Definitive Endoderm Formation Research Articles

Intestinal lineage commitment of embryonic stem cells

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.

A Distinct MicroRNA Signature for Definitive Endoderm Derived From Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the potential to differentiate into many adult cell types, and they are being explored as a resource for cell replacement therapies for multiple diseases. In order to optimize in vitro differentiation protocols, it will be necessary to elucidate regulatory mechanisms that contribute to lineage specification. MicroRNAs (miRNAs) are emerging as key regulators of hESC differentiation and embryonic development. In this study, we compare miRNA expression profiles between pluripotent hESCs and definitive endoderm (DE), an early step in the pathway toward the pancreatic lineage. Results from microarray analysis showed that DE can be distinguished by its unique miRNA profile, which consists of 37 significantly down-regulated and 17 up-regulated miRNAs in 2 different cell lines and in the presence/absence of feeder layers. Comparison to other hESC-derived lineages showed that most of the highly up-regulated miRNAs are specific to endoderm in early development. Notably, miR-375, which was previously implicated in regulating development and function of later stages of pancreatic development, is highly and specifically up-regulated during DE formation, suggesting that it may have a distinct role very early in development. Examination of potential mRNA targets showed that TIMM8A is repressed by ectopic miR-375 expression in pluripotent hESCs.

Hepatocyte-like cells derived from human embryonic stem cells specifically via definitive endoderm and a progenitor stage

Human embryonic stem cells offer a potential unlimited supply for functional hepatocytes, since they can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing various hepatic markers. These cells could be used in various applications such as studies of drug metabolism and hepatotoxicity, which however, would require a significant expression of drug metabolizing enzymes. To derive these cells we use a stepwise differentiation protocol where growth- and maturation factors are added. The first phase involves the formation of definitive endoderm. Next, these cells are treated with factors known to promote the induction and proliferation towards hepatic progenitor cell types. In the last phase the cells are terminally differentiated and maturated into functional hepatocyte-like cells. The cultures were characterized by analysis of endodermal or hepatic markers and compared to cultures derived without induction via definitive endoderm. Hepatic functions such as urea secretion, glycogen storage, indocyanine green uptake and secretion, and cytochrome P450-expression and activity were evaluated. The DE-Hep showed a hepatocyte morphology with sub-organized cells and exhibited many liver-functions including transporter activity and capacity to metabolize drugs specific for important cytochrome P450 sub-families. This represents an important step in differentiation of hESC into functional hepatocytes.

Foxh1 and Foxa2 are not required for formation of the midgut and hindgut definitive endoderm

The definitive endoderm forms during gastrulation and is rapidly transformed into the gut tube which is divided along the anterior-posterior axis into the foregut, midgut, and hindgut. Lineage tracing and genetic analysis have examined the origin of the definitive endoderm during gastrulation and demonstrated that the majority of definitive endoderm arises at the anterior end of the primitive streak (APS). Foxh1 and Foxa2 have been shown to play a role in specification of the APS and definitive endoderm. However, prior studies have focused on the role of these factors in specification of foregut definitive endoderm, while their role in the specification of midgut and hindgut definitive endoderm is less understood. Furthermore, previous analyses of these mutants have utilized definitive endoderm markers that are restricted to the anterior endoderm, expressed in extraembryonic endoderm, or present in other germ layers. Here, we characterized the expression of several novel definitive and visceral endoderm markers in Foxh1 and Foxa2 null embryos. In accordance with previous studies, we observed a deficiency of foregut definitive endoderm resulting in incorporation of visceral endoderm into the foregut. Interestingly, this analysis revealed that formation of midgut and hindgut definitive endoderm is unaffected by loss of Foxh1 or Foxa2. This finding represents a significant insight into specification and regionalization of mouse definitive endoderm.

Generation of Monoclonal Antibodies Specific for Cell Surface Molecules Expressed on Early Mouse Endoderm

The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.

