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.     

Protein synthesis in epiblast versus hypoblast during the critical stages of induction and growth of the primitive streak in the chick embryo.

In the chick the inducing power of the hypoblast for primitive streak was assumed to reach its maximum at the beginning of the primitive streak stage and to last until its completion. It was therefore of interest to trace the protein synthetic activity of the epiblast and hypoblast during five successive developmental stages and to correlate them with the known morphogenetic events.The investigation was done along two lines: 1) A quantitative survey was made of the uptake of tritiated phenylalanine into epiblasts versus hypoblasts and their incorporation into trichloroacetic acid-precipitable protein. 2) Incorporation of label into protein was followed by a comparative investigation of the electropherograms of epiblast versus hypoblast at the different stages.The quantitative survey has shown an almost uniform and rather low incorporation of label into protein in the hypoblast layer with a very short period of doubled activity between full hypoblast and initial primitive streak (p.s.). During this period the inductive capacity of the hypoblast for primitive streak was supposed to reach its maximal value.The qualitative survey indicated different patterns of incorporation in the two layers studied. Of special interest are two peaks (III and IV) which appear in the hypoblast previous to p.s. formation at the time of its augmented synthetic activity which also coincides with the onset of its inductive capacity. At later stages two similar peaks appear in the epiblast. It is suggested that a protein included in the above peaks might represent the inductor of the primitive streak.     

Differential heat shock gene expression in chick blastula

Summary The component areas of chick blastula show differential expression of heat shock genes. The area opaca (ao), marginal zone (mz) and central area (ca) components of the epiblast display distinct quantitative and minor qualitative differences in the heat-induced and heat-repressible proteins, but are clearly different from the primary hypoblast (endoderm) in their expression of a given stress protein (hsp) as a response to heat shock. The major proteins synthesized in the component areas of epiblast in response to heat shock include hsp 18, 24, 70 and 89 Kd. Two-dimensional electrophoresis shows that each of these proteins consists of multiple charged species. The hypoblast expresses only hsp 70 Kd at non-significant levels and shows marked inhibition in the level of synthesis of heat-shock-repressible proteins. Heat shock during the blastula stage results in an increase in the size of the blastoderm and disrupts normal embryonic development. The heat shock genes provide an important molecular marker, which attests to regional specification in the chick blastula.     

5-Bromodeoxyuridine inhibition of the epiblast competence for primitive streak formation in the young chick blastoderm

The competence of stage XIII chick epiblast which under the influence of an inductive hypoblast is directed to form a normal primitive streak, is affected by 5-bromodeoxyuridine (BUdR). The BUdR-treated epiblast forms an atypical primitive streak and no axial mesoderm. However, a nonorganized mesenchymal layer is formed between the epiblast and the hypoblast, and atypical neural tissue in the epiblast. BUdR interferes neither with hypoblast formation nor with its inductivity even when blastoderms are treated with BUdR as early as uterine stage VIII and later.     

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.     

Changes in the expression of the carbohydrate epitope HNK-1 associated with mesoderm induction in the chick embryo

We report that a monoclonal antibody, HNK-1, identifies specific regions and cell types during primitive streak formation in the chick blastoderm. Immunohistochemical studies show that the cells of the forming hypoblast are HNK-1 positive from the earliest time at which they can be identified. Some cells of the margin of the blastoderm are also positive. The mesoderm cells of the primitive streak stain strongly with the antibody from the time of their initial appearance. In the epiblast, some cells are positive and some negative at pre-primitive-streak stages, but as the primitive streak develops a gradient of staining intensity is seen within the upper layer, increasing towards the primitive streak. At later stages of development, the notochord and the mesenchyme of the headfold are positive, while the rest of the mesoderm (lateral plate) no longer expresses HNK-1 immunoreactivity. This antibody therefore reveals changes associated with mesodermal induction: before induction, it recognizes the 'inducing' tissue (the hypoblast) and reveals a mosaic pattern in the responding tissue (the epiblast); after primitive streak formation, the mesoderm of the primitive streak that results from the inductive interactions expresses the epitope strongly. Affinity purification of HNK-1-related proteins in various tissues was carried out, followed by SDS-PAGE to identify them. The hypoblast, mesoderm and epiblast of gastrulating chick embryos have some HNK-1-related proteins in common, while others are unique to specific tissues. Attempts have been made to identify these proteins using Western blots and antibodies known to recognize HNK-1-related molecules, but none of the antibodies used identify the bands unique to any of the tissues studied. We conclude that these proteins may be novel members of the HNK-1/L2 family, and that they may have a role in cell interactions during early development.     

