) 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, flt-1, tie-2, and VEGF, result in embryonic lethality due to aberrant vascular development. Interestingly, mutations in each gene gives rise to distinct phenotypic abnormalities. These data indicate that multiple signaling pathways, each with non-overlapping functions, are required for elaboration of the 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, has been shown 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 role in recruiting endothelial cells into the developing vasculature during embryogenesis and processes such as wound healing and tumor angiogenesis in the adult animal. To test this hypothesis, gene targeting in embryonic stem (ES) cells will be used to generate B61 knockout mice. Homozygous mutant embryos will be analyzed for defects in endothelial cell and blood vessel development. Second, the role of the B61/eck signaling pathway in adult animals will be assessed using three in vivo assays. B61 and eck knockout mice (providing that B61 mutant mice are viable; eck mutant mice are viable) will tested for wound healing capability and extent of tumor growth and tumorigenesis induced by carcinogen skin treatment. In addition, ES cells of defined B61 and eck genotypes (e.g., B61 -/- and/or eck -/- cell lines) will be tested for the ability to form vascularized teratocarcinomas after subcutaneous injection into wild type, B61 mutant, or eck mutant hosts. 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. In the future, the proposed investigations will help to determine whether B61 and eck molecules can be targeted for therapy.
