Dorsoventral differences in cell-cell interactions modulate the motile behaviour of cells from the Xenopus gastrula.

When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.

Bottle cell formation in relation to mesodermal patterning in the Xenopus embryo.

The appearance of bottle cells at the dorsal vegetal/marginal boundary of Xenopus embryos marks the onset of blastopore formation. The conditions leading to this epithelial activity were investigated by inducing bottle cells ectopically in the animal region with VegT or different members of the transforming growth factor (TGF)-beta family. Morphological studies on the ectopic bottle cells indicate their close similarity to the endogenous bottle cells at the dorsal blastopore lip. The subepithelial cells of the induced animal region express mesodermal genes in a pattern reminiscent to that observed on the dorsal lip. Relating this expression pattern to the position of the ectopic bottle cells leads to the conclusion that bottle cells form in regions of high TGF-beta signalling. The specific inhibitory effects of cerberus on ectopically induced bottle cells revealed that nodal related growth factors are the intrinsic signals that elicit bottle cell formation in the normal embryo. In addition, fibroblast growth factor signalling is an essential precondition for this epithelial response as it is for mesoderm formation. We conclude that bottle cell formation in the epithelial layer of the gastrula is closely linked to mesodermal patterning in the subepithelial tissues.

Assembly of tight junctions during early vertebrate development.

Tight junction formation during development is critical for embryonic patterning and organization. We consider mechanisms of junction biogenesis in cleaving mouse and Xenopus eggs. Junction assembly follows the establishment of cell polarity at 8-cell (mouse) or 2-cell (Xenopus) stages, characterized by sequential membrane delivery of constituents, coordinated by embryonic (mouse) or maternal (Xenopus) expression programmes. Cadherin adhesion is permissive for tight junction construction only in the mouse. Occludin post-translational modification and membrane delivery, mediated by delayed ZO-1 alpha(+)isoform expression in the mouse, provides a mechanism for completion of tight junction biogenesis and sealing, regulating the timing of blastocoel cavitation.

Tight junction biogenesis in the early Xenopus embryo.

Tight junctions (TJs) perform a critical role in the transport functions and morphogenetic activity of the primary epithelium formed during Xenopus cleavage. Biogenesis of these junctions was studied by immunolocalization of TJ-associated proteins (cingulin, ZO-1 and occludin) and by an in vivo biotin diffusion assay. Using fertilized eggs synchronized during the first division cycle, we found that membrane assembly of the TJ initiated at the animal pole towards the end of zygote cytokinesis and involved sequential incorporation of components in the order cingulin, ZO-1 and occludin. The three constituents appeared to be recruited from maternal stores and were targeted to the nascent TJ site by different pathways. TJ protein assembly was focused precisely to the border between the oolemma-derived apical membrane and newly-inserted basolateral membrane generated during cytokinesis and culminated in the formation of functional TJs in the two-cell embryo, which maintained a diffusion barrier. New membrane formation and the generation of cell surface polarity therefore precede initiation of TJ formation. Moreover, assembly of TJ marker protein precisely at the apical-basolateral membrane boundary was preserved in the complete absence of intercellular contacts and adhesion. Thus, the mechanism of TJ biogenesis in the Xenopus early embryo relies on intrinsic cues of a cell autonomous mechanism. These data reveal a distinction between Xenopus and mammalian early embryos in the origin and mechanisms of epithelial cell polarization and TJ formation during cleavage of the egg.

Immunocytochemical studies of the interactions of cadherins and catenins in the early Xenopus embryo.

Linkage of cadherins to the cytoskeleton is crucial for their adhesive function. Since alpha- and beta-catenin play a key role in this linkage, these proteins are possible targets for processes that control cell-cell adhesion. To achieve a better understanding of the regulation of cell-cell adhesion in embryonic morphogenesis, we used immunohistology to investigate how in Xenopus blastomeres catenins respond to disturbances in the expression of maternal cadherins. Overexpression of myc-tagged maternal cadherin leads to a proportionate increase of the level of beta-catenin. The two proteins colocalize in the endoplasmic reticulum, in cytoplasmic vesicles, and along the cell membrane, indicating that the beta-catenin binds to overexpressed cadherin early in its passage to the plasma membrane. Expression of cadherin is essential for the stable presence of beta-catenin, as depletion from maternal cadherin mRNA leads to a complete loss of beta-catenin from the blastomeres. alpha-Catenin behaves differently. Overexpression of cadherin leaves the amount and localization of alpha-catenin largely unaffected, and additional cadherin inserts itself into the membrane without a proportionate rise in the level of membrane-bound alpha-catenin. However, cadherin mRNA depletion leads to a redistribution of alpha-catenin from the membrane to the cytoplasm. Thus, cadherin is required to localize alpha-catenin to the membrane, but the amount of alpha-catenin along the membrane seems to be restricted to a certain level which cannot be exceeded. The relevance of these observations for the regulation of cadherin-mediated cell adhesion in the Xenopus embryo is discussed. Additionally, we demonstrate that plakoglobin, like beta-catenin an armadillo repeat protein, shows neither accumulation after overexpression nor colocalization with the overexpressed cadherin.

