Our goals are to identify and understand the function of genes involved in heart development and function. We are conducting these studies in zebrafish, a vertebrate model system well suited to embryological and genetic studies. Over the past few years, we have investigated topics such as myocardial differentiation, heart tube formation, endocardial cushion development and cardiac function. We have also conducted a large-scale forward genetic screen for additional genes regulating cardiovascular development and have identified several remarkable mutants. One of these, grinch, appears to lack all myocardial cells and another, s617, appears to define a new class of cardia bifida mutants. We will focus our efforts on these two mutants, as well as 3 genes regulating endocardial cushion development and 3 other genes regulating cardiac function. Thus, we propose to continue our studies of zebrafish heart development and function with the following specific aims: 1) Investigate myocardial differentiation. We will focus our efforts on the grinch mutation which appears to block myocardial differentiation. Detailed phenotypic analysis and gene isolation will allow us to formulate more precise hypotheses regarding the role of Grinch in this process. 2) Investigate heart tube formation. These studies will focus on the s617 gene which appears to regulate heart tube formation through a previously uncharacterized mechanism. Indeed, the myocardial epithelium and the overlying pharyngeal endoderm exhibit no apparent defects in s617 mutant embryos (apart from the migration defect of the myocardium). 3) Analyze endocardial cushion development by focusing on the role of 3 genes in this process, metronome, s225 and s482. These genes appear to regulate different aspects of endocardial cushion development and our initial studies will focus on detailed phenotypic analyses and gene isolation. 4) Investigate the role of doc-oc, slip jig and daredevil in cardiac function. In order to better characterize cardiac function phenotypes, we will utilize the newly developed 'single plane illumination microscopy1 in combination with a transgenic line that allows the monitoring of intracellular calcium concentrations in vivo. 5) Continue to develop reagents and techniques to facilitate the use of the zebrafish as a rapid secondary screening system for genes regulating heart development. ? ? ?
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