Abnormal development of the myocardial-smooth muscle junction at the arterial pole of the heart leads to congenital defects classified as conotruncal malformations such as double outlet right ventricle and tetralogy of Fallot. Recent work from our lab shows that the basic elements that build the arterial pole prior to septation are highly conserved during development. In development of a heart with divided pulmonary and systemic circulations, the myocardium and smooth muscle are added to the arterial pole long before the region is septated. Further work from this lab has shown definitively that arterial pole malalignments, i.e. conotruncal malalignment defects are due to abnormal pre-septation arterial pole development. The zebrafish is an ideal organism in which to study arterial pole development without the confounding event of septation. In addition, its strength as a genetic model makes it an excellent vertebrate in which to study genes that potentially underlie conotruncal malformations. Tbx1 is a gene associated with the DiGeorge phenotype of which conotruncal malformations are a major component. The zebrafish van gogh mutant has a null mutation in tbxl, but the heart defect associated with this mutation has never been analyzed for arterial pole development. In addition, FGF8 is reported to be a downstream effecter of TBX1 and we have evidence in the chick that outflow alignment is very sensitive to FGF8 signaling. Thus, the hypothesis for this proposal is that Tbx1 through FGF8 and other as yet unidentified genes regulates the contribution of precursors to the myocardial and smooth muscle cells that form the arterial pole of the zebrafish heart. To understand the gene-phenotype relationship of conotruncal malformations we need to have detailed information about the origin and developmental history of the arterial pole progenitors. Therefore, Aim 1 will use cell tracing and ablations to identify and determine the contribution of the progenitors of the myocardium and smooth muscle that form the zebrafish arterial pole. The type of cell tracing and discrete ablations of the arterial pole progenitors that are proposed are not possible in any other animal model.
Aim 2 will determine the sensitivity of these progenitors in forming the arterial pole to disrupted expression of tbxl and fgf8 using zebrafish mutants and antisense morpholino technology. These experiments will provide cellular and molecular information about normal and abnormal development of the arterial pole progenitors in a genetic background similar to that in DiGeorge patients. Because of the variability of the DiGeorge phenotype Aim 3 is designed to identify unknown genetic modifiers of tbxl function using a candidate gene approach and high throughput screening of double heterozygotes. Because the arterial pole is the site of conotruncal malformations, detailed knowledge of its development and the genes that influence it will ultimately allow better prediction of individuals at risk for having offspring with such malformations. ? ? ?
