Promoter analysis of the fish gene of slow/cardiac-type myosin heavy chain implicated in specification of muscle fiber types.

Vertebrate skeletal muscles consist of heterogeneous tissues containing various types of muscle fibers, where specification of the fiber type is crucial for muscle development. Fish are an attractive experimental model to study the mechanisms of such fiber type specification because of the separated localization of slow and fast muscles in the trunk myotome. We examined regulation of expression of the torafugu gene of slow/cardiac-type myosin heavy chain, MYH M5 , and isolated an operational promoter in order to force its tissue-specific expression across different fish species via the transgenic approach in zebrafish and medaka. This promoter activity was observed in adaxial cell-derived superficial slow muscle fibers under the control of a hedgehog signal. We also uncovered coordinated expression of MYH M5 and Sox6b, which is an important transcriptional repressor for specification of muscle fiber types and participates in hedgehog signaling. Sequence comparison in the 5'-flanking region identified three conserved regions, CSR1-CSR3, between torafugu MYH M5 and its zebrafish ortholog. Analysis of deletion mutants showed that CSR1 significantly stimulates gene expression in slow muscle fibers. In contrast, deletion of CSR3 resulted in ectopic expression of a reporter gene in fast muscle fibers. CSR3 was found to contain a putative Sox family protein-binding site. These results indicate that the dual mechanism causing inhibition in fast muscle fibers and activation in slow muscle fibers is essential for slow muscle fiber-specific gene expression in fish.

Evolution and Distribution of Teleost myomiRNAs: Functionally Diversified myomiRs in Teleosts.

Myosin heavy chain (MYH) genes belong to a multigene family, and the regulated expression of each member determines the physiological and contractile muscle properties. Among these, MYH6, MYH7, and MYH14 occupy unique positions in the mammalian MYH gene family because of their specific expression in slow/cardiac muscles and the existence of intronic micro(mi) RNAs. MYH6, MYH7, and MYH14 encode miR-208a, miR-208b, and miR-499, respectively. These MYH encoded miRNAs are designated as myomiRs because of their muscle-specific expression and functions. In mammals, myomiRs and host MYHs form a transcription network involved in muscle fiber-type specification; thus, genomic positions and expression patterns of them are well conserved. However, our previous studies revealed divergent distribution and expression of MYH14/miR-499 among teleosts, suggesting the unique evolution of myomiRs and host MYHs in teleosts. Here, we examined distribution and expression of myomiRs and host MYHs in various teleost species. The major cardiac MYH isoforms in teleosts are an intronless gene, atrial myosin heavy chain (amhc), and ventricular myosin heavy chain (vmhc) gene that encodes an intronic miRNA, miR-736. Phylogenetic analysis revealed that vmhc/miR-736 is a teleost-specific myomiR that differed from tetrapoda MYH6/MYH7/miR-208s. Teleost genomes also contain species-specific orthologs in addition to vmhc and amhc, indicating complex gene duplication and gene loss events during teleost evolution. In medaka and torafugu, miR-499 was highly expressed in slow/cardiac muscles whereas the expression of miR-736 was quite low and not muscle specific. These results suggest functional diversification of myomiRs in teleost with the diversification of host MYHs.

Multiple cis-elements in the 5'-flanking region of embryonic/larval fast-type of the myosin heavy chain gene of torafugu, MYH(M743-2), function in the transcriptional regulation of its expression.

The myosin heavy chain gene, MYH(M743-2), is highly expressed in fast muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional regulatory mechanism involved in its expression remained unknown. Here, we analyzed the 2075bp 5'-flanking region of torafugu MYH(M743-2) to examine the spatial and temporal regulation by using transgenic and transient expression techniques in zebrafish embryos. Combining both transient and transgenic analyses, we demonstrated that the 2075bp 5'-flanking sequences was sufficient for its expression in skeletal, craniofacial and pectoral fin muscles. The immunohistochemical observation revealed that the zebrafish larvae from the stable transgenic line consistently expressed enhanced green fluorescent protein (EGFP) in fast muscle fibers. Promoter deletion analyses demonstrated that the minimum 468bp promoter region could direct MYH(M743-2) expression in zebrafish larvae. We discovered that the serum response factor (SRF)-like binding sites are required for promoting MYH(M743-2) expression and myoblast determining factor (MyoD) and myocyte enhancer factor-2 (MEF2) binding sites participate in the transcriptional control of MYH(M743-2) expression in fast skeletal muscles. We further discovered that MyoD binding sites, but not MEF2, participate in the transcriptional regulation of MYH(M743-2) expression in pectoral fin and craniofacial muscles. These results clearly demonstrated that multiple cis-elements in the 5'-flanking region of MYH(M743-2) function in the transcriptional control of its expression.