Primitive Ectoderm Cells Research Articles

Characterization and neural differentiation of mouse embryonic and induced pluripotent stem cells on cadherin-based substrata

A suitable culture condition using advanced biomaterials has the potential to improve stem cell differentiation into selective lineages. In this study, we evaluated the effects of recombinant extracellular matrix (ECM) components on the mouse embryonic stem (mES) and induced pluripotent stem (miPS) cells' self-renewal and differentiation into neural progenitors, comparing conventional culture substrata. The recombinant ECMs were established by immobilizing two chimera proteins of cadherin molecules, E-cadherin-Fc and N-cadherin-Fc, either alone or in combination. We report that the completely homogeneous population of mES and miPS cells could be maintained on E-cadherin-based substrata under feeder- and serum-free culture conditions to initiate neural differentiation. Using defined monolayer differentiation conditions on E-cadherin and N-cadherin (E-/N-cad-Fc) hybrid substratum, we routinely obtained highly homogeneous population of primitive ectoderm and neural progenitor cells. Moreover, the differentiated cells with higher expression of βIII-tubulin, Pax6, and tyrosine hydroxylase (TH) in absence of GFAP (a glial cell marker) expression suggesting the presence of a lineage restricted to neural cells. Our improved culture method should provide a homogeneous microenvironment for differentiation and obviate the need for protocols based on stromal feeders or embryoid bodies.

Dual Roles of Oct4 in the Maintenance of Mouse P19 Embryonal Carcinoma Cells: As Negative Regulator of Wnt/β-Catenin Signaling and Competence Provider for Brachyury Induction

Transcription factor Oct4 is expressed in pluripotent cell lineages during mouse development, namely, in inner cell mass (ICM), primitive ectoderm, and primordial germ cells. Functional studies have revealed that Oct4 is essential for the maintenance of pluripotency in inner cell mass and for the survival of primordial germ cells. However, the function of Oct4 in the primitive ectoderm has not been fully explored. In this study, we investigated the role of Oct4 in mouse P19 embryonal carcinoma (EC) cells, which exhibit molecular and developmental properties similar to the primitive ectoderm, as an in vitro model. Knockdown of Oct4 in P19 EC cells upregulated several early mesoderm-specific genes, such as Wnt3, Sp5, and Fgf8, by activating Wnt/β-catenin signaling. Overexpression of Oct4 was sufficient to suppress Wnt/β-catenin signaling through its action as a transcriptional activator. However, Brachyury, a key regulator of early mesoderm development and a known direct target of Wnt/β-catenin signaling, was unable to be upregulated in the absence of Oct4, even with additional activation of Wnt/β-catenin signaling. Microarray analysis revealed that Oct4 positively regulated the expression of Tdgf1, a critical component of Nodal signaling, which was required for the upregulation of Brachyury in response to Wnt/β-catenin signaling in P19 EC cells. We propose a model that Oct4 maintains pluripotency of P19 EC cells through 2 counteracting actions: one is to suppress mesoderm-inducing Wnt/β-catenin signaling, and the other is to provide competence to Brachyury gene to respond to Wnt/β-catenin signaling.

Stem cell therapeutics—reality versus hype and hope

In the last decade medical science has started to make the first steps towards a paradigm shift from repair to regeneration [1]. The potential use of a range of stem cells in regenerative medicine is currently one of the most intensively researched areas worldwide [2]. In particular, embryonic stem cells (ESC) and more recently Induced Pluripotent Stemcells (iPS) [3] have provoked a lot of interest as a potential source of stem cells in the treatment of diseases such as Alzheimer’s and Parkinson’s diseases. Nevertheless, these hopes have not yet been fulfilled with either ESC or iPS. Murine ESC were first described in 1981 [4] and subsequent studies showed the regenerative potential of murine stem cells derived from both preand postimplantation embryos [5–7]. This early murine work was followed by the development of human ESC in 1998 [8]. Both murine and human pre-implantation ESC are derived from the inner cell mass (ICM) of the blastocyst at day 5/6 of growth. The ICM consists of primitive ectoderm cells, which subsequently develop into epiblast cells in postimplantation embryos. Despite these ontogenic similarities human and murine ESC do not resemble each other in many aspects [9]. For example, murine ESC rely on leukemia inhibitory factor (LIF) and bone morphogenic protein-4 (BMP4) for maintenance of pluripotency [10, 11], whilst human ESC are dependent on TGFβ/Activin/Nodal pathway activity and fibroblast growth factor-2 (FGF2) [12–17] and thus resemble epiblast stem cells (EpiSC). Usually EpiSC cannot be reverted to a state such that they respond to LIF and BMP4. Furthermore, primordial germ cells, which originate from pluripotent epiblast cells in mice, respond in a similar way to LIF and BMP4, such as murine ESC derived from the ICM [18]. Additionally, murine ESC can be cultured as single cells while human ESC are particularly sensitive to dissociation and should be cultured in clumps [19]. There are clearly many basic concepts still to be explored in the biology of ESC of various species. Thorough understanding of these cells is still in its’ infancy despite 20 years of basic research. Bearing this is mind there should be an air of caution when proposing future clinical applications. Can or should we move forward into thinking about clinical applications when the fundamentals Capsule Human cord blood stem cells have a realistic and proven potential for the treatment of various diseases compared to embryonic stem cells / iPS cells.

Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells

The pluripotent state, which is first established in the primitive ectoderm cells of blastocysts, is lost progressively and irreversibly during subsequent development. For example, development of post-implantation epiblast cells from primitive ectoderm involves significant transcriptional and epigenetic changes, including DNA methylation and X chromosome inactivation, which create a robust epigenetic barrier and prevent their reversion to a primitive-ectoderm-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signalling, but they respond to activin/basic fibroblast growth factor to form self-renewing epiblast stem cells (EpiSCs), which exhibit essential properties of epiblast cells and that differ from embryonic stem (ES) cells derived from primitive ectoderm. Here we show reprogramming of advanced epiblast cells from embryonic day 5.5-7.5 mouse embryos with uniform expression of N-cadherin and inactive X chromosome to ES-cell-like cells (rESCs) in response to LIF-STAT3 signalling. Cultured epiblast cells overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, resulting in DNA demethylation, X reactivation and expression of E-cadherin. The accompanying changes in the transcriptome result in a loss of phenotypic and epigenetic memory of epiblast cells. Using this approach, we report reversion of established EpiSCs to rESCs. Moreover, unlike epiblast and EpiSCs, rESCs contribute to somatic tissues and germ cells in chimaeras. Further studies may reveal how signalling-induced epigenetic reprogramming may promote reacquisition of pluripotency.

Hedgehog Serves as a Mitogen and Survival Factor During Embryonic Stem Cell Neurogenesis

Hedgehog (Hh) signaling is involved in a wide range of important biological activities. Within the vertebrate central nervous system, Sonic Hedgehog (Shh) can act as a morphogen or mitogen that regulates the patterning, proliferation, and survival of neural stem cells (NSCs). However, its role in embryonic stem cell (ESC) neurogenesis has not been explored in detail. We have previously shown that Hh signaling is required for ESC neurogenesis. In order to elucidate the underlying mechanism, we utilized the Sox1-GFP ESC line, which has a green fluorescent protein (GFP) reporter under the control of the Sox1 gene promoter, providing an easy means of detecting NSCs in live cell culture. We show here that ESC differentiation in adherent culture follows the ESC--> primitive ectoderm --> neurectoderm transitions observed in vivo. Selective death of the Sox1-GFP-negative cells contributes to the enrichment of Sox1-GFP-positive NSCs. Interestingly, Shh is expressed exclusively by the NSCs themselves and elicits distinct downstream gene expression in Sox1-GFP-positive and -negative cells. Suppression of Hh signaling by antagonist treatment leads to different responses from these two populations as well: increased apoptosis in Sox1-GFP-positive NSCs and decreased proliferation in Sox1-GFP-negative primitive ectoderm cells. Hedgehog agonist treatment, in contrast, inhibits apoptosis and promotes proliferation of Sox1-GFP-positive NSCs. These results suggest that Hh acts as a mitogen and survival factor during early ESC neurogenesis, and evidence is presented to support a novel autocrine mechanism for Hh-mediated effects on NSC survival and proliferation.

