Endothelial cells serve a broad range of functions, and are central figures in a variety of physiological and pathophysiological processes within the blood vessel wall. Characteristic features of endothelial cells have been determined through in- depth studies of these cells in culture, and a number of genes which mediate endothelial cell processes have been cloned. Experiments investigating pathological blood vessel growth in the adult animal and vascular development in the embryo have highlighted the dominant role of this cell type in all mechanisms of blood vessel formation. Despite the importance of this cell type, there has been very little research at the molecular level to investigate fundamental processes such as endothelial cell differentiation and the role of this process in blood vessel development.The goal of the proposed research is to investigate these critical processes through studies employing a transgenic mouse which contains a reporter transgene in a developmentally regulate endothelial cell-specific genetic locus, del-1. Given the pattern of expression of the transgene, it is hypothesized that the protein encoded in this locus is directly involved in the regulation of endothelial cell development.To investigate this possibility, del-1 has been characterized by genomic cloning, an exon has been identified by exon trapping and corresponding cDNAs isolated from an embryonic mouse library. The trapped exon and cDNAs encode a protein containing a discoidin-like domain which may be involved in intercellular interactions. Biochemical and genetic experiments are proposed to investigate the possible roles of this protein in endothelial cell development and vascular morphogenesis. Since the transgene is expressed in only one capillary circulation, the pulmonary microcirculation, tissue isolated from this mouse can be employed in epithelial-mesenchymal switch experiments to study the developmental basis for endothelial cell heterogeneity. Cultured cell lines have been established from yolk sac blood islands of transgenic embryos at 8 days of development. These cells are stable in culture, can be induced to form characteristic vascular- like structures in vitro, express a number of endothelial cell markers, and express the reporter transgene upon differentiation. Experiments will be conducted to further characterize the phenotype of these cells, and identify factors which will induce their further differentiation toward the definitive endothelial cell phenotype. Such experiments will provide a foundation for studies investigating the molecular basis of endothelial cell differentiation. Preliminary experiments indicate that these cells can contribute to the pulmonary circulation in embryonic lung in vitro. Thus, they can be used as an assay to study the mechanism of induction of features characteristic to lung capillary endothelial cells.
