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Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/beta-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/beta-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/beta-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/beta-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/beta-catenin signalling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine.

A truncated form of Xenopus E-cadherin (deltaE-cad) comprising the cytoplasmic and transmembrane domains was overexpressed generating a dominant negative mutation in the urodelan amphibian embryo Pleurodeles waltl. deltaE-cad mRNA and rhodamine-lysinated-dextran (RLDx) cell lineage tracer were microinjected into 32-cell stage blastomeres which contribute principally to the notochord and central nervous system. deltaE-cad expression causes defects in forebrain and hindbrain formation coupled with the development of supernumerary vesicles. Duplication of the notochord also occurs due to the retardation of medial cell intercalation with correlated duplications of spinal cord and somites. These results emphasize the role of cadherins in mediating cell-cell adhesion in early amphibian embryogenesis. They extend to Pleurodeles the observations made in Xenopus, illustrating that despite differences in morphogenetic processes, the molecular mechanisms are conserved in these two species.

Cell fate of the blastomeres at the 32-cell stage in the Pleurodeles waltl embryo was analyzed by injection of rhodamine or fluorescein lysinated-dextran (RLDx or FLDx). At the tailbud stage, the progeny of each blastomere contributed to more than one germ layer with unequal distribution along the anteroposterior and dorsoventral axis. Such a regionalized positioning of the descendants of the 32-cell blastomeres was found in the neuroectoderm, the epidermis, the notochord, the somites, the lateral plate, and the endoderm, but not in the head mesenchyme, the pronephros, or the blood islands. Results of double labeling of juxtaposed blastomeres showed that cell mixing and rearrangement take place during organ formation. Results are compared with those of the 32-cell stage fate map in Xenopus and Rana and reveal the more restricted fate of 32-cell stage blastomeres in Pleurodeles germ layers.

Embryogenesis implies a strict control of cell interaction and cell migration. The spatial and temporal regulation of morphogenetic movements occurring during gastrulation is directly dependent on the early cell interactions that take place in the blastula. The newt Pleurodeles waltl is a favorable model for the study of these early morphogenetic events. The combination of orthotopic grafting and fluorescent lineage tracers has led to precise early gastrula mesoderm fate maps. It is now clear that there are no sharp boundaries between germ layers at the onset of gastrulation but rather diffuse transition zones. The coordination of cell movements during gastrulation is closely related to the establishment of dorsoventral polarity. Ventralization by U.V. irradiation or dorsalization by lithium treatment modifies the capacity for autonomous migration on the fibronectin coated substratum of marginal zone cells accordingly. It is now firmly established that mesodermal cells need to adhere to a fibrillar extracellular matrix (ECM) to undergo migration during gastrulation. Extracellular fibrils contain laminin and fibronectin (FN). Interaction of cells with ECM involves receptors of the beta 1 integrin family. A Pleurodeles homolog of the alpha v integrin subunit has been recently identified. Protein alpha v expression is restricted to the surface of mesodermal cells during gastrulation. Integrin-mediated interactions of cells with FN are essential for ECM assembly and mesodermal cell migration. Intracellular injection of antibodies to the cytoplasmic domain of beta 1 into early cleavage embryos causes inhibition of FN fibril formation. Intrablastocoelic injections of several probes including antibodies to FN or integrin alpha 5 beta 1, competitive peptides to the major cell binding site of FN or the antiadhesive protein tenascin all block mesodermal cell migration. This results in a complete arrest of gastrulation indicating that mesodermal cell migration is a major driving force in urodele gastrulation. It is now possible to approach the role of fibroblast growth factor (FGF) during cell interactions taking place in urodele embryos. Four different FGF receptors (FGFR) have been cloned in Pleurodeles. Each of them has a unique mRNA expression pattern. FGFR-1, FGFR-3, and the variant of FGFR-2 containing the IIIb exon are maternally expressed and might be involved in mesodermal induction. During gastrulation, FGFR-3 and FGFR-4 have a restricted pattern of expression, whereas FGFR-1 mRNA is nearly uniformly distributed. Splicing variants FGFR-2IIIb and FGFR-2IIIc have exclusive expression patterns during neurulation. IIIb is expressed in epidermis and IIIc in neural tissue, suggesting a function in the differentiation of ectodermal derivatives.

