Distinct enhancers regulate skeletal and cardiac muscle-specific expression programs of the cardiac [alpha]-actin gene in xenopus embryos

During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the [alpha]-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac [alpha]-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box I faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC[(A/T).sub.6]GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac [alpha]-actin promoter, to confer appropriate cardiac a-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac a-actin gene promoter is to act solely as a high-affinity SRF binding site. Key Words: cardiac [alpha]-actin; muscle; SRF; Xenopus.