Specific roles of the alpha V beta 1, alpha V beta 3 and alpha V beta 5 integrins in avian neural crest cell adhesion and migration on vitronectin

Specific roles of the V1, V3 and V5 integrins in avian neural crest cell Muriel Delannet 2 Fabrice Martin 2 Blaise Bossy 1 David A. Cheresh 0 Louis F. Reichardt 1 Jean-Loup Duband 2 0 Department of Immunology, The Scripps Research Institute , La Jolla, California 92037 , USA 1 University of California and Howard Hughes Medical Institute , Parnassus and Third Avenues, San Francisco, California 94143- 0724 , USA 2 Laboratoire de Biologie Cellulaire du Developpement, Institut Jacques Monod, Universite Paris 7 , 2, place Jussieu, 75251 Paris Cedex 05 , France adhesion and migration on vitronectin neural crest; vitronectin; integrins; quail; cell adhesion; cell migration SUMMARY To identify potentially important extracellular matrix adhesive molecules in neural crest cell migration, the possible role of vitronectin and its corresponding integrin receptors was examined in the adhesion and migration of avian neural crest cells in vitro. Adhesion and migration on vitronectin were comparable to those found on fibronectin and could be almost entirely abolished by antibodies against vitronectin and by RGD peptides. Immunoprecipitation and immunocytochemistry analyses revealed that neural crest cells expressed primarily the V1, V3 and V5 integrins as possible vitronectin receptors. Inhibition assays of cellular adhesion and migration with functionperturbing antibodies demonstrated that adhesion of neural crest cells to vitronectin was mediated essentially by one or more of the different V integrins, with a possible preeminence of V1, whereas cell migration involved mostly the V3 and V5 integrins. Immunofluorescence labeling of cultured motile neural crest cells revealed that During early embryonic development, certain groups of cells, like neural crest cells, can transiently express locomotory properties that allow them to migrate long distances from their sites of origin and populate other areas of the embryo where they undergo differentiation (Le Douarin, 1982; Newgreen and Erickson, 1986; Levi et al., 1990; Erickson and Perris, 1993). During migration to their final destination, neural crest cells penetrate extracellular matrices that are known to contain fibronectin, collagens, laminin, tenascin and a variety of proteoglycans (Thiery et al., 1982; Krotoski et al., 1986; Duband and Thiery, 1987; Tan et al., 1987; Mackie et al., 1988; Perris et al., 1991a,b). The role of these matrix components in migration has been analyzed in detail in the avian embryo essentially in in vitro approaches. Neural crest cells cultured in the V integrins are differentially distributed on the cell surface. The 1 and V subunits were both diffuse on the surface of cells and in focal adhesion sites in association with vinculin, talin and -actinin, whereas the V3 and V5 integrins were essentially diffuse on the cell surface. Finally, vitronectin could be detected by immunoblotting and immunohistochemistry in the early embryo during the ontogeny of the neural crest. It was in particular closely associated with the surface of migrating neural crest cells. In conclusion, our study indicates that neural crest cells can adhere to and migrate on vitronectin in vitro by an RGDdependent mechanism involving at least the V1, V3 and V5 integrins and that these integrins may have specific roles in the control of cell adhesion and migration. vitro adhere to and migrate efficiently on fibronectin, laminin, and type I, IV and VI collagens (Newgreen et al., 1982; Rovasio et al., 1983; Tucker and Erickson, 1984; Perris et al., 1989, 1991a, 1993a). In addition, antibodies to fibronectin or to the integrin 1 subunit and RGD peptides can impair neural crest cell migration on fibronectin substrata (Rovasio et al., 1983; Boucaut et al., 1984; Bronner-Fraser, 1985; Duband et al., 1986). Likewise, antibodies to the 1 or to the 1 subunit of integrins can affect neural crest cell adhesion to laminin or collagens (Lallier and Bronner-Fraser, 1992; Perris et al., 1993b). These studies thus provide strong evidence that avian neural crest cells can adhere and migrate in vitro on a variety of extracellular matrix molecules through 1 integrins. In vivo, injection of RGD-containing peptides or antibodies to fibronectin, to a laminin-proteoglycan complex or to the integrin 1 subunit into the cranial region of avian embryos cause severe deficencies in neural crest cell migration (Boucaut et al., 1984; Bronner-Fraser, 1985; Poole and Thiery, 1986; Bronner-Fraser and Lallier, 1991). However, the same antibodies fail to perturb neural crest cell migration in trunk regions although they are able to inhibit strongly myoblast migration (Jaffredo et al., 1988; Bronner-Fraser, 1993). This indicates that, while cranial neural crest cells are likely to migrate in vivo primarily on fibronectin and laminin using 1 integrins, truncal neural crest cells may be able to interact with additional extracellular matrix molecules for migration using non-1 integrins, allowing them to overcome the inhibitory effect of the antibodies. Consistent with this, it has been shown recently that cranial and trunk neural crest cells may differ in their mechanisms of adhesion to selected extracellular matrix components in vitro (Lallier et al., 1992). Therefore, additional extracellular matrix components that promote truncal neural crest cell locomotion have to be determined. A possible candidate is vitronectin, a multifunctional adhesive glycoprotein of Mr of about 70103 (70K) found in the circulation and in the extracellular matrix of various tissues and which interacts with the surface of cells primarily through the V3 integrin, also called vitronectin receptor (for reviews, see Preissner, 1991; Felding-Habermann and Cheresh, 1993). Owing to its multidomain structure with binding sites for various integrins, heparin, collagen, plasminogen and plasminogen activator inhibitor 1, vitronectin plays a critical role in the substratum adhesion of a large variety of cell types, in hemostasis and in immune defense. Surprisingly, although considerable information has accumulated regarding its structural and adhesive properties, the involvement of vitronectin as a possible regulatory extracellular matrix molecule during development has been poorly investigated. In the present report, we examine in vitro the adhesive and migratory response of avian trunk neural crest cells to vitronectin. We also describe the distribution of vitronectin in neural crest cell migratory pathways and characterize the repertoire and functions of the vitronectin-binding integrins in these cells. MATERIALS AND METHODS Adhesive proteins and antibodies Vitronectin was purified from bovine serum by affinity chromatography on a heparin-Sepharose column as described by Yatohgo et al. (1988). Bovine plasma fibronectin was purified on a gelatinSepharose column as described previously (Rovasio et al., 1983). Rabbit polyclonal antibodies to chicken and bovine vitronectin were kindly provided by Dr M. (...truncated)