Primitive streak-forming cells of the chick invaginate through a basement membrane

Summary Recently fibronectin was shown to appear in the development of the chick for the first time as a thin band on the epiblastic side facing the hypoblast just prior to primitive streak formation. It was thus suggested that fibronectin might be instrumental in the migration of cells that lead to axis formation during primitive streak formation. In the present work we have examined simultaneously for the presence of fibronectin and the specific basement membrane glycoprotein laminin during primitive streak formation using immunofluorescence methods. Laminin was found to be expressed between the epiblast and the hypoblast of stage XIII¹ chick blastoderms. During the immediately following process of streak formation the laminin was found to be continuously detectable throughout the area covered by the hypoblast, but disrupted on the streak area. Fibronectin was found to co-distribute with laminin in stage XIII and in the early primitive streak chick blastoderms. It is concluded that at stage XIII laminin and fibronectin form part of a basement membrane that is partially disrupted during the immediately following process of primitive streak formation in order to allow the migration of the streak-forming epiblastic cells during this morphogenetic process.     

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     

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.     

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.     

Mesoblastic Cells in the Early Phase of Primitive Streak Formation in Chick Embryos. Observations by Scanning Electron Microscopy in ovo and time-Lapse Cinematography in vitro.

During primitive streak formation in the chick embryo, mesoblastic cells were observed by SEM after removal of the hypoblast layer. Before the primitive streak began to develop, numbers of bleb cells and bleb-like protrusions were seen on the ventral surface of the epiblast. From optical observation on the process of change of epiblastic cells into bleb cells in vitro , it was concluded that cells that had elongated became bleb cells when they emerged from the epiblast. Cell behavior during primitive streak formation is discussed on the basis of these findings.     

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.     

Nuclear beta-catenin and the development of bilateral symmetry in normal and LiCl-exposed chick embryos.

Studies in Xenopus laevis and zebrafish suggest a key role for beta-catenin in the specification of the axis of bilateral symmetry. In these organisms, nuclear beta-catenin demarcates the dorsalizing centers. We have asked whether beta-catenin plays a comparable role in the chick embryo and how it is adapted to the particular developmental constraints of chick development. The first nuclear localization of beta-catenin is observed in late intrauterine stages of development in the periphery of the blastoderm, the developing area opaca and marginal zone. Obviously, this early, radially symmetric domain does not predict the future organizing center of the embryo. During further development, cells containing nuclear beta-catenin spread under the epiblast and form the secondary hypoblast. The onset of hypoblast formation thus demarcates the first bilateral symmetry in nuclear beta-catenin distribution. Lithium chloride exposure also causes ectopic nuclear localization of beta-catenin in cells of the epiblast in the area pellucida. Embryos treated before primitive streak formation become completely radialized, as shown by the expression of molecular markers, CMIX and GSC. Lithium treatments performed during early or medium streak stages cause excessive development of the anterior primitive streak, node and notochord, and lead to a degeneration of prospective ventral and posterior structures, as shown by the expression of the molecular markers GSC, CNOT1, BMP2 and Ch-Tbx6L. In summary, we found that in spite of remarkable spatiotemporal differences, beta-catenin acts in the chick in a manner similar to that in fish and amphibia.     

Analysis of tissue flow patterns during primitive streak formation in the chick embryo

