The applicant has demonstrated that type beta1 transforming growth factor (TGFbeta1) controls the expression of that at least 16 tissue-restricted genes in neonatal rat cardiac muscle cells and is the prototype for an autocrine or paracrine factor mediating ventricular hypertrophy triggered by mechanical load. Induction of the skeletal alpha-actin promoter by TGFbeta unlike other genes, is dependent on serum response factor (SRF). However, the role of TGFbeta in vivo, and during ontogeny in particular, has not been determined. The first cells of the embryo to contain RNA for TGFbeta1 are cardiac precursor cells, and, in the course of cardiac organogenesis, TGFbetas and TGFbeta-like peptides are expressed in a elaborately complex spatial and temporal pattern. Functional studies in embryonal carcinoma cells and in phyla other than mammals suggest that members of the TGFbeta superfamily might direct two key aspects of cardiac organogenesis establishment of the cardiac muscle lineage, and transformation of endocardial precursor cells into valve-forming mesenchyme. Despite the abundance of descriptive information concerning TGFbeta expression in developing myocardium, and encouraging results in transformed cells or in species that are related only distantly, there has not been to date a genetic analysis of TGFbeta signal transduction, or growth factor signalling more generally, by means of loss-of- function mutation in the developing mammalian heart. Dominant- negative mutations are one means to this end, with certain theoretical and technical advantages which complement homologous recombination. Furthermore, procedures for """"""""binary"""""""" or """"""""two-stage"""""""" transgenic mice, described initially by Frank Ruddle and Philip Leder, hold the promise of targeting potentially lethal dominant- inhibitors to predetermined development and tissue-specific windows.
Specific Aims of the present project are to: (1) Define the role of SFR and accessory SRE-binding proteins in regulation of the SkA promoter by TGFbeta and other members of the TGFbeta superfamily. (2) Establish with dominant-negative mutations. whether ras and AP1 are necessary for induction of SkA expression in neonatal rat cardiac muscle, or during cardiac myogenesis in ES cells. (3) Determine whether the dominant negative mutations of ras and AP1 cause developmental cardiac defects in vivo, and provide evidence for the role of ras and AP1 signal transduction pathways in the developing heart. (4) Determine the ability of kinase-deficient TGFbeta and activin receptors to function in cultured cardiac muscle. ES cells, and transgenic mice as dominant-inhibitors of TGFbeta signal transduction. (5) Target dominant-inhibitory alleles of AP1, ras, and the TGFbeta receptor to valvular mesenchyme of transgenic mice, via TGFbeta3 or beta2 cis-acting sequences, to determine whether these mutations can cause development defects in septation or in formation of the cardiac valves. These investigations will comprise an initial genetic analysis of the role played in the intact animal by AP1, ras, and the TGFbeta receptor in establishment of the cardiac muscle lineage, cardiac muscle gene transcription, and cardiac organogenesis.
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