Cloning of the Xenopus integrin alpha(v) subunit and analysis of its distribution during early development.

One striking feature of the integrin alpha(v) subunit is its ability to associate with at least five different beta subunits (beta1, beta3, beta5, beta6 and beta8) to form functional receptors. These receptors are involved in diverse biological processes, such as differentiation, cell adhesion and migration. Here we report the cloning of the Xenopus homolog of the integrin alpha(v) subunit. Integrin alpha(v) mRNA and protein are maternally supplied and present throughout development. During gastrulation and neurulation alpha(v) protein appears on cell membranes of all three germ layers. In tailbud stage embryos great amounts of the alpha(v) protein can be observed in the inner layer of the ectoderm and in the endothelial cells lining the pharynx and gut.

Pre-MBT patterning of early gene regulation in Xenopus: the role of the cortical rotation and mesoderm induction.

Patterning events that occur before the mid-blastula transition (MBT) and that organize the spatial pattern of gene expression in the animal hemisphere have been analyzed in Xenopus embryos. We present evidence that genes that play a role in dorsoventral specification display different modes of activation. Using early blastomere explants (16-128-cell stage) cultured until gastrula stages, we demonstrate by RT-PCR analysis that the expression of goosecoid (gsc), wnt-8 and brachyury (bra) is dependent on mesoderm induction. In contrast, nodal-related 3 (nr3) and siamois (sia) are expressed in a manner that is independent of mesoderm induction, however their spatially correct activation does require cortical rotation. The pattern of sia and nr3 expression reveals that the animal half of the 16-cell embryo is already distinctly polarized along the dorsoventral axis as a result of rearrangement of the egg structure during cortical rotation. Similar to the antagonistic activity between the ventral and the dorsal mesoderm, the ventral animal blastomeres can attenuate the expression of nr3 and sia in dorsal animal blastomeres. Our data suggest that no Nieuwkoop center activity at the blastula stage is required for the activation of nr3 and sia in vivo.

A paired oocyte adhesion assay reveals the homophilic binding properties of the Xenopus maternal cadherins, XB/U- and EP-cadherin.

The homophilic nature of cadherin-mediated cell-cell adhesion provides an organism with the opportunity of altering the adhesive capabilities of its cells by selectively modulating the expression of different cadherin types. Differential cadherin expression is of major importance in regulating the cell rearrangements involved in the processes which shape tissues and organs during embryogenesis. The pregastrula embryo of Xenopus laevis expresses two maternally supplied cadherins: XB/U-cadherin and EP-cadherin. Since these two proteins are almost 92% identical at the amino acid level, it was unclear whether heterophilic interactions between them were possible. Different functional roles can only be ascribed to the two cadherins if the possibility of heterophilic binding between them can be excluded. We describe a simple and straightforward assay which can be used to assess interactions between adhesion molecules. A combination of antisense oligonucleotide and enzyme treatments eliminates endogenous cadherins in Xenopus oocytes and subsequent injection of a specific mRNA yields oocytes carrying only one or the other cadherin. After removal of the vitelline membranes, two oocytes expressing the appropriate cadherins will adhere to one another when they are placed in close contact. By scoring for adhesion in homotypic and heterotypic pairings, we demonstrate that XB/U-cadherin and EP-cadherin do not interact with one another.

Fine structural immunocytochemistry of catenins in amphibian and mammalian muscle.

In adherens junctions, alpha- and beta-catenin serve to link cadherins to the cortical cytoskeleton. Immunofluorescence and immuno-electron microscopy have been applied to elucidate the nature and localization of cadherin/catenin-mediated cell-cell adhesion sites in adult muscle tissues. Antibodies against alpha- and beta-catenin have been used as indicators of such sites in amphibian and mammalian muscle. Intercalated discs are prominent cell-cell adhesion sites in heart muscle. They contain large amounts of the two catenins, the distributions of which are disclosed. In addition and in contrast to their counterparts in guinea pig, cardiomyocytes of Xenopus are also interconnected laterally by catenin-containing cell-cell junctions. These are doublet structures that approach the intercellular contacts of two adjacent cells from both sides and occur in register with the Z-discs. We interpret these structures as catenin/cadherin-based costameres. Ultrastructural details of these structures are described. In addition to its presence in cell-cell adhesion sites, we have found beta-catenin, but not alpha-catenin, in the Z-discs of heart and skeletal striated muscles. In smooth muscle, actin filaments insert into the dense bodies, which are therefore regarded as functional equivalents of the Z-discs. Accordingly, beta-catenin is also found in these structures, again in the absence of alpha-catenin. These non-peripheral intracellular localizations in the Z-discs of striated muscles and the dense bodies of smooth muscle indicate a hitherto unknown function of beta-catenin in these specialized cells.

