Abstract
The genomic structure including the 5′-upstream regulatory region of the fast skeletal myosin light chain 2 gene (mlc2f) was characterized in the marine medaka (Oryzias dancena; Beloniformes). Molecular phylogenic analysis inferred that the marine medaka mlc2f should belong to the teleost mlc2fa group characterized by seven-exon organization. Bioinformatic analysis of the regulatory region represented the various transcription factor binding motifs especially including myogenic regulatory factor binding sites such as myocyte enhancer factor-2 site and E-box. Real-time RT-PCR assays revealed that the mlc2f mRNA was highly predominant in skeletal muscles and also that the muscular mlc2f expression was little modulated by environmental salinity ranging from 0 to 30 ppt. The mlc2f mRNA expression was differentially modulated during embryonic development of this species, in which the expression of mlc2f was stimulated from the retinal pigmentation stage and then markedly activated until hatching. From the microinjection-based introduction of a green fluorescent protein gene (gfp) driven by a 2.95-kb marine medaka mlc2f promoter into the marine medaka embryos, the onset of transgenic GFP expression was in accordance with that of endogenous mlc2f gene. Although all the microinjected embryos and resultant F0 fish showed a mosaic distribution of GFP expression with some ectopic expression pattern, the overall expression of transgenic GFP was enriched in the skeletal muscles. However, transgenic hemizygous F1 fish produced from the germline positive founders showed uniform, fast-skeletal muscle-specific expression of GFP, which resembled the pattern of the endogenous mlc2f gene expression. The expression of transgenic GFP was observable mainly in head region and weakly in caudal peduncle of the hatched larvae. The GFP expression was gradually intensified in these sites and became spread over other skeletal muscle parts with larval ages, such that the fish at 21 days post hatching acquired uniform GFP expression over nearly whole skeletal muscles. Data from this study suggest that the mlc2f promoter-driven transgenesis holds promising potential to monitor the differentiation of the fast skeletal muscles of this species in a real-time fashion and also to drive efficiently the expression of foreign proteins in the marine medaka skeletal muscles.
Similar content being viewed by others
References
Black BL, Olson EN (1998) Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu Rev Cell Dev Biol 14:167–196
Carvalho L, Heisenberg C-P (2010) The yolk syncytial layer in early zebrafish development. Trends Cell Biol 20:586–592
Chen X, Li L, Cheng J, Chan LL, Wang DZ, Wang KJ, Baker ME, Hardiman G, Schlenk D, Cheng SH (2011) Molecular staging of marine medaka: a model organism for marine ecotoxicity study. Mar Pollut Bull 63:309–317
Cho YS, Lee SY, Kim KY, Nam YK (2009) Two metallothionein genes from mud loach Misgurnus mizolepis (Teleostei; Cypriniformes): gene structure, genome organization, and mRNA expression analysis. Comp Biochem Physiol 153B:317–326
Cho YS, Lee SY, Kim DS, Nam YK (2010a) Spawning performance, embryonic development and early viability under different salinity conditions in a euryhaline medaka species, Oryzias dancena. Korean J Ichthyol 22:25–33
Cho YS, Lee SY, Kim DS, Nam YK (2010b) Tolerance capacity to salinity changes in adult and larva of Oryzias dancena, a euryhaline medaka. Korean J Ichthyol 22:9–16
Cho YS, Lee SY, Kim YK, Kim DS, Nam YK (2011) Functional ability of cytoskeletal β-actin regulator to drive constitutive and ubiquitous expression of a fluorescent reporter throughout the life cycle of transgenic marine medaka Oryzias dancena. Transgenic Res 20:1333–1355