A cDNA encoding a novel Xenopus fibroblast growth factor receptor, XFGFR4B, has been cloned. XFGFR4B mRNA is detected throughout embryogenesis. However, from the late gastrula stage on, XFGFR4B transcripts are expressed in two defined areas of the anterior neural plate. Interestingly, these two regions are fated to become parts of the retina, midbrain, hindbrain, and otic vesicle, all of which continue to express XFGFR4B mRNA in tailbud stage embryos and early tadpoles. Expression of XFGFR4B mRNA can be maintained at neurula stage in isolated blastula ectoderm in response to mesodermal induction by activin or neural induction by noggin, suggesting that XFGFR4B expression might be regulated by early cell interactions. Distribution of XFGFR4B mRNA suggests that XFGFR4B might serve an important function during patterning of neuroepithelium.

We have used in situ hybridization to survey the expression pattern of three fibroblast growth factor receptor (FGFR) mRNAs (PFR-1, PFR-3 and PFR-4, which we previously identified as the amphibian Pleurodeles waltl homologs of human FGFR-1, FGFR-3 and FGFR-4, respectively) during morphogenesis. Previous work suggests that these FGFR mRNAs exhibit a distinct pattern of expression at early developmental stages. In the present study we have tested the functional activity of these receptors and shown that both FGF-1 (acidic FGF) and FGF-2 (basic FGF), but not FGF-7 (keratinocyte growth factor), can lead to their activation, suggesting that the three cDNAs encode functional receptors. Results from in situ hybridization indicate that various FGFRs are involved in various developmental events. Their involvement in these processes is both overlapping and distinct. During the differentiation of the central nervous system (CNS), PFR-1 and PFR-4 mRNAs show high levels of redundant expression, while the sites of expression of PFR-3 mRNA correlate with regions, such as the diencephalon and the rhombencephalon, undergoing important anatomic changes. The three FGFR mRNAs are distinctly expressed in the cranial ganglia, the pigmented epithelia of retina and the otic vesicles. Most significantly, we found that they are strongly expressed at cranial and branchial mesenchymal condensation sites. PFR-3 mRNA is expressed earlier in this process than PFR-1 and PFR-4 mRNAs. Furthermore PFR-3 mRNA is detected in the mesenchyme of the limb bud, while PFR-1 and PFR-4 mRNAs are found in the primordia of the skeletal elements. In addition, PFR-1 mRNA is expressed in axial mesenchyme and PFR-4 mRNA is detected in the melanophores, xanthophores and in the pronephros. These results suggest that various FGFRs may be involved in distinct developmental events including cell proliferation and differentiation. We also discuss the functional redundancy of the FGFR system during amphibian morphogenesis.

Tenascin (Tn) protein and transcripts were analyzed in developing, adult and regenerating caudal spinal cord (SC) of Pleurodeles waltl. A polyclonal antibody (PAb) against Xenopus Tn and a newt Tn cDNA probe were used. In Western blots, anti-Tn PAb recognized Tn polypeptides of 200-220 kDa in tail regenerate extracts, but also the homolog of Tn/Cytotactin/J1 in brain and SC of adult newt. Immunofluorescence studies showed some reactivity around ependymoglial cells and strong labeling in the nervous tracts, in the developing as well as in the regenerating SC or adult SC. Immunogold electron microscopy revealed the presence of Tn throughout the ependymoglial cells, particularly near and along the plasma membrane of radial processes surrounding axons, especially growth cones. Tn could be more precisely found within rough endoplasmic reticulum and Golgi structures, or again in the surrounding extracellular space. This suggested that Tn was at least produced by radial glial profiles forming axonal compartments in which axons grew. Using the DNA probe for Tn, expression of Tn mRNA was also examined by Northern blot and RNAase protection analyses and by in situ hybridization, respectively. The levels of transcripts, barely detectable in adult tail, increased in regenerates from 3 days through 4-8 weeks post-amputation. In situ Tn mRNA were mainly localized in the mesenchyme, especially at the epithelial-mesenchymal interface, and in the developing cartilage, at the early regeneration stages, whereas high amounts of transcripts were seen not only at these stages, but also later, in the regenerating SC. Our main results supported the view that, in the caudal SC of newts, Tn, synthesized by radial ependymoglial cells, was similarly expressed during regeneration as well as larval development, and exhibited a sustained high accumulation level in the adult SC. On the basis of the multifunctional properties of Tn, the putative roles played by Tn as a substrate for neuronal pathfinding and boundary shaping were discussed.