Beta-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos.

The question of how dorsal-ventral polarity is established in vertebrates is central to our understanding of their early development. Several lines of evidence suggest that wnt-signaling is involved in the induction of dorsal-specific gene expression in the Spemann Organizer of amphibians. Here, we show that beta-catenin, acting as a component of the wnt-pathway, transiently accumulates in nuclei on the dorsal side of Xenopus and zebrafish blastulae. The spatially restricted nuclear translocation of beta-catenin precedes the expression of dorsal-specific genes. In experimentally ventralized frog embryos the dorsal ventral pattern of beta-catenin nuclear staining is abolished; in contrast, embryos hyperdorsalized by Li-ions or by injection of Xwnt8 mRNA exhibit an enhanced nuclear accumulation of beta-catenin. The results show that translocation of beta-catenin into nuclei in the wake of wnt-signaling is an early step in the establishment of the dorsal-ventral axis in frog and fish embryos.

Epithelial cell polarity in early Xenopus development.

The Xenopus blastula consists of two morphologically distinct cell types. Polarized epithelial cells build up the embryonic surface and fence off an inner non-polarized cell population. We examined the establishment of this early functional cell diversification in the embryo by single cell analysis, in vitro cell culture, and transplantation experiments. Single blastomeres from a 64-cell embryo (1/64 cells) exhibit several features of polarized cells. The plasma membrane of 1/64 cells consists of an apical domain, which is inherited from the original egg membrane, and a basolateral domain derived from newly formed membrane during cleavage. These are inherent, cell-autonomous properties of the blastomeres, as they form and are maintained in blastomeres raised in the absence of any cell interactions in calcium free medium. Upon in vitro culture a single 1/64 cell gives rise to an aggregate of two different cell types. Cells carrying a part of the former egg membrane domain differentiate into polarized epithelial cells, whereas cells lacking this membrane domain are not polarized. These results demonstrate that the inclusion of the egg membrane, rather than external signals related to the position of a cell in the intact embryo, is required for the apical/basolateral differentiation of the surface epithelium. This view is supported by cell transplantation studies. A single 1/64 cell was implanted into the blastocoel of a stage 8 blastula embryo. The progeny of the implanted cell proliferate within the host embryo and split into two morphologically distinct populations with different cell behaviours. Cells incorporating a part of the egg membrane form coherent patches of polarized epithelial cell sheets in the interior of the host embryo. In contrast, cells lacking egg membrane do not exhibit any characteristics of polarized cells and eventually spread into different regions of the host embryo. Our results show that the egg membrane and/or components of the submembrane cortex play a determinative role in the formation of the blastula epithelium.

Integrin alpha 5 during early development of Xenopus laevis.

The full length sequence of the Xenopus integrin alpha 5 subunit is reported. Analysis of cloned cDNA fragments reveals that alternative polyadenylation of alpha 5 mRNA occurs in the embryo. Furthermore, a variant form of the alpha 5 mRNA is expressed which encodes an integrin alpha 5 subunit with a truncated cytoplasmic domain. Integrin alpha 5 mRNA and protein are expressed in oocytes, eggs and throughout development. Spatial expression of alpha 5 mRNAs is first detected by whole mount in situ hybridization in presumptive neural crest cells and in the somitic mesoderm from the midgastrula stage onwards. In contrast, the alpha 5 protein is present on newly formed plasma membranes beginning at first cleavage. During neurulation, the integrin alpha 5 subunit disappears from the outer layer of the ectoderm, the notochord and the neural tube and accumulates in the sensorial layer of the ectoderm, the somites and the neural crest cells. These results provide evidence for the position specific regulation of alpha subunit expression in early vertebrate embryos.

A beta 1-integrin associated alpha-chain is differentially expressed during Xenopus embryogenesis.

