Vegf Signaling and Cardiac Chamber Morphogenesis in Zebrafish

Duke University, Durham NC

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Abstract

? DESCRIPTION (provided by applicant): Background: During cardiac morphogenesis, the embryonic heart changes in structure from a continuous linear tube to a multi-chambered pump composed of one to two atria and ventricles. Embryonic cardiomyocytes whose progeny will contribute to an atrium or the ventricle are specified early in development. How these cell populations change over time to produce chambers of distinct size and function is not well understood. In zebrafish, the ventricular wall thickens during juvenile development, the result of dynamic proliferative events by a small number of dominant cardiomyocytes. By contrast, we have observed that the atrial wall remains a monolayer of cardiomyocytes throughout life. The morphogenetic processes that build the adult atrium have yet to be characterized, and the molecular events responsible for chamber-specific wall thickening remain unclear. This study will contribute to our understanding of cardiac chamber morphogenesis through the following aims: Aim 1: Determine the cardiomyocyte proliferation dynamics that build the adult atrium. The hypothesis to be tested is: individual atrial cardiomyocytes undergo non-uniform proliferation to build the atrial wall, which occurs in a manner distinct from that of the ventricuar myocardium. Aim 2: Investigate the role of Vegf-A in ventricle-restricted wall thickening. The hypothesis to be tested is: Vegf- A expression is restricted to the ventricle, thereby generating chamber-specific differences in wall thickness Methods: Aim 1 will employ multicolor genetic labeling to trace clonal progeny of embryonic cardiomyocytes in the atrium from embryogenesis to maturity. Clone size, shape, and number will be analyzed in order to quantify relative contribution of embryonic atrial cardiomyocytes to the developing wall. Further, the relationship of atrial pectinate muscle to the adjacent wall will be characterized and compare to the analogous trabecular muscle in the ventricle. Aim 2 will utilize fluorescent reporters to map the expression domains of the zebrafish Vegf-A paralogs under normal conditions and in the setting of high cardiac load. Furthermore, conditional Vegf-A over- expression and knockout lines to assess whether Vegf-A is necessary and/or sufficient to initiate wall thickening in the cardiac chambers. Objectives: The results of Aim 1 will define how embryonic atrial cardiomyocytes proliferate and interact during cardiac morphogenesis to build the adult atrium. This, in conjunction with data obtained previously for the ventricle, will define the different dynamics tha build cardiac chambers of strikingly different structure. The results will provide a baseline for Aim 2, which will investigate a candidate mechanism, Vegf-A expression, through which these different outcomes arise. By tracing and later modulating Vegf-A expression in the cardiac chambers I will demonstrate whether Vegf-A, through initiation of coronary vasculogenesis, drives the observed differences in clonal cardiomyocyte proliferation that lead to chamber-specific wall thickness.