Progressive growth of tumors is dependent on continuous stimulation of new blood vessel formation. Considerable evidence indicates that receptor-protein tyrosine kinases (R-PTKs) and their ligands have crucial roles in the differentiation of endothelial cells and their subsequent organization into the vascular system. """"""""Knockout mice"""""""" carrying mutations in endothelial cell specific R-PTKs and ligands, such as flk-1, flk-1, tie-2, and VEGF, result in embryonic lethality due to aberrant vascular development. Interestingly, mutation in each gene gives rise to distinct phenotypic abnormalities. These data indicate that multiple signaling pathways, each with nonoverlapping functions, are required for elaboration of the endocardium and vasculature. It will be essential to identify and characterize other loci that contribute to endothelial cell development in order to define the multiple pathways required for vasculogenesis and angiogenesis. B61, the major ligand for the ECK R-PTK (B61 also binds other members of the eph family), was shown by others to possess angiogenic and endothelial cell attractant activity. Consistent with this notion, B61 is expressed at sites of blood vessel formation during mouse embryogenesis. These data raise the intriguing possibility that B61 has a major role in recruiting endothelial cells into the developing vasculature. To test this hypothesis, first, candidate receptors for the B61 ligand will be identified by screening each member of the eph family for expression in the developing mouse vascular system by whole mount immunohistochemistry. Second, gene targeting in embryonic stem cells will be used to generate mice that carry null mutations at the B61 locus. Homozygous mutant embryos will be analyzed for defects in endothelial cell and blood vessel development. The extent of vascular development in mutant embryos will be assessed by histological analysis and by examination of the expression of three genes, flk-1, flt-1 and tek , that are markers for early to late stages of endothelial cell differentiation. Third, to assess the role of B61 and eck in tumor angiogenesis, embryonic stem cell lines that carry double knockouts at the B61 and/or eck loci (i.e., B61-1- and/or eck-1 cell lines) will be used to generate teratocarcinomas in syngeneic or immunocompromised mice. Tumors will be examined after four weeks for extent of tumor growth and vascular development. It is anticipated that these data will provide insight into the function of the B61/ECK signaling pathway in vascular development during embryogenesis and tumorigenesis.
