Appearance and distribution of entactin in the early chick embryo

Abstract. Entactin is a sulfated glycoprotein of basement membranes and recent data indicate that it may play a major role in extracellular matrix (ECM) assembly and in modulating the activities of the other molecular components. We investigated the time of appearance and subsequent distribution of entactin during the earliest stages of morphogenesis and its involvement in the first major cellular migrations and interactions in the chick embryo. Entactin is first detected in the epiblast and in the hypoblast at the blastula stage. The accumulating ECM displays intense presence of entactin in the space between the epiblast and the hypoblast at late blastula. Entactin is increasingly abundant in the neural plate and in the ECM and also at least transiently in many mesodermal tissues such as the notochord, the developing heart and somites in the early chick embryo. Immuno-gold labeling revealed a punctate pattern of entactin distribution in the ECM during the gastrula, neurula and at later stages and at all levels within the embryo. Because of its early appearance in more than one germ layer, entactin may be important in the formation of most embryonic structures. Entactin is detected at the same developmental time and co-localizes with laminin. Antibodies to entactin do not interfere with triggering of the first major cell movements but perturb directional migration of these cells. It would seem that entactin plays a functional role in the directed migration of cells and does not seem to affect cell adhesion during the period of the first morphogenetic events in the early chick embryo.     

Appearance and distribution of entactin in the early chick embryo

Abstract. Entactin is a sulfated glycoprotein of basement membranes and recent data indicate that it may play a major role in extracellular matrix (ECM) assembly and in modulating the activities of the other molecular components. We investigated the time of appearance and subsequent distribution of entactin during the earliest stages of morphogenesis and its involvement in the first major cellular migrations and interactions in the chick embryo. Entactin is first detected in the epiblast and in the hypoblast at the blastula stage. The accumulating ECM displays intense presence of entactin in the space between the epiblast and the hypoblast at late blastula. Entactin is increasingly abundant in the neural plate and in the ECM and also at least transiently in many mesodermal tissues such as the notochord, the developing heart and somites in the early chick embryo. Immunogold labeling revealed a punctate pattern of entactin distribution in the ECM during the gastrula, neurula and at later stages and at all levels within the embryo. Because of its early appearance in more than one germ layer, entactin may be important in the formation of most embryonic structures. Entactin is detected at the same developmental time and co-localizes with laminin. Antibodies to entactin do not interfere with triggering of the first major cell movements but perturb directional migration of these cells. It would seem that entactin plays a functional role in the directed migration of cells and does not seem to affect cell adhesion during the period of the first morphogenetic events in the early chick embryo.     

Monensin inhibits the first cellular movements in early chick embryo

Summary In early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.     

Distribution and functional role of laminin during induction of the embryonic axis in the chick embryo

Laminin is a major glycoprotein of basement membranes and has been shown to promote cell adhesion, and movement of various nonepithelial cells and tumour cells. Using antibodies to laminin in paraffin sections and cultured embryos, we have studied the distribution of laminin and its involvement in the first morphogenetic events, beginning with the first extensive cellular migrations and interactions that result in the induction of the primitive streak (PS) and of the neural plate in the early chick embryo. Laminin immunogold labeling was not detected in the blastoderm at stage X. At stage XIII, laminin immunoreactivity was detected at the ventral surface of the epiblast and in the entire hypoblast. The intense labeling of the hypoblast indicated that these cells are active in laminin synthesis. Extracellular matrix (ECM) started accumulating as the first embryonic spaces were forming, before the morphogenetic movements of gastrulation were initiated. Immunogold labeling revealed a punctate pattern of laminin distribution in the ECM in the blastocoele, and in the space below the neural plate. Laminin, which is a multidomain molecule known to interact with other molecules of the ECM and with the cell surface, could serve as the scaffold for highly specific contact points of migrating cells and for the folding of epithelial sheets during this time in the developing embryo. We incubated blastoderms at stages X and XIII with laminin antibodies (1:30 dilution) for 4 h, then cultured the blastoderms further in plain egg albumin. The laminin antibodies did not interfere with triggering of PS cell movements, but perturbed the normal migration pattern of these cells. A normal PS did not form and, as a consequence, the embryonic axis was not induced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concanavalin A-binding by cells of the early chick embryo

