Analysis of Mural Cell Ontogeny and Gene Function During Vascular Development

描述（由申请人提供）：摘要/项目概述壁细胞在促进血管内皮细胞（EC）层的长期稳定中起着不可或缺的作用，并且对于发育期间和成年时循环系统的适当维护至关重要。壁细胞分化或与EC相互作用的缺陷与动脉粥样硬化/再狭窄、中风、视网膜病和其他人类血管病理学有关，并且理解这些细胞的发育起源和调节其行为的基因对于长期确定和理解疾病中的功能障碍特别重要。使用最近开发的斑马鱼转基因线标记限制壁细胞群体在体内，延时双光子显微镜将用于谱系追踪实验，以确定个体发育的壁细胞填充各种血管床。此外，先前描述的壁细胞功能调节剂，如PDGFR？，将通过条件性基因抑制策略进行操作，以验证这些细胞对哺乳动物中观察到的类似线索的反应，并提供对这些线索在壁细胞发育阶段中所起作用的更广泛理解，例如从其组织来源部位分层，细胞增殖，细胞募集到脉管系统和EC-壁细胞异型相互作用（Aim 1）。目的二是结合RiboTag技术，对壁细胞中调控的新基因进行分离和表达谱分析 通过RNA-Seq翻译来自不同发育阶段的壁细胞的mRNA，并对FACs分选的壁细胞群体进行蛋白质组学分析，以评估壁细胞发育期间总蛋白和磷蛋白水平的变化（目标2）。最后，除了全基因组ENU诱变筛选外，还将对鉴定的调控基因进行功能分析（对人类疾病具有影响的那些基因具有最高的分析优先级），以更广泛地了解发育和疾病期间指导壁细胞行为的基因程序（目的3）。以这种多方面的遗传学为基础的方法来了解在发育过程中控制壁细胞行为的信号，将拓宽对“正常”壁细胞生物学的认识，同时为疾病的分析提供一个起点， 在血管生物学界的一个独特的利基开始工作，作为一个终身研究教员。

DESCRIPTION (provided by applicant): ABSTRACT/PROJECT SUMMARY Mural cells play an indispensable role in promoting long-term stabilization of the endothelial cell (EC) layer linin the vascular tube and are critical for proper maintenance of the circulatory system both during development and as an adult. Defects in mural cell differentiation or interactions with ECs are implicated in atherosclerosis/restenosis, stroke, retinopathies and other human vascular pathologies, and understanding the developmental origins of these cells and the genes regulating their behavior is of particular importance long- term to determine and understand dysfunction in disease. Using recently developed zebrafish transgenic lines marking restricted mural cell populations in vivo, time-lapse two-photon microscopy will be used for lineage tracing experiments to determine the ontogeny of mural cells populating various vascular beds. Additionally, previously described regulators of mural cell function, such as PDGFR?, will be manipulated through conditional gene suppression strategies to validate that these cells are responding to similar cues to those noted in mammals and to provide a broader understanding to the role these cues play in stages of mural cell development, such as delamination from their tissue site of origin, cell proliferation, cell recruitment to the vasculature and EC-mural cell heterotypic interactions (Aim 1). Aim 2 will focus on the identification of novel gene programs regulated in mural cells by utilizing a combination of 'RiboTag' technology, to isolate and profile translating mRNAs from mural cells at various stages of development through RNA-Seq, with proteomics analysis of FACs sorted mural cell populations, to assess changes in total and phospho-protein levels during mural cell development (Aim 2). Finally, functional analysis will be carried out on identified regulated genes (with those that have implications in human disorders having highest priority for analysis) in addition to a genome wide ENU mutagenesis screen, to gain a broader understanding of gene programs directing mural cell behavior during both development and disease (Aim 3). Taking this multifaceted genetics based approach to understand signals governing mural cell behavior during development will broaden insights into 'normal' mural cell biology while providing a starting point for analysis of disease and provide me a unique niche in the vascular biology community to begin work as a tenure-track research faculty member.