Intercellular Contacts Between the Embryonic or Extraembryonic Ectoderm and the Primitive Endoderm in Rat Egg Cylinders Prior to the Formation of Primitive Streak

In pre-primitive streak-stage rat egg cylinders, both the embryonic and extraembryonic ectodermal cells projected cytoplasmic protrusions through gaps in the basal lamina and formed intimate cell-to-cell contact with the primitive endodermal cells. The 70 Å microfilaments were considered to participate in the production of these cytoplasmic protrusions. However, direct cell contact mediated by adherent junctions was occasionally found between the embryonic or extraembryonic ectodermal cells and the primitive endodermal cells. It has been proposed that these cell-to-cell contacts may play a role either in the supporting effect of primitive endodermal cells in the maintenance of cellular organization of the ectodermal cells, or in the facilitation of transport of nutritive materials from the primitive endodermal cells to both types of ectodermal cells.     

Molecular study of mouse peri-implantation development using the in vitro culture of aggregated inner cell mass

To study the mechanisms of mouse peri-implantation development, we explored the in vitro culture of the isolated inner cell mass (ICM) of the blastocyst. As previously reported, individually cultured ICM recapitulated several early embryological events, such as the formation of primitive endoderm, epiblast, and proamniotic cavity. However, we found that the timing and efficiency of these morphogenetic processes significantly varied among the ICM. Due to this unpredictability in developmental potential, individually cultured ICM may be unsuitable for further analysis. By contrast, we found that when five ICM were fused into a single mass, such aggregates (5x ICM) underwent efficient and synchronous morphogenesis. The synchronous nature of 5x ICM development was also demonstrated by the temporal and spatial pattern of apoptotic cell death. TUNEL assay showed that a number of the epiblast cells committed apoptosis in 48 hr of culture, which took place after primitive endoderm differentiation but prior to proamniotic cavity formation. In situ hybridization analysis showed that Oct4 was downregulated and alpha-fetoprotein was upregulated in the primitive endoderm of the cultured 5x ICM. In addition, RT-PCR analysis revealed the expression of various primitive endodermal genes, but not of extraembryonic ectodermal markers in the cultured 5x ICM. Taken together, we propose that the 5x ICM is a useful in vitro tool to study the mechanisms of peri-implantation development of the mouse embryo. Mol. Reprod. Dev. 67: 83-90, 2004.     

The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF)

LIF (leukaemia inhibitory factor) is commonly used to maintain mouse embryonic stem cells in an undifferentiated state. These cells spontaneously differentiate when allowed to aggregate in the absence of LIF, forming embryoid bodies in which early embryonic cell lineages develop. Using embryoid bodies cultured in the presence and absence of LIF, we show that although LIF inhibited the development of visceral and parietal endodermal cells, it did not affect the differentiation of the primitive endodermal cell precursors of these extraembryonic cell lineages. Furthermore, deposition of the basement membrane produced by the primitive endodermal cells, which separates them from the remaining cells of the embryoid body, still occurred. The differentiation of primitive ectodermal cells and their progeny was inhibited by LIF, as evidenced by the lack of expression of FGF-5, muscle, and neuronal markers. However, cavitation of the embryoid body and maintenance of the cells in contact with the primitive endodermal basement membrane as an epiblast epithelium still occurred normally in the presence of LIF. These results indicate that cavitation and formation of the epiblast epithelium are regulated by mechanisms distinct from those controlling the differentiation of epiblast cell lineages. Furthermore, although epithelium formation and cavitation do not require the differentiation of visceral endodermal cells, the results are consistent with the hypothesis that the primitive endodermal basement membrane is sufficient to induce the epithelialization of undifferentiated embryonic stem cells necessary for cavitation.     

