Eisa-Beygi, Shahram

The zebrafish has emerged as an excellent model for cardiovascular research thanks to its ex-utero and rapid embryonic development, its embryonic transparent nature, and its capacity to survive in the absence of a functional cardiovascular system during the first week of development, which enables functional characterization of mutations that would otherwise induce lethality in traditional murine models. The aim of this project is to identify and characterize novel genes involved in zebrafish cardiovascular development using mutagenic gene trapping, a technique that generates random insertional mutations across the genome. We use the well-defined RP2 gene-breaking transposon system, which not only mutates, but also fluorescently tags the trapped gene product(s) (Clark, Nat Methods, 2011). RP2 also introduces loxP sites into the mutant locus, which can be used for Cre-mediated phenotype rescue by microinjection of Cre recombinase, or by crossing to tissue-specific Cre lines. From over 3000 RP2-injected embryos, 141 fish showed germline transmission. Among them, 51 expressed strong fluorescence in different tissues, including the heart, vessels, notochord, central nervous system (CNS), and eyes. Three cardiovascular lines were selected for phenotypic characterization and functional studies. RP2#C2 strain expressed fluorescence in the heart valves, CNS, eyes and pectoral fin buds. Inverse PCR in RP2#C2 demonstrated a trapped gene at meis4.1a, which encodes a homeobox transcription factor that has not been previously studied in zebrafish. RP2#121 strain expressed strong fluorescence in cardiac and skeletal muscles. RP2#91 strain showed expression in the vasculature and demonstrated a trapped gene at pdgfra. Homozygous RP2#91 mutants showed severe defects in the heart, blood flow, and other body parts including the head and musculature. We are currently creating a panel of tissue-specific Cre lines targeting tissues such as cardiomyocytes, endothelial cells and smooth muscle cells for spatiotemporal rescuing of the mutant phenotype. The generated zebrafish protein-trap lines are invaluable tools to annotate gene function, dissect the molecular mechanisms of cardiovascular development, and potentially serve as disease models.

Cardiac development in vertebrates is a finely tuned process regulated by a set of conserved signaling pathways. Perturbations of these processes are often associated with congenital cardiac malformations. Platelet-derived growth factor receptor α (PDGFRα) is a highly conserved tyrosine kinase receptor, which is essential for development and organogenesis. Disruption of Pdgfrα function in murine models are embryonic lethal due to severe cardiovascular defects, suggesting a role in cardiac development, thus necessitating the use of alternative models to explore its precise function. In this study, we generated a zebrafish pdgfra mutant line by gene trapping, in which the Pdgfra protein is truncated and fused with mRFP (Pdgfra-mRFP). Our results demonstrate that pdgfra mutants have defects in cardiac morphology as a result of abnormal fusion of myocardial precursors. Expression analysis of the developing heart at later stages suggested that Pdgfra-mRFP is expressed in the endocardium. Further examination of the endocardium in pdgfra mutants revealed defective endocardial migration to the midline, where cardiac fusion eventually occurs. Together, our data suggests that pdgfra is required for proper medial migration of both endocardial and myocardial precursors, an essential step required for cardiac assembly and development.