The antigen of mAb 2F10 was identified as a Xenopus beta 1-integrin associated alpha-chain by the criteria (1) that it coprecipitates with anti beta 1-antibody, (2) that it changes molecular mass upon reduction in a way that is characteristic for integrin alpha-chains and (3) that it is present on cell membranes. This alpha-chain, termed alpha 2F10, is found in small amounts in the pregastrula stages of Xenopus development and accumulates thereafter in the embryo, alpha 2F10 can be detected by immunofluorescence first at stage 17 of embryogenesis on the cell membranes of the sensorial layer of the ectoderm, the notochord and the endoderm. This characteristic pattern of distribution is maintained throughout the following embryonic stages. Timed explanation experiments indicate that all cells of the pregastrula have the potency to express alpha 2F10. This potency becomes successively restricted during gastrulation to yield the ultimate pattern of expression.

Xenopus cadherins: the maternal pool comprises distinguishable members of the family.

Three maternal cadherins have been reported to occur in the pregastrula Xenopus embryo. EP- and XB-cadherin are distinguished by their distinct cDNA sequences. U-cadherin has been characterized by its reaction with a specific monoclonal antibody (mAb 6D5). Thus far, lack of specific probes that discriminate between these molecules has prevented their identification as distinct cadherins. We now demonstrate by means of RNase protection assays that both EP- and XB-cadherin mRNAs are present in oocytes and mature eggs. By use of the Xenopus cadherin proteins expressed in mammalian cell lines, we find that mAb 6D5 crossreacts with XB-cadherin, but not with EP-cadherin. The major fraction of the maternal cadherins does not contain the 6D5 epitope and probably represents EP-cadherin. A minor fraction carries the 6D5 epitope indicative for the XB- and U-type of cadherins. We have termed this fraction XB/U-cadherin. The function of maternal cadherins was examined by in vitro cell adhesion assays. A newly developed antiserum with a broad specificity for various Xenopus cadherins efficiently blocks all calcium dependent cell adhesion in the early embryo. We conclude that the maternal cadherins play a central role in interblastomere adhesion in the early embryo and comprise at least two discrete cadherin forms, EP- and XB/U-cadherin.

Planar polarity in the ciliated epidermis of Xenopus embryos.

The coordinated orientation of ciliary beat in the larval epidermis of amphibians, evident in an organized streamline pattern, suggests a planar polarity of the epithelium, i.e., a polarity within the plane of the cell sheet. It has been proposed that the direction of ciliary beat is determined at mid gastrula by a gradient of a diffusible factor produced by the mesoderm. To analyze whether ectoderm in isolation can establish a uniform direction of ciliary beat, and at what stage its polarity is specified in the embryo, ectoderm of Xenopus laevis embryos of different stages was cultured in vitro on substrates. On concanavalin A, ectoderm isolated at early gastrula stages, i.e., prior to any contact with mesoderm, can autonomously coordinate the direction of ciliary beat, at least in small regions. A uniform planar polarity is expressed by ectoderm explanted from the early mid gastrula onward. On fibronectin, which promotes migration, the direction of movement correlates well with the direction of ciliary beat, and directional migration can even override the inherent polarity specified prior to explantation. Embryos which lack dorsal mesoderm nevertheless develop a highly organized streamline pattern, excluding a strict requirement for dorsal mesoderm for the determination of planar polarity. However, in spite of the early specification of planar polarity found for isolated tissue, rotated ectodermal transplants in situ can readjust their polarity in accordance with that of the host.

Maturation induced internalization of beta 1-integrin by Xenopus oocytes and formation of the maternal integrin pool.

A pool of beta 1-integrin, ready to be inserted into the cleavage membranes, is present in the cytoplasm of the Xenopus egg, while its plasma membrane is devoid of this membrane protein (Gawantka et al., 1992). The underlying mechanisms that lead to this specific pattern of beta 1-integrin distribution in the egg have been investigated. beta 1-Integrin is present on the oocyte membrane throughout oogenesis. During maturation the oocyte membrane is cleared of beta 1-integrin via internalization of the protein by the oocyte. Synthesis of beta 1-integrin precursor is stimulated moderately in the maturing oocyte. At the same time processing of the precursor into the mature form of beta 1-integrin and its complexing with a putative alpha-chain is greatly accelerated. This way a maternal integrin pool accumulates in the mature oocyte. It is localized in conspicuous yolk free patches which contain large amounts of endoplasmic reticulum, Golgi complexes and smooth vesicles. We suggest that membrane vesicles harbouring the beta 1-integrin are generated in these cytoplasmic regions and that this store of vesicles provides the material source for the rapid membrane formation during cleavage.

Catenins in Xenopus embryogenesis and their relation to the cadherin-mediated cell-cell adhesion system.

