Tbx1 is required for cardiovascular development in human, mouse and zebra fish. Since the initial recognition of the role of this gene in development, there has been considerable progress in the analysis of mutants that led to novel hypotheses. Tbx1 is a T-box transcription factor required for segmentation of the embryonic pharynx, patterning of the aortic arch, and morphogenesis of the outflow tract of the heart. However, further knowledge acquisition about this gene would be limited if we do not address two fundamental questions: what are the critical players in the regulation of the Tbx1 gene, and what are the targets of the Tbx1 protein. The Tbx1 gene has an exquisitely dynamic regulation, and here we show that this is associated with time-specific roles during embryogenesis. For example, early expression is required for cardiovascular development while late expression is required for thymic organogenesis and palatogenesis. In addition, we show tissue specific requirements for Tbx1 function during cardiovascular development. This proposal builds upon these new data to search for regulators and targets of Tbx1 that are important at the critical times and tissues. The overall aim is to establish hard links in the genetic pathway within which Tbx1 functions. We believe that this work will lead to genes causing or modifying congenital heart disease. In the first specific aim we will use novel approaches to identify genetic elements necessary for Tbx1 regulation in the pharyngeal endoderm, a key tissue for Tbx1 function, and will also determine if Tbx1 expression in this tissue is sufficient to rescue the cardiovascular abnormalities seen in mutants. In the second specific aim, we will use a combination of transcriptome analysis and chromatin immunoprecipitation assay to identify transcriptional targets. Preliminary data show the power of this approach. In addition, we propose in vitro and in vivo validation of candidate target genes. This project addresses directly the function of Tbx1 in cardiovascular development as defined by upstream regulation of the gene and transcriptional targets of the protein in relevant tissues. Data should provide the dissection of a genetic network disrupted in DiGeorge and Velocardiofacial syndromes, and possibly in other genetic disorders affecting cardiovascular development.
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