Identifying Binding Sites, Target Genes, and Functional Domains of the T-box Transcription Factor midline.

摘要

Congenital heart defects are the most common birth defect and affect ∼1/100 newborns. While our knowledge of the underlying developmental mechanisms of the heart has greatly expanded in recent years, there are still many idiopathic conditions that must be explored. Conserved developmental mechanisms have made the fruit fly, Drosophila melanogaster, instrumental to our current understanding heart development. The T-box transcription factor midline (mid) and its vertebrate homologue, Tbx20, have demonstrated roles in the developing heart. This thesis explores the function of mid in the developing heart. To identify genes regulated by mid, I performed gene expression profiling on embryos ectopically expressing mid. This approach identified 565 genes at 1.5-fold p<0.05 that respond to ectopic mid.;To identify potential direct targets of mid, I identified a DNA motif recognized by Mid (CAAGGTGTCAAGG CG). Searching the gene-span +/- 2 kb of each D. melanogaster gene identified 2112 potential Mid targets. Of these, 86 were in common with hits from expression profiling.;An RNAi screen of genes most strongly affected by ectopic mid found that knockdown of CG32373, closely related to cell-surface or secreted proteins, led to an observable phenotype in the heart. However, CG32373 in situ hybridization appears limited to the trachea and does not appear induced in mid mutants, despite reductions in its transcripts in response ectopic mid detected by qPCR. Furthermore, only one of two CG32373 RNAi lines was able to induce a phenotype.;To identify mechanisms that Mid uses to regulate its targets, I examined the expression of known and novel targets in embryos ectopically expressing mid alleles with mutations in conserved domains. None of the three domains strongly affected the rescue of tin or svp . However, the response of some genes identified through expression profiling was altered when mutant alleles of mid were expressed.;The results contained in this thesis further our understanding of mid function and more importantly, our understanding of heart development. Further exploration of the identified genes in both flies and humans will likely expand the potential targets for treatment and diagnosis of congenital heart defects.