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During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscletissue 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 1 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)(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 alpha-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac alpha-actin gene promoter is to act solely as a high-affinity SRF binding site.
Fig1. A portion of cardiac alpha-actin promoter that is -580 bp is sufficient for correct expression in Xenopus embryos, tadpoles, and explants. (Top) Schematic presentation of -580 cardiac alpha-actin promoter. Transgene expression can be detected in the somites of the late neurulaembryo and in myotomes, facial muscle and heart (h) of the larval tadpole, either by whole-mount in situ for GFP RNA (A, B) or by GFP fluorescence (C, D). (E) GFP activity in the tail of 7-day-old tadpole. (F) Ventral view of a 7-day-old tadpole, showing strong GFP activity in the heart (boxed), body wall, and jaw muscles. Anterior is to the right. (G) Close-up of the heart region boxed in (G). ot, outflow tract; v, ventricle. (H, I) Animal cap explants from F1 transgenic embryos cultured in the absence (H) or presence (I) of 8 U/ml activin until sibling embryos reached stage 24. GFP activity is only seen in animal caps treated with activin, demonstrating that the transgene is activated by the mesoderm- inducing signal. GFP expression can be used to monitor heart and head muscles in explants from F1 transgenic embryos. (J) Stage 34 control embryo, ventral view, anterior to the right. Strong expres- sion is detected in the heart (arrow) and head muscles (arrow heads). (K) A heart field explant excised at stage 22, oriented in the same way as an embryo in (J).
FIG. 2. E-boxes are dispensable for normal expression of cardiac alpha-actin in embryos, while CArG box 1 and sequence immediately downstream of it are necessary for expression in the heart. (A) Deletion of sequences upstream of -216 results in expression indistinguishable from that obtained with the entire 580-bp pro- moter. (B) 5'delta-103 is expressed in the heart but not in the myotomes. It also gives inappropriate expression in the head. (C) Linker scan mutation LS25 shows no effect on expression. (D) In contrast, LS29, which destroys CArG box 1, results in loss of heart expression. (E) A sequence downstream of CArG box 1 is essential for expression in the heart, as revealed by linker scan mutation LS13.
FIG. 3. (A) Internal deletion LS10/13 removes CArG boxes 1-4, resulting in weak expression in the myotomes and loss of expression in the heart. (B) An oligonucleotide comprising CArG box 1 and the adjacent 10 bp downstream are sufficient to restore normal expression to LS10/13. (C), L-LS10/13, in which 5 bp most proximal to CArG box 1 are changed, is not expressed in the heart but is expressed normally in the myotomes. (D) Mutation of the more distal 5 bp has no effect on transgene activity in the heart. (E) Mutant T-LS10/13 shows expression in the myotomes, but not in the heart (arrow). (Transgenic constructs shown in B all contain two copies of the oligonucleotides; see Table 1.)
FIG. 4. CArG Box 1 + DS is sufficient for expression in myotomes and head and heart region. (A) A chimeric construct comprising oligonucleotide CArG Box 1 + DS adjacent to the minimal cytoskeletal actin promoter gave expression in the myotomes, heart (arrowed), and branchial arches of the stage 33 tadpole. (B) Similar expression was obtained with this oligonucleotide in front of the thymidine kinase minimal promoter. (C) However, at earlier stages, transgene expression directed by CArG Box 1 +DS is premature and too broad, as seen in the presumptive heart field of stage 22embryo. (D, F) Using either the cardiac actin or cytoskeletal actin basal promoter, an E-box placed adjacent to the CArG box 1 + DS restricts activity of transgene to the myotomes. No expression is seen in the heart (arrowed). (E) The CArG Box 1 + DS oligonucleotide also directs premature and broad expression in the stage 22embryo when placed in front of the 5'delta-103 truncation of the cardiac alpha-actin promoter. (G) Nucleotides spanning -75 to -91 of the cardiac alpha-actin promoter can act as a weak myotome-specific enhancer. Dorsolateral view of a stage. 25 embryo showing feint, but detectable fluorescence in the myotomes. (NB, Strong fluorescence in the forebrain, hindbrain, and eyes (arrows) results from the presence of a gamma crystallin/GFP cotransgene). (A) Anterior to left. (G) Anterior at top.
FIG5. E-boxes mediate silencing of cardiac alpha-actin promoter in serum-stimulated fibroblasts. NIH3T3 fibroblasts were transfected with test plasmids comprising the human c-fos gene driven by the full-length c-fosh promoter (Fos-711) or a chimeric promoter con- taining cardiac actin promoter sequences fused upstream of the c-fos h 5'delta-100. Three cardiac actin promoter regions were tested; the full-length promoter (-580), a 5'truncation that removes the E-box motifs (-246), and an internal deletion of the 580-bp pro- moter that removes 31 bp encompassing the three E-box motifs (deltaM). Cells were serum-starved (-) and then stimulated with serum for 40 min (+). Total RNA was analysed by RNase protections. M-DNA markers; P-undigested riboprobes; C-probes hybridised to tRNA. Diagrams showing essential features of test plas- mids are shown below the gel. Protected Fos and globin transcripts are indicated.
