Transcriptional regulation during heart development

Cardiac gene expression is commonly thought to be the result of combinatorial interactions between heart-specific and globally expressed transcription factors. Myocardin has recently been identified as a cardiac and smooth muscle specific cofactor for the globally expressed serum response factor (SRF) protein. Using a combination of gain-of-function and loss-of-function approaches, we show that myocardin is both necessary and sufficient for the transcriptional activation of a wide range of cardiac differentiation products. We also demonstrate that myocardin cooperates with additional factors to induce gene expression, fitting with previous models of cardiac gene regulation. Myocardin is able to induce ectopic expression of cTnI, cardiac α-actin, MHCα, and MLC2, in addition to smooth muscle markers in whole embryos or animal pole explants. Conversely, suppression of myocardin gene activity by morpholino knockdown results in the inhibition of cardiac differentiation products. The expression of the early cardiac specification gene Nkx2-5 however, is unaffected by either myocardin misexpression or loss-of-function. From this data, we conclude that myocardin is the key intermediate between cardiac specification and differentiation. The process of cardiac differentiation includes the formation of the atrial and ventricular chambers which display distinct contractile, physiological, conductive, and genetic properties. The atrial natriuretic factor (ANF) gene is initially expressed throughout the myocardial layer of the heart, but during subsequent development, expression becomes limited to the atrial chambers. We have isolated the Xenopus ANF promoter in order to examine the temporal and spatial regulation of the ANF gene in vivo using transgenic embryos. The mammalian and Xenopus ANF promoters show remarkable sequence similarity, including an Nkx2-5 binding site (NKE), two GATA sites, two SRF binding sites (SREs) and a Tbx binding site (TBE). Our transgenic studies show that mutations in either the NKE or proximal GATA elements result in ectopic ANF promoter activity in the kidneys, facial muscles, and aortic arch artery associated muscles, and causes persistent expression in the ventricle and outflow tract of the heart. We propose that the NKE and proximal GATA elements serve as crucial binding sites for assembly of a repressor complex, that is required for atrial-specific expression of the ANF gene.