Mutagenic Gene Trapping to Study Novel Genes in Zebrafish Cardiovascular Development

背景、策略和远期目标：斑马鱼具有胚胎透明、胚胎发育快、体外发育快、繁殖力强等特点，为功能基因组学和发育生物学研究提供了独特的模式系统。斑马鱼是心血管(CV)研究的一个特别强大的范例，因为它在早期发育过程中能够在缺乏功能CV系统的情况下存活。该项目的长期目标是使用突变基因捕获技术识别和表征斑马鱼CV发育中涉及的新基因，这是一种在整个基因组中随机引入插入突变的技术。我们使用了一个成熟的RP2基因断裂蛋白陷阱系统(Clark等人，自然方法，2011；8：506)，该系统突变并荧光标记捕获的基因产物(融合蛋白与RFP)。初步结果与特定目的：在总共3000多个注射RP2的胚胎中，350条鱼显示出转基因的种系传递。在已建立的品系中，141个品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。在这项拨款申请中，我们建议研究两个心血管基因陷阱系：(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位于20号染色体上的PDGFRA(阿尔法型血小板衍生生长因子受体)。(2)RP2#121菌株：RFP在心脏和骨骼肌中强烈表达。然而，捕获的基因还没有确定。假设：基因捕获是一种很好的策略，可以产生一组突变来促进基于假设的研究。我们将为每个已识别的突变基因(陷阱基因)形成一个特定的假设。对于品系RP2#91，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信号通路发挥作用，调节内皮细胞的增殖/凋亡和血管生成。研究目的和计划：具体研究目标和相关研究计划包括：目的1：基因捕获品系RP2#91(PdgfraRP2)的表型和分子特征。我们首先计划通过使用斑马鱼细胞类型特异性GFP系或免疫染色技术来确定PDGFRA表达的细胞类型-血管平滑肌细胞与周细胞。RNA原位杂交也将被用来建立时空基因表达谱。我们将充分研究PDGFRA CV突变的表型，并确定突变胚胎中的分子缺陷。目的2：通过挽救或表型复制pdgfraRP2(-/-)表型来验证功能基因中断。RP2阻断吗啡或Cre基因的微量注射实验将被用于挽救表型，而PDGFRA化学抑制剂将被用于表型复制。目的3：PDGFRA在斑马鱼血管发育和功能中作用的分子机制。我们计划研究pdfgra的磷酸化和随后激活的细胞信号通路在调节血管发育、成熟和血管生成中的作用。由于壁细胞和内皮细胞之间的相互干扰和相互作用，我们将应用多种技术来研究内皮细胞的迁移、增殖和凋亡。目的4：心脏基因陷阱系RP2#121的特征研究意义：对两个CV特异性基因捕获斑马鱼系的研究将有助于解释它们的功能，并使我们能够剖析脊椎动物心血管发育的分子机制。

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.