin M.; Lamperis, Sophia M.; Paner, Gladell P.

2016-01-01

A fundamental understanding of prostate development and tissue homeostasis has the high potential to reveal mechanisms for prostate disease initiation and identify novel therapeutic approaches for disease prevention and treatment. Our current understanding of prostate lineage specification stems from the use of developmental model systems that rely upon the embryonic preprostatic urogenital sinus mesenchyme to induce the formation of mature prostate epithelial cells. It is unclear, however, how the urogenital sinus epithelium can derive both adult urethral glands and prostate epithelia. Furthermore, the vast disparity in disease initiation between these two glands highlights key developmental factors that predispose prostate epithelia to hyperplasia and cancer. In this study we demonstrate that the caudal Müllerian duct mesenchyme (CMDM) drives prostate epithelial differentiation and is a key determinant in cell lineage specification between urethral glands and prostate epithelia. Utilizing both human embryonic stem cells and mouse embryonic tissues, we document that the CMDM is capable of inducing the specification of androgen receptor, prostate-specific antigen, NKX3.1, and Hoxb13-positive prostate epithelial cells. These results help to explain key developmental differences between prostate and urethral gland differentiation, and implicate factors secreted by the caudal Müllerian duct as novel targets for prostate disease prevention and treatment. PMID:27595922

Is fibroblast growth factor receptor 4 a suitable target of cancer therapy?

PubMed

Heinzle, Christine; Erdem, Zeynep; Paur, Jakob; Grasl-Kraupp, Bettina; Holzmann, Klaus; Grusch, Michael; Berger, Walter; Marian, Brigitte

2014-01-01

Fibroblast growth factors (FGF) and their tyrosine kinase receptors (FGFR) support cell proliferation, survival and migration during embryonic development, organogenesis and tissue maintenance and their deregulation is frequently observed in cancer development and progression. Consequently, increasing efforts are focusing on the development of strategies to target FGF/FGFR signaling for cancer therapy. Among the FGFRs the family member FGFR4 is least well understood and differs from FGFRs1-3 in several aspects. Importantly, FGFR4 deletion does not lead to an embryonic lethal phenotype suggesting the possibility that its inhibition in cancer therapy might not cause grave adverse effects. In addition, the FGFR4 kinase domain differs sufficiently from those of FGFRs1-3 to permit development of highly specific inhibitors. The oncogenic impact of FGFR4, however, is not undisputed, as the FGFR4-mediated hormonal effects of several FGF ligands may also constitute a tissue-protective tumor suppressor activity especially in the liver. Therefore it is the purpose of this review to summarize all relevant aspects of FGFR4 physiology and pathophysiology and discuss the options of targeting this receptor for cancer therapy.

Adipogenesis and epicardial adipose tissue: A novel fate of the epicardium induced by mesenchymal transformation and PPARγ activation

PubMed Central

Yamaguchi, Yukiko; Cavallero, Susana; Patterson, Michaela; Shen, Hua; Xu, Jian; Kumar, S. Ram; Sucov, Henry M.

2015-01-01

The hearts of many mammalian species are surrounded by an extensive layer of fat called epicardial adipose tissue (EAT). The lineage origins and determinative mechanisms of EAT development are unclear, in part because mice and other experimentally tractable model organisms are thought to not have this tissue. In this study, we show that mouse hearts have EAT, localized to a specific region in the atrial–ventricular groove. Lineage analysis indicates that this adipose tissue originates from the epicardium, a multipotent epithelium that until now is only established to normally generate cardiac fibroblasts and coronary smooth muscle cells. We show that adoption of the adipocyte fate in vivo requires activation of the peroxisome proliferator activated receptor gamma (PPARγ) pathway, and that this fate can be ectopically induced in mouse ventricular epicardium, either in embryonic or adult stages, by expression and activation of PPARγ at times of epicardium–mesenchymal transformation. Human embryonic ventricular epicardial cells natively express PPARγ, which explains the abundant presence of fat seen in human hearts at birth and throughout life. PMID:25646471

Wisdom teeth: mankind's future third vice-teeth?

PubMed

Zou, DuoHong; Zhao, Jun; Ding, WangHui; Xia, LunGuo; Jang, XinQuan; Huang, YuanLiang

2010-01-01

The third molar teeth (wisdom teeth) represent the last eruption of the teeth in the human dentition. Throughout evolution, the mandible has had a tendency to decrease in size; the third molar teeth are often impacted, resulting in incomplete tooth eruption that often causes clinical pericoronitis, dental caries, and pericemental abscess. Therefore, the wisdom teeth are often extracted. Moreover, wisdom teeth are often removed for clinical orthodontic treatment. On the other hand, tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect people's lives. Autologous tissues for dental tissue regeneration that could replace lost teeth could provide a vital alternative to currently available clinical treatments. To pursue this goal, we hypothesize that human third molar tooth buds can be obtained during development. Human wisdom tooth germination tissue could then be placed into an embryonic stem cell bank for storage. When the donor's other teeth are missing, embryonic stem cell and tissue engineering technologies, will permit the restoration of the missing teeth. Therefore wisdom teeth will be mankind's future third vice-teeth.

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs

PubMed Central

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A.

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial–mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues. PMID:22539926

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs.

PubMed

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial-mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues.

Scube3 Is Expressed in Multiple Tissues during Development but Is Dispensable for Embryonic Survival in the Mouse

PubMed Central

Xavier, Guilherme M.; Panousopoulos, Leonidas; Cobourne, Martyn T.

2013-01-01

The vertebrate Scube family consists of three independent members Scube1-3; which encode secreted cell surface-associated membrane glycoproteins that share a domain organization of at least five recognizable motifs and the ability to both homo- and heterodimerize. There is recent biochemical evidence to suggest that Scube2 is directly involved in Hedgehog signaling, acting co-operatively with Dispatched to mediate the release in soluble form of cholesterol and palmitate-modified Hedgehog ligand during long-range activity. Indeed, in the zebrafish myotome, all three Scube proteins can subtly promote Hedgehog signal transduction in a non-cell autonomous manner. In order to further investigate the role of Scube genes during development, we have generated mice with targeted inactivation of Scube3. Despite a dynamic developmental expression pattern, with transcripts present in neuroectoderm, endoderm and endochondral tissues, particularly within the craniofacial region; an absence of Scube3 function results in no overt embryonic phenotype in the mouse. Mutant mice are born at expected Mendelian ratios, are both viable and fertile, and seemingly retain normal Hedgehog signaling activity in craniofacial tissues. These findings suggest that in the mouse, Scube3 is dispensable for normal development; however, they do not exclude the possibility of a co-operative role for Scube3 with other Scube members during embryogenesis or a potential role in adult tissue homeostasis over the long-term. PMID:23383134

In Situ Histochemical Localisation of Alkaloids and Acetogenins in the Endosperm and Embryonic Axis of Annona Macroprophyllata Donn. Sm. Seeds During Germination

PubMed Central

Brechú-Franco, A.E.; Laguna-Hernández, G.; De la Cruz-Chacón, I.; González-Esquinca, A.R.

2016-01-01

Currently, the Annonaceae family is characterised by the production of acetogenins (ACGs), and also by the biosynthesis of alkaloids, primarily benzylisoquinolines derived from tyrosine. The objective of this study was to confirm the presence of alkaloids and acetogenins in the idioblasts of the endosperm and the embryonic axis of A. macroprophyllata seeds in germination. The Dragendorff, Dittmar, Ellram, and Lugol reagents were used to test for alkaloids, and Kedde’s reagent was used to determine the presence of acetogenins in fresh sections of the endosperm and embryonic axis of seeds after twelve days of germination. A positive reaction was observed for all the reagents, and the presence of alkaloids and acetogenins was confirmed in the idioblasts of the endosperm and those involved in the differentiation of the embryonic axis of the developing seedling. We concluded that the idioblasts store both metabolites, acetogenins and alkaloids. Beginning at differentiation, the idioblasts of the embryonic axis simultaneously biosynthesise acetogenins and alkaloids that are characteristic of the species during the development of the seedling. The method used here can be applied to histochemically confirm the presence of acetogenins and alkaloids in tissues and structures of the plant in different stages of its life cycle. PMID:26972713

Apical and basal epitheliomuscular F-actin dynamics during Hydra bud evagination

PubMed Central

Aufschnaiter, Roland; Wedlich-Söldner, Roland; Zhang, Xiaoming

2017-01-01

ABSTRACT Bending of 2D cell sheets is a fundamental morphogenetic mechanism during animal development and reproduction. A critical player driving cell shape during tissue bending is the actin cytoskeleton. Much of our current knowledge about actin dynamics in whole organisms stems from studies of embryonic development in bilaterian model organisms. Here, we have analyzed actin-based processes during asexual bud evagination in the simple metazoan Hydra. We created transgenic Hydra strains stably expressing the actin marker Lifeact-GFP in either ectodermal or endodermal epitheliomuscular cells. We then combined live imaging with conventional phalloidin staining to directly follow actin reorganization. Bending of the Hydra epithelial double layer is initiated by a group of epitheliomuscular cells in the endodermal layer. These cells shorten their apical-basal axis and arrange their basal muscle processes in a circular configuration. We propose that this rearrangement generates the initial forces to bend the endoderm towards the ectoderm. Convergent tissue movement in both epithelial layers towards the centre of evagination then leads to elongation and extension of the bud along its new body axis. Tissue movement into the bud is associated with lateral intercalation of epithelial cells, remodelling of apical septate junctions, and rearrangement of basal muscle processes. The work presented here extends the analysis of morphogenetic mechanisms beyond embryonic tissues of model bilaterians. PMID:28630355

The Lin28/Let-7 System in Early Human Embryonic Tissue and Ectopic Pregnancy

PubMed Central

Steffani, Liliana; Martínez, Sebastián; Monterde, Mercedes; Ferri, Blanca; Núñez, Maria Jose; AinhoaRomero-Espinós; Zamora, Omar; Gurrea, Marta; Sangiao-Alvarellos, Susana; Vega, Olivia; Simón, Carlos; Pellicer, Antonio; Tena-Sempere, Manuel

2014-01-01

Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs), in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases) and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls) were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7–9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (de)regulated pathways in the pathophysiology of ectopic pregnancy in humans. PMID:24498170

Mechanical analysis of a heat-shock induced developmental defect

NASA Astrophysics Data System (ADS)

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

2014-03-01

Embryonic development in Drosophila is a complex process involving coordinated movements of mechanically interacting tissues. Perturbing this system with a transient heat shock can result in a number of developmental defects. In particular, a heat shock applied during the earliest morphogenetic movements of gastrulation can lead to apparent recovery, but then subsequent morphogenetic failure 5-6 hours later during germ band retraction. The process of germ band retraction requires an intact amnioserosa - a single layered extra-embryonic epithelial tissue - and heat shock at gastrulation can induce the later opening of holes in the amnioserosa. These holes are highly correlated with failures of germ band retraction. These holes could be caused by a combination of mechanical weakness in the amnioserosa or local increases in mechanical stress. Here, we assess the role of mechanical stress using confocal imaging to compare cell and tissue morphology in the amnioserosa of normal and heat-shocked embryos and laser hole drilling to map the stress field around the times and locations at which heat-shock induced holes open.

Cloning of aged animals: a medical model for tissue and organ regeneration.

PubMed

Tian, X C; Kubota, C; Yang, X

2001-11-01

Cloning by nuclear transfer has great potential application in pharmaceutical protein production, xeno-transplantation, and perhaps most excitingly, therapeutic cloning. In therapeutic cloning a patient's own skin cells can be used to generate cloned embryos from which embryonic stem cells are isolated. Through targeted differentiation, embryonic stem cells can be directed to develop into the desired tissues/organs for replacement. The combination of homologous recombination of genes and nuclear transfer also offers the promise of correcting defective genes in humans. Demonstration of the successful cloning of aged animals is important for these future medical applications because degenerative diseases often afflict older adults. Our studies have demonstrated that skin fibroblast cells from aged adults, even after prolonged culture, provide nuclear donors equally as competent for cloning as cells from young adults or fetuses. These findings have paved the way for medically treating degenerative diseases of aged humans by tissue regeneration technologies made possible through cloning and homologous recombination.

Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.

PubMed

Whittington, Niteace C; Wray, Susan

2017-10-23

Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Unique patterns of organization and migration of FGF-expressing cells during Drosophila morphogenesis.

PubMed

Du, Lijuan; Zhou, Amy; Patel, Akshay; Rao, Mishal; Anderson, Kelsey; Roy, Sougata

2017-07-01

Fibroblast growth factors (FGF) are essential signaling proteins that regulate diverse cellular functions in developmental and metabolic processes. In Drosophila, the FGF homolog, branchless (bnl) is expressed in a dynamic and spatiotemporally restricted pattern to induce branching morphogenesis of the trachea, which expresses the Bnl-receptor, breathless (btl). Here we have developed a new strategy to determine bnl- expressing cells and study their interactions with the btl-expressing cells in the range of tissue patterning during Drosophila development. To enable targeted gene expression specifically in the bnl expressing cells, a new LexA based bnl enhancer trap line was generated using CRISPR/Cas9 based genome editing. Analyses of the spatiotemporal expression of the reporter in various embryonic stages, larval or adult tissues and in metabolic hypoxia, confirmed its target specificity and versatility. With this tool, new bnl expressing cells, their unique organization and functional interactions with the btl-expressing cells were uncovered in a larval tracheoblast niche in the leg imaginal discs, in larval photoreceptors of the developing retina, and in the embryonic central nervous system. The targeted expression system also facilitated live imaging of simultaneously labeled Bnl sources and tracheal cells, which revealed a unique morphogenetic movement of the embryonic bnl- source. Migration of bnl- expressing cells may create a dynamic spatiotemporal pattern of the signal source necessary for the directional growth of the tracheal branch. The genetic tool and the comprehensive profile of expression, organization, and activity of various types of bnl-expressing cells described in this study provided us with an important foundation for future research investigating the mechanisms underlying Bnl signaling in tissue morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulation of bone morphogenetic proteins in early embryonic development

NASA Astrophysics Data System (ADS)

Yamamoto, Yukiyo; Oelgeschläger, Michael

2004-11-01

Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

PubMed

Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

2016-07-01

The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general. © 2016. Published by The Company of Biologists Ltd.

Hypoxia delays hematopoiesis: retention of embryonic hemoglobin and erythrocytes in larval rainbow trout, Oncorhynchus mykiss, during chronic hypoxia exposure.

PubMed

Bianchini, Kristin; Wright, Patricia A

2013-12-01

In rainbow trout development, a switch occurs from high-affinity embryonic hemoglobin (Hb) and round, embryonic erythrocytes to lower-affinity adult Hb and oval, adult erythrocytes. Our study investigated the early ontogeny of rainbow trout blood properties and the hypoxia response. We hypothesized that hypoxia exposure would delay the ontogenetic turnover of Hb and erythrocytes because retention of high-affinity embryonic Hb would facilitate oxygen loading. To test this hypothesis we developed a method of efficiently extracting blood from individual embryos and larvae and optimized several techniques for measuring hematological parameters on microliter (0.5-2.0 μl) blood samples. In chronic hypoxia (30% of oxygen saturation), stage-matched embryos and larvae possessed half the Hb concentration, erythrocyte counts and hematocrit observed in normoxia. Hypoxia-reared larvae also had threefold to sixfold higher mRNA expression of the embryonic Hb α-1, β-1 and β-2 subunits relative to stage-matched normoxia-reared larvae. Furthermore, in hypoxia, the round embryonic erythrocytic shape persisted into later developmental stages. Despite these differences, Hb-oxygen affinity (P50), cooperativity and the Root effect were unaltered in hypoxia-reared O. mykiss. The data support our hypothesis that chronic hypoxia delays the ontogenetic turnover of Hb and erythrocytes, but without the predicted functional consequences (i.e. higher than expected P50). These results also suggest that the Hb-oxygen affinity is protected during development in chronic hypoxia to favor oxygen unloading at the tissues. We conclude that in early trout development, the blood-oxygen transport system responds very differently to chronic hypoxia relative to adults, possibly because respiration depends relatively more on oxygen diffusion than convection.

Early events in xenograft development from the human embryonic stem cell line HS181--resemblance with an initial multiple epiblast formation.

PubMed

Gertow, Karin; Cedervall, Jessica; Jamil, Seema; Ali, Rouknuddin; Imreh, Marta P; Gulyas, Miklos; Sandstedt, Bengt; Ahrlund-Richter, Lars

2011-01-01

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart.

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 distribution in the mouse germlines as in D. melanogaster. The proportion of mutations that are fixed during early embryonic development is greatly underestimated. For example, a DNA lesion in a postmeiotic gamete that becomes fixed as a dominant mutation during early embryonic development of the F1 may produce an individual completely mutant in the germ line and relevant somatic tissue or, alternatively, the F1 germline may be completely mutant but with no relevant somatic tissues for detecting the mutation until the F2. In both cases the mutation would be classified as complete in the F1 and F2, respectively, and not recognized as embryonic in origin. Because germ cells differentiate later in mammalian development, there are more opportunities for correlation between germline and soma in the mammal than Drosophila. However, because the germ cells and any somatic tissue, like blood, are derived from small samples, there may be many individuals that test negative in blood but have germlines that are either mosaic or entirely mutant.

Spinach and carrots: vitamin A and health

USDA-ARS?s Scientific Manuscript database

Vitamin A is an essential nutrient for the promotion of general growth, maintenance of visual function, regulation of the differentiation of epithelial tissues and immune function, and embryonic development. Vitamin A can only be supplied naturally, either as preformed vitamin A from foods of animal...

Mechanisms of embryonic stomach development.

PubMed

McCracken, Kyle W; Wells, James M

2017-06-01

The stomach is a digestive organ that has important roles in human physiology and pathophysiology. The developmental origin of the stomach is the embryonic foregut, which also gives rise a number of other structures. There are several signaling pathways and transcription factors that are known to regulate stomach development at different stages, including foregut patterning, stomach specification, and gastric regionalization. These developmental events have important implications in later homeostasis and disease in the adult stomach. Here we will review the literature that has shaped our current understanding of the molecular mechanisms that coordinate gastric organogenesis. Further we will discuss how developmental paradigms have guided recent efforts to differentiate stomach tissue from pluripotent stem cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

The bHLH transcription factor Hand is regulated by Alk in the Drosophila embryonic gut

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

Varshney, Gaurav K.; Palmer, Ruth H.

2006-12-29

During embryonic development the midgut visceral muscle is formed by fusion of cells within the visceral mesoderm, a process initiated by the specification of a specialised cell type, the founder cell, within this tissue. Activation of the receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) in the developing visceral muscle of Drosophila melanogaster initiates a signal transduction pathway required for muscle fusion. In this paper, we have investigated downstream components which are regulated by this novel signalling pathway. Here we show that Alk-mediated signal transduction drives the expression of the bHLH transcription factor Hand in vivo. Loss of Alk function resultsmore » in a complete lack of Hand expression in this tissue, whereas Alk gain of function results in an expansion of Hand expression. Finally, we have investigated the process of muscle fusion in the gut of Hand mutant animals and can find no obvious defects in this process, suggesting that Hand is not critical for visceral muscle fusion per se.« less

Embryonal rhabdomyosarcoma in an immature Baird's tapir (Tapirus bairdii).

PubMed

Bonar, Christopher J; Lewandowski, Albert H; Skowronek, Anthony J

2007-03-01

An immature Baird's tapir (Tapirus bairdii) with a history of seizure-like episodes developed signs of respiratory disease. The initial clinical diagnosis was pneumonia, and antibiotic therapy was started. The animal failed to improve after 14 days of therapy and developed unilateral, bloody nasal discharge. Endoscopic examination and radiography revealed a soft tissue mass in the nasopharynx depressing the soft palate. The tapir died 32 days after initial presentation. Histologic examination of the mass demonstrated a mesenchymal tumor composed of spindle cells with elongate nuclei forming densely packed fascicles. The neoplastic spindle cells showed prominent cross-striations. Immunohistochemistry revealed the cells to be positive for desmin and myoglobin, but negative for smooth muscle actin, confirming diagnosis of rhabdomyosarcoma. Embryonal rhabdomyosarcoma is the most common nasopharyngeal soft tissue tumor of humans, and it has been reported infrequently in dogs, horses, and pigs. Neoplasia should be a differential diagnosis in cases of unilateral nasal discharge and inspiratory stridor, even in young animals.

Programmed cell senescence during mammalian embryonic development.

PubMed

Muñoz-Espín, Daniel; Cañamero, Marta; Maraver, Antonio; Gómez-López, Gonzalo; Contreras, Julio; Murillo-Cuesta, Silvia; Rodríguez-Baeza, Alfonso; Varela-Nieto, Isabel; Ruberte, Jesús; Collado, Manuel; Serrano, Manuel

2013-11-21

Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-β/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence. Copyright © 2013 Elsevier Inc. All rights reserved.

Mapping the Extracellular and Membrane Proteome Associated with the Vasculature and the Stroma in the Embryo*

PubMed Central

Soulet, Fabienne; Kilarski, Witold W.; Roux-Dalvai, Florence; Herbert, John M. J.; Sacewicz, Izabela; Mouton-Barbosa, Emmanuelle; Bicknell, Roy; Lalor, Patricia; Monsarrat, Bernard; Bikfalvi, Andreas

2013-01-01

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments. PMID:23674615

The Evolution of Lineage-Specific Regulatory Activities in the Human Embryonic Limb

PubMed Central

Cotney, Justin; Leng, Jing; Yin, Jun; Reilly, Steven K.; DeMare, Laura E.; Emera, Deena; Ayoub, Albert E.; Rakic, Pasko; Noonan, James P.

2013-01-01

SUMMARY The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution. PMID:23827682

Stem Cells in Skeletal Tissue Engineering: Technologies and Models

PubMed Central

Langhans, Mark T.; Yu, Shuting; Tuan, Rocky S.

2017-01-01

This review surveys the use of pluripotent and multipotent stem cells in skeletal tissue engineering. Specific emphasis is focused on evaluating the function and activities of these cells in the context of development in vivo, and how technologies and methods of stem cell-based tissue engineering for stem cells must draw inspiration from developmental biology. Information on the embryonic origin and in vivo differentiation of skeletal tissues is first reviewed, to shed light on the persistence and activities of adult stem cells that remain in skeletal tissues after embryogenesis. Next, the development and differentiation of pluripotent stem cells is discussed, and some of their advantages and disadvantages in the context of tissue engineering is presented. The final section highlights current use of multipotent adult mesenchymal stem cells, reviewing their origin, differentiation capacity, and potential applications to tissue engineering. PMID:26423296

Robust cell tracking in epithelial tissues through identification of maximum common subgraphs.

PubMed

Kursawe, Jochen; Bardenet, Rémi; Zartman, Jeremiah J; Baker, Ruth E; Fletcher, Alexander G

2016-11-01

Tracking of cells in live-imaging microscopy videos of epithelial sheets is a powerful tool for investigating fundamental processes in embryonic development. Characterizing cell growth, proliferation, intercalation and apoptosis in epithelia helps us to understand how morphogenetic processes such as tissue invagination and extension are locally regulated and controlled. Accurate cell tracking requires correctly resolving cells entering or leaving the field of view between frames, cell neighbour exchanges, cell removals and cell divisions. However, current tracking methods for epithelial sheets are not robust to large morphogenetic deformations and require significant manual interventions. Here, we present a novel algorithm for epithelial cell tracking, exploiting the graph-theoretic concept of a 'maximum common subgraph' to track cells between frames of a video. Our algorithm does not require the adjustment of tissue-specific parameters, and scales in sub-quadratic time with tissue size. It does not rely on precise positional information, permitting large cell movements between frames and enabling tracking in datasets acquired at low temporal resolution due to experimental constraints such as phototoxicity. To demonstrate the method, we perform tracking on the Drosophila embryonic epidermis and compare cell-cell rearrangements to previous studies in other tissues. Our implementation is open source and generally applicable to epithelial tissues. © 2016 The Authors.

Robust cell tracking in epithelial tissues through identification of maximum common subgraphs

PubMed Central

Bardenet, Rémi; Zartman, Jeremiah J.; Baker, Ruth E.

2016-01-01

Tracking of cells in live-imaging microscopy videos of epithelial sheets is a powerful tool for investigating fundamental processes in embryonic development. Characterizing cell growth, proliferation, intercalation and apoptosis in epithelia helps us to understand how morphogenetic processes such as tissue invagination and extension are locally regulated and controlled. Accurate cell tracking requires correctly resolving cells entering or leaving the field of view between frames, cell neighbour exchanges, cell removals and cell divisions. However, current tracking methods for epithelial sheets are not robust to large morphogenetic deformations and require significant manual interventions. Here, we present a novel algorithm for epithelial cell tracking, exploiting the graph-theoretic concept of a ‘maximum common subgraph’ to track cells between frames of a video. Our algorithm does not require the adjustment of tissue-specific parameters, and scales in sub-quadratic time with tissue size. It does not rely on precise positional information, permitting large cell movements between frames and enabling tracking in datasets acquired at low temporal resolution due to experimental constraints such as phototoxicity. To demonstrate the method, we perform tracking on the Drosophila embryonic epidermis and compare cell–cell rearrangements to previous studies in other tissues. Our implementation is open source and generally applicable to epithelial tissues. PMID:28334699

Study of effects of radio-wave frequency radiation emitted from cellular telephones on embryonic development of danio rerio

NASA Astrophysics Data System (ADS)

Vagula, Mary; Harkless, Ryan

2013-05-01

Radio wave frequency (RF) radiation emitted from cellular telephones has become increasingly ubiquitous as a result of the popularity of these phones. With the increasing and unavoidable exposure to RF radiation a reality, it is imperative that the effects of such radiation on living tissue be well understood. In particular, it is critical to understand any effects that RF radiation may have as a carcinogen and on embryonic development, as pregnant women are not exempt from such exposure. As a model organism, zebrafish (Danio rerio) have been studied extensively, and their value in studies of gene expression cannot be overstated. This study observed the effects of RF radiation on the embryonic development of zebrafish. The expression of two genes, shha and hoxb9a, that are key to the early development of the fish was examined. Both genes have homologs in humans as well as in other model organisms. Preliminary results suggest that exposure to cell phone radiation might have an effect on the expression of shha in zebrafish embryos, causing under expression. More trials are necessary to validate these results.

Exposure of the eggs to 17alpha-methyl testosterone reduced hatching success and growth and elicited teratogenic effects in postembryonic life stages of crayfish.

PubMed

Vogt, Günter

2007-12-30

Testosterone is regularly found in the tissues of decapod crustaceans. Although this vertebrate-type sex hormone is not the principal factor of sex differentiation in crustaceans, it was shown to be capable of acting on the reproductive organs of shrimps and crabs. In the present study I have exposed developing eggs and stage 5 juveniles of the parthenogenetic all female marbled crayfish to 17alpha-methyl testosterone in order to test whether in freshwater crayfish sex can be changed from female to male by this androgen. MT did not elicit sex change, neither when administered during embryonic development nor during juvenile stage 5, the main period of proliferation of the oocytes. However, exposure to 100 microg/L MT from 64% to 84% embryonic development resulted in prolonged embryonic development, reduced hatching success, reduced growth of the juveniles, and severe malformations of the appendages in the juveniles. The marbled crayfish is recommended to be considered for toxicity tests due to its easy culture in the laboratory and its genotypical uniformity.

Promotion of hair follicle development and trichogenesis by Wnt-10b in cultured embryonic skin and in reconstituted skin

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

Ouji, Yukiteru; Yoshikawa, Masahide; Shiroi, Akira

2006-06-30

We previously showed that Wnt-10b promoted the differentiation of primary skin epithelial cells (MPSEC) toward hair shaft and inner root sheath of the hair follicle (IRS) cells in vitro. In the present study, we found that Wnt-10b promotes the development of hair follicles using a culture of mouse embryonic skin tissue and trichogenesis using a reconstitution experiment with nude mice. Hair follicle development was observed in skin taken from mouse embryos on embryonic day 10.5 following a 2-day culture with recombinant Wnt-10b (rWnt-10b), however, not without rWnt-10b. Brown hair growth was observed at the site of reconstituted skin in Balb/cmore » nude mice where dermal fibroblasts and keratinocytes, derived from C3H/HeN new born mice, were transplanted with Wnt-10b-producing COS cells (Wnt-COS). Without the co-transplantation of Wnt-COS, no hair growth was observed. Our results suggest an important role of Wnt-10b in the initiation of hair follicle development and following trichogenesis.« less

Psf2 plays important roles in normal eye development in Xenopus laevis

PubMed Central

Walter, Brian E.; Perry, Kimberly J.; Fukui, Lisa; Malloch, Erica L.; Wever, Jason

2008-01-01

Purpose Psf2 (partner of Sld5 2) represents a member of the GINS (go, ichi, ni, san) heterotetramer [1] and functions in DNA replication as a “sliding clamp.” Previous in situ hybridization analyses revealed that Psf2 is expressed during embryonic development in a tissue-specific manner, including the optic cup (retina) and the lens [2]. This article provides an analysis of Psf2 function during eye development in Xenopus laevis. Methods A morpholino targeted to Psf2 mRNA was designed to knockdown Psf2 translation and was injected into specific embryonic cells during early cleavage stages in the frog, Xenopus laevis. Injected embryos were assayed for specific defects in morphology, cell proliferation, and apoptosis. Synthetic Psf2 RNA was also co-injected with the morpholino to rescue morpholino-mediated developmental defects. It is well known that reciprocal inductive interactions control the development of the optic cup and lens. Therefore, control- and morpholino-injected embryos were used for reciprocal transplantation experiments to distinguish the intrinsic role of Psf2 in the development of the optic cup (retina) versus the lens. Results Morpholino-mediated knockdown of Psf2 expression resulted in dosage-dependent phenotypes, which included microphthalmia, incomplete closure of the ventral retinal fissure, and retinal and lens dysgenesis. Defects were also observed in other embryonic tissues that normally express Psf2 including the pharyngeal arches and the otic vesicle, although other tissues that express Psf2 were not found to be grossly defective. Eye defects could be rescued by co-injection of synthetic Psf2 RNA. Examination of cell proliferation via an antibody against phospho-histone H3 S10P revealed no significant differences in the retina and lens following Psf2 knockdown. However, there was a significant increase in the level of apoptosis in retinal as well as forebrain tissues, as revealed by TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay. Conclusions The results demonstrate intrinsic roles for Psf2 in both retinal and to a lesser extent, lens tissues. Observed lens defects can mainly be attributed to deficiencies in retinal development and consequently the late phase of lens induction, which involves instructive cues from the optic cup. Developmental defects were not observed in all tissues that express Psf2, which could be related to differences in the translation of Psf2 or redundant effects of related factors such as proliferating cell nuclear antigen (PCNA). PMID:18509549

Cancer: A Problem of Developmental Biology; Scientific Evidence for Reprogramming and Differentiation Therapy.

PubMed

Sell, Stewart; Nicolini, Andrea; Ferrari, Paola; Biava, Pier M

2016-01-01

Current medical literature acknowledges that embryonic micro-environment is able to suppress tumor development. Administering carcinogenic substances during organogenesis in fact leads to embryonic malformations, but not to offspring tumor growth. Once organogenesis has ended, administration of carcinogenic substances causes a rise in offspring tumor development. These data indicate that cancer can be considered a deviation in normal development, which can be regulated by factors of the embryonic microenvironment. Furthermore, it has been demonstrated that teratoma differentiates into normal tissues once it is implanted in the embryo. Recently, it has been shown that implanting a melanoma in Zebrafish embryo did not result in a tumor development; however, it did in the adult specimen. This demonstrates that cancer cells can differentiate into normal tissues when implanted in the embryo. In addition, it was demonstrated that other tumors can revert into a normal phenotype and/or differentiate into normal tissue when implanted in the embryo. These studies led some authors to define cancer as a problem of developmental biology and to predict the present concept of "cancer stem cells theory". In this review, we record the most important researches about the reprogramming and differentiation treatments of cancer cells to better clarify how the substances taken from developing embryo or other biological substances can induce differentiation of malignant cells. Lastly, a model of cancer has been proposed here, conceived by one of us, which is consistent with the reality, as demonstrated by a great number of researches. This model integrates the theory of the "maturation arrest" of cancer cells as conceived by B. Pierce with the theory which describes cancer as a process of deterministic chaos determined by genetic and/or epigenetic alterations in differentiated cells, which leads a normal cell to become cancerous. All the researches here described demonstrated that cancer can be considered a problem of developmental biology and that one of the most important hallmarks of cancer is the loss of differentiation as already described by us in other articles.

YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues.

PubMed

Liu, Ming; Zhao, Shuangyun; Lin, Qingjie; Wang, Xiu-Ping

2015-04-01

Yes-associated protein (YAP) is a Hippo signaling transcriptional coactivator that plays pivotal roles in stem cell proliferation, organ size control, and tumor development. The downstream targets of YAP have been shown to be highly context dependent. In this study, we used the embryonic mouse tooth germ as a tool to search for the downstream targets of YAP in ectoderm-derived tissues. Yap deficiency in the dental epithelium resulted in a small tooth germ with reduced epithelial cell proliferation. We compared the gene expression profiles of embryonic day 14.5 (E14.5) Yap conditional knockout and YAP transgenic mouse tooth germs using transcriptome sequencing (RNA-Seq) and further confirmed the differentially expressed genes using real-time PCR and in situ hybridization. We found that YAP regulates the expression of Hoxa1 and Hoxc13 in oral and dental epithelial tissues as well as in the epidermis of skin during embryonic and adult stages. Sphere formation assay suggested that Hoxa1 and Hoxc13 are functionally involved in YAP-regulated epithelial progenitor cell proliferation, and chromatin immunoprecipitation (ChIP) assay implies that YAP may regulate Hoxa1 and Hoxc13 expression through TEAD transcription factors. These results provide mechanistic insights into abnormal YAP activities in mice and humans. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Therapeutic cloning: far from application at this stage].

PubMed

De Both, N J

2001-11-03

Therapeutic cloning has become possible since the discovery that nuclei from somatic cells of adult animal tissue can successfully be used for cloning and the fact that human embryonic stem cell lines have been established from preimplantation embryos. When nuclei from healthy tissue of a patient are transplanted into enucleated oocytes, these oocytes can be artificially activated so that embryos develop from which embryonic stem cells of the donor can be derived. These embryonic stem cells can be cultured as permanent lines in unlimited numbers and remain pluripotent, i.e. they can be induced to differentiate into the required cell type by adding one or more specific factors. These cells can then be transplanted back into the patient suffering from either a lack or dysfunction of these cells. This approach prevents the rejection of the transplanted cells by the patient's immunological system. As this type of cloning has a very low efficiency, a large number of unfertilized donor oocytes is required. It is questionable whether enough donors are or will be available for this purpose. The cultured cells must satisfy certain conditions before they can be used for transplantation. They must be checked for chromosomal abnormalities, and a complete differentiation of the embryonic stem cells into the cells types needed by the patient is necessary as after the transplantation, undifferentiated stem cells will form teratomas. Furthermore, it is difficult to ensure that the cells end up in the right place and to ensure that they fully integrate into the existing tissue to form functional connections. Due to this array of technical problems the question remains as to whether therapeutic cloning will become feasible in the near future.

In vitro spatially organizing the differentiation in individual multicellular stem cell aggregates.

PubMed

Qi, Hao; Huang, Guoyou; Han, Yu Long; Lin, Wang; Li, Xiujun; Wang, Shuqi; Lu, Tian Jian; Xu, Feng

2016-01-01

With significant potential as a robust source to produce specific somatic cells for regenerative medicine, stem cells have attracted increasing attention from both academia and government. In vivo, stem cell differentiation is a process under complicated regulations to precisely build tissue with unique spatial structures. Since multicellular spheroidal aggregates of stem cells, commonly called as embryoid bodies (EBs), are considered to be capable of recapitulating the events in early stage of embryonic development, a variety of methods have been developed to form EBs in vitro for studying differentiation of embryonic stem cells. The regulation of stem cell differentiation is crucial in directing stem cells to build tissue with the correct spatial architecture for specific functions. However, stem cells within the three-dimensional multicellular aggregates undergo differentiation in a less unpredictable and spatially controlled manner in vitro than in vivo. Recently, various microengineering technologies have been developed to manipulate stem cells in vitro in a spatially controlled manner. Herein, we take the spotlight on these technologies and researches that bring us the new potential for manipulation of stem cells for specific purposes.

Derivation of Xeno-Free and GMP-Grade Human Embryonic Stem Cells – Platforms for Future Clinical Applications

PubMed Central

Aizenman, Einat; Kirshberg, Sophie; Ilouz, Nili; Gil, Yaniv; Berman-Zaken, Yael; Perlman, Temima Schnitzer; Geva, Nitshia; Levy, Ora; Arbell, Daniel; Simon, Alex; Ben-Meir, Assaf; Shufaro, Yoel; Laufer, Neri; Reubinoff, Benjamin E.

2012-01-01

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs. PMID:22745653

Imaging of mesoscopic-scale organisms using selective-plane optoacoustic tomography.

PubMed

Razansky, Daniel; Vinegoni, Claudio; Ntziachristos, Vasilis

2009-05-07

Mesoscopic-scale living organisms (i.e. 1 mm to 1 cm sized) remain largely inaccessible by current optical imaging methods due to intensive light scattering in tissues. Therefore, imaging of many important model organisms, such as insects, fishes, worms and similarly sized biological specimens, is currently limited to embryonic or other transparent stages of development. This makes it difficult to relate embryonic cellular and molecular mechanisms to consequences in organ function and animal behavior in more advanced stages and adults. Herein, we have developed a selective-plane illumination optoacoustic tomography technique for in vivo imaging of optically diffusive organisms and tissues. The method is capable of whole-body imaging at depths from the sub-millimeter up to centimeter range with a scalable spatial resolution in the order of magnitude of a few tenths of microns. In contrast to pure optical methods, the spatial resolution here is not determined nor limited by light diffusion; therefore, such performance cannot be achieved by any other optical imaging technology developed so far. The utility of the method is demonstrated on several whole-body models and small-animal extremities.

Dynamic integrated analysis of DNA methylation and gene expression profiles in in vivo and in vitro fertilized mouse post-implantation extraembryonic and placental tissues.

