Mutagenic Gene Trapping to Study Novel Genes in Zebrafish Cardiovascular Development

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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.