Cell patterns and cell movements during early development of an annual fish, Nothobranchius neumanni.

During epiboly stages the cells (called deep blastomeres) which will form the definitive embryo disperse over the surface of the yolk sphere, only later aggregating and developing an embryonic axis. Five different statistical tests were used to study the pattern formed by the deep blastomeres during epiboly and early dispersed stages. The two most reliable tests, based on the distance from each deep blastomere within a selected area to its nearest neighboring cell, indicate that the distribution pattern changes from regular during epiboly stages to random during dispersed stages 1 and 2. Careful observation and time-lapse microphotography revealed some aspects of how the cells set up the regular pattern. The deep blastomeres exhibit a variety of cell extensions, with which they often contact one another. When two deep blastomeres make contact during epiboly stages, they soon break the contact and move apart; they overlap one another only rarely. Deep blastomeres are frequently located at, and are even elongated along, borders of the overlying flat cells (enveloping layer cells). These two mechanisms, one similar to contact inhibition of cell movement, the other to contact guidance, may contribute to the rather regular spacing of the deep blastomeres as well as to their arrangement in rows during epiboly stages.     

Surface specializations of Fundulus cells and their relation to cell movements during gastrulation

Cell movements in Fundulus blastoderms during gastrulation were studied utilizing time-lapse cinemicrography and electron microscopy. Time-lapse films reveal that cells of the enveloping layer undulate and sometimes separate briefly but remain together in a cohesive layer. During epiboly, the marginal enveloping layer cells move over the periblast as it expands over the yolk sphere. Movement occurs as a result of ruffled membrane activity of the free borders of the marginal cells. Deep blastomeres become increasingly active during blastula and gastrula stages. Lobopodia project from the blastomeres in blastulae and adhere to other cells in gastrulae, giving the cells traction for movement. Contact specializations are formed by the lateral adjacent plasma membranes of enveloping layer cells. An apical junction is characterized by an intercellular gap of 60–75 A. Below this contact, the plasma membranes are separated by 120 A or more. In mid-gastrulae, cytoplasmic fibrils occur adjacent to some apical junctions, and small desmosomes appear below the apical junction. Septate desmosomes also appear at this time. A junction with an intercellular gap of 60 A occurs between marginal enveloping layer cells and periblast. Contacts between deep blastomeres become numerous in gastrulae and consist of contacts at the crests of surface undulations, short areas of contact in which the plasma membranes are 60 or 120 A apart, and long regions characterized by a 200-A intercellular gap. Lobopodia contact other blastomeres only in gastrulae. These junctions contain a 200-A intercellular space. Some deep blastomeres are in contact with the tips of periblast microvilli. The mechanism of epiboly in Fundulus is discussed and reevaluated in terms of these observations. The enveloping layer is adherent to the margin of the periblast and moves over it as a coherent cellular sheet. Periblast epiboly involves a controlled flow of cytoplasm from the thicker periblast into the thinner yolk cytoplasmic layer with which it is continuous. Deep cells move by adhering to each other, to the inner surface of the enveloping layer, and to the periblast.     

Embryogenesis in mouth-breeding cichlids (Osteichthyes,Teleostei) structure and fate of the enveloping layer

The eggs of African mouth-brooders are of unusual size and shape. Studying their development may help to more clearly understand epiboly, gastrulation, and the relation between enveloping layer (periderm) and epidermis. When epiboly has progressed over just one fifth of the yolk mass, the germ ring and embryonic shield are already well established. Behind the germ ring very few deep cells are present at this early stage of epiboly, except in the embryonic shield. When the blastodisc covers the animal half of the yolk mass, the future body is already well established with notochord, somites and developing neural keel. Apart from these structures, no deep cells can be detected between enveloping layer and yolk surface; not even a germ ring remains behind the advancing edge of the enveloping layer. Epiboly over the greater part of the yolk is achieved only by the enveloping layer and the yolk syncytial layer. As the margin of the enveloping layer begins to reduce its circumference when closing around the vegetal pole, groups of cells in the advancing edge become spindle-shaped, with a single cell in between of each of these groups broadening along the edge. The enveloping layer (called periderm after epiboly) remains intact until after hatching, when, together with the underlying ectoderm, it forms the double-layered skin of the larval fish. Thereafter, cells deriving from the subperipheral ectoderm gradually replace the decaying periderm cells to form the final epidermis. Thus, in the cichlids studied, the enveloping layer alone forms the yolk sac to begin with, and it covers the larval body until some days after hatching.     

