Ectopic expression of GATA6 bypasses requirement for Grb2 in primitive endoderm formation

Gene knockouts in mice have showed that Grb2 and GATA6 are essential for the formation of primitive endoderm in blastocysts. Here, we confirmed that implanted Grb2‐null blastocysts lack primitive or extraembryonic endoderm cells either at E4.5 or E5.5 stages. We analyzed the relationship between Grb2 and GATA6 in the differentiation of embryonic stem (ES) cells to primitive endoderm in embryoid body models. Upon transfection with GATA6 expression vector, Grb2‐null ES cells underwent endoderm differentiation as indicated by the expression of the extraembryonic endoderm markers Dab2 and GATA4. Transfection of GATA4 expression vector also had the same differentiation potency. When GATA6‐ or GATA4‐transfected Grb2‐null ES cells were allowed to aggregate, fragments of an endoderm layer formed on the surface of the spheroids. The results suggest that GATA6 is downstream of Grb2 in the inductive signaling pathway and the expression of GATA6 is sufficient to compensate for the defects caused by Grb2 deficiency in the development of the primitive and extraembryonic endoderm. Developmental Dynamics 240:566–576, 2011. © 2010 Wiley‐Liss, Inc.     

Differences in early lineage segregation between mammals.

Two lineage segregation events in mammalian development form the trophectoderm, primitive endoderm, and pluripotent primitive ectoderm. In mouse embryos, Oct4, Cdx2, Nanog, and Gata6 govern these events, but it is unknown whether this is conserved between mammals. Here, the expression patterns of these genes and their products were determined in porcine oocytes and embryos and in bovine embryos. CDX2 and GATA6 expression in porcine and bovine blastocysts resembled that of mouse, indicating conserved functions. However, NANOG expression was undetectable in porcine oocytes and embryos. Some inner cell mass cells in bovine blastocysts expressed NANOG protein. OCT4 protein was undetectable in porcine morulae, but present in both the trophectoderm and the inner cell mass of blastocysts, suggesting that downregulation of OCT4 in the trophectoderm does not precede trophectoderm formation. Combined, the results indicate differences in lineage segregation between mammals.     

Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo

Fgf8 and Fgf4 encode FGF family members that are coexpressed in the primitive streak of the gastrulating mouse embryo. We have analyzed the phenotype of Fgf8(-/-) embryos and discovered that they fail to express Fgf4 in the streak. In the absence of both FGF8 and FGF4, epiblast cells move into the streak and undergo an epithelial-to-mesenchymal transition, but most cells then fail to move away from the streak. As a consequence, no embryonic mesoderm- or endoderm-derived tissues develop, although extraembryonic tissues form. Patterning of the prospective neuroectoderm is greatly perturbed in the mutant embryos. Anterior neuroectoderm markers are widely expressed, at least in part because the anterior visceral endoderm, which provides signals that regulate their expression, is not displaced proximally in the absence of definitive endoderm. Posterior neuroectoderm markers are not expressed, presumably because there is neither mesendoderm underlying the prospective neuroectoderm nor a morphologically normal node to provide the inductive signals necessary for their expression. This study identifies Fgf8 as a gene essential for gastrulation and shows that signaling via FGF8 and/or FGF4 is required for cell migration away from the primitive streak.     

Distribution of p21ras in postimplantation rat embryos.

Expression of ras cellular oncogenes during the early postimplantation period in the rat was investigated using immunohistochemistry to p21ras. A broad spectrum polyclonal antibody recognizing N-, Ha- and Ki- forms of p21ras was used in an indirect avidin-biotin-peroxidase (ABC) technique. Positive staining indicating the presence of p21ras was found in embryos from 6.5 to 12 days embryonic age. In early egg cylinders (6.5 days), positive staining for p21ras was observed on the ectoplacental cone, primitive ectoderm and trophectoderm, while primitive endoderm and parietal endoderm appeared paler. In later egg cylinder stages (7.5 days), strong positive staining was observed in the primitive embryonic ectoderm and ectoplacental cone, but parietal and visceral endoderm still appeared to be devoid of positive staining. As development proceeded during primitive streak stages, the visceral and parietal endoderm became positively stained. By 10 days, all tissues appeared to be positive for p21ras, with strong staining appearing in the heart and neural elements. Therefore, p21ras does not appear to be ubiquitous in the rat conceptus prior to gastrulation, but shows differential distribution, appearing later in endodermal derivatives. Possibly p21ras is involved in determination of the ectodermal and endodermal lineages.     

