Cell movements during epiboly and gastrulation in zebrafish

Beginning during the late blastula stage in zebrafish, cells located beneath a surface epithelial layer of the blastoderm undergo rearrangements that accompany major changes in shape of the embryo. We describe three distinctive kinds of cell rearrangements. (1) Radial cell intercalations during epiboly mix cells located deeply in the blastoderm among more superficial ones. These rearrangements thoroughly stir the positions of deep cells, as the blastoderm thins and spreads across the yolk cell. (2) Involution at or near the blastoderm margin occurs during gastrulation. This movement folds the blastoderm into two cellular layers, the epiblast and hypoblast, within a ring (the germ ring) around its entire circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; the movement shears the positions of cells that were neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution. The epiblast is equivalent to embryonic ectoderm. (3) Mediolateral cell intercalations in both the epiblast and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this rearrangement, cells that were initially neighboring one another become dispersed along the anterior-posterior axis of the embryo. Epiboly, involution and convergent extension in zebrafish involve the same kinds of cellular rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.     

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.     

Slb/Wnt11 controls hypoblast cell migration and morphogenesis at the onset of zebrafish gastrulation

During vertebrate gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. In zebrafish, silberblick (slb)/wnt11 regulates normal gastrulation movements by activating a signalling pathway similar to the Frizzled-signalling pathway, which establishes epithelial planar cell polarity (PCP) in Drosophila. However, the cellular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still unclear. Using high-resolution two-photon confocal imaging followed by computer-assisted reconstruction and motion analysis, we have analysed the movement and morphology of individual cells in three dimensions during the course of gastrulation. We show that in slb-mutant embryos, hypoblast cells within the forming germ ring have slower, less directed migratory movements at the onset of gastrulation. These aberrant cell movements are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. We conclude that slb/wnt11-mediated orientation of cellular processes plays a role in facilitating and stabilising movements of hypoblast cells in the germ ring, thereby pointing at a novel function of the slb/wnt11 signalling pathway for the regulation of migratory cell movements at early stages of gastrulation.     

Transfer of extracellular matrix components between germ layers in chimaeric chicken-quail blastoderms

Summary A chemical basis for the transmission of signals during gastrulation has been investigated by using chimaeric embryos resulting from the combination of ³H-glucosamine-labelled and unlabelled hypoblast with epiblast taken from chicken and quail embryos at stage 3 of Vakaet (1970). The ability to distinguish chicken from quail cells on the basis of their different nuclear distribution of heterochromatin after Feulgen staining made it possible to determine the origin of the cells in the chimaerae. Tritiated quail hypoblast (after incubation of the embryo in the presence of ³H-glucosamine) was transplanted onto unlabelled chicken blastoderm deprived of its hypoblast. After culture of the chimaera for 5 h, the autoradiographic pattern shows silver grains not only over the graft, but also at the ventral surface of the epiblast of the host. Transfer of label may occur to mesoblast cells, but not between chicken and quail hypoblast cells. Chase experiments exclude the possibility that unprocessed, tritiated glucosamine is transferred. Chemical fixation of the host before transplantation of a labelled quail hypoblast also allows visualization of a transfer of macromolecules from hypoblast to the basement membrane of the epiblast, suggesting that an intervention of the epiblast cells in this process is not necessary. The morphology of the chimaeric embryos, as studied by scanning electron microscopy, suggests a direct deposition of these macromolecules by filopodia of the dorsal surface of the hypoblast. The possibility of diffusion of free macromolecules has been considered and can reasonably be discarded on the basis of several observations. The reverse experiment, in which unlabelled quail hypoblast and possibly some mesoblast have been combined with a tritiated host deprived of its hypoblast, also shows the transfer of label from the host to the cellular surface of the graft. A two-way exchange of glucosamine-containing molecules thus occurs in the blastoderm. It is hypothesized that: (1) low molecular weight compounds, macromolecular material, and/or catabolic products, are exchanged between the different germ layers during gastrulation; (2) the components of the extracellular matrix turn over and are continuously changing; (3) this transfer is a possible mechanism of transmission for developmental or inductive signals during embryonic development. The present results also demonstrate the participation of underlying tissue in the biosynthesis of basement membrane components of an epithelium.     

Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII–XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vilelline membranes at stages VII–IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX–XIV with and without an STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII–IX. When individual stage IX–XIV embryos were dispersed and cultured without a feeder layer, 25–45 PGCs/embryo were detected only with stage X–XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX–XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI–XIV. © 1996 Wiley-Liss, Inc.     

Dye-coupling and the formation and fate of the hypoblast in the teleost fish embryo, Barbus conchonius.

