Expression Of Endodermal Markers Research Articles

APE1 promotes embryonic stem cell proliferation and teratoma formation by regulating GDNF/GFRα1 axis

The teratomas formation has severely hindered the application of embryonic stem cells (ESCs) in clinical trials. Apurinic/apyrimidinic endonuclease 1 (APE1) is strongly involved in the development of tumors and differentiation process of stem cells. However, the role of APE1 in teratomas remains unknown. The expression of APE1 was examined in mouse ESCs (mESCs) by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot. The role and mechanism of APE1 in the proliferation, pluripotency and differentiation of E14 cells were determined by cell counting, flow cytometry and western blot assays. Besides, the role of APE1 in teratomas was also probed in xenografted mice. The expression of APE1 was upregulated in mESCs with differentiation. Knockdown of APE1 reduced the cell numbers, induced the arrest of the G2/M phase, and decreased the expression of cell cycle-related proteins in E14 cells. Besides, loss- and gain-of-function assays revealed that APE1 enhanced the levels of proteins involved in pluripotency, reduced the protein expression of ectoderm markers, and increased the protein levels of endoderm markers in E14 cells. Mechanically, inhibition of APE1 downregulated the expression of GDNF and GFRα1 in E14 cells. GDNF reversed the role of APE1 in the proliferation, pluripotency and embryogenesis of E14 cells. Moreover, suppression of APE1 reduced the teratoma volume and the relative protein expression of endoderm markers, but increased the relative protein expression of ectoderm markers in xenografted mice. Collectively, knockdown of APE1 attenuated proliferation, pluripotency and embryogenesis of mESCs via GDNF/GFRα1 axis.

A small molecule modulating monounsaturated fatty acids and Wnt signaling confers maintenance to induced pluripotent stem cells against endodermal differentiation

BackgroundStearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. Through the fatty acid acylation process, this enzyme orchestrates post-translational modifications to proteins involved in cell development and differentiation. In this study, we used biochemical methods, immunostaining, and covalent labeling to evaluate whether a small molecule modulating unsaturated fatty acids can influence the early endodermal differentiation of human-induced pluripotent stem cells (iPSCs).MethodsThe hiPSCs were cultured in an endoderm-inducing medium containing activin A and defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The cell cycles and the yields of the three germ layers (endoderm, mesoderm, and ectoderm) were assessed using flow cytometry. The expression of endoderm and pluripotency markers and the expressions of Wnt signaling pathway proteins were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction.ResultsWhen SCD1 was inhibited on the first day, the population of cells with endodermal features decreased at the end of differentiation. Moreover, early SCD1 inhibition preserved the properties of hiPSCs, preventing their shift toward mesodermal or ectodermal lineage. Also, first-day-only treatment of cells with the SCD1 inhibitor decreased β-catenin gene expression and the intensity of fluorescent emission in the click chemistry assay. The cells were effectively rescued from these effects by cotreatment with oleate. Late treatment with the inhibitor in the two subsequent days of endoderm induction did not have any significant effects on endoderm-specific markers or fluorescent intensity. Reproducible results were also obtained with human embryonic stem cells.ConclusionThe small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring the maintenance of hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in the endoderm commitment of pluripotent stem cells may be of importance in disorders of endoderm-derived organs and dysregulated metabolism.The schematic representation of the study design and main results. Activin A induces endoderm features through Smad2/3/4 and increases the expression of SCD1. SCD1 can produce MUFAs and subsequently modify the Wnt molecules. MUFA acylated/activated Wnts are secreted to interact with corresponding receptors on the target cells. β-catenin accumulates in the cytoplasm and is translocated into the nucleus after the interaction of Wnt with the receptor. Then, β-catenin increases the expression of the endoderm markers Sox17 and CXCR4.

Impaired Gene Expression Due to Iodine Excess in the Development and Differentiation of Endoderm and Thyroid Is Associated with Epigenetic Changes.

