Regulation of cell polarity, radial intercalation and epiboly in Xenopus: novel roles for integrin and fibronectin

Fibronectin (FN) is reported to be important for early morphogenetic movements in a variety of vertebrate embryos, but the cellular basis for this requirement is unclear. We have used confocal and digital time-lapse microscopy to analyze cell behaviors in Xenopus gastrulae injected with monoclonal antibodies directed against the central cell-binding domain of fibronectin. Among the defects observed is a disruption of fibronectin matrix assembly, resulting in a failure of radial intercalation movements, which are required for blastocoel roof thinning and epiboly. We identified two phases of FN-dependent cellular rearrangements in the blastocoel roof. The first involves maintenance of early roof thinning in the animal cap, and the second is required for the initiation of radial intercalation movements in the marginal zone. A novel explant system was used to establish that radial intercalation in the blastocoel roof requires integrin-dependent contact of deep cells with fibronectin. Deep cell adhesion to fibronectin is sufficient to initiate intercalation behavior in cell layers some distance from the substrate. Expression of a dominant-negative beta1 integrin construct in embryos results in localized depletion of the fibronectin matrix and thickening of the blastocoel roof. Lack of fibronectin fibrils in vivo is correlated with blastocoel roof thickening and a loss of deep cell polarity. The integrin-dependent binding of deep cells to fibronectin is sufficient to drive membrane localization of Dishevelled-GFP, suggesting that a convergence of integrin and Wnt signaling pathways acts to regulate radial intercalation in Xenopus embryos.     

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.     

Mutations in half baked/E-cadherin block cell behaviors that are necessary for teleost epiboly

Epiboly, the spreading of the blastoderm over the large yolk cell, is the first morphogenetic movement of the teleost embryo. Examining this movement as a paradigm of vertebrate morphogenesis, we have focused on the epiboly arrest mutant half baked (hab), which segregates as a recessive lethal, including alleles expressing zygotic-maternal dominant (ZMD) effects. Here we show that hab is a mutation in the zebrafish homolog of the adhesion protein E-cadherin. Whereas exclusively recessive alleles of hab produce truncated proteins, dominant alleles all contain transversions in highly conserved amino acids of the extracellular domains, suggesting these alleles produce dominant-negative effects. Antisense oligonucleotides that create specific splicing defects in the hab mRNA phenocopy the recessive phenotypes and, surprisingly, some of the ZMD phenotypes as well. In situ analyses show that during late epiboly hab is expressed in a radial gradient in the non axial epiblast, from high concentrations in the exterior layer of the epiblast to low concentrations in the interior layer of the epiblast. During epiboly, using an asymmetric variant of radial intercalation, epiblast cells from the interior layer sequentially move into the exterior layer and become restricted to that layer; there they participate in subtle cell shape changes that further expand the blastoderm. In hab mutants, when cells intercalate into the exterior layer, they tend to neither change cell shape nor become restricted, and many of these cells 'de-intercalate' and move back into the interior layer. Cell transplantation showed all these defects to be cell-autonomous. Hence, as for the expansion of the mammalian trophoblast at a similar developmental stage, hab/E-cadherin is necessary for the cell rearrangements that spread the teleost blastoderm over the yolk.     

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.     

Involvement of the guanine nucleotide exchange factor xLARG in the epiboly of <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryo animal pole cells

The development of multicellular organisms is a complicated coordinated process of the movement of groups of embryonic cells, which is controlled by many regulatory systems. At present little is known about the regulation of the earliest manifestations of movement in embryogenesis: epiboly and radial intercalation. The coordinators of these processes may be small GTPases of the Rho family and their activators, guanine nucleotide exchange factors. It has been shown in this work that overexpression of a guanine nucleotide exchange factor xLARG in Xenopus laevis embryos leads to an increase in the amount of the active form of xLARG. In addition, an increase in the expression of xLARG disturbs the process of radial intercalation. The data obtained suggest that xLARG is involved in maintaining the xLARG activation level necessary for the occurrence of epiboly.     

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.     

Involvement of guanine nucleotide exchange factor xLARG in epiboly of cells of the animal pole of Xenopus laevis embryos

The development of multicellular organisms is a complicated coordinated process of the movement of groups of embryonic cells, which is controlled by many regulatory systems. At present little is known about the regulation of the earliest manifestations of the movement in the embryogenesis: epiboly and radial intercalation. The coordinators of these processes may be small GTPases of the Rho family and their activators, the factors of exchange of guanylic nucleotides. It has been shown in this work that the overexpression of the factor of exchange of guanylic nucleotides xLARG in Xenopus laevis embryos leads to an increase in the amount of the active form of xLARG. In addition, an increase in the expression of xLARG disturbs the process of radial intercalation. The data obtained suggest that xLARG is involved in maintaining the xLARG activation level necessary for the occurrence of epiboly.     