The surfaces of cells from the early embryo of the chick were examined using electron microscope techniques for the visualization of concanavalin A-binding sites. Horseradish peroxidase and Ferritin labelled concanavalin A were used to determine the distribution of the binding sites. All surfaces of the epiblast and hypoblast layers which were accessible to concanavalin A showed the presence of binding sites in stage 1 embryos. The ventral surface of the epiblast showed a high lectin affinity which may reflect the development of a basal lamina on this surface. The individual hypoblast cells at this stage showed a non-uniform distribution of binding sites, having a greater affinity on the dorsal surface than the ventral. By the time of primitive streak formation (stage 4-5) the dorsal surface of the epiblast displayed increased binding sites, while the frequency of sites on the ventral surface of the endoblast was reduced. The latter may reflect a change from one cell population to another, which occurs in the lower layer of the embryo at this time. No consistent correlation could be drawn between changes in motility of cells actually invaginating through the primitive streak and changes in affinity for concanavalin A. An overall increase in affinity of the dorsal surface of the epiblast was revealed by Ferritin and may reflect the changes in surface structure occurring in readiness for the morphogenetic migrations of gastrulation.     

Induction of the primitive streak in the chick blastoderm embryo: patterns of protein synthesis

Summary Induction of the primitive streak is correlated with specific qualitative and quantitative changes in protein synthesis in the component areas of chick blastoderm. Blastoderm embryos at the initial to intermediate primitive streak stage were labeled with L-[³⁵S] methionine. Radioactively labeled proteins separated by two-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed differences in the number and density of spots among the component areas of the epiblast and hypoblast. Protein patterns of the area opaca, marginal zone and central area of the epiblast are very similar qualitatively but show distinct quantitative differences. A comparison between any of the component areas of the epiblast and the hypoblast in chick blastoderm embryos, however, reveals both qualitative and quantitative differences. A protein with a molecular weight of 30,000 unique to the component areas of the epiblast, and proteins with a molecular weight of 22,000 and 37,000 unique to the hypoblast are prominent and seem to be related to the initial appearance of the primitive streak.     

Induction of gastrulation in the chick embryo

Interaction between the epiblast and the primary hypoblast in chick blastula results in induction of the primitive streak (PS) in the epiblast. Alpha-amanitin, a specific inhibitor of poly A-containing RNA synthesis, inhibits formation of the definitive PS. This inhibition is associated with qualitative changes in the pattern of protein synthesis in the hypoblast but not in the epiblast. The protein pattern of the component areas of the epiblast shows increase in some polypeptides after treatment with alpha-amanitin. By contrast, alpha-amanitin resulted in a decrease in synthesis of several polypeptides, which are either undetectable or weakly present in the hypoblast. The alpha-amanitin-sensitive translational products of the embryonic genome that are observed in the hypoblast may have specific functions in the control of PS induction and stabilization.     

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.     

Intercellular contact in the unincubated chick embryo

Summary The unincubated chick blastoderm, which consists of a complete upper epithelial layer of one cell thickness (epiblast) and an incomplete lower layer (hypoblast), was examined with the electron microscope in order to define the types of cell contact present. The terminal contacts between the cells of the epiblast invariably involved several focal tight junctions, but only occasionally involved tight junctions. Desmosomes were not observed in these areas, but were encountered in various phases of development in the deeper contact regions between epiblast cells. This deeper region also showed sporadic focal tight junctions and frequent micropapillae. These micropapillae were also common on the surfaces of hypoblast cells. Intercellular spaces between epiblast and hypoblast cells and within the hypoblast were often wide, narrowing to occasional focal tight junctions. Tight junctions and desmosomes were not observed in association with hypoblast cells. Gap junctions were not observed in any region of the embryo.These observations are discussed in relation to the morphogenetic movements occurring in the forming hypoblast and also the influence of this layer on the subsequent development of the embryo. Comparisons are drawn between the contact morphology in the unincubated blastoderm and that in later stages of development.Supported by the Medical Research Council of Canada.     