The DVE changes distal epiblast fate from definitive endoderm to neurectoderm by antagonizing nodal signaling.

To assess the function of the distal visceral endoderm (DVE) of embryonic day 5.5 (E5.5) embryos, we established a system to directly ablate the DVE and observe the consequences after culture. When the DVE was successfully ablated, such embryos (DVE-ablated embryos) showed deregulated expression of Nodal and Wnt3 and ectopically formed the primitive streak at the proximal portion of the embryo. The DVE and anterior visceral endoderm (AVE) are implicated in the development of neurectoderm. We found that the distal epiblast of E5.5 embryo rotates anteriorly by the beginning of gastrulation. These cells remained to be anteriorly located during gastrulation and contributed to the ectoderm in the anterior side of the embryo. This indicates that the distal epiblast of E5.5 embryo becomes neurectoderm in normal development. In DVE-ablated embryos, the distal epiblast did not show any movement during culture and was abnormally fated to early definitive endoderm lineage. The data suggest that down-regulation of Nodal signaling in the distal epiblast of E5.5 embryo may be an initial step of neural development.     

Three-dimensional extracellular matrix stimulates gastrulation-like events in human embryoid bodies

Human embryonic stem (hES) cells cultured in suspension form aggregates called embryoid bodies (EBs), which structurally resemble the pregastrulation-stage embryo. We show that these EBs express genes characteristic of the visceral endoderm (VE) and form an outer boundary of VE-like cells, but only a minority expresses markers of the primitive streak and the definitive endoderm in patterns suggestive of gastrulation-like events. EBs embedded in a Matrigel matrix, however, lack the presumptive VE but up-regulate expression of gastrulation-related genes, which is correlated with the expression of these genes in a greater proportion of individual EBs. Over time, Matrigelembedded EBs form internally organized structures and exhibit higher expression of markers of the definitive endoderm and mesoderm. We hypothesize that a three-dimensional adhesive support provides an organizing influence analogous to the early basal lamina surrounding the epiblast of the pregastrulation embryo. Furthermore, we identify a synthetic three-dimensional scaffold capable of supporting EB formation that partially mimics the effects of Matrigel.     

Differentiation of the embryonic disc,amnion, and yolk sac in the rhesus monkey

The inner cell mass of the blastocyst has differentiated into epiblast and hypoblast (primitive endoderm) prior to implantation. Since endoderm cells extend beyond the epiblast, it can be considered that both parietal and visceral endoderm are present. At implantation, epiblast cells begin to show marked evidence of polarity. They form a spherical aggregate with their basal ends toward the basal lamina and apical ends toward the interior. The potential for an internal space is formed by this change in polarity of the cells. No cytological evidence of separation of those cells that will form amniotic epithelium from the rest of the epiblast is seen until a cavity begins to form. The amniotic epithelium is originally contiguous with overlying cytotrophoblast, and a diverticulum remains in this position during early development. Epiblast forms a pseudostratified columnar epithelium, but dividing cells are situated toward the amniotic cavity rather than basally. The first evidence of a trilaminar disc occurs when a strand of cells contiguous with epiblast is found extending toward visceral endoderm. These presumptive mesoderm cells are undifferentiated, whereas extraembryonic mesoderm cells are already a distinct population forming extracellular materials. After implantation, visceral endoderm cells proliferate forming an irregular layer one to three cells thick. Visceral endoderm cells have smooth apical surfaces, but very irregular basal surfaces, and no basal lamina. At the margins of the disc, visceral endoderm is continuous with parietal endoderm and reflects back over the apices of the marginal visceral endoderm cells. This sacculation by visceral endoderm cells precedes pinching off of the secondary yolk sac from the remaining primary yolk sac.     

Expression of a novel antigen, EEM-1, in the mouse embryo and embryonic stem cell-derived embryoid bodies.