We have studied the fate of presumptive mesodermal cells in the early Rana pipiens gastrula. We labeled superficial cells of the early gastrula with 125I. We also labeled all cells in a gastrula with rhodamine-lysine-dextran cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the notochord and somite regions are both derived from the superficial and deep portions of circumblastoporal regions. In contrast, pronephros, lateral plate, cardiac anlagen, and blood cells only arise from deep cells in circumblastoporal regions. Such observations indicate that the fate map for R. pipiens is different from that of Xenopus laevis, where mesodermal derivatives appear to be restricted to the deep layers, and from that of Pleurodeles waltl, where all mesodermal derivatives are formed both from superficial and deep layers. We also have shown that from the neurula stage there is substantial mixing between cells due to the ingression of cells in the dorsal region so that superficial labeled cells, initially located in the roof of the archenteron, contribute to ventral regions of both the somites and notochord.

There is increasing evidence that distinct fibroblast growth factor receptor (FGFR) genes are involved in embryonic development and that unique expression patterns of individual FGFRs correlate with tissue-specific functions. In addition, alternative splicing of mRNA transcripts from at least two of these genes (FGFR-1 and FGFR-2) can generate receptor variants with different ligand-binding specificity. By polymerase chain reaction methods and by screening a cDNA library, we have isolated five amphibian FGFR-2 splice variants which share a high degree of identity to their human counterparts. These mRNAs are developmentally regulated and are expressed in a tissue-specific manner. In particular, two alternative exons (termed IIIb and IIIc) in the second half of the third immunoglobulin-like loop are remarkably conserved and have a distinct pattern of regulation during development. Either aFGF or bFGF can activate IIIb- or IIIc-containing receptors. In contrast, KGF only activates IIIb-containing receptors. Exon IIIb-containing receptors are maternally derived mRNAs, whereas exon IIIc-containing receptors are zygotically expressed. Furthermore, their tissue distribution pattern was mutually exclusive. From the beginning of the neurula stage onward, IIIb transcripts are expressed in the epidermis, while IIIc transcripts are activated in the neuroectoderm by neural induction. At the late tail-bud stage, in situ hybridization revealed expression of IIIc mRNA in the telencephalon and the diencephalon, as well as in the head mesenchyme condensation sites originating from the proliferating neural crest cells. IIIb mRNA was detected in the epidermis and in the epithelium of the pharynx. Our data suggest that exon IIIb-containing receptors may play a role in the development of epithelial tissues, while exon IIIc-containing receptors may play a role during neural tissue formation.

Partial cDNA clones encoding approximately the carboxy terminal half of Pleurodeles fibronectin (FN) were isolated. They account for 4.7 Kbp of the 3' region of the FN mRNA. The cDNA nucleotide sequence comprises all three alternatively spliced segments designated EIIIA, EIIIB and V-segment, respectively. All three segments are included in FN mRNA synthesized during early embryogenesis whereas, from the tailbud stage onward the V-region was partially excluded. The isolation of Pleurodeles cDNA clones including the three different spliced EIIIA, EIIIB and V segment raises new possibilities for the study of the precise role of specific regions of FN in early amphibian development.

Tenascin (TN) is an extracellular matrix (ECM) glycoprotein which possesses antiadhesive properties and thus is able to modulate cell interactions with molecules of the ECM. In Xenopus embryos, TN is expressed dorsally in a very restricted pattern. We have studied the distribution of TN mRNA in tailbud-stage embryos by in situ hybridization. We show that TN transcripts are principally expressed in myotome cells. No TN mRNA could be detected in lateral plate mesoderm. This had led us to study TN expression in response to mesodermal inducers. Analysis of the distribution of TN after mesodermal induction of blastula animal caps with activin A and bFGF or after ectopic expression of XBra shows that TN expression is elicited when dorsal or posterior structures are formed. Our results show that TN regionalization in Xenopus embryos depends on mesoderm patterning.

