As described in the goals and objectives section of this report, this project consists of two specific aims: Isolation of novel genes regulating vascular integrity via forward genetics We use forward-genetic approaches to identify and characterize new mutants that specifically disrupt cranial vascular integrity in the zebrafish, with the aim of identifying novel candidate genes influencing the susceptibility to hemorrhagic stroke. We are carrying out an F2 genetic screen for ENU-induced dominant hemorrhage-prone mutants. We have screened over 500 genomes to date, and identified 15 new vascular mutants showing increased susceptibility to intracranial hemorrhage. A bulked segregant mapping pipeline is in place to rapidly determine the rough position of newly identified mutants on the zebrafish genetic map, and fine mapping and molecular cloning is in progress for many of the mutants. We are also using exome sequencing and SNP analysis with a newly developed SNP database to perform higher-throughout cloning of mutants. We have previously cloned many novel genes influencing vascular development from our genetic screens, and these new vascular integrity mutants promise to bring to light new pathways important in the maintenance of vascular barrier function. Functional analysis of zebrafish orthologs of human stroke disease genes Together with the Laboratory of Dr. Elizabeth Tournier-Lasserve, we are carrying out experimental analysis on zebrafish orthologs of human hemorrhagic stroke genes. We are studying the defective genes from patients with Cerebral Cavernous Malformations (CCM), Moya-Moya, and other cerebrovascular diseases. Our goal is to better understand the roles of these genes in maintaining vascular integrity by using the genetic and experimental tools available in the fish to probe their functions in vivo. We have recently shown that the Brcc1 gene (lesions in which cause human syndromic Moya-Moya, a cerebrovascular disease resulting in excess fragile vessel formation) is necessary for angiogenesis in vivo.

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