PubMed

Tan, Kun; Zhang, Zhenni; Miao, Kai; Yu, Yong; Sui, Linlin; Tian, Jianhui; An, Lei

2016-07-01

How does in vitro fertilization (IVF) alter promoter DNA methylation patterns and its subsequent effects on gene expression profiles during placentation in mice? IVF-induced alterations in promoter DNA methylation might have functional consequences in a number of biological processes and functions during IVF placentation, including actin cytoskeleton organization, hematopoiesis, vasculogenesis, energy metabolism and nutrient transport. During post-implantation embryonic development, both embryonic and extraembryonic tissues undergo de novo DNA methylation, thereby establishing a global DNA methylation pattern, and influencing gene expression profiles. Embryonic and placental tissues of IVF conceptuses can have aberrant morphology and functions, resulting in adverse pregnancy outcomes such as pregnancy loss, low birthweight, and long-term health effects. To date, the IVF-induced global profiling of DNA methylation alterations, and their functional consequences on aberrant gene expression profiles in IVF placentas have not been systematically studied. Institute for Cancer Research mice (6 week-old females and 8-9 week-old males) were used to generate in vivo fertilization (IVO) and IVF blastocysts. After either IVO and development (IVO group as control) or in vitro fertilization and culture (IVF group), blastocysts were collected and transferred to pseudo-pregnant recipient mice. Extraembryonic (ectoplacental cone and extraembryonic ectoderm) and placental tissues from both groups were sampled at embryonic day (E) 7.5 (IVO, n = 822; IVF, n = 795) and E10.5 (IVO, n = 324; IVF, n = 278), respectively. The collected extraembryonic (E7.5) and placental tissues (E10.5) were then used for high-throughput RNA sequencing (RNA-seq) and methylated DNA immunoprecipitation sequencing (MeDIP-seq). The main dysfunctions indicated by bioinformatic analyses were further validated using molecular detection, and morphometric and phenotypic analyses. Dynamic functional profiling of high-throughput data, together with molecular detection, and morphometric and phenotypic analyses, showed that differentially expressed genes dysregulated by DNA methylation were functionally involved in: (i) actin cytoskeleton disorganization in IVF extraembryonic tissues, which may impair allantois or chorion formation, and chorioallantoic fusion; (ii) disturbed hematopoiesis and vasculogenesis, which may lead to abnormal placenta labyrinth formation and thereby impairing nutrition transport in IVF placentas; (iii) dysregulated energy and amino acid metabolism, which may cause placental dysfunctions, leading to delayed embryonic development or even lethality; (iv) disrupted genetic information processing, which can further influence gene transcriptional and translational processes. Findings in mouse placental tissues may not be fully representative of human placentas. Further studies are necessary to confirm these findings and determine their clinical significance. Our study is the first to provide the genome-wide analysis of gene expression dysregulation caused by DNA methylation during IVF placentation. Systematic understanding of the molecular mechanisms implicated in IVF placentation can be useful for the improvement of existing assisted conception systems to prevent these IVF-associated safety concerns. This work was supported by grants from the National Natural Science Foundation of China (No. 31472092), and the National High-Tech R&D Program (Nos. 2011|AA100303, 2013AA102506). There was no conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Qualitative research of alternatively splice variants of fibronectin in different development stage of mice heart.

PubMed

Lu, Feng; Ma, Fang-Fang; Zhang, Wei; Li, Ying; Wei, Fei-Yu; Zhou, Lei

2015-12-01

Fibronectin (FN) plays vital roles in cell adhesion, differentiation, proliferation and migration. It is involved in the process of embryonic development and is highly conserved during evolution. The EIIIA and EIIIB of FN show a very high degree of homology among vertebrates. Embryos deleting both EIIIA and EIIIB displayed multiple embryonic cardiovascular defects, implying their crucial role during embryogenesis. The correlation of spliced EIIIB, EIIIA, and IIICS of FN to heart development was studied by observing their chronological expression in mice heart. C57 mice embryos at E11.5, E12.5, E13.5, E14.5, E15.5, E16.5, E17.5, E18.5, E19.5 days, postnatal day 1 (P1d), and adult male mice (3 months) were used. For each alternatively spliced FN1 domain (EIIIB, EIIIA and IIICS), primer pairs were designed for specific amplification. Total RNA was extracted from the heart tissue, reverse transcripted to cDNA, followed by RT-PCR with specific primers. The PCR amplification was verified by agarose gel electrophoresis, showing specific fragments of the expected sizes. In adult mice heart, only alternatively splice variants of EIIIA-, EIIIB-, IIICS+ were expressed. While in embryonic mice, spliced variant of EIIIA+/-, EIIIB+/-, IIICS+ were observed. The expression of EIIIA and EIIIB changed during heart development. FN is crucial for the normal development of the embryonic heart by modulating cardiac neural crest (CNC) proliferation and survival, and maintenance of CNC cells. FN1 gene seems to play a significant role by expression of highly conserved EIIIA and EIIIB in embryonic heart development.

Determining the impact of cell mixing on signaling during development.

PubMed

Uriu, Koichiro; Morelli, Luis G

2017-06-01

Cell movement and intercellular signaling occur simultaneously to organize morphogenesis during embryonic development. Cell movement can cause relative positional changes between neighboring cells. When intercellular signals are local such cell mixing may affect signaling, changing the flow of information in developing tissues. Little is known about the effect of cell mixing on intercellular signaling in collective cellular behaviors and methods to quantify its impact are lacking. Here we discuss how to determine the impact of cell mixing on cell signaling drawing an example from vertebrate embryogenesis: the segmentation clock, a collective rhythm of interacting genetic oscillators. We argue that comparing cell mixing and signaling timescales is key to determining the influence of mixing. A signaling timescale can be estimated by combining theoretical models with cell signaling perturbation experiments. A mixing timescale can be obtained by analysis of cell trajectories from live imaging. After comparing cell movement analyses in different experimental settings, we highlight challenges in quantifying cell mixing from embryonic timelapse experiments, especially a reference frame problem due to embryonic motions and shape changes. We propose statistical observables characterizing cell mixing that do not depend on the choice of reference frames. Finally, we consider situations in which both cell mixing and signaling involve multiple timescales, precluding a direct comparison between single characteristic timescales. In such situations, physical models based on observables of cell mixing and signaling can simulate the flow of information in tissues and reveal the impact of observed cell mixing on signaling. © 2017 Japanese Society of Developmental Biologists.

Engineering human cell spheroids to model embryonic tissue fusion in vitro

PubMed Central

Wolf, Cynthia J.; Wood, Carmen; Ren, Hongzu; Grindstaff, Rachel; Padgett, William; Swank, Adam; MacMillan, Denise; Fisher, Anna; Winnik, Witold; Abbott, Barbara D.

2017-01-01

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications. PMID:28898253

Extracting cardiac shapes and motion of the chick embryo heart outflow tract from four-dimensional optical coherence tomography images

NASA Astrophysics Data System (ADS)

Yin, Xin; Liu, Aiping; Thornburg, Kent L.; Wang, Ruikang K.; Rugonyi, Sandra

2012-09-01

Recent advances in optical coherence tomography (OCT), and the development of image reconstruction algorithms, enabled four-dimensional (4-D) (three-dimensional imaging over time) imaging of the embryonic heart. To further analyze and quantify the dynamics of cardiac beating, segmentation procedures that can extract the shape of the heart and its motion are needed. Most previous studies analyzed cardiac image sequences using manually extracted shapes and measurements. However, this is time consuming and subject to inter-operator variability. Automated or semi-automated analyses of 4-D cardiac OCT images, although very desirable, are also extremely challenging. This work proposes a robust algorithm to semi automatically detect and track cardiac tissue layers from 4-D OCT images of early (tubular) embryonic hearts. Our algorithm uses a two-dimensional (2-D) deformable double-line model (DLM) to detect target cardiac tissues. The detection algorithm uses a maximum-likelihood estimator and was successfully applied to 4-D in vivo OCT images of the heart outflow tract of day three chicken embryos. The extracted shapes captured the dynamics of the chick embryonic heart outflow tract wall, enabling further analysis of cardiac motion.

Seed Coating Increases Seed Moisture Uptake and Restricts Embryonic Oxygen Availability in Germinating Cereal Seeds.

PubMed

Gorim, Linda; Asch, Folkard

2017-05-24

Seed coating is a technology to improve germination and homogenize stand establishment. Although coating often results in lower germination rates, seeds that do germinate grow more vigorously and show strongly reduced respiratory losses during reserve mobilization. We hypothesize that the higher mobilization efficiency is due to a shift in the enzymatic cleavage of sucrose from invertase to sucrose synthase in the embryonic tissue caused by a reduced oxygen availability induced by oversaturation with water caused by the coating during early germination. We investigated the effect of coating on barley, rye, and wheat seed imbibition during the first 30 h after seeds were placed in moisture. We profiled oxygen in the embryos and measured sucrose and acid invertase levels as imbibition progressed. We found that seeds within coatings absorbed significantly more moisture than uncoated seeds. Coating resulted in near anoxic oxygen concentrations in the developing embryonic tissues in all three species. In barley, sucrose was not cleaved via the invertase pathway, despite the fact that invertase activity in coated seeds was increased. In rye and wheat, invertase activities were significantly lower in embryos from coated seeds without significantly changing the sugar composition.

Seed Coating Increases Seed Moisture Uptake and Restricts Embryonic Oxygen Availability in Germinating Cereal Seeds

PubMed Central

Gorim, Linda; Asch, Folkard

2017-01-01

Seed coating is a technology to improve germination and homogenize stand establishment. Although coating often results in lower germination rates, seeds that do germinate grow more vigorously and show strongly reduced respiratory losses during reserve mobilization. We hypothesize that the higher mobilization efficiency is due to a shift in the enzymatic cleavage of sucrose from invertase to sucrose synthase in the embryonic tissue caused by a reduced oxygen availability induced by oversaturation with water caused by the coating during early germination. We investigated the effect of coating on barley, rye, and wheat seed imbibition during the first 30 h after seeds were placed in moisture. We profiled oxygen in the embryos and measured sucrose and acid invertase levels as imbibition progressed. We found that seeds within coatings absorbed significantly more moisture than uncoated seeds. Coating resulted in near anoxic oxygen concentrations in the developing embryonic tissues in all three species. In barley, sucrose was not cleaved via the invertase pathway, despite the fact that invertase activity in coated seeds was increased. In rye and wheat, invertase activities were significantly lower in embryos from coated seeds without significantly changing the sugar composition. PMID:28538658

Human embryonic stem cell lines: socio-legal concerns and therapeutic promise.

PubMed

McLaren, Anne

2002-10-01

Stem cell lines would be very valuable for the repair of diseased or damaged organs. Stem cells derived from adult tissues raise few ethical problems, and would not be rejected if derived from the patient. They show considerable plasticity and might be appropriate for some clinical conditions, but they tend not to grow well in culture. Stem cells derived from the early human embryo proliferate indefinitely in culture and can give rise to many different tissues, but their derivation requires destruction of the embryo, which is not ethically acceptable in some countries. Other countries allow strictly regulated destructive research on human embryos, usually those that have been produced for infertile couples in infertility clinics. Embryos that are no longer required for the couple's own reproductive project could be donated for research rather than just discarded. Different approaches are being developed to avoid immunological rejection of embryonic stem cells used for therapy. Derivation of embryonic stem cell lines by somatic cell nuclear transfer ('cloning') from the patients themselves might be one possible approach, but is unlikely to be used in routine clinical practice if more cost-effective methods are available.

Re-expression of pro-fibrotic, embryonic preserved mediators in irradiated arterial vessels of the head and neck region.

PubMed

Möbius, Patrick; Preidl, Raimund H M; Weber, Manuel; Amann, Kerstin; Neukam, Friedrich W; Wehrhan, Falk

2017-11-01

Surgical treatment of head and neck malignancies frequently includes microvascular free tissue transfer. Preoperative radiotherapy increases postoperative fibrosis-related complications up to transplant loss. Fibrogenesis is associated with re-expression of embryonic preserved tissue developmental mediators: osteopontin (OPN), regulated by sex-determining region Y‑box 9 (Sox9), and homeobox A9 (HoxA9) play important roles in pathologic tissue remodeling and are upregulated in atherosclerotic vascular lesions; dickkopf-1 (DKK1) inhibits pro-fibrotic and atherogenic Wnt signaling. We evaluated the influence of irradiation on expression of these mediators in arteries of the head and neck region. DKK1, HoxA9, OPN, and Sox9 expression was examined immunohistochemically in 24 irradiated and 24 nonirradiated arteries of the lower head and neck region. The ratio of positive cells to total cell number (labeling index) in the investigated vessel walls was assessed semiquantitatively. DKK1 expression was significantly decreased, whereas HoxA9, OPN, and Sox9 expression were significantly increased in irradiated compared to nonirradiated arterial vessels. Preoperative radiotherapy induces re-expression of embryonic preserved mediators in arterial vessels and may thus contribute to enhanced activation of pro-fibrotic downstream signaling leading to media hypertrophy and intima degeneration comparable to fibrotic development steps in atherosclerosis. These histopathological changes may be promoted by HoxA9-, OPN-, and Sox9-related inflammation and vascular remodeling, supported by downregulation of anti-fibrotic DKK1. Future pharmaceutical strategies targeting these vessel alterations, e. g., bisphosphonates, might reduce postoperative complications in free tissue transfer.

Regulation of macrophage development and function in peripheral tissues

PubMed Central

Lavin, Yonit; Mortha, Arthur; Rahman, Adeeb; Merad, Miriam

2015-01-01

Macrophages are immune cells of haematopoietic origin that provide crucial innate immune defence and have tissue-specific functions in the regulation and maintenance of organ homeostasis. Recent studies of macrophage ontogeny, as well as transcriptional and epigenetic identity, have started to reveal the decisive role of the tissue stroma in the regulation of macrophage function. These findings suggest that most macrophages seed the tissues during embryonic development and functionally specialize in response to cytokines and metabolites that are released by the stroma and drive the expression of unique transcription factors. In this Review, we discuss how recent insights into macrophage ontogeny and macrophage–stroma interactions contribute to our understanding of the crosstalk that shapes macrophage function and the maintenance of organ integrity. PMID:26603899

Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken

PubMed Central

Yvernogeau, Laurent

2017-01-01

Hematopoietic stem cells (HSCs), which are responsible for blood cell production, are generated during embryonic development. Human and chicken embryos share features that position the chicken as a reliable and accessible alternative model to study developmental hematopoiesis. However, the existence of HSCs has never been formally proven in chicken embryos. Here, we have established a complete cartography and quantification of hematopoietic cells in the aorta during development. We demonstrate the existence of bona fide HSCs, originating from the chicken embryo aorta (and not the yolk sac, allantois or head), through an in vivo transplantation assay. Embryos transplanted in ovo with GFP embryonic tissues on the chorio-allantoic membrane provided multilineage reconstitution in adulthood. Historically, most breakthrough discoveries in the field of developmental hematopoiesis were first made in birds and later extended to mammals. Our study sheds new light on the avian model as a valuable system to study HSC production and regulation in vivo. PMID:28526756

Cell differentiation: therapeutical challenges in diabetes.

PubMed

Roche, Enrique; Vicente-Salar, Nestor; Arribas, Maribel; Paredes, Beatriz

2012-01-01

Stem cells, derived from either embryonic or adult tissues, are considered to be potential sources of insulin-secreting cells to be transplanted into type 1 and advanced stages of type 2 diabetic patients. Many laboratories have considered this possibility, resulting in a large amount of published protocols, with a wide degree of complexity among them. Our group was the first to report that it was possible to obtain insulin-secreting cells from mouse embryonic stem cells, proving the feasibility of this new challenge. The same observation was immediately reported using human embryonic stem cells. However, the resulting cell product was not properly characterised, affecting the reproducibility of the protocol by other groups. A more elaborated protocol was developed by Lumelsky and co-workers, demonstrating that neuroectodermal cells could be an alternative source for insulin-producing cells. However, the resulting cells of this protocol produced low amounts of the hormone. This aimed other groups to perform key changes in order to improve the insulin content of the resulting cells. Recently, Baetge's group has published a new protocol based on the knowledge accumulated in pancreatic development. In this protocol, human embryonic stem cells were differentiated into islet-like structures through a five step protocol, emulating the key steps during embryonic development of the endocrine pancreas. The final cell product, however, seemed to be in an immature state, thus further improvement is required. Despite this drawback, the protocol represents the culmination of work performed by different groups and offers new research challenges for the investigators in this exciting field. Concerning adult stem cells, the possibility of identifying pancreatic precursors or of reprogramming extrapancreatic derived cells are key possibilities that may circumvent the problems that appear when using embryonic stem cells, such as immune rejection and tumour formation.

Disruption of epithelial cell migration as a potential mechanism of cleft palate induction

EPA Science Inventory

Cleft palate occurs in about one in seven hundred births per year, making it the most prevalent craniofacial birth defect in the world. During embryonic development, tissue fusion is a critical step in the formation of the palate, cornea, urethra, and neural tube. Epithelial cell...

An Engineered Organoid Culture Model That Incorporates Human Mesenchymal and Epithelial Cells to Model Palatal Fusion.

EPA Science Inventory

During embryonic development, fusion events are critical to morphogenesis of organs and tissues, including the iris, urethra, heart, neural tube, and secondary palate. Modeling this process in vitro is challenging as the interactions of mesenchymal and epithelial cells can be cr...

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

PubMed

Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

2010-11-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats

PubMed Central

Matveyenko, Aleksey V.; Georgia, Senta; Bhushan, Anil

2010-01-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant. PMID:20587750

Histone Deacetylase Inhibitors in Cell Pluripotency, Differentiation, and Reprogramming

PubMed Central

Kretsovali, Androniki; Hadjimichael, Christiana; Charmpilas, Nikolaos

2012-01-01

Histone deacetylase inhibitors (HDACi) are small molecules that have important and pleiotropic effects on cell homeostasis. Under distinct developmental conditions, they can promote either self-renewal or differentiation of embryonic stem cells. In addition, they can promote directed differentiation of embryonic and tissue-specific stem cells along the neuronal, cardiomyocytic, and hepatic lineages. They have been used to facilitate embryo development following somatic cell nuclear transfer and induced pluripotent stem cell derivation by ectopic expression of pluripotency factors. In the latter method, these molecules not only increase effectiveness, but can also render the induction independent of the oncogenes c-Myc and Klf4. Here we review the molecular pathways that are involved in the functions of HDAC inhibitors on stem cell differentiation and reprogramming of somatic cells into pluripotency. Deciphering the mechanisms of HDAC inhibitor actions is very important to enable their exploitation for efficient and simple tissue regeneration therapies. PMID:22550500

Interdependence of Bad and Puma during ionizing-radiation-induced apoptosis.

PubMed

Toruno, Cristhian; Carbonneau, Seth; Stewart, Rodney A; Jette, Cicely

2014-01-01

Ionizing radiation (IR)-induced DNA double-strand breaks trigger an extensive cellular signaling response that involves the coordination of hundreds of proteins to regulate DNA repair, cell cycle arrest and apoptotic pathways. The cellular outcome often depends on the level of DNA damage as well as the particular cell type. Proliferating zebrafish embryonic neurons are highly sensitive to IR-induced apoptosis, and both p53 and its transcriptional target puma are essential mediators of the response. The BH3-only protein Puma has previously been reported to activate mitochondrial apoptosis through direct interaction with the pro-apoptotic Bcl-2 family proteins Bax and Bak, thus constituting the role of an "activator" BH3-only protein. This distinguishes it from BH3-only proteins like Bad that are thought to indirectly promote apoptosis through binding to anti-apoptotic Bcl-2 family members, thereby preventing the sequestration of activator BH3-only proteins and allowing them to directly interact with and activate Bax and Bak. We have shown previously that overexpression of the BH3-only protein Bad in zebrafish embryos supports normal embryonic development but greatly sensitizes developing neurons to IR-induced apoptosis. While Bad has previously been shown to play only a minor role in promoting IR-induced apoptosis of T cells in mice, we demonstrate that Bad is essential for robust IR-induced apoptosis in zebrafish embryonic neural tissue. Moreover, we found that both p53 and Puma are required for Bad-mediated radiosensitization in vivo. Our findings show the existence of a hierarchical interdependence between Bad and Puma whereby Bad functions as an essential sensitizer and Puma as an essential activator of IR-induced mitochondrial apoptosis specifically in embryonic neural tissue.

Exogenous transforming growth factor-β1 enhances smooth muscle differentiation in embryonic mouse jejunal explants.

PubMed

Coletta, Riccardo; Roberts, Neil A; Randles, Michael J; Morabito, Antonino; Woolf, Adrian S

2017-01-13

An ex vivo experimental strategy that replicates in vivo intestinal development would in theory provide an accessible setting with which to study normal and dysmorphic gut biology. The current authors recently described a system in which mouse embryonic jejunal segments were explanted onto semipermeable platforms and fed with chemically defined serum-free media. Over 3 days in organ culture, explants formed villi and they began to undergo spontaneous peristalsis. As defined in the current study, the wall of the explanted gut failed to form a robust longitudinal smooth muscle (SM) layer as it would do in vivo over the same time period. Given the role of transforming growth factor β1 (TGFβ1) in SM differentiation in other organs, it was hypothesized that exogenous TGFβ1 would enhance SM differentiation in these explants. In vivo, TGFβ receptors I and II were both detected in embryonic longitudinal jejunal SM cells and, in organ culture, exogenous TGFβ1 induced robust differentiation of longitudinal SM. Microarray profiling showed that TGFβ1 increased SM specific transcripts in a dose dependent manner. TGFβ1 proteins were detected in amniotic fluid at a time when the intestine was physiologically herniated. By analogy with the requirement for exogenous TGFβ1 for SM differentiation in organ culture, the TGFβ1 protein that was demonstrated to be present in the amniotic fluid may enhance intestinal development when it is physiologically herniated in early gestation. Future studies of embryonic intestinal cultures should include TGFβ1 in the defined media to produce a more faithful model of in vivo muscle differentiation. Copyright © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd. Copyright © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, 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.

Copine1 regulates neural stem cell functions during brain development.

PubMed

Kim, Tae Hwan; Sung, Soo-Eun; Cheal Yoo, Jae; Park, Jae-Yong; Yi, Gwan-Su; Heo, Jun Young; Lee, Jae-Ran; Kim, Nam-Soon; Lee, Da Yong

2018-01-01

Copine 1 (CPNE1) is a well-known phospholipid binding protein in plasma membrane of various cell types. In brain cells, CPNE1 is closely associated with AKT signaling pathway, which is important for neural stem cell (NSC) functions during brain development. Here, we investigated the role of CPNE1 in the regulation of brain NSC functions during brain development and determined its underlying mechanism. In this study, abundant expression of CPNE1 was observed in neural lineage cells including NSCs and immature neurons in human. With mouse brain tissues in various developmental stages, we found that CPNE1 expression was higher at early embryonic stages compared to postnatal and adult stages. To model developing brain in vitro, we used primary NSCs derived from mouse embryonic hippocampus. Our in vitro study shows decreased proliferation and multi-lineage differentiation potential in CPNE1 deficient NSCs. Finally, we found that the deficiency of CPNE1 downregulated mTOR signaling in embryonic NSCs. These data demonstrate that CPNE1 plays a key role in the regulation of NSC functions through the activation of AKT-mTOR signaling pathway during brain development. Copyright © 2017 Elsevier Inc. All rights reserved.

Wnt affects symmetry and morphogenesis during post-embryonic development in colonial chordates.

PubMed

Di Maio, Alessandro; Setar, Leah; Tiozzo, Stefano; De Tomaso, Anthony W

2015-01-01

Wnt signaling is one of the earliest and most highly conserved regulatory pathways for the establishment of the body axes during regeneration and early development. In regeneration, body axes determination occurs independently of tissue rearrangement and early developmental cues. Modulation of the Wnt signaling in either process has shown to result in unusual body axis phenotypes. Botryllus schlosseri is a colonial ascidian that can regenerate its entire body through asexual budding. This processes leads to an adult body via a stereotypical developmental pathway (called blastogenesis), without proceeding through any embryonic developmental stages. In this study, we describe the role of the canonical Wnt pathway during the early stages of asexual development. We characterized expression of three Wnt ligands (Wnt2B, Wnt5A, and Wnt9A) by in situ hybridization and qRT-PCR. Chemical manipulation of the pathway resulted in atypical budding due to the duplication of the A/P axes, supernumerary budding, and loss of the overall cell apical-basal polarity. Our results suggest that Wnt signaling is used for equivalent developmental processes both during embryogenesis and asexual development in an adult organism, suggesting that patterning mechanisms driving morphogenesis are conserved, independent of embryonic, or regenerative development.

Utilization of microgravity bioreactors for differentiation of mammalian skeletal tissue

NASA Technical Reports Server (NTRS)

Klement, B. J.; Spooner, B. S.

1993-01-01

Bioreactor cell and tissue culture vessels can be used to study bone development in a simulated microgravity environment. These vessels will also provide an advantageous, low maintenance culture system on space station Freedom. Although many types of cells and tissues can potentially utilize this system, our particular interest is in developing bone tissue. We have characterized an organ culture system utilizing embryonic mouse pre-metatarsal mesenchyme, documenting morphogenesis and differentiation as cartilage rods are formed, with subsequent terminal chondrocyte differentiation to hypertrophied cells. Further development to form bone tissue is achieved by supplementation of the culture medium. Research using pre-metatarsal tissue, combined with the bioreactor culture hardware, could give insight into the advantages and/or disadvantages of conditions experienced in microgravity. Studies such as these have the potential to enhance understanding of bone development and adult bone physiology, and may help define the processes of bone demineralization experienced in space and in pathological conditions here on earth.

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression.

PubMed

Klochendler-Yeivin, A; Fiette, L; Barra, J; Muchardt, C; Babinet, C; Yaniv, M

2000-12-01

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri-implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild-type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types.

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression

PubMed Central

Klochendler-Yeivin, Agnes; Fiette, Laurence; Barra, Jaqueline; Muchardt, Christian; Babinet, Charles; Yaniv, Moshe

2000-01-01

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri-implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild-type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types. PMID:11263494

Msx homeobox gene family and craniofacial development.

PubMed

Alappat, Sylvia; Zhang, Zun Yi; Chen, Yi Ping

2003-12-01

Vertebrate Msx genes are unlinked, homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene. These genes are expressed at multiple sites of tissue-tissue interactions during vertebrate embryonic development. Inductive interactions mediated by the Msx genes are essential for normal craniofacial, limb and ectodermal organ morphogenesis, and are also essential to survival in mice, as manifested by the phenotypic abnormalities shown in knockout mice and in humans. This review summarizes studies on the expression, regulation, and functional analysis of Msx genes that bear relevance to craniofacial development in humans and mice. Key words: Msx genes, craniofacial, tooth, cleft palate, suture, development, transcription factor, signaling molecule.

Random X inactivation in the mule and horse placenta.

PubMed

Wang, Xu; Miller, Donald C; Clark, Andrew G; Antczak, Douglas F

2012-10-01

In eutherian mammals, dosage compensation of X-linked genes is achieved by X chromosome inactivation. X inactivation is random in embryonic and adult tissues, but imprinted X inactivation (paternal X silencing) has been identified in the extra-embryonic membranes of the mouse, rat, and cow. Few other species have been studied for this trait, and the data from studies of the human placenta have been discordant or inconclusive. Here, we quantify X inactivation using RNA sequencing of placental tissue from reciprocal hybrids of horse and donkey (mule and hinny). In placental tissue from the equid hybrids and the horse parent, the allelic expression pattern was consistent with random X inactivation, and imprinted X inactivation can clearly be excluded. We characterized horse and donkey XIST gene and demonstrated that XIST allelic expression in female hybrid placental and fetal tissues is negatively correlated with the other X-linked genes chromosome-wide, which is consistent with the XIST-mediated mechanism of X inactivation discovered previously in mice. As the most structurally and morphologically diverse organ in mammals, the placenta also appears to show diverse mechanisms for dosage compensation that may result in differences in conceptus development across species.

Chromatin Immunoprecipitation in Early Mouse Embryos.

PubMed

García-González, Estela G; Roque-Ramirez, Bladimir; Palma-Flores, Carlos; Hernández-Hernández, J Manuel

2018-01-01

Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.

Is Fibroblast Growth Factor Receptor 4 a Suitable Target of Cancer Therapy?

PubMed Central

Heinzle, Christine; Erdem, Zeynep; Paur, Jakob; Grasl-Kraupp, Bettina; Holzmann, Klaus; Grusch, Michael; Berger, Walter; Marian, Brigitte

2017-01-01

Fibroblast growth factors (FGF) and their tyrosine kinase receptors (FGFR) support cell proliferation, survival and migration during embryonic development, organogenesis and tissue maintenance and their deregulation is frequently observed in cancer development and progression. Consequently, increasing efforts are focusing on the development of strategies to target FGF/FGFR signaling for cancer therapy. Among the FGFRs the family member FGFR4 is least well understood and differs from FGFRs1-3 in several aspects. Importantly, FGFR4 deletion does not lead to an embryonic lethal phenotype suggesting the possibility that its inhibition in cancer therapy might not cause grave adverse effects. In addition, the FGFR4 kinase domain differs sufficiently from those of FGFRs1-3 to permit development of highly specific inhibitors. The oncogenic impact of FGFR4, however, is not undisputed, as the FGFR4-mediated hormonal effects of several FGF ligands may also constitute a tissue-protective tumor suppressor activity especially in the liver. Therefore it is the purpose of this review to summarize all relevant aspects of FGFR4 physiology and pathophysiology and discuss the options of targeting this receptor for cancer therapy. PMID:23944363

Distributional shift of urea production site from the extraembryonic yolk sac membrane to the embryonic liver during the development of cloudy catshark (Scyliorhinus torazame).

PubMed

Takagi, Wataru; Kajimura, Makiko; Tanaka, Hironori; Hasegawa, Kumi; Ogawa, Shuntaro; Hyodo, Susumu

2017-09-01

Urea is an essential osmolyte for marine cartilaginous fishes. Adult elasmobranchs and holocephalans are known to actively produce urea in the liver, muscle and other extrahepatic organs; however, osmoregulatory mechanisms in the developing cartilaginous fish embryo with an undeveloped urea-producing organ are poorly understood. We recently described the contribution of extraembryonic yolk sac membranes (YSM) to embryonic urea synthesis during the early developmental period of the oviparous holocephalan elephant fish (Callorhinchus milii). In the present study, to test whether urea production in the YSM is a general phenomenon among oviparous Chondrichthyes, we investigated gene expression and activities of ornithine urea cycle (OUC) enzymes together with urea concentrations in embryos of the elasmobranch cloudy catshark (Scyliorhinus torazame). The intracapsular fluid, in which the catshark embryo develops, had a similar osmolality to seawater, and embryos maintained a high concentration of urea at levels similar to that of adult plasma throughout development. Relative mRNA expressions and activities of catshark OUC enzymes were significantly higher in YSM than in embryos until stage 32. Concomitant with the development of the embryonic liver, the expression levels and activities of OUC enzymes were markedly increased in the embryo from stage 33, while those of the YSM decreased from stage 32. The present study provides further evidence that the YSM contributes to embryonic urea homeostasis until the liver and other extrahepatic organs become fully functional, and that urea-producing tissue shifts from the YSM to the embryonic liver in the late developmental period of oviparous marine cartilaginous fishes. Copyright © 2017 Elsevier Inc. All rights reserved.

Ectodermal dysplasia (ED) syndrome.

PubMed

Chee, Siew-Yin; Wanga, Chung-Hsing; Lina, Wei-De; Tsaia, Fuu-Jen

2014-01-01

Ectodermal dysplasia (ED) syndrome comprises a large, heterogeneous group of inherited disorders that are defined by primary defects in the development of 2 or more tissues derived from the embryonic ectoderm. The tissues primarily involved are the skin and its appendages (including hair follicles, eccrine glands, sebaceous glands, nails) and teeth. The clinical features include sparse hair, abnormal or missing teeth, and an inability to sweat due to lack of sweat glands. One such case report of ectodermal dysplasia is presented here.

Comparative Chondrogenesis of Human Cell Sources in 3D Scaffolds

PubMed Central

Tıg̑lı, R. Seda; Ghosh, Sourabh; Laha, Michael M.; Shevde, Nirupama K.; Daheron, Laurence; Gimble, Jeffrey; Gümüşdereliog̑lu, Menemşe; Kaplan, David L.

2009-01-01

Cartilage tissue can be engineered by starting from a diversity of cell sources, including stem-cell based and primary cell-based platforms. Selecting an appropriate cell source for the process of cartilage tissue engineering or repair is critical and challenging due to the variety of cell options available. In this study, cellular responses of isolated human chondrocytes, human embryonic stem cells and mesenchymal stem cells (MSCs) derived from three sources, human embryonic stem cells, bone marrow and adipose tissue, were assessed for chondrogenic potential in 3D culture. All cell sources were characterized by FACS analysis to compare expression of some surface markers. The cells were differentiated in two different biomaterial matrices, silk and chitosan scaffolds, in the presence and absence of bone morphogenetic protein 6 (BMP-6) along with the standard chondrogenic differentiating factors. Embryonic stem cells derived MSCs showed unique characteristics with preserved chondrogenic phenotype in both scaffolds with regard to chondrogenesis, as determined by real time RT-PCR, histological and microscopic analyses. After 4 weeks of cultivation, embryonic stem cells derived MSCs were promising for chondrogenesis, particularly in the silk scaffolds with BMP-6. The results suggest that cell source differences are important to consider with regard to chondrogenic outcomes and with the variables addressed here, the human embryonic stem cells derived MSCs were the preferred cell source. PMID:19382119

Rhabdomyosarcoma of Cervix: A Case Report.

PubMed

Hosseini, Maryam Sadat; Ashrafganjoei, Tahereh; Sourati, Ainaz; Tabatabeifar, Morteza; Mohamadianamiri, Mahdiss

2016-06-01

Rhabdomyosarcoma has known as a highly malignant soft tissue sarcoma. It has been the most common soft tissue sarcoma in childhood, accounting for about 3 to 4 % of all cases of childhood cancer. Rhabdomyosarcoma was rare in adults, accounting for 3% of all soft-tissue sarcomas. embryonal rhabdomyosarcoma of female genital tract including uterine cervix in an adult was rare. This study has reported a 33-year-old woman presented with abnormal vaginal discharge. Gynecologic examination revealed a cervical mass with grape- like feature protruding into vagina with posterior- superior vaginal wall involvement. Biopsy has performed and pathologic examination was consistent with embryonal botryoid type rhabdomyosarcoma. She has undergone the staging work up measurements including thoracic computed tomography (CT) scan, abdominopelvic magnetic resonance imaging (MRI), bone scan and bone marrow examination. In exception of abdominopelvic MRI, with 2 suspicious pelvic lymph nodes in addition of cervical mass, all others were normal. Radical hysterectomy with lymph node debulking and ovarian preservation has performed. Final results have shown embryonal botryoid type rhabdomyosarcoma of cervix. ovaries, endometrium, parametrium, and follopian tubes were unremarkable. Pelvic lymph nodes pathology and intraabdominal fluid cytology were negative for malignancy. Lymphovascular invasion was identified. She has advised for adjuvant chemotherapy. This case has reminded that embryonal rhabdomyosarcoma could occur in uncommon site and older female. Longer follow up of these cases has required due to lack of survival data for embryonal rhabdomyosarcoma of this site and age group.

Normal development of the tomato clownfish Amphiprion frenatus: live imaging and in situ hybridization analyses of mesodermal and neurectodermal development.

PubMed

Ghosh, J; Wilson, R W; Kudoh, T

2009-12-01

The normal embryonic development of the tomato clownfish Amphiprion frenatus was analysed using live imaging and by in situ hybridization for detection of mesodermal and neurectodermal development. Both morphology of live embryos and tissue-specific staining revealed significant differences in the gross developmental programme of A. frenatus compared with better-known teleost fish models, in particular, initiation of somitogenesis before complete epiboly, initiation of narrowing of the neurectoderm (neurulation) before somitogenesis, relatively early pigmentation of melanophores at the 10-15 somite stage and a distinctive pattern of melanophore distribution. These results suggest evolutionary adaptability of the teleost developmental programme. The ease of obtaining eggs, in vitro culture of the embryo, in situ staining analyses and these reported characteristics make A. frenatus a potentially important model marine fish species for studying embryonic development, physiology, ecology and evolution.

Amniotic fluid stem cells: a promising therapeutic resource for cell-based regenerative therapy.

PubMed

Antonucci, Ivana; Pantalone, Andrea; Tete, Stefano; Salini, Vincenzo; Borlongan, Cesar V; Hess, David; Stuppia, Liborio

2012-01-01

Stem cells have been proposed as a powerful tool in the treatment of several human diseases, both for their ability to represent a source of new cells to replace those lost due to tissue injuries or degenerative diseases, and for the ability of produce trophic molecules able to minimize damage and promote recovery in the injured tissue. Different cell types, such as embryonic, fetal or adult stem cells, human fetal tissues and genetically engineered cell lines, have been tested for their ability to replace damaged cells and to restore the tissue function after transplantation. Amniotic fluid -derived Stem cells (AFS) are considered a novel resource for cell transplantation therapy, due to their high renewal capacity, the "in vitro" expression of embryonic cell lineage markers, and the ability to differentiate in tissues derived from all the three embryonic layers. Moreover, AFS do not produce teratomas when transplanted into animals and are characterized by a low antigenicity, which could represent an advantage for cell transplantation or cell replacement therapy. The present review focuses on the biological features of AFS, and on their potential use in the treatment of pathological conditions such as ischemic brain injury and bone damages.

Isolation, characterization, and differentiation of stem cells for cartilage regeneration.

PubMed

Beane, Olivia S; Darling, Eric M

2012-10-01

The goal of tissue engineering is to create a functional replacement for tissues damaged by injury or disease. In many cases, impaired tissues cannot provide viable cells, leading to the investigation of stem cells as a possible alternative. Cartilage, in particular, may benefit from the use of stem cells since the tissue has low cellularity and cannot effectively repair itself. To address this need, researchers are investigating the chondrogenic capabilities of several multipotent stem cell sources, including adult and extra-embryonic mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Comparative studies indicate that each cell type has advantages and disadvantages, and while direct comparisons are difficult to make, published data suggest some sources may be more promising for cartilage regeneration than others. In this review, we identify current approaches for isolating and chondrogenically differentiating MSCs from bone marrow, fat, synovium, muscle, and peripheral blood, as well as cells from extra-embryonic tissues, ESCs, and iPSCs. Additionally, we assess chondrogenic induction with growth factors, identifying standard cocktails used for each stem cell type. Cell-only (pellet) and scaffold-based studies are also included, as is a discussion of in vivo results.

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

PubMed Central

Sehgal, Poonam; Chaturvedi, Pankaj; Kumaran, R. Ileng; Kumar, Satish; Parnaik, Veena K.

2013-01-01

Background Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways. Methodology and Principal Findings We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna+/− embryonic stem cells. Conclusions We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development. PMID:23451281

Growth and Electrophysiological Properties of Rat Embryonic Cardiomyocytes on Hydroxyl- and Carboxyl-Modified Surfaces

PubMed Central

NATARAJAN, ANUPAMA; CHUN, CHANGJU; HICKMAN, JAMES J.; MOLNAR, PETER

2010-01-01

Biodegradable scaffolds such as poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) or poly(glycolic acid) (PGA) are commonly used materials in tissue engineering. The chemical composition of these scaffolds changes during degradation which provides a changing environment for the seeded cells. In this study we have developed a simple and relatively high-throughput method in order to test the physiological effects of this varying chemical environment on rat embryonic cardiac myocytes. In order to model the different degradation stages of the scaffold, glass coverslips were functionalized with 11-mercaptoundecanoic acid (MUA) and 11-mercapto-1-undecanol (MUL) as carboxyl- and hydroxyl-group presenting surfaces and also with trimethoxysilylpropyldiethylenetriamine (DETA) and (3-aminopropyl)triethoxysilane (APTES) as controls. Embryonic cardiac myocytes formed beating islands on all tested surfaces but the number of attached cells and beating patches was significantly lower on MUL compared to any of the other functionalized surfaces. Moreover, whole cell patch clamp experiments showed that the average length of action potentials generated by the beating cardiac myocytes were significantly longer on MUL compared to the other surfaces. Our results, using our simple test system, are in agreement with earlier observations that utilized the complex 3D biodegradable scaffold. Thus, surface functionalization with self-assembled monolayers combined with histological/physiological testing could be a relatively high throughput method for biocompatibility studies and for the optimization of the material/tissue interface in tissue engineering. PMID:18854125

Identification and embryonic expression of a new AP-2 transcription factor, AP-2 epsilon.

