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At least 19 recordsLinked to original sources

PubMed1996

The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo.

The developmental potency of cells in the proximal and distal regions of the epiblast of pre- and early-primitive-streak-stage mouse embryos was assessed by their differentiation in the host embryo following orthotopic and heterotopic cell transplantation. Normally, cells in the distal epiblast differentiate predominantly into neuroectoderm and surface ectoderm. However, when they were transplanted to proximal regions of the epiblast, distal epiblast cells behaved like proximal epiblast cells: they colonised the extraembryonic mesoderm and other mesodermal tissues in the posterior region of the host embryo. In addition, about 3.7% of the transplanted distal epiblast cells differentiated into primordial germ cells. This proportion is comparable to the 3.9% of orthotopically transplanted proximal epiblast cells that became primordial germ cells. When proximal epiblast cells were transplanted heterotopically to distal sites, their descendants were generally absent from the extraembryonic mesoderm and the germ cell population of the host embryo. Like cells in the distal epiblast, they mostly colonised the neural plate and surface ectoderm. This plasticity of cell fate suggests that the epiblast cells are not irreversibly allocated to any specific lineages, including the germ line. The adoption of developmental fate that is typical of the cell population at the site of transplantation suggests that the specification of cell lineages is subject to certain site-specific influences in the epiblast. Allocation of cells to the ectodermal and germ cell lineages may be subject to local tissue interactions and the restriction of morphogenetic tissue movement of different epiblast cell populations during gastrulation.

Animals↗

PubMed1991

Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.

The fate of cells in the epiblast at prestreak and early primitive streak stages has been studied by injecting horseradish peroxidase (HRP) into single cells in situ of 6.7-day mouse embryos and identifying the labelled descendants at midstreak to neural plate stages after one day of culture. Ectoderm was composed of descendants of epiblast progenitors that had been located in the embryonic axis anterior to the primitive streak. Embryonic mesoderm was derived from all areas of the epiblast except the distal tip and the adjacent region anterior to it: the most anterior mesoderm cells originated posteriorly, traversing the primitive streak early; labelled cells in the posterior part of the streak at the neural plate stage were derived from extreme anterior axial and paraxial epiblast progenitors; head process cells were derived from epiblast at or near the anterior end of the primitive streak. Endoderm descendants were most frequently derived from a region that included, but extended beyond, the region producing the head process: descendants of epiblast were present in endoderm by the midstreak stage, as well as at later stages. Yolk sac and amnion mesoderm developed from posterolateral and posterior epiblast. The resulting fate map is essentially the same as those of the chick and urodele and indicates that, despite geometrical differences, topological fate relationships are conserved among these vertebrates. Clonal descendants were not necessarily confined to a single germ layer or to extraembryonic mesoderm, indicating that these lineages are not separated at the beginning of gastrulation. The embryonic axis lengthened up to the neural plate stage by (1) elongation of the primitive streak through progressive incorporation of the expanding lateral and initially more anterior regions of epiblast and, (2) expansion of the region of epiblast immediately cranial to the anterior end of the primitive streak. The population doubling time of labelled cells was 7.5 h; a calculated 43% were in, or had completed, a 4th cell cycle, and no statistically significant regional differences in the number of descendants were found. This clonal analysis also showed that (1) growth in the epiblast was noncoherent and in most regions anisotropic and directed towards the primitive streak and (2) the midline did not act as a barrier to clonal spread, either in the epiblast in the anterior half of the axis or in the primitive streak. These results taken together with the fate map indicate that, while individual cells in the epiblast sheet behave independently with respect to their neighbours, morphogenetic movement during germ layer formation is coordinated in the population as a whole.

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PubMed2005

Mouse epiblasts change responsiveness to BMP4 signal required for PGC formation through functions of extraembryonic ectoderm.