Epiblast and primitive-streak origins of the endoderm in the gastrulating chick embryo

Gastrulation is characterized by the extensive movements of cells. Fate mapping is used to follow such cell movements as they occur over time, and prospective fate maps have been constructed for several stages of the model organisms used in modern studies in developmental biology. In chick embryos, detailed fate maps have been constructed for both prospective mesodermal and ectodermal cells. However, the origin and displacement of the prospective endodermal cells during crucial periods in gastrulation remain unclear. This study had three aims. First, we determined the primitive-streak origin of the endoderm using supravital fluorescent markers, and followed the movement of the prospective endodermal cells as they dispersed to generate the definitive endodermal layer. We show that between stages 3a/b and 4, the intraembryonic definitive endoderm receives contributions mainly from the rostral half of the primitive streak, and that endodermal movements parallel those of ingressing adjacent mesodermal subdivisions. Second, the question of the epiblast origin of the endodermal layer was addressed by precisely labeling epiblast cells in a region known to give rise to prospective somitic cells, and following their movement as they underwent ingression through the primitive streak. We show that the epiblast clearly contributes prospective endodermal cells to the primitive streak, and subsequently to definitive endoderm of the area pellucida. Finally, the relationship between the hypoblast and the definitive endoderm was defined by following labeled rostral primitive-streak cells over a short period of time as they contributed to the definitive endoderm, and combining this with in situ hybridization with a riboprobe for Crescent, a marker of the hypoblast. We show that as the definitive endodermal layer is laid down, there is cell-cell intercalation at its interface with the displaced hypoblast cells. These data were used to construct detailed prospective fate maps of the endoderm in the chick embryo, delineating the origins and migrations of endodermal cells in various rostrocaudal levels of the primitive streak during key periods in early development.     

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.     

Involvement of Ras in extraembryonic endoderm differentiation of embryonic stem cells

Embryonic stem (ES) cells, derived from the inner cell mass of blastocyst can differentiate into multiple cell lineages. In this study, we examined the possible involvement of Ras in ES cell differentiation. We found that Ras was activated upon formation of embryoid bodies (EBs), an initial step in ES cell differentiation. When expressed during EB differentiation, a dominant-negative mutant of Ras suppressed induction of marker genes for extraembryonic endoderm differentiation, including GATA-4, GATA-6, alpha-fetoprotein, and hepatocyte nuclear factor 3beta, while an activated mutant promoted their induction. Expression of a Ras mutant that selectively activates the Raf/MEK/Erk pathway also enhanced induction of extraembryonic endoderm markers, and treatment with a MEK inhibitor resulted in their decreased expression. In addition, Ras stimulated downregulation of Nanog, a suppressor of endoderm differentiation in ES cells. These data suggest that Ras activation during EB differentiation plays a crucial role in initiation of extraembryonic endoderm differentiation.     

Wnt6 induces the specification and epithelialization of F9 embryonal carcinoma cells to primitive endoderm

Epithelial-to-mesenchymal transitions (EMTs) play key roles in the normal development of an organism as well as its demise following the metastasis of a malignant tumour. An EMT during early mouse development results in the differentiation of primitive endoderm into the parietal endoderm that forms part of the parietal yolk sac. In the embryo, primitive endoderm develops from cells in the inner cell mass, but the signals that instruct these cells to become specified and adopt an epithelial fate are poorly understood. The mouse F9 teratocarcinoma cell line, a model that can recapitulate the in vivo primitive to parietal endoderm EMT, has been used extensively to elucidate the signalling cascades involved in extraembryonic endoderm differentiation. Here, we identified Wnt6 as a gene up-regulated in F9 cells in response to RA and show that Wnt6 expressing cells or cells exposed to Wnt6 conditioned media form primitive endoderm. Wnt6 induction of primitive endoderm is accompanied by beta-catenin and Snail1 translocation to the nucleus and the appearance of cytokeratin intermediate filaments. Attenuating glycogen synthase kinase 3 activity using LiCl gave similar results, but the fact that cells de-differentiate when LiCl is removed reveals that other signalling pathways are required to maintain cells as primitive endoderm. Finally, Wnt6-induced primitive endodermal cells were tested to determine their competency to complete the EMT and differentiate into parietal endoderm. Towards that end, results show that up-regulating protein kinase A activity is sufficient to induce markers of parietal endoderm. Together, these findings indicate that undifferentiated F9 cells are responsive to canonical Wnt signalling, which negatively regulates glycogen synthase kinase 3 activity leading to the epithelialization and specification of primitive endoderm competent to receive additional signals required for EMT. Considering the ability of F9 cells to mimic an in vivo EMT, the identification of this Wnt6-beta-catenin-Snail signalling cascade has broad implications for understanding EMT mechanisms in embryogenesis and metastasis.     