New observations on thin strips of cells from the leading edge of the involuting presumptive mesoderm explanted onto FN-coated substrate show a striking preferential cellular emigration from the leading edge of explants. Microinjected probes (Fab' anti-FN, Fab' anti-integrin and RGD-peptides) that disrupt cell adhesion to the FN-matrix on basal surface of the blastocoel roof also disrupt normal anuran gastrulation, producing blocked embryos with no adhesion of leading edge mesodermal cells to the blastocoel roof, abnormal epiboly, and defects of mesodermal cell spreading across the basal surface of the blastocoel roof toward the animal pole. These results show that the FN-rich fibrillar extracellular matrix on the basal surface of the blastocoel roof is required for normal gastrulation in Rana pipiens embryos.

In early vertebrate development, the proper assembly of fibronectin into fibrils is crucial for embryonic cells to adhere and to migrate on the extracellular matrix. The molecular mechanisms by which such a process occurs in vivo are poorly understood. In the amphibian embryo Pleurodeles waltl fibronectin fibrils appear first at the blastula stage. They form a fibrillar matrix on the basal surface of animal cells facing the blastocoel. Using competition and perturbation experiments with purified proteolytic fragments and domain-specific monoclonal antibodies, we demonstrate that at least three fibronectin sites are essential for assembly of fibronectin fibrils in the blastula of Pleurodeles waltl. Two sites, the RGDS sequence and the synergistic domain in the 10th type III repeat, are both involved in receptor recognition. A third site that spans the 9th type I and 1st type III homology sequences is also likely to participate in fibronectin-fibronectin interactions.

During early development of the urodele Ambystoma maculatum, the appearance and distribution of fibronectin-containing fibrillar extracellular materials were studied by immunocytochemistry. Fibronectin (FN) first appears in the early blastula (stage 7) as thin punctate fibrils on the cell surface concentrated in the marginal zone. In late blastula (stage 9), thin fibrils are found throughout the blastocoel roof. Early gastrulae (stage 10) have numerous fibrils and multifibrillar strands concentrated in the dorsal lip region and oriented preferentially along a line parallel to the dorsal lip-animal pole axis. There is a striking increase in the amount of FN fibrils during the rest of gastrulation. This FN-containing network can be transferred to plastic substrata with preservation of the preferential orientation observed in vivo. Dorsal marginal zone explants placed on such conditioned substrata show polarized outgrowth toward the animal pole region of conditioned areas when placed on the dorsal lip side or the ventral marginal zone side of conditioned substrata. This outgrowth occurs symmetrically on bovine plasma FN-coated substrata, is prevented by Fab' fragments of antibodies to FN but fails to occur on laminin coated substrata. When migrating mesodermal cells from early gastrulae are cultured on substrata conditioned by deposition of the fibrillar matrix, these cells exhibit striking contact inhibition of locomotion, a phenomenon that may explain dispersal of migrating mesodermal cells across the blastocoel roof. When leading edges of mesodermal cells collide, cells abruptly change direction. When leading edges collide with trailing edges, the trailing edges detach from the substratum and cells move apart in the direction of the leading edge.

Tenascin is a large oligomeric extracellular matrix (ECM) glycoprotein whose expression is highly restricted during vertebrate development. It has a characteristic hexameric quaternary structure with six arms linked to a central globular domain. Each arm contains a single polypeptide with the central globular domain formed by the covalent association of the N-terminal ends of the six polypeptides. Tenascin first appears during development, associated with the neural crest cell migration pathways of mammalian, avian and amphibian embryos. During later development, it is observed at sites of cartilage, bone and tendon formation. Tenascin expression also occurs in defined areas in the developing nervous system and in condensing mesenchyme, in response to epithelio-mesenchymal interactions. The function of tenascin in these different morphogenetic processes is not yet clearly understood. Tenascin can promote neurite outgrowth in vitro and can inhibit cell interactions with fibronectin. Results based on antibody mapping and molecular cloning indicate that these properties involve two distinct cell binding sites. Together with its highly regulated expression in the embryo, these properties suggest that tenascin plays a key role in the control of cell migration and differentiation during development.

The early amphibian embryo provides an attractive model for the in vivo analysis of cell interactions with extracellular matrix components. During gastrulation, mesodermal cells use an anastomosing network of extracellular fibrils as substratum for their migration. These fibrils contain glycosaminoglycans and non collagenous proteins including laminin and fibronectin. The function of these extracellular components in the mesodermal cell migration process has been inferred from grafting experiments and microinjection of probes such as specific antibodies or GRGDS-containing peptides. Using the amphibian embryo as an experimental system, combination of microsurgical, cell behavioral and molecular approaches will provide new insights into cell-extracellular matrix interactions directing morphogenetic cell movements.