In the course of an analysis of cell-cell adhesion in the Xenopus embryo, antibodies directed against alpha- and beta-catenin were applied to investigate their relation to the cadherins occurring early in this system. The results demonstrate that alpha- and beta-catenin are provided maternally and increase in amount throughout embryogenesis. Immunoprecipitations indicate that both of the catenins are complexed to U-cadherin in the early phase of embryogenesis and to E-cadherin, when it appears during gastrulation. An excess of alpha-catenin occurs in free form in the early embryo, whereas all of the beta-catenin seems to be complexed to cadherin. Synthesis of the two components throughout early embryogenesis and their binding to newly synthesized cadherins were demonstrated by metabolic labelling. The spatial distribution of alpha-catenin was analysed by immunohistology. During cleavage alpha-catenin is deposited evenly along the plasma membranes within the embryo, while the cell peripheries at the surface of the embryo remain devoid of alpha-catenin. At later stages, the pattern of alpha-catenin distribution becomes more complex. Quantitative differences in the intensity of staining along the plasma membranes in the different regions of the embryo can be distinguished. Particularly the appearance of E-cadherin in the gastrula ectoderm is accompanied by conspicuous depositions of alpha-catenin along the respective plasma membranes in this layer. All cells in the later embryo, apart from the neural crest cells, carry alpha-catenin on their plasma membranes indicating the universal character of cadherin-mediated cell-cell adhesion in the Xenopus embryo.

Beta 1-integrin is a maternal protein that is inserted into all newly formed plasma membranes during early Xenopus embryogenesis.

A monoclonal antibody (mAb 8C8) that recognizes the Xenopus beta 1-integrin chain was used to study the appearance, synthesis and distribution of this integrin subunit during the early development of Xenopus. Both the precursor and the mature form of beta 1-integrin are provided maternally. They do not increase significantly in amount until early gastrula when the level of both forms begins to rise gradually. Synthesis of beta 1-integrin from maternal mRNA is observed throughout the pregastrula phase, though it seems to add only little to the total beta 1-integrin of the embryo. Until late blastula only small amounts of precursor are processed into the mature form. Starting with the formation of the first cleavage membrane, mature beta 1-integrin is inserted into the newly formed plasma membranes of all cells. The membrane domains forming the outer surface of the embryo remain devoid of the antigen. The data suggest an as yet unknown function of beta 1-integrin during the cleavage phase.

U-cadherin in Xenopus oogenesis and oocyte maturation.

U-cadherin is a member of the cadherin family in Xenopus that participates in interblastomere adhesion in the early embryo from the first cleavage onwards. Though a maternal pool of U-cadherin is available in the egg, it is not present on the egg membrane (Angres et al., 1991. Development 111, 829-844). To assess the origin of this unexpected distribution in the egg, the accumulation and localization of the cadherin during oogenesis and oocyte maturation were investigated. We report here that U-cadherin is present in Xenopus oocytes throughout oogenesis. It is localized at the oocyte-follicle cell contacts suggesting that it functions in the adhesion of the two cell types. When oocytes mature and the contacts to the follicle cells break, U-cadherin disappears from the oocyte surface. Evidence for a translocation of U-cadherin from the membrane to the inside of the oocyte was obtained when the fate of membrane-bound U-cadherin, which was labelled on the surface of oocytes prior to maturation, was followed through maturation. The total U-cadherin content of the oocyte increases during maturation. Metabolic labelling experiments indicate that at maturation the translation of U-cadherin is elevated well above the level that one would expect from the general increase in protein synthesis is presumably the main source of the maternal pool of U-cadherin in the egg.

Differential expression of two cadherins in Xenopus laevis.

Using a cadherin fraction from Xenopus tissue culture cells as an immunogen, two monoclonal antibodies were obtained that allowed the characterization of two distinct cadherins in the Xenopus embryo. The two cadherins differ in molecular weight, in their time of appearance during development and in their spatial pattern of expression. One of the antigens was identified as E-cadherin. It appears in the embryonic ectoderm during gastrulation when epidermal differentiation commences and it disappears from the neural plate area upon neural induction. The second antigen could not be allocated to any of the known cadherin subtypes and was termed U-cadherin. It is present in the egg and becomes deposited in newly formed inner cell membranes during cleavage, the outer apical membranes of the embryo remaining devoid of the cadherin throughout development. U-cadherin is found on membranes of all cells up to the late neurula stages. A conspicuous polarized expression of the antigen on the membranes of individual inner cells suggests its participation in the segregation of cell layers and organ anlagen. These findings are discussed in the context of current hypotheses on the role of cadherins in establishing the spatial structure of the embryo.