Formation of a Polarised Primitive Endoderm Layer in Embryoid Bodies Requires Fgfr/Erk Signalling

The primitive endoderm arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. Fgf receptor/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier, which normally blocks free diffusion across the epithelial cell layer, occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.     

Extraembryonic Endoderm cells as a model of endoderm development

In recent years the multipotent extraembryonic endoderm (XEN) stem cells have been the center of much attention. In vivo, XEN cells contribute to the formation of the extraembryonic endoderm, visceral and parietal endoderm and later on, the yolk sac. Recent data have shown that the distinction between embryonic and extraembryonic endoderm is not as strict as previously thought due to the integration, and not the displacement, of the visceral endoderm into the definitive embryonic endoderm. Therefore, cells from the extraembryonic endoderm also contribute to definitive endoderm. Many research groups focused on unraveling the potential and ability of XEN cells to both support differentiation and/or differentiate into endoderm‐like tissues as an alternative to embryonic stem (ES) cells. Moreover, the conversion of ES to XEN cells, shown recently without genetic manipulations, uncovers significant and novel molecular mechanisms involved in extraembryonic endoderm and definitive endoderm development. XEN cell lines provide a unique model for an early mammalian lineage that complements the established ES and trophoblast stem cell lines. Through the study of essential genes and signaling requirements for XEN cells in vitro, insights will be gained about the developmental program of the extraembryonic and embryonic endodermal lineage in vivo. This review will provide an overview on the current literature focusing on XEN cells as a model for primitive endoderm and possibly definitive endoderm as well as the potential of using these cells for therapeutic applications.     

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)? Xsox17alpha and beta 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.     

Fate of the inner cell mass in mouse embryos as studied by microinjection of lineage tracers

We microinjected horseradish peroxidase and rhodamine-conjugated dextran into single inner cell mass (ICM) cells of preimplantation mouse embryos to study their fate in culture. Simultaneous iontophoresis of both lineage markers allowed immediate localization of the injected cell by epifluorescence, followed by microdrop culture of individual embryos. After 24 hr in culture, labeled descendants were found in the polar trophectoderm, ICM, and parietal endoderm, providing direct evidence that the ICM contributes descendants to the trophectoderm and the endoderm in the intact mouse embryo. Our results substantiate the totipotency of the ICM during the expanding blastocyst stage and further demonstrate that the ICM is a stem cell population from which cells are recruited into these tissue lineages during growth of the blastocyst.     

Stage-specific embryonic antigen 3 as a marker of visceral extraembryonic endoderm

The distribution of the stage-specific embryonic antigen SSEA-3 was studied immunohistochemically on postimplantation mouse embryos. This carbohydrate antigen, identified as an epitope of a globo-series ganglioside isolated from human teratocarcinoma cells (Kannagi et al., 1983, J. Biol. Chem.258, 8934–8942) was originally detected on the zygote and mouse early cleavage-stage embryos. It disappears on the early blastocyst and reappears on the primitive endoderm of the implanting blastocyst (Shevinsky et al., 1982, Cell30, 697–705). We now show in the early egg cylinder (on the sixth day of pregnancy) SSEA-3 is present in the entire visceral endoderm but not in any other part of the conceptus. From Day 7 of pregnancy onward, SSEA-3 is restricted to the extraembryonic visceral endoderm and the visceral yolk sac cells. Therefore, SSEA-3 is a useful marker for this endodermal cell lineage in midgestational mouse embryos.     

