Extraembryonic Endoderm Research Articles

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.

Stem cells and lineage development in the mammalian blastocyst

The mammalian blastocyst is the source of the most pluripotent stem cells known: embryonic stem (ES) cells. However, ES cells are not totipotent; in mouse chimeras, they do not contribute to extra-embryonic cell types of the trophectoderm (TE) and primitive endoderm (PrE) lineages. Understanding the genetic pathways that control pluripotency v. extra-embryonic lineage restriction is key to understanding not only normal embryonic development, but also how to reprogramme adult cells to pluripotency. The trophectoderm and primitive endoderm lineages also provide the first signals that drive patterned differentiation of the pluripotent epiblast cells of the embryo. My laboratory has produced permanent mouse cell lines from both the TE and the PrE, termed trophoblast stem (TS) and eXtra-embryonic ENdoderm (XEN) cells. We have used these cells to explore the genetic and molecular hierarchy of lineage restriction and identify the key factors that distinguish the ES cell v. the TS or XEN cell fate. The major molecular pathways of lineage commitment defined in mouse embryos and stem cells are probably conserved across mammalian species, but more comparative studies of lineage development in embryos of non-rodent mammals will likely yield interesting differences in terms of timing and details.

Constitutive Gene Expression Predisposes Morphogen-Mediated Cell Fate Responses of NT2/D1 and 27X-1 Human Embryonal Carcinoma Cells

Human embryonal carcinoma (EC) cell lines exhibit considerable heterogeneity in their levels of pluripotency. Thus, NT2/D1 cells differentiate into neural lineages upon exposure to all-trans retinoic acid (ATRA) and non-neural epithelial lineages upon exposure to bone morphogenetic protein-2 (BMP-2). In contrast, 27X-1 cells differentiate into extra-embryonic endodermal (ExE) cells upon treatment with either morphogen. To understand the molecular basis for the differential responses of the two cell lines, we performed gene expression profiling at the undifferentiated EC cell line state to identify constitutive differences in gene expression. NT2/D1 cells preferentially expressed transcripts associated with neurectodermal development, whereas 27X-1 cells expressed high levels of transcripts associated with mesendodermal characteristics. We then determined temporal expression profiles of 27X-1 cells during ExE differentiation upon treatment with ATRA and BMP-2 and compared the data with changes in gene expression observed during BMP-2- and ATRA-induced differentiation of NT2/D1 cells. ATRA and BMP-2 induced distinct sets of transcription factors and phenotypic markers in the two EC cell lines, underlying distinct lineage choices. Although 27X-1 differentiation yielded comprehensive gene expression profiles of parietal endodermal lineages, we were able to use the combined analysis of 27X-1 data with data derived from yolk sac tumors for the identification of transcripts associated with visceral endoderm formation. Our results demonstrate constitutive differences in the levels of pluripotency between NT2/D1 and 27X-1 cells that correlate with lineage potential. This study also demonstrates that EC cells can serve as robust models to investigate early lineage choices during both embryonic and extra-embryonic human development.

A Heme Export Protein Is Required for Red Cell Differentiation and May Facilitate Iron Homeostasis.

