We have recently identified a novel signaling pathway containing the tyrosine kinase Syk and the adaptor SLP-76 that is required for separation of blood and lymphatic vessels during lymphatic vascular development. Loss of this signaling pathway in mice results in blood-filled lymphatics and the creation of arterio-venous shunts mediated by aberrant lymphatic connections. How Syk signaling regulates separation of blood and lymphatic vessels is unknown and is the focus of this proposal. During lymphatic vascular development Syk is expressed exclusively in hematopoietic cells and reconstitution of lethally irradiated wild-type mice with Syk-deficient fetal liver is sufficient to confer the vascular phenotype, suggesting that Syk signaling may influence vascular development indirectly through circulating cells. Recent phenotypic studies, however, have led us to a novel, unifying hypothesis: we propose that Syk operates in a celt autonomous signaling pathway that is required by hematopoietic endothelial progenitors to create separate blood and lymphatic vessels. To test this model we will further characterize the sites and natural history of vascular mixing in Syk-deficient mice to determine if blood-lymphatic connections form exclusively at early stages of lymphatic growth. Our model predicts that the endothelial cells that mediate vascular mixing in the absence of Syk arise from Syk-expressing progenitors. We will test this prediction by expressing Cre from the syk locus and crossing syk-Cre mice to the ROSA26R Cre reporter strain to fate-map the progeny of Syk-expressing cells. To functionally test the role of Syk in endothelial cells and endothelial progenitors we will rescue Syk expression in those cells using transgenic promoters and excise syk from those cells using mice carrying a conditional syk allele. Finally, genetic studies suggest that Syk may be required downstream of integrins during vascular development. We will test this hypothesis by rescuing Syk-deficiency with mutant Syk proteins capable of interacting with integrin but not ITAM-containing receptors. These studies will determine if Syk signaling defines a pathway required by hematopoietic progenitors to contribute to vascular development in the embryo and vascular repair in the adult. Identification of such a pathway has broad implications for our understanding of vascular development and for angiogenic therapies utilizing hematopioetic endothelial precursors.