HEART DEVELOPMENT AND GENETICS

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This proposal is to continue our studies of cardiovascular development. The long-term goal of this work is to identify the genes responsible for fashioning of the cardiovascular system, genes that are critical both to its form and function. We seek to do so by genetic dissection, in other words by using mutations to define the important steps in development of the heart and ultimately to define the responsible genes at the molecular level. We have selected the zebrafish as the appropriate model system. This small fresh water teleost is easily raised, and produces hundreds of large eggs daily, with all development occurring externally in a transparent embryo. It is particularly amenable to genetic screens. Our focus is specifically upon the cardiovascular system. Within 3 days of fertilization we have found that the zebrafish heart forms and loops, becomes subdivided into chambers which are separated by valves, and begins to generate an effective circulation. By single cell injection we have begun to track the cardiac progenitor cells as they arise in the blastula, and to track their migration and fate within the nascent heart tube. In addition, we have identified mutations that affect the heart. For example, we are studying fish strains in which the homozygote lacks any heart beat. The specific aims of this proposal are: (1) To delineate the lineage of the heart, and determine the cell fate decisions which give rise to its various components. For example, preliminary results suggest that the progeny of separate precursor cells populate the endocardium as opposed to myocardium, and the atrium rather than the ventricle; (2) To generate monoclonal markers, in addition to the chamber-specific ones we have currently, in order to identify epitopes that provide patterning information, as well as ones that identify subpopulations of cardiac progenitor cells; (3) To continue the molecular analysis of zebrafish mutations that affect the heart, with special focus upon the silent heart mutation. Our preliminary evidence strongly suggests that the cell biology of this disorder resides at the coupling interface between electrical activity at the cell membrane and contraction (and that the deficit is one of "electromechanical dissociation"). Our preliminary evidence suggests that we may have identified the molecular defect, which we believe to be in troponin T, a component of the actomyosin regulatory complex. We seek to test this hypothesis specifically by molecular analysis. The genetics of cardiovascular disease is only poorly understood, even though many cardiac disorders have an important hereditary component. The zebrafish system provides a potential model system for molecular dissection of such disorders.