CELLULAR BASIS OF EPIBOLY OF THE ENVELOPING LAYER IN THE EMBRYO OF MEDAKA, ORYZIAS LATIPES. I. CELL ARCHITECTURE REVEALED BY SILVER STAINING METHOD

A modification of silver nitrate staining, when applied to embryos of Oryzias latipes , was found to make clear not only the boundary of enveloping layer during epiboly, but also the outline of individual cells of the layer. The linear speed of advance of the enveloping layer was constant at fixed temperatures (20°, 25° or 30°C), except for the start and the end of the epibloy. Observation of the shape and arrangement of cells in the layer stained at successive stages of epiboly revealed that the enveloping layer expands uniformly over the yolk until late gastrula (3/4 epiboly). No cytokinetic figure was observed during epiboly until the blastopore was going to close. Total cell number of the layer remained constant during epiboly. Thus the expansion of the enveloping layer is accomplished without an accompanying increase in the number of constituent cells. In the last phase of epiboly, the surface area occupied by individual cells reduced locally at the region above the embryonic body, which suggests the occurrence in teleost of the convergence of cell sheets commonly observed in amphibian embryos.     

The Epithelial Cell Adhesion Molecule EpCAM Is Required for Epithelial Morphogenesis and Integrity during Zebrafish Epiboly and Skin Development

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers.     

Zebrafish Dynamin is required for maintenance of enveloping layer integrity and the progression of epiboly

Epiboly, the first morphogenetic cell movement that occurs in the zebrafish embryo, is the process by which the blastoderm thins and spreads to engulf the yolk cell. This process requires the concerted actions of the deep cells, the enveloping layer (EVL) and the extra-embryonic yolk syncytial layer (YSL). The EVL is mechanically coupled to the YSL which acts as an epiboly motor, generating the force necessary to draw the blastoderm towards the vegetal pole though actomyosin flow and contraction of the actomyosin ring. However, it has been proposed that the endocytic removal of yolk cell membrane just ahead of the advancing blastoderm may also play a role. To assess the contribution of yolk cell endocytosis in driving epiboly movements, we used a combination of drug- and dominant-negative-based approaches to inhibit Dynamin, a large GTPase with a well-characterized role in vesicle scission. We show that Dynamin-dependent endocytosis in the yolk cell is dispensable for epiboly of the blastoderm. However, global inhibition of Dynamin function revealed that Dynamin plays a fundamental role within the blastoderm during epiboly, where it maintains epithelial integrity and the transmission of tension across the EVL. The epithelial defects were associated with disrupted tight junctions and a striking reduction of cortically localized phosphorylated ezrin/radixin/moesin (P-ERM), key regulators of epithelial integrity in other systems. Furthermore, we show that Dynamin maintains EVL and promotes epiboly progression by antagonizing Rho A activity.     

Flotillins control zebrafish epiboly through their role in cadherin‐mediated cell–cell adhesion

Zebrafish gastrulation and particularly epiboly that involves coordinated movements of several cell layers is a dynamic process for which regulators remain to be identified. We show here that Flotillin 1 and 2, ubiquitous and highly conserved proteins, are required for epiboly. Flotillins knockdown compromised embryo survival, strongly delayed epiboly and impaired deep cell radial intercalation and directed collective migration without affecting enveloping layer cell movement. At the molecular level, we identified that Flotillins are required for the formation of E‐cadherin‐mediated cell–cell junctions. These results provide the first in vivo evidence that Flotillins regulate E‐cadherin‐mediated cell–cell junctions to allow epiboly progression.     