We have investigated the patterns of tissue flow underlying the formation of the primitive streak in the chick embryo. Analysis of time-lapse sequences of brightfield images to extract the tissue velocity field and of fluorescence images of small groups of DiI-labelled cells have shown that epiblast cells move in two large-scale counter-rotating streams, which merge at the site of streak formation. Despite the large-scale tissue flows, individual cells appear to move little relative to their neighbours. As the streak forms, it elongates in both the anterior and posterior directions. Inhibition of actin polymerisation via local application of the inhibitor latrunculin A immediately terminates anterior extension of the streak tip, but does not prevent posterior elongation. Inhibition of actin polymerisation at the base of the streak completely inhibits streak formation, implying that continuous movement of cells into the base of the forming streak is crucial for extension. Analysis of cycling cells in the early embryo shows that cell-cycle progression in the epiblast is quite uniform before the primitive streak forms then decreases in the central epiblast and incipient streak and increases at the boundary between the area pellucida and area opaca during elongation. The cell-cycle inhibitor aphidicolin, at concentrations that completely block cell-cycle progression, permits initial streak formation but arrests development during extension. Our analysis suggests that cell division maintains the cell-flow pattern that supplies the streak with cells from the lateral epiblast, which is critical for epiblast expansion in peripheral areas, but that division does not drive streak formation or the observed tissue flow.     

The sorting-out of thymidine-labelled chick hypoblast cells in mixed epiblast--hypoblast aggregates.

Tritium-labelled disaggregated chick hypoblast cells were mixed with non-labelled epiblast cells and vice-versa. The mixtures were allowed to aggregate in a gyratory shaker and were transferred on to a solid culture medium for further incubation. The aggregates were fixed after various incubation times, sectioned and examined for sorting-out. There was already a tendency to sort out after 10 h of incubation, a process which was completed after 25 h. The hypoblast cells formed a continuous layer adjacent to the vitelline membrane, while the epiblast cells moved out to form the upper external layer. The position of the two layers was normal as far as the substrate and external environment are concerned, and reversed in relation to their relative position to the vitelline membrane. The hypoblast cells tended to migrate to the margins of the aggregate. The latter phenomenon seems to parallel the migration of hypoblast cells towards the extra-embryonal area during the formation of the primitive streak.     

The Sorting-out of Thymidine-labelled Chick Hypoblast Cells in Mixed Epiblast–Hypoblast Aggregates

Tritium-labelled disaggregated chick hypoblast cells were mixed with non-labelled epiblast cells and vice-versa . The mixtures were allowed to aggregate in a gyratory shaker and were transferred on to a solid culture medium for further incubation. The aggregates were fixed after various incubation times, sectioned and examined for sorting-out. There was already a tendency to sort out after 10 h of incubation, a process which was completed after 25 h. The hypoblast cells formed a continuous layer adjacent to the vitelline membrane, while the epiblast cells moved out to form the upper external layer. The position of the two layers was normal as far as the substrate and external environment are concerned, and reversed in relation to their relative position to the vitelline membrane. The hypoblast cells tended to migrate to the margins of the aggregate. The latter phenomenon seems to parallel the migration of hypoblast cells towards the extra-embryonal area during the formation of the primitive streak.     

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.     

Morphological and Gene Expression Changes in Cattle Embryos from Hatched Blastocyst to Early Gastrulation Stages after Transfer of In Vitro Produced Embryos

A detailed morphological staging system for cattle embryos at stages following blastocyst hatching and preceding gastrulation is presented here together with spatiotemporal mapping of gene expression for BMP4, BRACHYURY, CERBERUS1 (CER1), CRIPTO, EOMESODERMIN, FURIN and NODAL. Five stages are defined based on distinct developmental events. The first of these is the differentiation of the visceral hypoblast underlying the epiblast, from the parietal hypoblast underlying the mural trophoblast. The second concerns the formation of an asymmetrically positioned, morphologically recognisable region within the visceral hypoblast that is marked by the presence of CER1 and absence of BMP4 expression. We have termed this the anterior visceral hypoblast or AVH. Intra-epiblast cavity formation and the disappearance of the polar trophoblast overlying the epiblast (Rauber’s layer) have been mapped in relation to AVH formation. The third chronological event involves the transition of the epiblast into the embryonic ectoderm with concomitant onset of posterior NODAL, EOMES and BRACHYURY expression. Lastly, gastrulation commences as the posterior medial embryonic ectoderm layer thickens to form the primitive streak and cells ingress between the embryonic ectoderm and hypoblast. At this stage a novel domain of CER1 expression is seen whereas the AVH disappears. Comparison with the mouse reveals that while gene expression patterns at the onset of gastrulation are well conserved, asymmetry establishment, which relies on extraembryonic tissues such as the hypoblast and trophoblast, has diverged in terms of both gene expression and morphology.     

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.