Heart formation involves a complex series of events that are initiated when mesodermal precursor cells adopt a cardiac cell fate. Subsequent migration, patterning, growth and differentiation of cardiac myocytes give rise to the embryonic myocardium, which undergoes looping morphogenesis, septation and interconnection with the vascular system to ultimately yield the multi-chambered heart. The complexity of heart formation and the large number of genes involved in this process are reflected in the high frequency of congenital heart defects in humans, which can be ascribed to abnormalities in specific steps in the cardiac developmental pathway. Our laboratory has discovered two distinct families of basic helix-loop-helix (bHLH) transcription factors, referred to as HANDs and HRTs, with central roles in cardiac growth and development. HAND1 and HAND2 are expressed in distinct patterns in the developing heart, as well as in the neural crest and limb buds, and knockout mice lacking these genes show severe abnormalities in growth and patterning of the corresponding embryonic structures. There are three members of the HRT family (HRT1, 2, 3), which also exhibit highly specific and dynamic expression patterns during cardiovascular development. The HRT genes are regulated by Notch signaling, and HRT2 has been shown to be required for cardiac and arterial development. The overall objective of this project is to use HANDs and HRTs as entry points into the developmental circuits that control the formation and function of the cardiovascular system and to further define the mechanisms of action of these transcription factors. As a complement to our primary focus on mammalian cardiovascular development, we will use the Drosophila HAND gene as an entry point into the pathways that control heart formation. Toward that end, we have generated a strain of transgenic fruit flies that expresses green fluorescent protein (GFP) under control of cardiac-specific regulatory elements from the Drosophila HAND gene. These flies have enabled us to undertake an unbiased search for genes that regulate multiple steps in cardiac development, including cardiac cell specification, differentiation, patterning, growth, morphogenesis, and contractility. The cloning of these mutant genes and the ultimate elucidation of their functions should provide new insights into the fundamental developmental circuitry of cardiac development and provide candidate genes for further exploration in mammalian heart development and congenital heart disease.