PubMed

Wang, Hao-Ven; Vaupel, Kristina; Buettner, Reinhard; Bosserhoff, Anja-Katrin; Moser, Markus

2004-09-01

AP-2 proteins comprise a family of highly related transcription factors, which are expressed during mouse embryogenesis in a variety of ectodermal, neuroectodermal, and mesenchymal tissues. AP-2 transcription factors were shown to be involved in morphogenesis of craniofacial, urogenital, neural crest-derived, and placental tissues. By means of a partial cDNA fragment identified during an expressed sequence tag search for AP-2 genes, we identified a fifth, previously unknown AP-2-related gene, AP-2 epsilon. AP-2 epsilon encodes an open reading frame of 434 amino acids, which reveals the typical modular structure of AP-2 transcription factors with highly conserved C-terminal DNA binding and dimerization domains. Although the N-terminally localized activation domain is less homologous, position and identity of amino acids essential for transcriptional transactivation are conserved. Reverse transcriptase-polymerase chain reaction analyses of murine embryos revealed AP-2 epsilon expression from gestational stage embryonic day 7.5 throughout all later embryonic stages until birth. Whole-mount in situ hybridization using a specific AP-2 epsilon cDNA fragment demonstrated that during embryogenesis, expression of AP-2 epsilon is mainly restricted to neural tissue, especially the midbrain, hindbrain, and olfactory bulb. This expression pattern was confirmed by immunohistochemistry with an AP-2 epsilon-specific antiserum. By using this antiserum, we could further localize AP-2 epsilon expression in a hypothalamic nucleus and the neuroepithelium of the vomeronasal organ, suggesting an important function of AP-2 epsilon for the development of the olfactory system.

A survey of children affected by ectomermal dysplasia syndromes shows an increased prevalence of atopic disorders and immune deficiency

USDA-ARS?s Scientific Manuscript database

Ectodermal dysplasia (ED) syndromes are rare genetic disorders that affect the development of tissues derived from the embryonic ectoderm. Studies and anecdotal experience have indicated that atopic disorders (AD) and immune deficiencies (ID) may be associated with ED in children. Some ED genotypes ...

Protocorms and Protocorm-Like Bodies Are Molecularly Distinct from Zygotic Embryonic Tissues in Phalaenopsis aphrodite1[OPEN

PubMed Central

Chen, Jhun-Chen; Wei, Miao-Ju

2016-01-01

The distinct reproductive program of orchids provides a unique evolutionary model with pollination-triggered ovule development and megasporogenesis, a modified embryogenesis program resulting in seeds with immature embryos, and mycorrhiza-induced seed germination. However, the molecular mechanisms that have evolved to establish these unparalleled developmental programs are largely unclear. Here, we conducted comparative studies of genome-wide gene expression of various reproductive tissues and captured the molecular events associated with distinct reproductive programs in Phalaenopsis aphrodite. Importantly, our data provide evidence to demonstrate that protocorm-like body (PLB) regeneration (the clonal regeneration practice used in the orchid industry) does not follow the embryogenesis program. Instead, we propose that SHOOT MERISTEMLESS, a class I KNOTTED-LIKE HOMEOBOX gene, is likely to play a role in PLB regeneration. Our studies challenge the current understanding of the embryonic identity of PLBs. Taken together, the data obtained establish a fundamental framework for orchid reproductive development and provide a valuable new resource to enable the prediction of gene regulatory networks that is required for specialized developmental programs of orchid species. PMID:27338813

Embryologic and Fetal Development of the Human Eyelid

PubMed Central

Abdulhafez, Mohamed H.; Fouad, Yousef A.; Dutton, Jonathan J.

2016-01-01

Purpose: To review the recent data about eyelid morphogenesis, and outline a timeline for eyelid development from the very early stages during embryonic life till final maturation of the eyelid late in fetal life. Methods: The authors extensively review major studies detailing human embryologic and fetal eyelid morphogenesis. These studies span almost a century and include some more recent cadaver studies. Numerous studies in the murine model have helped to better understand the molecular signals that govern eyelid embryogenesis. The authors summarize the current findings in molecular biology, and highlight the most significant studies in mice regarding the multiple and interacting signaling pathways involved in regulating normal eyelid morphogenesis. Results: Eyelid morphogenesis involves a succession of subtle yet strictly regulated morphogenetic episodes of tissue folding, proliferation, contraction, and even migration, which may occur simultaneously or in succession. Conclusions: Understanding the extraordinary process of building eyelid tissue in embryonic life, and deciphering its underlying signaling machinery has far reaching clinical implications beyond understanding the developmental abnormalities involving the eyelids, and may pave the way for achieving scar-reducing therapies in adult mammalian wounds, or control the spread of malignancies. PMID:27124372

Variations in brain defects result from cellular mosaicism in the activation of heat shock signalling.

PubMed

Ishii, Seiji; Torii, Masaaki; Son, Alexander I; Rajendraprasad, Meenu; Morozov, Yury M; Kawasawa, Yuka Imamura; Salzberg, Anna C; Fujimoto, Mitsuaki; Brennand, Kristen; Nakai, Akira; Mezger, Valerie; Gage, Fred H; Rakic, Pasko; Hashimoto-Torii, Kazue

2017-05-02

Repetitive prenatal exposure to identical or similar doses of harmful agents results in highly variable and unpredictable negative effects on fetal brain development ranging in severity from high to little or none. However, the molecular and cellular basis of this variability is not well understood. This study reports that exposure of mouse and human embryonic brain tissues to equal doses of harmful chemicals, such as ethanol, activates the primary stress response transcription factor heat shock factor 1 (Hsf1) in a highly variable and stochastic manner. While Hsf1 is essential for protecting the embryonic brain from environmental stress, excessive activation impairs critical developmental events such as neuronal migration. Our results suggest that mosaic activation of Hsf1 within the embryonic brain in response to prenatal environmental stress exposure may contribute to the resulting generation of phenotypic variations observed in complex congenital brain disorders.

Cyclin-dependent kinase inhibitor p21 does not impact embryonic endochondral ossification in mice

PubMed Central

CHINZEI, NOBUAKI; HAYASHI, SHINYA; HASHIMOTO, SHINGO; KANZAKI, NORIYUKI; IWASA, KENJIRO; SAKATA, SHUHEI; KIHARA, SHINSUKE; FUJISHIRO, TAKAAKI; KURODA, RYOSUKE; KUROSAKA, MASAHIRO

2015-01-01

Endochondral ossification at the growth plate is regulated by a number of factors and hormones. The cyclin-dependent kinase inhibitor p21 has been identified as a cell cycle regulator and its expression has been reported to be essential for endochondral ossification in vitro. However, to the best of our knowledge, the function of p21 in endochondral ossification has not been evaluated in vivo. Therefore, the aim of this study was to investigate the function of p21 in embryonic endochondral ossification in vivo. Wild-type (WT) and p21 knockout (KO) pregnant heterozygous mice were sacrificed on embryonic days E13.5, E15.5 and E18.5. Sagittal histological sections of the forearms of the embryos were collected and stained with Safranin O and 5-bromo-2′-deoxyuridine (BrdU). Additionally, the expression levels of cyclin D1, type II collagen, type X collagen, Sox9, and p16 were examined using immunohistochemistry, and the expression levels of p27 were examined using immunofluorescence. Safranin O staining revealed no structural change between the cartilage tissues of the WT and p21KO mice at any time point. Type II collagen was expressed ubiquitously, while type X collagen was only expressed in the hypertrophic zone of the cartilage tissues. No differences in the levels of Sox9 expression were observed between the two groups at any time point. The levels of cyclin D1 expression and BrdU uptake were higher in the E13.5 cartilage tissue compared with those observed in the embryonic cartilage tissue at subsequent time points. Expression of p16 and p27 was ubiquitous throughout the tissue sections. These results indicate that p21 may not be essential for embryonic endochondral ossification in articular cartilage of mice and that other signaling networks may compensate for p21 deletion. PMID:25376471

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells

PubMed Central

West, Michael D.; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B.; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-01

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1, encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro-derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition. PMID:29487692

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells.

PubMed

West, Michael D; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-30

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1 , encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro -derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition.

Molecular cloning and functional analysis of ESGP, an embryonic stem cell and germ cell specific protein.

PubMed

Chen, Yan-Mei; Du, Zhong-Wei; Yao, Zhen

2005-12-01

Several putative Oct-4 downstream genes from mouse embryonic stem (ES) cells have been identified using the suppression-subtractive hybridization method. In this study, one of the novel genes encoding an ES cell and germ cell specific protein (ESGP) was cloned by rapid amplification of cDNA ends. ESGP contains 801 bp encoding an 84 amino acid small protein and has no significant homology to any known genes. There is a signal peptide at the N-terminal of ESGP protein as predicted by SeqWeb (GCG) (SeqWeb version 2.0.2, http://gcg.biosino.org:8080/). The result of immunofluorescence assay suggested that ESGP might encode a secretory protein. The expression pattern of ESGP is consistent with the expression of Oct-4 during embryonic development. ESGP protein was detected in fertilized oocyte, from 3.5 day postcoital (dpc) blastocyst to 17.5 dpc embryo, and was only detected in testis and ovary tissues in adult. In vitro, ESGP was only expressed in pluripotent cell lines, such as embryonic stem cells, embryonic caoma cells and embryonic germ cells, but not in their differentiated progenies. Despite its specific expression, forced expression of ESGP is not indispensable for the effect of Oct-4 on ES cell self-renewal, and does not affect the differentiation to three germ layers.

Integration of immunological aspects in the European Human Embryonic Stem Cell Registry.

PubMed

Borstlap, Joeri; Kurtz, Andreas

2008-05-01

The immunological properties of stem cells are of increasing importance in regenerative medicine. Immunomodulatory mechanisms seem to play an important role not only with respect to the understanding of underlying mechanisms of autologous versus allogenic therapeutic approaches, but also for endogeneous tissue regeneration. The newly established European human embryonic stem cell registry (hESCreg) offers an international database for the registration, documentation and characterisation of human embryonic stem cells (hESC) and their use. By doing so, hESCreg aims to develop a model procedure for further standardisation efforts in the field of stem cell research and regenerative medicine, and eventually the registry may lead to a repository of therapy-related information. Currently the stem cell characterisation data acquired by the registry are divided into several categories such as cell derivation, culture conditions, genetic constitution, stem cell marker expression and degree of modification. This article describes immunological aspects of stem cell characterisation and explores the layout and relevance of a possible additional section to the hESCreg repository to include immunological characteristics of human embryonic stem cells.

Hmga2 is required for canonical WNT signaling during lung development

PubMed Central

2014-01-01

Background The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. Here we deciphered the role of the high-mobility-group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2-deficient mice (Hmga2 −/− ). Results We found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2 −/− mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA-binding protein 6 and frizzled homolog 2. Analysis of siRNA-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of Hmga2 as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical WNT signaling in mediating the effect of Hmga2-loss-of-fuction. Finally, we found that HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression. Conclusions Our results support that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium. Our results are the starting point for future studies investigating the relevance of Hmga2-mediated regulation of WNT signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury. PMID:24661562

Human Development VI: Supracellular Morphogenesis. The Origin of Biological and Cellular Order

PubMed Central

Ventegodt, Søren; Hermansen, Tyge Dahl; Flensborg-Madsen, Trine; Nielsen, Maj Lyck; Merrick, Joav

2006-01-01

Uninterrupted morphogenesis shows the informational potentials of biological organisms. Experimentally disturbed morphogenesis shows the compensational dynamics of the biological informational system, which is the rich informational redundancy. In this paper, we use these data to describe morphogenesis in terms of the development of supracellular levels of the organism, and we define complex epigenesis and supracellular differentiation. We review the phenomena of regeneration and induction of Hydra and amphibians, and the higher animals informational needs for developing their complex nervous systems. We argue, also building on the NO-GO theorem for ontogenesis as chemistry, that the traditional chemical explanations of high-level informational events in ontogenesis, such as transmutation, regeneration, and induction, are insufficient. We analyze the informational dynamics of three embryonic compensatory reactions to different types of disturbances: (1) transmutations of the imaginal discs of insects, (2) regeneration after removal of embryonic tissue, and (3) embryonic induction, where two tissues that normally are separated experimentally are made to influence each other. We describe morphogenesis as a complex bifurcation, and the resulting morphological levels of the organism as organized in a fractal manner and supported by positional information. We suggest that some kind of real nonchemical phenomenon must be taking form in living organisms as an information-carrying dynamic fractal field, causing morhogenesis and supporting the organisms morphology through time. We argue that only such a phenomenon that provides information-directed self-organization to the organism is able to explain the observed dynamic distribution of biological information through morphogenesis and the organism's ability to rejuvenate and heal. PMID:17115082

Smad4 is required for the development of cardiac and skeletal muscle in zebrafish.

PubMed

Yang, Jie; Wang, Junnai; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Jiang, Lijun; Wei, Juncheng; Ma, Quanfu; Wu, Mingfu; Ye, Shuangmei; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan

Transforming growth factor-beta (TGF-beta) regulates cellular functions and plays key roles in development and carcinogenesis. Smad4 is the central intracellular mediator of TGF-beta signaling and plays crucial roles in tissue regeneration, cell differentiation, embryonic development, regulation of the immune system and tumor progression. To clarify the role of smad4 in development, we examined both the pattern of smad4 expression in zebrafish embryos and the effect of smad4 suppression on embryonic development using smad4-specific antisense morpholino-oligonucleotides. We show that smad4 is expressed in zebrafish embryos at all developmental stages examined and that embryonic knockdown of smad4 results in pericardial edema, decreased heartbeat and defects in the trunk structure. Additionally, these phenotypes were associated with abnormal expression of the two heart-chamber markers, cmlc2 and vmhc, as well as abnormal expression of three makers of myogenic terminal differentiation, mylz2, smyhc1 and mck. Furthermore, a notable increase in apoptosis was apparent in the smad4 knockdown embryos, while no obvious reduction in cell proliferation was observed. Collectively, these data suggest that smad4 plays an important role in heart and skeletal muscle development. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

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

Formation of Stomach Tissue by Organoid Culture Using Mouse Embryonic Stem Cells.

PubMed

Noguchi, Taka-Aki K; Kurisaki, Akira

2017-01-01

In this chapter, we describe a method for the induction of stomach organoids from mouse embryonic stem (ES) cells. We used an embryoid body-based differentiation method to induce gastric primordial epithelium covered with mesenchyme and further differentiate it in Matrigel by 3D culture. The differentiated organoid contains both corpus- and antrum-specific mature gastric tissue cells. This protocol may be useful for a variety of studies in developmental biology and disease modeling of the stomach.

Pazopanib Hydrochloride in Treating Young Patients With Solid Tumors That Have Relapsed or Not Responded to Treatment

ClinicalTrials.gov

2013-09-27

Childhood Central Nervous System Choriocarcinoma; Childhood Central Nervous System Embryonal Tumor; Childhood Central Nervous System Germ Cell Tumor; Childhood Central Nervous System Germinoma; Childhood Central Nervous System Mixed Germ Cell Tumor; Childhood Central Nervous System Teratoma; Childhood Central Nervous System Yolk Sac Tumor; Metastatic Childhood Soft Tissue Sarcoma; Recurrent Childhood Brain Stem Glioma; Recurrent Childhood Central Nervous System Embryonal Tumor; Recurrent Childhood Soft Tissue Sarcoma; Recurrent Childhood Visual Pathway Glioma; Unspecified Childhood Solid Tumor, Protocol Specific

Evidence in duck for supporting alteration of incubation temperature may have influence on methylation of genomic DNA.

PubMed

Yan, Xi-Ping; Liu, He-He; Liu, Jun-Ying; Zhang, Rong-Ping; Wang, Guo-Song; Li, Qing-Qing; Wang, Ding-Min-Cheng; Li, Liang; Wang, Ji-Wen

2015-10-01

Incubation temperature has an immediate and long-term influence on the embryonic development in birds. DNA methylation as an important environment-induced mechanism could serve as a potential link between embryos' phenotypic variability and temperature variation, which reprogrammed by DNA (cytosine-5)-methyltransferases (DNMTS) and Methyl-CpG binding domain proteins (MBPS) 3&5 (MBD3&5). Five genes in DNMTS and MBPS gene families were selected as target genes, given their important role in epigenetic modification. In this study, we aimed to test whether raising incubation temperature from 37.8°C to 38.8°C between embryonic days (ED) 1-10, ED10-20 and ED20-27 have effect on DNA methylation and whether DNMTS, MBPS play roles in thermal epigenetic regulation of early development in duck. Real-time quantitative PCR analysis showed that increased incubation temperature by 1°C has remarkably dynamic effect on gene expression levels of DNMTS and MBPS. Slight changes in incubation temperature significantly increased mRNA levels of target genes in breast muscle tissue during ED1-10, especially for DNMT1, DNMT3A and MBD5. In addition, higher temperature significantly increased enzyme activities of DNMT1 in leg muscle during ED10-20, liver tissue during ED1-10, ED20-27 and DNMT3A in leg muscle and breast muscle tissue during ED10-20. These results suggest that incubation temperature has an extended effect on gene expression levels and enzyme activities of DNMTS and MBPS, which provides evidence that incubation temperature may influence DNA methylation in duck during early developmental stages. Our data indicated that DNMTS and MBPS may involved in thermal epigenetice regulation of embryos during the early development in duck. The potential links between embryonic temperature and epigenetic modification need further investigation. © The Author 2015. Published by Oxford University Press on behalf of the Poultry Science Association.

Autonomous assembly of epithelial structures by subrenal implantation of dissociated embryonic inner-ear cells.

PubMed

Wang, Li; Zhang, Kaiqing; Zhu, Helen He; Gao, Wei-Qiang

2015-05-27

Microenvironment and cell-cell interactions play an important role during embryogenesis and are required for the stemness and differentiation of stem cells. The inner-ear sensory epithelium, containing hair cells and supporting cells, is derived from the stem cells within the otic vesicle at early embryonic stages. However, whether or not such microenvironment or cell-cell interactions within the embryonic otic tissue have the capacity to regulate the proliferation and differentiation of stem cells and to autonomously reassemble the cells into epithelial structures is unknown. Here, we report that on enzymatic digestion and dissociation to harvest all the single cells from 13.5-day-old rat embryonic (E13.5) inner-ear tissue as well as on implantation of these cells under renal capsules; the dissociated cells are able to reassemble themselves to form epithelial structures as early as 7 days after implantation. By 25 days after implantation, more mature epithelial structures are formed. Immunostaining with cell-type-specific markers reveals that hair cells and supporting cells are not only formed, but are also well aligned with the hair cells located in the apical layer surrounded by the supporting cells. These findings suggest that microenvironment and cell-cell interactions within the embryonic inner-ear tissue have the autonomous signals to induce the formation of sensory epithelial structures. This method may also provide a useful system to study the potential of stem cells to differentiate into hair cells in vivo.

Based serum metabolomics analysis reveals simultaneous interconnecting changes during chicken embryonic development.

PubMed

Peng, M L; Li, S N; He, Q Q; Zhao, J L; Li, L L; Ma, H T

2018-05-28

Metabolic disorder is a major health problem and is associated with a number of metabolic diseases. Due to native hyperglycaemia and resistance to exogenous insulin, chickens as a model had used in the studies of adipose tissue biology, metabolism and obesity. But no detailed information is available about the comprehensive changes of serum metabolites at different stages of chicken embryonic development. This study employed LC/MS-QTOF to determine the changes of major functional metabolites at incubation day 14 (E14d), 19 (E19d) and hatching day 1 (H1d), and the associated pathways of differential metabolites during chicken embryonic development were analysed using Metabolite Set Enrichment Analysis method. Results showed that 39 metabolites were significantly changed from E14d to E19d and 68 metabolites were significantly altered from E19d to H1d in chicken embryos. Protein synthesis was promoted by increasing the concentrations of L-glutamine and threonine, and gonadal development was promoted through increasing oestrone content from E14d to E19d in chicken embryos, which indicated that serum glutamine, threonine and oestrone contents may be considered as the candidate indicators for assessment of early embryonic development. 2-oxoglutaric acid mainly contributed to enhancing the citric cycle, and it plays an important role in improving the growth of chicken embryos at the late development; the decreasing of L-glutamine, L-isoleucine and L-leucine contents from E19d to H1d in chicken embryonic development implied their possible functions as the feed additive during early posthatch period of broiler chickens to satisfy the growth. These results provided insights into understand the roles of serum metabolites at different developmental stages of chicken embryos, it also provides available information for chicken as a model to study metabolic disease or human obesity. © 2018 Blackwell Verlag GmbH.

Growth and differentiation of mammalian embryonic tissues exposed to hypergravity in vivo and in vitro

NASA Technical Reports Server (NTRS)

Duke, J.; Janer, L.; Moore, J.

1985-01-01

Decreased cartilage areas in embryonic limbs developing under excess g in vitro, is reported, as well as delayed skeletal development in embryos and fetuses exposed to excess g in utero. 12.5-day mouse limb buds were cultured at 2.6 g, and fixed at two days and six days of culture. In vivo experiments used alizarin-stained 18-day fetuses exposed to 2.3 g. In all studies, cartilage areas were determined using a digitized tablet. Form factor analysis determined that the main effect of in vitro centrifugation was a reduction in length of the limb elements, probably due to the precocious chondrogenesis seen in the upper regions of centrifuged limbs. Similar reductions in length of ossified areas was seen in the in utero studies.

In vitro control of fasciation in proliferating nucellar embryos of Mangifera indica L. var totapari red small for cloning.

PubMed

Chaturvedi, H C; Agnihotri, S; Sharma, M; Sharma, A K; Jain, M; Chourasia, A

2003-11-01

Nucellar tissue contained in ovular halves of young fruits of Mangifera indica L. totapari red small, a dwarfing rootstock, differentiated fasciated embryonal structures in presence of 6-benzylaminopurine [BAP(0.15 mg l(-1))], 6-(gamma-gamma-dimethylallylamino) purine [2iP(0.15 mg l(-1))] and indole-3-acetic acid [(IAA(0.5 mg l(-1))] incorporated in the semisolid medium during 50-60 days. Due to embryonal fasciation, hardly 2-3 well-formed embryos could be obtained per culture of proliferating embryos. Of the 3 ethylene inhibitors [L-alpha-(2-aminoethoxyvinyl)-glycine-HCl (AVG), AgNO3 and salicylic acid (SA)] used, embryonal fasciation and necrosis of intervening tissue was completely controlled by 3-4 subcultures of fasciated mass of embryos under the influence of AVG (0.05 mg l(-1)) in presence of adenine sulphate [AdS (50 mg l(-1))] incorporated in the same medium. Almost synchronized development of isolated embryos, measuring ca 2 cm in length, was observed in a different medium used in liquid stationary state and supplemented, particularly with stress-producing substances [abscisic acid (ABA, 0.01 mg l(-1)); and polyethylene glycol (PEG, 100 mg l(-1))] besides certain other modifications. About 34% convertibility of processed embryos was obtained during a period of 90 days. The plantlets had well-developed roots along with laterals which were longer than leafy shoots. In vitro raised plants survived ex vitro for about 2 months.

Human Fetal Keratocytes Have Multipotent Characteristics in the Developing Avian Embryo

PubMed Central

Chao, Jennifer R.; Bronner, Marianne E.

2013-01-01

The human cornea contains stem cells that can be induced to express markers consistent with multipotency in cell culture; however, there have been no studies demonstrating that human corneal keratocytes are multipotent. The objective of this study is to examine the potential of human fetal keratocytes (HFKs) to differentiate into neural crest-derived tissues when challenged in an embryonic environment. HFKs were injected bilaterally into the cranial mesenchyme adjacent to the neural tube and the periocular mesenchyme in chick embryos at embryonic days 1.5 and 3, respectively. The injected keratocytes were detected by immunofluorescence using the human cell-specific marker, HuNu. HuNu-positive keratocytes injected along the neural crest pathway were localized adjacent to HNK-1-positive migratory host neural crest cells and in the cardiac cushion mesenchyme. The HuNu-positive cells transformed into neural crest derivatives such as smooth muscle in cranial blood vessels, stromal keratocytes, and corneal endothelium. However, they failed to form neurons despite their presence in the condensing trigeminal ganglion. These results show that HFKs retain the ability to differentiate into some neural crest-derived tissues. Their ability to respond to embryonic cues and generate corneal endothelium and stromal keratocytes provides a basis for understanding the feasibility of creating specialized cells for possible use in regenerative medicine. PMID:23461574

ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV

PubMed Central

Huynh, Julie M.; Dang, Hope; Munoz-Tucker, Isabel A.; O’Ketch, Marvin; Liu, Ian T.; Perno, Savannah; Bhuyan, Natasha; Crain, Allison; Borbon, Ivan; Fares, Hanna

2016-01-01

Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5. PMID:26596346

Temozolomide, Cixutumumab, and Combination Chemotherapy in Treating Patients With Metastatic Rhabdomyosarcoma

ClinicalTrials.gov

2017-07-31

Adult Rhabdomyosarcoma; Childhood Alveolar Rhabdomyosarcoma; Childhood Embryonal Rhabdomyosarcoma; Metastatic Childhood Soft Tissue Sarcoma; Stage IV Adult Soft Tissue Sarcoma; Untreated Childhood Rhabdomyosarcoma

Endochondral bone formation in embryonic mouse pre-metatarsals

NASA Technical Reports Server (NTRS)

Klement, B. J.; Spooner, B. S.

1992-01-01

Long term exposure to a reduced gravitational environment has a deleterious effect on bone. The developmental events which occur prior to initial bone deposition will provide insight into the regulation of mature bone physiology. We have characterized a system in which the events preceding bone formation take place in an isolated in vitro organ culture environment. We show that cultured pre-metatarsal tissue parallels development of pre-metatarsal tissue in the embryo. Both undergo mesenchyme differentiation and morphogenesis to form a cartilage rod, which resembles the future bone, followed by terminal chondrocyte differentiation in a definite morphogenetic pattern. These sequential steps occur prior to osteoblast maturation and bone matrix deposition in the developing organism. Alkaline phosphatase (ALP) activity is a distinctive enzymatic marker for mineralizing tissues. We have measured this activity throughout pre-metatarsal development and show (a) where in the tissue it is predominantly found, and (b) that this is indeed the mineralizing isoform of the enzyme.

Real-time Visualization of Tissue Dynamics during Embryonic Development and Malignant Transformation

NASA Astrophysics Data System (ADS)

Yamada, Kenneth

Tissues undergo dramatic changes in organization during embryonic development, as well as during cancer progression and invasion. Recent advances in microscopy now allow us to visualize and track directly the dynamic movements of tissues, their constituent cells, and cellular substructures. This behavior can now be visualized not only in regular tissue culture on flat surfaces (`2D' environments), but also in a variety of 3D environments that may provide physiological cues relevant to understanding dynamics within living organisms. Acquisition of imaging data using various microscopy modalities will provide rich opportunities for determining the roles of physical factors and for computational modeling of complex processes in living tissues. Direct visualization of real-time motility is providing insight into biology spanning multiple spatio-temporal scales. Many cells in our body are known to be in contact with connective tissue and other forms of extracellular matrix. They do so through microscopic cellular adhesions that bind to matrix proteins. In particular, fluorescence microscopy has revealed that cells dynamically probe and bend the matrix at the sites of cell adhesions, and that 3D matrix architecture, stiffness, and elasticity can each regulate migration of the cells. Conversely, cells remodel their local matrix as organs form or tumors invade. Cancer cells can invade tissues using microscopic protrusions that degrade the surrounding matrix; in this case, the local matrix protein concentration is more important for inducing the micro-invasive protrusions than stiffness. On the length scales of tissues, transiently high rates of individual cell movement appear to help establish organ architecture. In fact, isolated cells can self-organize to form tissue structures. In all of these cases, in-depth real-time visualization will ultimately provide the extensive data needed for computer modeling and for testing hypotheses in which physical forces interact closely with cell signaling to form organs or promote tumor invasion.

Establishment of Functional Genomics Pipeline in Mouse Epiblast-Like Tissue by Combining Transcriptomic Analysis and Gene Knockdown/Knockin/Knockout, Using RNA Interference and CRISPR/Cas9.

PubMed

Takata, Nozomu; Sakakura, Eriko; Kasukawa, Takeya; Sakuma, Tetsushi; Yamamoto, Takashi; Sasai, Yoshiki

2016-06-01

The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. However, regulation of epiblast gene expression is poorly understood because of the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of mouse embryonic stem cell (ESC), we generated and characterized epiblast-like tissue in three-dimensional culture. We identified significant genome-wide gene expression changes in this epiblast-like tissue by transcriptomic analysis. In addition, we identified the particular significance of the Erk/Mapk and integrin-linked kinase pathways, and genes related to ectoderm/epithelial formation, using the bioinformatics resources IPA and DAVID. Here, we focused on Fgf5, which ranked in the top 10 among the discovered genes. To develop a functional analysis of Fgf5, we created an efficient method combining CRISPR/Cas9-mediated genome engineering and RNA interference (RNAi). Notably, we show one-step generation of various Fgf5 reporter lines including heterozygous and homozygous knockins (the GET method). For time- and dose-dependent depletion of fgf5 over the course of development, we generated an ESC line harboring Tol2 transposon-mediated integration of an inducible short hairpin RNA interference system (pdiRNAi). Our findings raised the possibility that Fgf/Erk signaling and apicobasal epithelial integrity are important factors in epiblast development. In addition, our methods provide a framework for a broad array of applications in the areas of mammalian genetics and molecular biology to understand development and to improve future therapeutics.

In situ hybridization analysis of the temporospatial expression of the midkine/pleiotrophin family in rat embryonic pituitary gland.

PubMed

Fujiwara, Ken; Maliza, Rita; Tofrizal, Alimuddin; Batchuluun, Khongorzul; Ramadhani, Dini; Tsukada, Takehiro; Azuma, Morio; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi

2014-07-01

Pituitary gland development is controlled by numerous signaling molecules, which are produced in the oral ectoderm and diencephalon. A newly described family of heparin-binding growth factors, namely midkine (MK)/pleiotrophin (PTN), is involved in regulating the growth and differentiation of many tissues and organs. Using in situ hybridization with digoxigenin-labeled cRNA probes, we detected cells expressing MK and PTN in the developing rat pituitary gland. At embryonic day 12.5 (E12.5), MK expression was localized in Rathke's pouch (derived from the oral ectoderm) and in the neurohypophyseal bud (derived from the diencephalon). From E12.5 to E19.5, MK mRNA was expressed in the developing neurohypophysis, and expression gradually decreased in the developing adenohypophysis. To characterize MK-expressing cells, we performed double-staining of MK mRNA and anterior pituitary hormones. At E19.5, no MK-expressing cells were stained with any hormone. In contrast, PTN was expressed only in the neurohypophysis primordium during all embryonic stages. In situ hybridization clearly showed that MK was expressed in primitive (immature/undifferentiated) adenohypophyseal cells and neurohypophyseal cells, whereas PTN was expressed only in neurohypophyseal cells. Thus, MK and PTN might play roles as signaling molecules during pituitary development.

Skull Development, Ossification Pattern, and Adult Shape in the Emerging Lizard Model Organism Pogona vitticeps: A Comparative Analysis With Other Squamates.

PubMed

Ollonen, Joni; Da Silva, Filipe O; Mahlow, Kristin; Di-Poï, Nicolas

2018-01-01

The rise of the Evo-Devo field and the development of multidisciplinary research tools at various levels of biological organization have led to a growing interest in researching for new non-model organisms. Squamates (lizards and snakes) are particularly important for understanding fundamental questions about the evolution of vertebrates because of their high diversity and evolutionary innovations and adaptations that portrait a striking body plan change that reached its extreme in snakes. Yet, little is known about the intricate connection between phenotype and genotype in squamates, partly due to limited developmental knowledge and incomplete characterization of embryonic development. Surprisingly, squamate models have received limited attention in comparative developmental studies, and only a few species examined so far can be considered as representative and appropriate model organism for mechanistic Evo-Devo studies. Fortunately, the agamid lizard Pogona vitticeps (central bearded dragon) is one of the most popular, domesticated reptile species with both a well-established history in captivity and key advantages for research, thus forming an ideal laboratory model system and justifying his recent use in reptile biology research. We first report here the complete post-oviposition embryonic development for P. vitticeps based on standardized staging systems and external morphological characters previously defined for squamates. Whereas the overall morphological development follows the general trends observed in other squamates, our comparisons indicate major differences in the developmental sequence of several tissues, including early craniofacial characters. Detailed analysis of both embryonic skull development and adult skull shape, using a comparative approach integrating CT-scans and gene expression studies in P. vitticeps as well as comparative embryology and 3D geometric morphometrics in a large dataset of lizards and snakes, highlights the extreme adult skull shape of P. vitticeps and further indicates that heterochrony has played a key role in the early development and ossification of squamate skull bones. Such detailed studies of embryonic character development, craniofacial patterning, and bone formation are essential for the establishment of well-selected squamate species as Evo-Devo model organisms. We expect that P. vitticeps will continue to emerge as a new attractive model organism for understanding developmental and molecular processes underlying tissue formation, morphology, and evolution.

Skull Development, Ossification Pattern, and Adult Shape in the Emerging Lizard Model Organism Pogona vitticeps: A Comparative Analysis With Other Squamates

PubMed Central

Ollonen, Joni; Da Silva, Filipe O.; Mahlow, Kristin; Di-Poï, Nicolas

2018-01-01

The rise of the Evo-Devo field and the development of multidisciplinary research tools at various levels of biological organization have led to a growing interest in researching for new non-model organisms. Squamates (lizards and snakes) are particularly important for understanding fundamental questions about the evolution of vertebrates because of their high diversity and evolutionary innovations and adaptations that portrait a striking body plan change that reached its extreme in snakes. Yet, little is known about the intricate connection between phenotype and genotype in squamates, partly due to limited developmental knowledge and incomplete characterization of embryonic development. Surprisingly, squamate models have received limited attention in comparative developmental studies, and only a few species examined so far can be considered as representative and appropriate model organism for mechanistic Evo-Devo studies. Fortunately, the agamid lizard Pogona vitticeps (central bearded dragon) is one of the most popular, domesticated reptile species with both a well-established history in captivity and key advantages for research, thus forming an ideal laboratory model system and justifying his recent use in reptile biology research. We first report here the complete post-oviposition embryonic development for P. vitticeps based on standardized staging systems and external morphological characters previously defined for squamates. Whereas the overall morphological development follows the general trends observed in other squamates, our comparisons indicate major differences in the developmental sequence of several tissues, including early craniofacial characters. Detailed analysis of both embryonic skull development and adult skull shape, using a comparative approach integrating CT-scans and gene expression studies in P. vitticeps as well as comparative embryology and 3D geometric morphometrics in a large dataset of lizards and snakes, highlights the extreme adult skull shape of P. vitticeps and further indicates that heterochrony has played a key role in the early development and ossification of squamate skull bones. Such detailed studies of embryonic character development, craniofacial patterning, and bone formation are essential for the establishment of well-selected squamate species as Evo-Devo model organisms. We expect that P. vitticeps will continue to emerge as a new attractive model organism for understanding developmental and molecular processes underlying tissue formation, morphology, and evolution. PMID:29643813

Tissue fusion over nonadhering surfaces

PubMed Central

Nier, Vincent; Deforet, Maxime; Duclos, Guillaume; Yevick, Hannah G.; Cochet-Escartin, Olivier; Marcq, Philippe; Silberzan, Pascal

2015-01-01

Tissue fusion eliminates physical voids in a tissue to form a continuous structure and is central to many processes in development and repair. Fusion events in vivo, particularly in embryonic development, often involve the purse-string contraction of a pluricellular actomyosin cable at the free edge. However, in vitro, adhesion of the cells to their substrate favors a closure mechanism mediated by lamellipodial protrusions, which has prevented a systematic study of the purse-string mechanism. Here, we show that monolayers can cover well-controlled mesoscopic nonadherent areas much larger than a cell size by purse-string closure and that active epithelial fluctuations are required for this process. We have formulated a simple stochastic model that includes purse-string contractility, tissue fluctuations, and effective friction to qualitatively and quantitatively account for the dynamics of closure. Our data suggest that, in vivo, tissue fusion adapts to the local environment by coordinating lamellipodial protrusions and purse-string contractions. PMID:26199417

Optimal development of matrix elasticity

PubMed Central

Majkut, Stephanie; Idema, Timon; Swift, Joe; Krieger, Christine; Liu, Andrea; Discher, Dennis E.

2014-01-01

Summary In development and differentiation, morphological changes often accompany mechanical changes [1], but it is unclear if or when cells in embryos sense tissue elasticity. The earliest embryo is uniformly pliable while adult tissues vary widely in mechanics from soft brain and stiff heart to rigid bone [2], but the sensitivity of cells to microenvironment elasticity is debated [3]. Regenerative cardiology provides strong motivation because rigid post-infarct regions limit pumping by the adult heart [4]. Here we focus on embryonic heart and isolated cardiomyocytes, which both beat spontaneously. Tissue elasticity, Et, increases daily for heart to 1-2 kiloPascal by embryonic day-4 (E4), and although this is ∼10-fold softer than adult heart, the beating contractions of E4-cardiomyocytes prove optimal at ∼Et,E4 both in vivo and in vitro. Proteomics reveals daily increases in a small subset of proteins, namely collagen plus cardiac-specific excitation-contraction proteins. Rapid softening of the heart's matrix with collagenase or stiffening it with enzymatic crosslinking suppresses beating. Sparsely cultured E4-cardiomyocytes on collagen-coated gels likewise show maximal contraction on matrices with native E4 stiffness, highlighting cell-intrinsic mechanosensitivity. While an optimal elasticity for striation proves consistent with the mathematics of force-driven sarcomere registration, contraction wave-speed is linear in Et as theorized for Excitation-Contraction Coupled to Matrix Elasticity. Mechanosensitive stem cell cardiogenesis helps generalize tissue results, which demonstrate how myosin-II organization and contractile function is optimally matched to the load presented by matrix elasticity. PMID:24268417

Technique: imaging earliest tooth development in 3D using a silver-based tissue contrast agent.

PubMed

Raj, Muhammad T; Prusinkiewicz, Martin; Cooper, David M L; George, Belev; Webb, M Adam; Boughner, Julia C

2014-02-01

Looking in microscopic detail at the 3D organization of initiating teeth within the embryonic jaw has long-proved technologically challenging because of the radio-translucency of these tiny un-mineralized oral tissues. Yet 3D image data showing changes in the physical relationships among developing tooth and jaw tissues are vital to understand the coordinated morphogenesis of vertebrate teeth and jaws as an animal grows and as species evolve. Here, we present a new synchrotron-based scanning solution to image odontogenesis in 3D and in histological detail using a silver-based contrast agent. We stained fixed, intact wild-type mice aged embryonic (E) day 10 to birth with 1% Protargol-S at 37°C for 12-32 hr. Specimens were scanned at 4-10 µm pixel size at 28 keV, just above the silver K-edge, using micro-computed tomography (µCT) at the Canadian Light Source synchrotron. Synchrotron µCT scans of silver-stained embryos showed even the earliest visible stages of tooth initiation, as well as many other tissue types and structures, in histological detail. Silver stain penetration was optimal for imaging structures in intact embryos E15 and younger. This silver stain method offers a powerful yet straightforward approach to visualize at high-resolution and in 3D the earliest stages of odontogenesis in situ, and demonstrates the important of studying the tooth organ in all three planes of view. Copyright © 2013 Wiley Periodicals, Inc.

Use of fibroblast growth factor 2 for expansion of chondrocytes and tissue engineering

NASA Technical Reports Server (NTRS)

Vunjak-Novakovic, Gordana (Inventor); Martin, Ivan (Inventor); Freed, Lisa E. (Inventor); Langer, Robert (Inventor)

2003-01-01

The present invention provides an improved method for expanding cells for use in tissue engineering. In particular the method provides specific biochemical factors to supplement cell culture medium during the expansion process in order to reproduce events occurring during embryonic development with the goal of regenerating tissue equivalents that resemble natural tissues both structurally and functionally. These specific biochemical factors improve proliferation of the cells and are capable of de-differentiation mature cells isolated from tissue so that the differentiation potential of the cells is preserved. The bioactive molecules also maintain the responsiveness of the cells to other bioactive molecules. Specifically, the invention provides methods for expanding chondrocytes in the presence of fibroblast growth factor 2 for use in regeneration of cartilage tissue.

