This proposal seeks to establish correlations -- in response to a defined induction -- between chromatin changes, the appearance of cell-specific markers, and cell surface changes which result in three-dimensional heart morphogenesis. In the first of two unique methodological approaches, an affinity method is being developed to isolate active myocyte chromatin, which will be characterized in terms of two nuclear proteins which markedly change during skeletal myogenesis: ubiquitinated histone H2A and newly synthesized histone H3.3. The accumulation of H3.3 messenger RNA and protein levels during muscle development and maturation will help elucidate its role in maintenance of the striated muscle differentiated state. The second, related approach utilizes a novel heart development culture system which enables analysis of the effects of interacting embryonic tissues during morphogenesis. Using techniques of in situ hybridization histochemistry and immunocytochemistry, this model, which faithfully recapitulates in vivo cardiogenesis, will be used to test the hypothesis that the anterior foregut endoderm secretes proteins, to be characterized, which induce terminal differentiation and morphogenesis in precardiac mesoderm. It is proposed that this induction elicits the following sequence of events: (i) expression of histone H3.3 mRNA and its translation products, signaling chromatin structural rearrangements permissive for cardiogenesis, (ii) expression of mRNA's for cardiac-specific contractile proteins, and (iii) expression of cell:cell (CAM's) and cell: substrate (SAM's) adhesion molecules which mediate cellular movements in morphogenesis, culminating in the development of a three-dimensional, contractile organ. These studies will help elucidate mechanisms which regulate the induction and maintenance of the cardiac differentiated state, providing insights into the etiology of disorders including congenital anomalies and cardiomyopathies.
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