Background: Thyroid hormone (TH) synthesis is essential for the control of development, growth, and metabolism in vertebrates and depends on a sufficient dietary iodine intake. Importantly, both iodine deficiency and iodine excess (IE) impair TH synthesis, causing serious health problems especially during fetal/neonatal development. While it is known that IE disrupts thyroid function by inhibiting thyroid gene expression, its effects on thyroid development are less clear. Accordingly, this study sought to investigate the effects of IE during the embryonic development/differentiation of endoderm and the thyroid gland. Methods: We used the murine embryonic stem (ES) cell model of in vitro directed differentiation to assess the impact of IE on the generation of endoderm and thyroid cells. Additionally, we subjected endoderm and thyroid explants obtained during early gestation to IE and evaluated gene and protein expression of endodermal markers in both models. Results: ES cells were successfully differentiated into endoderm cells and, subsequently, into thyrocytes expressing the specific thyroid markers Tshr, Slc5a5, Tpo, and Tg. IE exposure decreased the messenger RNA (mRNA) levels of the main endoderm markers Afp, Crcx4, Foxa1, Foxa2, and Sox17 in both ES cell-derived endoderm cells and embryonic explants. Interestingly, IE also decreased the expression of the main thyroid markers in ES cell-derived thyrocytes and thyroid explants. Finally, we demonstrate that DNA methyltransferase expression was increased by exposure to IE, and this was accompanied by hypermethylation and hypoacetylation of histone H3, pointing to an association between the gene repression triggered by IE and the observed epigenetic changes. Conclusions: These data establish that IE treatment is deleterious for embryonic endoderm and thyroid gene expression.

Unique and redundant roles of SOX2 and SOX17 in regulating the germ cell tumor fate.

Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma-like TCam-2 cells to SOX17 in somatic cells and SOX2 in EC-like 2102EP cells. In seminoma-like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17-binding sites overlap in seminoma-like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma-like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC-like cells SOX2 regulates pluripotency-associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.

Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells.

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells 2019;37:306-317.

Hematopoietic Developmental Potential of Human Pluripotent Stem Cell Lines Is Accompanied by the Morphology of Embryoid Bodies and the Expression of Endodermal and Hematopoietic Markers.

The potential clinical applications of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs) are limited by the difficulty of recapitulating embryoid hematopoiesis and by the unknown differentiation potential of hPSC lines. To evaluate their hematopoietic developmental potential, available hPSC lines were differentiated by an embryoid body (EB) suspension culture in serum-free medium supplemented with three different cytokine mixes (CMs). The hPSC differentiation status was investigated by the flow cytometry expression profiles of cell surface molecules, and the gene expression of pluripotency and differentiation markers over time was evaluated by real-time reverse transcription polymerase chain reaction (qRT-PCR). hPSC lines differed in several aspects of the differentiation process, including the absolute yield of hematopoietic progenitors, the proportion of hematopoietic progenitor populations, and the effect of various CMs. The ability to generate hematopoietic progenitors was then associated with the morphology of the developing EBs, the expression of the endodermal markers AFP and SOX17, and the hematopoietic transcription factor RUNX1. These findings deepen the knowledge about the hematopoietic propensity of hPSCs and identify its variability as an aspect that must be taken into account before the usage of hPSC-derived HSCs in downstream applications.

Investigation of endoderm marker-genes during gastrulation and gut-development in the velvet worm Euperipatoides kanangrensis

The ancestral state of animal gastrulation and its bearing for our understanding of bilaterian evolution still is one of the most controversially discussed topics in the field of evolutionary and developmental biology. One hypothesis, the so-called amphistomy scenario, suggests the presence of a slit-like blastopore in the last common ancestor of Bilateria.Onychophoran ontogeny at least superficially appears to support this scenario since a ventral groove clearly forms during gastrulation. The origin and nature of this groove, however, is another matter of ongoing controversy; i.e. the question of whether this structure actually represents the blastopore, or at least part of it. Recent research using genetic markers argued against the furrow representing a blastoporal structure. Here we investigate the origin of endoderm, which usually originates from the blastopore. We find conserved expression patterns of the endoderm- and gut-marker genes GATA456, GATA123, Hnf4 and fkh during gut development, and discuss the formation of the onychophoran gut in comparison with that in a range of arthropods.Despite expression of endodermal markers in and around the furrow we do not find convincing evidence that the furrow may be part of the blastopore, and thus we suggest that onychophoran development does not yield support for the amphistomy scenario.

