We have focused on a murine cardiac specific homeodomain gene, Nkappax2-5 which is instrumental in the patterning of the embryonic heart. We showed that Nkappax2-5 forms combinatorial binding complexes with other transcription factors, such as serum response factor to cardiac, skeletal, and smooth muscle tissues. Acting in concert with these factors, GATA-4 factor stimulated cardiac actin gene between several cardiac specified transacting factors. We isolated a new Nkappax family member, Nkx-28, which is also expressed during very early heart cell commitment, but unlike Nkappax2-5, Nkappax2-8 was down regulated as the heart tube underwent looping, until it reappeared in the inflow and outflow tracts and in the pharyngeal arches associated with thymus formation. Nkappax2-8 displayed striking similarities with genes thought to be associated with DiGeorge's Syndrome. In the proposed studies, we are trying to determine the regulatory role of Nkappax2-8 and whether Nkx2-5 and Nkx2-8 play redundant roles in driving heart cell differentiation and morphogenesis.
The Specific Aims of the proposal are: To determine the temporal and spatial expression of Nkappax2-8 during cardiac morphogenesis in mouse. To define functional protein domainal regions of Nkappax2-5 required for association and interaction with GATA, SRF and other regulatory factors. To characterize the murine genomic locus encoding Nkappax2-5 to identify the cis-acting sequences and trans-acting factors responsible for the high level expression of Nkappax2-5 in primitive myocardium and by peptide growth factors. To investigate the consequence of homologous recombinant DNA knockouts of Nkappax2-5, Nkappax2-8, SRF, and GATA 4 and their inter crosses as a way to test their combinatorial regulatory roles in early embryonic heart formation. To characterize the phenotype of homozygous Nkappax2-8 defective mutants and whether Nkappax2-8 plays a role in CATCH- 22 and DiGeorge syndrome? To develop an Nkappax-2-5 driven cardiac- specific gene knock-out system based on the cre-lox recombinase strategy to investigate the role of SRF and other accessory transcription factors on cardiac development. We believe that defining the molecular basis underlying the establishment and maintenance of cardiac muscle differentiation may eventually lead to an improved understanding of cardiovascular disease.
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