Contractility,differential tension and membrane removal lead zebrafish epiboly biomechanics

Precise tissue remodeling during development is essential for shaping embryos and optimal organ function. Epiboly is an early gastrulation event by which the blastoderm expands around the yolk to engulf it. Three different layers are involved in this process, an epithelial layer (the enveloping layer, EVL), the embryo proper, constituted by the deep cells (DCs), and the yolk cell. Although teleost epiboly has been studied for many years, a clear understanding of its mechanics was still missing. Here we present new information on the cellular, molecular and mechanical elements involved in epiboly that, together with some other recent data and upon comparison with previous biomechanical models, lets conclude that the expansion of the epithelia is passive and driven by active cortical contraction and membrane removal in the adjacent layer, the External Yolk Syncytial Layer (E-YSL). The isotropic actomyosin contraction of the E-YSL cortex generates an anisotropic stress pattern and a directional net movement consequence of the differences in the deformation response of the 2 opposites adjacent domains (EVL and the Yolk Cytoplasmic Layer - YCL). Contractility is accompanied by the local formation of membrane folds and its removal by Rab5ab dependent macropinocytosis. The increase in area of the epithelia during the expansion is achieved by cell-shape changes (flattening) responding to spherical geometrical cues. The counterbalance between the geometry of the embryo and forces dissipation among different elements is therefore essential for epiboly global coordination.     

Coordinated cell-shape changes control epithelial movement in zebrafish and Drosophila

Epithelial morphogenesis depends on coordinated changes in cell shape, a process that is still poorly understood. During zebrafish epiboly and Drosophila dorsal closure, cell-shape changes at the epithelial margin are of critical importance. Here evidence is provided for a conserved mechanism of local actin and myosin 2 recruitment during theses events. It was found that during epiboly of the zebrafish embryo, the movement of the outer epithelium (enveloping layer) over the yolk cell surface involves the constriction of marginal cells. This process depends on the recruitment of actin and myosin 2 within the yolk cytoplasm along the margin of the enveloping layer. Actin and myosin 2 recruitment within the yolk cytoplasm requires the Ste20-like kinase Msn1, an orthologue of Drosophila Misshapen. Similarly, in Drosophila, actin and myosin 2 localization and cell constriction at the margin of the epidermis mediate dorsal closure and are controlled by Misshapen. Thus, this study has characterized a conserved mechanism underlying coordinated cell-shape changes during epithelial morphogenesis.     

Bipartite axiation follows incomplete epiboly in zebrafish embryos treated with chemical teratogens

Medial clefts in the axis of the trunk region are malformations known from many chordates and are mostly referred to as rachischisis anterior. In teleosts, rachischisis was previously ascribed either to secondary rifting of a single uniform axial rudiment, or to the establishment of two (half) axes and body halves physically separate from the very beginning. In order to decide between these conflicting interpretations, we treated zebrafish embryos during blastodisc stages and epiboly with several chemical teratogens causing rachischisis anterior. Treatment with ethanol, Colcemid, hydroxyurea, or cycloheximide was found to delay the proliferation and movements of the deep cells more strongly than the timing of cell differentiation, so that the deep cells embark on organogenesis before having reached their destinations in the uniform germ shield. Treatment with alpha-amanitin, on the other hand, seems primarily to affect the periblast and enveloping layer; the incomplete epiboly observed in these layers appears to restrain deep cell epiboly physically and thus to cause rachischisis. In both instances, the split condition of the embryo's trunk region is clearly due to the ectopic formation of physically separate body halves right from the beginning, a mode we call bipartite axiation. We also describe secondary anomalies specific for individual teratogens, and briefly discuss the possible origins of rachischisis anterior among other chordates including man.     

Time-lapse cinemicrographic analysis of superficial cell behavior during and prior to gastrulation in Xenopus laevis

Time-lapse cinemicrography was used to show what changes in the number, size, shape, arrangement and what movements of apices of superficial cells occur during epiboly, extension, convergence and blastopore formation in the blastula or gastrula of Xenopus laevis. Epiboly of the animal region occurs by apical expansion of superficial cells at a nearly constant rate from the midblastula to the midgastrula stage. Egression of deep cells into the superficial layer does not occur. Extension of the dorsal marginal zone begins in the late blastula stage with the rapid spreading of the apices of cells in this region and this continues until the onset of neurulation when rapid shrinkage begins. Extension and convergence of the dorsal marginal zone occurs by a rearrangement in which individual cells exchange neighbors and by a change in the shape of the cell apices. Regional differences in apical expansion are accompanied by differences in rate of anticlinal division of superficial cells such that cells in all sectors of the animal region and the marginal zone show similar patterns of decrease in apparent apical area. Shrinkage of the apices of bottle cells during blastopore formation is described. From this and other studies, a model of the cellular behavior of epiboly, extension and convergence is constructed and several hypotheses as to how these activities might generate the mechanical forces of the gastrulation movements are presented.     