Retinoic Acid signaling regulates Krt5 and Krt14 independently of stem cell markers in submandibular salivary gland epithelium

PubMed Central

Abashev, Timur M.; Metzler, Melissa A.; Wright, Diana M.; Sandell, Lisa L.

2017-01-01

Background Retinoic Acid (RA), the active metabolite of Vitamin A, has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA regulation have not been identified. Results Here we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and non-neuronal mesenchyme. By culturing epithelium explants in isolation from other tissues we demonstrate that RA influences epithelium morphogenesis by direct action in that tissue. Moreover, we demonstrate that inhibition of RA signaling represses cell proliferation and expression of FGF10 signaling targets, and upregulates expression of basal epithelial keratins Krt5 and Krt14. Importantly, we show that the stem cell gene Kit is regulated inversely from Krt5/Krt14 by RA signaling. Conclusions RA regulates Krt5 and Krt14 expression independently of stem cell character in developing salivary epithelium. RA, or chemical inhibitors of RA signaling, could potentially be used for modulating growth and differentiation of epithelial stem cells for the purpose of re-populating damaged glands or generating bioengineered organs. PMID:27884045

Identification of new regulators of embryonic patterning and morphogenesis in Xenopus gastrulae by RNA sequencing.

PubMed

Popov, Ivan K; Kwon, Taejoon; Crossman, David K; Crowley, Michael R; Wallingford, John B; Chang, Chenbei

2017-06-15

During early vertebrate embryogenesis, cell fate specification is often coupled with cell acquisition of specific adhesive, polar and/or motile behaviors. In Xenopus gastrulae, tissues fated to form different axial structures display distinct motility. The cells in the early organizer move collectively and directionally toward the animal pole and contribute to anterior mesendoderm, whereas the dorsal and the ventral-posterior trunk tissues surrounding the blastopore of mid-gastrula embryos undergo convergent extension and convergent thickening movements, respectively. While factors regulating cell lineage specification have been described in some detail, the molecular machinery that controls cell motility is not understood in depth. To gain insight into the gene battery that regulates both cell fates and motility in particular embryonic tissues, we performed RNA sequencing (RNA-seq) to investigate differentially expressed genes in the early organizer, the dorsal and the ventral marginal zone of Xenopus gastrulae. We uncovered many known signaling and transcription factors that have been reported to play roles in embryonic patterning during gastrulation. We also identified many uncharacterized genes as well as genes that encoded extracellular matrix (ECM) proteins or potential regulators of actin cytoskeleton. Co-expression of a selected subset of the differentially expressed genes with activin in animal caps revealed that they had distinct ability to block activin-induced animal cap elongation. Most of these factors did not interfere with mesodermal induction by activin, but an ECM protein, EFEMP2, inhibited activin signaling and acted downstream of the activated type I receptor. By focusing on a secreted protein kinase PKDCC1, we showed with overexpression and knockdown experiments that PKDCC1 regulated gastrulation movements as well as anterior neural patterning during early Xenopus development. Overall, our studies identify many differentially expressed signaling and cytoskeleton regulators in different embryonic regions of Xenopus gastrulae and imply their functions in regulating cell fates and/or behaviors during gastrulation. Copyright © 2016 Elsevier Inc. All rights reserved.

Meis2 is essential for cranial and cardiac neural crest development.

PubMed

Machon, Ondrej; Masek, Jan; Machonova, Olga; Krauss, Stefan; Kozmik, Zbynek

2015-11-06

TALE-class homeodomain transcription factors Meis and Pbx play important roles in formation of the embryonic brain, eye, heart, cartilage or hematopoiesis. Loss-of-function studies of Pbx1, 2 and 3 and Meis1 documented specific functions in embryogenesis, however, functional studies of Meis2 in mouse are still missing. We have generated a conditional allele of Meis2 in mice and shown that systemic inactivation of the Meis2 gene results in lethality by the embryonic day 14 that is accompanied with hemorrhaging. We show that neural crest cells express Meis2 and Meis2-defficient embryos display defects in tissues that are derived from the neural crest, such as an abnormal heart outflow tract with the persistent truncus arteriosus and abnormal cranial nerves. The importance of Meis2 for neural crest cells is further confirmed by means of conditional inactivation of Meis2 using crest-specific AP2α-IRES-Cre mouse. Conditional mutants display perturbed development of the craniofacial skeleton with severe anomalies in cranial bones and cartilages, heart and cranial nerve abnormalities. Meis2-null mice are embryonic lethal. Our results reveal a critical role of Meis2 during cranial and cardiac neural crest cells development in mouse.

Angiogenesis of the uterus and chorioallantois in the eastern water skink Eulamprus quoyii.

PubMed

Murphy, Bridget F; Parker, Scott L; Murphy, Christopher R; Thompson, Michael B

2010-10-01

We have discovered a modification of the uterus that appears to facilitate maternal-fetal communication during pregnancy in the scincid lizard Eulamprus quoyii. A vessel-dense elliptical area (VDE) on the mesometrial side of the uterus expands as the embryo grows, providing a large vascular area for physiological exchange between mother and embryo. The VDE is already developed in females with newly ovulated eggs, and is situated directly adjacent to the chorioallantois of the embryo when it develops. It is likely that signals from the early developing embryo determine the position of the VDE, as the VDE is off-centre in cases where the embryo sits obliquely in the uterus. The VDE is not a modification of the uterus over the entire chorioallantoic placenta, as the VDE is smaller than the chorioallantois after embryonic stage 33, but expansion of the VDE and growth of the chorioallantois during pregnancy are strongly correlated. The expansion of the VDE is also strongly correlated with embryonic growth and increasing embryonic oxygen demand (Vo2). We propose that angiogenic stimuli are exchanged between the VDE and the chorioallantois in E. quoyii, allowing the simultaneous growth of both tissues.

In vitro 3D regeneration-like growth of human patient brain tissue.

PubMed

Tang-Schomer, M D; Wu, W B; Kaplan, D L; Bookland, M J

2018-05-01

In vitro culture of primary neurons is widely adapted with embryonic but not mature brain tissue. Here, we extended a previously developed bioengineered three-dimensional (3D) embryonic brain tissue model to resected normal patient brain tissue in an attempt to regenerate human neurons in vitro. Single cells and small sized (diameter < 100 μm) spheroids from dissociated brain tissue were seeded into 3D silk fibroin-based scaffolds, with or without collagen or Matrigel, and compared with two-dimensional cultures and scaffold-free suspension cultures. Changes of cell phenotypes (neuronal, astroglial, neural progenitor, and neuroepithelial) were quantified with flow cytometry and analyzed with a new method of statistical analysis specifically designed for percentage comparison. Compared with a complete lack of viable cells in conventional neuronal cell culture condition, supplements of vascular endothelial growth factor-containing pro-endothelial cell condition led to regenerative growth of neurons and astroglial cells from "normal" human brain tissue of epilepsy surgical patients. This process involved delayed expansion of Nestin+ neural progenitor cells, emergence of TUJ1+ immature neurons, and Vimentin+ neuroepithelium-like cell sheet formation in prolonged cultures (14 weeks). Micro-tissue spheroids, but not single cells, supported the brain tissue growth, suggesting importance of preserving native cell-cell interactions. The presence of 3D scaffold, but not hydrogel, allowed for Vimentin+ cell expansion, indicating a different growth mechanism than pluripotent cell-based brain organoid formation. The slow and delayed process implied an origin of quiescent neural precursors in the neocortex tissue. Further optimization of the 3D tissue model with primary human brain cells could provide personalized brain disease models. Copyright © 2018 John Wiley & Sons, Ltd.

Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update

PubMed Central

Miele, Lucio; Harris, Pamela Jo; Jeong, Woondong; Bando, Hideaki; Kahn, Michael; Yang, Sherry X.

2015-01-01

During the past decade, cancer stem cells (CSCs) have been increasingly identified in many malignancies. Although the origin and plasticity of these cells remain controversial, tumour heterogeneity and the presence of small populations of cells with stem-like characteristics is established in most malignancies. CSCs display many features of embryonic or tissue stem cells, and typically demonstrate persistent activation of one or more highly conserved signal transduction pathways involved in development and tissue homeostasis, including the Notch, Hedgehog (HH), and Wnt pathways. CSCs generally have slow growth rates and are resistant to chemotherapy and/or radiotherapy. Thus, new treatment strategies targeting these pathways to control stem-cell replication, survival and differentiation are under development. Herein, we provide an update on the latest advances in the clinical development of such approaches, and discuss strategies for overcoming CSC-associated primary or acquired resistance to cancer treatment. Given the crosstalk between the different embryonic developmental signalling pathways, as well as other pathways, designing clinical trials that target CSCs with rational combinations of agents to inhibit possible compensatory escape mechanisms could be of particular importance. We also share our views on the future directions for targeting CSCs to advance the clinical development of these classes of agents. PMID:25850553

Monoamine Oxidases Regulate Telencephalic Neural Progenitors in Late Embryonic and Early Postnatal Development

PubMed Central

Cheng, Aiwu; Scott, Anna L.; Ladenheim, Bruce; Chen, Kevin; Ouyang, Xin; Lathia, Justin D.; Mughal, Mohamed; Cadet, Jean Lud; Mattson, Mark P.; Shih, Jean C.

2010-01-01

Monoamine neurotransmitters play major roles in regulating a range of brain functions in adults and increasing evidence suggests roles for monoamines in brain development. Here we show that mice lacking the monoamine metabolic enzymes MAO A and MAO B (MAO AB-deficient mice) exhibit diminished proliferation of neural stem cells (NSC) in the developing telencephalon beginning in late gestation [embryonic day (E) 17.5], a deficit that persists in neonatal and adult mice. These mice showed significantly increased monoamine levels and anxiety-like behaviors as adults. Assessments of markers of intermediate progenitor cells (IPC) and mitosis showed that NSC in the subventricular zone (SVZ), but not in the ventricular zone, are reduced in MAO AB-deficient mice. A developmental time course of monoamines in frontal cortical tissues revealed increased serotonin levels as early as E14.5, and a further large increase was found between E17.5 and postnatal day 2. Administration of an inhibitor of serotonin synthesis (parachlorophenylalanine) between E14.5 and E19.5 restored the IPC numbers and SVZ thickness, suggesting the role of serotonin in the suppression of IPC proliferation. Studies of neurosphere cultures prepared from the telencephalon at different embryonic and postnatal ages showed that serotonin stimulates proliferation in wild-type, but not in MAO AB-deficient, NSC. Together, these results suggest that a MAO-dependent long-lasting alteration in the proliferation capacity of NSC occurs late in embryonic development and is mediated by serotonin. Our findings reveal novel roles for MAOs and serotonin in the regulation of IPC proliferation in the developing brain. PMID:20702706

Expression and activity of the 5'-AMP-activated protein kinase pathway in selected tissues during chicken embryonic development.

USDA-ARS?s Scientific Manuscript database

The 5’-AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine protein kinase and a key part of a kinase signaling cascade that senses cellular energy status (AMP/ATP ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating m...

Human embryonic stem cells and therapeutic cloning.

PubMed

Hwang, Woo Suk; Lee, Byeong Chun; Lee, Chang Kyu; Kang, Sung Keun

2005-06-01

The remarkable potential of embryonic stem (ES) cells is their ability to develop into many different cell types. ES cells make it possible to treat patients by transplanting specialized healthy cells derived from them to repair damaged and diseased cells or tissues, known as "stem cell therapy". However, the issue of immunocompatibility is one of considerable significance in ES cell transplantation. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from nuclear transfer of the patient's own cells. This concept is known as "therapeutic cloning". In this review, we describe the derivations of ES cells and cloned ES cells by somatic cell nuclear transfer, and their potential applications in transplantation medicine.

Pre-metatarsal skeletal development in tissue culture at unit- and microgravity

NASA Technical Reports Server (NTRS)

Klement, B. J.; Spooner, B. S.

1994-01-01

Explant organ culture was used to demonstrate that isolated embryonic mouse pre-metatarsal mesenchyme is capable of undergoing a series of differentiative and morphogenetic developmental events. Mesenchyme differentiation into chondrocytes, and concurrent morphogenetic patterning of the cartilage tissue, and terminal chondrocyte differentiation with subsequent matrix mineralization show that cultured tissue closely parallels in vivo development. Whole mount alizarin red staining of the cultured tissue demonstrates that the extracellular matrix around the hypertrophied chondrocytes is competent to support mineralization. Intensely stained mineralized bands are similar to those formed in pre-metatarsals developing in vivo. We have adapted the culture strategy for experimentation in a reduced gravity environment on the Space Shuttle. Spaceflight culture of pre-metatarsals, which have already initiated chondrogenesis and morphogenetic patterning, results in an increase in cartilage rod size and maintenance of rod shape, compared to controls. Older pre-metatarsal tissue, already terminally differentiated to hypertrophied cartilage, maintained rod structure and cartilage phenotype during spaceflight culture.

Identification and expression analyses of a novel serotonin receptor gene, 5-HT2β, in the field cricket, Gryllus bimaculatus.

PubMed

Watanabe, T; Aonuma, H

2012-01-01

Biogenic amine serotonin (5-HT) modulates various aspects of behaviors such as aggressive behavior and circadian behavior in the cricket. In our previous report, in order to elucidate the molecular basis of the cricket 5-HT system, we identified three genes involved in 5-HT biosynthesis, as well as four 5-HT receptor genes (5-HT1A, 5-HT1B, 5-HT2α, and 5-HT7) expressed in the brain of the field cricket Gryllus bimaculatus DeGeer [7]. In the present study, we identified Gryllus 5-HT2β gene, an additional 5-HT receptor gene expressed in the cricket brain, and examined its tissue-specific distribution and embryonic stage-dependent expression. Gryllus 5-HT2β gene was ubiquitously expressed in the all examined adult tissues, and was expressed during early embryonic development, as well as during later stages. This study suggests functional differences between two 5-HT2 receptors in the cricket.

Ras-dva Is a Novel Pit-1- and Glucocorticoid-Regulated Gene in the Embryonic Anterior Pituitary Gland

PubMed Central

Ellestad, Laura E.

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5′-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5′-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland. PMID:23161868

Ras-dva is a novel Pit-1- and glucocorticoid-regulated gene in the embryonic anterior pituitary gland.

PubMed

Ellestad, Laura E; Porter, Tom E

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5'-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5'-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo.

PubMed

Havens, Aaron M; Sun, Hongli; Shiozawa, Yusuke; Jung, Younghun; Wang, Jingcheng; Mishra, Anjali; Jiang, Yajuan; O'Neill, David W; Krebsbach, Paul H; Rodgerson, Denis O; Taichman, Russell S

2014-04-01

The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that ∼60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders.

The Gpr1/Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals

PubMed Central

Duffié, Rachel; Ajjan, Sophie; Greenberg, Maxim V.; Zamudio, Natasha; Escamilla del Arenal, Martin; Iranzo, Julian; Okamoto, Ikuhiro; Barbaux, Sandrine; Fauque, Patricia; Bourc'his, Déborah

2014-01-01

Many loci maintain parent-of-origin DNA methylation only briefly after fertilization during mammalian development: Whether this form of transient genomic imprinting can impact the early embryonic transcriptome or even have life-long consequences on genome regulation and possibly phenotypes is currently unknown. Here, we report a maternal germline differentially methylated region (DMR) at the mouse Gpr1/Zdbf2 (DBF-type zinc finger-containing protein 2) locus, which controls the paternal-specific expression of long isoforms of Zdbf2 (Liz) in the early embryo. This DMR loses parental specificity by gain of DNA methylation at implantation in the embryo but is maintained in extraembryonic tissues. As a consequence of this transient, tissue-specific maternal imprinting, Liz expression is restricted to the pluripotent embryo, extraembryonic tissues, and pluripotent male germ cells. We found that Liz potentially functions as both Zdbf2-coding RNA and cis-regulatory RNA. Importantly, Liz-mediated events allow a switch from maternal to paternal imprinted DNA methylation and from Liz to canonical Zdbf2 promoter use during embryonic differentiation, which are stably maintained through somatic life and conserved in humans. The Gpr1/Zdbf2 locus lacks classical imprinting histone modifications, but analysis of mutant embryonic stem cells reveals fine-tuned regulation of Zdbf2 dosage through DNA and H3K27 methylation interplay. Together, our work underlines the developmental and evolutionary need to ensure proper Liz/Zdbf2 dosage as a driving force for dynamic genomic imprinting at the Gpr1/Zdbf2 locus. PMID:24589776

Developmental bias in cleavage-stage mouse blastomeres

PubMed Central

Tabansky, Inna; Lenarcic, Alan; Draft, Ryan W.; Loulier, Karine; Keskin, Derin B; Rosains, Jacqueline; Rivera-Feliciano, José; Lichtman, Jeff W.; Livet, Jean; Stern, Joel NH; Sanes, Joshua R.; Eggan, Kevin

2012-01-01

Summary Introduction The cleavage stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, as preimplantation genetic diagnosis (PGD) requires removal of blastomeres from the early human embryo. To determine if blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, non-invasive combinatorial fluorescent labeling method for embryonic lineage tracing. Results When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into post-implantation stages, and therefore has relevance for subsequent development. Discussion The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues. PMID:23177476

Bioprinting of a functional vascularized mouse thyroid gland construct.

PubMed

Bulanova, Elena A; Koudan, Elizaveta V; Degosserie, Jonathan; Heymans, Charlotte; Pereira, Frederico DAS; Parfenov, Vladislav A; Sun, Yi; Wang, Qi; Akhmedova, Suraya A; Sviridova, Irina K; Sergeeva, Natalia S; Frank, Georgy A; Khesuani, Yusef D; Pierreux, Christophe E; Mironov, Vladimir A

2017-08-18

Bioprinting can be defined as additive biofabrication of three-dimensional (3D) tissues and organ constructs using tissue spheroids, capable of self-assembly, as building blocks. The thyroid gland, a relatively simple endocrine organ, is suitable for testing the proposed bioprinting technology. Here we report the bioprinting of a functional vascularized mouse thyroid gland construct from embryonic tissue spheroids as a proof of concept. Based on the self-assembly principle, we generated thyroid tissue starting from thyroid spheroids (TS) and allantoic spheroids (AS) as a source of thyrocytes and endothelial cells (EC), respectively. Inspired by mathematical modeling of spheroid fusion, we used an original 3D bioprinter to print TS in close association with AS within a collagen hydrogel. During the culture, closely placed embryonic tissue spheroids fused into a single integral construct, EC from AS invaded and vascularized TS, and epithelial cells from the TS progressively formed follicles. In this experimental setting, we observed formation of a capillary network around follicular cells, as observed during in utero thyroid development when thyroid epithelium controls the recruitment, invasion and expansion of EC around follicles. To prove that EC from AS are responsible for vascularization of the thyroid gland construct, we depleted endogenous EC from TS before bioprinting. EC from AS completely revascularized depleted thyroid tissue. The cultured bioprinted construct was functional as it could normalize blood thyroxine levels and body temperature after grafting under the kidney capsule of hypothyroid mice. Bioprinting of functional vascularized mouse thyroid gland construct represents a further advance in bioprinting technology, exploring the self-assembling properties of tissue spheroids.

Growth and differentiation of mammalian embryonic tissues exposed to hypergravity in vivo and in vitro

NASA Technical Reports Server (NTRS)

Duke, P. J.

1984-01-01

In about 10 years or so, men and women from Earth will be long-term inhabitants of a space station aboard which plants and animals will be growing and developing in gravities other than that of Earth. The effect of gravitational changes on development was examined. It is indicated that differentiation is speeded up under excess G and slowed in low or null G. The effects of exposure to excess gravity on fusion of the embryonic mouse secondary palate were studied. During fusion, the palatal shelves first adhere by means of glycoproteins appearing along the medial epithelial edge (MEE). The contacting epithelia then reorganize and undergo programmed cell death, allowing the underlying mesenchymes to come in contact. The process of cell death occurs in vitro at about the same rate that it occurs in vivo.

The role of flow in the morphodynamics of embryonic heart

NASA Astrophysics Data System (ADS)

Gharib, Morteza

2017-11-01

Nature has shown us that some hearts do not require valves to achieve unidirectional flow. In its earliest stages, the vertebrate heart consists of a primitive tube that drives blood through a simple vascular network nourishing tissues and other developing organ systems. We have shown that in the case of the embryonic zebrafish heart, an elastic wave resonance mechanism based on impedance mismatches at the boundaries of the heart tube is the likely mechanism responsible for the valveless pumping behavior. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop there is considerable regurgitation, resulting in oscillatory flow between the atrium and ventricle. We show that reversing flows are particularly strong stimuli to endothelial cells and that heart valves form as a developmental response to oscillatory blood flow through the maturing heart.

An Embryonic and Induced Pluripotent Stem Cell Model for Ovarian Granulosa Cell Development and Steroidogenesis.

PubMed

Lipskind, Shane; Lindsey, Jennifer S; Gerami-Naini, Behzad; Eaton, Jennifer L; O'Connell, Daniel; Kiezun, Adam; Ho, Joshua W K; Ng, Nicholas; Parasar, Parveen; Ng, Michelle; Nickerson, Michael; Demirci, Utkan; Maas, Richard; Anchan, Raymond M

2018-05-01

Embryoid bodies (EBs) can serve as a system for evaluating pluripotency, cellular differentiation, and tissue morphogenesis. In this study, we use EBs derived from mouse embryonic stem cells (mESCs) and human amniocyte-derived induced pluripotent stem cells (hAdiPSCs) as a model for ovarian granulosa cell (GC) development and steroidogenic cell commitment. We demonstrated that spontaneously differentiated murine EBs (mEBs) and human EBs (hEBs) displayed ovarian GC markers, such as aromatase (CYP19A1), FOXL2, AMHR2, FSHR, and GJA1. Comparative microarray analysis identified both shared and unique gene expression between mEBs and the maturing mouse ovary. Gene sets related to gonadogenesis, lipid metabolism, and ovarian development were significantly overrepresented in EBs. Of the 29 genes, 15 that were differentially regulated in steroidogenic mEBs displayed temporal expression changes between embryonic, postnatal, and mature ovarian tissues by polymerase chain reaction. Importantly, both mEBs and hEBs were capable of gonadotropin-responsive estradiol (E2) synthesis in vitro (217-759 pg/mL). Live fluorescence-activated cell sorting-sorted AMHR2 + granulosa-like cells from mEBs continued to produce E2 after purification (15.3 pg/mL) and secreted significantly more E2 than AMHR2 - cells (8.6 pg/mL, P < .05). We conclude that spontaneously differentiated EBs of both mESC and hAdiPSC origin can serve as a biologically relevant model for ovarian GC differentiation and steroidogenic cell commitment. These cells should be further investigated for therapeutic uses, such as stem cell-based hormone replacement therapy and in vitro maturation of oocytes.

The notochord: structure and functions.

PubMed

Corallo, Diana; Trapani, Valeria; Bonaldo, Paolo

2015-08-01

The notochord is an embryonic midline structure common to all members of the phylum Chordata, providing both mechanical and signaling cues to the developing embryo. In vertebrates, the notochord arises from the dorsal organizer and it is critical for proper vertebrate development. This evolutionary conserved structure located at the developing midline defines the primitive axis of embryos and represents the structural element essential for locomotion. Besides its primary structural function, the notochord is also a source of developmental signals that patterns surrounding tissues. Among the signals secreted by the notochord, Hedgehog proteins play key roles during embryogenesis. The Hedgehog signaling pathway is a central regulator of embryonic development, controlling the patterning and proliferation of a wide variety of organs. In this review, we summarize the current knowledge on notochord structure and functions, with a particular emphasis on the key developmental events that take place in vertebrates. Moreover, we discuss some genetic studies highlighting the phenotypic consequences of impaired notochord development, which enabled to understand the molecular basis of different human congenital defects and diseases.

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

Defective pulmonary innervation and autonomic imbalance in congenital diaphragmatic hernia

PubMed Central

Lath, Nikesh R.; Galambos, Csaba; Rocha, Alejandro Best; Malek, Marcus; Gittes, George K.

2012-01-01

Congenital diaphragmatic hernia (CDH) is associated with significant mortality due to lung hypoplasia and pulmonary hypertension. The role of embryonic pulmonary innervation in normal lung development and lung maldevelopment in CDH has not been defined. We hypothesize that developmental defects of intrapulmonary innervation, in particular autonomic innervation, occur in CDH. This abnormal embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH. To define patterns of pulmonary innervation in CDH, human CDH and control lung autopsy specimens were stained with the pan-neural marker S-100. To further characterize patterns of overall and autonomic pulmonary innervation during lung development in CDH, the murine nitrofen model of CDH was utilized. Immunostaining for protein gene product 9.5 (a pan-neuronal marker), tyrosine hydroxylase (a sympathetic marker), vesicular acetylcholine transporter (a parasympathetic marker), or VIP (a parasympathetic marker) was performed on lung whole mounts and analyzed via confocal microscopy and three-dimensional reconstruction. Peribronchial and perivascular neuronal staining pattern is less complex in human CDH than control lung. In mice, protein gene product 9.5 staining reveals less complex neuronal branching and decreased neural tissue in nitrofen-treated lungs from embryonic day 12.5 to 16.5 compared with controls. Furthermore, nitrofen-treated embryonic lungs exhibited altered autonomic innervation, with a relative increase in sympathetic nerve staining and a decrease in parasympathetic nerve staining compared with controls. These results suggest a primary defect in pulmonary neural developmental in CDH, resulting in less complex neural innervation and autonomic imbalance. Defective embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH. PMID:22114150

Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

PubMed Central

Fraga, Amanda; Ribeiro, Lupis; Lobato, Mariana; Santos, Vitória; Silva, José Roberto; Gomes, Helga; da Cunha Moraes, Jorge Luiz; de Souza Menezes, Jackson

2013-01-01

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen. PMID:23750237

Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

PubMed

Cases, Olivier; Perea-Gomez, Aitana; Aguiar, Diego P; Nykjaer, Anders; Amsellem, Sabine; Chandellier, Jacqueline; Umbhauer, Muriel; Cereghini, Silvia; Madsen, Mette; Collignon, Jérôme; Verroust, Pierre; Riou, Jean-François; Creuzet, Sophie E; Kozyraki, Renata

2013-06-07

Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.

MRG15 Regulates Embryonic Development and Cell Proliferation

PubMed Central

Tominaga, Kaoru; Kirtane, Bhakti; Jackson, James G.; Ikeno, Yuji; Ikeda, Takayoshi; Hawks, Christina; Smith, James R.; Matzuk, Martin M.; Pereira-Smith, Olivia M.

2005-01-01

MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15−/− embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15−/− mouse embryonic fibroblasts. The hearts of the Mrg15−/− embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15−/− embryos appeared pale, and microarray analysis revealed that α-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the α-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation. PMID:15798182

Embryonic control of epidermal cell patterning in the root and hypocotyl of Arabidopsis.

PubMed

Lin, Y; Schiefelbein, J

2001-10-01

A position-dependent pattern of epidermal cell types is produced during the development of the Arabidopsis seedling root and hypocotyl. To understand the origin and regulation of this patterning mechanism, we have examined the embryonic expression of the GLABRA2 (GL2) gene, which encodes a cell-type-specific transcription factor. Using in situ RNA hybridization and a sensitive GL2::GFP reporter, we discovered that a position-dependent pattern of GL2 expression is established within protodermal cells at the heart stage and is maintained throughout the remainder of embryogenesis. In addition, we show that an exceptional GL2 expression character and epidermal cell pattern arises during development of the root-hypocotyl junction, which represents an anatomical transition zone. Furthermore, we find that two of the genes regulating seedling epidermal patterning, TRANSPARENT TESTA GLABRA (TTG) and WEREWOLF (WER), also control the embryonic GL2 pattern, whereas the CAPRICE (CPC) and GL2 genes are not required to establish this pattern. These results indicate that position-dependent patterning of epidermal cell types begins at an early stage of embryogenesis, before formation of the apical meristems and shortly after the cellular anatomy of the protoderm and outer ground tissue layer is established. Thus, epidermal cell specification in the Arabidopsis seedling relies on the embryonic establishment of a patterning mechanism that is perpetuated postembryonically.

Tissue interactions in the induction of anterior pituitary: role of the ventral diencephalon, mesenchyme, and notochord.

PubMed

Gleiberman, A S; Fedtsova, N G; Rosenfeld, M G

1999-09-15

Rathke's pouch, the epithelial primordium of the anterior pituitary, differentiates in close topographical and functional association with the ventral diencephalon. It is still not known whether the ventral diencephalon acts as the initial inducer of pituitary development. The roles of the adjacent mesenchyme and notochord, two other tissues located in close proximity to Rathke's pouch, in this process are even less clear. In this report we describe an in vitro experimental system that reproduces the earliest steps of anterior pituitary development. We provide evidence that the ventral diencephalon from 2- to 4-day-old chick embryos is able to function as an inducer of pituitary development and can convert early chick embryonic head ectoderm, which is not involved normally in pituitary development, into typical anterior pituitary tissue. This induction is contact-dependent. In our experimental system, there is a requirement for the supporting action of mesenchyme, which is independent of the mesenchyme source. Transplantation of the notochord into the lateral head region of a six-somite chick embryo induces an epithelial invagination, suggesting that the notochord induces the outpouching of the roof of the stomodeal ectoderm that results in formation of Rathke's pouch and causes the close contact between this ectoderm and the ventral diencephalon. Finally, we demonstrate that the ventral diencephalon from e9.5-e11.5 mouse embryos is also an efficient inducer of anterior pituitary differentiation in chick embryonic lateral head ectoderm, suggesting that the mechanism of anterior pituitary induction is conserved between mammals and birds, using the same, or similar, signaling pathways. Copyright 1999 Academic Press.

Reprogrammed mouse astrocytes retain a "memory" of tissue origin and possess more tendencies for neuronal differentiation than reprogrammed mouse embryonic fibroblasts.

PubMed

Tian, Changhai; Wang, Yongxiang; Sun, Lijun; Ma, Kangmu; Zheng, Jialin C

2011-02-01

Direct reprogramming of a variety of somatic cells with the transcription factors Oct4 (also called Pou5f1), Sox2 with either Klf4 and Myc or Lin28 and Nanog generates the induced pluripotent stem cells (iPSCs) with marker similarity to embryonic stem cells. However, the difference between iPSCs derived from different origins is unclear. In this study, we hypothesized that reprogrammed cells retain a "memory" of their origins and possess additional potential of related tissue differentiation. We reprogrammed primary mouse astrocytes via ectopic retroviral expression of OCT3/4, Sox2, Klf4 and Myc and found the iPSCs from mouse astrocytes expressed stem cell markers and formed teratomas in SCID mice containing derivatives of all three germ layers similar to mouse embryonic stem cells besides semblable morphologies. To test our hypothesis, we compared embryonic bodies (EBs) formation and neuronal differentiation between iPSCs from mouse embryonic fibroblasts (MEFsiPSCs) and iPSCs from mouse astrocytes (mAsiPSCs). We found that mAsiPSCs grew slower and possessed more potential for neuronal differentiation compared to MEFsiPSCs. Our results suggest that mAsiPSCs retain a "memory" of the central nervous system, which confers additional potential upon neuronal differentiation.

The Sox17CreERT2 knock-in mouse line displays spatiotemporal activation of Cre recombinase in distinct Sox17 lineage progenitors.

PubMed

Engert, Silvia; Burtscher, Ingo; Kalali, Behnam; Gerhard, Markus; Lickert, Heiko

2013-11-01

The HMG-box transcription factor Sox17 is essential for endoderm formation, vascular development, and definitive hematopoiesis. To investigate the fate of distinct Sox17-expressing progenitor cells in a spatiotemporal manner, we generated a hormone-inducible CreERT2 knock-in mouse line. By homologous recombination we fused a codon improved, ligand-dependent estrogen receptor Cre recombinase by an intervening viral T2A sequence for co-translational cleavage to the 3' coding region of Sox17. Induction of Cre activity by administration of tamoxifen at defined time points of early mouse development and subsequent genetic lineage tracing confirmed the inducibility and tissue specificity of Cre recombination. Furthermore, Cre activity could be selectively induced in extra-embryonic and embryonic endoderm lineages, the primitive gut tube, and in endothelial cells of the vascular system as well as in the hemogenic endothelium of the dorsal aorta. The Sox17CreERT2 mouse line therefore represents a new tool for genetic lineage tracing in a tissue-specific manner and in addition enables lineage-restricted functional analysis. Copyright © 2013 Wiley Periodicals, Inc.

The ontogeny of allorecognition in a colonial hydroid and the fate of early established chimeras.

PubMed

Fuchs, Marc-Aurel; Mokady, Ofer; Frank, Uri

2002-08-01

Colonies of the marine hydroid, Hydractinia, are able to discriminate between their own tissues and those belonging to unrelated conspecifics. We have studied the ontogeny of this allorecognition system by a series of allogeneic transplantations along a developmental gradient, including two-cell-stage embryos, 8 h morulae, planula larvae and metamorphosed polyps. Allograft acceptance of incompatible tissue was observed in all embryonic and larval stages, whereas metamorphosed polyps rejected incompatible transplanted allografts. Most of the chimeras established at the two-cell-stage, although composed of two allogeneic, incompatible entities with mismatching allorecognition loci, developed normally and remained stable through metamorphosis. The results of post metamorphic transplantation assays among the chimeras and the naive ramets, suggested that both incompatible genotypes were still represented in the chimera despite the onset of alloimmune maturation. The naive colonies always rejected each other. Chimeras established from later embryonic and larval stages did not develop into adult chimeric entities, but rather separated immediately post metamorphosis. We thus show that (1) allorecognition in this species matures during metamorphosis and (2) genetically incompatible entities may coexist in one immunologically mature, chimeric soma, provided that they were grafted early enough in ontogeny.

Eyeing the Cyr61/CTGF/NOV (CCN) group of genes in development and diseases: highlights of their structural likenesses and functional dissimilarities.

PubMed

Krupska, Izabela; Bruford, Elspeth A; Chaqour, Brahim

2015-09-23

"CCN" is an acronym referring to the first letter of each of the first three members of this original group of mammalian functionally and phylogenetically distinct extracellular matrix (ECM) proteins [i.e., cysteine-rich 61 (CYR61), connective tissue growth factor (CTGF), and nephroblastoma-overexpressed (NOV)]. Although "CCN" genes are unlikely to have arisen from a common ancestral gene, their encoded proteins share multimodular structures in which most cysteine residues are strictly conserved in their positions within several structural motifs. The CCN genes can be subdivided into members developmentally indispensable for embryonic viability (e.g., CCN1, 2 and 5), each assuming unique tissue-specific functions, and members not essential for embryonic development (e.g., CCN3, 4 and 6), probably due to a balance of functional redundancy and specialization during evolution. The temporo-spatial regulation of the CCN genes and the structural information contained within the sequences of their encoded proteins reflect diversity in their context and tissue-specific functions. Genetic association studies and experimental anomalies, replicated in various animal models, have shown that altered CCN gene structure or expression is associated with "injury" stimuli--whether mechanical (e.g., trauma, shear stress) or chemical (e.g., ischemia, hyperglycemia, hyperlipidemia, inflammation). Consequently, increased organ-specific susceptibility to structural damages ensues. These data underscore the critical functions of CCN proteins in the dynamics of tissue repair and regeneration and in the compensatory responses preceding organ failure. A better understanding of the regulation and mode of action of each CCN member will be useful in developing specific gain- or loss-of-function strategies for therapeutic purposes.

Subcutaneous transplantation of embryonic pancreas for correction of type 1 diabetes

PubMed Central

Gunawardana, Subhadra C.; Benninger, Richard K. P.; Piston, David W.

2009-01-01

Islet transplantation is a promising therapeutic approach for type 1 diabetes. However, current success rates are low due to progressive graft failure in the long term and inability to monitor graft development in vivo. Other limitations include the necessity of initial invasive surgery and continued immunosuppressive therapy. We report an alternative transplantation strategy with the potential to overcome these problems. This technique involves transplantation of embryonic pancreatic tissue into recipients’ subcutaneous space, eliminating the need for invasive surgery and associated risks. Current results in mouse models of type 1 diabetes show that embryonic pancreatic transplants in the subcutaneous space can normalize blood glucose homeostasis and achieve extensive endocrine differentiation and vascularization. Furthermore, modern imaging techniques such as two-photon excitation microscopy (TPEM) can be employed to monitor transplants through the intact skin in a completely noninvasive manner. Thus, this strategy is a convenient alternative to islet transplantation in diabetic mice and has the potential to be translated to human clinical applications with appropriate modifications. PMID:19066321

CBX7 gene expression plays a negative role in adipocyte cell growth and differentiation

PubMed Central

Forzati, Floriana; Federico, Antonella; Pallante, Pierlorenzo; Colamaio, Marianna; Esposito, Francesco; Sepe, Romina; Gargiulo, Sara; Luciano, Antonio; Arra, Claudio; Palma, Giuseppe; Bon, Giulia; Bucher, Stefania; Falcioni, Rita; Brunetti, Arturo; Battista, Sabrina; Fedele, Monica; Fusco, Alfredo

2014-01-01

ABSTRACT We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation. PMID:25190058

Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency.

PubMed

Polesskaya, Anna; Cuvellier, Sylvain; Naguibneva, Irina; Duquet, Arnaud; Moss, Eric G; Harel-Bellan, Annick

2007-05-01

Lin-28 is a highly conserved, RNA-binding, microRNA-regulated protein that is involved in regulation of developmental timing in Caenorhabditis elegans. In mammals, Lin-28 is stage-specifically expressed in embryonic muscle, neurons, and epithelia, as well as in embryonic carcinoma cells, but is suppressed in most adult tissues, with the notable exception of skeletal and cardiac muscle. The specific function and mechanism of action of Lin-28 are not well understood. Here we used loss-of-function and gain-of-function assays in cultured myoblasts to show that expression of Lin-28 is essential for skeletal muscle differentiation in mice. In order to elucidate the specific function of Lin-28, we used a combination of biochemical and functional assays, which revealed that, in differentiating myoblasts, Lin-28 binds to the polysomes and increases the efficiency of protein synthesis. An important target of Lin-28 is IGF-2, a crucial growth and differentiation factor for muscle tissue. Interaction of Lin-28 with translation initiation complexes in skeletal myoblasts and in the embryonic carcinoma cell line P19 was confirmed by localization of Lin-28 to the stress granules, temporary structures that contain stalled mRNA-protein translation complexes. Our results unravel novel mechanisms of translational regulation in skeletal muscle and suggest that Lin-28 performs the role of "translational enhancer" in embryonic and adult cells and tissues.

Developments in medical law in the United Kingdom in 2005 and 2006.

PubMed

Gertz, Renate; Harmon, Shawn; Laurie, Graeme; Pradella, Geoff

2006-06-01

This article highlights and summarises the key developments in medical law in the jurisdictions of the United Kingdom in 2005 and to April 2006. Topics are mental health and mental capacity, data protection, freedom of information and the impact on health data, the Human Tissue Act, genetic research databanks, Human Fertilisation and Embryology Act--Review of the legislation, consultations and related case law, developments in embryo and embryonic stem cell research, clinical trials and human subject research, medical futility, and physician assisted dying.

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry.