The present report describes Lucifer Yellow (LY) transfer between the syncytial layer of the yolk cell (YSL) and blastodermal cells during epiboly in the teleost fish Barbus conchonius. The fate of a group of labeled cells is described until germ layer formation. At the onset of epiboly, LY seems to be transferred from the YSL to all blastodermal cells. Between 10% and 40% epiboly, dye-coupling appears to be restricted to the marginal region. Within 60 min individually labeled cells are distributed among unlabeled cells within the blastoderm. Between 40% and 60% epiboly, we observed a ring-shaped group of labeled cells, which probably have involuted during early gastrulation. Consequently, this cell group may correlate with the leading edge of the hypoblast layer within the germ ring. At 60% epiboly and later, the blastodermal cells are dye-uncoupled from the YSL. A gradual translocation of the ring-shaped hypoblast towards a dorsally located bar-like structure is observed between 50% and 100% epiboly. At 100% epiboly, fluorescent cells were located in contact with the YSL within the embryo proper, with the brightest fluorescence in the future head region. The translocation is due to dorsalwards convergent cell movements during the gastrulation process. The appearance of the hypoblast as a dye-coupled cell layer may correlate with some restriction in cell fate since the hypoblast differs in fate from the epiblast.     

Polyingression, an Important Morphogenetic Movement in Chick Gastrulation

Three lines of evidence are presented to support the conceptthat polyingression represents a major morphogenetic movementin the genesis of the primary hypoblast, definitive endodermand the mesoderm layers in chick gastrulation. Colchicine treatedembryos produce a primary hypoblast layer thereby indicatingthat cell proliferation in the posterior marginal zone and elsewherein the early embryo does not make a necessary contribution tohypoblast formation. Scanning electron microscopy provides morphologiccomplementary evidence that polyingression from the epiblastis a principal source of cells for the hypoblast. Finally, themorphology of cells with acetylcholinesterase activity suggeststhat this enzyme is related to release of cells from the epiblastso that ingression can occur.     

Studies on the migration of primordial germ cells in early chick embryos: cultivation of hypoblast explants from the germinal crescent area

The hypoblast from the germinal crescent area of stage 4 to 5 chick embryos, when grown on a glass surface, spreads out as a thin and continuous cellular sheet. Primordial germ cells (PCCs) are found to migrate individually over this hypoblast monolayer (as they do in vivo) and, perhaps even more striking, their morphological features during migration are remarkably similar to those observed in vivo. Inasmuch as this in vitro system permits direct observations of PGCs migrating on their natural, rather than artificial, substratum undoubtedly it will prove to be an effective means of studying the mode of PGC migration in early chick embryos.     

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.     

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 hypoblast of the chick embryo positions the primitive streak by antagonizing nodal signaling

The hypoblast (equivalent to the mouse anterior visceral endoderm) of the chick embryo plays a role in regulating embryonic polarity. Surprisingly, hypoblast removal causes multiple embryonic axes to form, suggesting that it emits an inhibitor of axis formation. We show that Cerberus (a multifunctional antagonist of Nodal, Wnt, and BMP signaling) is produced by the hypoblast and inhibits primitive streak formation. This activity is mimicked by Cerberus-Short (CerS), which only inhibits Nodal. Nodal misexpression can initiate an ectopic primitive streak, but only when the hypoblast is removed. We propose that, during normal development, the primitive streak forms only when the hypoblast is displaced away from the posterior margin by the endoblast, which lacks Cerberus.     

Analysis of germ line development in the chick embryo using an anti-mouse EC cell antibody

We have found that EMA-1, a monoclonal antibody originally raised against mouse embryonal carcinoma (Nulli SCC1) cells (Hahnel & Eddy, 1982), also labels chick primordial germ cells (PGCs). We have used this antibody in immunohistological studies to follow the development of PGCs in the chick embryo from the time of their initial appearance beneath the epiblast, through their migratory phase and subsequent colonization of the germinal epithelium. During hypoblast formation, individual EMA-1-labelled cells appeared to separate from the basal surface of the epiblast and enter the blastocoel, coincident with the appearance of morphologically identifiable PGCs in this same area. EMA-1 continued to label germ cells until the initiation of gametogenesis in each sex; specifically, labelling was absent by 7-8 days of incubation in females and started to decrease at 11 days of incubation in males. There was a recurrence of the epitope on oogonia at 15 days of incubation, but not on spermatogonia during the remainder of development through hatching. These observations are consistent with an epiblast origin for the avian germ line, and are strikingly similar to those reported for the early mouse embryo using the same antibody (Hahnel & Eddy, 1986).     

