Mutagenic Gene Trapping to Study Novel Genes in Zebrafish Cardiovascular Development

诱变基因捕获研究斑马鱼心血管发育中的新基因

• 批准号: RGPIN-2014-05389
• 负责人: Wen, XiaoYan
• 金额: $ 2.99万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611612
• 关键词: Mutagenic; Gene; Trapping; Study; Novel

Background, strategy and long-term goal: Zebrafish provides a unique model system for functional genomics and developmental biology studies, owing to its embryonic transparency, rapid and external embryonic development and high fecundity. The zebrafish is especially a powerful paradigm for cardiovascular (CV) research due to its capability of surviving in the absence of a functional CV system during early development. The long-term goal of this project is to identify and characterize novel genes involved in zebrafish CV development using mutagenic gene trapping, a technique that randomly introduces insertional mutations across the genome. We have employed a well-established RP2 gene-breaking protein trap system (Clark et al, Nature Methods, 2011;8:506) that mutates and fluorescently tags the trapped gene product (fusion protein with RFP). It also introduces loxP sites into the mutant locus, which can be used for Cre-mediated phenotype rescue.Preliminary results & specific goals: From a total of over 3000 RP2-injected embryos, 350 fish showed germline transmission of the transgene. Among the established lines, 141 lines expressed strong RFP in specific tissues, including the central nervous system, heart, vessels, muscle, and eyes. In this grant application, we propose to study two cardiovascular gene trap lines: (1) RP2#91 strain: RFP is strongly expressed in the vascular mural cells but not the endothelial cells. The trapped gene is identified as pdgfra (alpha-type platelet-derived growth factor receptor) on chromosome 20. (2) RP2#121 strain: RFP is strongly expressed in the ventricle of the heart and in skeletal muscles. However, the trapped genes have yet to be identified.Hypothesis: Gene trapping is an excellent strategy to generate a panel of mutations to foster hypothesis-based research. We will form a specific hypothesis for each identified mutant gene (trapped gene). For line RP2#91, we hypothesize that (1) pdgfra is required for zebrafish cardiovascular development and function; (2) In zebrafish mural cells, pdgfra exerts its function through Ras-MAPK signaling pathway to regulate endothelial cell proliferation/apoptosis and angiogenesis.Research Aims and Plan: The specific research aims and associated research plan include: Aim 1: Phenotypic and molecular characterization of gene-trapped line RP2#91 (pdgfraRP2). We first plan to define the cell types of pdgfra expression - vascular smooth muscles cells vs. pericytes, by employing zebrafish cell-type specific GFP lines or immnunostaining techniques. RNA in-situ hybridization will also be used to establish spatio-temporal gene expression profile. We will fully characterize pdgfra CV mutant phenotypes and define the molecular defects in mutant embryos. Aim 2: Validation of functional gene disruption by rescuing or phenocopying pdgfraRP2(-/-) phenotype. RP2-blocking morpholino or Cre mRNA microinjection experiments will be used to rescue the phenotype and PDGFRA chemical inhibitors will be used to phenocopy the mutation. Aim 3: Molecular mechanisms of pdgfra function in zebrafish vascular development and function. We plan to study pdfgra phosphorylation and the subsequent activation of cellular signaling pathways in regulation of vascular development, maturation and angiogenesis. Because of the crosstalk and interactions between mural and endothelial cells, we will apply various techniques to study endothelial cell migration, proliferation and apoptosis. Aim 4: Characterization of cardiac gene trap line RP2#121.Significance: The proposed studies of the two CV-specific gene-trapped zebrafish lines will help annotate their function and enable us to dissect the molecular mechanisms underlying cardiovascular development in vertebrates.

背景、策略和长期目标：斑马鱼由于其胚胎透明、快速的外部胚胎发育和高繁殖力，为功能基因组学和发育生物学研究提供了独特的模型系统。斑马鱼尤其是心血管（CV）研究的有力范例，因为它能够在早期发育过程中在缺乏功能性心血管系统的情况下生存。该项目的长期目标是利用诱变基因捕获（一种在基因组中随机引入插入突变的技术）来识别和表征斑马鱼 CV 发育中涉及的新基因。我们采用了完善的 RP2 基因破坏蛋白捕获系统（Clark 等人，NatureMethods，2011；8:506），该系统可以突变并荧光标记捕获的基因产物（与 RFP 的融合蛋白）。它还将 loxP 位点引入突变位点，可用于 Cre 介导的表型拯救。初步结果和具体目标：在总共 3000 多个注射 RP2 的胚胎中，有 350 条鱼显示出转基因的种系传递。在已建立的细胞系中，有 141 个细胞系在特定组织中表达强 RFP，包括中枢神经系统、心脏、血管、肌肉和眼睛。在本次资助申请中，我们建议研究两种心血管基因捕获株系：（1）RP2#91株：RFP在血管壁细胞中强烈表达，但在内皮细胞中不表达。捕获的基因被鉴定为20号染色体上的pdgfra（α型血小板衍生生长因子受体）。 (2)RP2#121株：RFP在心室和骨骼肌中强烈表达。然而，捕获的基因尚未被识别。假设：基因捕获是生成一组突变以促进基于假设的研究的绝佳策略。我们将为每个识别的突变基因（捕获基因）形成一个特定的假设。对于 RP2#91 系，我们假设 (1) pdgfra 是斑马鱼心血管发育和功能所必需的； （2）在斑马鱼壁细胞中，pdgfra通过Ras-MAPK信号通路发挥其功能，调节内皮细胞增殖/凋亡和血管生成。 研究目的和计划：具体研究目的和相关研究计划包括： 目的1：基因捕获系RP2#91（pdgfraRP2）的表型和分子表征。我们首先计划通过采用斑马鱼细胞类型特异性 GFP 系或免疫染色技术来定义 pdgfra 表达的细胞类型 - 血管平滑肌细胞与周细胞。 RNA原位杂交也将用​​于建立时空基因表达谱。我们将全面表征 pdgfra CV 突变表型并定义突变胚胎中的分子缺陷。目标 2：通过拯救或表型复制 pdgfraRP2(-/-) 表型来验证功能性基因破坏。 RP2 阻断吗啉或 Cre mRNA 显微注射实验将用于挽救表型，PDGFRA 化学抑制剂将用于对突变进行表型复制。 目标 3：pdgfra 在斑马鱼血管发育和功能中发挥作用的分子机制。我们计划研究 pdfgra 磷酸化和随后激活的细胞信号通路在血管发育、成熟和血管生成调节中的作用。由于壁细胞和内皮细胞之间的串扰和相互作用，我们将应用各种技术来研究内皮细胞的迁移、增殖和凋亡。目标 4：心脏基因捕获系 RP2#121 的表征。意义：对两种 CV 特异性基因捕获斑马鱼系的拟议研究将有助于注释它们的功能，并使我们能够剖析脊椎动物心血管发育的分子机制。