αE-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly

αE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of αE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of αE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of αE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of αE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of αE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and αE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and αE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.     

Extracellular matrix assembly and organization during zebrafish gastrulation

Zebrafish gastrulation entails morphogenetic cell movements that shape the body plan and give rise to an embryo with defined anterior–posterior and dorsal–ventral axes. Regulating these cell movements are diverse signaling pathways and proteins including Wnts, Src-family tyrosine kinases, cadherins, and matrix metalloproteinases. While our knowledge of how these proteins impact cell polarity and migration has advanced considerably in the last decade, almost no data exist regarding the organization of extracellular matrix (ECM) during zebrafish gastrulation. Here, we describe for the first time the assembly of a fibronectin (FN) and laminin containing ECM in the early zebrafish embryo. This matrix was first detected at early gastrulation (65% epiboly) in the form of punctae that localize to tissue boundaries separating germ layers from each other and the underlying yolk cell. Fibrillogenesis increased after mid-gastrulation (80% epiboly) coinciding with the period of planar cell polarity pathway-dependent convergence and extension cell movements. We demonstrate that FN fibrils present beneath deep mesodermal cells are aligned in the direction of membrane protrusion formation. Utilizing antisense morpholino oligonucleotides, we further show that knockdown of FN expression causes a convergence and extension defect. Taken together, our data show that similar to amphibian embryos, the formation of ECM in the zebrafish gastrula is a dynamic process that occurs in parallel to at least a portion of the polarized cell behaviors shaping the embryonic body plan. These results provide a framework for uncovering the interrelationship between ECM structure and cellular processes regulating convergence and extension such as directed migration and mediolateral/radial intercalation.     

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.     

Contact inhibition of overlapping: one of the factors involved in deep cell epiboly of Nothobranchius korthausae

Living eggs of the annual fish Nothobranchius korthausae were studied during epiboly either by direct observation or with time-lapse cinematography. Before epiboly the deep cells are tightly packed on top of each other between the enveloping cell layer and the periblast and they are stationary. From stage 17a (half-epiboly) to stage 21, deep cells build up a monolayer of moving and colliding cells. In the course of epiboly all the deep cells acquire the ability to show contact inhibition of overlapping. This was demonstrated directly by recording the outcomes of many cellular collisions. Neither cellular overlapping nor nuclear overlapping was observed. The dispersion of the deep cells during epiboly over the yolk is due both to contact inhibition and to the fact that the deep cells are attached to the undersurface of the spreading enveloping cell layer.     

Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants.

We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured explants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the explants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the explants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anterior-posterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.     

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.     

Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1

The directed movement of cells is critical for numerous developmental and disease processes. A developmentally reiterated form of migration is radial intercalation; the process by which cells move in a direction orthogonal to the plane of the tissue from an inner layer to an outer layer. We use the radial intercalation of cells into the skin of Xenopus laevis embryos as a model to study directed cell migration within an epithelial tissue. We identify a novel function for both the microtubule-binding protein CLAMP and members of the microtubule-regulating Par complex during intercalation. Specifically, we show that Par3 and aPKC promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration. We propose a model in which the Par complex defines the orientation of apical migration during intercalation and in which subcellular localization of CLAMP promotes the establishment of an axis of microtubule stability required for the active migration of cells into the outer epithelium.     

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.     

Dorso-ventral polarity of the zebrafish embryo is distinguishable prior to the onset of gastrulation

We describe a set of observations on developing zebrafish embryos and discuss the main conclusions they allow:(1) the embryonic dorso-ventral polarity axis is morphologically distinguishable prior to the onset of gastrulation; and (2) the involution of deep layer cells starts on the prospective dorsal side of the embryo. An asymmetry can be distinguished in the organization of the blastomeres in the zebrafish blastula at the 30% epiboly stage, in that one sector of the blastoderm is thicker than the other. Dye-labelling experiments with DiI and DiO and histological analysis allow us to conclude that the embryonic shield will form on the thinner side of the blastoderm. Therefore, this side corresponds to the prospective dorsal side of the embryo. Simultaneous injections of dyes on the thinner side of the blastoderm and on the opposite side show that involution of deep layer cells during gastrulation starts at the site at which the embryonic shield will form and extends from here to the prospective ventral regions of the germ ring.     

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     

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.