Analysis of Mural Cell Ontogeny and Gene Function During Vascular Development

血管发育过程中壁细胞个体发育和基因功能分析

基本信息

批准号：
9812864
负责人：
Amber Nicole Stratman
金额：
$ 24.9万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-12-15 至 2021-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9812864
关键词：
Adult Animals Atherosclerosis Basic Science Behavior Biology Blood Vessels Candidate Disease Gene Cardiovascular system Cell Communication Cell Differentiation process Cell Ontogeny Cell Proliferation Cell physiology Cells Cellular biology Communities Cues Defect Development Diabetic Retinopathy Disease Embryo Endothelial Cells Ethylnitrosourea Functional disorder Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Genetic Screening Goals Growth Human Larva Ligands Maintenance Mammals Messenger RNA Modeling Mutagenesis Onset of illness Optics PDGFRB gene Pathology Pericytes Phase Phase Transition Phosphoproteins Platelet-Derived Growth Factor Play Population Process Proteomics Regulation Research Retinal Diseases RiboTag Role Signal Pathway Signal Transduction Site Smooth Muscle Myocytes Stroke Technology Therapeutic Intervention Time Tissues Transgenic Organisms Translating Tube Work Zebrafish base cell behavior cellular imaging expectation experimental study faculty research gene function genetic approach genome-wide high resolution imaging in vivo insight interest live cell imaging member novel programs protein profiling recruit restenosis tenure track tool transcriptome sequencing two photon microscopy vascular bed

项目摘要

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.

描述（由申请人提供）：摘要/项目摘要壁细胞在促进血管内皮细胞（EC）层的长期稳定方面发挥着不可或缺的作用，并且对于在发育过程中和发育过程中正确维持循环系统至关重要。一个成年人。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、视网膜病变和其他人类血管病变有关，从长远来看，了解这些细胞的发育起源和调节其行为的基因尤为重要。疾病中的功能障碍。使用最近开发的斑马鱼转基因系标记体内限制的壁细胞群，延时双光子显微镜将用于谱系追踪实验，以确定填充不同血管床的壁细胞的个体发育。此外，之前描述的壁细胞功能调节因子，例如 PDGFR？，将通过条件基因抑制策略进行操纵，以验证这些细胞对与哺乳动物中注意到的类似提示做出反应，并为这些提示所起的作用提供更广泛的理解在壁细胞发育阶段，例如从组织起源部位分层、细胞增殖、细胞募集到脉管系统以及 EC-壁细胞异型相互作用（目标 1）。目标 2 将侧重于利用“RiboTag”技术的组合来识别壁细胞中调节的新基因程序，以分离和分析通过 RNA-Seq 翻译不同发育阶段壁细胞的 mRNA，并对 FAC 分选的壁细胞群进行蛋白质组学分析，以评估壁细胞发育过程中总蛋白和磷酸化蛋白水平的变化（目标 2）。最后，除了全基因组 ENU 诱变筛选之外，还将对已识别的调节基因（其中对人类疾病有影响的基因具有最高优先级的分析）进行功能分析，以获得对指导壁细胞行为的基因程序的更广泛的了解。发育和疾病（目标 3）。采用这种基于多方面遗传学的方法来了解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，同时为疾病分析提供一个起点，并为我提供帮助。在血管生物学界有一个独特的利基，可以开始担任终身教职研究教员。