Enforced Expression of Mixl1 During Mouse ES Cell Differentiation Suppresses Hematopoietic Mesoderm and Promotes Endoderm Formation

The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse embryonic stem cell (ESC) differentiation. We show that three independently derived ESC lines constitutively expressing Mixl1 (Mixl1(C) ESCs) differentiate into embryoid bodies (EBs) containing a higher proportion of E-cadherin (E-Cad)(+) cells. Our analysis also shows that this differentiation occurs at the expense of hematopoietic mesoderm differentiation, with Mixl1(C) ESCs expressing only low levels of Flk1 and failing to develop hemoglobinized cells. Immunohistochemistry and immunofluorescence studies revealed that Mixl1(C) EBs have extensive areas containing cells with an epithelial morphology that express E-Cad, FoxA2, and Sox17, consistent with enhanced endoderm formation. Luciferase reporter transfection experiments indicate that Mixl1 can transactivate the Gsc, Sox17, and E-Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. Taken together, these studies suggest that high levels of Mixl1 preferentially allocate cells to the endoderm during ESC differentiation.

Production of β‐cells from human embryonic stem cells

The making of functional pancreatic islets in renewable numbers has been a goal of stem cell biologists since early 2000. Since that time, many studies have reported successful creation of glucose-responsive pancreatic beta-cells. Not until the more recent systematic application of developmental principles to stem cell biology systems were breakthroughs achieved on directed specification of the required early developmental intermediates. The most important first step is the formation of the definitive endoderm (DE) lineage which is compulsory for production of the epithelium of the pancreas and the other important endoderm-derived organs such as the liver, intestine and lung. The formation of DE from embryonic stem cells made possible additional experimentation aimed at directing the endoderm to further specified foregut and pancreatic endoderm lineages. With these discoveries came the first production of immature pancreatic endocrine cells. Most recently, the production in vivo of glucose-responsive insulin-producing cells with the capacity to correct Steptozotocin-induced hyperglycaemia in mice has been achieved. The work leading up to this achievement, in relation to the other principle human stem cell studies conducted in this area, will be briefly described. The necessary steps and ideal characteristics of embryonic stem cell-based differentiation to pancreatic beta-cells capable of glucose stimulated insulin secretion will be underscored.

Towards Regenerative Therapy for Thymic Insufficiency after Hematopoietic Stem Cell Transplantation: Generation of MTS24 Positive Definitive Endoderm from Murine Embryonic Stem Cells.

Following hematopoietic stem cell transplantation (HSCT), longterm T-cell reconstitution should be established by thymus-dependent de-novo generation of naïve T-cells (thymopoiesis), which is especially important for generating a naïve T-cell pool with a broad T-cell receptor (TCR) repertoire. However, while erythroid and myeloid hematopoietic cell lineages recover rapidly following HSCT, T-cell development may severely lag behind due to thymic insufficiency. Recent studies in fetal mice have identified common thymic epithelial progenitor cells (TEPC) that were capable to re-establish a thymus in-vivo upon transplantation into a-thymic nude mice. These TEPC are characterized by expression of the transcription factor Foxn1 and by cell surface expression of MTS24. These TEPC arise exclusively from progenitors originating from the anterior foregut endoderm during embryogenesis. Therefore, we hypothesized that common TEPC may be generated in-vitro from embryonic stem (ES) cells that have differentiated towards definitive endoderm. Currently, the mechanisms underlying commitment of definitive endoderm towards a thymic fate are unknown. In order to differentiate murine ES cells towards definitive endoderm and TEPC and to identify the factors involved in the commitment of endoderm towards a thymic fate we investigated the expression of MTS24 and of genes associated with thymic differentiation in ES-cell derived endoderm using a Gcs–GFP/Sox17–huCD25 reporter ES cell line. Culture of these GscgfpSox17huCD25 ES cells in the presence of Activin A resulted in a rapid induction of mesendodermal differentiation. After 6 days of culture the majority of cells differentiated towards mesoderm (Gsc+Sox17−, 60%) and definitive endoderm (GSC+Sox17+, 35%). Apart from the addition of Activin A, the use of low passage number ES-cells and a seeding density between 200–300 cells/cm2 were the most important factors determining efficient differentiation towards definitive endoderm. Addition of insulin or WNT-3a had no significant effect on differentiation, while usage of a high passage number of ES-cells and/or a high seeding density mainly promoted development of visceral endoderm. Real-time quantitative PCR of the definitive endoderm fraction of these cultures not only showed expression of genes associated with definitive endoderm and gut tube formation (i.e. Sox17, Foxa2, Hnf4a and TCF2) but also of genes associated with anterior foregut endoderm (i.e. Hhex, Pax9) and a low, but significant, expression of Foxn1. Analysis of MTS24 expression within these cultures showed the presence of this antigen on all three cell types. The percentage of cells expressing MTS24 was highest in visceral endoderm (30–50%) and lowest in mesoderm (5–10%). The expression was approximately 12% in definitive endoderm. We conclude that murine ES cells cultured in the presence of Activin A can efficiently differentiate towards gut-tube like endoderm, including anterior forgut endoderm, and that a fraction of the generated endoderm also expresses the surface marker MTS24, suggesting the generation of epithelial progenitors with phenotypic characteristics of TEPC.

