Germ Layers Endoderm Research Articles

Gut-like ectodermal tissue in a sea anemone challenges germ layer homology

Cnidarians (for example, sea anemones and jellyfish) develop from an outer ectodermal and inner endodermal germ layer, whereas bilaterians (for example, vertebrates and flies) additionally have a mesodermal layer as intermediate germ layer. Currently, cnidarian endoderm (that is, ‘mesendoderm’) is considered homologous to both bilaterian endoderm and mesoderm. Here we test this hypothesis by studying the fate of germ layers, the localization of gut cell types, and the expression of numerous ‘endodermal’ and ‘mesodermal’ transcription factor orthologues in the anthozoan sea anemone Nematostella vectensis. Surprisingly, we find that the developing pharyngeal ectoderm and its derivatives display a transcription-factor expression profile (foxA, hhex, islet, soxB1, hlxB9, tbx2/3, nkx6 and nkx2.2) and cell-type combination (exocrine and insulinergic) reminiscent of the developing bilaterian midgut, and, in particular, vertebrate pancreatic tissue. Endodermal derivatives, instead, display cell functions and transcription-factor profiles similar to bilaterian mesoderm derivatives (for example, somatic gonad and heart). Thus, our data supports an alternative model of germ layer homologies, where cnidarian pharyngeal ectoderm corresponds to bilaterian endoderm, and the cnidarian endoderm is homologous to bilaterian mesoderm. The cnidarian endoderm has been considered homologous to the bilaterian endoderm and mesoderm. Here, a gut-like ectoderm is revealed in a sea anemone, corresponding to the bilaterian endoderm, supporting an alternative model of germ layer homology.

Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human amniotic fluid stem cells.

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and β-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.

Comparative induction of pluripotency in human umbilical vein endothelial cells and dermal fibroblasts and further differentiation into Hepatocytes

Generating induced pluripotent stem cells (iPSCs) is a standardized technique. To date iPS cells have been generated from HUVECs using viral-based approaches. We employ an episomal plasmid-based approach to generate integration-free iPSCs (E-iPSCs) from human somatic cells. In this study, we compared the generation of E-iPSC lines from human umbilical vein endothelial cells (HUVEC) and fetal foreskin derived fibroblast (HFF1). The efficiency of inducing pluripotency in HFF1 was 0.03% compared to 2.5% in HUVEC. This implies that the efficiency of reprogramming HUVECs is 83-fold higher than in HFF1 cells. Additionally, the kinetics of reprogramming was much faster using HUVECs, i.e. three weeks for the stabilization of E-iPSC colonies compared to HFF1 cells which needed four months. The E-iPSCs from both somatic cell types were fully characterized and are comparable to human embryonic stem cells (hESCs). Both E-iPSC lines express pluripotency associated transcription factors as OCT4, NANOG, SOX2 and also the surface markers SSEA-4, TRA-l-60, TRA-1 – 81 and TRA-2 – 49 but not SSEA-1. Additionally, they have the ability to differentiate to all cell types representative of the three germ layers endoderm, ectoderm and mesoderm in vitro (by formation of embryoid bodies) and in vivo (teratoma formation in immunodeficient mice).

SnapShot: GI Tract Development

The endoderm germ layer contributes to the respiratory and gastrointestinal (GI) lineages during development, giving rise to an array of specialized epithelial cell types lining organs, including the thyroid, thymus, lungs, liver, biliary system, pancreas, and intestines. This SnapShot timelines and summarizes key stages following gastrulation, including endoderm patterning, organ specification, and organogenesis. A lineage tree of the developing endocrine pancreas is outlined to further illustrate this process.

Spatiotemporal transcriptomics reveals the evolutionary history of the endoderm germ layer.