Mouse primordial germ cells (PGCs) are initially identified as a cluster of alkaline phosphatase (AP)-positive cells within the extraembryonic mesoderm near the posterior part of the primitive streak at embryonic day (E) 7.25. Clonal analysis of epiblast cells has revealed that the putative precursors of PGCs are localized in the proximal epiblast, and we demonstrated that the conditions required for PGC formation are induced in the proximal region of epiblasts by extraembryonic ectoderm. Bone morphogenetic protein (BMP) 4 and BMP8b, which belong to the transforming growth factor-beta (TGF-beta) superfamily, might generate induction signals from extraembryonic ectoderm. Smad1 and Smad5, which are intracellular signaling molecules for BMP4, might also play a critical role in stimulating epiblasts to form PGC. However, how pluripotential epiblasts temporally and spatially respond to BMP signals to form PGCs remains unclear. The present study examines changes of responsiveness to BMP4 for PGC formation in epiblasts and their molecular mechanisms. We initially examined the effect of recombinant human (rh) BMP4 upon cultured epiblasts at different developmental stages, and found that they acquire the ability to respond to BMP4 signals for PGC formation between E5.25 and E5.5. In addition, such competence was conferred upon epiblasts by the extraembryonic ectoderm. We also showed that the increased expression of Smad1 and the onset of Smad5 expression induced by extraembryonic ectoderm might be responsible for quick acquisition of this competence. Furthermore, we show that only proximal epiblast cells maintain responsiveness to BMP4 for PGC formation at E6.0, and that this is associated with the proximal epiblast-specific expression of Smad5. These results explain why only the proximal region of epiblasts can sustain the ability to form PGCs.

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PubMed1995

In vitro pluripotency of epiblasts derived from bovine blastocysts.

Two experiments were conducted to compare the utility of in vitro- and in vivo-derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo-collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo-collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte-like cells, and macrophage-like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo-derived blastocysts, especially from early-hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state.

Alkaline Phosphatase↗

PubMed2004

Ultrastructural and immunohistochemical characterization of the bovine epiblast.

The epiblast represents the final embryonic founder cell population with the potential for giving rise to all cell types of the adult body. The pluripotency of the epiblast is lost during the process of gastrulation. Large animal species have a lack of specific markers for pluripotency. The aim of the present study was to characterize the bovine epiblast cell population and to provide such markers. Bovine Day 12 and Day 14 embryos were processed for transmission-electron microscopy or immunohistochemistry. In Day 12 embryos, two cell populations of the epiblast were identified: one constituting a distinctive basal layer apposing the hypoblast, and one arranged inside or above the former layer, including cells apposing the Rauber layer. Immunohistochemically, staining for the octamer-binding transcription factor 4 (OCT4, also known as POU5F1), revealed a specific and exclusive staining of nuclei of the complete epiblast. Colocalization of vimentin and OCT4 was demonstrated. Only trophectodermal cells stained for alkaline phosphatase. Staining for the proliferation marker Ki-67 was localized to most nuclei throughout the epiblast. A continuous staining for zonula occludens-1 protein was found between cells of the trophectoderm and hypoblast but was not evident in the epiblast. A basement membrane, detected by staining for laminin, formed a "cup-like" structure in which the epiblast was located. The ventrolateral sides of the cup appeared to be incomplete. In conclusion, the bovine epiblast includes at least two cell subpopulations, and OCT4 was shown, to our knowledge for the first time, to be localized exclusively to epiblast cells in this species.

Alkaline Phosphatase↗

PubMed1995

The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast.