Hepatic population derived from human pluripotent stem cells is effectively increased by selective removal of undifferentiated stem cells using YM155

BackgroundPluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells are promising target cells for cell regenerative medicine together with recently advanced technology of in-vitro differentiation. However, residual undifferentiated stem cells (USCs) during in-vitro differentiation are considered a potential risk for development of cancer cells and nonspecific lineage cell types. In this study we observed that USCs still exist during hepatic differentiation, consequently resulting in poor quality of the hepatic population and forming teratoma in vivo. Therefore, we hypothesized that effectively removing USCs from in-vitro differentiation could improve the quality of the hepatic population and guarantee safety from risk of teratoma formation.MethodsHuman PSCs were differentiated to hepatocytes via four steps. YM155, a known BIRC5 inhibitor, was applied for removing the residual USCs on the hepatic differentiation. After YM155 treatment, hepatocyte development was evaluated by measuring gene expression, immunostaining and hepatic functions at each stage of differentiation, and forming teratomas were confirmed by cell transplantation with or without YM155.ResultsThe selected concentrations of YM155 removed USCs (NANOG+ and OCT4+) in a dose-dependent manner. As a result, expression of endodermal markers (SOX17, FOXA2 and CXCR4) at stage II of differentiation and hepatic markers (ALB, AFP and HNF4A) at stage III was up-regulated by YM155 treatment as well as the hepatic population (ALB+), and functions (ALB/urea secretion and CYP450 enzyme activity) were enhanced at the final stage of differentiation (stage IV). Furthermore, we demonstrated that NANOG and OCT4 expression remaining until stage III (day 15 of differentiation) completely disappeared when treated with YM155 and teratoma formation was effectively prevented by YM155 pretreatment in the in-vitro study.ConclusionsWe suggest that the removal of USCs using YM155 could improve the quantity and quality of induced hepatocytes and eliminate the potential risk of teratoma formation.

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes.

Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK(-/-) EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation.

Hypoxia promotes thyroid differentiation of native murine induced pluripotent stem cells.

Hypothyroidism is a very common hormonal deficiency and the stem cell technology which developed in the recent years may offer a therapeutic strategy for treating this disorder. Hypoxia has been demonstrated to play an important role in embryonic formation and development and to modulate stem cell differentiation. However, the influence of oxygen tension on thyroid differentiation has not been studied. In this study, we used murine induced pluripotent stem (iPS) cells for thyroid cell differentiation under normoxic and hypoxic conditions and compared differentiation efficiency in morphology, function, gene and protein expression under both conditions. We found that hypoxia promoted adhesion and outgrowth of embryoid bodies (EBs) derived from murine iPS cells. Expression of endodermal markers (Foxa2 and Gata4) and thyroid transcription factors (Pax8 and Nkx2.1) was increased by hypoxia at both gene and protein levels during early-mid differentiation stages (p<0.05). And so were the thyroid specific markers NIS and TSHR at the end of the experiment (p<0.05). In addition, functional iodide uptake by differentiated cells was also increased after hypoxia. Thyroid differentiation from iPS cells is enhanced under hypoxia and this may involve hypoxia inducible factors (HIFs) and their downstream gene FGF2. Our data offer a foundation for understanding thyroid development and provide a potentially more efficient way to use cell therapy for treating thyroid deficiency.

Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in embryonic stem cells.

Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.

Smad2 and smad3 Are Required for Mesendoderm Induction by Transforming Growth Factor-β/Nodal Signals in Zebrafish

The transforming growth factor-beta ligands Nodal, activin, and Vg1 play important roles in mesendoderm induction and patterning during vertebrate embryogenesis. These ligands are believed to transduce the signal through the receptor-activated transcription factors Smad2 and Smad3. However, the roles of smad2/3 genes in development of zebrafish embryos are largely unknown because the presence of multiple smad2/3 genes and their maternal expression have hampered the investigation of their developmental roles. We generated potent and specific dominant-negative forms of zebrafish Smad2, Smad3a, and Smad3b by mutating multiple amino acids. Overexpression of these mutants abolished mesendoderm induction by ectopic Nodal signaling in zebrafish embryos. Expression of dominant-negative smad2/3 abrogated Smad2/3 activities in wild-type embryos and caused various mesendodermal defects similar to those in Nodal-deficient embryos. Smad2/3-deficient cells transplanted into the blastodermal margin of wild-type hosts preferentially differentiated into ectodermal tissues rather than mesendodermal tissues, supporting the idea that response of cells to mesendoderm inducers requires Smad2/3 activities. Interference with Smad2/3 activities in Zoep, Moep, and MZoep mutant embryos resulted in more severe mesendodermal defects. Thus, our data reveal that Nodal signaling and mesendoderm induction depend on Smad2/3 and suggest that transforming growth factor-beta signals other than Nodal also contribute to Smad2/3 signaling and embryonic patterning.

