A 5’-flanking region of embryonic-type myosin heavy chain gene, MYH(M)(7)(4)(3)(−)(2), from torafugu Takifugu rubripes regulates developmental muscle-specific expression
The myosin heavy chain gene, MYH(M)₇₄₃₋₂, is highly expressed in fast muscle fibers of torafugu embryos. However, the regulatory mechanisms involved in its expression have been unclear. In this study, we examined spatio-temporal expression patterns of this gene during development by injecting expression vectors containing the GFP reporter gene fused to the 5'-flanking region of MYH(M)₇₄₃₋₂ into fertilized eggs of zebrafish and medaka. Although the -2.1kb 5'-flanking region of torafugu MYH(M)₇₄₃₋₂ showed no homology with the corresponding regions of zebrafish and medaka orthologous genes on the rVISTA analysis, the torafugu 5'-flanking region activated the GFP expression which was detected in the myotomal compartment for both zebrafish and medaka embryos. The GFP expression was localized to fast and slow muscle fibers in larvae as revealed by immunohistochemical analysis. In addition to the above tissues, GFP was also expressed in jaw, eye and pectoral fin muscles in embryos and larvae. These results clearly demonstrated that the 2.1 kb 5'-flanking region of MYH(M)₇₄₃₋₂ contains essential cis-regulatory sequences for myogenesis that are conserved among torafugu, zebrafish and medaka.
Available from: PubMed Central
- "In zebrafish, at least 3 fast skeletal MYHs, myhz1, myhz2 and myhc4, are dominantly expressed in fast fibers of embryos [9-11]; orthologs of torafugu , medaka  and common carp [7,8] are also expressed in embryos from the respective fish species. In addition, the proximal promoters of the above-listed MYHs from torafugu [43,44] and medaka  drive the reporter gene expression in the fast fibers of zebrafish embryos. These teleost MYHs are all classified as fast C type , suggesting that the proximal promoters of fast skeletal MYHs of lampreys and fast C-type MYHs of teleosts have a common ancestor and share common regulatory mechanisms, even though no synteny (see Figures S1 and S2)  or strict orthology (see Figure 2) was found between lamprey and teleost MYHs. "
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ABSTRACT: Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains. In the case of humans (tetrapod), a total of 6 fast skeletal-type MYH genes (MYHs) are clustered on a single chromosome. In contrast, torafugu (teleost) contains at least 13 fast skeletal MYHs, which are distributed in 5 genomic regions; the MYHs are clustered in 3 of these regions. In the present study, the evolutionary relationship among fast skeletal MYHs is elucidated by comparing the MYHs of teleosts and tetrapods with those of cyclostome lampreys, one of two groups of extant jawless vertebrates (agnathans). We found that lampreys contain at least 3 fast skeletal MYHs, which are clustered in a head-to-tail manner in a single genomic region. Although there was apparent synteny in the corresponding MYH cluster regions between lampreys and tetrapods, phylogenetic analysis indicated that lamprey and tetrapod MYHs have independently duplicated and diversified. Subsequent transgenic approaches showed that the 5'-flanking sequences of Japanese lamprey fast skeletal MYHs function as a regulatory sequence to drive specific reporter gene expression in the fast skeletal muscle of zebrafish embryos. Although zebrafish MYH promoters showed apparent activity to direct reporter gene expression in myogenic cells derived from mice, promoters from Japanese lamprey MYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny.
Available from: Shigeharu Kinoshita
- "Interestingly, sequence comparison analysis showed highly conserved 5′-flanking regions between torafugu MYHM5 and medaka miR-499 (see Figure 5A). The spatio-temporal expression of the major skeletal MYHs in teleost fish is regulated by small regions scattered throughout the 5′-flanking sequence [18,30,34,35]. Recently, Yeung et al.  reported promoter activity in a 6.2-kb upstream sequence of mouse MYH14 that mimics endogenous MYH14 and miR-499 expression. "
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ABSTRACT: A novel sarcomeric myosin heavy chain gene, MYH14, was identified following the completion of the human genome project. MYH14 contains an intronic microRNA, miR-499, which is expressed in a slow/cardiac muscle specific manner along with its host gene; it plays a key role in muscle fiber-type specification in mammals. Interestingly, teleost fish genomes contain multiple MYH14 and miR-499 paralogs. However, the evolutionary history of MYH14 and miR-499 has not been studied in detail. In the present study, we identified MYH14/miR-499 loci on various teleost fish genomes and examined their evolutionary history by sequence and expression analyses.
Synteny and phylogenetic analyses depict the evolutionary history of MYH14/miR-499 loci where teleost specific duplication and several subsequent rounds of species-specific gene loss events took place. Interestingly, miR-499 was not located in the MYH14 introns of certain teleost fish. An MYH14 paralog, lacking miR-499, exhibited an accelerated rate of evolution compared with those containing miR-499, suggesting a putative functional relationship between MYH14 and miR-499. In medaka, Oryzias latipes, miR-499 is present where MYH14 is completely absent in the genome. Furthermore, by using in situ hybridization and small RNA sequencing, miR-499 was expressed in the notochord at the medaka embryonic stage and slow/cardiac muscle at the larval and adult stages. Comparing the flanking sequences of MYH14/miR-499 loci between torafugu Takifugu rubripes, zebrafish Danio rerio, and medaka revealed some highly conserved regions, suggesting that cis-regulatory elements have been functionally conserved in medaka miR-499 despite the loss of its host gene.
This study reveals the evolutionary history of the MYH14/miRNA-499 locus in teleost fish, indicating divergent distribution and expression of MYH14 and miR-499 genes in different teleost fish lineages. We also found that medaka miR-499 was even expressed in the absence of its host gene. To our knowledge, this is the first report that shows the conversion of intronic into non-intronic miRNA during the evolution of a teleost fish lineage.
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