The role of E-cadherin and integrins in mesoderm differentiation and migration at the mammalian primitive streak

Abstract

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-perturbing antibodies. Explants of epiblast or mesoderm tissue dissected from the primitive streak of 7.5- to 7.8-day mouse embryos were cultured on a fibronectin substratum in serum-free, chemically defined medium. After 16-24 hours in culture, cells in explants of epiblast exhibited the typical close-packed morphology of epithelia, and the tissue remained 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 explants of primitive streak mesoderm exhibited a greatly flattened, fibroblastic morphology, did not express Endo B transcripts, and migrated away from the center of the explant. As epiblast cells in vivo undergo the epithelial-mes-enchymal transition at the primitive streak, they cease expressing the prominent calcium-sensitive cell adhesion molecule E-cadherin (uvomorulin, Cell-CAM 120/80). We asked whether the loss of E-cadherin expression was a passive result of differentiation or if it might play a more causative role in mesoderm differentiation and migration. Culture with function-perturbing antibodies against E-cadherin caused cells within epiblast explants to lose cell-cell contacts, to flatten, and to assume a mes-enchymal morphology; they were also induced to migrate. Anti-E-cadherin antibodies had no effect on explants of primitive streak mesoderm. In immunofluo-rescence studies, anti-E-cadherin-treated epiblast cells ceased to express SSEA-1, a carbohydrate moiety that is lost as mesoderm differentiates from the epiblast in vivo, and they also ceased to express E-cadherin itself. In contrast, these cells began to express the intermediate filament protein vimentin, a cytoskeletal protein characteristic of the primitive streak mesoderm at this stage of development. As epiblast cells differentiate into mesoderm, their predominant adhesive interactions change from cell-cell to cell-substratum. Therefore, we also investigated the adhesive interactions between primitive streak tissues and extracellular matrix (ECM) components. Epiblast explants adhered well to fibronectin, more poorly to laminin and type IV collagen, and not at all to vitronectin. In contrast, mesoderm explants attached well to all these proteins. Furthermore, 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. Adhesion of epiblast to fibronectin and of primitive streak mesoderm to fibronectin, vitronectin, laminin, and type IV collagen was completely blocked by incubation with a broad-spectrum polyclonal serum, anti-ECM receptor (anti-ECMR) antiserum, which recognizes β1 and β3 integrins. Anti-ECMR-treated mesodermal explants recovered and attached, spread, and migrated normally after antibodies were removed. In addition, an antibody specific for α6β1 integrin, which mediates adhesion to laminin, selectively blocked attachment of mesoderm to laminin but not to fibronectin, indicating that α6β1 is a major laminin receptor for these cells. We conclude that disruption of E-cadherin function in mammalian epiblast cells at the primitive streak in vitro causes them to acquire a phenotype characteristic of mesoderm, and we propose 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 of receptors and that developmentally regulated changes in cell-ECM adhesion accompany the epithelial-mesenchymal transition at the mammalian primitive streak.