The applicant hopes to understand the role of cardiogenic transcription factors in the determination and development of cardiac muscle cells. He has identified and cloned a cDNA encoding a cardiac regulatory transcription factor, called Clp-1, that may play an important role in the development of cardiogenic progenitor cells. The stated goal of the current application is to understand the role of Clp-1 in cardiogenesis by addressing when, where and in what capacity Clp-1 performs its function as a regulator of cardiogenesis. The proposal has four specific aims.
Aim 1 is directed towards additional characterization of the structural and functional properties of Clp-1 as it relates to its potential cardiogenic regulatory properties. The applicant and his colleagues will attempt to identify the structural domains of Clp-1 that are responsible for binding the B element MEF-2 binding site in the cardiac MLC-2 gene promoter and determine if these same structural domains mediate Clp-1 activation of the cardiac MLC-2 gene in vivo.
Aim 2 is directed towards investigating the cellular functions of Clp-1 in order to establish its role as a regulator of cardiac genes and cardiogenic cell differentiation. These studies will focus on determining 1) the potential of Clp-1 to act as a cardiogenic transcriptional activator capable of participating in the activation of cardiac lineage-determining genes, 2) the ability of Clp-1 to irreversibly commit precursor cells to the cardiogenic cell lineage, and 3) the hierarchal relationship of Clp-1 to BMP-2, another developmental signal which is known to be important in commitment of stem cells to the cardiac lineage.
Aim 3 will focus on the characterization of promoter elements required for control of Clp-1 expression. The applicant will examine the promoter region of the mouse Clp-1 gene in an effort to define the sequence(s) required for directing expression in cardiogenic mesodermal cells.
Aim 4 will attempt to determine the importance and necessity of Clp-1 in the actual elaboration of the cardiogenic developmental program in vivo. This will be evaluated in knockout mice following genetic ablation of the Clp-1 locus. The development of these mice will be followed primarily with respect to the morphological and histological status of the developing heart and secondarily with regard to non-cardiac phenotypes should they arise. Abnormal heart development will be analyzed at the molecular level by assaying for the expression of normal heart genetic markers presumably under the control of the Clp-1 transcription factor. In addition, physiological analysis of transgenic adult hearts will be undertaken to look for subtle perturbations that lead to detectable changes in heart function.