Immunohistochemical analysis of the segregation process of the quail germ cell lineage

An antiserum against quail 7 day gonadal germ cells was found to react specifically with gonadal germ cells of both sexes. Transverse sections from a range of early quail developmental stages were submitted to the antibody PAP reaction. Blastodiscs from the earliest uterine stages (II to X E.G. & K) reacted very strongly, while the overall reaction gradually decreased in older blastoderms. At stage XIII both epiblast and hypoblast were weakly stained, but some large, PGC-like cells stained intensively. During gastrulation (PS formation) the reaction of the epiblast disappears quicker than that of the hypoblast. The newly formed mesoderm and entoderm do not react at all and the reaction gradually becomes limited mainly to the PGCs and somewhat to the primary hypoblast which is moving into the germinal crescent. The widely spread reaction at the early stages is thus gradually being restricted to the PGCs.     

Characterization of stage-specific embryonic antigen-1 (SSEA-1) expression during early development of the turkey embryo.

SSEA-1 is a carbohydrate epitope associated with cell adhesion, migration and differentiation. In the present study, SSEA-1 expression was characterized during turkey embryogenesis with an emphasis on its role in primordial germ cell development. During hypoblast formation, SSEA-1 positive cells were identified in the blastocoel and hypoblast and later in the germinal crescent. Based on location and morphology, these cells were identified, as PGCs. Germ cells circulating through embryonic blood vessels were also SSEA-1 positive. During the active phase of migration, PGCs in the dorsal mesentery and gonad could no longer be identified using the SSEA-1 antibody. The presence of PGCs at corresponding stages was verified using periodic acid Schiff stain. Pretreatment of PGCs with trypsin, alpha-galactosidase and neuraminidase did not restore immunoreactivity to SSEA-1. In general, expression was not limited to the germ cell lineage. SSEA-1 was also detected on the ectoderm, yolk sac endoderm, gut and mesonephric tubules. During neural tube closure, SSEA-1 was expressed by the neural epithelium of the fusing neural folds. Later SSEA-1 was detected in regions of the developing spinal cord. Enzyme pretreatment unmasked the epitope on some neural crest cells and cells in the sympathetic ganglion. The temporal and spatial distribution of SSEA-1 in the turkey embryo suggests a role in early germ cell and neural cell development. The absence of SSEA-1 on turkey gonadal germ cells was different from that observed for the chick. Therefore, while features of avian germ cell development appear to be conserved, expression of SSEA-1 can vary with the species.     

Turkey embryo staging from cleavage through hypoblast formation

The progressive development of the turkey embryo from first cleavage through hypoblast formation was examined in order to determine the applicability of a chicken embryo staging procedure. It was concluded that the temporal and spatial events associated with the development of the early turkey embryo are sufficiently different from those of the chicken embryo to warrant a separate staging procedure. Cleavage is asynchronous and often results in asymmetrical segmentation. Unlike the chicken embryo, which at oviposition has already formed the area pellucida and area opaca and is classified as a Stage X embryo, the turkey embryo at oviposition is only at the beginning of area pellucida formation and is classified as a Stage VII embryo. After about 3 hr of incubation and prior to completion of the area pellucida, hypoblast formation begins at the posterior end, thereby establishing the bilaterally symmetrical pattern of the embryo. When viewed from the dorsal surface, an opaque region is observed at the center of the area pellucida. This opacity is unique to the turkey embryo and is referred to as the area alba. When viewed from the ventral surface, the area alba appears to be composed of large whitish cells. To conclude, the rate of turkey embryo development through the completion of hypoblast formation, which consists of 11 stages, lags behind that of the chicken. Furthermore, the organization as well as origin of the area pellucida and hypoblast observed in the turkey embryo differ from that of the chicken embryo. © 1993 Wiley-Liss, Inc.     

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.     

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.     

Conserved patterns of cell movements during vertebrate gastrulation

Vertebrate embryogenesis entails an exquisitely coordinated combination of cell proliferation, fate specification and movement. After induction of the germ layers, the blastula is transformed by gastrulation movements into a multilayered embryo with head, trunk and tail rudiments. Gastrulation is heralded by formation of a blastopore, an opening in the blastula. The axial side of the blastopore is marked by the organizer, a signaling center that patterns the germ layers and regulates gastrulation movements. During internalization, endoderm and mesoderm cells move via the blastopore beneath the ectoderm. Epiboly movements expand and thin the nascent germ layers. Convergence movements narrow the germ layers from lateral to medial while extension movements elongate them from head to tail. Despite different morphology, parallels emerge with respect to the cellular and genetic mechanisms of gastrulation in different vertebrate groups. Patterns of gastrulation cell movements relative to the blastopore and the organizer are similar from fish to mammals, and conserved molecular pathways mediate gastrulation movements.