Heme is a component of cytochromes, catalases, glutathione peroxidase, hydroxylases, and nitric oxide synthase, as well as myoglobin and hemoglobin, and thus is critical to all aerobic cells. It is also a transcriptional and translational initiator of globin synthesis. However, free heme is toxic so that cells must balance synthesis with use. We recently determined that the cell surface receptor for Feline Leukemia Virus, subgroup C, (FLVCR), which causes pure red cell aplasia in cats, is a heme export protein (Cell; 118:6, 2004). To understand its physiologic significance, we generated constitutive (Flvcr+/−) and inducible (Flvcrflox/flox;Mx-cre) FLVCR null mice by targeted deletion of exon 3, and confirmed this produced a nonfunctional protein by zinc mesoporphyrin (ZnMP) and 55Fe-heme export studies. Intercrossing Flvcr+/− mice produced 0 null animals among 109 progeny genotyped. FLVCR null embryos die either before E8.5 or at E14.5, suggesting two separate mechanisms for their demise. In situ hybridization studies in normal mouse embryos show highest Flvcr transcript levels in the ectoplacental cone which forms placenta (E8.5), the placenta (E9.5), and high levels in the fetal liver (E12.5). These data plus studies with Flvcrflox/floxx Meox2-Cre mice (Cre inactivates Flvcr in all embryonic tissue, but not extraembryonic visceral endoderm and placenta) suggest that the early lethality of null mice is due to impaired maternal-embryo heme-iron transport. At E14.5, residual null mice were small with deformed limb buds and craniofacial abnormalities resembling the congenital anomalies of Diamond-Blackfan anemia. Flow cytometric analyses of fetal liver cells from deleted animals with Ter119 and CD71 demonstrated a block in erythroid differentiation at the CFU-E/proerythroblast stage, implying that their death results from a stage-specific failure in definitive erythropoiesis. Heterozygous null animals (Flvcr+/−) were viable with normal hematologic parameters. These mice had 30.9% of normal Flvcr mRNA by q RT-PCR, however, they demonstrated normal protein by western blot analysis and normal heme export function by ZnMP studies. Thus, heterozygous null mice compensate at the protein level, confirming FLVCR is critical in vivo. We next utilized our inducible mutant line (Flvcrflox/flox;Mx-cre) to generate a viable null mouse for study. Flvcr deleted mice developed pure red cell aplasia characterized by macrocytic anemia (deleted: HCT 13.8±1.2, MCV 68.35± 1.6; control: HCT 50.24 ± 2.0, MCV 51.3±1.5), reticulocytopenia, and reactive thrombocytosis. Marrow and spleen had excess cells with a proerythroblast morphology and lacked maturing red cells. Flow cytometry confirmed maturation arrest at the CFU-E/proerythroblast stage. CFU-E from FLVCR null mice were undetectable in culture. Deleted mice also demonstrated iron loading of hepatocytes. FLVCR is highly expressed in multiple human tissues with high heme-flux by western blot analyses, including liver, placenta, duodenum, and uterus, in addition to the bone marrow. Our studies prove that FLVCR is required for the survival or differentiation of proerythroblasts (that stage where heme synthesis intensifies). These results, plus additional data not shown, suggest that FLVCR may also protect nonerythroid tissues with high free heme exposure (uterus, duodenum), may mediate maternal-embryo heme-iron transport, and may mediate the transport of heme from hepatocyte to bile which would imply a novel mechanism for iron export from the body.

Murine inner cell mass-derived lineages depend on Sall4 function

Sall4 is a mammalian Spalt transcription factor expressed by cells of the early embryo and germ cells, an expression pattern similar to that of both Oct4 and Sox2, which play essential roles during early murine development. We show that the activity of Sall4 is cell-autonomously required for the development of the epiblast and primitive endoderm from the inner cell mass. Furthermore, no embryonic or extraembryonic endoderm stem cell lines could be established from Sall4-deficient blastocysts. In contrast, neither the development of the trophoblast lineage nor the ability to generate trophoblast cell lines from murine blastocysts was impaired in the absence of Sall4. These data establish Sall4 as an essential transcription factor required for the early development of inner cell mass-derived cell lineages.

Gα13 activation rescues moesin-depletion induced apoptosis in F9 teratocarcinoma cells

Mouse F9 cells differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm in response to RA and dibutyryl (db-) cAMP. G protein signaling either blocks or mimics RA-induced differentiation, the latter signaling through the Wnt-β-catenin pathway. In our study, we found that a constitutively active Gα13 mutant induces F9 cells to differentiate into parietal endoderm in the absence of exogenous agents. Gα13 expression and subsequent differentiation are accompanied by β-catenin translocation to the nucleus. Differentiation and changes in cell morphology are supported by rearrangements to the F-actin cytoskeleton. ERM (ezrin–radixin–moesin) proteins, known to link F-actin to transmembrane receptors, are also redistributed during differentiation. Furthermore, morpholino antisense and shRNA approaches show that moesin expression is essential since its knockdown leads to altered F-actin distribution and subsequent apoptosis. Moesin-depleted cells, however, remain attached to the substrate when Gα13 is constitutively expressed, but they do not differentiate into extraembryonic endoderm. Our study demonstrates a link between Gα13 signaling that regulates differentiation of F9 cells through primitive to parietal endoderm and a moesin requirement for cell survival.

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.

Definitive endoderm of the mouse embryo: Formation, cell fates, and morphogenetic function

The endoderm is one of the primary germ layers but, in comparison to ectoderm and mesoderm, has received less attention. The definitive endoderm forms during gastrulation and replaces the extraembryonic visceral endoderm. It participates in the complex morphogenesis of the gut tube and contributes to the associated visceral organs. This review highlights the role of the definitive endoderm as a source of patterning cues for the morphogenesis of other germ-layer tissues, such as the anterior neurectoderm and the pharyngeal region, and also emphasizes the intricate patterning that the endoderm itself undergoes enabling the acquisition of regionalized cell fates.

