THE ROLE OF E-CADHERIN AND INTEGRINS IN MESODERM DIFFERENTIATION AND MIGRATION AT THE MAMMALIAN PRIMITIVE STREAK

We have examined the role of cell-cell and cell-extracellular matrix ( ECM) interactions during mesoderm differentiation and migration at the primitive streak of the mouse embryo with the use of function-perturb ing antibodies. Explants of epiblast or mesoderm tissue dissected from the primitive streak of 7.5- to 7.8-day mouse embryos were cultured o n a fibronectin substratum in serum-free, chemically defined medium. A fter 1624 hours in culture, cells in explants of epiblast exhibited th e typical close-packed morphology of epithelia, and the tissue remaine d as a coherent patch of cells that were shown to express transcripts of the cytokeratin Endo B by in situ analysis. In contrast, cells in e xplants of primitive streak mesoderm exhibited a greatly flattened, fi broblastic morphology, did not express Endo B transcripts, and migrate d away from the center of the explant. As epiblast cells in vivo under go the epithelial-mesenchymal transition at the primitive streak, they cease expressing the prominent calcium-sensitive cell adhesion molecu le E-cadherin (uvomorulin, Cell-CAM 120/80). We asked whether the loss of E-cadherin expression was a passive result of differentiation or i f it might play a more causative role in mesoderm differentiation and migration. Culture with function-perturbing antibodies against E-cadhe rin caused cells within epiblast explants to lose cell-cell contacts, to flatten, and to assume a mesenchymal morphology; they were also ind uced to migrate. Anti-E-cadherin antibodies had no effect on explants of primitive streak mesoderm. In immunofluorescence studies, anti-E-ca dherin-treated epiblast cells ceased to express SSEA-1, a carbohydrate moiety that is lost as mesoderm differentiates from the epiblast in v ivo, and they also ceased to express F-cadherin itself in contrast, th ese cells began to express the intermediate filament protein vimentin, a cytoskeletal protein characteristic of the primitive streak mesoder m at this stage of development. As epiblast cells differentiate into m esoderm, their predominant adhesive interactions change from cell-cell to cell-substratum. Therefore, we also investigated the adhesive inte ractions between primitive streak tissues and extracellular matrix (EC M) components. Epiblast explants adhered well to fibronectin, more poo rly to laminin and type IV collagen, and not at all to vitronectin. In contrast, mesoderm explants attached well to all these proteins. Furt hermore, epiblast, but not mesoderm, displayed an anchorage-dependent viability in culture. After anti-E-cadherin treatment, epiblast cells that had assumed the mesenchymal morphology did attach to vitronectin, another characteristic shared with primitive streak mesoderm. Adhesio n of epiblast to fibronectin and of primitive streak mesoderm to fibro nectin, vitronectin, laminin, and type IV collagen was completely bloc ked by incubation with a broad-spectrum polyclonal serum, anti-ECM rec eptor (anti-ECMR) antiserum, which recognizes beta1 and beta3 integrin s. Anti-ECMR-treated mesodermal explants recovered and attached, sprea d, and migrated normally after antibodies were removed. In addition, a n antibody specific for alpha6beta1 integrin, which mediates adhesion to laminin, selectively blocked attachment of mesoderm to laminin but not to fibronectin, indicating that alpha6beta1 is a major laminin rec eptor for these cells. We conclude that disruption of E-cadherin funct ion in mammalian epiblast cells at the primitive streak in vitro cause s them to acquire a phenotype characteristic of mesoderm, and we propo se that similar mechanisms act during mesoderm differentiation in the intact embryo. Our results also show that the cell-substratum adhesion of primitive streak tissues is mediated by the integrin superfamily o f receptors and that developmentally regulated changes in cell-ECM adh esion accompany the epithelial-mesenchymal transition at the mammalian primitive streak.