Studies are proposed to analyze the several discrete steps by which the most primitive precursors of endothelial cells can, during mammalian embryonic development, generate the diverse blood vessels that comprise the microvasculature of the emerging organs. These steps are believed to include: 1) the emergence from postulated hemangioblasts of hematopoietic stem cells which give rise to tahe various lineages of the circulating blood, and angioblasts which give rise to blood vessels. Angioblasts are first detectable in the extraembryonic yolk sac where they can be shown to coalesce to give rise to the first visible microvessels. Although subsequently the initial blood vessels may branch and penetrate different organ rudiments (angiogenesis), a significant number of angioblasts migrate as individual cells into developing organ rudiments to participate there in the formation of specific microvascular beds (vasculogenesis). To what extent there is also differentiation and recruitment of locally arising mesenchymal cells in the formation of the microvessels in different organ sites is not known. To study the sequence of events underlying the development of the microvasculature experiments are proposed to: 1. a. Identify and isolate yolk sac angioblasts; b. Characterize these angioblasts in terms of their cell surface phenotype, response to growth factors, expression of specific kinases and synthesis of cytokines; c. Develop methods that permit angioblast proliferation without differentiation, thus establishing angioblast cell lines; d. Establish methods that permit angioblasts to form three-dimensional tubes or patent vessels in vitro. 2. a. determine the emergence of lung-specific properties in the developing lung rudiment seeded by yolk sac angioblasts (1a or 1c; vasculogenesis) or penetrated by yolk sac microvessels (1d; angiogenesis) b. similarly determine the pattern of differentiation of myocardium associated microvessels during heart development c. delineate the appearance of brain-specific properties in the developing microvasculature of the forebrain/cerebrum 3. analyze, using the information generated above, the earliest emergence of angioblast-specific properties in cells from the inner cell mass of the early embryo or generated from embryonic stem cell (ES cell) precursors. Together, these studies will provide a picture of how organ-specific heterogeneity of the microvasculature occurs in the mammalian embryo. Understanding of this process may lead to development of strategies for selective augmentation or inhibition of blood vessel formation and provide insight into such specialized vascular differentiations as those associated with inflammation, establishment of tumors, or the pathogenesis of a wide array of angiogenic diseases.