Dynamic expression of Thyrotropin‐releasing hormone in the mouse definitive endoderm

Thyrotropin-releasing hormone (TRH) is a well-characterized regulator of the hypothalamic-pituitary-thyroid endocrine axis. Here, we describe the expression of Trh during early embryonic development in the mouse. We find Trh to be highly expressed during postimplantation stages in the mouse embryo, with expression first observed in the epiblast at embryonic day (E) 6.5. During gastrulation, Trh is expressed in the newly formed definitive endoderm cells, and at embryonic day (E) 7.75, marks the entire definitive endoderm. Subsequently, Trh mRNA levels rapidly decrease such that, by E9.0, expression in the definitive endoderm is no longer detected, after which neural expression predominates. Thus, Trh is expressed dynamically and specifically in the developing mouse definitive endoderm from E7.0 to E8.5. Trh is unique among definitive endoderm markers as it transiently marks the entire definitive endoderm population and is not expressed in the extraembryonic endoderm. Trh will be a valuable tool to study definitive endoderm formation in the mouse embryo.

The Differentiation of Distal Lung Epithelium from Embryonic Stem Cells

The potential for embryonic stem (ES) cells to differentiate into cells with a distal lung epithelial phenotype has been demonstrated using different in vitro culture methods. Three separate protocols are described here that utilize both murine and human ES cells. The distal lung epithelial phenotype is induced through the use of embryonic distal lung mesenchyme in coculture systems with differentiating embryoid bodies or the use of soluble factors in defined media to maximize definitive endoderm formation and select and maintain the desired phenotype. Phenotypic analysis is demonstrated using immunocytochemistry and SP-C promoter-eGFP reporter gene expression in transgenic ES cells. These methods provide an increased efficiency of distal lung epithelial derivation from ES cells and, therefore, they provide the foundation for the development of a cell replacement product to treat chronic lung disease or a useful in vitro model for the study of lung disease and development.

Isolating endoderm and understanding developmental signals: defining sequential steps of embryonic stem cell differentiation to β cells.

False starts have marked early work towards efficiently differentiating embryonic stem cells to β cells. Recent research has returned to foundations of developmental biology by focusing first on achieving definitive endoderm differentiation as a requisite step to ultimately producing high-quality islet endocrine cells. The present review will highlight recent demonstrations of definitive endoderm differentiation from embryonic stem cells and emphasize how mesenchymal signals, learned from embryological studies, may be used to further induce pancreatic specification from embryonic stem cells. Recently, advances have been made in the identification and purification of cells having the expected characteristics of definitive endoderm, from which all pancreatic cell types are derived. These studies have defined putative markers of definitive endoderm and provided insights into embryological signals controlling endoderm formation in mice and humans. Emerging data about the relationship between developing definitive endoderm and the surrounding mesenchyme now suggest possible methods of replicating the pancreatic developmental process in embryonic stem cells in vitro. Lessons learned from understanding developmental mechanisms of definitive endoderm formation and pancreas specification in lower organisms and adapted in application to human embryonic stem cells will drive further advances in this field.

