Cardiac-specific activity of an Nkx2-5 enhancer requires an evolutionarily conserved Smad binding site

Heart formation in vertebrates and fruit flies requires signaling by bone morphogenetic proteins (BMPs) to cardiogenic mesodermal precursor cells. The vertebrate homeobox gene Nkx2-5 and its Drosophila ortholog, tinman, are the earliest known markers for the cardiac lineage. Transcriptional activation of tinman expression in the cardiac lineage is dependent on a mesoderm-specific enhancer that binds Smad proteins, which activate transcription in response to BMP signaling, and Tinman, which maintains its own expression through an autoregulatory loop. Here, we show that an evolutionarily conserved, cardiac-specific enhancer of the mouse Nkx2-5 gene contains multiple Smad binding sites, as well as a binding site for Nkx2-5. A single Smad site is required for enhancer activity at early and late stages of heart development in vivo, whereas the Nkx2-5 site is not required for enhancer activity. These findings demonstrate that Nkx2-5, like tinman, is a direct target for transcriptional activation by Smad proteins; however, the independence of this Nkx2-5 enhancer of Nkx2-5 binding suggests a fundamental difference in the transcriptional circuitry for activation of Nkx2-5 and tinman expression during cardiogenesis in vertebrates and fruit flies.