Early development of the chick embryo

The ultrastructure of the early chick embryo was investigated, using scanning (SEM) and transmission electron microscopy (TEM). Eggs were obtained from the shell gland by injecting hens intravenously with a synthetic prostaglandin or arginine vasopressin. Embryos were examined during late cleavage (stages IV–VI, Eyal-Giladi and Kochav, '76), formation of the area pellucida (stages VII–XI), and formation of the hypoblast (stages X–XIV). SEM highlighted the reduction in cell number at the underside of the embryo during formation of the area pellucida although it became apparent that the thickness of the embryo is not reduced to a single layer of cells at stage X. In addition, blastomeres at the perimeter of embryos (stages V–VI) project filopodial extensions onto a smooth membrane that separates the sub-embryonic cavity from the yolk. During hypoblast formation, epiblast cells generate stellate projections at their basal aspect, thus providing a meshwork for the advancing secondary hypoblast cells. By stage XII the epiblast was one cell thick and reminiscent of a columnar epithelium when viewed transversely. Cells of the deep portion of the posterior marginal zone were distinguished morphologically in the stage XII embryo by their many cell surface projections and ruffled appearance. Blastomeres at the perimeter of stage V–VI embryos projected filopodial extensions onto a smooth membrane which separates the sub-embryonic cavity from the yolk. This membrane is presumed to be confluent with the cytolemma. Evidence is presented demonstrating the presence of intracellular membrane-bound droplets which are hypothesised to contain sub-embryonic fluid. © 1993 Wiley-Liss, Inc.     

An electron-microscopical analysis of embryonic chick tissues explanted in culture

Summary Three types of tissue (hypoblast, germ wall and epiblast) were dissected from early chick embryos and explanted on Falcon plastic dishes. After they had settled and spread, the explants were fixed, usually within 18–24 h after explantation, and sections were cut through the tissue and the Falcon dish. The closeness of the cells to the substrate varied even within the same explant, but the epiblast tended to be closer to the substrate than did the hypoblast or germ wall. Plaques were present in all three tissues in regions where the cell processes contacted the substrate. Extensive desmosomes were visible in the epiblast explants, small desmosomes were present in the germ wall explants, but desmosomes were never seen in hypoblast explants. These differences in cell/substrate and cell/cell morphology are discussed in relation to the different behavioural characteristics of the three tissues. Some mixed cultures were also examined by electron microscopy. When the epiblast was confronted with either hypoblast or germ wall, it underlapped them at the region of contact.     

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.     

Activation of epiblast gene expression by the hypoblast layer in the prestreak chick embryo

Axis formation is a highly regulated process in vertebrate embryos. In mammals, inductive interactions between an extra-embryonic layer, the visceral endoderm, and the embryonic layer before gastrulation are critical both for anterior neural patterning and normal primitive streak formation. The role(s) of the equivalent extra-embryonic endodermal layer in the chick, the hypoblast, is still less clear, and dramatic effects of hypoblast on embryonic gene expression have yet to be demonstrated. We present evidence that two genes later associated with the gastrula organizer (Gnot-1 and Gnot-2) are induced by hypoblast signals in prestreak embryos. The significance of this induction by hypoblast is discussed in terms of possible hypoblast functions and the regulation of axis formation in the early embryo. Several factors known to be expressed in hypoblast, and retinoic acid, synergistically induce Gnot-1 and Gnot-2 expression in blastoderm cell culture. The presence of retinoic acid in prestreak embryos has not yet been directly demonstrated, but exogenous retinoic acid appears to mimic the effects of hypoblast rotation on primitive streak extension, raising the possibility that retinoid signaling plays some role in the pregastrula embryo.     