A novel monoclonal antibody designated EEM-1 is described. EEM-1 recognizes an intracellular protein with an apparent molecular weight of >250 kDa. Expression of the EEM-1 antigen is largely confined to extra-embryonic membranes, but some expression does occur in the embryo. In the embryonic day 6 (E6) and E7 embryo it is expressed in visceral and parietal endoderm; later derivatives of these structures in the yolk sac are negative. The outer layer of the amnion also stains. Within the embryo proper, antigen is expressed in limited regions of the gut, kidney, and pancreas. EEM-1 is also expressed in embryonic stem (ES) cells differentiating in vitro. Undifferentiated ES cells do not express the antigen. Embryoid bodies (EBs) derived from ES cells have patches of EEM-1-positive cells on their surface at 2 days in culture. Older EBs have increasing numbers of positive cells which are confined to the surface. A special class of EBs, termed "cystic EBs," are covered by a cell layer which strongly express EEM-1 antigen. The EEM-1 antibody will be useful for investigating the development of extra-embryonic membranes and their counterparts in the ES cell in vitro model.     

Distribution of p21ras in postimplantation rat embryos

Expression of ras cellular oncogenes during the early postimplantation period in the rat was investigated using immunohistochemistry to p21^ras was used in an indirect avidin-biotin-peroxidase (ABC) technique. Positive staining indicating the presence of p21^ras was found in embryos from 6.5 to 12 days embryonic age. In early egg cylinders (6.5 days), positive staining for p21^ras was observed on the ectoplacental cone, primitive ectoderm and trophectoderm, while primitive endoderm and parietal endoderm appeared paler. In later egg cylinder stages (7.5 days), strong positive staining was observed in the primitive embryonic ectoderm and ectoplacental cone, but parietal and visceral endoderm still appeared to be devoid of positive staining. As development proceeded during primitive streak stages, the visceral and parietal endoderm became positively stained. By 10 days, all tissues appeared to be positive for p21^ras, with strong staining appearing in the heart and neural elements. Therefore, p21^ras does not appear to be ubiquitous in the rat conceptus prior to gastrulation, but shows differential distribution, appearing later in endodermal derivatives. Possibly p21^ras is involved in determination of the ectodermal and endodermal lineages.© Willey-Liss, Inc.     

Localization of Mg++-dependent adenosine triphosphatase and alkaline phosphatase activities in the postimplantation mouse embryos in day 5 and 6

Summary Mg⁺⁺-dependent adenosine triphosphatase (Mg-ATPase) and alkaline phosphatase (ALPase) activities were histo- and cytochemically investigated in postimplantation mouse embryos from day 5 to day 6. In day 5 postimplantation embryos, Mg-ATPase activity was detected in the embryonic ectoderm and weakly in the visceral endoderm. Weak ALPase activity was found in the embryonic ectoderm and visceral endoderm. Parietal endoderm, both in day 5 and in day 6 embryos, had very weak or no Mg-ATPase and ALPase activities. Mg-ATPase activity in day 6 embryos was found with the same localization as that in day 5 embryos. No ALPase activity was observed in their embryonic ectoderm. Extraembryonic ectodermal cell mass had the strongest Mg-ATPase activity in these stage embryos.These results suggest that the localization of both enzyme activities in postimplantation mouse embryos is closely related to the morphogenesis. As regards the proamniotic cavity formation, the fact that Mg-ATPase activity was still observed in the embryonic ectoderm in these stages suggests the involvement of active transport system on the production of nascent proamniotic cavity fluid.     

c‐fos elimination compensates for disabled‐2 requirement in mouse extraembryonic endoderm development

Disabled‐2 (Dab2) is expressed in primitive endoderm cells as they are differentiating from the inner cell mass and dab2 deficiency in mice results in lethality at E5.5–E6.5 due to the disorganization of the endoderm layers. Here we show that Dab2 suppresses c‐Fos expression in endoderm cells. A morphological normal primitive endoderm layer was observed in putative E5.5 dab2 (?/?):c‐fos (?/?) embryos, indicating that the primitive endoderm defect due to the loss of Dab2 is rescued by deletion of the c‐fos gene. The lethality of the double knockout embryos was delayed until E9.5–E10.5 and the defective embryos failed to undergo organogenesis. We conclude that Dab2 plays a role in epithelial organization by suppression of c‐Fos expression and suggest that unsuppressed c‐Fos can lead to disruption of primitive endoderm epithelial organization, yet an additional dab2 function is required for later organogenesis. Developmental Dynamics 238:514–523, 2009. © 2009 Wiley‐Liss, Inc.     

