The neurovascular unit (NVU) consists of the brain endothelium, along with associated glia, pericytes and neurons. The molecular definition of mediators of cell-cell interaction between these cell types should yield significant insight into vascular function during physiologic and pathologic processes in the CNS. We have constructed a mouse knockout of GPR124, an orphan G-protein coupled receptor originally identified by its overexpression in tumor endothelium. Inactivation of GPR124 by replacement with a lacZ reporter gene results in embryonic lethality with hemorrhage which is strikingly restricted to the brain. Initial analysis of the mutant CNS microvasculature has revealed profound defects of angiogenic migration and sprouting, while expression analysis of GPR124 indicates expression is highly selective for CNS vasculature. The GPR124 phenotype is highly suggestive of defective paracrine signaling within cells of the neurovascular unit, resulting in improper angiogenesis. In the current proposal, the functions of GPR124 will be explored taking advantage of GPR124 knockout mice which we have created. First, the structure of aberrant vasculature in GPR124-/- mice will be studied with respect to qualitative and quantitative deficiencies of with other cells of the NVU, cellular junctions and extracellular matrix. Second, genetic epistatic relationships will be sought between GPR124 and other genes whose knockouts have similar phenotypes, such as integrins and Id transcriptional repressers. Third, cell types of the neurovascular unit, such as glia, pericytes and neurons, will be systematically tested for the ability to elicit GPR124-dependent responses in tissue culture. Finally, conditional approaches to GPR124 inactivation will be pursued using adenoviral and conditional knockout strategies, to facilitate the eventual study of GPR124 function in fully adult organisms. ? ?
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