Generation of mice harbouring a conditional loss-of-function allele of Gata6

The zinc finger transcription factor GATA6 is believed to have important roles in the development of several organs including the liver, gastrointestinal tract and heart. However, analyses of the contribution of GATA6 toward organogenesis have been hampered because Gata6-/- mice fail to develop beyond gastrulation due to defects in extraembryonic endoderm function. We have therefore generated a mouse line harbouring a conditional loss-of-function allele of Gata6 using Cre/loxP technology.LoxP elements were introduced into introns flanking exon 2 of the Gata6 gene by homologous recombination in ES cells. Mice containing this altered allele were bred to homozygosity and were found to be viable and fertile. To assess the functional integrity of the loxP sites and to confirm that we had generated a Gata6 loss-of-function allele, we bred Gata6 'floxed' mice to EIIa-Cre mice in which Cre is ubiquitously expressed, and to Villin-Cre mice that express Cre in the epithelial cells of the intestine. We conclude that we have generated a line of mice in which GATA6 activity can be ablated in a cell type specific manner by expression of Cre recombinase. This line of mice can be used to establish the role of GATA6 in regulating embryonic development and various aspects of mammalian physiology.

Parietal endoderm secreted S100A4 promotes early cardiomyogenesis in embryoid bodies

Cardiomyogenesis is influenced by factors secreted by anterior-lateral and extra-embryonic endoderm. Differentiation of embryonic stem cells in embryoid bodies allows to study the influence of growth factors on cardiomyogenesis. By these means SPARC was identified as a new factor enhancing cardiomyogenesis [M. Stary, W. Pasteiner, A. Summer, A. Hrdina, A. Eger, G. Weitzer, Parietal endoderm secreted SPARC promotes early cardiomyogenesis in vitro, Exp. Cell Res. 310 (2005) 331–341]. Here we report a similar and new function for S100A4, a calcium-binding protein of the EF-hand type. S100A4 is secreted by parietal endoderm and promotes early differentiation and proliferation of cardiomyocytes. Oligomeric S100A4 supports cardiomyogenesis in a concentration-dependent manner, whereas inhibition of autocrine S100A4 severely attenuates cardiomyogenesis. S100A4 specifically influences transcription in differentiating cardiomyocytes, as evident from increased expression of cardiac transcription factor genes nkx2.5 and mef2C. These data suggest that S100A4, like SPARC, plays a supportive role in early in vitro cardiomyogenesis.

Novel role for the orphan nuclear receptor Dax1 in embryogenesis, different from steroidogenesis

Cytomegalic adrenal hypoplasia congenita (AHC) is an X-linked disease caused by mutations in DAX1-encoding gene NR0B1, previously thought to function primarily in steroidogenesis. We sought to determine the expression pattern for Dax1 along with known network partners in early embryogenesis and to determine a steroidogenic capacity for the embryo prior to the establishment of the urogenital ridge at embryonic day 9 (E9). Here, we report that murine Dax1 is a unique marker in early embryonic development, distinguishing the extraembryonic (proximal) endoderm from the remainder of the developing embryo. We showed that Wilms tumor 1, steroidogenic factor 1, and estrogen receptor β were expressed throughout the embryo, but the progesterone, estrogen α and androgen receptors, cytochrome P450 (Cyp11a1) and Nur77 were not observed in any of the embryonic layers. Lack of Cyp11A1 expression at this stage confirmed an absence of inherent steroidogenic capacity for the early embryo. The role of Nr0b1 in embryonic stem (ES) cells was investigated using siRNA knockdown, resulting in differentiation toward endoderm-like fate. Nr0b1 conditional knockout in ES cells led to differentiation, confirming our knockdown results. Our investigations suggest that Nr0b1 functions in a novel role in the maintenance of a relatively undifferentiated state. Our results further suggest that the failure of conventional murine Nr0b1 knockout attempts may be due to disregulated differentiation.

