Tinman is a direct activator of midline in the Drosophila dorsal vessel

Heart development requires a conserved core of transcription factors comprised of Nkx2.5, GATA and T-box family transcription factors. In Drosophila melanogaster, the Nkx2.5 gene tinman acts upstream of many cardiac genes including the Tbx20 homolog midline, a critical regulator of heart development in both flies and vertebrates. By testing genomic fragments containing clusters of consensus Tinman-binding sites, we identified a 4.3 kb fragment 5' of midline that directs reporter expression in all midline-expressing heart cells and a 1.7 kb subfragment that drives reporter expression in mid-expressing heart cells that maintain tin expression. Both fragments direct reporter gene expression in response to tinman in transgenic embryos and in transient transfection assays in Drosophila S2 cells. Mutation of two Tinman binding sites (Tin1 and Tin2) reduces or abolishes cardiac expression in derivatives of the 1.7 kb fragment. We conclude that Tin is a direct regulator of midline in fly heart development.