Analysis of Mural Cell Ontogeny and Gene Function During Vascular Development

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，对发育和成年后循环系统的正常维持至关重要。壁细胞分化缺陷或与内皮细胞相互作用与动脉粥样硬化/再狭窄、中风、视网膜病变和其他人类血管病变有关，了解这些细胞的发育起源和调节其行为的基因对确定和理解疾病功能障碍具有特别重要的长期意义。利用最近开发的斑马鱼转基因系标记体内受限的壁细胞群体，延时双光子显微镜将用于谱系追踪实验，以确定壁细胞在各种血管床上的个体发生。此外，先前描述的壁细胞功能调节因子，如PDGFR？将通过条件基因抑制策略来操纵这些细胞，以验证这些细胞对哺乳动物中发现的类似信号的反应，并提供更广泛的理解这些信号在壁细胞发育阶段所起的作用，例如从其组织起源部位剥离、细胞增殖、细胞向脉管系统募集和ec -壁细胞异型相互作用（目的1）。目标2将专注于鉴定在壁细胞中调节的新基因程序，利用“RiboTag”技术的组合，分离和分析

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.