The germ layer concept has been one of the foremost organizing principles in developmental biology, classification, systematics and evolution for 150 years1-3. Of the three germ layers, the mesoderm is found in bilaterian animals but is absent in species in the phyla Cnidaria and Ctenophora, which has been taken as evidence that the mesoderm was the final germ layer to evolve1,4,5. The origin of the ectoderm and endoderm germ layers, however, remains unclear with models supporting the antecedence of each as well as a simultaneous origin4,6-9. Here, we determine the temporal and spatial components of gene expression spanning embryonic development for all Caenorhabditis elegans genes and use it to determine the evolutionary ages of the germ layers. The gene expression program of the mesoderm is induced after those of the ectoderm and endoderm, thus making it the last germ layer to both evolve and develop. Strikingly, the C. elegans endoderm and ectoderm expression programs do not co-induce; rather the endoderm activates earlier, and this is observed also in the expression of endoderm orthologs during the embryology of Xenopus tropicalis, Nematostella vectensis, and the sponge Amphimedon queenslandica. Querying for the phylogenetic ages of specifically expressed genes revealed that the endoderm is comprised of older genes. Taken together, we propose that the endoderm program dates back to the origin of multicellularity, while the ectoderm originated as a secondary germ layer freed from ancestral feeding functions.

Characterisation of human induced pluripotent stem cell-derived hepatocyte-like cells and endodermal progenitors

Background Human embryonic stem cells (hESCs) have two fundamental characteristics. First is pluripotency, they have the ability to differentiate to all cell types of the three germ layers endoderm, ectoderm and mesoderm in vitro (by formation of embryoid bodies) and in vivo (by teratoma formation in immune deficient mice). Second, hESCs have the capability to self-renew indefinitely. Embryonic stem cells express pluripotency-associated markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, TRA-181 and alkaline phosphatase. The use of hESCs in research and future regenerative medicine approaches is hampered by ethical and moral concerns as these cells are derived from blastocysts. Besides the derivation of lineage-restricted cells from hESCs as well as the immune rejection of hESCs derived cells are still problematic. To avoid ethical and immune rejection concerns, scientists searched for alternative ways to derive pluripotent cells from mouse somatic cells [1]. Shortly after that two groups managed reprogramming of human adult fibroblasts with viral transduction mediated over-expression of four transcription factors OCT4, SOX2, KLF4 and cMYC or OCT4, SOX2, NANOG and LIN28. In general, derivation of induced pluripotent stem cells (iPSCs) from somatic cells and differentiating these into a donor cell type of interest are promising approaches for (i) modelling human diseases in vitro, (ii) toxicology and drug screening, (iii) future application in tissue replacement therapies. We and others have shown that iPSCs can be differentiated into hepatocyte-like cells that model in vitro the patient’s genetic disease or metabolic capability, thereby adding a further dimension to existing toxicity testing platforms. An iPSC-based strategy thus allows large scale studies impossible to perform on primary cell cultures or from biopsies and also enables studies on hepatocytes genetically susceptible to drug-induced liver injury (DILI) as in vitro models with genotypic relevance for toxicology screening. Furthermore, these patient-specific iPSC-derived hepatocytes can be used for characterising the metabolism of a candidate drug.

The generation of definitive endoderm from human embryonic stem cells is initially independent from activin A but requires canonical Wnt-signaling.

The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.

Histopathological Disposition of Tissues During Malignant Carcinoma

Carcinoma is a type of cancer that develops from epithelial cells. Specifically, a carcinoma is a cancer that begins in a tissue that lines the inner or outer surfaces of the body, and that generally arises from cells originating in the endodermal or ectodermal germ layer during embryogenesis. Carcinomas occur when the DNA of a cell is damaged or altered and the cell begins to grow uncontrollably and become malignant.

