Mutagenic Gene Trapping to Study Novel Genes in Zebrafish Cardiovascular Development

背景、策略和长期目标：斑马鱼以其胚胎透明、胚胎发育快速、体外发育和繁殖力高的特点，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。斑马鱼是心血管（CV）研究的一个强有力的范例，因为它在早期发育过程中没有功能心血管系统的情况下存活的能力。该项目的长期目标是利用诱变基因诱捕技术（一种在基因组中随机引入插入突变的技术）鉴定和表征与斑马鱼CV发育有关的新基因。我们采用了一种完善的RP2基因断裂蛋白捕获系统（Clark等人，Nature Methods, 2011;8:506），该系统对捕获的基因产物（与RFP融合的蛋白）进行突变和荧光标记。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多个注射了rp2的胚胎中，350条鱼显示出转基因的种系传播。在已建立的细胞系中，141个细胞系在特定组织中表达强烈的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，我们拟研究两种心血管基因诱捕系：(1)RP2#91菌株：RFP在血管壁细胞中强烈表达，而在内皮细胞中不表达。被捕获的基因在20号染色体上被鉴定为pdgfra （α型血小板衍生生长因子受体）。(2) RP2#121菌株：RFP在心脏心室和骨骼肌中强烈表达。然而，被困的基因尚未被识别。假设：基因诱捕是一种极好的策略，可以产生一系列突变，以促进基于假设的研究。我们将对每一个鉴定出的突变基因（诱捕基因）形成一个特定的假设。对于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，调控内皮细胞增殖/凋亡和血管生成。研究目标和计划：具体研究目标和相关研究计划包括：目标1：基因捕获系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原位杂交也将用于建立基因的时空表达谱。我们将充分表征pdgfra CV突变表型，并定义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破坏。将使用rp2阻断morpholino或Cre mRNA显微注射实验来挽救表型，并使用PDGFRA化学抑制剂来表型突变。目的3:pdgfra在斑马鱼血管发育和功能中的分子机制。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、成熟和血管生成中的调控作用。由于壁细胞和内皮细胞之间的相互作用和相互作用，我们将应用各种技术来研究内皮细胞的迁移、增殖和凋亡。目的4：心脏基因诱捕系RP2#121的鉴定。意义：对两种cv特异性基因捕获的斑马鱼品系的研究将有助于说明它们的功能，并使我们能够解剖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.