PubMed

Epshtein, Alona; Sakhneny, Lina; Landsman, Limor

2017-01-28

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

In vivo quantification of spatially-varying mechanical properties in developing tissues

PubMed Central

Serwane, Friedhelm; Mongera, Alessandro; Rowghanian, Payam; Kealhofer, David A.; Lucio, Adam A.; Hockenbery, Zachary M.; Campàs, Otger

2017-01-01

It is generally believed that the mechanical properties of the cellular microenvironment and their spatiotemporal variations play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has been performed yet. Here we introduce a technique that employs biocompatible ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo. Using this technique, we show that vertebrate body elongation entails spatially-varying tissue mechanics along the anteroposterior axis. Specifically, we find that the zebrafish tailbud is viscoelastic (elastic below a few seconds and fluid after just one minute) and displays decreasing stiffness and increasing fluidity towards its posterior elongating region. This method opens new avenues to study mechanobiology in vivo, both in embryogenesis and in disease processes, including cancer. PMID:27918540

Acute alcohol exposure during mouse gastrulation alters lipid metabolism in placental and heart development: Folate prevention

PubMed Central

Han, Mingda

2016-01-01

Background Embryonic acute exposure to ethanol (EtOH), lithium, and homocysteine (HCy) induces cardiac defects at the time of exposure; folic acid (FA) supplementation protects normal cardiogenesis (Han et al., 2009, 2012; Serrano et al., 2010). Our hypothesis is that EtOH exposure and FA protection relate to lipid and FA metabolism during mouse cardiogenesis and placentation. Methods On the morning of conception, pregnant C57BL/6J mice were placed on either of two FA‐containing diets: a 3.3 mg health maintenance diet or a high FA diet of 10.5 mg/kg. Mice were injected a binge level of EtOH, HCy, or saline on embryonic day (E) 6.75, targeting gastrulation. On E15.5, cardiac and umbilical blood flow were examined by ultrasound. Embryonic cardiac tissues were processed for gene expression of lipid and FA metabolism; the placenta and heart tissues for neutral lipid droplets, or for medium chain acyl‐dehydrogenase (MCAD) protein. Results EtOH exposure altered lipid‐related gene expression on E7.5 in comparison to control or FA‐supplemented groups and remained altered on E15.5 similarly to changes with HCy, signifying FA deficiency. In comparison to control tissues, the lipid‐related acyl CoA dehydrogenase medium length chain gene and its protein MCAD were altered with EtOH exposure, as were neutral lipid droplet localization in the heart and placenta. Conclusion EtOH altered gene expression associated with lipid and folate metabolism, as well as neutral lipids, in the E15.5 abnormally functioning heart and placenta. In comparison to controls, the high FA diet protected the embryo and placenta from these effects allowing normal development. Birth Defects Research (Part A) 106:749–760, 2016. © 2016 The Authors Birth Defects Research Part A: Clinical and Molecular Teratology Published by Wiley Periodicals, Inc. PMID:27296863

New gene targets for glucagon-like peptide-1 during embryonic development and in undifferentiated pluripotent cells.

PubMed

Sanz, Carmen; Blázquez, Enrique

2011-09-01

In humans, glucagon-like peptide (GLP-1) functions during adult life as an incretin hormone with anorexigenic and antidiabetogenic properties. Also, the therapeutic potential of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and in bolstering the maintenance of human mesenchymal stem cell (hMSC) stores by promoting the proliferation and cytoprotection of hMSC seems to be relevant. Since these observations suggest a role for GLP-1 during developmental processes, the aim of the present work was to characterize GLP-1 in early development as well as its gene targets in mouse embryonic stem (mES) cells. Mouse embryos E6, E8, and E10.5 and pluripotent mES were used for the inmunodetection of GLP-1 and GLP-1 receptor. Quantitative real-time PCR was used to determine the expression levels of GLP-1R in several tissues from E12.5 mouse embryos. Additionally, GLP-1 gene targets were studied in mES by multiple gene expression analyses. GLP-1 and its receptors were identified in mES and during embryonic development. In pluripotent mES, GLP-1 modified the expression of endodermal, ectodermal, and mesodermal gene markers as well as sonic hedgehog, noggin, members of the fibroblast and hepatic growth factor families, and others involved in pancreatic development. Additionally, GLP-1 promoted the expression of the antiapoptotic gene bcl2 and at the same time reduced proapoptotic caspase genes. Our results indicate that apart from the effects and therapeutic benefits of GLP-1 in adulthood, it may have additional gene targets in mES cells during embryonic life. Furthermore, the pathophysiological implications of GLP-1 imbalance in adulthood may have a counterpart during development.

Local and long-range endogenous resting potential gradients antagonistically regulate apoptosis and proliferation in the embryonic CNS.

PubMed

Pai, Vaibhav P; Lemire, Joan M; Chen, Ying; Lin, Gufa; Levin, Michael

2015-01-01

Bioelectric signals, particularly transmembrane voltage potentials (Vmem), play an important role in large-scale patterning during embryonic development. Endogenous bioelectric gradients across tissues function as instructive factors during eye, brain, and other morphogenetic processes. An important and still poorly-understood aspect is the control of cell behaviors by the voltage states of distant cell groups. Here, experimental alteration of endogenous Vmem was induced in Xenopus laevis embryos by misexpression of well-characterized ion channel mRNAs, a strategy often used to identify functional roles of Vmem gradients during embryonic development and regeneration. Immunofluorescence analysis (for activated caspase 3 and phosphor-histone H3P) on embryonic sections was used to characterize apoptosis and proliferation. Disrupting local bioelectric signals (within the developing neural tube region) increased caspase 3 and decreased H3P in the brain, resulting in brain mispatterning. Disrupting remote (ventral, non-neural region) bioelectric signals decreased caspase 3 and highly increased H3P within the brain, with normal brain patterning. Disrupting both the local and distant bioelectric signals produced antagonistic effects on caspase 3 and H3P. Thus, two components of bioelectric signals regulate apoptosis-proliferation balance within the developing brain and spinal cord: local (developing neural tube region) and distant (ventral non-neural region). Together, the local and long-range bioelectric signals create a binary control system capable of fine-tuning apoptosis and proliferation with the brain and spinal cord to achieve correct pattern and size control. Our data suggest a roadmap for utilizing bioelectric state as a diagnostic modality and convenient intervention parameter for birth defects and degenerative disease states of the CNS.

Maternal Diabetes Leads to Adaptation in Embryonic Amino Acid Metabolism during Early Pregnancy.

PubMed

Gürke, Jacqueline; Hirche, Frank; Thieme, René; Haucke, Elisa; Schindler, Maria; Stangl, Gabriele I; Fischer, Bernd; Navarrete Santos, Anne

2015-01-01

During pregnancy an adequate amino acid supply is essential for embryo development and fetal growth. We have studied amino acid composition and branched chain amino acid (BCAA) metabolism at day 6 p.c. in diabetic rabbits and blastocysts. In the plasma of diabetic rabbits the concentrations of 12 amino acids were altered in comparison to the controls. Notably, the concentrations of the BCAA leucine, isoleucine and valine were approximately three-fold higher in diabetic rabbits than in the control. In the cavity fluid of blastocysts from diabetic rabbits BCAA concentrations were twice as high as those from controls, indicating a close link between maternal diabetes and embryonic BCAA metabolism. The expression of BCAA oxidizing enzymes and BCAA transporter was analysed in maternal tissues and in blastocysts. The RNA amounts of three oxidizing enzymes, i.e. branched chain aminotransferase 2 (Bcat2), branched chain ketoacid dehydrogenase (Bckdha) and dehydrolipoyl dehydrogenase (Dld), were markedly increased in maternal adipose tissue and decreased in liver and skeletal muscle of diabetic rabbits than in those of controls. Blastocysts of diabetic rabbits revealed a higher Bcat2 mRNA and protein abundance in comparison to control blastocysts. The expression of BCAA transporter LAT1 and LAT2 were unaltered in endometrium of diabetic and healthy rabbits, whereas LAT2 transcripts were increased in blastocysts of diabetic rabbits. In correlation to high embryonic BCAA levels the phosphorylation amount of the nutrient sensor mammalian target of rapamycin (mTOR) was enhanced in blastocysts caused by maternal diabetes. These results demonstrate a direct impact of maternal diabetes on BCAA concentrations and degradation in mammalian blastocysts with influence on embryonic mTOR signalling.

Maternal Diabetes Leads to Adaptation in Embryonic Amino Acid Metabolism during Early Pregnancy

PubMed Central

Gürke, Jacqueline; Hirche, Frank; Thieme, René; Haucke, Elisa; Schindler, Maria; Stangl, Gabriele I.; Fischer, Bernd; Navarrete Santos, Anne

2015-01-01

During pregnancy an adequate amino acid supply is essential for embryo development and fetal growth. We have studied amino acid composition and branched chain amino acid (BCAA) metabolism at day 6 p.c. in diabetic rabbits and blastocysts. In the plasma of diabetic rabbits the concentrations of 12 amino acids were altered in comparison to the controls. Notably, the concentrations of the BCAA leucine, isoleucine and valine were approximately three-fold higher in diabetic rabbits than in the control. In the cavity fluid of blastocysts from diabetic rabbits BCAA concentrations were twice as high as those from controls, indicating a close link between maternal diabetes and embryonic BCAA metabolism. The expression of BCAA oxidizing enzymes and BCAA transporter was analysed in maternal tissues and in blastocysts. The RNA amounts of three oxidizing enzymes, i.e. branched chain aminotransferase 2 (Bcat2), branched chain ketoacid dehydrogenase (Bckdha) and dehydrolipoyl dehydrogenase (Dld), were markedly increased in maternal adipose tissue and decreased in liver and skeletal muscle of diabetic rabbits than in those of controls. Blastocysts of diabetic rabbits revealed a higher Bcat2 mRNA and protein abundance in comparison to control blastocysts. The expression of BCAA transporter LAT1 and LAT2 were unaltered in endometrium of diabetic and healthy rabbits, whereas LAT2 transcripts were increased in blastocysts of diabetic rabbits. In correlation to high embryonic BCAA levels the phosphorylation amount of the nutrient sensor mammalian target of rapamycin (mTOR) was enhanced in blastocysts caused by maternal diabetes. These results demonstrate a direct impact of maternal diabetes on BCAA concentrations and degradation in mammalian blastocysts with influence on embryonic mTOR signalling. PMID:26020623

Establishment and Characterization of an Embryonic Cell Line from Sarconesiopsis magellanica

PubMed Central

Cruz, Mónica; Bello, Felio J.

2013-01-01

Sarconesiopsis magellanica (Le Guillou) (Diptera: Calliphoridae) is a necrophagous fly that is important in both human and veterinary medicines. This insect has been registered in Colombia as a biological indicator in estimating post-mortem interval. Insect cell cultures are an important biotechnological tool for basic and applied studies, and cell cultures derived from S. magellanica embryonic tissues are described in this study. S. magellanica embryonated eggs were taken for tissue explants. These were seeded in L-15, Grace/L-15, Eagle MEM, MM, VP12, MM/VP12, and Schneider culture media. The morphological, cytogenetic, biochemical, and molecular characteristics of the cell cultures were examined. Cell growth was achieved in the L15, Grace/L15, and Schneider culture media, and the confluent monolayers were obtained 8, 10, and 19 days after the embryonated eggs were explanted. However, the Schneider medium was the most efficient to develop the subcultures, and 21 passages have been maintained. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer and in the subcultures there was greater cell morphology uniformity, fibroblastoid types being predominant. Cultured cells had a chromosomal number of 12, and the karyotypic complement consisted of five pairs of somatic chromosomes and one sexual pair. The cell culture isozyme patterns of S. magellanica coincided with adult samples from the same species. The molecular analysis, using RAPD-PCR, demonstrated the authentication of the cell cultures of this fly and their differentiation from other cultures derived from two sand flies species. This cell line is a new in vitro model that will be used in biomedical and biotechnological studies. PMID:24766352

Immuno- and gene expression analysis of EGFR and Nestin during mice skin development.

PubMed

Falodah, Fawaz Adnan; Al-Karim, Saleh

2016-06-01

Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. The main objectives of the present study were to identify the precise anatomical localization of stem cells in mice during skin developing; and to determine the expression levels by using immuno- and gene expression analysis. In this comparative cross sectional study, six ages been chosen and divided into: embryonic days (E12.5, E14.5 and E19.5) and litter days (L7, L14 and L19). Skin were removed from the back side and processed to assess both immuno- and gene-expression of EGFR and Nestin surface antigen markers. Data of the different studied age groups was compared using the SPSS software. EGFR was mainly expressed in the outer root sheath (ORS), in basal and, to a lesser extent, in suprabasal keratinocytes and tend to lie where the dermis comes closest to the skin surface, while Nestin expressed throughout the dermis in the early embryo, but it is subsequently restricted to the follicular connective tissue sheaths later in development and to hair follicles after birth. Immunoexpression analysis showed a strong EGFR expression in all group ages except E12.5 which recorded as moderate, while Nestin showed strong expression level for all embryonic stages, while in the litters it was moderate. The qRT-PCR results were consistent with those of the immunohistochemical study. The Pearson correlation analyze present a correlation between the cases of study with age (p≤0.01), which indicated to the effect of age to mice development. EGFR and Nestin showed to have vital role during mice development, and considered to be suitable markers for the study of skin stem cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell-accurate optical mapping across the entire developing heart.

PubMed

Weber, Michael; Scherf, Nico; Meyer, Alexander M; Panáková, Daniela; Kohl, Peter; Huisken, Jan

2017-12-29

Organogenesis depends on orchestrated interactions between individual cells and morphogenetically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighboring cells, which is established and fine-tuned during embryonic development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca 2+ -mapping of all cells in the entire electro-mechanically uncoupled heart during the looping stage of live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs.

Cell-accurate optical mapping across the entire developing heart

PubMed Central

Meyer, Alexander M; Panáková, Daniela; Kohl, Peter

2017-01-01

Organogenesis depends on orchestrated interactions between individual cells and morphogenetically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighboring cells, which is established and fine-tuned during embryonic development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca2+-mapping of all cells in the entire electro-mechanically uncoupled heart during the looping stage of live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs. PMID:29286002

Identification and expression analysis of zebrafish glypicans during embryonic development.

PubMed

Gupta, Mansi; Brand, Michael

2013-01-01

Heparan sulfate Proteoglycans (HSPG) are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG's, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.

Gallus gallus orthologous to human alpha-dystroglycanopathies candidate genes: Gene expression and characterization during chicken embryogenesis.

PubMed

Izquierdo-Lahuerta, Adriana; de Luis, Oscar; Gómez-Esquer, Francisco; Cruces, Jesús; Coloma, Antonio

2016-09-23

Alpha-dystroglycanopathies are a heterogenic group of human rare diseases that have in common defects of α-dystroglycan O-glycosylation. These congenital disorders share common features as muscular dystrophy, malformations on central nervous system and more rarely altered ocular development, as well as mutations on a set of candidate genes involved on those syndromes. Severity of the syndromes is variable, appearing Walker-Warburg as the most severe where mutations at protein O-mannosyl transferases POMT1 and POMT2 genes are frequently described. When studying the lack of MmPomt1 in mouse embryonic development, as a murine model of Walker-Warburg syndrome, MmPomt1 null phenotype was lethal because Reitchert's membrane fails during embryonic development. Here, we report gene expression from Gallus gallus orthologous genes to human candidates on alpha-dystroglycanopathies POMT1, POMT2, POMGnT1, FKTN, FKRP and LARGE, making special emphasis in expression and localization of GgPomt1. Results obtained by quantitative RT-PCR, western-blot and immunochemistry revealed close gene expression patterns among human and chicken at key tissues affected during development when suffering an alpha-dystroglycanopathy, leading us to stand chicken as a useful animal model for molecular characterization of glycosyltransferases involved in the O-glycosylation of α-Dystroglycan and its role in embryonic development. Copyright © 2016 Elsevier Inc. All rights reserved.

A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing.

PubMed

Zhou, Ruoji; Xu, An; Wang, Donghui; Zhu, Dandan; Mata, Helen; Huo, Zijun; Tu, Jian; Liu, Mo; Mohamed, Alaa M T; Jewell, Brittany E; Gingold, Julian; Xia, Weiya; Rao, Pulivarthi H; Hung, Mien-Chie; Zhao, Ruiying; Lee, Dung-Fang

2018-03-01

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancers. Many hot-spot mutations of TP53 confer novel functions not found in wild-type p53 and contribute to tumor development and progression. We report on the generation of a H1 human embryonic stem cell line carrying a homozygous TP53 R282W mutation using TALEN-mediated genome editing. The generated cell line demonstrates normal karyotype, maintains a pluripotent state, and is capable of generating a teratoma in vivo containing tissues from all three germ layers. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Induced Pluripotent Stem Cells in Dermatology: Potentials, Advances, and Limitations

PubMed Central

Bilousova, Ganna; Roop, Dennis R.

2014-01-01

The discovery of methods for reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) has raised the possibility of producing truly personalized treatment options for numerous diseases. Similar to embryonic stem cells (ESCs), iPSCs can give rise to any cell type in the body and are amenable to genetic correction by homologous recombination. These ESC properties of iPSCs allow for the development of permanent corrective therapies for many currently incurable disorders, including inherited skin diseases, without using embryonic tissues or oocytes. Here, we review recent progress and limitations of iPSC research with a focus on clinical applications of iPSCs and using iPSCs to model human diseases for drug discovery in the field of dermatology. PMID:25368014

Molecular preservation in Late Cretaceous sauropod dinosaur eggshells.

PubMed

Schweitzer, M H; Chiappe, L; Garrido, A C; Lowenstein, J M; Pincus, S H

2005-04-22

Exceptionally preserved sauropod eggshells discovered in Upper Cretaceous (Campanian) deposits in Patagonia, Argentina, contain skeletal remains and soft tissues of embryonic Titanosaurid dinosaurs. To preserve these labile embryonic remains, the rate of mineral precipitation must have superseded post-mortem degradative processes, resulting in virtually instantaneous mineralization of soft tissues. If so, mineralization may also have been rapid enough to retain fragments of original biomolecules in these specimens. To investigate preservation of biomolecular compounds in these well-preserved sauropod dinosaur eggshells, we applied multiple analytical techniques. Results demonstrate organic compounds and antigenic structures similar to those found in extant eggshells.

Molecular preservation in Late Cretaceous sauropod dinosaur eggshells

PubMed Central

Schweitzer, M.H; Chiappe, L; Garrido, A.C; Lowenstein, J.M; Pincus, S.H

2005-01-01

Exceptionally preserved sauropod eggshells discovered in Upper Cretaceous (Campanian) deposits in Patagonia, Argentina, contain skeletal remains and soft tissues of embryonic Titanosaurid dinosaurs. To preserve these labile embryonic remains, the rate of mineral precipitation must have superseded post-mortem degradative processes, resulting in virtually instantaneous mineralization of soft tissues. If so, mineralization may also have been rapid enough to retain fragments of original biomolecules in these specimens. To investigate preservation of biomolecular compounds in these well-preserved sauropod dinosaur eggshells, we applied multiple analytical techniques. Results demonstrate organic compounds and antigenic structures similar to those found in extant eggshells. PMID:15888409

Electrophoretic separation and analysis of living cells from solid tissues by several methods - Human embryonic kidney cell cultures as a model

NASA Technical Reports Server (NTRS)

Todd, Paul; Plank, Lindsay D.; Kunze, M. Elaine; Lewis, Marian L.; Morrison, Dennis R.

1986-01-01

The use of free-fluid electrophoresis methods to separate tissue cells having a specific function is discussed. It is shown that cells suspended by trypsinization from cultures of human embryonic kidney are electrophoretically heterogeneous and tolerate a wide range of electrophoresis buffers and conditions without significant attenuation of function. Moreover, these cells do not separate electrophoretically on the basis of size or cell position alone and can be separated according to their ability to give rise to progeny that produce specific plasminogen activators.

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

Retinoic acid signaling regulates Krt5 and Krt14 independently of stem cell markers in submandibular salivary gland epithelium.

PubMed

Abashev, Timur M; Metzler, Melissa A; Wright, Diana M; Sandell, Lisa L

2017-02-01

Retinoic acid (RA), the active metabolite of vitamin A, has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA regulation have not been identified. Here, we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and nonneuronal mesenchyme. By culturing epithelium explants in isolation from other tissues, we demonstrate that RA influences epithelium morphogenesis by direct action in that tissue. Moreover, we demonstrate that inhibition of RA signaling represses cell proliferation and expression of FGF10 signaling targets, and upregulates expression of basal epithelial keratins Krt5 and Krt14. Importantly, we show that the stem cell gene Kit is regulated inversely from Krt5/Krt14 by RA signaling. RA regulates Krt5 and Krt14 expression independently of stem cell character in developing salivary epithelium. RA, or chemical inhibitors of RA signaling, could potentially be used for modulating growth and differentiation of epithelial stem cells for the purpose of re-populating damaged glands or generating bioengineered organs. Developmental Dynamics 246:135-147, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Morphogenetic Implications of Peristalsis-Driven Fluid Flow in the Embryonic Lung

PubMed Central

Bokka, Kishore K.; Jesudason, Edwin C.; Lozoya, Oswaldo A.; Guilak, Farshid; Warburton, David; Lubkin, Sharon R.

2015-01-01

Epithelial organs are almost universally secretory. The lung secretes mucus of extremely variable consistency. In the early prenatal period, the secretions are of largely unknown composition, consistency, and flow rates. In addition to net outflow from secretion, the embryonic lung exhibits transient reversing flows from peristalsis. Airway peristalsis (AP) begins as soon as the smooth muscle forms, and persists until birth. Since the prenatal lung is liquid-filled, smooth muscle action can transport fluid far from the immediately adjacent tissues. The sensation of internal fluid flows has been shown to have potent morphogenetic effects, as has the transport of morphogens. We hypothesize that these effects play an important role in lung morphogenesis. To test these hypotheses in a quantitative framework, we analyzed the fluid-structure interactions between embryonic tissues and lumen fluid resulting from peristaltic waves that partially occlude the airway. We found that if the airway is closed, fluid transport is minimal; by contrast, if the trachea is open, shear rates can be very high, particularly at the stenosis. We performed a parametric analysis of flow characteristics' dependence on tissue stiffnesses, smooth muscle force, geometry, and fluid viscosity, and found that most of these relationships are governed by simple ratios. We measured the viscosity of prenatal lung fluid with passive bead microrheology. This paper reports the first measurements of the viscosity of embryonic lung lumen fluid. In the range tested, lumen fluid can be considered Newtonian, with a viscosity of 0.016 ± 0.008 Pa-s. We analyzed the interaction between the internal flows and diffusion and conclude that AP has a strong effect on flow sensing away from the tip and on transport of morphogens. These effects may be the intermediate mechanisms for the enhancement of branching seen in occluded embryonic lungs. PMID:26147967

Towards a global human embryonic stem cell bank.

PubMed

Lott, Jason P; Savulescu, Julian

2007-08-01

An increasingly unbridgeable gap exists between the supply and demand of transplantable organs. Human embryonic stem cell technology could solve the organ shortage problem by restoring diseased or damaged tissue across a range of common conditions. However, such technology faces several largely ignored immunological challenges in delivering cell lines to large populations. We address some of these challenges and argue in favor of encouraging contribution or intentional creation of embryos from which widely immunocompatible stem cell lines could be derived. Further, we argue that current immunological constraints in tissue transplantation demand the creation of a global stem cell bank, which may hold particular promise for minority populations and other sub-groups currently marginalized from organ procurement and allocation systems. Finally, we conclude by offering a number of practical and ethically oriented recommendations for constructing a human embryonic stem cell bank that we hope will help solve the ongoing organ shortage problem.

Collective cell migration in development

PubMed Central

Scarpa, Elena

2016-01-01

During embryonic development, tissues undergo major rearrangements that lead to germ layer positioning, patterning, and organ morphogenesis. Often these morphogenetic movements are accomplished by the coordinated and cooperative migration of the constituent cells, referred to as collective cell migration. The molecular and biomechanical mechanisms underlying collective migration of developing tissues have been investigated in a variety of models, including border cell migration, tracheal branching, blood vessel sprouting, and the migration of the lateral line primordium, neural crest cells, or head mesendoderm. Here we review recent advances in understanding collective migration in these developmental models, focusing on the interaction between cells and guidance cues presented by the microenvironment and on the role of cell–cell adhesion in mechanical and behavioral coupling of cells within the collective. PMID:26783298

Mouse androgenetic embryonic stem cells differentiated to multiple cell lineages in three embryonic germ layers in vitro.

PubMed

Teramura, Takeshi; Onodera, Yuta; Murakami, Hideki; Ito, Syunsuke; Mihara, Toshihiro; Takehara, Toshiyuki; Kato, Hiromi; Mitani, Tasuku; Anzai, Masayuki; Matsumoto, Kazuya; Saeki, Kazuhiro; Fukuda, Kanji; Sagawa, Norimasa; Osoi, Yoshihiko

2009-06-01

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.

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

NASA Astrophysics Data System (ADS)

Sato, Yuki; Takahashi, Yoshiko

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

Pteropod eggs released at high pCO2 lack resilience to ocean acidification

NASA Astrophysics Data System (ADS)

Manno, Clara; Peck, Victoria L.; Tarling, Geraint A.

2016-05-01

The effects of ocean acidification (OA) on the early recruitment of pteropods in the Scotia Sea, was investigated considering the process of spawning, quality of the spawned eggs and their capacity to develop. Maternal OA stress was induced on female pteropods (Limacina helicina antarctica) through exposure to present day pCO2 conditions and two potential future OA states (750 μatm and 1200 μatm). The eggs spawned from these females, both before and during their exposure to OA, were incubated themselves in this same range of conditions (embryonic OA stress). Maternal OA stress resulted in eggs with lower carbon content, while embryonic OA stress retarded development. The combination of maternal and embryonic OA stress reduced the percentage of eggs successfully reaching organogenesis by 80%. We propose that OA stress not only affects the somatic tissue of pteropods but also the functioning of their gonads. Corresponding in-situ sampling found that post-larval L. helicina antarctica concentrated around 600 m depth, which is deeper than previously assumed. A deeper distribution makes their exposure to waters undersaturated for aragonite more likely in the near future given that these waters are predicted to shoal from depth over the coming decades.

Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

PubMed

Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W; Burt, David W; Kaiser, Pete; Hume, David A; Sang, Helen M

2014-08-01

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. © 2014. Published by The Company of Biologists Ltd.

Efficient electroporation of DNA and protein into confluent and differentiated epithelial cells in culture.

PubMed

Deora, Ami A; Diaz, Fernando; Schreiner, Ryan; Rodriguez-Boulan, Enrique

2007-10-01

Electroporation-mediated delivery of molecules is a procedure widely used for transfecting complementary DNA in bacteria, mammalian and plant cells. This technique has proven very efficient for the introduction of macromolecules into cells in suspension culture and even into cells in their native tissue environment, e.g. retina and embryonic tissues. However, in spite of several attempts to date, there are no well-established procedures to electroporate polarized epithelial cells adhering to a tissue culture substrate (glass, plastic or filter). We report here the development of a simple procedure that uses available commercial equipment and works efficiently and reproducibly for a variety of epithelial cell lines in culture.

Proteomic Profile of Mabuya sp. (Squamata: Scincidae) Ovary and Placenta During Gestation.

PubMed

Hernández-Díaz, Nathaly; Torres, Rodrigo; Ramírez-Pinilla, Martha Patricia

2017-06-01

Reptiles are one of the most diverse groups of vertebrates, providing an integrated system for comparative studies on metabolic, animal physiology, and developmental biology. However, the molecular data available are limited and only recently have started to call attention in the "omics" sciences. Mabuya sp. is a viviparous placentrotrophic skink with particular reproductive features, including microlecithal eggs, early luteolysis, prolonged gestation, and development of a highly specialized placenta. This placenta is responsible for respiratory exchange and the transference of all nutrients necessary for embryonic development. Our aim was to identify differentially expressed proteins in the ovary and placenta of Mabuya sp. during early, mid, and late gestation; their possible metabolic pathways; and biological processes. We carried out a comparative proteomic analysis during gestation in both tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization. Differential protein expression in both tissues (Student's t-test P < 0.05) was related to several processes such as cell structure, cell movement, and energy. Proteins found in ovary are mainly associated with follicular development and its regulation. In the placenta, particularly during mid and late gestation, protein expression is involved in nutrient metabolism, transport, protein synthesis, and embryonic development. This work provides new insights about the proteins expressed and their physiological mechanisms in Mabuya sp. placenta and ovary during gestation. © 2017 Wiley Periodicals, Inc.

Generation of a mouse model with a reversible hypomorphic cytochrome P450 reductase gene: utility for tissue-specific rescue of the reductase expression, and insights from a resultant mouse model with global suppression of P450 reductase expression in extrahepatic tissues.

PubMed

Wei, Yuan; Zhou, Xin; Fang, Cheng; Li, Lei; Kluetzman, Kerri; Yang, Weizhu; Zhang, Qing-Yu; Ding, Xinxin

2010-07-01

A mouse model termed Cpr-low (CL) was recently generated, in which the expression of the cytochrome P450 reductase (Cpr) gene was globally down-regulated. The decreased CPR expression was accompanied by phenotypical changes, including reduced embryonic survival, decreases in circulating cholesterol, increases in hepatic P450 expression, and female infertility (accompanied by elevated serum testosterone and progesterone levels). In the present study, a complementary mouse model [named reversible-CL (r-CL)] was generated, in which the reduced CPR expression can be reversed in an organ-specific fashion. The neo cassette, which was inserted into the last Cpr intron in r-CL mice, can be deleted by Cre recombinase, thus returning the structure of the Cpr gene (and hence CPR expression) to normal in Cre-expressing cells. All previously identified phenotypes of the CL mice were preserved in the r-CL mice. As a first application of the r-CL model, we have generated an extrahepatic-CL (xh-CL) mouse for testing of the functions of CPR-dependent enzymes in all extrahepatic tissues. The xh-CL mice, generated by mating of r-CL mice with albumin-Cre mice, had normal CPR expression in hepatocytes but down-regulated CPR expression elsewhere. They were indistinguishable from wild-type mice in body and liver weights, circulating cholesterol levels, and hepatic microsomal P450 expression and activities; however, they still showed elevated serum testosterone and progesterone levels and sterility in females. Embryonic lethality was prevented in males, but apparently not in females, indicating a critical role for fetal hepatic CPR-dependent enzymes in embryonic development, at least in males.

Skeletogenesis in the swell shark Cephaloscyllium ventriosum.

PubMed

Eames, B Frank; Allen, Nancy; Young, Jonathan; Kaplan, Angelo; Helms, Jill A; Schneider, Richard A

2007-05-01

Extant chondrichthyans possess a predominantly cartilaginous skeleton, even though primitive chondrichthyans produced bone. To gain insights into this peculiar skeletal evolution, and in particular to evaluate the extent to which chondrichthyan skeletogenesis retains features of an osteogenic programme, we performed a histological, histochemical and immunohistochemical analysis of the entire embryonic skeleton during development of the swell shark Cephaloscyllium ventriosum. Specifically, we compared staining properties among various mineralizing tissues, including neural arches of the vertebrae, dermal tissues supporting oral denticles and Meckel's cartilage of the lower jaw. Patterns of mineralization were predicted by spatially restricted alkaline phosphatase activity earlier in development. Regarding evidence for an osteogenic programme in extant sharks, a mineralized tissue in the perichondrium of C. ventriosum neural arches, and to a lesser extent a tissue supporting the oral denticle, displayed numerous properties of bone. Although we uncovered many differences between tissues in Meckel's cartilage and neural arches of C. ventriosum, both elements impart distinct tissue characteristics to the perichondral region. Considering the evolution of osteogenic processes, shark skeletogenesis may illuminate the transition from perichondrium to periosteum, which is a major bone-forming tissue during the process of endochondral ossification.

From Embryonic Development to Human Diseases: The Functional Role of Caveolae/Caveolin

PubMed Central

Sohn, Jihee; Brick, Rachel M.; Tuan, Rocky S.

2017-01-01

Caveolae, an almost ubiquitous, structural component of the plasma membrane, play a critical role in many functions essential for proper cell function, including membrane trafficking, signal transduction, extracellular matrix remodeling, and tissue regeneration. Three main types of caveolin proteins have been identified from caveolae since the discovery of caveolin-1 in the early 1990s. All three (Cav-1, Cav-2, and Cav-3) play crucial roles in mammalian physiology, and can effect pathogenesis in a wide range of human diseases. While many biological activities of caveolins have been uncovered since its discovery, their role and regulation in embryonic develop remain largely poorly understood, although there is increasing evidence that caveolins may be linked to lung and brain birth defects. Further investigations are clearly needed to decipher how caveolae/caveolins mediate cellular functions and activities of normal embryogenesis and how their perturbations contribute to developmental disorders. PMID:26991990

Bmp2 and Notch cooperate to pattern the embryonic endocardium.

PubMed

Papoutsi, T; Luna-Zurita, L; Prados, B; Zaffran, S; de la Pompa, J L

2018-05-31

Signaling interactions between myocardium and endocardium pattern embryonic cardiac regions, instructing their development to fulfill specific functions in the mature heart. We show that ectopic Bmp2 expression in the mouse chamber myocardium changes the transcriptional signature of adjacent chamber endocardial cells into valve tissue, and enables them to undergo epithelial-mesenchyme transition. This induction is independent of valve myocardium specification and requires high levels of Notch1 activity. Biochemical experiments suggest that Bmp2-mediated Notch1 induction is achieved through transcriptional activation of the Notch ligand Jag1, and physical interaction of Smad1/5 with the intracellular domain of the Notch1 receptor. Thus, widespread myocardial Bmp2 and endocardial Notch signaling drive presumptive ventricular endocardium to differentiate into valve endocardium. Understanding the molecular basis of valve development is instrumental to designing therapeutic strategies for congenital heart valve defects. © 2018. Published by The Company of Biologists Ltd.

Msx-2 expression and glucocorticoid-induced overexpression in embryonic mouse submandibular glands.

PubMed

Jaskoll, T; Luo, W; Snead, M L

1998-01-01

It is well known that the process of branching morphogenesis requires epithelial-mesenchymal interactions. One outstanding model for the study of tissue interactions during branching morphogenesis is the embryonic mouse submandibular gland (SMG). Although it has been clearly demonstrated that the branching pattern is dependent on interactions between the epithelium and the surrounding mesenchyme, little is known about the molecular mechanism underlying the branching process. One group of transcription factors that likely participates in the control of epithelial-mesenchymal inductive interactions are the Msx-class of homeodomain-containing proteins. In this paper, we focus on Msx-2 because its developmental expression is correlated with inductive interactions, suggesting that Msx-2 may play a functional role during cell-cell interactions. We demonstrate the expression of Msx-2 mRNA and protein to be primarily in the branching epithelia with progressive embryonic (E13 to E15) SMG development and, to a lesser extent, in the mesenchyme. We also show that Msx-2 is expressed by embryonic SMG primordia cultured under defined conditions. In addition, to begin to delineate a functional role for Msx-2, we employed an experimental strategy by using exogenous glucocorticoid (CORT) treatment of embryonic SMGs in vitro and in vivo to significantly enhance branching morphogenesis and evaluate the effect of CORT treatment on embryonic SMG Msx-2 expression. A marked increase in Msx-2 transcripts and protein is detected with in vitro and in vivo CORT treatment. Our studies indicate that one mechanism of CORT regulation of salivary gland morphogenesis is likely through the modulation of Msx-2 gene expression.

Ribociclib and Doxorubicin in Treating Patients With Metastatic or Advanced Soft Tissue Sarcomas That Cannot Be Removed by Surgery

ClinicalTrials.gov

2018-05-09

Metastatic Angiosarcoma; Metastatic Epithelioid Sarcoma; Metastatic Fibrosarcoma; Metastatic Leiomyosarcoma; Metastatic Liposarcoma; Metastatic Malignant Peripheral Nerve Sheath Tumor; Metastatic Synovial Sarcoma; Metastatic Undifferentiated Pleomorphic Sarcoma; Myxofibrosarcoma; Pleomorphic Rhabdomyosarcoma; Stage III Soft Tissue Sarcoma; Stage IV Soft Tissue Sarcoma; Undifferentiated (Embryonal) Sarcoma

Human pancreas development.

PubMed

Jennings, Rachel E; Berry, Andrew A; Strutt, James P; Gerrard, David T; Hanley, Neil A

2015-09-15

A wealth of data and comprehensive reviews exist on pancreas development in mammals, primarily mice, and other vertebrates. By contrast, human pancreatic development has been less comprehensively reviewed. Here, we draw together those studies conducted directly in human embryonic and fetal tissue to provide an overview of what is known about human pancreatic development. We discuss the relevance of this work to manufacturing insulin-secreting β-cells from pluripotent stem cells and to different aspects of diabetes, especially permanent neonatal diabetes, and its underlying causes. © 2015. Published by The Company of Biologists Ltd.

Physicochemical processes in embryonic plant tissue during the transition to the state of cold anabiosis and storage at liquid nitrogen temperature

NASA Astrophysics Data System (ADS)

Khodko, A. T.; Lysak, Yu. S.

2017-10-01

Critical opalescence phenomenon was observed in the cytoplasm of garlic embryonic tissue—meristem—upon cooling in liquid nitrogen vapor, indicating liquid-liquid phase transition in the system. It was established that cells of the meristem tissue survive the cooling-thawing cycle. We suggest that the transition of meristem tissue to the state of anabiosis is mainly due to a drastic slowing of the diffusion in the cytoplasm caused by the passage of the solution through the critical point, followed by the formation of a dispersed system—a highly concentrated emulsion—as a result of a liquid-liquid phase transition. This macrophase separation is characteristic of polymer-solvent systems. We established the regime of cooling down to liquid nitrogen temperature and subsequent thawing in the cryopreservation cycle for the biological object under study, which ensures the preservation of tissue viability.

Let's push things forward: disruptive technologies and the mechanics of tissue assembly.

PubMed

Varner, Victor D; Nelson, Celeste M

2013-09-01

Although many of the molecular mechanisms that regulate tissue assembly in the embryo have been delineated, the physical forces that couple these mechanisms to actual changes in tissue form remain unclear. Qualitative studies suggest that mechanical loads play a regulatory role in development, but clear quantitative evidence has been lacking. This is partly owing to the complex nature of these problems - embryonic tissues typically undergo large deformations and exhibit evolving, highly viscoelastic material properties. Still, despite these challenges, new disruptive technologies are enabling study of the mechanics of tissue assembly in unprecedented detail. Here, we present novel experimental techniques that enable the study of each component of these physical problems: kinematics, forces, and constitutive properties. Specifically, we detail advances in light sheet microscopy, optical coherence tomography, traction force microscopy, fluorescence force spectroscopy, microrheology and micropatterning. Taken together, these technologies are helping elucidate a more quantitative understanding of the mechanics of tissue assembly.

Let's push things forward: disruptive technologies and the mechanics of tissue assembly

PubMed Central

Varner, Victor D.; Nelson, Celeste M.