Embryonic stem cells of the non-human primateCallithrix jacchuscan be differentiated into definitive endoderm by Activin-A but not IDE-1/2

Pluripotent stem cells hold great promise for regenerative medicine, due to their unlimited self-renewal potential and the ability to differentiate into all somatic cell types. Differences between the rodent disease models and the situation in humans can be narrowed down with non-human primate models. The common marmoset monkey (Callithrix jacchus) is an interesting model for biomedical research because these animals are easy to breed, get relatively old (≤ 13 years), are small in size, are relatively cost-effective and have a high genetic proximity to the human. In particular, diseases of the liver and pancreas are interesting for cell replacement therapies but the in vitro differentiation of ESCs into the definitive endoderm germ layer is still a demanding task. Membrane-permeable, chemically defined small molecules can possibly replace recombinant growth factors used in most directed differentiation protocols. However, the potent small molecules IDE-1 and IDE-2 were not able to induce definitive endoderm-like cells when ESCs from the common marmoset were treated with these compounds, whereas the recombinant growth factor Activin A could force the differentiation into this lineage. Our results indicate that ESCs from the common marmoset are less sensitive or even insensitive to these small molecules. Thus, differences between the species of human ESCs and ESCs of this non-human primate might be a useful model to further evaluate the exact mode of action of these compounds.

Effect of 3D Cultivation Conditions on the Differentiation of Endodermal Cells

Cellular therapy of endodermal organs is one of the most important issues in modern cellular biology and biotechnology. One of the most promising directions in this field is the study of the transdifferentiation abilities of cells within the same germ layer. A method for anin vitroinvestigation of the cell differentiation potential (the cell culture in a three-dimensional matrix) is described in this article. Cell cultures of postnatal salivary gland cells and postnatal liver progenitor cells were obtained; their comparative analysis under 2D and 3D cultivation conditions was carried out. Both cell types have high proliferative abilities and can be cultivated for more than 20 passages. Under 2D cultivation conditions, the cells remain in an undifferentiated state. Under 3D conditions, they undergo differentiation, which was confirmed by a lower cell proliferation and by an increase in the differentiation marker expression. Salivary gland cells can undergo hepatic and pancreatic differentiation under 3D cultivation conditions. Liver progenitor cells also acquire a pancreatic differentiation capability under conditions of 3D cultivation. Thus, postnatal salivary gland cells exhibit a considerable differentiation potential within the endodermal germ layer and can be used as a promising source of endodermal cells for the cellular therapy of liver pathologies. Cultivation of cells under 3D conditions is a useful model for thein vitroanalysis of the cell differentiation potential.

Generating cells of the gastrointestinal system: current approaches and applications for the differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are defined by their abilities to self-renew and to differentiate into any cell type of the human body. Due to these unique properties, hPSCs represent a potentially unlimited source of cells/tissues for cell replacement therapies. Use of these cells may also revolutionize the way drugs are discovered, designed, and tested. Furthermore, the study of how cells differentiate can also change our understanding of complex human biology and disease. For these reasons, scientists have dedicated significant time and effort to generate specific cell types from hPSCs with therapeutic potential, including cells derived from the definitive endoderm germ layer such as liver cells (hepatocytes) and pancreatic β cells. In this review, we will focus broadly on the most advanced differentiation strategies currently employed to differentiate hPSCs to endodermal lineages such as the liver, pancreas, and intestine as well as the principles of developmental biology around which these protocols were designed. This will be followed by a brief discussion of the vast potential of these systems as suitable in vitro models for human embryonic development and disease.

