Cellular phenotype transformation during early embryogenesis: a role for focal adhesion kinase?

We have used the gastrulating chick embryo as a model for studying the potential role of focal adhesion kinase (FAK) in phenotypic transformation. In the gastrulating embryo, there is a well-defined epithelial to mesenchymal transformation as the upper epithelial epiblast layer of cells ingresses at the primitive streak to form the invasive mesenchymal mesoderm layer and the epithelioid endoderm layer. Immunolocalization showed that FAK was expressed primarily in the apical cytoplasm of the epiblast layer, together with some regions of the mesoderm and endoderm. Hensen's node and the primitive streak, where the transformation occurs, showed very low immunoreactivity. Levels of FAK in these individual tissues were quantified by densitometric analysis of Western blots, and FAK activation was quantified by stripping these blots and reprobing for phosphotyrosine. Immunoprecipitation indicated that the phosphotyrosine bands corresponded with the FAK bands on the blots. Although the blots confirmed that FAK was highly expressed in the epiblast, the level of FAK activation was highest in the endoderm, despite relatively low expression of the protein, Similar quantitative blotting was carried out using cells from each of the three layers cultured on different substrata. The results indicated that cells cultures on fibronectin, laminin, and Matrigel expressed differing levels of FAK, with differing levels of tyrosine phosphorylation, depending on the cell type and the substratum. We conclude that FAK is developmentally regulated during gastrulation, and that this regulation could be influenced by the changing substratum encountered by the differentiating cells during this process. However, the apical localization of FAK in much of the epiblast appears to preclude a consistent focal contact-like association of this molecule with integrins in vivo, and we therefore suggest that in the embryo, FAK may be involved in integrin-mediated signalling pathways without physical association with cell-substratum contacts.