The overall focus of the research in my laboratory is to examine the processes of commitment, differentiation and diversification of cardiac myocytes during avian heart development. Cardiac progenitor cells have been mapped to the anterior lateral plate mesoderm at stage 4 (day 1) of development. These progenitor cells become committed to the cardiogenic lineage at stage 4. They differentiate through stage 15. Immunohistochemical and molecular biological techniques have detected the expression of muscle specific proteins and message as early as stage 7, before the first heart beat or myofibril is formed at stage 10. We hypothesize that between stages 4 and 15 cell-type-specific gene expression begins. Consequently, this is the appropriate time to study the regulation of expression of these genes. Trans-acting factors have been identified in a variety of systems and in both vertebrate and invertebrate species. In the skeletal muscle system the first regulatory factor to be identified was MYOD1. Many of these factors contain a highly conserved basic-helix-loop-helix domain, which is involved in homo-or heterodimer formation and subsequent binding to DNA to activate gene expression. Several similarities and differences exist between the skeletal and cardiac systems. Both systems share a number of contractile proteins and both sets of genes are known to contain the """"""""canonical"""""""" E box enhancer sequence. Yet, the timing of maturation in these two systems is very different with the cardiac system maturing much earlier. Also, skeletal cells are derived from somitic mesoderm and cardiac cells from splanchnic mesoderm. We expected that gene expression in both cell types may be conducted in similar but not identical ways. We present strong preliminary data which suggests that cardiac myocytes contain proteins antigenically related to the HLH family of regulatory factors. We have identified 24 cDNA clones that are reactive with an antibody specific to the second helix of MyoD1. One of these clones, c22 has the characteristics of a DNA binding protein. We propose to (1) characterize the spatial and temporal expression of c22, (2) determine and analyze its nucleotide and amino acid structure, (3) identify in vivo DNA- binding sites and (4) determine its function.
