MYH14 (MYH7b) is the most recently identified sarcomeric myosin heavy chain gene (MYH) from the human genome. Recent observations have revealed that MYH M5 , a torafugu orthologue of MYH14, is one of the major components of the MYH repertoire expressed in torafugu embryos and adults, suggesting its pivotal role in fish muscle formation. We have examined the expression pattern of MYH14 in zebrafish, a model organism for the study of vertebrate development. In situ hybridization studies revealed that zebrafish MYH14 was expressed in the most myotomal region containing fast muscle fibers in the embryonic stage and in superficial slow muscle fibers in the adult stage. Weak signals of MYH14 transcripts were also detected in intermediate muscle fibers located between superficial slow and inner fast muscle fibers in the adult. Reverse transcription-PCR studies showed that MYH14 was not only expressed in skeletal muscles but also in cardiac muscles. These expression patterns are in a marked contrast to that of torafugu MYH M5 , which is solely expressed in slow and cardiac muscle fibers in both the embryonic and adult stages. Our results imply species-specific functions of MYH14 in fish muscle formation.

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.

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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 , 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 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 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.