Pluripotent stem cells in development and disease

Learning Outcome 1: The origins and characteristics of ESCs and iPSCs. Learning Outcome 2: How to control pluripotent stem cell differentiation to direct their development along specific lineages. Learning Outcome 3: The power of pluripotent stem cell technology to study development, disease mechanisms and therapy. Abstract: Recent breakthroughs in the stem cell biology field have demonstrated the potential of pluripotent Embryonic Stem Cells (ESCs) to generate any cell type in the body. This technology has been underpinned by advances in elucidating the regulatory networks involved in stemcell self-renewal andpluripotency, but also by insights provided by developmental biology regarding the transcriptional regulators and signaling pathways that set up the body plan and control organogenesis in the embryo. Efficient ESC differentiation protocols have been established, and continue to be modified and improved, that model mammalian development by recapitulating gastrulation in vitro through the induction of ectoderm, mesoderm and endoderm germ layers, and subsequently directing their further differentiation along their respective downstream lineages. The field of pluripotent stem cell biology has also been pushed to the forefront of biomedical researchby the important discovery of reprogramming somatic cells to anESClike state, generating induced Pluripotent Stem cells (iPSCs). The ability to reprogram cells has offered the unique opportunity to generate patient-specific iPSCs, and combine this with lineage-specific differentiation protocols to further model human disease in a dish and understand disease mechanisms. Taken together, pluripotent stem cell technology has provided the unique tools to not only model mammalian development, but also to generate functional cell types and tissues in vitro that can be used for drug discovery, for gaining insights into disease, as well as genetic rescue and potential autologous cell-based regenerative and replacement therapies. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

GFP reporters as tools for investigating bone cell biology and regenerative medicine

Learning Outcome 1: The origins and characteristics of ESCs and iPSCs. Learning Outcome 2: How to control pluripotent stem cell differentiation to direct their development along specific lineages. Learning Outcome 3: The power of pluripotent stem cell technology to study development, disease mechanisms and therapy. Abstract: Recent breakthroughs in the stem cell biology field have demonstrated the potential of pluripotent Embryonic Stem Cells (ESCs) to generate any cell type in the body. This technology has been underpinned by advances in elucidating the regulatory networks involved in stemcell self-renewal andpluripotency, but also by insights provided by developmental biology regarding the transcriptional regulators and signaling pathways that set up the body plan and control organogenesis in the embryo. Efficient ESC differentiation protocols have been established, and continue to be modified and improved, that model mammalian development by recapitulating gastrulation in vitro through the induction of ectoderm, mesoderm and endoderm germ layers, and subsequently directing their further differentiation along their respective downstream lineages. The field of pluripotent stem cell biology has also been pushed to the forefront of biomedical researchby the important discovery of reprogramming somatic cells to anESClike state, generating induced Pluripotent Stem cells (iPSCs). The ability to reprogram cells has offered the unique opportunity to generate patient-specific iPSCs, and combine this with lineage-specific differentiation protocols to further model human disease in a dish and understand disease mechanisms. Taken together, pluripotent stem cell technology has provided the unique tools to not only model mammalian development, but also to generate functional cell types and tissues in vitro that can be used for drug discovery, for gaining insights into disease, as well as genetic rescue and potential autologous cell-based regenerative and replacement therapies. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

Population Based Model of Human Embryonic Stem Cell (hESC) Differentiation during Endoderm Induction

The mechanisms by which human embryonic stem cells (hESC) differentiate to endodermal lineage have not been extensively studied. Mathematical models can aid in the identification of mechanistic information. In this work we use a population-based modeling approach to understand the mechanism of endoderm induction in hESC, performed experimentally with exposure to Activin A and Activin A supplemented with growth factors (basic fibroblast growth factor (FGF2) and bone morphogenetic protein 4 (BMP4)). The differentiating cell population is analyzed daily for cellular growth, cell death, and expression of the endoderm proteins Sox17 and CXCR4. The stochastic model starts with a population of undifferentiated cells, wherefrom it evolves in time by assigning each cell a propensity to proliferate, die and differentiate using certain user defined rules. Twelve alternate mechanisms which might describe the observed dynamics were simulated, and an ensemble parameter estimation was performed on each mechanism. A comparison of the quality of agreement of experimental data with simulations for several competing mechanisms led to the identification of one which adequately describes the observed dynamics under both induction conditions. The results indicate that hESC commitment to endoderm occurs through an intermediate mesendoderm germ layer which further differentiates into mesoderm and endoderm, and that during induction proliferation of the endoderm germ layer is promoted. Furthermore, our model suggests that CXCR4 is expressed in mesendoderm and endoderm, but is not expressed in mesoderm. Comparison between the two induction conditions indicates that supplementing FGF2 and BMP4 to Activin A enhances the kinetics of differentiation than Activin A alone. This mechanistic information can aid in the derivation of functional, mature cells from their progenitors. While applied to initial endoderm commitment of hESC, the model is general enough to be applicable either to a system of adult stem cells or later stages of ESC differentiation.

Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60–90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27–90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.

Strabismus-mediated primary archenteron invagination is uncoupled from Wnt/β-catenin-dependent endoderm cell fate specification in Nematostella vectensis (Anthozoa, Cnidaria): Implications for the evolution of gastrulation.

BackgroundGastrulation is a uniquely metazoan character, and its genesis was arguably the key step that enabled the remarkable diversification within this clade. The process of gastrulation involves two tightly coupled events during embryogenesis of most metazoans. Morphogenesis produces a distinct internal epithelial layer in the embryo, and this epithelium becomes segregated as an endoderm/endomesodermal germ layer through the activation of a specific gene regulatory program. The developmental mechanisms that induced archenteron formation and led to the segregation of germ layers during metazoan evolution are unknown. But an increased understanding of development in early diverging taxa at the base of the metazoan tree may provide insights into the origins of these developmental mechanisms.ResultsIn the anthozoan cnidarian Nematostella vectensis, initial archenteron formation begins with bottle cell-induced buckling of the blastula epithelium at the animal pole. Here, we show that bottle cell formation and initial gut invagination in Nematostella requires NvStrabismus (NvStbm), a maternally-expressed core component of the Wnt/Planar Cell Polarity (PCP) pathway. The NvStbm protein is localized to the animal pole of the zygote, remains asymmetrically expressed through the cleavage stages, and becomes restricted to the apical side of invaginating bottle cells at the blastopore. Antisense morpholino-mediated NvStbm-knockdown blocks bottle cell formation and initial archenteron invagination, but it has no effect on Wnt/ß-catenin signaling-mediated endoderm cell fate specification. Conversely, selectively blocking Wnt/ß-catenin signaling inhibits endoderm cell fate specification but does not affect bottle cell formation and initial archenteron invagination.ConclusionsOur results demonstrate that Wnt/PCP-mediated initial archenteron invagination can be uncoupled from Wnt/ß-catenin-mediated endoderm cell fate specification in Nematostella, and provides evidence that these two processes could have evolved independently during metazoan evolution. We propose a two-step model for the evolution of an archenteron and the evolution of endodermal germ layer segregation. Asymmetric accumulation and activation of Wnt/PCP components at the animal pole of the last common ancestor to the eumetazoa may have induced the cell shape changes that led to the initial formation of an archenteron. Activation of Wnt/ß-catenin signaling at the animal pole may have led to the activation of a gene regulatory network that specified an endodermal cell fate in the archenteron.

Control of Directed Cell Migration In Vivo by Membrane-to-Cortex Attachment

Author SummaryCell migration, like any event involving shape changes, is a mechanical process controlled by complex biochemical pathways. Here, we examine cell migration in developing embryos with a combination of cell biological tools and atomic force microscopy, so as to investigate how cellular mechanical properties control migration. A fundamental step during migration is the formation of a protrusion at the leading edge of the cell. In three-dimensional environments, and particularly in vivo, cells use different protrusion types: spike-like filopodia and flattened lamellipodia, whose growth is driven by actin polymerization, and spherical blebs, which grow because of intracellular pressure pushing on the membrane. It is important to understand how the formation of different protrusion types is mechanically and molecularly controlled, and how the different protrusions specifically contribute to migration. We have addressed this using the zebrafish embryo as a model system. We show that reducing the strength of the attachment between the plasma membrane and the underlying cortical network of actin filaments, or increasing intracellular pressure, increases the proportion of cellular blebs and reduces the directionality of cell migration. Our work reveals that blebs, lamellipodia, and filopodia are not interchangeable and that the relative proportion of each type of protrusion, under the control of mechanical parameters, determines migration directionality during zebrafish gastrulation.