Germ Line, Stem Cells, and Epigenetic Reprogramming

The germ cell lineage has the unique attribute of generating the totipotent state. Development of blastocysts from the totipotent zygote results in the establishment of pluripotent primitive ectoderm cells in the inner cell mass of blastocysts, which subsequently develop into epiblast cells in postimplantation embryos. The germ cell lineage in mice originates from these pluripotent epiblast cells of postimplantation embryos in response to specific signals. Pluripotent stem cells and unipotent germ cells share some fundamental properties despite significant phenotypic differences between them. Additionally, early primordial germ cells can be induced to undergo dedifferentiation into pluripotent embryonic germ cells. Investigations on the relationship between germ cells and pluripotent stem cells may further elucidate the nature of the pluripotent state. Furthermore, comprehensive epigenetic reprogramming of the genome in early germ cells, including extensive erasure of epigenetic modifications, is a critical step toward establishment of totipotency. The mechanisms involved may be relevant for gaining insight into events that lead to reprogramming of somatic cells into pluripotent stem cells.

Comparative integromics on JMJD2A, JMJD2B and JMJD2C: Preferential expression of JMJD2C in undifferentiated ES cells

Fertilized egg or totipotent zygote undergoes cleavage divisions to form a blastocyst, consisting of outer trophoectoderm cells and inner cell mass with pluripotent primitive ectoderm cells. Epigenetic reprogramming, erasure and maintenance of epigenetic modification, occurs during early embryogenesis. In 2004, we identified and characterized JMJD2A/JHDM3A, JMJD2B, JMJD2C, JMJD2D, JMJD2E and JMJD2F. JMJD2A, JMJD2B and JMJD2C share the common domain architecture with JmjN, JmjC, two PHD, and two TUDOR domains. In 2006, other groups characterized JMJD2 family members as the H3K9 and/or H3K36 histone demethylases. Here, comparative integromics analyses on JMJD2A, JMJD2B and JMJD2C were carried out. Mouse Jmjd2a was expressed in fertilized egg and 2-cell embryos, while human JMJD2A was expressed in undifferentiated and differentiated ES cells. AP1-binding site and six bHLH-binding sites within intron 13 of human JMJD2A gene were conserved in mouse Jmjd2a gene. Mouse Jmjd2b was expressed in 8-cell embryos and undifferentiated ES cells, while human JMJD2B was expressed in undifferentiated and differentiated ES cells. Two GATA-binding sites within intron 6 of human JMJD2B gene were conserved in mouse Jmjd2b gene. Mouse Jmjd2c and human JMJD2C were preferentially expressed in undifferentiated ES cells. Four NANOG-binding sites, one TCF/ LEF-binding site, and one bHLH-binding site were located within evolutionary conserved region at the 3'-flanking region of human JMJD2C gene. NANOG- TCF/LEF-, and bHLH-binding sites within the 3'-flanking region of human JMJD2C gene were conserved in chimpanzee, cow, mouse and rat JMJD2C othologs. Together these facts indicate that JMJD2C is the evolutionarily conserved target of Homeo-domain transcription factor NANOG, and that JMJD2C is the histone demethylase implicated in the epigenetic reprogramming during the early embryogenesis.

Ceramide Regulates Atypical PKCζ/λ-mediated Cell Polarity in Primitive Ectoderm Cells: A NOVEL FUNCTION OF SPHINGOLIPIDS IN MORPHOGENESIS

In mammals, the primitive ectoderm is an epithelium of polarized cells that differentiates into all embryonic tissues. Our study shows that in primitive ectoderm cells, the sphingolipid ceramide was elevated and co-distributed with the small GTPase Cdc42 and cortical F-actin at the apicolateral cell membrane. Pharmacological or RNA interference-mediated inhibition of ceramide biosynthesis enhanced apoptosis and impaired primitive ectoderm formation in embryoid bodies differentiated from mouse embryonic stem cells. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog. Ceramide depletion prevented plasma membrane translocation of PKCzeta/lambda, its interaction with Cdc42, and phosphorylation of GSK-3beta, a substrate of PKCzeta/lambda. Recombinant PKCzeta formed a complex with the polarity protein Par6 and Cdc42 when bound to ceramide containing lipid vesicles. Our data suggest a novel mechanism by which a ceramide-induced, apicolateral polarity complex with PKCzeta/lambda regulates primitive ectoderm cell polarity and morphogenesis.

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.

A germ cell origin of embryonic stem cells?