In the amniotes, two unique layers of cells, the epiblast and the hypoblast, constitute the embryo at the blastula stage. All the tissues of the adult will derive from the epiblast, whereas hypoblast cells will form extraembryonic yolk sac endoderm. During gastrulation, the endoderm and the mesoderm of the embryo arise from the primitive streak, which is an epiblast structure through which cells enter the interior. Previous investigations by others have led to the conclusion that the avian hypoblast, when rotated with regard to the epiblast, has inductive properties that can change the fate of competent cells in the epiblast to form an ectopic embryonic axis. Thus, it has been suggested that the hypoblast normally induces the epiblast to form a primitive streak at a specific locus. In the work reported here, an attempt was made to reexamine the issue of induction. In contrast to previous reports, it was found that the rotated hypoblast of the chicken embryo does not initiate formation of an ectopic axis in the epiblast. The embryonic axis always initiates and develops according to the basic polarity of the epiblast layer. These results provoke a reinterpretation of the issues of mesoderm induction and primitive streak initiation in the avian embryo.

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PubMed1997

The allocation of epiblast cells to the embryonic heart and other mesodermal lineages: the role of ingression and tissue movement during gastrulation.

The cardiogenic potency of cells in the epiblast of the early primitive-streak stage (early PS) embryo was tested by heterotopic transplantation. The results of this study show that cells in the anterior and posterior epiblast of the early PS-stage embryos have similar cardiogenic potency, and that they differentiated to heart cells after they were transplanted directly to the heart field of the late PS embryo. That the epiblast cells can acquire a cardiac fate without any prior act of ingression through the primitive streak or movement within the mesoderm suggests that neither morphogenetic event is critical for the specification of the cardiogenic fate. The mesodermal cells that have recently ingressed through the primitive streak can express a broad cell fate that is characteristic of the pre-ingressed cells in the host when they were returned to the epiblast. However, mesoderm cells that have ingressed through the primitive streak did not contribute to the lateral plate mesoderm after transplantation back to the epiblast, implying that some restriction of lineage potency may have occurred during ingression. Early PS stage epiblast cells that were transplanted to the epiblast of the mid PS host embryos colonised the embryonic mesoderm but not the extraembryonic mesoderm. This departure from the normal cell fate indicates that the allocation of epiblast cells to the mesodermal lineages is dependent on the timing of their recruitment to the primitive streak and the morphogenetic options that are available to the ingressing cells at that instance.

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PubMed2000

Correlation between the expression of the HNK-1 epitope and cellular invasiveness in prestreak epiblast cells of chick embryos.

During avian gastrulation, certain cells present in the epiblast layer ingress through the basement membrane sealing the basal surface of themselves. Previously we reported that chick prestreak epiblast cells show two different behavioral phenotypes upon reconstituted basement membrane and laminin gel in vitro. Half of the dissociated epiblast cells invade the gel substratum after one-day of culture, whereas the others attach to the gel but do not invade. It is expected that such heterogeneity in the behavior of the epiblast cells reflects some mechanism that sorts the cells into those that will ingress into the blastocoelic cavity and those that will remain in the epiblast layer. To test this hypothesis, we dissociated chick prestreak epiblast cells into single cells, cultured them on the laminin gel, and then stained them with anti-HNK-1 antibody. This antibody binds to an epitope present on half of the prestreak epiblast cells which are thought to differentiate into presumptive mesoendodermal cells. We found that 80% of the invasive epiblast cells were HNK-1-positive whereas 77% of the non-invasive cells were HNK-1 negative. In the case of invasive cells, the edges of the proteolytic holes made by the invasive cells were often stained. These results suggest that the cells expressing the HNK-1 carbohydrate chain are preferentially invasive, and this induces selective ingression of the carrier cells for mesoendodermal differentiation in vivo.

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PubMed1993

Locations of the ectodermal and nonectodermal subdivisions of the epiblast at stages 3 and 4 of avian gastrulation and neurulation.