We report here the purification of tenascin, an extracellular matrix molecule involved in the control of morphogenesis, from the conditioned medium of the Xenopus XTC cell line. Tenascin was purified by affinity chromatography on a column of the monoclonal antibody mAb TnM1; the molecule eluted from this column has a relative molecular mass of 210 kDa after reduction. Electrophoretic analysis under non-reducing conditions shows that the purified components are oligomeric disulfide-linked complexes which barely enter a 4% polyacrylamide gel. Upon rotary shadowing these molecules appear to possess a central globular domain to which pairs or triplets of arms are attached. Polyclonal antibodies have been raised against purified Xenopus tenascin. They recognise specifically the antigen on Western blots of XTC conditioned medium and adult brain, by immunofluorescence, these antibodies reveal large amounts of tenascin in the secretory vesicles as well as in the extracellular matrix of XTC cells. In the Xenopus tadpole, they stain the developing cartilage, the basal lamina of skin epidermis, myotendinous ligaments and restricted regions of the central nervous system.

We have reviewed the evidence supporting the notion that the fibrillar extracellular matrix on the basal surface of the blastocoel roof in amphibian embryos directs and guides mesodermal cell migration during gastrulation. Based on extensive experimental evidence in several different systems, we conclude the following: (i) the fibrillar extracellular matrix contains fibronectin (FN) and laminin. (ii) The fibrils are oriented in such a way as to promote directional migration of mesodermal cells during migration. (iii) We have used several different probes to disrupt the interaction between migrating mesodermal cells and the fibrillar extracellular matrix. These probes include: (a) nucleocytoplasmic and interspecific hybridization. Such embryos have defects in FN synthesis and gastrulation. (b) Fab' fragments of anti-FN and anti-integrin VLA-5 IgGs prohibit mesodermal cell adhesion both in vitro and in vivo and gastrulation is arrested. (c) Peptides containing the RGDS sequence specifically inhibit interactions between migrating mesodermal cells and the FN-fibrillar matrix. (d) Tenascin blocks cell adhesion to FN in vitro and gastrulation in vivo. (e) Antibodies against the cytoplasmic domain of beta 1 integrin, when injected into blastomeres, prevent FN-fibrillogenesis in progeny of injected blastomeres and delay mesodermal cell migration selectively in the progeny of injected blastomeres but not in the uninjected blastomere progeny.

We have used amphibian gastrulation as a model system to study the action of the extracellular matrix (ECM) glycoprotein tenascin on mesodermal cell migration. Tenascin function was assayed in vitro during spreading of isolated cells from the dorsal marginal zone (DMZ) and during cell migration from DMZ explants. Plastic coated with bovine fibronectin or gastrula ECM was used as a substratum. In both cases, tenascin added to the medium inhibited spreading and migration of mesodermal cells. In addition, a substratum coated with a mixture of fibronectin and tenascin was found to prevent mesodermal cell migration. Tenascin was also microinjected into the blastocoel cavity of living embryos at the late blastula stage. This led to a complete arrest of gastrulation in more than 80% of the cases. Scanning electron microscopy of fractures from arrested gastrulae showed that mesodermal cell migration was blocked. Similar injection experiments carried out at the middle gastrula stage demonstrated that tenascin is able to inhibit cell migration after cells have already contacted the ECM. Mesodermal cell migration in the presence of tenascin could be restored in vitro and in vivo by the monoclonal antibody mAb Tn68 which is known to mask a cell binding site of the molecule. Finally, tenascin microinjected into the blastocoel of blastula or gastrula stage embryos bound within 15 min to the ECM fibrils at all the stages studied. Our results show that exogenous tenascin can be incorporated into embryonic ECM and interferes in vivo with the interactions of cells with a fibronectin-rich matrix.