Activin A Efficiently Specifies Definitive Endoderm from Human Embryonic Stem Cells Only When Phosphatidylinositol 3-Kinase Signaling Is Suppressed

Human ESCs (hESCs) respond to signals that determine their pluripotency, proliferation, survival, and differentiation status. In this report, we demonstrate that phosphatidylinositol 3-kinase (PI3K) antagonizes the ability of hESCs to differentiate in response to transforming growth factor beta family members such as Activin A and Nodal. Inhibition of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then definitive endoderm (DE) by allowing them to be specified by Activin/Nodal signals present in hESC cultures. Under conditions where hESCs are grown in mouse embryo fibroblast-conditioned medium under feeder-free conditions, approximately 70%-80% are converted into DE following 5 days of treatment with inhibitors of the PI3K pathway, such as LY 294002 and AKT1-II. Microarray and quantitative polymerase chain reaction-based gene expression profiling demonstrates that definitive endoderm formation under these conditions closely parallels that following specification with elevated Activin A and low fetal calf serum (FCS)/knockout serum replacement (KSR). Reduced insulin/insulin-like growth factor (IGF) signaling was found to be critical for cell fate commitment into DE. Levels of insulin/IGF present in FCS/KSR, normally used to promote self-renewal of hESCs, antagonized differentiation. In summary, we show that generation of hESC-DE requires two conditions: signaling by Activin/Nodal family members and release from inhibitory signals generated by PI3K through insulin/IGF. These findings have important implications for our understanding of hESC self-renewal and early cell fate decisions.

Targeted disruption of cubilin reveals essential developmental roles in the structure and function of endoderm and in somite formation

BackgroundCubilin is a peripheral membrane protein that interacts with the integral membrane proteins megalin and amnionless to mediate ligand endocytosis by absorptive epithelia such as the extraembryonic visceral endoderm (VE).ResultsHere we report the effects of the genetic deletion of cubilin on mouse embryonic development. Cubilin gene deletion is homozygous embryonic lethal with death occurring between 7.5–13.5 days post coitum (dpc). Cubilin-deficient embryos display developmental retardation and do not advance morphologically beyond the gross appearance of wild-type 8–8.5 dpc embryos. While mesodermal structures such as the allantois and the heart are formed in cubilin mutants, other mesoderm-derived tissues are anomalous or absent. Yolk sac blood islands are formed in cubilin mutants but are unusually large, and the yolk sac blood vessels fail to undergo remodeling. Furthermore, somite formation does not occur in cubilin mutants. Morphological abnormalities of endoderm occur in cubilin mutants and include a stratified epithelium in place of the normally simple columnar VE epithelium and a stratified cuboidal epithelium in place of the normally simple squamous epithelium of the definitive endoderm. Cubilin-deficient VE is also functionally defective, unable to mediate uptake of maternally derived high-density lipoprotein (HDL).ConclusionIn summary, cubilin is required for embryonic development and is essential for the formation of somites, definitive endoderm and VE and for the absorptive function of VE including the process of maternal-embryo transport of HDL.