Differentiation of Mouse Embryonic Stem Cells after RNA Interference-Mediated Silencing of OCT4 and Nanog

RNA interference (RNAi) holds great promise as a tool to study the basic biology of stem cells or to direct differentiation in a specific manner. Barriers to achieving efficient and specific gene silencing in RNAi experiments include limitations in transfection efficiency and in the efficacy and specificity of RNAi silencing effectors. Here, we combine methods of efficient lipid-mediated delivery with chemically modified RNAi compounds to silence genes related to pluripotency, in order to direct differentiation of mouse embryonic stem cells. After transfection of embryonic stem cells with OCT4- or Nanog-targeted RNAi compounds, levels of OCT4 or Nanog transcript and protein were reduced accordingly. Reduction in OCT4 expression correlated with induction of trophectoderm genes Cdx2, Hand1, and PL-1, with formation of cells with trophoblast giant cell phenotype after 6 days. Reduction in Nanog expression correlated with induction of extraembryonic endoderm genes GATA4, GATA6, and laminin B1, with subsequent generation of groups of cells with parietal endoderm phenotype. Our results indicate that transient inhibition of OCT4 or Nanog by RNAi compounds is sufficient to induce differentiation toward extraembryonic lineages, which supports the model that these transcription factors function in a dose-dependent manner to influence cell fate.

257 EXPRESSION PROFILING OF GENES CRUCIAL FOR LINEAGE DETERMINATION IN IN VITRO-DERIVED EARLY BOVINE EMBRYOS

In the early blastocyst, lineage segregation depends on the expression of several key specific transcription factors. In the mouse, commitment to inner cell mass (ICM), lineage is positively regulated by Oct-4, a repressor of trophectoderm (TE) cell fate, and Nanog, which inhibits the formation of extra-embryonic and primitive endoderm. Cdx2, a caudal-type homeodomain protein, is specifically expressed in the nascent TE. The mechanisms that drive Cdx2 segregation to the outside cells are still unclear. However, the expression of Fgf Receptor 2 (FgfR2), restricted to the outside cells, and the role for its ligand, Fgf4, in promoting TE development, suggest that this signalling pathway may act upstream or in parallel with Cdx2. Little information is available on these genes in bovine; therefore the aims of the present study were as follows: (a) to identify and characterize the expression profiles of Cdx2 and FgfR2 variants (IIIc and IIIb) in bovine oocytes and pre-implantation embryos; and (b) to compare their expression patterns in ICM and TE with that of Oct-4 and Nanog. Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. RNA was isolated from MII oocytes; 2-, 4-, 8-, and 16-cell embryos; morulae; blastocysts; ICMs; and TEs. Semi-quantitative analysis of Cdx2 and FgfR2 expression in oocytes and embryos was performed in the exponential phase of PCR amplification with rabbit globin as exogenous control. In order to exclude false negative results, PCR amplification in isolated TE and ICM was extended to the plateau phase for all genes considered. Fragment identity was confirmed by sequencing. Comparison of bovine Cdx2 cDNA sequence (EMBL AM293662) with databases revealed a 91% and 87% homology with human and mouse, respectively. Cdx2 expression was not detectable in MII oocytes, but increased in 2-cell embryos. Transcript levels decreased at the 4- and 8-cell stages and then increased again in the blastocyst. FgfR2 variants were present as both maternal and embryonic transcripts, because they were detectable throughout pre-implantation development. Cdx2 and FgfR2 IIIc and IIIb expression was restricted to TE cells. Nanog was detected only in ICM, whereas Oct-4 was expressed in both lineages, as previously described in bovine (van Eijk et al. 1999 Bio. Reprod. 60, 1093-1103). In conclusion, the expression profiles of Nanog, Cdx2, and FgfR2 in bovine pre-implantation embryos follow the pattern previously described in the mouse. Their differentially segregated expression is consistent with their role as selector factors of ICM vs. TE fates. The significance of Oct-4 ubiquitous distribution still remains to be elucidated. This work was supported by FIRB RBNE01HPMX_005, TECLA-MIUR, and EUROSTELLS-ESF.

Effect of leukemia inhibitory factor on embryonic stem cell differentiation: implications for supporting neuronal differentiation1

Leukemia inhibitory factor (LIF), a pleiotropic cytokine, has been used extensively in the maintenance of mouse embryonic stem cell pluripotency. In this current work, we examined the effect of the LIF signaling pathway in embryonic stem (ES) cell differentiation to a neural fate. In the presence of LIF (1000 U/mL), the production of neuronal cells derived from embryoid bodies (EB) was tested under various culture conditions. Inhibition of the LIF pathway was examined with specific inhibitors. The effects of cell apoptosis and proliferation on neural differentiation were examined. ES cell differentiation into three-germ layers was compared. Under various culture conditions, neuronal differentiation was increased in the presence of LIF. Blocking the LIF-activated STAT3 signaling pathway with specific inhibitors abolished the neuronal differentiation of ES cells, whereas inhibition of the LIF-activated MEK signaling pathway impaired the differentiation of ES cells toward a glial fate. LIF suppressed cell apoptosis and promoted cell proliferation during ES cell differentiation. LIF inhibited the differentiation of ES cells to both mesoderm and extraembryonic endoderm fates, but enhanced the determination of neural progenitors. These results suggest that LIF plays a positive role during the differentiation of ES cells into neuronal cells.