Zic3 Is Required for Maintenance of Pluripotency in Embryonic Stem Cells

Embryonic stem (ES) cell pluripotency is dependent upon sustained expression of the key transcriptional regulators Oct4, Nanog, and Sox2. Dissection of the regulatory networks downstream of these transcription factors has provided critical insight into the molecular mechanisms that regulate ES cell pluripotency and early differentiation. Here we describe a role for Zic3, a member of the Gli family of zinc finger transcription factors, in the maintenance of pluripotency in ES cells. We show that Zic3 is expressed in ES cells and that this expression is repressed upon differentiation. The expression of Zic3 in pluripotent ES cells is also directly regulated by Oct4, Sox2, and Nanog. Targeted repression of Zic3 in human and mouse ES cells by RNA interference-induced expression of several markers of the endodermal lineage. Notably, the expression of Nanog, a key pluripotency regulator and repressor of extraembryonic endoderm specification in ES cells, was significantly reduced in Zic3 knockdown cells. This suggests that Zic3 may prevent endodermal marker expression through Nanog-regulated pathways. Thus our results extend the ES cell transcriptional network beyond Oct4, Nanog, and Sox2, and further establish that Zic3 plays an important role in the maintenance of pluripotency by preventing endodermal lineage specification in embryonic stem cells.

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.

Frizzled5/8 is required in secondary mesenchyme cells to initiate archenteron invagination during sea urchin development

Wnt signaling pathways play key roles in numerous developmental processes both in vertebrates and invertebrates. Their signals are transduced by Frizzled proteins, the cognate receptors of the Wnt ligands. This study focuses on the role of a member of the Frizzled family, Fz5/8, during sea urchin embryogenesis. During development, Fz5/8 displays restricted expression, beginning at the 60-cell stage in the animal domain and then from mesenchyme blastula stage, in both the animal domain and a subset of secondary mesenchyme cells (SMCs). Loss-of-function analyses in whole embryos and chimeras reveal that Fz5/8 is not involved in the specification of the main embryonic territories. Rather, it appears to be required in SMCs for primary invagination of the archenteron, maintenance of endodermal marker expression and apical localization of Notch receptors in endodermal cells. Furthermore, among the three known Wnt pathways, Fz5/8 appears to signal via the planar cell polarity pathway. Taken together, the results suggest that Fz5/8 plays a crucial role specifically in SMCs to control primary invagination during sea urchin gastrulation.

An endoderm-specific GATA factor gene, dGATAe, is required for the terminal differentiation of the Drosophila endoderm

GATA factors play an essential role in endodermal specification in both protostomes and deuterostomes. In Drosophila, the GATA factor gene serpent ( srp) is critical for differentiation of the endoderm. However, the expression of srp disappears around stage 11, which is much earlier than overt differentiation occurs in the midgut, an entirely endodermal organ. We have identified another endoderm-specific Drosophila GATA factor gene, dGATAe. Expression of dGATAe is first detected at stage 8 in the endoderm, and its expression continues in the endodermal midgut throughout the life cycle. srp is required for expression of dGATAe, and misexpression of srp resulted in ectopic dGATAe expression. Embryos that either lacked dGATAe or were injected with double-stranded RNA (dsRNA) corresponding to dGATAe failed to express marker genes that are characteristic of differentiated midgut. Conversely, overexpression of dGATAe induced ectopic expression of endodermal markers even in the absence of srp activity. Transfection of the dGATAe cDNA also induced endodermal markers in Drosophila S2 cells. These studies provide an outline of the genetic pathway that establishes the endoderm in Drosophila. This pathway is triggered by sequential signaling through the maternal torso gene, a terminal gap gene, huckebein ( hkb), and finally, two GATA factor genes, srp and dGATAe.