Endoderm and Pancreatic Islet Lineage Differentiation from Human Embryonic Stem Cells

Human embryonic stem cells (HESCs) are a potential source of insulin-producing tissue for transplantation. Recent studies have begun to define factors that promote definitive endoderm formation from HESCs, but conditions permitting complete islet specification in vitro have not been described. Here, we study spontaneous differentiation of HESCs to definitive endoderm and pancreatic progenitor cells, and begin to determine which aspects of the protocol are required for this cell fate commitment. HESCs were differentiated in culture for up to 10 weeks, including an embryoid body (EB) formation step. Modifications to the protocol included elimination of the EB phase, varying initial cell cluster size when forming EBs, and addition of mesoderm-derived cells to EBs. Differentiated cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. HESCs are capable of spontaneous differentiation to cells expressing the definitive endoderm and pancreatic progenitor markers Foxa2, Sox17, and Pdx1, and ultimately, some cells express islet endocrine hormones. This differentiation occurs to a much greater extent when an EB formation step is included. Increased expression of endoderm markers during and after EB formation also correlated strongly with the size of cell clusters used to start EBs, as well as the addition of mesoderm- derived embryonic cells. This study demonstrates that a subset of differentiated HESC progeny adopt an endoderm fate and exhibit the capacity for further pancreatic lineage specification in vitro. Basal conditions were established for examining factors that can commit HESC-derived endoderm cells to specific pancreatic lineages.

Sox17 influences the differentiation of respiratory epithelial cells

The Sry-related HMG box transcription factor, Sox17, is required for formation of definitive endoderm that gives rise to various organs, including thyroid, lung, liver, pancreas, and intestine. While expressed at high levels in the embryonic endoderm, Sox17 is also expressed in mature tissues, including the lung. Sox17 expression in respiratory epithelial cells was first detected in the fetal lung at embryonic day 18. Thereafter, Sox17 expression was restricted primarily to ciliated cells, suggesting its potential role in airway cell differentiation. When expressed in epithelial cells of the embryonic lung, Sox17 inhibited peripheral epithelial cell differentiation and disrupted branching morphogenesis. In vitro, Sox17 inhibited Sftpc and enhanced Foxj1 promoter activity, consistent with its expression in proximal airway cells. Conditional expression of Sox17 in peripheral respiratory epithelial cells of adult lung induced hyperplastic clusters of cells expressing increased levels of β-catenin and differentiation markers representing multiple proximal respiratory epithelial cell types. Sox17 prolonged survival and enhanced growth and differentiation of respiratory epithelial cells in vitro. Sox17 induced plasticity of respiratory epithelial cells, reprogramming alveolar cells into epithelial cells with characteristics more typical of the proximal airway. Sites of expression and the effects of Sox17 in vivo and in vitro are consistent with a role for Sox17 or other members of the Sox family of transcription factors in differentiation of the conducting airway epithelium.

The Vg1-related protein Gdf3 acts in a Nodal signaling pathway in the pre-gastrulation mouse embryo

The formation of the anterior visceral endoderm (AVE) in the pre-gastrulation mouse embryo represents a crucial event in patterning of the anterior-posterior axis. Here, we show that the transforming growth factor beta (Tgfbeta) family member Gdf3 (growth-differentiation factor 3), a close relative of Xenopus Vg1, resembles the Tgfbeta ligand Nodal in both its signaling activity and its role in AVE formation in vivo. Thus, in cell culture, Gdf3 signaling requires the EGF-CFC co-receptor Cripto and can be inhibited by Lefty antagonists. In Xenopus embryos, Gdf3 misexpression results in secondary axis formation, and induces morphogenetic elongation and mesendoderm formation in animal caps. In mouse embryos, Gdf3 is expressed in the inner cell mass and epiblast, and null mutants frequently exhibit abnormal formation or positioning of the AVE. This phenotype correlates with defects in mesoderm and definitive endoderm formation, as well as abnormal Nodal expression levels. Our findings indicate that Gdf3 acts in a Nodal-like signaling pathway in pre-gastrulation development, and provide evidence for the functional conservation of Vg1 activity in mice.

Efficient differentiation of human embryonic stem cells to definitive endoderm

The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of development.