Beta-catenin signaling marks the prospective site of primitive streak formation in the mouse embryo.

Beta-catenin signaling has been shown to be involved in triggering axis formation in several organisms, including Xenopus and zebrafish. Genetic analysis has demonstrated that the Wnt/beta-catenin signaling pathway is also involved in axis formation in the mouse, since a targeted deletion of beta-catenin results in embryos that have a block in anterior-posterior axis formation, fail to initiate gastrulation, and do not form mesoderm. However, because beta-catenin is ubiquitously expressed, the precise time and cell types in which this signaling pathway is active during early embryonic development remain unknown. Thus, to better understand the role of the Wnt/beta-catenin signaling pathway in axis formation and mesoderm specification, we have examined both the distribution and signaling activity of beta-catenin during early embryonic development in the mouse. We show that the N-terminally nonphosphorylated form of beta-catenin as well as beta-catenin signaling is first detectable in the extraembryonic visceral endoderm in day 5.5 embryos. Before the initiation of gastrulation at day 6.0, beta-catenin signaling is asymmetrically distributed within the epiblast and is localized to a small group of cells adjacent to the embryonic--extraembryonic junction. At day 6.5 and onward, beta-catenin signaling was detected in the primitive streak and mature node. Thus, beta-catenin signaling precedes primitive streak formation and is present in epiblast cells that will go on to form the primitive streak. These results support a critical role for the Wnt/beta-catenin pathway in specifying cells to form the primitive streak and node in the mammalian embryo as well as identify a novel domain of Wnt/beta-catenin signaling activity during early embryogenesis.     

The tyrosine hydroxylase gene is expressed in endoderm and pancreas of early quail embryos

The initial expression of the gene encoding tyrosine hydroxylase (TH) was studied in the trunk of quail embryos by in situ hybridization. We detected the presence of quail TH mRNA on embryonic day 3.5 (E3.5) in the sympathetic ganglia and aortic plexus, both neural crest derived structures. In contrast, the TH gene was expressed much earlier in the endodermal layer of E2 embryos, i. e. from the 8-somite stage onwards. TH mRNA was found also in the pancreatic bud, an endoderm-derived structure. The TH protein and catecholamines were subsequently looked for in these structures. TH immunoreactivity was found in cells of E2 explanted endoderm, but no catecholamine histofluorescence was observed before or after a few days in culture. TH-positive cells were also detected in cultures of pancreatic rudiments, explanted from E3 to E6 quail embryos. We suggest that the TH-positive cells of the endoderm are the progenitors of the catecholaminergic cells of the pancreas and of the enterochromaffin cells of the gut. The hypothesis that the TH-positive cells of the endoderm are involved in the expression of the catecholaminergic phenotype by neural crest cells is discussed.     

Germ layer differentiation during early hindgut and cloaca formation in rabbit and pig embryos

Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human‐like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal–epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two‐step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron‐dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam‐like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid‐somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations.     

Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.

Wilms' tumor 1-associating protein (WTAP) was previously identified as a protein associated with Wilms' tumor-1 (WT-1) protein that is essential for the development of the genitourinary system. Although WTAP has been suggested to function in alternative splicing, stabilization of mRNA, and cell growth, its in vivo function is still unclear. We generated Wtap mutant mice using a novel gene-trap approach and showed that Wtap mutant embryos exhibited defective egg-cylinder formation at the gastrulation stage and died by embryonic day 10.5. Although they could form extraembryonic tissues and anterior visceral endoderm, Wtap mutant embryos and embryonic stem cells failed to differentiate into endoderm and mesoderm. The chimera analysis showed that Wtap in extraembryonic tissues was required for the formation of mesoderm and endoderm in embryonic tissues. Taken together, our findings indicate that Wtap is indispensable for differentiation of mesoderm and endoderm in the mouse embryo.