This project is directed at understanding the role of the homeobox transcription factor Nkx2.5 in the regulation of vertebrate cardiac development. There is compelling evidence that Nkx2.5-like genes are mediators of commitment and differentiation in the cardiac cell lineage, and of cardiac morphogenesis. This investigation is broadly based on the hypothesis that normal cardiac ontogeny depends on the timely expression of appropriate Nkx2.5 activity, and that perturbation of that activity will result in abnormal cardiac phenotypes. The proposal is organized around three experimental Aims, each designed to answer a specific set of questions about the biology of Nkx2.5 as it pertains to cardiac development. Aim l is to define the gene and cDNA sequences of the zebrafish Nkx2.5 ortholog, zNkx2.5. How many NK and homeodomain genes are present in zebrafish, and which of these is the true Nkx2.5 ortholog? Which sequences can be used for the construction of specific probes for the true ortholog? Aim 2 is to determine the pattern of expression of zNkx2.5 in zebrafish. How early and in what cardiac precursors is zNkx2.5 expressed? How does the pattern of expression of zNkx2.5 vary with time? Are there temporal or spatial differences between mRNA and protein expression for zNkx2.5? How does timing of expression compare to that of other cardiac determining gene products? Aim 3 is to determine the dependence of cardiac development and overall body plan on appropriate zNkx2.5 expression in zebrafish by perturbing mRNA levels via direct injection of zNkx2.5 mRNA or anti-zNkx2.5 mRNA ribozyme. What is the aim of function phenotype? What is the loss of function phenotype? To what extent can we alter the timing and location of cardiac precursor determination? This research applies molecular strategies to the analysis of vertebrate cardiac development. It is being pursued in zebrafish because a variety of critical features makes this experimental animal exceptionally well- suited to this purpose. The applicant expects to gain some expertise in basic vertebrate embryology, and to learn specific techniques of embryo manipulation including collection, manipulation, microinjection of RNA or protein agents, and in situ assay for gene product expression.