Mice exclusively expressing the short isoform of Smad2 develop normally and are viable and fertile

Smad2 and Smad3 are closely related effectors of TGFbeta/Nodal/Activin-related signaling. Smad3 mutant mice develop normally, whereas Smad2 plays an essential role in patterning the embryonic axis and specification of definitive endoderm. Alternative splicing of Smad2 exon 3 gives rise to two distinct protein isoforms. The short Smad2(Deltaexon3) isoform, unlike full-length Smad2, Smad2(FL), retains DNA-binding activity. Here, we show that Smad2(FL) and Smad2(Deltaexon3) are coexpressed throughout mouse development. Directed expression of either Smad2(Deltaexon3) or Smad3, but not Smad2(FL), restores the ability of Smad2-deficient embryonic stem (ES) cells to contribute descendants to the definitive endoderm in wild-type host embryos. Mice engineered to exclusively express Smad2(Deltaexon3) correctly specify the anterior-posterior axis and definitive endoderm, and are viable and fertile. Moreover, introducing a human Smad3 cDNA into the mouse Smad2 locus similarly rescues anterior-posterior patterning and definitive endoderm formation and results in adult viability. Collectively, these results demonstrate that the short Smad2(Deltaexon3) isoform or Smad3, but not full-length Smad2, activates all essential target genes downstream of TGFbeta-related ligands, including those regulated by Nodal.

Angiomotin Regulates Visceral Endoderm Movements during Mouse Embryogenesis

In pregastrula stage mouse embryos, visceral endoderm (VE) migrates from a distal to anterior position to initiate anterior identity in the adjacent epiblast [1, 2]. This anterior visceral endoderm (AVE) is then displaced away from the epiblast by the definitive endoderm to become associated with the extra-embryonic ectoderm and subsequently contributes to the yolk sac. Little is known about the molecules that regulate this proximal displacement. Here we describe a role for mouse angiomotin (amot) in VE movements. amot expression is initially detected in the AVE and subsequently in the VE associated with the extra-embryonic ectoderm. Most amot mutant mice die soon after gastrulation with distinct furrows of VE located at the junction of the embryonic and extra-embryonic regions. Mutant analysis suggests that VE accumulation in these furrows is caused by defects in cell migration into proximal extra-embryonic regions, although distal-to-anterior movements associated with the epiblast, definitive endoderm formation, and anterior specification of the epiblast appear to be normal. These results suggest that amot acts within subregions of the VE to regulate morphogenetic movements that are required for embryo viability.

Mouse Mix gene is activated early during differentiation of ES and F9 stem cells and induces endoderm in frog embryos.

In frog and zebrafish, the Mix/Bix family of paired type homeodomain proteins play key roles in specification and differentiation of mesendoderm. However, in mouse, only a single Mix gene (mMix) has been identified to date and its function is unknown. We have analyzed the expression of mouse Mix RNA and protein in embryos, embryoid bodies formed from embryonic stem cells and F9 teratocarcinoma cells, as well as several differentiated cell types. Expression in embryoid bodies in culture mirrors that in embryos, where Mix is transcribed transiently in primitive (visceral) endoderm (VE) and in nascent mesoderm. In F9 cells induced by retinoic acid to differentiate to VE, mMix is coordinately expressed with three other endodermal transcription factors, well before AFP, and its protein product is localized to the nucleus. In a subpopulation of nascent mesodermal cells from embryonic stem cell embryoid bodies, mMix is coexpressed with Brachyury. Intriguingly, mMix mRNA is detected in a population (T+Flk1+) of cells which may contain hemangioblasts, before the onset of hematopoiesis and activation of hematopoietic markers. In vitro and in vivo, mMix expression in nascent mesoderm is rapidly down-regulated and becomes undetectable in differentiated cell types. In the region of the developing gut, mMix expression is confined to the mesoderm of mid- and hindgut but is absent from definitive endoderm. Injection of mouse mMix RNA into early frog embryos results in axial truncation of developing tadpoles and, in animal cap assays, mMix alone is sufficient to activate expression of several endodermal (but not mesodermal) markers. Although these observations do not exclude a possible cell-autonomous function for mMix in mesendodermal progenitor cells, they do suggest an additional, non-cell autonomous role in nascent mesoderm in the formation and/or patterning of adjacent definitive endoderm.