Molecular Genetic Dissection of Fenestrated Vascular Development in the Choroid Plexus

项目摘要/摘要 脑血管疾病仍然是导致长期严重残疾的主要原因。这些疾病中的许多 至少部分起源于影响血管发育和成熟的遗传缺陷 中枢神经系统(CNS)和脑膜。尽管我们对中枢神经系统的理解取得了实质性进展 血管生成和屏障生成，我们对高渗透性或有窗口的微血管系统的了解 在脉络丛(CP)和室周器官中形成的细胞仍然有限。这个项目的总体目标是 建议通过了解人类免疫缺陷的分子遗传机制来填补科学知识的这一空白。 开窗的脑血管发育，特别是CP。我们最近发现CP 斑马鱼的血管化需要一种独特的血管生成机制，斑马鱼的血管内皮细胞 生长因子(VEGF)同源基因，特别是Vegfab和VEGFC，在功能上扮演着多余的角色。令人惊讶的是， 这一机制是CP血管形成所特有的，因为邻近的非开窗脑血管是 在蔬菜中不受影响；VEGFC双突变。这些结果导致我们制定了一种范式转换 有开窗CP和无开窗脑血管发育的假说需要不同组的 血管内皮生长因子/血管内皮生长因子受体编码和信号转导。考虑到Vegf在不同的病理生理过程中的关键作用 此外，在任何脊椎动物物种中都没有报道过驱动CP血管形成的分子线索， 本研究结果将对CP生理和血管领域产生重大的积极影响 CP血管化以外的生物学。我们进一步的遗传、药理学和表达数据表明 CP上皮细胞旁分泌VEGFC和内皮细胞自分泌VEGFC共同作用的模型 通过PI3K和ERK信号通路控制CP的血管形成。以下两个具体目标是 来检验我们的假设。目标1将检验开窗脑和非开窗脑的假说 血管发育需要不同的血管内皮生长因子/血管内皮生长因子受体编码和信号。目标2将检验这一假设 CP上皮细胞旁分泌VEGFb和自分泌VEGFC的时空协同作用 内皮细胞共同驱动有窗孔的CP血管发育。我们将通过以下方式实现这些目标 具有先进成像技术和遗传镶嵌分析的新型斑马鱼分子遗传工具。这个 建议的工作是这一领域的一项创新性的分子遗传学研究，因为它利用了 斑马鱼模型以克服技术限制并解决以下基本过程：1) 开窗CP血管发育的细胞和分子基础；2)发育机制 异质脑血管网络形成的背后。从这里收集到的机械性信息 这项工作将为CP血管发病机制的潜在分子途径提供新的线索 此外，它还可以帮助设计预防或治疗中枢神经系统血管疾病的疗法。

PROJECT SUMMARY/ABSTRACT Cerebrovascular diseases remain a leading cause of serious long-term disability. Many of these diseases originate, at least in part, from genetic defects that impact the development and maturation of blood vessels in the central nervous system (CNS) and meninges. Despite substantial progress in our understanding of CNS angiogenesis and barriergenesis, our knowledge of the highly permeable, or fenestrated, microvasculature formed in the choroid plexus (CP) and circumventricular organs remains limited. The overall goal of this proposal is to fill this gap in scientific knowledge by understanding the molecular genetic mechanisms of fenestrated brain vascular development, specifically focusing on the CP. We recently found that CP vascularization in zebrafish requires a unique angiogenic mechanism whereby zebrafish vascular endothelial growth factor (Vegf) orthologs, specifically Vegfab and Vegfc, play a functionally redundant role. Surprisingly, this mechanism is specific to CP vascularization, since the neighboring non-fenestrated brain vasculature is not affected in the vegfab;vegfc double mutants. These results led us to formulate a paradigm-shifting hypothesis that fenestrated CP and non-fenestrated brain vascular development requires distinct sets of the Vegf/Vegfr codes and signaling. Given the critical roles for Vegfs in diverse pathophysiological processes and also the fact that no molecular cue that drives CP vascularization has been reported in any vertebrate species, the results of this study will have a significant positive impact on the fields of CP physiology and vascular biology beyond CP vascularization. Our further genetic, pharmacological, and expression data propose a model in which paracrine Vegfab from CP epithelial cells and autocrine Vegfc from the endothelium together controls CP vascularization via PI3K and ERK signaling pathways. The following two Specific Aims are proposed to test our hypotheses. Aim 1 will test the hypothesis that fenestrated CP and non-fenestrated brain vascular development requires distinct Vegf/Vegfr codes and signaling. Aim 2 will test the hypothesis that the spatiotemporal coordination of paracrine Vegfab from CP epithelial cells and autocrine Vegfc from the endothelium together drives fenestrated CP vascular development. We will pursue these aims by combining novel zebrafish molecular genetic tools with advanced imaging technologies and genetic mosaic analyses. The proposed work is an innovative molecular genetics study in this field because it exploits various strengths of the zebrafish model to overcome technical limitations and address the following fundamental processes: 1) the cellular and molecular basis of fenestrated CP vascular development; and 2) the developmental mechanisms behind the formation of heterogeneous brain vascular networks. Mechanistic information gleaned from this work will shed new light on potential molecular pathways involved in the pathogenesis of CP vascular malformations and also help to design therapeutics for preventing or treating CNS vascular diseases.