A vHNF1/TCF2-HNF6 Cascade Regulates the Transcription Factor Network That Controls Generation of Pancreatic Precursor Cells

Generation of pancreatic precursor cells in the endoderm is controlled by a network of transcription factors. Hepatocyte nuclear factor-6 (HNF6) is a key player in this network, because it controls the initiation of the expression of pancreatic and duodenal homeobox 1 (Pdx1), the earliest marker of pancreatic precursor cells. To further characterize this network, we have investigated how the expression of HNF6 is controlled in mouse endoderm, by using in vitro and in vivo protein-DNA interaction techniques combined with endoderm electroporation, transgenesis, and gene inactivation in embryos. We delineated Hnf6 regulatory regions that confer expression of a reporter gene in the embryonic endoderm but not in extraembryonic visceral endoderm. HNF6 expression in the embryonic endoderm was found to depend on an intronic enhancer. This enhancer contains functional binding sites for the tissue-specific factors of the forkhead box A and HNF1 families. Among the latter, variant HNF1 (vHNF1)/TCF2, which is expressed before HNF6 in the endoderm, was found to be critical for HNF6 expression. Therefore, the sequential activation of vHNF1, HNF6, and Pdx1 in the endoderm appears to control the generation of pancreatic precursors. This cascade may be used to benchmark in vitro differentiation of pancreatic precursor cells from embryonic stem cells, for cell therapy of diabetes.

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

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

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

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

Perception of differentiation cues by GATA factors in primitive endoderm lineage determination of mouse embryonic stem cells

The formation of the primitive endoderm covering the inner cell mass of early mouse embryos can be simulated in vitro by the differentiation of mouse embryonic stem (ES) cells in culture following either aggregation of suspended cells or stimulation of cell monolayers with retinoic acid. The developmentally regulated transcription factors GATA-4 and GATA-6 have determining role in mouse extraembryonic endoderm development. We analyzed the in vitro differentiation of mouse embryonic stem cells deficient of GATA factors and conclude that GATA-4 is required for ES cells to perceive a cell positioning (cell aggregation) signal and GATA-6 is required to sense morphogenic (retinoic acid) signal. The collaboration between GATA-6 and GATA-4, or GATA-6 and GATA-5 which can substitute for GATA-4, is involved in the perception of differentiation cues by embryonic stem cells in their determination of endoderm lineage. This study indicates that the lineage differentiation of ES cells can be manipulated by the expression of GATA factors.

Germ layer induction from embryonic stem cells

Embryonic stem (ES) cells have the potential to develop into all cell types of the adult body. This capability provides the basis for considering the ES cell system as a novel and unlimited source of cells for replacement therapies for the treatment of a wide range of diseases. Before the cell-based therapy potential of ES cells can be realized, a better understanding of the pathways regulating lineage-specific differentiation is required. Current studies suggest that the bone morphogenic protein, transforming growth factor-beta, Wnt, and fibroblast growth factor pathways that are required for gastrulation and germ layer induction in the embryo are also essential for differentiation of ES cells in culture. The current understanding of how these factors influence germ layer induction in both the embryo and in the ES cell differentiation system is addressed in this review.

Downregulation of NANOG Induces Differentiation of Human Embryonic Stem Cells to Extraembryonic Lineages

The homeobox transcription factor Nanog has been proposed to play a crucial role in the maintenance of the undifferentiated state of murine embryonic stem cells. A human counterpart, NANOG, has been identified, but its function and localization have not hitherto been described. We have used a combination of RNA interference and quantitative real-time polymerase chain reaction to study NANOG in human embryonic stem and embryonic carcinoma cells. Transfection of NANOG-specific small interfering RNAs reduced levels of NANOG transcript and protein and induced activation of the extraembryonic endoderm-associated genes GATA4, GATA6, LAMININ B1, and AFP as well as upregulation of trophectoderm-associated genes CDX2, GATA2, hCG-alpha, and hCG-beta. Immunostaining of preimplantation human embryos showed that NANOG was expressed in the inner cell mass of expanded blastocysts but not in earlier-stage embryos, consistent with a role in the maintenance of pluripotency. Taken together, our findings suggest that NANOG acts as a gatekeeper of pluripotency in human embryonic stem and carcinoma cells by preventing their differentiation to extraembryonic endoderm and trophectoderm lineages.