A prospective fate map of the avian epiblast at late gastrula and early neurula stages has been generated through the construction of quail/chick transplantation chimeras. This map shows the subdivisions of the prospective ectoderm, mesoderm, and endoderm, both within the epiblast prior to their ingression and within the primitive streak. The map demarcates the locations and extents of the prospective surface ectoderm, otic placodes, neural crest, and neural plate--including its postnodal levels--in prospective ectoderm of the epiblast; prospective foregut, within the prospective endoderm of the epiblast and primitive streak; and prospective notochord, somites, intermediate mesoderm, lateral plate mesoderm, and extraembryonic mesoderm in the prospective mesoderm of the epiblast and/or primitive streak. Prospective cardiogenic cells are apparently absent from the primitive streak at these stages, and contributions of the epiblast to the heart are relatively scant and inconsistent with the expected timing and directions of migrations of prospective cardiogenic cells. Mapping of the primitive streak at earlier stages in another study (García-Martinez and Schoenwolf: Developmental Biology, in press) reveals that the ingression of cardiogenic cells through the primitive streak occurs prior to late gastrula stages, suggesting that contributions of epiblast to the heart at later stages are artifactual. Tests of prospective potency, based on the projected locations of origin of various cell groups provided by the new prospective fate map, are underway.

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PubMed1993

Alkaline phosphatase staining of pig and sheep epiblast cells in culture.

To define better the characteristics of pig and sheep epiblast cells in culture, the cells were tested for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of mouse embryonic stem cells. Pig and sheep epiblast cells were positive for AP staining both at isolation from the blastocyst and after primary in vitro culture. The innermost portion of the attendant endoderm surrounding the epiblast was also positive for AP staining during primary culture. AP staining was lost upon differentiation or senescence of the epiblast cells. Also, all differentiated epiblast-derived cell cultures were negative for AP staining, with the exception of neuron-like cultures. Epiblast-like cells were cultured from day 10 (pig) and day 13 (sheep) embryonic discs, and these cells were also AP positive until they differentiated. Trophectoderm-endoderm-like cells from embryonic discs were AP negative or weakly positive. AP is a convenient marker for undifferentiated pig and sheep epiblast cells in culture when used in conjunction with cell morphology analysis.

Alkaline Phosphatase↗

PubMed1996

Skeletal myogenesis: the preferred pathway of chick embryo epiblast cells in vitro.

The epiblast layer of the chick embryo gives rise to all embryonic tissues. In vitro analyses were carried out to determine whether epiblast cells could form skeletal muscle prior to entry into the primitive streak. Epiblasts were separated from the mesoderm, hypoblast, and primitive streak, dissociated to produce a single cell suspension, and plated at high density. Myogenesis began on the first day in culture, and by the fifth day most cells had differentiated into skeletal muscle. Some cells differentiated without replicating. MyoD messenger RNA was present in epiblast tissue and translated in practically all cells in culture. Cells from regions of the epiblast which do not form muscle later in the embryo did so in vitro. Epiblasts cultured for 2 days as an intact epithelium, or in the presence of the mesoderm and hypoblast, did not undergo myogenesis. These findings demonstrate that myogenic potential is wide-spread within the primitive streak stage epiblast, and that muscle differentiation, which occurs relatively autonomously in culture, can be prevented by cell and tissue interactions.

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PubMed2001

Localization of cells of the prospective neural plate, heart and somites within the primitive streak and epiblast of avian embryos at intermediate primitive-streak stages.

