We have taken advantage of an enhancer trap event in a line of transgenic mice to identify a unique developmentally regulated endothelial cell-specific locus (Dell). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Dell has been shown to be a matrix protein, and to promote adhesion and migration of endothelial cells through interaction with the alphavbeta3 integrin receptor. Adhesion of cells to Dell is associated with integrin clustering in focal complexes, and recruitment and phosphorylation of cytoplasmic signaling proteins such as p125FAK in these structures. These data suggest that Dell may be the primary ligand for alphavbeta3 during embryonic vascular development supporting the migration and survival functions of this integrin receptor. Functional evaluation in a chick chorioallantoic membrane (CAM) assay showed Dell to be a potent angiogenic factor, suggesting that it must activate signaling pathways in addition to those associated with integrins. Experiments proposed in this application will investigate through in vivo and in vitro studies the functional role of Dell in vascular development, and the molecular mechanisms that mediate these functions. The first series of experiments evaluate the in vivo role of Dell protein in early vascular formation, through gene targeting in the mouse. Complementary gain-of-function studies in the chick CAM assay will employ recombinant proteins and retroviruses to investigate the mechanism of Dell's angiogenic activity. In vitro experiments will determine the effects of Dell on endothelial cell division and differentiation, as well as further characterize the integrin and non-integrin cell surface proteins that mediate Dell binding. Finally, the ability of Dell to protect the endothelial cell from apoptosis will be evaluated, as well as the integrin and growth factor signaling pathways that mediate Dell's angiogenic activity. These experiments should establish the in vivo functions of Dell, and provide an initial understanding of the molecular mechanisms involved.