Because embryonic stem (ES) cells are generally derived by the culture of inner cell mass (ICM) cells, they are often assumed to be the equivalent of ICM cells. However, various evidence indicates that ICM cells transition to a different cell type during ES-cell derivation. Historically, ES cells have been believed to most closely resemble pluripotent primitive ectoderm cells derived directly from the ICM. However, differences between ES cells and primitive ectoderm cells have caused developmental biologists to question whether ES cells really have an in vivo equivalent, or whether their properties merely reflect their tissue culture environment. Here, we review recent evidence that the closest in vivo equivalent of an ES cell is an early germ cell.

Pluripotency of mouse embryonic cells on germline at 3.5-8.5 and 11.5 days post-coitum after aggregation with precompacted embryos.

Albino mouse embryonic cells (Gpi-la/a) at 3.5-8.5 and 11.5 days were aggregated with zona cut 8-16 cell stage embryos from F1 females (Gpi-1 b/b), respectively. The aggregated embryos were transferred to pseudopregnant female mice. The recipients were allowed to go to term or were dissected at mid-gestation to assess the donor contribution in the conceptuses using glucose phosphate isomerase (GPI) analysis. The donor cells, which were previously labeled with fluorescent latex microparticles, were aggregated with embryos, and the allocation of the donor cells at the compacted morula and blastocyst stages were observed under a fluorescence microscope. When 3.5 and 45 day old inner-cell-mass (ICM) cells were used, fertile chimeric mice were obtained (50 and 19%, respectively), and when 5.5 days old primitive ectoderm cells were aggregated, they did not form chimeras but contributed to the fetuses, placenta and membrane after 13.5 days of pregnancy. However, cells from further stages never contributed to the conceptuses even though they were analyzed after 10.5 days of pregnancy. The labeled donor cells at these stages were not positively incorporated in the interior part of the compacted morula and the ICM of the blastocyst stage unlike the ICM at 3.5 days post-coitum after overnight culture.

Developmental potency of mouse primitive ectoderm cells, embryonic ectoderm cells and primordial germ cells after blastocyst injection

Developmental potency of primitive and embryonic ectoderm cells from 4.50-day to 6.25-day post-coitum (p.c.) mouse embryos and primordial germ cells from 12.50-day p.c. male genital ridges of fetal mice were studied by direct introducing them into 3.50-day p.c. blastocysts. Sixteen (61.5%) overt chimaeras out of 26(50%) offsprings were obtained after transfer of 52 blastocysts injected with 4.50-day primitive ectoderm cells; four (16.0%) overt chimaeras were obtained out of 25 (51.0%) offsprings with 4.75-day primitive ectoderm cells from 49 transferred blastocysts. However, no overt chimaera was obtained with either 5.25-dayor 6.25-day embryonic ectoderm cells or 12.50-day male primordial germ cells. GPI analysis of mid-gestation conceptuses developed from injected blastocysts showed that 5.25-day embryonic ectoderm cells could only contributed to yolk sac of conceptus. Results suggested that implantation acts as a trigger for the determination of primitive ectoderm cells, and their developmental potency becomes limited within a short period of time in normal development.

Characterization of a highly unstable mouse minisatellite locus: Evidence for somatic mutation during early development

A highly unstable mouse minisatellite locus, Ms6-hm, has been identified in mouse DNA fingerprints produced by cross-hybridization with human minisatellite probe 33.6. A 7-kb allele of Ms6-hm was cloned from a C57BL 6J mouse and collapsed to a 400-bp plasmid insert on propagation in Escherichia coli due to loss of the majority of minisatellite repeat units. Sequence analysis revealed that Ms6-hm has evolved by amplification within a member of the MT (mouse transcript) family of interspersed repetitive elements. Linkage analysis localized Ms6-hm near the brown coat color gene ( b) on chromosome 4. Multiallelism and heterozygosity at this locus within inbred strains result from a high germline mutation rate to newlength alleles (2.5% per gamete). Mice mosaic for cells carrying a nonparental allele in somatic tissue, and in some cases also in the germline, provide evidence for additional, somatic, mutation events at Ms6-hm. In two mosaic mice the fraction of cells containing the nonparental allele has been shown to be indistinguishable in different adult tissues. These somatic mutation events at Ms6-hm must therefore occur very early in development, preceding the allocation of somatic lineages, and the same pool of primitive ectoderm cells must contribute equally to all somatic tissues. Under low-stringency hybridization conditions the collapsed subclone of Ms6-hm cross-hybridizes to other unstable loci in the mouse genome to generate a novel and highly individual specific mouse DNA fingerprint.

