This grant application focuses on the regulation and function of Pax3 in neural crest ceils. Neural crest plays important roles in cardiovascular development since derivatives contribute to the outflow tract of the heart and to the great vessels. Mutations in neural crest genes, including Pax3, lead to congenital heart disease. This competitive renewal application builds upon significant data and resources that we have developed during the first granting cycle in order to elucidate the molecular pathways responsible for regulating Pax3 expression in neural crest. Neural crest cells represent an exciting and tractable embryonic example of a multipotent precursor cell population that is capable of differentiating along multiple cell fate pathways. Pax3 is expressed by multipotent neural crest progenitors, and our preliminary data indicate that it is also expressed by some adult neural crest-derived stem cells. However, Pax3 expression always abates prior to terminal differentiation of multipotent progenitors. Previous studies have documented the requirement for Pax3 expression during development. Here, we propose that Pax3 inactivation is also critical. We will test the novel hypothesis that Pax3 is required to maintain the multipotent, undifferentiated phenotype of neural crest cells. This will be examined in animal models, in cell-based systems, and at the transcriptional level, taking advantage of our identification of a novel downstream target of Pax3, dopachrome tautomerase (DCT). DCT is an enzyme required for melanin synthesis in melanocytes (neural crest derivatives) that we now show is also expressed in a pattern reminiscent of the developing AV node. In the absence of Pax3, DCT expression is lost in both melanocytes and the heart. We suggest that Pax3 and neural crest are required for development of some aspects of the cardiac conduction system. We will test whether Pax3 regulates DCT directly or indirectly during cardiac development, and we will define the role of DCT in the heart. Therefore, aim 1 will define upstream molecular pathways regulating Pax3 in neural crest while aims 2 and 3 define mechanisms of Pax3 function during cardiac development at the molecular and whole organ level. The approaches in aim 2 are relevant to an understanding of how neural crest stem cell multipotency is maintained during critical periods of cardiac and embryonic development.
Aim 3 examines the role of neural crest during development of the conduction system, and specifically examines Pax3 regulation of DCT. ? ?
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