2013-01-01

Although many of the molecular mechanisms that regulate tissue assembly in the embryo have been delineated, the physical forces that couple these mechanisms to actual changes in tissue form remain unclear. Qualitative studies suggest that mechanical loads play a regulatory role in development, but clear quantitative evidence has been lacking. This is partly owing to the complex nature of these problems – embryonic tissues typically undergo large deformations and exhibit evolving, highly viscoelastic material properties. Still, despite these challenges, new disruptive technologies are enabling study of the mechanics of tissue assembly in unprecedented detail. Here, we present novel experimental techniques that enable the study of each component of these physical problems: kinematics, forces, and constitutive properties. Specifically, we detail advances in light sheet microscopy, optical coherence tomography, traction force microscopy, fluorescence force spectroscopy, microrheology and micropatterning. Taken together, these technologies are helping elucidate a more quantitative understanding of the mechanics of tissue assembly. PMID:23907401

Variable phenotypic penetrance of thrombosis in adult mice after tissue-selective and temporally controlled Thbd gene inactivation

PubMed Central

van Mens, Thijs E.; Liang, Hai-Po H.; Basu, Sreemanti; Hernandez, Irene; Zogg, Mark; May, Jennifer; Zhan, Min; Yang, Qiuhui; Foeckler, Jamie; Kalloway, Shawn; Sood, Rashmi; Karlson, Caren Sue

2017-01-01

Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd−/−) mice to term. Half of the Thbd−/− offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd−/− animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd−/− mice. However, Thbd−/− females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy. PMID:28920104

TGF-β Family Signaling in Embryonic and Somatic Stem Cell Renewal and Differentiation

PubMed Central

Mullen, Alan C.; Wrana, Jeffrey L.

2017-01-01

Soon after the discovery of Transforming Growth Factor-beta (TGF-β), seminal work in vertebrate and invertebrate models revealed the TGF-β family to be central regulators of tissue morphogenesis. Members of the family direct some of the earliest cell fate decisions in animal development, coordinate complex organogenesis and contribute to tissue homeostasis in the adult. Here we focus on the role of the TGF-β family in mammalian stem cell biology and discuss its wide and varied activities both in the regulation of pluripotency and in cell fate commitment. PMID:28108485

Molecular controls of arterial morphogenesis

PubMed Central

Simons, Michael; Eichmann, Anne

2015-01-01

Formation of arterial vasculature, here termed arteriogenesis, is a central process in embryonic vascular development as well as in adult tissues. While the process of capillary formation, angiogenesis, is relatively well understood, much remains to be learned about arteriogenesis. Recent discoveries point to the key role played by vascular endothelial growth factor receptor 2 (VEGFR2) in control of this process and to newly identified control circuits that dramatically influence its activity. The latter can present particularly attractive targets for a new class of therapeutic agents capable of activation of this signaling cascade in a ligand-independent manner, thereby promoting arteriogenesis in diseased tissues. PMID:25953926

A novel, tissue-specific, Drosophila homeobox gene.

PubMed

Barad, M; Jack, T; Chadwick, R; McGinnis, W

1988-07-01

The homeobox gene family of Drosophila appears to control a variety of position-specific patterning decisions during embryonic and imaginal development. Most of these patterning decisions determine groups of cells on the anterior-posterior axis of the Drosophila germ band. We have isolated a novel homeobox gene from Drosophila, designated H2.0. H2.0 has the most diverged homeobox so far characterized in metazoa, and, in contrast to all previously isolated homeobox genes, H2.0 exhibits a tissue-specific pattern of expression. The cells that accumulate transcripts for this novel gene correspond to the visceral musculature and its anlagen.

An update to space biomedical research: tissue engineering in microgravity bioreactors.

PubMed

Barzegari, Abolfazl; Saei, Amir Ata

2012-01-01

The severe need for constructing replacement tissues in organ transplanta-tion has necessitated the development of tissue engineering approaches and bioreactors that can bring these approaches to reality. The inherent limitations of conventional bioreactors in generating realistic tissue constructs led to the devise of the microgravity tissue engineering that uses Rotating Wall Vessel (RWV) bioreactors initially developed by NASA. In this review article, we intend to highlight some major advances and accomplishments in the rapidly-growing field of tissue engineering that could not be achieved without using microgravity. Research is now focused on assembly of 3 dimensional (3D) tissue fragments from various cell types in human body such as chon-drocytes, osteoblasts, embryonic and mesenchymal stem cells, hepatocytes and pancreas islet cells. Hepatocytes cultured under microgravity are now being used in extracorporeal bioartificial liver devices. Tissue constructs can be used not only in organ replacement therapy, but also in pharmaco-toxicology and food safety assessment. 3D models of vari-ous cancers may be used in studying cancer development and biology or in high-throughput screening of anticancer drug candidates. Finally, 3D heterogeneous assemblies from cancer/immune cells provide models for immunotherapy of cancer. Tissue engineering in (simulated) microgravity has been one of the stunning impacts of space research on biomedical sciences and their applications on earth.

Whole-organism clone tracing using single-cell sequencing.

PubMed

Alemany, Anna; Florescu, Maria; Baron, Chloé S; Peterson-Maduro, Josi; van Oudenaarden, Alexander

2018-04-05

Embryonic development is a crucial period in the life of a multicellular organism, during which limited sets of embryonic progenitors produce all cells in the adult body. Determining which fate these progenitors acquire in adult tissues requires the simultaneous measurement of clonal history and cell identity at single-cell resolution, which has been a major challenge. Clonal history has traditionally been investigated by microscopically tracking cells during development, monitoring the heritable expression of genetically encoded fluorescent proteins and, more recently, using next-generation sequencing technologies that exploit somatic mutations, microsatellite instability, transposon tagging, viral barcoding, CRISPR-Cas9 genome editing and Cre-loxP recombination. Single-cell transcriptomics provides a powerful platform for unbiased cell-type classification. Here we present ScarTrace, a single-cell sequencing strategy that enables the simultaneous quantification of clonal history and cell type for thousands of cells obtained from different organs of the adult zebrafish. Using ScarTrace, we show that a small set of multipotent embryonic progenitors generate all haematopoietic cells in the kidney marrow, and that many progenitors produce specific cell types in the eyes and brain. In addition, we study when embryonic progenitors commit to the left or right eye. ScarTrace reveals that epidermal and mesenchymal cells in the caudal fin arise from the same progenitors, and that osteoblast-restricted precursors can produce mesenchymal cells during regeneration. Furthermore, we identify resident immune cells in the fin with a distinct clonal origin from other blood cell types. We envision that similar approaches will have major applications in other experimental systems, in which the matching of embryonic clonal origin to adult cell type will ultimately allow reconstruction of how the adult body is built from a single cell.

Functional Dicer Is Necessary for Appropriate Specification of Radial Glia during Early Development of Mouse Telencephalon

PubMed Central

Nowakowski, Tomasz Jan; Mysiak, Karolina Sandra; Pratt, Thomas; Price, David Jonathan

2011-01-01

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1-/- dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon. PMID:21826226

Functional dicer is necessary for appropriate specification of radial glia during early development of mouse telencephalon.

PubMed

Nowakowski, Tomasz Jan; Mysiak, Karolina Sandra; Pratt, Thomas; Price, David Jonathan

2011-01-01

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1⁻/⁻ dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon.

Effects of low-dose heavy ions on embryonic development in mice and on melanocyte differentiation in the epidermis and hair bulb.

PubMed

Hirobe, Tomohisa; Eguchi-Kasai, Kiyomi; Sugaya, Kimihiko; Murakami, Masahiro

2013-05-01

The effects of prenatal low-dose irradiation with heavy ions on embryonic development in mice and on melanocyte differentiation are not well understood. We performed whole-body irradiation of pregnant C57BL/10J mice at embryonic Day 9 (E9) with a single dose of γ-rays, silicon, argon or iron ions. The number of living embryos and embryonic body weight at E18 decreased after exposure to heavy ions at high doses. Malformations such as small eyes and limb anomalies were observed in heavy-ion-treated embryos, but not in γ-ray-treated embryos. The frequency of abnormally curved tails was increased by exposure to γ-rays and argon and iron ions even at a dose of 0.1 Gy (P < 0.05). In contrast, a dose-dependent decrease in the number of epidermal melanoblasts/melanocytes and hair bulb melanocytes was observed after 0.1 Gy irradiation with γ-rays or heavy ions (P < 0.01). The decrease in the number of dorsal hair bulb melanocytes, dorsal and ventral epidermal melanoblasts/melanocytes and ventral hair bulb melanocytes was not necessarily correlated with the linear energy transfer of the radiation tested. Moreover, the effects of heavy ions were larger on the ventral skin than on the dorsal skin, indicating that the sensitivity of melanocytes to heavy ions differs between the dorsal and ventral skin. Taken together, these results suggest that the effects of the low-dose heavy ions differ between cell types and tissues, and the effects on the prenatal development of mice and melanocyte development are not necessarily greater than those of γ-rays.

Effects of low-dose heavy ions on embryonic development in mice and on melanocyte differentiation in the epidermis and hair bulb

PubMed Central

Hirobe, Tomohisa; Eguchi-Kasai, Kiyomi; Sugaya, Kimihiko; Murakami, Masahiro

2013-01-01

The effects of prenatal low-dose irradiation with heavy ions on embryonic development in mice and on melanocyte differentiation are not well understood. We performed whole-body irradiation of pregnant C57BL/10J mice at embryonic Day 9 (E9) with a single dose of γ-rays, silicon, argon or iron ions. The number of living embryos and embryonic body weight at E18 decreased after exposure to heavy ions at high doses. Malformations such as small eyes and limb anomalies were observed in heavy-ion-treated embryos, but not in γ-ray-treated embryos. The frequency of abnormally curved tails was increased by exposure to γ-rays and argon and iron ions even at a dose of 0.1 Gy (P < 0.05). In contrast, a dose-dependent decrease in the number of epidermal melanoblasts/melanocytes and hair bulb melanocytes was observed after 0.1 Gy irradiation with γ-rays or heavy ions (P < 0.01). The decrease in the number of dorsal hair bulb melanocytes, dorsal and ventral epidermal melanoblasts/melanocytes and ventral hair bulb melanocytes was not necessarily correlated with the linear energy transfer of the radiation tested. Moreover, the effects of heavy ions were larger on the ventral skin than on the dorsal skin, indicating that the sensitivity of melanocytes to heavy ions differs between the dorsal and ventral skin. Taken together, these results suggest that the effects of the low-dose heavy ions differ between cell types and tissues, and the effects on the prenatal development of mice and melanocyte development are not necessarily greater than those of γ-rays. PMID:23230241

Modeling human development in 3D culture.

PubMed

Ader, Marius; Tanaka, Elly M

2014-12-01

Recently human embryonic stem cell research has taken on a new dimension - the third dimension. Capitalizing on increasing knowledge on directing pluripotent cells along different lineages, combined with ECM supported three-dimensional culture conditions, it has become possible to generate highly organized tissues of the central nervous system, gut, liver and kidney. Each system has been used to study different aspects of organogenesis and function including physical forces underlying optic cup morphogenesis, the function of disease related genes in progenitor cell control, as well as interaction of the generated tissues with host tissue upon transplantation. Pluripotent stem cell derived organoids represent powerful systems for the study of how cells self-organize to generate tissues with a given shape, pattern and form. Copyright © 2014 Elsevier Ltd. All rights reserved.

Role of adiponectin in delayed embryonic development of the short-nosed fruit bat, Cynopterus sphinx.

PubMed

Anuradha; Krishna, Amitabh

2014-12-01

The aim of this study was to evaluate the role of adiponectin in the delayed embryonic development of Cynopterus sphinx. Adiponectin receptor (ADIPOR1) abundance was first observed to be lower during the delayed versus non-delayed periods of utero-embryonic unit development. The effects of adiponectin treatment on embryonic development were then evaluated during the period of delayed development. Exogenous treatment increased the in vivo rate of embryonic development, as indicated by an increase in weight, ADIPOR1 levels in the utero-embryonic unit, and histological changes in embryonic development. Treatment with adiponectin during embryonic diapause showed a significant increase in circulating progesterone and estradiol concentrations, and in production of their receptors in the utero-embryonic unit. The adiponectin-induced increase in estradiol synthesis was correlated with increased cell survival (BCL2 protein levels) and cell proliferation (PCNA protein levels) in the utero-embryonic unit, suggesting an indirect effect of adiponectin via estradiol synthesis by the ovary. An in vitro study further confirmed the in vivo findings that adiponectin treatment increases PCNA levels together with increased uptake of glucose by increasing the abundance of glucose transporter 8 (GLUT8) in the utero-embryonic unit. The in vitro study also revealed that adiponectin, together with estradiol but not alone, significantly increased ADIPOR1 protein levels. Thus, adiponectin works in concert with estradiol to increase glucose transport to the utero-embryonic unit and promote cell proliferation, which together accelerate embryonic development. © 2014 Wiley Periodicals, Inc.

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.

Nervous-Tissue-Specific Elimination of Microtubule-Actin Crosslinking Factor 1a Results in Multiple Developmental Defects in the Mouse Brain

PubMed Central

Goryunov, Dmitry; He, Cui-Zhen; Lin, Chyuan-Sheng; Leung, Conrad L.; Liem, Ronald K. H.

2010-01-01

The microtubule-actin crosslinking factor 1 (MACF1) is a ubiquitous cytoskeletal linker protein with multiple spliced isoforms expressed in different tissues. The MACF1a isoform contains microtubule and actin binding regions and is expressed at high levels in the nervous system. Macf1−/− mice are early embryonic lethal and hence the role of MACF1 in the nervous system could not be determined. We have specifically knocked out MACF1a in the developing mouse nervous system using Cre/loxP technology. Mutant mice died within 24–36 hrs after birth of apparent respiratory distress. Their brains displayed a disorganized cerebral cortex with a mixed layer structure, heterotopia in the pyramidal layer of the hippocampus, disorganized thalamocortical and corticofugal fibers, and aplastic anterior and hippocampal commissures. Embryonic neurons showed a defect in traversing the cortical plate. Our data suggest a critical role for MACF1 in neuronal migration that is dependent on its ability to interact with both microfilaments and microtubules. PMID:20170731

Nervous-tissue-specific elimination of microtubule-actin crosslinking factor 1a results in multiple developmental defects in the mouse brain.

PubMed

Goryunov, Dmitry; He, Cui-Zhen; Lin, Chyuan-Sheng; Leung, Conrad L; Liem, Ronald K H

2010-05-01

The microtubule-actin crosslinking factor 1 (MACF1) is a ubiquitous cytoskeletal linker protein with multiple spliced isoforms expressed in different tissues. The MACF1a isoform contains microtubule and actin-binding regions and is expressed at high levels in the nervous system. Macf1-/- mice are early embryonic lethal and hence the role of MACF1 in the nervous system could not be determined. We have specifically knocked out MACF1a in the developing mouse nervous system using Cre/loxP technology. Mutant mice died within 24-36h after birth of apparent respiratory distress. Their brains displayed a disorganized cerebral cortex with a mixed layer structure, heterotopia in the pyramidal layer of the hippocampus, disorganized thalamocortical and corticofugal fibers, and aplastic anterior and hippocampal commissures. Embryonic neurons showed a defect in traversing the cortical plate. Our data suggest a critical role for MACF1 in neuronal migration that is dependent on its ability to interact with both microfilaments and microtubules. Copyright 2010 Elsevier Inc. All rights reserved.

Transcriptional control of stem cell fate by E2Fs and pocket proteins

PubMed Central

Julian, Lisa M.; Blais, Alexandre

2015-01-01

E2F transcription factors and their regulatory partners, the pocket proteins (PPs), have emerged as essential regulators of stem cell fate control in a number of lineages. In mammals, this role extends from both pluripotent stem cells to those encompassing all embryonic germ layers, as well as extra-embryonic lineages. E2F/PP-mediated regulation of stem cell decisions is highly evolutionarily conserved, and is likely a pivotal biological mechanism underlying stem cell homeostasis. This has immense implications for organismal development, tissue maintenance, and regeneration. In this article, we discuss the roles of E2F factors and PPs in stem cell populations, focusing on mammalian systems. We discuss emerging findings that position the E2F and PP families as widespread and dynamic epigenetic regulators of cell fate decisions. Additionally, we focus on the ever expanding landscape of E2F/PP target genes, and explore the possibility that E2Fs are not simply regulators of general ‘multi-purpose’ cell fate genes but can execute tissue- and cell type-specific gene regulatory programs. PMID:25972892

Genetic heterogeneity of skin microvasculature

PubMed Central

Liu, Fang; Smith, Jason; Zhang, Zhen; Cole, Richard; Herron, Bruce J

2010-01-01

Angiogenesis, the formation of new blood vessels from existing vasculature, is a complex process that is essential for normal embryonic development. Current models for experimental evaluation of angiogenesis often use tissue from large vessels like the aorta and umbilical vein, which are phenotypically distinct from microvasculature. We demonstrate that the utilization of skin to measure microvascular angiogenesis in embryonic and adult tissues is an efficient way to quantify microvasculature angiogenesis. We validate this approach and demonstrate its added value by showing significant differences in angiogenesis in monogenic and polygenic mouse models. We discovered that the pattern of angiogenic response among inbred mouse strains in this ex vivo assay differ from the strain distributions of previous in vivo angiogenesis assays. The difference between the ex vivo and in vivo assays may be related to systemic factors present in whole animals. Expression analysis of cultured skin biopsies from strains of mice with opposing angiogenic response were performed to identify pathways that contribute to differential angiogenic response. Increased expression of negative regulators of angiogenesis in C57Bl/6J mice was associated with lower growth rates. PMID:20170648

Developmental expression of membrane type 4-matrix metalloproteinase (Mt4-mmp/Mmp17) in the mouse embryo

PubMed Central

Clemente, Cristina; Montalvo, María Gregoria; Seiki, Motoharu; Arroyo, Alicia G.

2017-01-01

Matrix metalloproteinases (MMPs) constitute a large group of endoproteases that play important functions during embryonic development, tumor metastasis and angiogenesis by degrading components of the extracellular matrix. Within this family, we focused our study on Mt4-mmp (also called Mmp17) that belongs to a distinct subset that is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety and with the catalytic site exposed to the extracellular space. Information about its function and substrates is very limited to date, and little has been reported on its role in the developing embryo. Here, we report a detailed expression analysis of Mt4-mmp during mouse embryonic development by using a LacZ reporter transgenic mouse line. We showed that Mt4-mmp is detected from early stages of development to postnatal stages following a dynamic and restricted pattern of expression. Mt4-mmp was first detected at E8.5 limited to the intersomitic vascularization, the endocardial endothelium and the dorsal aorta. Mt4-mmpLacZ/+ cells were also observed in the neural crest cells, somites, floor plate and notochord at early stages. From E10.5, expression localized in the limb buds and persists during limb development. A strong expression in the brain begins at E12.5 and continues to postnatal stages. Specifically, staining was observed in the olfactory bulb, cerebral cortex, hippocampus, striatum, septum, dorsal thalamus and the spinal cord. In addition, LacZ-positive cells were also detected during eye development, initially at the hyaloid artery and later on located in the lens and the neural retina. Mt4-mmp expression was confirmed by quantitative RT-PCR and western blot analysis in some embryonic tissues. Our data point to distinct functions for this metalloproteinase during embryonic development, particularly during brain formation, angiogenesis and limb development. PMID:28926609

Random Walk of Single Gold Nanoparticles in Zebrafish Embryos Leading to Stochastic Toxic Effects on Embryonic Developments

PubMed Central

Browning, Lauren M.; Lee, Kerry J.; Huang, Tao; Nallathamby, Prakash D.; Lowman, Jill E.; Xu, Xiao-Hong Nancy

2010-01-01

We have synthesized and characterized stable (non-aggregation, non-photobleaching and non-blinking), nearly monodisperse and highly-purified Au nanoparticles, and used them to probe transport of cleavage-stage zebrafish embryos and to study their effects on embryonic development in real time. We found that single Au nanoparticles (11.6 ± 0.9 nm in diameter) passively diffused into chorionic space of the embryos via their chorionic-pore-canals and continued their random-walk through chorionic space and into inner mass of embryos. Diffusion coefficients of single nanoparticles vary dramatically (2.8×10-11 to 1.3×10-8 cm2/s) as nanoparticles diffuse through various parts of embryos, suggesting highly diverse transport barriers and viscosity gradients of embryos. The amount of Au nanoparticles accumulated in embryos increase with its concentration. Interestingly, their effects on embryonic development are not proportionally related to the concentration. Majority of embryos (74% on average) incubated chronically with 0.025-1.2 nM Au nanoparticles for 120 h developed to normal zebrafish, with some (24%) being dead and few (2%) deformed. We developed a new approach to image and characterize individual Au nanoparticles embedded in tissues using histology sample preparation methods and LSRP spectra of single nanoparticles. We found that Au nanoparticles in various parts of normally developed and deformed zebrafish, suggesting that random-walk of nanoparticles in embryos during their development might have led to stochastic effects on embryonic development. These results show that Au nanoparticles are much more biocompatible (less toxic) to the embryos than Ag nanoparticles that we reported previously, suggesting that they are better suited as biocompatible probes for imaging embryos in vivo. The results provide powerful evidences that biocompatibility and toxicity of nanoparticles highly depend on their chemical properties, and the embryos can serve as effective in-vivo assays to screen their biocompatibility. PMID:20644873

Hyperforin inhibits cell proliferation and differentiation in mouse embryonic stem cells.

PubMed

Nakamura, K; Aizawa, K; Yamauchi, J; Tanoue, A

2013-10-01

Hyperforin, a phloroglucinol derivative of St. John's Wort, has been identified as the major molecule responsible for this plant's products anti-depressant effects. It can be expected that exposure to St. John's Wort during pregnancy occurs with some frequency although embryotoxic or teratogenic effects of St. John's Wort and hyperforin have not yet been experimentally examined in detail. In this study, to determine any embryotoxic effects of hyperforin, we have attempted to determine whether hyperforin affects growth and survival processes of employing mouse embryonic stem (mES) cells (representing embryonic tissue) and fibroblasts (representing adult tissues). We used a modified embryonic stem cell test, which has been validated as an in vitro developmental toxicity protocol, mES cells, to assess embryotoxic potential of chemicals under investigation. We have identified that high concentrations of hyperforin inhibited mouse ES cell population growth and induced apoptosis in fibroblasts. Under our cell culture conditions, ES cells mainly differentiated into cardiomyocytes, although various other cell types were also produced. In this condition, hyperforin affected ES cell differentiation into cardiomyocytes in a dose-dependent manner. Analysis of tissue-specific marker expression also revealed that hyperforin at high concentrations partially inhibited ES cell differentiation into mesodermal and endodermal lineages. Hyperforin is currently used in the clinic as a safe and effective antidepressant. Our data indicate that at typical dosages it has only a low risk of embryotoxicity; ingestion of large amounts of hyperforin by pregnant women, however, may pose embryotoxic and teratogenic risks. © 2013 John Wiley & Sons Ltd.

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

PubMed

Lefevre, James G; Chiu, Han S; Combes, Alexander N; Vanslambrouck, Jessica M; Ju, Ali; Hamilton, Nicholas A; Little, Melissa H

2017-03-15

Human pluripotent stem cells, after directed differentiation in vitro , can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation. © 2017. Published by The Company of Biologists Ltd.

The Emergence of Blood and Blood Vessels in the Embryo and Its Relevance to Postnatal Biology and Disease

NASA Astrophysics Data System (ADS)

Sills, Tiffany M.; Hirschi, Karen K.

Blood and blood vessels develop in parallel within mammalian systems, and this temporal and spatial association has led to the confirmation of an endothelial origin of hematopoiesis. The extraembryonic yolk sac and aorto-gonado-mesonephros (AGM) region both contain a specialized population of endothelial cells ("hemogenic endothelium") that function to produce hematopoietic stem and progenitor cells, which then differentiate to provide the full complement of blood cells within the developing embryo and furthermore in the adult system. Therefore, this population has great therapeutic potential in the fields of regenerative medicine and tissue engineering. This chapter reviews the development of the vascular and hematopoietic systems, characterization and function of the hemogenic endothelium within embryonic and embryonic stem cell (ES cell) models, and speculate on the presence of such a population within the adult system. In order to harness this endothelial subtype for clinical application, we must understand both the normal functions of these cells and the potential for misregulation in disease states.

Essential Role of Cyclin-G–associated Kinase (Auxilin-2) in Developing and Mature Mice

PubMed Central

Lee, Dong-won; Zhao, Xiaohong; Yim, Yang-In; Eisenberg, Evan

2008-01-01

Hsc70 with its cochaperone, either auxilin or GAK, not only uncoats clathrin-coated vesicles but also acts as a chaperone during clathrin-mediated endocytosis. However, because synaptojanin is also involved in uncoating, it is not clear whether GAK is an essential gene. To answer this question, GAK conditional knockout mice were generated and then mated to mice expressing Cre recombinase under the control of the nestin, albumin, or keratin-14 promoters, all of which turn on during embryonic development. Deletion of GAK from brain, liver, or skin dramatically altered the histology of these tissues, causing the mice to die shortly after birth. Furthermore, by expressing a tamoxifen-inducible promoter to express Cre recombinase we showed that deletion of GAK caused lethality in adult mice. Mouse embryonic fibroblasts in which the GAK was disrupted showed a lack of clathrin-coated pits and a complete block in clathrin-mediated endocytosis. We conclude that GAK deletion blocks development and causes lethality in adult animals by disrupting clathrin-mediated endocytosis. PMID:18434600

Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice.

PubMed

Bruin, Jennifer E; Rezania, Alireza; Xu, Jean; Narayan, Kavitha; Fox, Jessica K; O'Neil, John J; Kieffer, Timothy J

2013-09-01

Islet transplantation is a promising cell therapy for patients with diabetes, but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However, the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore, we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo. The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte, Laguna Hills, CA, USA) devices into diabetic mice. Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing >80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices, despite lacking direct contact with the host environment, and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells. Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.

Differential developmental ability of embryos cloned from tissue-specific stem cells.

PubMed

Inoue, Kimiko; Noda, Shinichi; Ogonuki, Narumi; Miki, Hiromi; Inoue, Shinichi; Katayama, Kazufumi; Mekada, Kazuyuki; Miyoshi, Hiroyuki; Ogura, Atsuo

2007-05-01

Although cloning animals by somatic cell nuclear transfer is generally inefficient, the use of certain nuclear donor cell types may significantly improve or deteriorate outcomes. We evaluated whether two multipotent stem cell lines produced in vitro--neural stem cells (NSCs) and mesenchymal stem cells (MSCs)--could serve as nuclear donors for nuclear transfer cloning. Most (76%) NSC-derived embryos survived the two-cell-to-four-cell transition, the stage when the major zygotic gene activation occurs. Consistent with this observation, the expression patterns of zygotically active genes were better in NSC-derived embryos than in fibroblast clone embryos, which arrested at the two-cell stage more frequently. Embryo transfer experiments demonstrated that at least some of these NSC embryos had the ability to develop to term fetuses (1.6%, 3/189). In contrast, embryos reconstructed using MSCs showed a low rate of in vitro development and never underwent implantation in vivo. Chromosomal analysis of the donor MSCs revealed very frequent aneuploidy, which probably impaired the potential for development of their derived clones. This is the first demonstration that tissue-specific multipotent stem cells produced in vitro can serve as donors of nuclei for cloning mice; however, these cells may be prone to chromosomal aberrations, leading to high embryonic death rates. We found previously that hematopoietic stem cells (HSCs) are very inefficient donor cells because of their failure to activate the genes essential for embryonic development. Taken together, our data led us to conclude that tissue-specific stem cells in mice, namely NSCs, MSCs, and HSCs, exhibited marked variations in the ability to produce cloned offspring and that this ability varies according to both the epigenetic and genetic status of the original genomes. Disclosure of potential conflicts of interest is found at the end of this article.

Postnatal extra-embryonic tissues as a source of multiple cell types for regenerative medicine applications.

PubMed

Gubar, O S; Rodnichenko, A E; Vasyliev, R G; Zlatska, A V; Zubov, D O

2017-09-01

We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73 + CD90 + CD105 + CD146 + CD166+CD34 - CD45 - HLA-DR - . HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use.

Polythelia is not a mere aesthetic issue.

PubMed

Kokavec, R; Macúch, J; Fedeles, J; Ondriás, F

2002-01-01

Anomalies in breast development include an increase in the number of breast (polymastia), nipples (polythelia), areolas (polythelia areolaris) and the tissue of the breast gland. Of the many forms, the most common is an accessory nipple, polythelia. It commonly develops along the embryonic "milk line" (Fig. 1). It constitutes a diagnostic problem only sporadically. Excision is indicated for diagnostic, treatment or cosmetic reasons. In the literature, the evidence of these anomalies suggests that they could be markers for others, most notably urologic malformations and malignancies.

Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.

PubMed

Shaker, Mohammed R; Kim, Joo Yeon; Kim, Hyun; Sun, Woong

2015-05-15

Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

The Most Prevalent Freeman-Sheldon Syndrome Mutations in the Embryonic Myosin Motor Share Functional Defects.

PubMed

Walklate, Jonathan; Vera, Carlos; Bloemink, Marieke J; Geeves, Michael A; Leinwand, Leslie

2016-05-06

The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5-9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2-35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of embryo-specific genes

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

Not Available

1989-01-01

The objective of the proposed research is to characterize the structure and function of a set of genes whose expression is regulated in embryo development, and that is not expressed in mature tissues -- the embryonic genes. In the last two years, using cDNA clones, we have isolated 22 cDNA clones, and characterized the expression pattern of their corresponding RNA. At least 4 cDNA clones detect RNAs of embryonic genes. These cDNA clones detect RNAs expressed in somatic as well as zygotic embryos of carrot. Using the cDNA clones, we screened the genomic library of carrot embryo DNA, and isolatedmore » genomic clones for three genes. The structure and function of two genes DC 8 and DC 59 have been characterized and are reported in this paper.« less

[Intestinal polyp of the umbilical cord].

PubMed

Guschmann, M; Janda, J; Wenzelides, K; Vogel, M

2002-02-01

The morphology, pathogenesis, complications and differential diagnosis of an intestinal polyp of the umbilical cord are presented. The polyp were detected postnatal on the umbilical cord in an healthy male newborn. The presents of intestinal tissue upon the umbilical cord ist possible about the persistence from remnants of the ductus omphalomesentericus with prolapse and differentiation of the intestinal cells. The ductus omphalomesentericus is a tubular structure, a communication between the developing embryonic gut and the yolk sac, forming during the early embryonic life. Obliteration of the omphalomesenteric duct is usually complete by the 10(th) week of gestation. Various portions of the duct may persist, however, giving rise to polyps, fistulas or cysts of the umbilical cord with potentially dangerous clinical consequences. Other tumors of the umbilical cord are myxoma, angioma and teratoma are differential diagnosis.

Coordinating cell and tissue behavior during zebrafish neural tube morphogenesis.

PubMed

Araya, Claudio; Ward, Laura C; Girdler, Gemma C; Miranda, Miguel

2016-03-01

The development of a vertebrate neural epithelium with well-organized apico-basal polarity and a central lumen is essential for its proper function. However, how this polarity is established during embryonic development and the potential influence of surrounding signals and tissues on such organization has remained less understood. In recent years the combined superior transparency and genetics of the zebrafish embryo has allowed for in vivo visualization and quantification of the cellular and molecular dynamics that govern neural tube structure. Here, we discuss recent studies revealing how co-ordinated cell-cell interactions coupled with adjacent tissue dynamics are critical to regulate final neural tissue architecture. Furthermore, new findings show how the spatial regulation and timing of orientated cell division is key in defining precise lumen formation at the tissue midline. In addition, we compare zebrafish neurulation with that of amniotes and amphibians in an attempt to understand the conserved cellular mechanisms driving neurulation and resolve the apparent differences among animals. Zebrafish neurulation not only offers fundamental insights into early vertebrate brain development but also the opportunity to explore in vivo cell and tissue dynamics during complex three-dimensional animal morphogenesis. © 2015 Wiley Periodicals, Inc.

Rax : developmental and daily expression patterns in the rat pineal gland and retina.

PubMed

Rohde, Kristian; Klein, David C; Møller, Morten; Rath, Martin F

2011-09-01

Retina and anterior neural fold homeobox (Rax) gene encodes a transcription factor essential for vertebrate eye development. Recent microarray studies indicate that Rax is expressed in the adult rat pineal gland and retina. The present study reveals that Rax expression levels in the rat change significantly during retinal development with a peak occurring at embryonic day 18, whereas Rax expression in the pineal is relatively delayed and not detectable until embryonic day 20. In both tissues, Rax is expressed throughout postnatal development into adulthood. In the mature rat pineal gland, the abundance of Rax transcripts increases 2-fold during the light period with a peak occurring at dusk. These findings are consistent with the evidence that Rax is of functional importance in eye development and suggest a role of Rax in the developing pineal gland. In addition, it would appear possible that Rax contributes to phenotype maintenance in the mature retina and pineal gland and may facilitate 24-h changes in the pineal transcriptome. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Changes in the antioxidant metabolism in the embryonic development of the common South American toad Bufo arenarum: differential responses to pesticide in early embryos and autonomous-feeding larvae.

PubMed

Ferrari, Ana; Anguiano, Liliana; Lascano, Cecilia; Sotomayor, Verónica; Rosenbaum, Enrique; Venturino, Andrés

2008-01-01

Amphibians may be critically challenged by aquatic contaminants during their embryonic development. Many classes of compounds, including organophosphorus pesticides, are able to cause oxidative stress that affects the delicate cellular redox balance regulating tissue modeling. We determined the progression of antioxidant defenses during the embryonic development of the South American common toad, Bufo arenarum. Superoxide dismutase (SOD) and catalase (CAT) activities were high in the unfertilized eggs, and remained constant during the first stages of development. SOD showed a significant increase when the gills were completely active and opercular folds began to form. Reductase (GR) activity was low in the oocytes and increased significantly when gills and mouth were entirely developed and the embryos presented a higher exposure to pro-oxidant conditions suggesting an environmental control. Reduced glutathione (GSH) content was also initially low, and rose continuously pointing out an increasing participation of GSH-related enzymes in the control of oxidative stress. GSH peroxidases and GSH-S-transferases showed relatively high and constant activities, probably related to lipid peroxide control. B. arenarum embryos have plenty of yolk platelets containing lipids, which provide the energy and are actively transferred to the newly synthesized membranes during the early embryonic development. Exposure to the pro-oxidant pesticide malathion during 48 h did not significantly affect the activity of antioxidant enzymes in early embryos, but decreased the activities of CAT, GR, and the pool of GSH in larvae. Previous work indicated that lipid peroxide levels were kept low in malathion-exposed larvae, thus we conclude that oxidative stress is overcome by the antioxidant defenses. The increase in the antioxidant metabolism observed in the posthatching phase of development of B. arenarum embryo, thus constitutes a defense against natural and human-generated pro-oxidants present in the aquatic environment.

Telomere correlations during early life in a long-lived seabird.

PubMed

Schmidt, Jacob E; Sirman, Aubrey E; Kittilson, Jeffrey D; Clark, Mark E; Reed, Wendy L; Heidinger, Britt J

2016-12-01

Telomere dynamics in blood cells have been linked to aging in a variety of organisms. However, whether blood telomeres are correlated with telomeres in other parts of the body is not well known, especially during early life when telomere loss is expected to be most rapid. We investigated this question in Franklin's gulls (Leucophaeus pipixcan) by measuring telomere lengths in blood and several other tissues including: heart, liver, and skeletal muscle at the end of embryonic (n=31) and post-natal development (n=20). In late-stage embryos, blood telomeres were significantly positively correlated with heart and skeletal muscle, but not liver telomeres. However, at the end of post-natal development, there were no significant correlations among blood telomeres and telomeres in any other tissues. In late-stage embryos, heart telomeres were significantly longer than blood, liver, and skeletal muscle telomeres, but at the end of post-natal development telomere lengths did not significantly differ among tissues. These results suggest that blood telomere length is not necessarily indicative of other tissues at all stages of development and highlights the importance of understanding any functional consequences of tissue specific telomere dynamics in early life. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparison of In Vitro- and Chorioallantoic Membrane (CAM)-Culture Systems for Cryopreserved Medulla-Contained Human Ovarian Tissue

PubMed Central

Isachenko, Vladimir; Mallmann, Peter; Petrunkina, Anna M.; Rahimi, Gohar; Nawroth, Frank; Hancke, Katharina; Felberbaum, Ricardo; Genze, Felicitas; Damjanoski, Ilija; Isachenko, Evgenia

2012-01-01

At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5–2.0×1.0–1.2×0.8–1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips. PMID:22479331

Maintaining embryonic stem cell pluripotency with Wnt signaling.

PubMed

Sokol, Sergei Y

2011-10-01

Wnt signaling pathways control lineage specification in vertebrate embryos and regulate pluripotency in embryonic stem (ES) cells, but how the balance between progenitor self-renewal and differentiation is achieved during axis specification and tissue patterning remains highly controversial. The context- and stage-specific effects of the different Wnt pathways produce complex and sometimes opposite outcomes that help to generate embryonic cell diversity. Although the results of recent studies of the Wnt/β-catenin pathway in ES cells appear to be surprising and controversial, they converge on the same conserved mechanism that leads to the inactivation of TCF3-mediated repression.

Epigenetic modulation by TFII-I during embryonic stem cell differentiation.

PubMed

Bayarsaihan, Dashzeveg; Makeyev, Aleksandr V; Enkhmandakh, Badam

2012-10-01

TFII-I transcription factors play an essential role during early vertebrate embryogenesis. Genome-wide mapping studies by ChIP-seq and ChIP-chip revealed that TFII-I primes multiple genomic loci in mouse embryonic stem cells and embryonic tissues. Moreover, many TFII-I-bound regions co-localize with H3K4me3/K27me3 bivalent chromatin within the promoters of lineage-specific genes. This minireview provides a summary of current knowledge regarding the function of TFII-I in epigenetic control of stem cell differentiation. Copyright © 2012 Wiley Periodicals, Inc.

The ECM moves during primitive streak formation--computation of ECM versus cellular motion.

PubMed

Zamir, Evan A; Rongish, Brenda J; Little, Charles D

2008-10-14

Galileo described the concept of motion relativity--motion with respect to a reference frame--in 1632. He noted that a person below deck would be unable to discern whether the boat was moving. Embryologists, while recognizing that embryonic tissues undergo large-scale deformations, have failed to account for relative motion when analyzing cell motility data. A century of scientific articles has advanced the concept that embryonic cells move ("migrate") in an autonomous fashion such that, as time progresses, the cells and their progeny assemble an embryo. In sharp contrast, the motion of the surrounding extracellular matrix scaffold has been largely ignored/overlooked. We developed computational/optical methods that measure the extent embryonic cells move relative to the extracellular matrix. Our time-lapse data show that epiblastic cells largely move in concert with a sub-epiblastic extracellular matrix during stages 2 and 3 in primitive streak quail embryos. In other words, there is little cellular motion relative to the extracellular matrix scaffold--both components move together as a tissue. The extracellular matrix displacements exhibit bilateral vortical motion, convergence to the midline, and extension along the presumptive vertebral axis--all patterns previously attributed solely to cellular "migration." Our time-resolved data pose new challenges for understanding how extracellular chemical (morphogen) gradients, widely hypothesized to guide cellular trajectories at early gastrulation stages, are maintained in this dynamic extracellular environment. We conclude that models describing primitive streak cellular guidance mechanisms must be able to account for sub-epiblastic extracellular matrix displacements.

Eph regulates dorsoventral asymmetry of the notochord plate and convergent extension-mediated notochord formation.