Systematic elimination of parthenogenetic cells in mouse chimeras.

The developmental potential of primitive ectoderm cells lacking paternal chromosomes was investigated by examining the distribution of parthenogenetic cells in chimeras. Using GPI-1 allozymes as marker, parthenogenetic cells were detected in most organs and tissues in adult chimeras. However, these cells were under severe selective pressure compared with cells from normal fertilized embryos. In the majority of chimeras, parthenogenetic cells in individual animals were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. Nevertheless, parthenogenetic cells were detected more consistently in some organs, especially the brain, heart, kidney and spleen. In contrast, there was apparently a systematic selection against parthenogenetic cells in some tissues, most notably in skeletal muscle, liver and pancreas. These results suggest that paternally derived genes are probably required not only for the development of extraembryonic structures but also for subsequent development of embryonic tissues derived from the primitive ectoderm lineage.

Erratum

Regeneration of endoderm from primitive ectoderm in the mouse embryo: fact or artifact?The publishers regret that there is an error on page 323 of this article. The last paragraph should read as follows:The fact that individual 5th day primitive ectoderm cells can give rise to both somatic cells and functional gametes (Gardner, 1978a) argues that they retain genomic totipotency. Nevertheless, results obtained in the present study and in earlier blastocyst injection experiments (Gardner & Rossant, 1979; Gardner, 1982) do not support the conclusion that these cells remain undetermined until 2 days or so after implantation. Rather, they suggest that both primitive ectoderm and primitive endoderm cells become restricted to mutually exclusive lineages more or less synchronously, some time between and days post coitum. So far, extraembryonic-endoderm-like cells have only been obtained unequivo-cally from primitive ectoderm following its exposure to conditions of terato-carcinogenesis (Graham, 1977). In view of current ignorance of events underlying this process and uncertainty as to whether the target for neoplastic conversion is the primitive ectoderm cell itself or one of its lineal descendants, it is not clear what relevance, if any, such findings have to the problem of stability of ectodermal determination. There are also indications that primitive-endoderm-derived cells may exhibit metaplasia in certain circumstances (Lu, Sobis, Van Hove & Vandeputte, 1984). Nevertheless, it would appear that both types of mature ICM cell have undergone sufficiently stable differentiation by the late blastocyst stage to render their interconversion during subsequent embryogenesis a most unlikely event.

Clonal analysis of early mammalian development.

Various extrinsic markers have been used to label single cells in the early mouse embryo. However, they are appropriate only for short-term experiments because of their susceptibility to dilution. Studies on cell lineage and commitments have therefore depended mainly on exploiting genes as markers by combining cells from embryos that differ in genotype at particular loci. Tissue recombination and transplantation experiments using such indelible intrinsic markers have enabled the fate of different cell populations in the blastocyst to be determined with reasonable precision. The trophectoderm and inner cell mass (i.c.m.) give rise to distinct complementary groups of tissues in the later conceptus, as do the primitive endodermal and primitive ectodermal components of the more mature i.c.m. When cloned by blastocyst injection, single i.c.m. cells colonize only those parts of host conceptuses that are derived from their tissue of origin. Thus, while clonal descendants of early i.c.m. cells can contribute to all tissues other than those of trophectodermal origin, primitive endodermal and primitive ectodermal clones are restricted, respectively, to the extraembryonic endoderm versus all i.c.m. derivatives except the extraembryonic endoderm. Interestingly, individual primitive ectoderm cells can include both germ cells and somatic cells among their mitotic descendants. By using the genetically determined presence versus absence of cytoplasmic malic enzyme activity as a cell marker, the deployment of clones has been made visible in situ in whole-mount preparations of extraembryonic membranes. Very little mixing of donor and host cells was seen in either the endoderm of the visceral yolk sac or the mesodermal and ectodermal layers of the amnion. In contrast, mosaicism in the parietal endoderm was so fine grained that, in all except 1 of 15 fields from several specimens that were analysed, the arrangement of donor and host cells did not differ significantly from that expected on the basis of their random association.