Rana pipiens eggs fertilized by Rana esculenta sperm (ESC) hybrid embryos develop until gastrulation in control Rana pipiens embryos (PIP) and then show morphogenetic arrest. After arrest, ESC do not gastrulate but live for 5 days as blastula-like embryos. We studied the distribution of fibronectin (FN)-containing fibrils and integrin (INT) in PIP and ESC. There are many FN-fibrils in PIP organized in anastomosing networks radiating away from the center of individual cells and across intercellular boundaries. ESC have fewer fibrils compared to PIP. These fibrils are first located between cells in disorganized arrays. After arrest in ESC, when PIP are Stage 14 neurulae, many more FN-fibrils appear. INT-staining occurs in both embryos in similar patterns. In xenoplastic transplantations, we found that the extracellular matrix on the inner surface of the ESC blastocoel roof serves as a substratum for PIP cell migration. In an in vitro assay, we found more cell adhesion to FN-substrata in PIP than in ESC. Cell locomotion rates on FN-substrata were 1.70 +/- 0.85 microns/min for PIP but only 0.46 +/- 0.56 microns/min for ESC. We also found that the inner surface of the blastocoel roof from ESC can not promote cell adhesion and locomotion when Stage 11 fragments are used for conditioning but that Stage 14 fragments can deposit a FN-fibril-rich extracellular matrix which supports PIP mesodermal cell migration at a rate of 1.26 +/- 0.38 microns/min.

We have studied the localization and function of a 140-kDa glycoprotein complex implicated in cell adhesion to fibronectin- and laminin-rich extracellular matrices in Pleurodeles waltlii gastrulae. In particular, we have shown that antibodies directed against highly purified avian fibronectin (FN) receptor complex cross-react with two major polypeptides of apparent molecular weights of 140,000 and 100,000 and a third minor component of 90,000. Using sections of embryos or whole mounts, we have also discovered that the putative FN receptor is widely distributed on the early embryonic cell surface. We have also found that the basal surface of the roof of the blastocoel, a region particularly enriched in an extracellular matrix consisting of fibronectin- and laminin-rich fibrils, is rich in receptor complex. We have prepared monovalent Fab' fragments of this antibody and have found that they cause detachment of cells previously attached to substrata coated with fibronectin, and they also arrest gastrulation when injected into the blastocoel of early gastrulae. Thus, it appears that the fibronectin receptor complex plays a significant functional role in cell attachment of gastrula-stage cells in vitro and in cell migration in vivo during gastrulation.

The protein synthesis manifested by aggregates of blastula and gastrula dissociated cells were studied by means of polyacylamide gradient gel electrophoresis. This method permits a comparative analysis of protein electrophoretic mobilities in aggregates and controls. In all cases, the data obtained establish the identity of protein banding and confirm an egg laying polymorphism.

The three-dimensional organisation of fibronectin (FN) in early amphibian embryos (Ambystoma mexicanum, Pleurodeles waltlii) was studied with the use of antibodies against amphibian-FN. Immunofluorescence labelling was performed on whole-mount specimens. It was shown that before gastrulation an extensive extracellular network consisting of anastomosed FN-fibrils underlies the roof of the blastocoel prior to the migration of mesodermal cells. Initially, FN-fibrils develop radially on the inner surface of ectodermal cells (early blastula stage) and become confluent there to elaborate a fibrillar network. During the late blastula stage the entire surface of the blastocoel roof (presumptive ectoderm and mesoderm) is covered by the FN-rich extracellular matrix. As gastrulation proceeds, the migrating mesodermal cells interact with the FN-fibrillar network. The results suggest that the FN-containing extracellular matrix plays an important role in mediating mesodermal cell-substratum contacts in gastrulating embryos. No FN-fibrils were found in other parts of the embryo.

Ectodermic blastomeres from arrested nucleocytoplasmic blastulae obtained by nuclear graft between Ambystoma mexicanum (A. m.) and Ambystoma dumerilii (A. d.), are transplanted in the blastocoele of recipient Pleurodeles waltlii blastulae. Histo-autoradiographic analysis shows that hybrid cells have cellular affinities and multiplication capacities very different from normal Ambystoma mexicanum blastulae isolated cells. The possibility that a revitalisation of lethal blastomeres can occur should not be retained.

In amphibian embryos, fibronectin (FN) assembles as a fibrillar network on the roof of the blastocoel cavity, preceding mesodermal cell migration. Local inversion of the ectoderm to produce a site where no FN is available prevents mesodermal cell migration. Microinjection of monovalent antibodies to FN arrests gastrulation. A complete inhibition of mesodermal cell migration is obtained after microinjection of a synthetic peptide containing the cell binding site sequence of FN. Prevention of interactions between receptors and FN appears to be the primary cause for blockage of gastrulation.