PubMed

Oda-Ishii, Izumi; Ishii, Yasuo; Mikawa, Takashi

2010-10-29

The notochord is a signaling center required for the patterning of the vertebrate embryonic midline, however, the molecular and cellular mechanisms involved in the formation of this essential embryonic tissue remain unclear. The urochordate Ciona intestinalis develops a simple notochord from 40 specific postmitotic mesodermal cells. The precursors intercalate mediolaterally and establish a single array of disk-shaped notochord cells along the midline. However, the role that notochord precursor polarization, particularly along the dorsoventral axis, plays in this morphogenetic process remains poorly understood. Here we show that the notochord preferentially accumulates an apical cell polarity marker, aPKC, ventrally and a basement membrane marker, laminin, dorsally. This asymmetric accumulation of apicobasal cell polarity markers along the embryonic dorsoventral axis was sustained in notochord precursors during convergence and extension. Further, of several members of the Eph gene family implicated in cellular and tissue morphogenesis, only Ci-Eph4 was predominantly expressed in the notochord throughout cell intercalation. Introduction of a dominant-negative Ci-Eph4 to notochord precursors diminished asymmetric accumulation of apicobasal cell polarity markers, leading to defective intercalation. In contrast, misexpression of a dominant-negative mutant of a planar cell polarity gene Dishevelled preserved asymmetric accumulation of aPKC and laminin in notochord precursors, although their intercalation was incomplete. Our data support a model in which in ascidian embryos Eph-dependent dorsoventral polarity of notochord precursors plays a crucial role in mediolateral cell intercalation and is required for proper notochord morphogenesis.

First echinoderm trehalase from a tropical sea cucumber (Holothuria leucospilota): Molecular cloning and mRNA expression in different tissues, embryonic and larval stages, and under a starvation challenge.

PubMed

Huo, Da; Jiang, Xiao; Wu, Xiaofen; Ren, Chunhua; Yu, Zonghe; Liu, Jinshang; Li, Hongmei; Ruan, Yao; Wen, Jin; Chen, Ting; Hu, Chaoqun

2018-04-29

Trehalases are a group of enzymes that catalyse the conversion of trehalose to glucose, and they are observed in most organisms. In this study, the first echinoderm trehalase, designated Hl-Tre, was identified from a tropical sea cucumber, Holothuria leucospilota. The full-length cDNA of H. leucospilota trehalase (Hl-Tre) is 2461 bp in length with an open reading frame (ORF) of 1788 bp that encodes a 595-amino-acid protein with a deduced molecular weight of 67.95 KDa. The Hl-Tre protein contains a signal peptide at the N-terminal and a functional trehalase domain, which includes the signature motifs 1 and 2. The mRNA expression of Hl-Tre was ubiquitously detected in all selected tissues, with the highest level being detected in the intestine. By in situ hybridization (ISH), the positive Hl-Tre signals were observed in the brush borders of the intestinal mucosa. In embryonic and larval stages, the transcript levels of Hl-Tre decreased during embryonic development and increased after the pentactula stage. After a challenge of starvation, the intestinal Hl-Tre mRNA levels were observed to be first decreased and partially recovered thereafter. Overall, our study provided the first evidence for trehalase in echinoderms and showed that this enzyme was potentially linked to a trehalose metabolic pathway in sea cucumbers. Copyright © 2017. Published by Elsevier B.V.

Dose-dependent Effect of Boric Acid on Myogenic Differentiation of Human Adipose-derived Stem Cells (hADSCs).

PubMed

Apdik, Hüseyin; Doğan, Ayşegül; Demirci, Selami; Aydın, Safa; Şahin, Fikrettin

2015-06-01

Boron, a vital micronutrient for plant metabolism, is not fully elucidated for embryonic and adult body development, and tissue regeneration. Although optimized amount of boron supplement has been shown to be essential for normal gestational development in zebrafish and frog and beneficial for bone regeneration in higher animals, effects of boron on myogenesis and myo-regeneration remains to be solved. In the current study, we investigated dose-dependent activity of boric acid on myogenic differentiation of human adipose-derived stem cells (hADSCs) using immunocytochemical, gene, and protein expression analysis. The results revealed that while low- (81.9 μM) and high-dose (819.6 μM) boron treatment increased myogenic gene expression levels such as myosin heavy chain (MYH), MyoD, myogenin, and desmin at day 4 of differentiation, high-dose treatment decreased myogenic-related gene and protein levels at day 21 of differentiation, confirmed by immunocytochemical analysis. The findings of the study present not only an understanding of boron's effect on myogenic differentiation but also an opportunity for the development of scaffolds to be used in skeletal tissue engineering and supplements for embryonic muscle growth. However, fine dose tuning and treatment period arranging are highly warranted as boron treatment over required concentrations and time might result in detrimental outcomes to myogenesis and myo-regeneration.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

PubMed

Muñoz, M; Martin, D; Carrocera, S; Alonso-Guervos, M; Mora, M I; Corrales, F J; Peynot, N; Giraud-Delville, C; Duranthon, V; Sandra, O; Gómez, E

2017-10-01

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Notochord isolation using laser capture microdissection.

PubMed

Santegoeds, R G C; Yakkioui, Y; Jahanshahi, A; Raven, G; Van Overbeeke, J J; Herrler, A; Temel, Y

2017-03-01

Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

The dual-specificity protein phosphatase DUSP9/MKP-4 is essential for placental function but is not required for normal embryonic development.

PubMed

Christie, Graham R; Williams, David J; Macisaac, Fiona; Dickinson, Robin J; Rosewell, Ian; Keyse, Stephen M

2005-09-01

To elucidate the physiological role(s) of DUSP9 (dual-specificity phosphatase 9), also known as MKP-4 (mitogen-activated protein kinase [MAPK] phosphatase 4), the gene was deleted in mice. Crossing male chimeras with wild-type females resulted in heterozygous (DUSP9(+/-)) females. However, when these animals were crossed with wild-type (DUSP9(+/y)) males none of the progeny carried the targeted DUSP9 allele, indicating that both female heterozygous and male null (DUSP9(-/y)) animals die in utero. The DUSP9 gene is on the X chromosome, and this pattern of embryonic lethality is consistent with the selective inactivation of the paternal X chromosome in the extraembryonic tissues of the mouse, suggesting that DUSP9/MKP4 performs an essential function during placental development. Examination of embryos between 8 and 10.5 days postcoitum confirmed that lethality was due to a failure of labyrinth development, and this correlates exactly with the normal expression pattern of DUSP9/MKP-4 in the trophoblast giant cells and labyrinth of the placenta. Finally, when the placental defect was rescued, male null (DUSP9(-/y)) embryos developed to term, appeared normal, and were fertile. Our results indicate that DUSP9/MKP-4 is essential for placental organogenesis but is otherwise dispensable for mammalian embryonic development and highlights the critical role of dual-specificity MAPK phosphatases in the regulation of developmental outcomes in vertebrates.

The life and works of S100P - from conception to cancer

PubMed Central

Prica, Filip; Radon, Tomasz; Cheng, Yuzhu; Crnogorac-Jurcevic, Tatjana

2016-01-01

Since its discovery in 1992, the small, 10.4 kDa calcium-binding protein S100P has gained the attention of researchers from different scientific fields due to its potential roles in both healthy and neoplastic tissues. Although not ubiquitously expressed, in tissues where it is present, S100P is associated with distinct changes in cellular behaviour. In this review we have summarized the evolutionary history of S100P, its expression and involvement in implantation and human embryonic development, as well as important functions in normal tissue and cancer. Finally, we have demonstrated its pivotal role as a potential diagnostic and therapeutic target, which opens promising avenues for further fruitful research on S100P. PMID:27186425

Extraembryonic origin of circulating endothelial cells.

PubMed

Pardanaud, Luc; Eichmann, Anne

2011-01-01

Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells.

PubMed

Pettinato, Giuseppe; Vanden Berg-Foels, Wendy S; Zhang, Ning; Wen, Xuejun

2014-01-01

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

Uniform Embryoid Body Production and Enhanced Mesendoderm Differentiation with Murine Embryonic Stem Cells in a Rotary Suspension Bioreactor.

PubMed

Lei, Xiaohua; Deng, Zhili; Duan, Enkui

2016-01-01

Embryonic stem cells (ESCs) are capable of differentiating into almost all cell types in vitro and hold great promise for drug screening, developmental studies and have a huge potential in many therapeutic areas. ESCs can aggregate to form embryoid body (EB) in static suspension culture by spontaneous differentiation, which resembles an intact embryo; while static suspension culture cannot prevent agglomeration of cells and offers little control over the size and shape of EBs, it results in aggregation of EBs into large, irregular masses, which prejudice the efficiency of differentiation of cells. Recently, bioreactor-based platforms have been shown to not only offer a beneficial effect on increasing diffusion of nutrients and oxygen which promotes cell viability and proliferation but also display local biomechanical properties (e.g., low fluid shear stresses and hydrodynamic force) in tissue development and organogenesis. This chapter describes a protocol for using a rotary suspension bioreactor to produce embryoid bodies and process the differentiation of mouse embryonic stem cells (mESCs), and to assess the efficiency of EB differentiation in the bioreactor by real-time PCR and immunostaining.

Three-dimensional bioprinting using self-assembling scalable scaffold-free “tissue strands” as a new bioink

PubMed Central

Yu, Yin; Moncal, Kazim K.; Li, Jianqiang; Peng, Weijie; Rivero, Iris; Martin, James A.; Ozbolat, Ibrahim T.

2016-01-01

Recent advances in bioprinting have granted tissue engineers the ability to assemble biomaterials, cells, and signaling molecules into anatomically relevant functional tissues or organ parts. Scaffold-free fabrication has recently attracted a great deal of interest due to the ability to recapitulate tissue biology by using self-assembly, which mimics the embryonic development process. Despite several attempts, bioprinting of scale-up tissues at clinically-relevant dimensions with closely recapitulated tissue biology and functionality is still a major roadblock. Here, we fabricate and engineer scaffold-free scalable tissue strands as a novel bioink material for robotic-assisted bioprinting technologies. Compare to 400 μm-thick tissue spheroids bioprinted in a liquid delivery medium into confining molds, near 8 cm-long tissue strands with rapid fusion and self-assemble capabilities are bioprinted in solid form for the first time without any need for a scaffold or a mold support or a liquid delivery medium, and facilitated native-like scale-up tissues. The prominent approach has been verified using cartilage strands as building units to bioprint articular cartilage tissue. PMID:27346373

Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

PubMed Central

Davies, Erin L; Lei, Kai; Seidel, Christopher W; Kroesen, Amanda E; McKinney, Sean A; Guo, Longhua; Robb, Sofia MC; Ross, Eric J; Gotting, Kirsten; Alvarado, Alejandro Sánchez

2017-01-01

Planarian neoblasts are pluripotent, adult somatic stem cells and lineage-primed progenitors that are required for the production and maintenance of all differentiated cell types, including the germline. Neoblasts, originally defined as undifferentiated cells residing in the adult parenchyma, are frequently compared to embryonic stem cells yet their developmental origin remains obscure. We investigated the provenance of neoblasts during Schmidtea mediterranea embryogenesis, and report that neoblasts arise from an anarchic, cycling piwi-1+ population wholly responsible for production of all temporary and definitive organs during embryogenesis. Early embryonic piwi-1+ cells are molecularly and functionally distinct from neoblasts: they express unique cohorts of early embryo enriched transcripts and behave differently than neoblasts in cell transplantation assays. Neoblast lineages arise as organogenesis begins and are required for construction of all major organ systems during embryogenesis. These subpopulations are continuously generated during adulthood, where they act as agents of tissue homeostasis and regeneration. DOI: http://dx.doi.org/10.7554/eLife.21052.001 PMID:28072387

Pluripotent hybrid cells contribute to extraembryonic as well as embryonic tissues.

PubMed

Do, Jeong Tae; Choi, Hyun Woo; Choi, Youngsok; Schöler, Hans R

2011-06-01

The restricted gene expression of a differentiated cell can be reversed by forming hybrid with embryonic stem cells (ESCs). The resulting hybrid cells showed not only an ESC-specific marker expression but also a differentiation potential similar to the pluripotent fusion partner. Here, we evaluated whether the tetraploid fusion hybrid cells have a unique differentiation potential compared with diploid pluripotent cells. The first Oct4-GFP-positive cells were observed at day 2 following fusion between ESCs and neurosphere cells (OG2(+/-)/ROSA26(+/-)). Reprogramming efficiency was as high as 94.5% at passage 5 and 96.4% at passage 13. We have found that the tetraploid hybrid cells could form chimera with contribution to placenta after blastocyst injection. This result indicates that the tetraploid pluripotent fusion hybrid cells have wide range of differentiation potential. Therefore, we suggest that once the somatic cells are reprogrammed by fusion with ESCs, the tetraploid hybrid cells contributed to the extraembryonic as well as embryonic tissues.

Mechanical signaling coordinates the embryonic heart

NASA Astrophysics Data System (ADS)

Chiou, Kevin; Rocks, Jason; Prosser, Benjamin; Discher, Dennis; Liu, Andrea

The heart is an active material which relies on robust signaling mechanisms between cells in order to produce well-timed, coordinated beats. Heart tissue is composed primarily of active heart muscle cells (cardiomyocytes) embedded in a passive extracellular matrix. During a heartbeat, cardiomyocyte contractions are coordinated across the heart to form a wavefront that propagates through the tissue to pump blood. In the adult heart, this contractile wave is coordinated via intercellular electrical signaling.Here we present theoretical and experimental evidence for mechanical coordination of embryonic heartbeats. We model cardiomyocytes as mechanically excitable Eshelby inclusions embedded in an overdamped elastic-fluid biphasic medium. For physiological parameters, this model replicates recent experimental measurements of the contractile wavefront which are not captured by electrical signaling models. We additionally challenge our model by pharmacologically blocking gap junctions, inhibiting electrical signaling between myocytes. We find that while adult hearts stop beating almost immediately after gap junctions are blocked, embryonic hearts continue beating even at significantly higher concentrations, providing strong support for a mechanical signaling mechanism.

Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

PubMed

Yang, Yang; Liu, Bei; Xu, Jun; Wang, Jinlin; Wu, Jun; Shi, Cheng; Xu, Yaxing; Dong, Jiebin; Wang, Chengyan; Lai, Weifeng; Zhu, Jialiang; Xiong, Liang; Zhu, Dicong; Li, Xiang; Yang, Weifeng; Yamauchi, Takayoshi; Sugawara, Atsushi; Li, Zhongwei; Sun, Fangyuan; Li, Xiangyun; Li, Chen; He, Aibin; Du, Yaqin; Wang, Ting; Zhao, Chaoran; Li, Haibo; Chi, Xiaochun; Zhang, Hongquan; Liu, Yifang; Li, Cheng; Duo, Shuguang; Yin, Ming; Shen, Huan; Belmonte, Juan Carlos Izpisua; Deng, Hongkui

2017-04-06

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT. Copyright © 2017 Elsevier Inc. All rights reserved.

Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

PubMed

Morizane, Ryuji; Monkawa, Toshiaki; Fujii, Shizuka; Yamaguchi, Shintaro; Homma, Koichiro; Matsuzaki, Yumi; Okano, Hideyuki; Itoh, Hiroshi

2014-01-01

Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

Functional tooth restoration by next-generation bio-hybrid implant as a bio-hybrid artificial organ replacement therapy

PubMed Central

Oshima, Masamitsu; Inoue, Kaoru; Nakajima, Kei; Tachikawa, Tetsuhiko; Yamazaki, Hiromichi; Isobe, Tomohide; Sugawara, Ayaka; Ogawa, Miho; Tanaka, Chie; Saito, Masahiro; Kasugai, Shohei; Takano-Yamamoto, Teruko; Inoue, Takashi; Tezuka, Katsunari; Kuboki, Takuo; Yamaguchi, Akira; Tsuji, Takashi

2014-01-01

Bio-hybrid artificial organs are an attractive concept to restore organ function through precise biological cooperation with surrounding tissues in vivo. However, in bio-hybrid artificial organs, an artificial organ with fibrous connective tissues, including muscles, tendons and ligaments, has not been developed. Here, we have enveloped with embryonic dental follicle tissue around a HA-coated dental implant, and transplanted into the lower first molar region of a murine tooth-loss model. We successfully developed a novel fibrous connected tooth implant using a HA-coated dental implant and dental follicle stem cells as a bio-hybrid organ. This bio-hybrid implant restored physiological functions, including bone remodelling, regeneration of severe bone-defect and responsiveness to noxious stimuli, through regeneration with periodontal tissues, such as periodontal ligament and cementum. Thus, this study represents the potential for a next-generation bio-hybrid implant for tooth loss as a future bio-hybrid artificial organ replacement therapy. PMID:25116435

Non-conventional protrusions: the diversity of cell interactions at short and long distance.

PubMed

Caviglia, Sara; Ober, Elke A

2018-06-08

Cells use different means to communicate within and between tissues and thereby coordinate their behaviours. Following the initial observations of enigmatic long filopodia unrelated to cell movement, it became clear that the roles of cellular protrusions are not restricted to sensing functions or motility and are much more diverse than previously appreciated. Advances in live-imaging and genetic tools revealed several types of non-conventional cell protrusions and their functions, ranging from tissue patterning, proliferation and differentiation control, tissue matching and cell spacing to more unexpected roles such as priming of cell adhesion as well as bidirectional coordination of tissue movements. Here, we will highlight exciting new insights into highly diverse cell behaviours elicited by protrusions and contact-dependent cell communication, essential for embryonic development across species. Copyright © 2018. Published by Elsevier Ltd.

Non-invasive structural and biomechanical imaging of the developing embryos (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhang, Jitao; Wu, Chen; Raghunathan, Raksha; Larin, Kirill V.; Scarcelli, Giuliano

2017-02-01

Embryos undergo dramatic changes in size, shape, and mechanical properties during development, which is regulated by both genetic and environmental factors. Quantifying mechanical properties of different embryonic tissues may represent good metrics for the embryonic health and proper development. Alternations and structure coupled with biomechanical information may provide a way for early diagnosis and drug treatment of various congenital diseases. Many methods have been developed to determine the mechanical properties of the embryo, such as atomic force microscopy (AFM), ultrasound elastography (UE), and optical coherent elastography (OCE). However, AFM is invasive and time-consuming. While UE and OCE are both non-invasive methods, the spatial resolutions are limited to mm to sub-mm, which is not enough to observe the details inside the embryo. Brillouin microscopy can potentially enable non-invasive measurement of the mechanical properties of a sample by measuring the spectra of acoustically induced light scattering therein. It has fast speed ( 0.1 second per point) and high resolution (sub-micron), and thus has been widely investigated for biomedical application, such as single cell and tissue. In this work, we utilized this technique to characterize the mechanical property of an embryo. A 2D elasticity imaging of the whole body of an E8 embryo was acquired by a Brillouin microscopy, and the stiffness changes between different organs (such as brain, heart, and spine) were shown. The elasticity maps were correlated with structural information provided by OCT.

High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring.

PubMed

Bahous, Renata H; Jadavji, Nafisa M; Deng, Liyuan; Cosín-Tomás, Marta; Lu, Jessica; Malysheva, Olga; Leung, Kit-Yi; Ho, Ming-Kai; Pallàs, Mercè; Kaliman, Perla; Greene, Nicholas D E; Bedell, Barry J; Caudill, Marie A; Rozen, Rima

2017-03-01

Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-week-old male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippocampus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deficiency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These findings highlight the unintended negative consequences of supplemental folic acid. © The Author 2017. Published by Oxford University Press.

Early embryonic sensitivity to cyclophosphamide in cardiac differentiation from human embryonic stem cells.

PubMed

Zhu, Ming-Xia; Zhao, Jin-Yuan; Chen, Gui-An; Guan, Li

2011-09-01

hESCs (human embryonic stem cells) can differentiate into tissue derivatives of all three germ layers in vitro and mimic the development of the embryo in vivo. In this study, we have investigated the potential of an hESC-based assay for the detection of toxicity to cardiac differentiation in embryonic development. First of all, we developed the protocol of cardiac induction from hESCs according to our previous work and distinguished cardiac precursor cells and late mature cardiomyocytes from differentiated cells, demonstrated by the Q-PCR (quantitative real-time PCR), immunocytochemistry and flow cytometry analysis. In order to test whether CPA (cyclophosphamide) induces developmental and cellular toxicity in the human embryo, we exposed the differentiating cells from hESCs to CPA (a well-known proteratogen) at different stages. We have found that a high concentration of CPA could inhibit cardiac differentiation of hESCs. Two separate exposure intervals were used to determine the effects of CPA on cardiac precursor cells and late mature cardiomyocytes respectively. The cardiac precursor cells were sensitive to CPA in non-cytotoxic concentrations for the expression of the cardiac-specific mRNA markers Nkx2.5 (NK2 transcription factor related, locus 5), GATA-4 (GATA binding protein 4 transcription factor) and TNNT2 (troponin T type 2). Non-cytotoxic CPA concentrations did not affect the mRNA markers' expression in late mature cardiomyocytes, indicating that cardiac precursors were more sensitive to CPA than late cardiomyocytes in cardiogenesis. We set up the in vitro developmental toxicity test model so as to reduce the number of test animals and expenses without compromising the safety of consumers and patients. Furthermore, such in vitro methods may be possibly suited to test a large number of chemicals than the classical employed in vivo tests.

HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development.

PubMed

Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie; Barozzi, Iros; Osterwalder, Marco; Akiyama, Jennifer A; Lincoln, Joy; Lopez-Rios, Javier; Visel, Axel; Zuniga, Aimée; Zeller, Rolf

2017-05-23

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development

DOE PAGES

Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie; ...

2017-05-23

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost frommore » Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.« less

HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development

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

Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost frommore » Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.« less

Spatially resolved RNA-sequencing of the embryonic heart identifies a role for Wnt/β-catenin signaling in autonomic control of heart rate

PubMed Central

Burkhard, Silja Barbara

2018-01-01

Development of specialized cells and structures in the heart is regulated by spatially -restricted molecular pathways. Disruptions in these pathways can cause severe congenital cardiac malformations or functional defects. To better understand these pathways and how they regulate cardiac development we used tomo-seq, combining high-throughput RNA-sequencing with tissue-sectioning, to establish a genome-wide expression dataset with high spatial resolution for the developing zebrafish heart. Analysis of the dataset revealed over 1100 genes differentially expressed in sub-compartments. Pacemaker cells in the sinoatrial region induce heart contractions, but little is known about the mechanisms underlying their development. Using our transcriptome map, we identified spatially restricted Wnt/β-catenin signaling activity in pacemaker cells, which was controlled by Islet-1 activity. Moreover, Wnt/β-catenin signaling controls heart rate by regulating pacemaker cellular response to parasympathetic stimuli. Thus, this high-resolution transcriptome map incorporating all cell types in the embryonic heart can expose spatially restricted molecular pathways critical for specific cardiac functions. PMID:29400650

Conference Scene: Induced pluripotent cells: a new path for regenerative medicine. 7 October 2010, BioPark, Welwyn Garden City, Hertfordshire, UK.

PubMed

Crutzen, Hélène S G

2011-01-01

Embryonic stem cells and induced pluripotent stem (iPS) cells, which are embryonic stem-like cells derived from adult tissues, have the broadest differentiation potential. These cells are unique in their ability to self-renew, to be maintained in an undifferentiated state for long periods of culturing and to give rise to many different cell lineages including germ-line cells. They therefore represent an invaluable tool for facilitating research towards the realization of regenerative medicine. The recent developments in embryonic stem cell and iPS cell technology have allowed human cell models to be developed that will hopefully provide novel platforms for disease analysis not only at the basic science level, but also for drug discovery and screening, and other clinical applications. This 1-day conference, chaired by Professor Peter Andrews from the University of Sheffield, UK, and Dr Chris Denning from the University of Nottingham, UK, focused on generation of iPS cells, their differentiation into specific fates and applications to disease modeling. It consisted of 11 talks by UK-based and international researchers, and three posters; Ms Azra Fatima from Cologne University, Germany, won the competition for her poster on the derivation of iPS cells from a patient with arrhythmogenic right ventricular cardiomyopathy.

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster.

PubMed

Cantera, Rafael; Lüer, Karin; Rusten, Tor Erik; Barrio, Rosa; Kafatos, Fotis C; Technau, Gerhard M

2002-12-01

The gene spalt is expressed in the embryonic central nervous system of Drosophila melanogaster but its function in this tissue is still unknown. To investigate this question, we used a combination of techniques to analyse spalt mutant embryos. Electron microscopy showed that in the absence of spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the beta-catenin Armadillo, its ligand N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organisation of the central nervous system.

Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

PubMed

Kitta, Ryo; Kuwamoto, Marina; Yamahama, Yumi; Mase, Keisuke; Sawada, Hiroshi

2016-12-01

To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori. © 2016 Japanese Society of Developmental Biologists.

Pteropod eggs released at high pCO2 lack resilience to ocean acidification

PubMed Central

Manno, Clara; Peck, Victoria L.; Tarling, Geraint A.

2016-01-01

The effects of ocean acidification (OA) on the early recruitment of pteropods in the Scotia Sea, was investigated considering the process of spawning, quality of the spawned eggs and their capacity to develop. Maternal OA stress was induced on female pteropods (Limacina helicina antarctica) through exposure to present day pCO2 conditions and two potential future OA states (750 μatm and 1200 μatm). The eggs spawned from these females, both before and during their exposure to OA, were incubated themselves in this same range of conditions (embryonic OA stress). Maternal OA stress resulted in eggs with lower carbon content, while embryonic OA stress retarded development. The combination of maternal and embryonic OA stress reduced the percentage of eggs successfully reaching organogenesis by 80%. We propose that OA stress not only affects the somatic tissue of pteropods but also the functioning of their gonads. Corresponding in-situ sampling found that post-larval L. helicina antarctica concentrated around 600 m depth, which is deeper than previously assumed. A deeper distribution makes their exposure to waters undersaturated for aragonite more likely in the near future given that these waters are predicted to shoal from depth over the coming decades. PMID:27181210

Identifying the architecture of a supracellular actomyosin network that induces tissue folding

NASA Astrophysics Data System (ADS)

Yevick, Hannah; Stoop, Norbert; Dunkel, Jorn; Martin, Adam

During embryonic development, the establishment of correct tissue form ensures proper tissue function. Yet, how the thousands of cells within a tissue coordinate force production to sculpt tissue shape is poorly understood. One important tissue shape change is tissue folding where a cell sheet bends to form a closed tube. Drosophila (fruit fly) embryos undergo such a folding event, called ventral furrow formation. The ventral furrow is associated with a supracellular network of actin and myosin, where actin-myosin fibers assemble and connect between cells. It is not known how this tissue-wide network grows and connects over time, how reproducible it is between embryos, and what determines its architecture. Here, we used topological feature analysis to quantitatively and dynamically map the connections and architecture of this supracellular network across hundreds of cells in the folding tissue. We identified the importance of the cell unit in setting up the tissue-scale architecture of the network. Our mathematical framework allows us to explore stereotypic properties of the myosin network such that we can investigate the reproducibility of mechanical connections for a morphogenetic process. NIH F32.

Contact inhibition of locomotion determines cell-cell and cell-substrate forces in tissues.

PubMed

Zimmermann, Juliane; Camley, Brian A; Rappel, Wouter-Jan; Levine, Herbert

2016-03-08

Cells organized in tissues exert forces on their neighbors and their environment. Those cellular forces determine tissue homeostasis as well as reorganization during embryonic development and wound healing. To understand how cellular forces are generated and how they can influence the tissue state, we develop a particle-based simulation model for adhesive cell clusters and monolayers. Cells are contractile, exert forces on their substrate and on each other, and interact through contact inhibition of locomotion (CIL), meaning that cell-cell contacts suppress force transduction to the substrate and propulsion forces align away from neighbors. Our model captures the traction force patterns of small clusters of nonmotile cells and larger sheets of motile Madin-Darby canine kidney (MDCK) cells. In agreement with observations in a spreading MDCK colony, the cell density in the center increases as cells divide and the tissue grows. A feedback between cell density, CIL, and cell-cell adhesion gives rise to a linear relationship between cell density and intercellular tensile stress and forces the tissue into a nonmotile state characterized by a broad distribution of traction forces. Our model also captures the experimentally observed tissue flow around circular obstacles, and CIL accounts for traction forces at the edge.

Skeletogenesis in the swell shark Cephaloscyllium ventriosum

PubMed Central

Eames, B Frank; Allen, Nancy; Young, Jonathan; Kaplan, Angelo; Helms, Jill A; Schneider, Richard A

2007-01-01

Extant chondrichthyans possess a predominantly cartilaginous skeleton, even though primitive chondrichthyans produced bone. To gain insights into this peculiar skeletal evolution, and in particular to evaluate the extent to which chondrichthyan skeletogenesis retains features of an osteogenic programme, we performed a histological, histochemical and immunohistochemical analysis of the entire embryonic skeleton during development of the swell shark Cephaloscyllium ventriosum. Specifically, we compared staining properties among various mineralizing tissues, including neural arches of the vertebrae, dermal tissues supporting oral denticles and Meckel's cartilage of the lower jaw. Patterns of mineralization were predicted by spatially restricted alkaline phosphatase activity earlier in development. Regarding evidence for an osteogenic programme in extant sharks, a mineralized tissue in the perichondrium of C. ventriosum neural arches, and to a lesser extent a tissue supporting the oral denticle, displayed numerous properties of bone. Although we uncovered many differences between tissues in Meckel's cartilage and neural arches of C. ventriosum, both elements impart distinct tissue characteristics to the perichondral region. Considering the evolution of osteogenic processes, shark skeletogenesis may illuminate the transition from perichondrium to periosteum, which is a major bone-forming tissue during the process of endochondral ossification. PMID:17451531

Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3.

PubMed

Kapacee, Zoher; Yeung, Ching-Yan Chloé; Lu, Yinhui; Crabtree, David; Holmes, David F; Kadler, Karl E

2010-10-01

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7days. Copyright © 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Continuum theory of gene expression waves during vertebrate segmentation.

PubMed

Jörg, David J; Morelli, Luis G; Soroldoni, Daniele; Oates, Andrew C; Jülicher, Frank

2015-09-01

The segmentation of the vertebrate body plan during embryonic development is a rhythmic and sequential process governed by genetic oscillations. These genetic oscillations give rise to traveling waves of gene expression in the segmenting tissue. Here we present a minimal continuum theory of vertebrate segmentation that captures the key principles governing the dynamic patterns of gene expression including the effects of shortening of the oscillating tissue. We show that our theory can quantitatively account for the key features of segmentation observed in zebrafish, in particular the shape of the wave patterns, the period of segmentation and the segment length as a function of time.

Continuum theory of gene expression waves during vertebrate segmentation

PubMed Central

Jörg, David J; Morelli, Luis G; Soroldoni, Daniele; Oates, Andrew C; Jülicher, Frank

2015-01-01

Abstract The segmentation of the vertebrate body plan during embryonic development is a rhythmic and sequential process governed by genetic oscillations. These genetic oscillations give rise to traveling waves of gene expression in the segmenting tissue. Here we present a minimal continuum theory of vertebrate segmentation that captures the key principles governing the dynamic patterns of gene expression including the effects of shortening of the oscillating tissue. We show that our theory can quantitatively account for the key features of segmentation observed in zebrafish, in particular the shape of the wave patterns, the period of segmentation and the segment length as a function of time. PMID:28725158

Iridovirus infection of cell cultures from the Diaprepes root weevil, Diaprepes abbreviatus

PubMed Central

Hunter, W. B.; Lapointe, S. L.

2003-01-01

We here report the development and viral infection of a Diaprepes root weevil cell culture. Embryonic tissues of the root weevil were used to establish cell cultures for use in screening viral pathogens as potential biological control agents. Tissues were seeded into a prepared solution of insect medium and kept at a temperature of 24°C. The cell culture had primarily fibroblast-like morphology with some epithelial monolayers. Root weevil cells were successfully infected in vitro with a known insect virus, Invertebrate Iridescent Virus 6. Potential uses of insect cell cultures and insect viruses are discussed. Abbreviation: IIV-6 Invertebrate Iridescent Virus 6 PMID:15841252

Molecular controls of arterial morphogenesis.

PubMed

Simons, Michael; Eichmann, Anne

2015-05-08

Formation of arterial vasculature, here termed arteriogenesis, is a central process in embryonic vascular development as well as in adult tissues. Although the process of capillary formation, angiogenesis, is relatively well understood, much remains to be learned about arteriogenesis. Recent discoveries point to the key role played by vascular endothelial growth factor receptor 2 in control of this process and to newly identified control circuits that dramatically influence its activity. The latter can present particularly attractive targets for a new class of therapeutic agents capable of activation of this signaling cascade in a ligand-independent manner, thereby promoting arteriogenesis in diseased tissues. © 2015 American Heart Association, Inc.

Nanoengineered surfaces for focal adhesion guidance trigger mesenchymal stem cell self-organization and tenogenesis.

PubMed

Iannone, Maria; Ventre, Maurizio; Formisano, Lucia; Casalino, Laura; Patriarca, Eduardo J; Netti, Paolo A

2015-03-11

The initial conditions for morphogenesis trigger a cascade of events that ultimately dictate structure and functions of tissues and organs. Here we report that surface nanopatterning can control the initial assembly of focal adhesions, hence guiding human mesenchymal stem cells (hMSCs) through the process of self-organization and differentiation. This process self-sustains, leading to the development of macroscopic tissues with molecular profiles and microarchitecture reminiscent of embryonic tendons. Therefore, material surfaces can be in principle engineered to set off the hMSC program toward tissuegenesis in a deterministic manner by providing adequate sets of initial environmental conditions.

α5β1 Integrin-Fibronectin Interactions Specify Liquid to Solid Phase Transition of 3D Cellular Aggregates

PubMed Central

Caicedo-Carvajal, Carlos E.; Shinbrot, Troy; Foty, Ramsey A.

2010-01-01

Background Tissue organization during embryonic development and wound healing depends on the ability of cells on the one hand to exchange adhesive bonds during active rearrangement and on the other to become fixed in place as tissue homeostasis is reached. Cells achieve these contradictory tasks by regulating either cell-cell adhesive bonds, mediated by cadherins, or cell-extracellular matrix (ECM) connections, regulated by integrins. Integrin α5β1 and soluble fibronectin (sFN) are key players in cell-ECM force generation and in ECM polymerization. Here, we explore the interplay between integrin α5β1 and sFN and its influence on tissue mechanical properties and cell sorting behavior. Methodology/Principal Findings We generated a series of cell lines varying in α5β1 receptor density. We then systematically explored the effects of different sFN concentrations on aggregate biomechanical properties using tissue surface tensiometry. We found previously unreported complex behaviors including the observation that interactions between fibronectin and integrin α5β1 generates biphasic tissue cohesion profiles. Specifically, we show that at constant sFn concentration, aggregate cohesion increases linearly as α5β1 receptor density is increased from low to moderate levels, producing a transition from viscoelastic-liquid to pseudo viscoelastic-solid behavior. However, further increase in receptor density causes an abrupt drop in tissue cohesion and a transition back to viscoelastic-liquid properties. We propose that this may be due to depletion of sFn below a critical value in the aggregate microenvironment at high α5β1 levels. We also show that differential expression of α5β1 integrin can promote phase-separation between cells. Conclusions/Significance The interplay between α5-integrin and sFn contributes significantly to tissue cohesion and, depending on their level of expression, can mediate a shift from liquid to elastic behavior. This interplay represents a tunable level of control between integrins and the ECM that can influence tissue cohesion and other mechanical properties, which may translate to the specification of tissue structure and function. These studies provide insights into important biological processes such as embryonic development, wound healing, and for tissue engineering applications. PMID:20686611

Integrated annotation and analysis of in situ hybridization images using the ImAnno system: application to the ear and sensory organs of the fetal mouse.

PubMed

Romand, Raymond; Ripp, Raymond; Poidevin, Laetitia; Boeglin, Marcel; Geffers, Lars; Dollé, Pascal; Poch, Olivier

2015-01-01

An in situ hybridization (ISH) study was performed on 2000 murine genes representing around 10% of the protein-coding genes present in the mouse genome using data generated by the EURExpress consortium. This study was carried out in 25 tissues of late gestation embryos (E14.5), with a special emphasis on the developing ear and on five distinct developing sensory organs, including the cochlea, the vestibular receptors, the sensory retina, the olfactory organ, and the vibrissae follicles. The results obtained from an analysis of more than 11,000 micrographs have been integrated in a newly developed knowledgebase, called ImAnno. In addition to managing the multilevel micrograph annotations performed by human experts, ImAnno provides public access to various integrated databases and tools. Thus, it facilitates the analysis of complex ISH gene expression patterns, as well as functional annotation and interaction of gene sets. It also provides direct links to human pathways and diseases. Hierarchical clustering of expression patterns in the 25 tissues revealed three main branches corresponding to tissues with common functions and/or embryonic origins. To illustrate the integrative power of ImAnno, we explored the expression, function and disease traits of the sensory epithelia of the five presumptive sensory organs. The study identified 623 genes (out of 2000) concomitantly expressed in the five embryonic epithelia, among which many (∼12%) were involved in human disorders. Finally, various multilevel interaction networks were characterized, highlighting differential functional enrichments of directly or indirectly interacting genes. These analyses exemplify an under-represention of "sensory" functions in the sensory gene set suggests that E14.5 is a pivotal stage between the developmental stage and the functional phase that will be fully reached only after birth.

Intermediate filament protein nestin is expressed in developing meninges.

PubMed

Yay, A; Ozdamar, S; Canoz, O; Baran, M; Tucer, B; Sonmez, M F

2014-01-01

Nestin is a type VI intermediate filament protein known as a marker for progenitor cells that can be mostly found in tissues during the embryonic and fetal periods. In our study, we aimed to determine the expression of nestin in meninges covering the brain tissue at different developmental stages and in the new born. In this study 10 human fetuses in different development stages between developmental weeks 9-34 and a newborn brain tissue were used. Fetuses in paraffin section were stained with H+E and nestin immunohistochemical staining protocol was performed. In this study, in the human meninges intense nestin expression was detected as early as in the 9th week of development. Intensity of this expression gradually decreased in later stages of development and nestin expression still persisted in a small population of newborn meningeal cells. In the present study, nestin positive cells gradually diminished in the developing and maturing meninges during the fetal period. This probably depends on initiation of a decrease in nestin expression and replacement with other tissue-specific intermediate filaments while the differentiation process continues. These differences can make significant contributions to the investigation and diagnosis of various pathological disorders (Tab. 1, Fig. 3, Ref. 36).

Embryonic toxico-pathological effects of meglumine antimoniate using a chick embryo model.

PubMed

Khosravi, Ahmad; Sharifi, Iraj; Tavakkoli, Hadi; Derakhshanfar, Amin; Keyhani, Ali Reza; Salari, Zohreh; Mosallanejad, Seyedeh Saedeh; Bamorovat, Mehdi

2018-01-01

Leishmaniasis is one of the diverse and neglected tropical diseases. Embryo-toxicity of drugs has always been a major concern. Chick embryo is a preclinical model relevant in the assessment of adverse effects of drugs. The current study aimed to assess embryonic histopathological disorders and amniotic fluid biochemical changes following meglumine antimoniate treatment. The alteration of vascular branching pattern in the chick's extra-embryonic membrane and exploration of molecular cues to early embryonic vasculogenesis and angiogenesis were also quantified. Embryonated chicken eggs were treated with 75 or 150 mg/kg of meglumine antimoniate. Embryo malformations, growth retardation and haemorrhages on the external body surfaces were accompanied by histopathological lesions in the brain, kidney, liver and heart in a dose-dependent manner. Significant rise occurred in the biochemical indices of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and amylase in the amniotic fluid. Quantification of the extra-embryonic membrane vasculature showed that the anti-angiogenic and anti-vasculogenic effects of the drug were revealed by a significant decrease in fractal dimension value and mean capillary area. The relative expression levels of vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 mRNA also significantly reduced. Concerns of a probable teratogenicity of meglumine antimoniate were established by data presented in this study. It is concluded that tissue lesions, amniotic fluid disturbance, altered early extra-embryonic vascular development and gene expression as well as the consecutive cascade of events, might eventually lead to developmental defects in embryo following meglumine antimoniate treatment. Therefore, the use of meglumine antimoniate during pregnancy should be considered as potentially embryo-toxic. Hence, physicians should be aware of such teratogenic effects and limit the use of this drug during the growing period of the fetus, particularly in rural communities. Further pharmaceutical investigations are crucial for planning future strategies.