Clonal analysis of X-chromosome inactivation and the origin of the germ line in the mouse embryo

Cloning of cells from peri-implantation embryos by blastocyst injection was used to investigate the time of X-chromosome inactivation in that part of the ectoderm lineage giving rise to foetal tissues of the mouse. Matings were arranged so that the two X-chromosomes of female donor cells controlled two distinct coat colours and host blastocysts were of a third colour genotype. No coat chimaeras were obtained in experiments using donor cells from the primitive ectoderm of 6th or 7th day embryos or from lactationally delayed implanting or reactivated blastocysts. In contrast, a minimum of 80 unequivocal coat chimaeras were obtained in experiments in which primitive ectoderm cells from 5th day implanting blastocysts were used for injection. The majority of these chimaeras that had received a female cell exhibited both donor colours in addition to host colour in their coats, suggesting that the donor cell had not undergone X-inactivation until one or more cycles after transplantation. The remainder of such chimaeras exhibited only one or other donor coat colour. Determination of the parental origin of the allocyclic X-chromosome in donor metaphase preparations in internal tissues of several chimaeras revealed that the coat pattern did not always reflect the X-activity status of the donor cell clone as a whole. Nevertheless, the findings suggest that X-inactivation takes place shortly after implantation in the primitive ectoderm cell population from which the foetus is derived. Of the 68 chimaeras in which the sex of both the donor and host component was established 62 proved to be fertile. Furthermore, 21 of the 37 fertile chimaeras whose sex corresponded with that of the donor cell yielded functional gametes of donor origin. Injection of cells from a single donor blastocyst into a series of host blastocysts established that at least 2 cells in 5th day primitive ectoderm can give rise to both somatic cells and functional germ cells among their mitotic descendants.

Growth factors in mammalian embryogenesis.

Malignant murine embryonal carcinoma (EC) cells have been studied as a means of characterizing the identity and action of growth regulatory molecules in the early postimplantation mouse embryo. The differentiation of EC cells in vitro is accompanied by significant changes in the control of cell proliferation, including the acquisition of dependence on specific exogenous growth factors for cell multiplication. This is at least partly controlled by the developmentally regulated expression of specific growth factor receptors and their intracellular response systems. The development of defined media has allowed the identification of the principal factors required for EC cell proliferation in vitro. These factors are synthesized in vivo by the extra-embryonic tissues of the developing embryo and by the differentiated progeny of EC cells in vitro. EC cells secrete a potent growth factor (embryonal carcinoma-derived growth factor, ECDGF) which has been purified and partly characterized. ECDGF induces proliferation of the differentiated progeny of EC cells and specific normal embryonic cell types in vitro, suggesting that ECDGF may act as an embryonic growth factor in vivo. Together these findings suggest that proliferation in the embryo may be controlled by reciprocal interaction between primitive ectoderm cells and their differentiated derivatives, mediated by the developmentally regulated expression of specific soluble growth factors.

Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo

The technique of injecting genetically labelled cells into blastocysts was used in an attempt to determine whether the parietal and visceral endoderm originate from the same or different cell populations in the early embryo. When the developmental potential of 5th day primitive ectoderm and primitive endoderm cells was compared thus, only the latter were found to colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded unequivocal colonization of both the parietal and the visceral endoderm in a proportion of chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were detected in the parietal endoderm only, cases of exclusively visceral colonization being rare. Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a striking tendency to contribute exclusively to the parietal endoderm following blastocyst injection. The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-endodermal cells.

Investigation of the fate of 4·5 daypost-coitummouse inner cell mass cells by blastocyst injection

Two distinct patterns of chimaerism were found in conceptuses produced by injecting dissociated 4.5-day inner cell mass cells into genetically dissimilar blastocysts. Pattern 1: donor cells were found in the endoderm layer of the visceral yolk sac, but not in the adjacent mesoderm layer of this organ or in the foetus itself. Pattern 2: donor cells were found in the mesoderm layer of the visceral yolk sac and/or foetus, but never in the yolk-sac endoderm as well. Primitive endoderm cells of donor inner cell masses are responsible for the first pattern and primitive ectoderm cells for the second. These results, together with those of previous studies, suggest that the entire foetus, including its endodermal components, is formed from the primitive ectoderm, and that primitive endoderm forms only extra-embryonic endoderm of the conceptus.