A Comparative Analysis of Extra-Embryonic Endoderm Cell Lines

Prior to gastrulation in the mouse, all endodermal cells arise from the primitive endoderm of the blastocyst stage embryo. Primitive endoderm and its derivatives are generally referred to as extra-embryonic endoderm (ExEn) because the majority of these cells contribute to extra-embryonic lineages encompassing the visceral endoderm (VE) and the parietal endoderm (PE). During gastrulation, the definitive endoderm (DE) forms by ingression of cells from the epiblast. The DE comprises most of the cells of the gut and its accessory organs. Despite their different origins and fates, there is a surprising amount of overlap in marker expression between the ExEn and DE, making it difficult to distinguish between these cell types by marker analysis. This is significant for two main reasons. First, because endodermal organs, such as the liver and pancreas, play important physiological roles in adult animals, much experimental effort has been directed in recent years toward the establishment of protocols for the efficient derivation of endodermal cell types in vitro. Conversely, factors secreted by the VE play pivotal roles that cannot be attributed to the DE in early axis formation, heart formation and the patterning of the anterior nervous system. Thus, efforts in both of these areas have been hampered by a lack of markers that clearly distinguish between ExEn and DE. To further understand the ExEn we have undertaken a comparative analysis of three ExEn-like cell lines (END2, PYS2 and XEN). PYS2 cells are derived from embryonal carcinomas (EC) of 129 strain mice and have been characterized as parietal endoderm-like [1], END2 cells are derived from P19 ECs and described as visceral endoderm-like, while XEN cells are derived from blastocyst stage embryos and are described as primitive endoderm-like. Our analysis suggests that none of these cell lines represent a bona fide single in vivo lineage. Both PYS2 and XEN cells represent mixed populations expressing markers for several ExEn lineages. Conversely END2 cells, which were previously characterized as VE-like, fail to express many markers that are widely expressed in the VE, but instead express markers for only a subset of the VE, the anterior visceral endoderm. In addition END2 cells also express markers for the PE. We extended these observations with microarray analysis which was used to probe and refine previously published data sets of genes proposed to distinguish between DE and VE. Finally, genome-wide pathway analysis revealed that SMAD-independent TGFbeta signaling through a TAK1/p38/JNK or TAK1/NLK pathway may represent one mode of intracellular signaling shared by all three of these lines, and suggests that factors downstream of these pathways may mediate some functions of the ExEn. These studies represent the first step in the development of XEN cells as a powerful molecular genetic tool to study the endodermal signals that mediate the important developmental functions of the extra-embryonic endoderm. Our data refine our current knowledge of markers that distinguish various subtypes of endoderm. In addition, pathway analysis suggests that the ExEn may mediate some of its functions through a non-classical MAP Kinase signaling pathway downstream of TAK1.     

A teratocarcinoma-derived endoderm stem cell line (1H5) that can differentiate into extra-embryonic endoderm cell types

We investigated the ability of the teratocarcinoma-derived, epithelial-type cell line 1H5 to differentiate into either of the two pathways to primary endoderm, and tested the hypothesis that 1H5 represents a state similar to primitive endoderm in the late 4th-day blastocyst. Like other endodermal cell types, 1H5 cells mixed with embryonal-carcinoma cells sort out into "embryoid bodies" or structures that resemble 4th-day mouse embryos. The epithelial line conforms morphologically and biochemically to the few known characteristics typical of primitive endoderm. The present study demonstrates that the formation in vitro of overt visceral endoderm is readily achieved. The spontaneous arrangement of the cells into a cystic form is followed by the appearance of several markers of visceral endoderm, most notably alphafetoprotein, which is detected when 1H5 cells are cultured either in the presence of retinoic acid or when the cells interact with embryonal-carcinoma cells in a specific spatial arrangement after sorting out. However, some less specific properties of visceral endoderm are not expressed. Although 1H5 differentiates histologically into parietal-like endoderm in the tumor form, parietal cells cannot yet be identified with certainty in vitro because of the paucity of parietal-specific markers. The 1H5 cell line could provide a useful system for studying the characteristics and mechanisms underlying visceral-endoderm differentiation in vitro, since it has the distinct advantage that homogeneous cultures are produced, in contrast to other teratocarcinoma cell lines such as F9 which differentiate into a mixture of cell types.     