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     

Rearrangement of enveloping layer cells without disruption of the epithelial permeability barrier as a factor in Fundulus epiboly

Silver nitrate staining of blastoderms of Fundulus heteroclitus gastrulae shows that the number of marginal cells of the enveloping layer (EVL) is reduced from 160 to 25 during epiboly. To determine whether this decrease in the number of marginal cells was due to ingression, cell death, or rearrangement of cells, marginal and submarginal regions of the late gastrula were observed directly by time-lapse cinemicrography. Marginal cells rearrange to occupy submarginal positions by first narrowing their boundary with the external yolk syncytial layer (E-YSL), thus becoming tapered in shape. Then, the narrowed marginal boundary retracts from the E-YSL and moves submarginally in the plane of the epithelium. Concurrently, the marginal cells on both sides come into apposition; no gap or break appears in the circum-apical continuity of the epithelial sheet. Marginal cells leave the margin of the EVL during epiboly at a rate of about six per hour. The rate of movement of the EVL cells with respect to one another is about 0.5 to 1.0 micron/min at 21 degrees C. Submarginal cells rearrange in a similar fashion. Although no protrusive activity was seen at the lateral aspects of rearranging cells, the tapering or narrowing associated with rearrangement was accompanied by formation of microfolds on their apical surfaces, and separating or recently separated submarginal cells form "flowers" of microfolds on their apices adjacent to the site of separation. Morphometric analysis shows that about half the narrowing of the margin of the EVL during epiboly is accounted for by cell rearrangement and the other half by the associated tapering and narrowing. These results suggest that epiboly of the EVL may have an active component as well as a passive one.     

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.     

Origin and organization of the zebrafish fate map

We have analyzed lineages of cells labeled by intracellular injection of tracer dye during early zebrafish development to learn when cells become allocated to particular fates during development, and how the fate map is organized. The earliest lineage restriction was described previously, and segregates the yolk cell from the blastoderm in the midblastula. After one or two more cell divisions, the lineages of epithelial enveloping layer (EVL) cells become restricted to generate exclusively periderm. Following an additional division in the late blastula, deep layer (DEL) cells generate clones that are restricted to single deep embryonic tissues. The appearance of both the EVL and DEL restrictions could be causally linked to blastoderm morphogenesis during epiboly. A fate map emerges as the DEL cell lineages become restricted in the late blastula. It is similar in organization to that of an amphibian embryo. DEL cells located near the animal pole of the early gastrula give rise to ectodermal fates (including the definitive epidermis). Cells located near the blastoderm margin give rise to mesodermal and endodermal fates. Dorsal cells in the gastrula form dorsal and anterior structures in the embryo, and ventral cells in the gastrula form dorsal, ventral and posterior structures. The exact locations of progenitors of single cell types and of local regions of the embryo cannot be mapped at the stages we examined, because of variable cell rearrangements during gastrulation.     

E-cadherin is required for gastrulation cell movements in zebrafish

E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.     

Multicellular computer simulation of morphogenesis: blastocoel roof thinning and matrix assembly in Xenopus laevis

In the blastocoel roof (BCR) of the Xenopus laevis embryo, epibolic movements are driven by the radial intercalation of deep cell layers and the coordinate spreading of the overlying superficial cell layer. Thinning of the lateral margins of the BCR by radial intercalation requires fibronectin (FN), which is produced and assembled into fibrils by the inner deep cell layer of the BCR. A cellular automata (CA) computer model was developed to analyze the spatial and temporal movements of BCR cells during epiboly. Simulation parameters were defined based on published data and independent results detailing initial tissue geometry, cell numbers, cell intercalation rates, and migration rates. Hypotheses regarding differential cell adhesion and FN assembly were also considered in setting system parameters. A 2-dimensional model simulation was developed that predicts BCR thinning time of 4.8 h, which closely approximates the time required for the completion of gastrulation in vivo. Additionally, the model predicts a temporal increase in FN matrix assembly that parallels fibrillogenesis in the embryo. The model is capable of independent predictions of cell rearrangements during epiboly, and here was used to predict successfully the lateral dispersion of a patch of cells implanted in the BCR, and increased assembly of FN matrix following inhibition of radial intercalation by N-cadherin over-expression.