Regulation of Vascular Integrity

如本报告的目标和目的部分所述，该项目包括两个具体目标： 利用正向遗传学分离调控血管完整性的新基因 我们使用正向遗传方法来识别和表征新的突变体，这些突变体专门破坏斑马鱼的脑血管完整性，目的是识别影响出血性中风易感性的新候选基因。我们正在对ENU诱导的显性出血易出血突变进行F2遗传筛选。到目前为止，我们已经筛选了500多个基因组，并鉴定了15个新的血管突变，这些突变显示出对颅内出血的易感性增加。一个庞大的分离株定位管道已经到位，以快速确定新发现的突变体在斑马鱼遗传图谱上的大致位置，许多突变体的精细定位和分子克隆正在进行中。我们还使用新开发的SNP数据库中的外显子测序和SNP分析来进行突变体的高通量克隆。我们之前已经从我们的遗传筛选中克隆了许多影响血管发育的新基因，这些新的血管完整性突变有望揭示在维持血管屏障功能方面重要的新途径。 人类中风病基因斑马鱼同源基因的功能分析 与Elizabeth Tournier-LasServe博士的实验室一起，我们正在对人类出血性中风基因的斑马鱼同源基因进行实验分析。我们正在研究脑海绵状血管畸形(CCM)、烟雾病和其他脑血管疾病患者的缺陷基因。我们的目标是通过使用鱼类中可用的遗传和实验工具来探索它们在体内的功能，从而更好地了解这些基因在维持血管完整性方面的作用。我们最近已经证明Brcc1基因(其中的病变导致人类综合征Moya-Moya，一种导致过度脆弱的血管形成的脑血管疾病)是体内血管生成所必需的。

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.