Targeting SOX17 in human embryonic stem cells creates unique strategies for isolating and analyzing developing endoderm

Human embryonic stem cells (hESCs) can provide insights into development of inaccessible human tissues such as embryonic endoderm. Progress in this area has been hindered by a lack of methods for isolating endodermal cells and tracing fates of their differentiated progeny. By using homologous recombination in human ESCs, we inserted an enhanced green fluorescent protein (eGFP) transgene into the SOX17 locus, a postulated marker of human endoderm. FACS purification and gene expression profiling confirmed that SOX17(+)-hESC progeny expressed endodermal markers and unveiled specific cell surface protein combinations that permitted FACS-based isolation of primitive gut tube endodermal cells produced from unmodified human ESCs and from induced pluripotent stem cells (iPSC). Differentiating SOX17(+) endodermal cells expressed markers of liver, pancreas, and intestinal epithelium in vitro and gave rise to endodermal progeny in vivo. Thus, prospective isolation, lineage tracing, and developmental studies of SOX17(+) hESC progeny have revealed fundamental aspects of human endodermal biology.     

Fine structural analysis of the effect of trypan blue on the visceral endoderm of the mouse egg cylinder

The effects of a maternal injection of trypan blue on primitive streak mouse embryos were studied with electron microscopy. Commercial trypan blue was purified by descending paper chromatography, and pregnant females received an intraperitoneal injection of the collected blue fraction on the evening of the 7th day of gestation. Ultrastructurally, the changes in the visceral endoderm were apparent 10 min after the injection and included an increase in the number of fuzzy-coated vesicles in the apical cytoplasm. By 20 min the apical cytoplasm of the extraembryonic visceral endodermal cells was filled with many fuzzy-coated vesicles and numerous vacuoles of various size and electron density. 30 min after the injection, the extraembryonic visceral endodermal cells were relatively smooth lacking a microvillous border and evidence of endocytic activity was rare. Many embryonic visceral endodermal cells were observed in various stages of degeneration although the underlying embryonic ectoderm appeared unaffected. Morphologically, it appears that trypan blue exerts its first effect by altering the endocytic activity of the visceral endoderm.     

Disabled-2 is essential for endodermal cell positioning and structure formation during mouse embryogenesis

The signal transduction adapter protein Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The brain-specific Disabled-1 (Dab1) functions in positional organization of brain cells during development. Dab2 is widely distributed and is highly expressed in many epithelial cell types. The dab2 gene was interrupted by in-frame insertion of beta-galactosidase (LacZ) in embryonic stem cells and transgenic mice were produced. Dab2 expression was first observed in the primitive endoderm at E4.5, immediately following implantation. The homozygous Dab2-deficient mutant is embryonic lethal (earlier than E6.5) due to defective cell positioning and structure formation of the visceral endoderm. In E5.5 dab2 (-/-) conceptus, visceral endoderm-like cells are present in the deformed primitive egg cylinder; however, the visceral endoderm cells are not organized, the cells of the epiblast have not expanded, and the proamniotic cavity fails to form. Disorganization of the visceral endodermal layer is evident, as cells with positive visceral endoderm markers are scattered throughout the dab2 (-/-) conceptus. Only degenerated remains were observed at E6.5 for dab2 (-/-) embryos, and by E7.5, the defective embryos were completely reabsorbed. In blastocyst in vitro culture, initially cells with characteristics of endoderm, trophectoderm, and inner cell mass were observed in the outgrowth of the hatched dab2 (-/-) blastocysts. However, the dab2 (-/-) endodermal cells are much more dispersed and disorganized than those from wild-type blastocysts, the inner cell mass fails to expand, and the outgrowth degenerates by day 7. Thus, Dab2 is required for visceral endodermal cell organization during early mouse development. The absence of an organized visceral endoderm in Dab2-deficient conceptus leads to the growth failure of the inner cell mass. We suggest that Dab2 functions in a signal pathway to regulate endodermal cell organization using endocytosis of ligands from the blastocoel cavity as a positioning cue.     

Scanning Electron Microscopy (SEM) of the Chick Embryo Primitive Streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2–5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the primitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arranged cells with long, thin processes.
Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.