The marginal zone and its contribution to the hypoblast and primitive streak of the chick embryo

The marginal zone of the chick embryo has been shown to play an important role in the formation of the hypoblast and of the primitive streak. In this study, time-lapse filming, fate mapping, ablation and transplantation experiments were combined to study its contribution to these structures. It was found that the deep (endodermal) portion of the posterior marginal zone contributes to the hypoblast and to the junctional endoblast, while the epiblast portion of the same region contributes to the epiblast of the primitive streak and to the definitive (gut) endoderm derived from it. Within the deep part of the posterior marginal zone, a subpopulation of HNK-1-positive cells contributes to the hypoblast. Removal of the deep part of the marginal zone prevents regeneration of the hypoblast but not the formation of a primitive streak. Removal of both layers of the marginal zone leads to a primitive streak of abnormal morphology but mesendodermal cells nevertheless differentiate. These results show that the two main properties of the posterior marginal zone (contributing to the hypoblast and controlling the site of primitive streak formation) are separable, and reside in different germ layers. This conclusion does not support the idea that the influence of the posterior marginal zone on the development of axial structures is due to it being the source of secondary hypoblast cells.     

Involvement of fibronectin during epiboly and gastrulation in embryos of the common carp,Cyprinus carpio

Summary The present report firstly describes a pilot study in which, during early development of embryos of the common carp, Cyprinus carpio, the cellular adhesion to fibronectin (FN) was blocked by administration of GRGDS peptide (which binds to the FN-receptor). As this treatment resulted in developmental aberrations, suggesting a functional role for FN, the major part of the work was focussed on the distribution of reactivity of anti-FN antibodies during epiboly and gastrulation. GRGDS treatment had a concentration dependent effect on development. Incubation of embryos in 1.5 mg/ml from the 32-cell stage onwards caused a retardation of epiboly, which did not proceed beyond 60%. The embryos did not show involution, as was confirmed by histological study. These preliminary results suggest that FN is involved in both epiboly and gastrulation of carp embryos. During cleavage, no specific extracellular binding of anti-FN antiserum could be observed. However, binding to a number of cell membranes took place from early epiboly onwards. After the onset of gastrulation, we observed a gradually increasing number of the deepest epiblast cells, showing immunostaining on part of their surface, facing the yolk syncytial layer (YSL) or the involuted cells. During early epiboly, anti-FN binding was restricted to areas in front of the migratory hypoblast cells. Later on, binding was found at the border of hypoblast and epiblast cells. At 100% epiboly, some contact areas of epiblast and hypoblast showed a discontinuous lining of reactivity, whilst other areas appeared devoid of anti-FN binding sites. The results indicate that FN is involved in the migration and guidance of hypoblast cells during gastrulation in carp. Correspondence to: P. Gevers     

Anterior identity is established in chick epiblast by hypoblast and anterior definitive endoderm

Previous studies of head induction in the chick have failed to demonstrate a clear role for the hypoblast and anterior definitive endoderm (ADE) in patterning the overlying ectoderm, whereas data from both mouse and rabbit suggest patterning roles for anterior visceral endoderm (AVE) and ADE. Based on similarity of gene expression patterns, fate and a dual role in 'protecting' the prospective forebrain from caudalising influences of the organiser, the chick hypoblast has been suggested to be the homologue of the mouse anterior visceral endoderm. In support of this, when transplanted to chick embryos, the rabbit AVE induces anterior markers in the chick epiblast. To reevaluate the role of the hypoblast/ADE (lower layer) in patterning the chick ectoderm, we used rostral blastoderm isolates (RBIs) as an assay, that is, rostral regions of blastoderms transected at levels rostral to the node. RBIs are, therefore, free from the influences of Hensen's node and ingressing axial mesoderm - tissues that are able to induce Ganf, the earliest specific marker of anterior neural plate. We demonstrate, using such RBIs (or RBIs dissected to remove the lower layer with or without tissue replacement), that the hypoblast/ADE (lower layer) is required and sufficient for patterning anterior positional identity in the overlying ectoderm, leading to expression of Ganf in neuroectoderm. Our results suggest that patterning of anterior positional identity and specification of neural identity are separable events operating to pattern the rostral end of the early chick embryo. Based on this new evidence we propose a revised model for establishing anteroposterior polarity, neural specification and head patterning in the early chick that is consonant with that occurring in other vertebrates.     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz