Major outstanding questions in cardiac biology involve the origin of the cardiac cell lineage and the production as well as the maintenance of the differentiated phenotype. An important drawback to the answer of these questions and the analysis of cardiac morphogenesis has been the absence of genetic approaches to study the critical events in cell lineage formation and organogenesis. Three complementary approaches can be used to address these questions: 1) To isolate and characterize transcription factors that are specific for the myocardial tissues. Once isolated, these sequences will be tested for their lineage-determining capacity and their mode of action. In particular, we will concentrate on those cardiac specific factors that are expressed from the early stages of cardiogenesis and control atrial- and ventricular-specific gene expression. For this the well defined cis-regulatory elements of the alpha and beta-MHC genes will serve as probes for the identification, purification and cloning of the factors. Even if the role of these molecules is restricted to the maintenance, rather the establishment of the cardiac phenotype, an understanding of their mode of action will serve to elucidate the mechanisms involved in the production of the different cell types in the myocardium. 2) So far, the only muscle-specific determining genes characterized are the members of the MyoD multigene family. However, none of these genes is expressed in the atrial and ventricular myocardium. Based on the many similarities between cardiac and skeletal muscle and, more specifically, on the observation that an embryonal carcinoma cell line can be induced to differentiate at high frequency into cardiac myogenic cells, the isolation of the cardiac lineage determining gene(s) will be attempted. For this, cDNAs will be amplified from mRNAs expressed during cardiac development that will be selected either for their homologies to the skeletal myogenic determining genes or by substraction hybridization against a battery of RNAs. By using a combination of efficient gene transfer procedures and selective markers, the clones able to convert multipotent cells to the cardiogenic lineage will be isolated and characterized. 3) The cis-acting elements of the cardiac MHC genes will be used to specifically target the expression of a selectable conditional lethal marker in the atrial or the in ventricular cells. Strains of 'transgenic hypomorphs' (mice that can be induced to develop cardiac defects at preselected stages of development) will be produced. This approach could constitute the basis for developing a model system in which the relative importance of different cell lineages and their origin could be analyzed.
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