ACTION OF SERUM ON FIBROBLASTS IN VITRO

PubMed Central

Carrel, Alexis; Ebeling, Albert H.

1923-01-01

It may be concluded that, under the conditions of the experiments: 1. The duration of life of fibroblasts is not altered by the presence of 7 per cent serum in a medium composed of fibrin and Tyrode solution, but is slightly decreased when the concentration of the serum reaches 25 per cent. 2. Fibroblasts cultivated in serum or in Tyrode solution are only in a condition of survival; they do not build up new protoplasm from the serum proteins and their mass does not increase. 3. When embryonic tissue juice is added to the medium, the tissues increase in mass. But the rate of growth is the same in media containing 0 per cent and 10 per cent serum. In 25 per cent serum, however, the rate of growth slightly decreases. Even in the presence of embryonic tissue juice, serum does not increase the rate of growth of connective tissue. 4. The nitrogenous compounds contained in serum are not used as food material by fibroblasts growing in vitro. PMID:19868757

Gene expression dynamics during embryonic development in rainbow trout

USDA-ARS?s Scientific Manuscript database

The supply of maternal RNAs in fertilized egg and activation of embryonic genome during maternal-zygotic transition (MZT) are important for normal embryonic development. In order to identify genes and gene products that are essential in the regulation of embryonic development in rainbow trout, RNA-S...

Familial ectodermal dysplasia: a peers' agony.

PubMed

Hegde, Karthik; Kashyap, Roopashri Rajesh; Nair, Gopakumar; Nair, Preeti P

2013-07-23

Ectodermal dysplasias include a various group of inherited disorders which share primary defect in the development of two or more tissues of embryonic ectodermal origin. Though there are many subtypes, ectodermal dysplasias are mainly hidrotic ectodermal dysplasia and hypohidrotic ectodermal dysplasia, among which the most common variety is X linked hypohidrotic ectodermal dysplasia. We report a rare case of X linked hypohidrotic ectodermal dysplasia occurring in a family with various skin, hair and oral abnormalities.

Regulatory RNA Key Player in p53-Mediated Apoptosis in Embryonic Stem Cells | Center for Cancer Research

Cancer.gov

Embryonic stem cells (ESCs) must maintain the integrity of their genomes or risk passing potentially deleterious mutations on to numerous tissues. Thus, ESCs have a unique genome surveillance system and easily undergo apoptosis or differentiation when DNA damage is detected. The protein p53 is known to promote differentiation in mouse ESCs (mESCs), but its role in DNA

Ontogeny and tissue distribution of the chicken Bu-1a antigen.

PubMed Central

Houssaint, E; Diez, E; Pink, J R

1987-01-01

By using a sensitive technique of immunofluorescence on polyethylene glycol-embedded tissue sections, we could better determine the distribution of L22+ cells in embryonic and adult chickens. L22 mAb was originally described as reacting with bursa and bursa-derived lymphocytes. We now present evidence to suggest that this mAb also reacts with a subset of macrophages found in bursa, thymus, spleen, liver, intestine and peritoneum. The L22+ cells appear early during embryonic life, simultaneously in yolk sac, bursa, thymus, spleen and bone marrow. At all steps of their ontogeny, thymocytes were L22-, while most, if not all, bursal lymphoid cells were L22+. Moreover, L22 antigen can be detected on haemopoietic cells probably precursors, before and during their entry into the bursal rudiment on Day 9 or 10 of embryonic life. We conclude from these data that L22 is not restricted to the B-cell lineage as it is shared with a subset of macrophages. Furthermore, our observations of L22+ cells during embryonic life favour the hypothesis of separate lineages for B-cell and T-cell precursors in chick embryo, which was previously put forward on the basis of different sets of experiments. Images Figure 1 Figure 3 Figure 4 PMID:3499381

Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues

PubMed Central

Peyer, Suzanne M.; Pankey, M. Sabrina; Oakley, Todd H.; McFall-Ngai, Margaret J.

2014-01-01

The squid Euprymna scolopes has evolved independent sets of tissues capable of light detection, including a complex eye and a photophore or ‘light organ’, which houses the luminous bacterial symbiont Vibrio fischeri. As the eye and light organ originate from different embryonic tissues, we examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated in the light organ by symbiosis. We obtained sequences of the four genes with PCR, confirmed orthology with phylogenetic analysis, and determined that each was expressed in the eye and light organ. With in situ hybridization (ISH), we localized the gene transcripts in developing embryos, comparing the patterns of expression in the two organs. The four transcripts localized to similar tissues, including those associated with the visual system ~1/4 into embryogenesis (Naef stage 18) and the light organ ~3/4 into embryogenesis (Naef stage 26). We used ISH and quantitative real-time PCR to examine transcript expression and differential regulation in postembryonic light organs in response to the following colonization conditions: wild-type, luminescent V. fischeri; a mutant strain defective in light production; and as a control, no symbiont. In ISH experiments light organs showed down regulation of the pax6, eya, and six transcripts in response to wild-type V. fischeri. Mutant strains also induced down regulation of the pax6 and eya transcripts, but not of the six transcript. Thus, luminescence was required for down regulation of the six transcript. We discuss these results in the context of symbiont-induced light-organ development. Our study indicates that the eye-specification genes are expressed in light-interacting tissues independent of their embryonic origin and are capable of responding to bacterial cues. These results offer evidence for evolutionary tinkering or the recruitment of eye development genes for use in a light-sensing photophore. PMID:24157521

Neural Differentiation of Embryonic Stem Cells In Vitro: A Road Map to Neurogenesis in the Embryo

PubMed Central

Abranches, Elsa; Silva, Margarida; Pradier, Laurent; Schulz, Herbert; Hummel, Oliver; Henrique, Domingos; Bekman, Evguenia

2009-01-01

Background The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. Methodology/Principal Findings In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation, and three gene signatures for subsequent stages of neural progenitor development, from an early stage that follows neural induction to a final stage preceding terminal differentiation. Conclusions/Significance Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons. PMID:19621087

Three-dimensional epithelial tissues generated from human embryonic stem cells.

PubMed

Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

2009-11-01

The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain.

PubMed

Iwashita, Misato; Kataoka, Noriyuki; Toida, Kazunori; Kosodo, Yoichi

2014-10-01

Accumulating evidence implicates the significance of the physical properties of the niche in influencing the behavior, growth and differentiation of stem cells. Among the physical properties, extracellular stiffness has been shown to have direct effects on fate determination in several cell types in vitro. However, little evidence exists concerning whether shifts in stiffness occur in vivo during tissue development. To address this question, we present a systematic strategy to evaluate the shift in stiffness in a developing tissue using the mouse embryonic cerebral cortex as an experimental model. We combined atomic force microscopy measurements of tissue and cellular stiffness with immunostaining of specific markers of neural differentiation to correlate the value of stiffness with the characteristic features of tissues and cells in the developing brain. We found that the stiffness of the ventricular and subventricular zones increases gradually during development. Furthermore, a peak in tissue stiffness appeared in the intermediate zone at E16.5. The stiffness of the cortical plate showed an initial increase but decreased at E18.5, although the cellular stiffness of neurons monotonically increased in association with the maturation of the microtubule cytoskeleton. These results indicate that tissue stiffness cannot be solely determined by the stiffness of the cells that constitute the tissue. Taken together, our method profiles the stiffness of living tissue and cells with defined characteristics and can therefore be utilized to further understand the role of stiffness as a physical factor that determines cell fate during the formation of the cerebral cortex and other tissues. © 2014. Published by The Company of Biologists Ltd.

Ethics and policy in embryonic stem cell research.

PubMed

Robertson, John A

1999-06-01

Embryonic stem cells, which have the potential to save many lives, must be recovered from aborted fetuses or live embyros. Although tissue from aborted fetuses can be used without moral complicity in the underlying abortion, obtaining stem cells from embryos necessarily kills them, thus raising difficult questions about the use of embryonic human material to save others. This article draws on previous controversies over embryo research and distinctions between intrinsic and symbolic moral status to analyze these issues. It argues that stem cell research with spare embryos produced during infertility treatment, or even embryos created specifically for research or therapeutic purposes, is ethically acceptable and should receive federal funding.

Planar Cell Polarity Pathway – Coordinating morphogenetic cell behaviors with embryonic polarity

PubMed Central

Gray, Ryan S.; Roszko, Isabelle; Solnica-Krezel, Lilianna

2011-01-01

Planar cell polarization entails establishment of cellular asymmetries within the tissue plane. An evolutionarily conserved Planar Cell Polarity (PCP) signaling system employs intra- and intercellular feedback interactions between its core components, including Frizzled, Van Gogh, Flamingo, Prickle and Dishevelled, to establish their characteristic asymmetric intracellular distributions and coordinate planar polarity of cell populations. By translating global patterning information into asymmetries of cell membranes and intracellular organelles, PCP signaling coordinates morphogenetic behaviors of individual cells and cell populations with the embryonic polarity. In vertebrates, by polarizing cilia in the node/Kupffer’s vesicle, PCP signaling links the anteroposterior to left-right embryonic polarity. PMID:21763613

A trade-off between embryonic development rate and immune function of avian offspring is concealed by embryonic temperature

USGS Publications Warehouse

Martin, Thomas E.; Arriero, Elena; Majewska, Ania

2011-01-01

Long embryonic periods are assumed to reflect slower intrinsic development that are thought to trade off to allow enhanced physiological systems, such as immune function. Yet, the relatively rare studies of this trade-off in avian offspring have not found the expected trade-off. Theory and tests have not taken into account the strong extrinsic effects of temperature on embryonic periods of birds. Here, we show that length of the embryonic period did not explain variation in two measures of immune function when temperature was ignored, based on studies of 34 Passerine species in tropical Venezuela (23 species) and north temperate Arizona (11 species). Variation in immune function was explained when embryonic periods were corrected for average embryonic temperature, in order to better estimate intrinsic rates of development. Immune function of offspring trades off with intrinsic rates of embryonic development once the extrinsic effects of embryonic temperatures are taken into account.

Unipotent, Atoh1+ progenitors maintain the Merkel cell population in embryonic and adult mice

PubMed Central

Wright, Margaret C.; Reed-Geaghan, Erin G.; Bolock, Alexa M.; Fujiyama, Tomoyuki; Hoshino, Mikio

2015-01-01

Resident progenitor cells in mammalian skin generate new cells as a part of tissue homeostasis. We sought to identify the progenitors of Merkel cells, a unique skin cell type that plays critical roles in mechanosensation. We found that some Atoh1-expressing cells in the hairy skin and whisker follicles are mitotically active at embryonic and postnatal ages. Genetic fate-mapping revealed that these Atoh1-expressing cells give rise solely to Merkel cells. Furthermore, selective ablation of Atoh1+ skin cells in adult mice led to a permanent reduction in Merkel cell numbers, demonstrating that other stem cell populations are incapable of producing Merkel cells. These data identify a novel, unipotent progenitor population in the skin that gives rise to Merkel cells both during development and adulthood. PMID:25624394

Kindler syndrome protein Kindlin-1 is mainly expressed in adult tissues originating from ectoderm/endoderm.

PubMed

Zhan, Jun; Yang, Mei; Zhang, Jing; Guo, YongQing; Liu, Wei; Zhang, HongQuan

2015-05-01

Mutations of integrin-interacting protein Kindlin-1 cause Kindler syndrome and deregulation of Kindlin-1 is implicated in human cancers. The Kindlin-1-related diseases are confined in limited tissue types. However, Kindlin-1 tissue distribution and the dogma that governs Kindlin-1 expression in normal human body are elusive. This study examined Kindlin-1 expression in normal human adult organs, human and mouse embryonic organs by immunohistochemical analyses. We identified a general principle that the level of Kindlin-1 expression in tissues is tightly correlated with the corresponding germ layers from which these tissues originate. We compared the expression of Kindlin-1 with Kindlin-2 and found that Kindlin-1 is highly expressed in epithelial tissues derived from ectoderm and endoderm, whereas Kindlin-2 is mainly expressed in mesoderm-derived tissues. Likewise, Kindlin-1 was also found highly expressed in endoderm/ectoderm-derived tissues in human and mouse embryos. Our findings indicate that Kindlin-1 may play an importance role in the development of endoderm/ectoderm related tissues.

Glassy dynamics in three-dimensional embryonic tissues

PubMed Central

Schötz, Eva-Maria; Lanio, Marcos; Talbot, Jared A.; Manning, M. Lisa

2013-01-01

Many biological tissues are viscoelastic, behaving as elastic solids on short timescales and fluids on long timescales. This collective mechanical behaviour enables and helps to guide pattern formation and tissue layering. Here, we investigate the mechanical properties of three-dimensional tissue explants from zebrafish embryos by analysing individual cell tracks and macroscopic mechanical response. We find that the cell dynamics inside the tissue exhibit features of supercooled fluids, including subdiffusive trajectories and signatures of caging behaviour. We develop a minimal, three-parameter mechanical model for these dynamics, which we calibrate using only information about cell tracks. This model generates predictions about the macroscopic bulk response of the tissue (with no fit parameters) that are verified experimentally, providing a strong validation of the model. The best-fit model parameters indicate that although the tissue is fluid-like, it is close to a glass transition, suggesting that small changes to single-cell parameters could generate a significant change in the viscoelastic properties of the tissue. These results provide a robust framework for quantifying and modelling mechanically driven pattern formation in tissues. PMID:24068179

Tributyltin impairs dentin mineralization and enamel formation in cultured mouse embryonic molar teeth.

PubMed

Salmela, Eija; Sahlberg, Carin; Alaluusua, Satu; Lukinmaa, Pirjo-Liisa

2008-11-01

Tributyltin (TBT), earlier used as an antifouling agent in marine paints, causes damage to the aquatic ecosystem, for example, impaired shell calcification in oysters. TBT affects hard tissue mineralization even in mammals: delayed bone mineralization has been observed in rodents exposed to TBT in utero. To see if TBT interferes with tooth development, especially dental hard tissue formation, we exposed mouse E18 mandibular first and second molars to 0.1, 0.5, 1.0, and 2.0 microM TBT chloride in organ culture for 7-12 days. The amount of enamel was assessed and the sizes of the first molars were measured from photographs taken after the culture. TBT concentration dependently impaired enamel formation (p < 0.001) and reduced tooth size (p < 0.001). Histological analysis showed slight arrest of dentin mineralization and enamel formation in first molars exposed to 0.1 microM TBT. At the concentration of 1.0 microM the effect was overt. The differentiation of ameloblasts in the mesial cusps was retarded but TBT had no effect on odontoblast morphology. The dental epithelium showed enhanced apoptosis. The failure of ameloblasts to form enamel was likely to be secondary to the effect of TBT on dentin mineralization. In the second molars, where predentin deposition had not started, ameloblasts and odontoblasts were nonpolarized and proliferative. The results showed that TBT concentration dependently impairs dental hard tissue formation and reduces tooth size in cultured mouse embryonic molars. The effects depend on the stage of tooth development at the start of exposure and may involve epithelial-mesenchymal interactions.

Invited review: mesenchymal progenitor cells in intramuscular connective tissue development.

PubMed

Miao, Z G; Zhang, L P; Fu, X; Yang, Q Y; Zhu, M J; Dodson, M V; Du, M

2016-01-01

The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) β signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.

Impact of collection season and storage of semen on methylation activity in swine placental and fetal tissues derived from summer or winter breedings

USDA-ARS?s Scientific Manuscript database

DNA methylation patterns in extra-embryonic tissues have been linked to irregular fetal growth and early pregnancy loss. The objective of the current study was to evaluate methylation profiles of placental and fetal tissue collected from pregnancies derived using cooled-extended (ExT) or cryopreserv...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples.

PubMed

Rehimi, Rizwan; Bartusel, Michaela; Solinas, Francesca; Altmüller, Janine; Rada-Iglesias, Alvaro

2017-08-29

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 10 4 - 5 x 10 5 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

The relationship of parthenogenesis in virgin Chinese Painted quail (Coturnix chinensis) hens with embryonic mortality and hatchability following mating.

PubMed

Parker, H M; Kiess, A S; Robertson, M L; Wells, J B; McDaniel, C D

2012-06-01

Unfertilized chicken, turkey, and quail eggs are capable of developing embryos by parthenogenesis. However, it is unknown if the physiological mechanisms regulating parthenogenesis in virgin hens may actually work against fertilization, embryonic development, and hatchability of eggs from these same hens following mating. Additionally, because most parthenogenic development closely resembles early embryonic mortality in fertilized eggs during the first 2 to 3 d of incubation, it is possible that many unhatched eggs classified as containing early embryonic mortality may actually be unfertilized eggs that contain parthenogens. Therefore, the objective of this study was to examine the relationship of parthenogenesis before mating with embryonic development and hatchability characteristics after mating. Based upon their ability to produce unfertilized eggs that contain parthenogens, 372 virgin Chinese Painted quail hens were divided into 7 groups, according to their incidence of parthenogenesis: 0, 10, 20, 30, 40, 50, and greater than 50% parthenogenesis. Males were then placed with these hens so that fertility, embryonic mortality, and hatchability could be evaluated for each hen. Hatchability of eggs set, hatchability of fertile eggs, and late embryonic mortality declined dramatically as the incidence of parthenogenesis increased. On the other hand, early embryonic mortality increased as parthenogenesis increased. Fertility was not different across the 7 parthenogenesis hen groups, perhaps because unfertilized eggs that exhibited parthenogenesis resembled and were therefore classified as early embryonic mortality. In conclusion, virgin quail hens that exhibit parthenogenesis appear to have impaired embryonic development and hatchability following mating. Additional sperm-egg interaction and embryonic research is needed to determine if a large portion of the early embryonic mortality experienced by mated hens that exhibit parthenogenesis as virgin hens is in fact embryonic development in unfertilized eggs.

Molecular Signatures of Tissue-Specific Microvascular Endothelial Cell Heterogeneity in Organ Maintenance and Regeneration

PubMed Central

Nolan, Daniel J.; Ginsberg, Michael; Israely, Edo; Palikuqi, Brisa; Poulos, Michael G.; James, Daylon; Ding, Bi-Sen; Schachterle, William; Liu, Ying; Rosenwaks, Zev; Butler, Jason M.; Xiang, Jenny; Rafii, Arash; Shido, Koji; Rabbany, Sina Y.; Elemento, Olivier; Rafii, Shahin

2013-01-01

SUMMARY Microvascular endothelial cells (ECs) within different tissues are endowed with distinct but as yet unrecognized structural, phenotypic, and functional attributes. We devised EC purification, cultivation, profiling, and transplantation models that establish tissue-specific molecular libraries of ECs devoid of lymphatic ECs or parenchymal cells. These libraries identify attributes that confer ECs with their organotypic features. We show that clusters of transcription factors, angiocrine growth factors, adhesion molecules, and chemokines are expressed in unique combinations by ECs of each organ. Furthermore, ECs respond distinctly in tissue regeneration models, hepatectomy, and myeloablation. To test the data set, we developed a transplantation model that employs generic ECs differentiated from embryonic stem cells. Transplanted generic ECs engraft into regenerating tissues and acquire features of organotypic ECs. Collectively, we demonstrate the utility of informational databases of ECs toward uncovering the extravascular and intrinsic signals that define EC heterogeneity. These factors could be exploited therapeutically to engineer tissue-specific ECs for regeneration. PMID:23871589

Micro/nano-computed tomography technology for quantitative dynamic, multi-scale imaging of morphogenesis.

PubMed

Gregg, Chelsea L; Recknagel, Andrew K; Butcher, Jonathan T

2015-01-01

Tissue morphogenesis and embryonic development are dynamic events challenging to quantify, especially considering the intricate events that happen simultaneously in different locations and time. Micro- and more recently nano-computed tomography (micro/nanoCT) has been used for the past 15 years to characterize large 3D fields of tortuous geometries at high spatial resolution. We and others have advanced micro/nanoCT imaging strategies for quantifying tissue- and organ-level fate changes throughout morphogenesis. Exogenous soft tissue contrast media enables visualization of vascular lumens and tissues via extravasation. Furthermore, the emergence of antigen-specific tissue contrast enables direct quantitative visualization of protein and mRNA expression. Micro-CT X-ray doses appear to be non-embryotoxic, enabling longitudinal imaging studies in live embryos. In this chapter we present established soft tissue contrast protocols for obtaining high-quality micro/nanoCT images and the image processing techniques useful for quantifying anatomical and physiological information from the data sets.

The Cdk4-E2f1 pathway regulates early pancreas development by targeting Pdx1+ progenitors and Ngn3+ endocrine precursors

PubMed Central

Kim, So Yoon; Rane, Sushil G.

2011-01-01

Cell division and cell differentiation are intricately regulated processes vital to organ development. Cyclin-dependent kinases (Cdks) are master regulators of the cell cycle that orchestrate the cell division and differentiation programs. Cdk1 is essential to drive cell division and is required for the first embryonic divisions, whereas Cdks 2, 4 and 6 are dispensable for organogenesis but vital for tissue-specific cell development. Here, we illustrate an important role for Cdk4 in regulating early pancreas development. Pancreatic development involves extensive morphogenesis, proliferation and differentiation of the epithelium to give rise to the distinct cell lineages of the adult pancreas. The cell cycle molecules that specify lineage commitment within the early pancreas are unknown. We show that Cdk4 and its downstream transcription factor E2f1 regulate mouse pancreas development prior to and during the secondary transition. Cdk4 deficiency reduces embryonic pancreas size owing to impaired mesenchyme development and fewer Pdx1+ pancreatic progenitor cells. Expression of activated Cdk4R24C kinase leads to increased Nkx2.2+ and Nkx6.1+ cells and a rise in the number and proliferation of Ngn3+ endocrine precursors, resulting in expansion of the β cell lineage. We show that E2f1 binds and activates the Ngn3 promoter to modulate Ngn3 expression levels in the embryonic pancreas in a Cdk4-dependent manner. These results suggest that Cdk4 promotes β cell development by directing E2f1-mediated activation of Ngn3 and increasing the pool of endocrine precursors, and identify Cdk4 as an important regulator of early pancreas development that modulates the proliferation potential of pancreatic progenitors and endocrine precursors. PMID:21490060

Fetal loss in homozygous mutant Norrie disease mice: a new role of Norrin in reproduction.

PubMed

Luhmann, Ulrich F O; Meunier, Dominique; Shi, Wei; Lüttges, Angela; Pfarrer, Christiane; Fundele, Reinald; Berger, Wolfgang

2005-08-01

Mutations in the Norrie disease pseudoglioma gene (NDP) are known to cause X-linked recessive Norrie disease. In addition, NDP mutations have been found in other vasoproliferative retinopathies such as familial exudative vitreoretinopathy, retinopathy of prematurity, and Coats disease, suggesting a role for Norrin in vascular development. Here we report that female mice homozygous for the Norrie disease pseudoglioma homolog (Ndph) knockout allele exhibit almost complete infertility, while heterozygous females and hemizygous males are fertile. Histological examinations and RNA in situ hybridization analyses revealed defects in vascular development and decidualization in pregnant Ndph-/- females from embryonic day 7 (E7) onwards, resulting in embryonic loss. Using RT-PCR analyses we also demonstrate, for the first time, the expression of Ndph in mouse uteri and deciduae as well as the expression of NDP in human placenta. Taken together, these data provide strong evidence for Norrin playing an important role in female reproductive tissues. Copyright 2005 Wiley-Liss, Inc

Molecular basis of embryonic stem cell self-renewal: from signaling pathways to pluripotency network

PubMed Central

Huang, Guanyi; Ye, Shoudong; Zhou, Xingliang; Liu, Dahai

2016-01-01

Embryonic stem cells (ESCs) can be maintained in culture indefinitely while retaining the capacity to generate any type of cell in the body, and therefore not only hold great promise for tissue repair and regeneration, but also provide a powerful tool for modeling human disease and understanding biological development. In order to fulfill the full potential of ESCs, it is critical to understand how ESC fate, whether to self-renew or to differentiate into specialized cells, is regulated. On the molecular level, ESC fate is controlled by the intracellular transcriptional regulatory networks that respond to various extrinsic signaling stimuli. In this review, we discuss and compare important signaling pathways in the self-renewal and differentiation of mouse, rat, and human ESCs with an emphasis on how these pathways integrate into ESC-specific transcription circuitries. This will be beneficial for understanding the common and conserved mechanisms that govern self-renewal, and for developing novel culture conditions that support ESC derivation and maintenance. PMID:25595304

Control of metazoan heme homeostasis by a conserved multidrug resistance protein

PubMed Central

Korolnek, Tamara; Zhang, Jianbing; Beardsley, Simon; Scheffer, George L; Hamza, Iqbal

2014-01-01

Several lines of evidence predict that specific pathways must exist in metazoans for the escorted movement of heme, an essential but cytotoxic iron-containing organic ring, within and between cells and tissues, but these pathways remain obscure. In Caenorhabditis elegans, embryonic development is inextricably dependent on both maternally-derived heme and environmentally-acquired heme. Here, we show that the multidrug resistance protein, MRP-5/ABCC5, likely acts as a heme exporter and targeted depletion of mrp-5 in the intestine causes embryonic lethality. Transient knockdown of mrp5 in zebrafish leads to morphological defects and failure to hemoglobinize red blood cells. MRP5 resides on the plasma membrane and endosomal compartments and regulates export of cytosolic heme. Together, our genetic studies in worms, yeast, zebrafish, and mammalian cells identify a conserved, physiological role for a multidrug resistance protein in regulating systemic heme homeostasis. We envision other MRP family members may play similar unanticipated physiological roles in animal development. PMID:24836561

Precise chronology of differentiation of developing human primary dentition.

PubMed

Hu, Xuefeng; Xu, Shan; Lin, Chensheng; Zhang, Lishan; Chen, YiPing; Zhang, Yanding

2014-02-01

While correlation of developmental stage with embryonic age of the human primary dentition has been well documented, the available information regarding the differentiation timing of the primary teeth was largely based on the observation of initial mineralization and varies significantly. In this study, we aimed to document precise differentiation timing of the developing human primary dentition. We systematically examined the expression of odontogenic differentiation markers along with the formation of mineralized tissue in each developing maxillary and mandibular teeth from human embryos with well-defined embryonic age. We show that, despite that all primary teeth initiate development at the same time, odontogenic differentiation begins in the maxillary incisors at the 15th week and in the mandibular incisors at the 16th week of gestation, followed by the canine, the first primary premolar, and the second primary premolar at a week interval sequentially. Despite that the mandibular primary incisors erupt earlier than the maxillary incisors, this distal to proximal sequential differentiation of the human primary dentition coincides in general with the sequence of tooth eruption. Our results provide an accurate chronology of odontogenic differentiation of the developing human primary dentition, which could be used as reference for future studies of human tooth development.

Formation of the Embryonic Head in the Mouse: Attributes of a Gene Regulatory Network.

PubMed

Tam, Patrick P L; Fossat, Nicolas; Wilkie, Emilie; Loebel, David A F; Ip, Chi Kin; Ramialison, Mirana

2016-01-01

The embryonic head is the first major body part to be constructed during embryogenesis. The allocation and the assembly of the progenitor tissues, which start at gastrulation, are accompanied by the spatiotemporal activity of transcription factors and signaling pathways that drives lineage specification, germ layer formation, and cell/tissue movement. The morphogenesis, regionalization, and patterning of the brain and craniofacial structures rely on the function of LIM-domain, homeodomain, and basic helix-loop-helix transcription factors. These factors constitute the central nodes of a gene regulatory network (GRN) which encompasses and intersects with signaling pathways involved with head formation. It is predicted that the functional output of this "head GRN" impacts on cellular function and cell-cell interactions that are essential for lineage differentiation and tissue modeling, which are key processes underpinning the formation of the head. © 2016 Elsevier Inc. All rights reserved.

Electromechanical integration of cardiomyocytes derived from human embryonic stem cells.

PubMed

Kehat, Izhak; Khimovich, Leonid; Caspi, Oren; Gepstein, Amira; Shofti, Rona; Arbel, Gil; Huber, Irit; Satin, Jonathan; Itskovitz-Eldor, Joseph; Gepstein, Lior

2004-10-01

Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.

Microgravity, stem cells, and embryonic development: challenges and opportunities for 3D tissue generation

NASA Astrophysics Data System (ADS)

Andreazzoli, Massimiliano; Angeloni, Debora; Broccoli, Vania; Demontis, Gian C.

2017-04-01

Space is a challenging environment for the human body, due to the combined effects of reduced gravity (microgravity) and cosmic radiation. Known effects of microgravity range from the blood redistribution that affects the cardiovascular system and the eye to muscle wasting, bone loss, anemia and immune depression. About cosmic radiation, the shielding provided by the spaceship hull is far less efficient than that afforded at ground level by the combined effects of the Earth atmosphere and magnetic field. The eye and its nervous layer (the retina) are affected by both microgravity and heavy ions exposure. Considering the importance of sight for long-term manned flights, visual research aimed at devising measures to protect the eye from environmental conditions of the outer space represents a special challenge to meet. In this review we focus on the impact of microgravity on embryonic development, discussing the roles of mechanical forces in the context of the neutral buoyancy the embryo experiences in the womb. At variance with its adverse effects on the adult human body, simulated microgravity may provide a unique tool for understanding the biomechanical events involved in the development and assembly in vitro of three-dimensional (3D) ocular tissues. Prospective benefits are the development of novel safety measures to protect the human eye from cosmic radiation in microgravity during long-term manned spaceflights in the outer space, as well as the generation of human 3D-retinas with its supporting structures to develop innovative and effective therapeutic options for degenerative eye diseases.

Shaping sound in space: the regulation of inner ear patterning.

PubMed

Groves, Andrew K; Fekete, Donna M

2012-01-01

The inner ear is one of the most morphologically elaborate tissues in vertebrates, containing a group of mechanosensitive sensory organs that mediate hearing and balance. These organs are arranged precisely in space and contain intricately patterned sensory epithelia. Here, we review recent studies of inner ear development and patterning which reveal that multiple stages of ear development - ranging from its early induction from the embryonic ectoderm to the establishment of the three cardinal axes and the fine-grained arrangement of sensory cells - are orchestrated by gradients of signaling molecules.

Shaping sound in space: the regulation of inner ear patterning

PubMed Central

Groves, Andrew K.; Fekete, Donna M.

2012-01-01

The inner ear is one of the most morphologically elaborate tissues in vertebrates, containing a group of mechanosensitive sensory organs that mediate hearing and balance. These organs are arranged precisely in space and contain intricately patterned sensory epithelia. Here, we review recent studies of inner ear development and patterning which reveal that multiple stages of ear development – ranging from its early induction from the embryonic ectoderm to the establishment of the three cardinal axes and the fine-grained arrangement of sensory cells – are orchestrated by gradients of signaling molecules. PMID:22186725

Friction forces position the neural anlage

PubMed Central

Smutny, Michael; Ákos, Zsuzsa; Grigolon, Silvia; Shamipour, Shayan; Ruprecht, Verena; Čapek, Daniel; Behrndt, Martin; Papusheva, Ekaterina; Tada, Masazumi; Hof, Björn; Vicsek, Tamás; Salbreux, Guillaume; Heisenberg, Carl-Philipp

2017-01-01

During embryonic development, mechanical forces are essential for cellular rearrangements driving tissue morphogenesis. Here, we show that in the early zebrafish embryo, friction forces are generated at the interface between anterior axial mesoderm (prechordal plate, ppl) progenitors migrating towards the animal pole and neurectoderm progenitors moving in the opposite direction towards the vegetal pole of the embryo. These friction forces lead to global rearrangement of cells within the neurectoderm and determine the position of the neural anlage. Using a combination of experiments and simulations, we show that this process depends on hydrodynamic coupling between neurectoderm and ppl as a result of E-cadherin-mediated adhesion between those tissues. Our data thus establish the emergence of friction forces at the interface between moving tissues as a critical force-generating process shaping the embryo. PMID:28346437

Friction forces position the neural anlage.

PubMed

Smutny, Michael; Ákos, Zsuzsa; Grigolon, Silvia; Shamipour, Shayan; Ruprecht, Verena; Čapek, Daniel; Behrndt, Martin; Papusheva, Ekaterina; Tada, Masazumi; Hof, Björn; Vicsek, Tamás; Salbreux, Guillaume; Heisenberg, Carl-Philipp

2017-04-01

During embryonic development, mechanical forces are essential for cellular rearrangements driving tissue morphogenesis. Here, we show that in the early zebrafish embryo, friction forces are generated at the interface between anterior axial mesoderm (prechordal plate, ppl) progenitors migrating towards the animal pole and neurectoderm progenitors moving in the opposite direction towards the vegetal pole of the embryo. These friction forces lead to global rearrangement of cells within the neurectoderm and determine the position of the neural anlage. Using a combination of experiments and simulations, we show that this process depends on hydrodynamic coupling between neurectoderm and ppl as a result of E-cadherin-mediated adhesion between those tissues. Our data thus establish the emergence of friction forces at the interface between moving tissues as a critical force-generating process shaping the embryo.

Expression of cardiac neural crest and heart genes isolated by modified differential display.

PubMed

Martinsen, Brad J; Groebner, Nathan J; Frasier, Allison J; Lohr, Jamie L

2003-08-01

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent outflow tract septation are critical events during vertebrate heart development. We have performed four modified differential display screens in the chick embryo to identify genes that may be involved in CNC, OFT, secondary heart field, and heart development. The screens included differential display of RNA isolated from three different axial segments containing premigratory cranial neural crest cells; of RNA from distal outflow tract, proximal outflow tract, and atrioventricular tissue of embryonic chick hearts; and of RNA isolated from left and right cranial tissues, including the early heart fields. These screens have resulted in the identification of the five cDNA clones presented here, which are expressed in the cardiac neural crest, outflow tract and developing heart in patterns that are unique in heart development.

Minimal Phenotype of Mice Homozygous for a Null Mutation in the Forkhead/Winged Helix Gene, Mf2

PubMed Central

Kume, Tsutomu; Deng, Keyu; Hogan, Brigid L. M.

2000-01-01

Mf2 (mesoderm/mesenchyme forkhead 2) encodes a forkhead/winged helix transcription factor expressed in numerous tissues of the mouse embryo, including paraxial mesoderm, somites, branchial arches, vibrissae, developing central nervous system, and developing kidney. We have generated mice homozygous for a null mutation in the Mf2 gene (Mf2lacZ) to examine its role during embryonic development. The lacZ allele also allows monitoring of Mf2 gene expression. Homozygous null mutants are viable and fertile and have no major developmental defects. Some mutants show renal abnormalities, including kidney hypoplasia and hydroureter, but the penetrance of this phenotype is only 40% or lower, depending on the genetic background. These data suggest that Mf2 can play a unique role in kidney development, but there is functional redundancy in this organ and other tissues with other forkhead/winged helix genes. PMID:10648626

Minimal phenotype of mice homozygous for a null mutation in the forkhead/winged helix gene, Mf2.

PubMed

Kume, T; Deng, K; Hogan, B L

2000-02-01

Mf2 (mesoderm/mesenchyme forkhead 2) encodes a forkhead/winged helix transcription factor expressed in numerous tissues of the mouse embryo, including paraxial mesoderm, somites, branchial arches, vibrissae, developing central nervous system, and developing kidney. We have generated mice homozygous for a null mutation in the Mf2 gene (Mf2(lacZ)) to examine its role during embryonic development. The lacZ allele also allows monitoring of Mf2 gene expression. Homozygous null mutants are viable and fertile and have no major developmental defects. Some mutants show renal abnormalities, including kidney hypoplasia and hydroureter, but the penetrance of this phenotype is only 40% or lower, depending on the genetic background. These data suggest that Mf2 can play a unique role in kidney development, but there is functional redundancy in this organ and other tissues with other forkhead/winged helix genes.

Monitoring p53 by MDM2 and MDMX is required for endocrine pancreas development and function in a spatio-temporal manner.

PubMed

Zhang, Yiwei; Zeng, Shelya X; Hao, Qian; Lu, Hua

2017-03-01

Although p53 is not essential for normal embryonic development, it plays a pivotal role in many biological and pathological processes, including cell fate determination-dependent and independent events and diseases. The expression and activity of p53 largely depend on its two biological inhibitors, MDM2 and MDMX, which have been shown to form a complex in order to tightly control p53 to an undetectable level during early stages of embryonic development. However, more delicate studies using conditional gene-modification mouse models show that MDM2 and MDMX may function separately or synergistically on p53 regulation during later stages of embryonic development and adulthood in a cell and tissue-specific manner. Here, we report the role of the MDM2/MDMX-p53 pathway in pancreatic islet morphogenesis and functional maintenance, using mouse lines with specific deletion of MDM2 or MDMX in pancreatic endocrine progenitor cells. Interestingly, deletion of MDM2 results in defects of embryonic endocrine pancreas development, followed by neonatal hyperglycemia and lethality, by inducing pancreatic progenitor cell apoptosis and inhibiting cell proliferation. However, unlike MDM2-knockout animals, mice lacking MDMX in endocrine progenitor cells develop normally. But, surprisingly, the survival rate of adult MDMX-knockout mice drastically declines compared to control mice, as blockage of neonatal development of endocrine pancreas by inhibition of cell proliferation and subsequent islet dysfunction and hyperglycemia eventually lead to type 1 diabetes-like disease with advanced diabetic nephropathy. As expected, both MDM2 and MDMX deletion-caused pancreatic defects are completely rescued by loss of p53, verifying the crucial role of the MDM2 and/or MDMX in regulating p53 in a spatio-temporal manner during the development, functional maintenance, and related disease progress of endocrine pancreas. Also, our study suggests a possible mouse model of advanced diabetic nephropathy, which is complementary to other established diabetic models and perhaps useful for the development of anti-diabetes therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

PubMed

Behringer, Richard; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, Andras

2016-12-01

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size. © 2016 Cold Spring Harbor Laboratory Press.

4-N-pyridin-2-yl-benzamide nanotubes compatible with mouse stem cell and oral delivery in Drosophila

NASA Astrophysics Data System (ADS)

Yadav, Jhillu S.; Lavanya, Madugula P.; Das, Pragna P.; Bag, Indira; Krishnan, Anita; Jagannadh, Bulusu; Mohapatra, Debendra K.; Pal Bhadra, Manika; Bhadra, Utpal

2010-04-01

p-aminobenzoic acid (PABA), a structural moiety of many commercial drugs, is self-assembled with linker alkyl side chains to form tubular nanostructures. The tubes exhibited fluorescence either intrinsic or from fluorescent molecules embedded in the wall during self-assembly. Uptake and inter-cellular delivery of the conjugated nanotubes in human cancer cells and in mouse embryonic stem cells were demonstrated by fluorescence imaging and flow cytometry. Biocompatibility, cytotoxicity and clearance were monitored both ex vivo in mouse multipotent embryonic stem cells and in vivo in adult Drosophila. Accumulation of nanotubes had no adverse effects and abnormalities on stem cell morphology and proliferation rate. A distinct distribution of two separate nanotubes in various internal organs of Drosophila interprets that accumulation of nanomaterials might be interdependent on the side chain modifications and physiological settings of cell or tissue types. Unlike carbon nanomaterials, exposure of PABA nanotubes does not produce any hazards including locomotion defects and mortality of adult flies. Despite differential uptake and clearance from multiple live tissues, the use of self-assembled nanotubes can add new dimensions and scope to the development of dual-purpose oral carriers for the fulfilment of many biological promises.