Vertebrate Endoderm Development and Organ Formation

The endoderm germ layer contributes to the respiratory and gastrointestinal tracts and to all of their associated organs. Over the past decade, studies in vertebrate model organisms, including frog, fish, chick, and mouse, have greatly enhanced our understanding of the molecular basis of endoderm organ development. We review this progress with a focus on early stages of endoderm organogenesis including endoderm formation, gut tube morphogenesis and patterning, and organ specification. Lastly, we discuss how developmental mechanisms that regulate endoderm organogenesis are used to direct differentiation of embryonic stem cells into specific adult cell types, which function to alleviate disease symptoms in animal models.

Sox17 Expression Patterns during Gastrulation and Early Neurulation in the Rabbit Suggest Two Sources of Endoderm Formation

Most gastrointestinal tract and associated gland epithelia originate from the endoderm germ layer discovered by Pander in 1817. The recent surge in stem cell concepts revived interest in the findings of 30 years ago that the endoderm layer itself originates from the epiblast (which since Pander’s time had been held to be the forerunner of the ectoderm and mesoderm germ layers only). However, the question as to which parts of the mammalian gastrulation-stage embryonic disc generate endoderm cells is still unresolved. Therefore, the expression of the gene coding for the transcription factor Sox17, a key transcription factor involved in endoderm formation in mouse, chick, frog, and zebrafish, was analyzed in the rabbit, a model organism for mammalian gastrulation morphology, using whole-mount in situ hybridization and high-resolution histological analysis of embryos at gastrulation and early neurulation stages. Sox17 mRNA in the mesoderm and lower layer (hypoblast) compartments within and adjacent to Hensen’s node and the anterior segment of the primitive streak confirmed the validity of this approach, as this region had previously been shown to form endoderm in mouse and chick. However, Sox17 expression in central and posterior epiblast at pregastrulation stages together with a transient expression at the posterior extremity of the primitive streak suggest that endoderm (possibly hindgut) may be formed close to the emerging cloacal membrane, as well.

Foxa2 regulates polarity and epithelialization in the endoderm germ layer of the mouse embryo

In the mouse, one of the earliest events in the determination of cell fate is the segregation of cells into germ layers during gastrulation; however, the cellular and molecular details are not well defined due to intrauterine development. We were able to visualize a clear sequence of events occurring in the process of germ-layer formation, using immunohistochemistry and time-lapse confocal imaging. The T-box transcription factor brachyury (T) and the Forkhead transcription factor Foxa2 specify mesoderm and endoderm in the posterior epiblast. Fate-specified epiblast cells lose their polarity and undergo epithelial-mesenchymal transition to invade into the primitive streak region, where these cell populations quickly separate and differentiate into morphologically and molecularly distinct Foxa2-positive endoderm and T-positive mesoderm populations. The endoderm cells flatten and acquire apical-basal polarity during intercalation into the outside epithelium in order to establish proper intracellular junctions with pre-existing cells. By contrast, the mesodermal cells become spherical during migration and acquire a mesenchymal fate. Interestingly, axial mesodermal cells are descended from Foxa2-positive epiblast cells that upregulate T protein in the anterior primitive streak region. These cells, as well as Foxa2-positive endoderm cells, are highly polarized and epithelialized, suggesting that Foxa2 promotes an epithelial fate and suppresses a mesenchymal fate. This observation is supported by the fact that Foxa2 mutant endodermal cells fail to maintain polarity and do not establish proper cellular junctions, and are thus unable to functionally integrate into the endoderm epithelium. We propose that Foxa2 regulates a molecular program that induces an epithelial cellular phenotype.