By constructing avian transplantation chimeras using fluorescently-labeled grafts and antibodies specific for grafted cells, we have generated a prospective fate map of the primitive streak and epiblast of the avian blastoderm at intermediate primitive-streak stages (stages 3a/3b). This high-resolution map confirms our previous study on the origin of the cardiovascular system from the primitive streak at these stages and provides new information on the epiblast origin of the neural plate, heart and somites. In addition, the origin of the rostral endoderm is now documented in more detail. The map shows that the prospective neural plate arises from the epiblast in close association with the rostral end of the primitive streak and lies within an area extending 250 microm rostral to the streak, 250 microm lateral to the streak and 125 microm caudal to the rostral border of the streak. The future floor plate of the neural tube arises within the midline just rostral to the streak, confirming our earlier study, but unlike at the late-primitive streak stages when both Hensen's node and the midline area rostral to Hensen's node contribute to the floor plate, only the area rostral to the primitive streak contributes to the floor plate at intermediate primitive-streak stages. Instead of contributing to the floor plate of the neural tube, the rostral end of the primitive streak at intermediate primitive-streak stages forms the notochord as well as the rostromedial endoderm, which lies beneath the prechordal plate mesoderm and extends caudolaterally on each side toward the cardiogenic areas. The epiblast lateral to the primitive streak and caudal to the neural plate contributes to the heart and it does so in rostrocaudal sequence (i.e., rostral grafts contribute to rostral levels of the straight heart tube, whereas progressively more caudal grafts contribute to progressively more caudal levels of the straight heart tube), and individual epiblast grafts contribute cells to both the myocardium and endocardium. The prospective somites (i.e., paraxial mesoderm) lie within the epiblast just lateral to the prospective heart mesoderm. Comparing this map with that constructed at late primitive-streak stages reveals that by the late primitive-streak stages, prospective heart mesoderm has moved from the epiblast through the primitive streak and into the mesodermal mantle, and that some of the prospective somitic mesoderm has entered the primitive streak and is undergoing ingression.

Animals↗

PubMed1981

Interaction of epiblast and hypoblast in the formation of the primitive streak and the embryonic axis in chick, as revealed by hypoblast-rotation experiments.

Three types of experiments were performed to determine the interaction between the epiblast and hypoblast for primitive streak formation: (1) Hypoblasts of blastoderms from stages XIII E.G & K to 3 H & H were separated from the epiblasts and rotated by 90 degrees counterclockwise; (2) hypoblasts from stages XIII E.G & K to 3 H & H blastoderms were rotated by 180 degrees; (3) hypoblasts were exchanged between blastoderms of different developmental stages and placed at 90 degrees counterclockwise to the axis of the recipient epiblast. In all blastoderms studied only a single PS developed. After rotation of the hypoblast by 90 degrees, the direction of the PS was according to the orientation of the hypoblast at stage XIII, whereas at older stages it gradually shifted towards the axis of the epiblast. At stage 3- H & H the PS is already imprinted in the epiblast and cannot be shifted. After rotation of the hypoblast by 180 degrees the PS originated at the point near the marginal zone at which the inductive part of the hypoblast interacted with a component epiblast. Conclusions are drawn about the dynamics of the inductiveness of the hypoblast and the competence of the epiblast for the PS formation and orientation.

Animals↗

PubMed2000

Regulation of epiblast cell movements by chondroitin sulfate during gastrulation in the chick.

Avian gastrulation is dependent on the ingression of outer layer cells into the interior of the embryo by means of a transient structure referred to as the primitive streak. As the growing streak progresses through the central area pellucida of the blastoderm, selective de-epithelialization of epiblast cells results in the initial migratory cells of the primitive mesoderm and endoderm. Here, we have examined the possibility that extracellular matrix molecules of the epiblast basal lamina influence the selection of streak-specific epiblast cells. By using whole embryo culture, we have found that removal of chondroitin sulfate glycosaminoglycans at gastrulation stages leads to defective streak formation. In situ hybridization with streak-specific markers in these embryos reveals ectopic patterns of gene expression, suggesting that differentiation of primitive streak precursors in the pregastrula epiblast is independent of normal streak morphogenesis. In addition, in vitro assays with chondroitin sulfate containing matrices suggest that specific cells of the epiblast are inhibited from joining the streak during gastrulation. Taken together, these results indicate that the presence of chondroitin sulfate in the epiblast basal lamina facilitates the allocation of cells to the primary germ layers by preventing ectopic axis formation.

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PubMed2005

Induction of initial cardiomyocyte alpha-actin--smooth muscle alpha-actin--in cultured avian pregastrula epiblast: a role for nodal and BMP antagonist.