The bHLH protein MyoR inhibits the differentiation of early embryonic endoderm

MyoR is a bHLH protein whose expression was reported to be almost exclusively restricted to the precursors of the skeletal muscle lineage where it was postulated to function as a transcriptional repressor of myogenesis. However, previous studies in our laboratory suggested a much broader role for MyoR in embryonic cell differentiation. We demonstrated that, besides being expressed in several adult tissues of non-muscle lineage, MyoR was expressed at a much earlier stage in mammalian development than had previously been reported, that is, as early as the blastocyst stage, well before skeletal muscle specification. We also found that, as in skeletal muscle precursor cells, MyoR expression is inversely correlated with the cellular differentiative state of ectodermal, non-muscle embryonal carcinoma (EC) cells. Retinoic acid (RA) treatment of ectodermal EC or embryonal stem (ES) cells promotes their differentiation into primitive endoderm. However, in the present study, we show that the RA-induced expression of endodermal markers such as EndoA, collagen IV, and t-PA are inhibited by exogenous MyoR expression and that the level of inhibition of these markers correlates with the level of MyoR expressed. Conversely, knock-down of MyoR expression via RNA interference enhances RA-induced differentiation of EC cells, promoting earlier and much higher expression of the above-mentioned endodermal markers following RA treatment. Finally, we have narrowed the period of exogenous MyoR-induced embryonic lethality to between 3.5 and 5.5 days post-coitum (dpc), the period during which embryonic endoderm differentiates from the embryonic ectoderm. Our results suggest, therefore, that inhibition of endodermal differentiation between 3.5 and 5.5 dpc contributes to the embryonic death of mouse embryos overexpressing exogenous MyoR and consequently that MyoR may serve as a repressor of embryonal endoderm differentiation.

Changes in embryonic cell fate produced by expression of an endodermal transcription factor, Xsox17

Many molecules induce the ectopic expression of tissue-specific genes in Xenopus embryos. Conversely, interfering with their activity disrupts patterns of gene expression, implicating them in normal development. Does this mean that they control cell fate (i.e. position, as well as differentiation)? Xsox17α and β can induce ectopic expression of endodermal markers; inhibiting their function suppresses expression of endodermal marker genes in the developing gut (Cell 91 (1997) 397). Here we show the effect of these manipulations on cell lineage. Expressing Xsox17 in a cells normally fated to become ectoderm causes their descendants either to relocate into the embryonic gut or to die at a late developmental stage. Conversely, disrupting Xsox17 activity in cells normally fated to be endodermal causes them to enter mesodermal and ectodermal lineages.

A post-mid-blastula transition requirement for TGFβ signaling in early endodermal specification

In Xenopus, endodermal cell fate is determined gradually from late blastula to early gastrula stages; cell–cell interaction plays an important role in this process. Here we use a cell dissociation assay to show that extracellular signaling is required continuously before endoderm determination. Activin and Vg1, but not BMP2 or basic FGF, rescue the expression of endodermal markers in dissociated cells when provided at the mid-blastula transition (MBT, the time in which zygotic transcription begins). Removal of exogenously added activin or Vg1 before MBT results in reduction of endodermal gene expression in dissociated vegetal cells. In vivo, endogenous endodermal markers are reduced in vegetal explants when activin-like signaling is blocked with dominant negative receptors. VegT, a maternal transcription factor shown to be critical for endoderm specification, relies on an active TGFβ pathway to induce endoderm in animal caps. These results indicate that TGFβ signaling may be activated by the maternally expressed VegT to participate in endoderm determination. In addition, VegT function seems to be required in parallel with the TGFβ pathway, as overexpression of activin does not relieve endoderm repression by a dominant negative VegT mutant in vegetal cells. Our data suggest that maternal VegT first activates a zygotic TGFβ signal, then cooperates with this signal to determine the endodermal cell fate.

Mode of action of VegT in mesoderm and endoderm formation

mRNA encoding the T-box transcription factor VegT is located throughout the vegetal pole of the Xenopus egg and is believed to play an important part in endoderm and mesoderm formation. We find that VegT generates endoderm both by cell-autonomous action and by generating TGF-beta family signals, the latter being entirely responsible for its mesoderm-inducing activity. Signalling molecules induced cell-autonomously by VegT include derrière, Xnr4 and activin B. Xnr1 and Xnr2 are also induced, but primarily in a non-autonomous manner. All of these signalling molecules are found in the blastula and gastrula vegetal pole and induce both endoderm and mesoderm in the animal cap assay, and hence are good candidates both for the endogenous zygotic mesoderm-inducing signal and for reinforcing the vegetal expression of endoderm markers.