During early cardiogenesis, endoderm-derived bone morphogenetic protein (BMP) induces the expression of both heart-specific transcription factors and sarcomeric proteins. However, BMP antagonists do not inhibit the expression of the "initial heart alpha-actin"--smooth muscle alpha-actin (SMA)--which is first expressed in the anterior lateral mesoderm and then recruited into the initial myofibrils (Nakajima et al. [2002] Dev. Biol. 245:291-303). Therefore, mechanisms that regulate the expression of SMA in the heart-forming mesoderm are not well-understood. Regional explantation experiments using chick blastoderm showed that the posterolateral region of the epiblast differentiated into cardiomyocytes. Posterior epiblast cultured with or without the associated hypoblast showed that interaction between the tissues of these two germ layers at the early pregastrula stage (stages X-XI) was a prerequisite for the expression of SMA. Posterior epiblast that is cultured without hypoblast could also be induced to express SMA if TGF-beta or activin was added to the culture medium. However, neither neutralizing antibodies against TGF-betas nor follistatin perturbed the expression of SMA in cultured blastoderm. Adding BMP to the cultured blastoderm inhibited the expression of SMA, whereas BMP antagonists, such as chordin, were able to induce the expression of SMA in cultured posterior epiblast. Furthermore, adding lefty-1, a nodal antagonist, to the blastoderm inhibited the expression of SMA, and nodal plus BMP antagonist up-regulated the expression of SMA in cultured posterior epiblast. Results indicate that the interaction between the tissues of the posterior epiblast and hypoblast is necessary to initiate the expression of SMA during early cardiogenesis and that nodal and BMP antagonist may play an important role in the regulation of SMA expression.

Actins↗

PubMed1999

Hepatocyte growth factor/scatter factor promotes a switch from E- to N-cadherin in chick embryo epiblast cells.

Epiblast cells downregulate E-cadherin and upregulate N-cadherin as they ingress through the primitive streak and when placed in culture. The factors that promote the alteration in cadherin expression during gastrulation are unknown. The effects of hepatocyte growth factor/scatter factor (HGF/SF) on cadherin expression were tested in cultures of prestreak epiblast cells. HGF/SF decreased the expression of E-cadherin and increased the percentage of cells with N-cadherin and sarcomeric myosin. Cells with N-cadherin but not E-cadherin differentiated into skeletal muscle. HGF/SF also stimulated proliferation and the formation of cellular aggregates. Sensitivity to HGF/SF in vitro depended on the original position of cells within the epiblast. More cells from the lateral epiblast switched cadherins and proliferated in response to HGF/SF than medial epiblast cells. HGF/SF may affect gastrulation by altering cadherin expression, modulating cell adhesion, and stimulating proliferation within the epiblast.

Animals↗

PubMed1993

Culturing the epiblast cells of the pig blastocyst.

Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.

Animals↗

PubMed1989

Adhesion and fusion of the extraembryonic epiblast.

A tissue culture model system has been devised to examine the attachment, expansion, and fusion of epithelial cell sheets. A normal embryonic epithelial tissue, the extraembryonic epiblast of the chick, is isolated mechanically and cultured on its natural substratum, the vitelline membrane. This persistently migratory tissue has distinct adhesive and non-adhesive regions. A serum-free chemically-defined culture medium has been formulated that permits determination of the effects of individual growth and trophic factors. Attachment of transferred epiblasts is dependent upon the presence of mineralocorticoids in the medium. This suggests that fluid transport is required for the cell sheet to make its initial attachment to the culture substratum. Expansion of the cell sheet following attachment, and the fusion of epiblasts advancing toward each other, does not require the presence of mineralocorticoid. No exogenous adhesive glycoproteins are required for attachment, expansion, or fusion. Antibody localization shows that endogenous laminin is present on the attachment surface of the specialized adhesive edge region of the extraembryonic epiblast. Following fusion of confronted epiblasts into one coherent cell sheet, the laminin disappears. Throughout these studies the adhesive and non-adhesive regions of the epiblast are identified by their characteristic distributions of actin microfilaments, localized using rhodamine-phalloidin staining.

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