Chu WY, Chen J, Zhou RX, Zhao FL, Meng T, Chen DX, Nong XX, Liu Z, Lu SQ, Zhang JS (2011) Characterization and ontogenetic expression analysis of the myosin light chains from the fast white muscle of mandarin fish Siniperca chuatsi. J Fish Biol 78:1225–1238
Czosnek H, Nudel U, Shani M, Barker PE, Pravtcheva DD, Ruddle FH, Yaffe D (1982) The genes coding for the muscle contractile proteins, myosin heavy chain, myosin light chain 2, and skeletal muscle actin are located on three different mouse chromosomes. EMBO J 1:1299–1305
Du SJ, Gao J, Anyangwe V (2003) Muscle-specific expression of myogenin in zebrafish embryos is controlled by multiple regulatory elements in the promoter. Comp Biochem Physiol 134B:123–134
Fujita K, Fujita K, Ye L, Sato M, Okagaki T, Nagamachi Y, Kohama K (1999) Myosin light chain kinase from skeletal muscle regulates an ATP-dependent interaction between actin and myosin by binding to actin. Mol Cell Biochem 190:85–90
Funkenstein B, Skopal T, Rapoport B, Rebhan Y, Du SJ, Radaelli G (2007) Characterization and functional analysis of the 5′ flanking region of myosin light chain-2 gene expressed in white muscle of the gilthead sea bream (Sparus aurata). Comp Biochem Physiol 2D:187–199
Funkenstein B, Balas V, Rebhan Y, Pliatner A (2009) Characterization and functional analysis of the 5′ flanking region of Sparus aurata myostatin-1 gene. Comp Biochem Physiol 153A:55–62
Gabillard JC, Ralliere C, Sabin N, Rescan PY (2010) The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation. BMC Biotechnol 10:39
Galloway TF, Bardal T, Kvam SN, Dahle SW, Nesse G, Randøl M, Kjørsvik E, Andersen Ø (2006) Somite formation and expression of MyoD, myogenin and myosin in Atlantic halibut (Hippoglossus hippoglossus L.) embryos incubated at different temperatures: transient asymmetric expression. J Exp Biol 209:2432–2441
Gong Z, Wan H, Tay TL, Wang H, Chen M, Yan T (2003) Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle. Biochem Biophys Res Commun 308:58–63
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows95/98/NT. Nucleic Acids Symp Ser 41:95–98
Hall TE, Cole NJ, Johnston IA (2003) Temperature and the expression of seven muscle-specific protein genes during embryogenesis in the Atlantic cod Gadus morhua L. J Exp Biol 206:3187–3200
Ikebe M, Kambara T, Stafford WF, Sata M, Katayama E, Ikebe R (1998) A hinge at the central helix of the regulatory light chain of myosin is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. J Biol Chem 273:17702–17707
Inoue K, Takei Y (2003) Asian medaka fishes offer new models for studying mechanisms of seawater adaptation. Comp Biochem Physiol 136B:635–645
James J, Osinska H, Hewett TE, Kimball T, Klevitsky R, Witt S, Hall DG, Gulick J, Robbins J (1999) Transgenic overexpression of a motor protein at high levels results in severe cardiac pathology. Transgenic Res 8:9–22
Ju B, Xu Y, He J, Liao J, Yan T, Hew CL, Lam TJ, Gong Z (1999) Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters. Dev Genet 25:158–167
Ju B, Chong SW, He J, Wang X, Xu Y, Wan H, Tong Y, Yan T, Korzh V, Gong Z (2003) Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter. Dev Dyn 227:14–26
Kang C-K, Tsai S-C, Lee T-H, Hwang P-P (2008) Differential expression of branchial Na+/K+-ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater. Comp Biochem Physiol 151A:566–575
Kassahn KS, Dang VT, Wilkins SJ, Perkins AC, Ragan MA (2009) Evolution of gene function and regulatory control after whole-genome duplication: comparative analyses in vertebrates. Genome Res 19:1404–1418
Koumans JTM, Akster HA, Booms GHR, Osse JWM (1993) Growth of carp (Cyprinus carpio) white axial muscle; hyperplasia and hypertrophy in relation to the myonucleus/sarcoplasm ratio and the occurrence of different subclasses of myogeniccells. J Fish Biol 43:69–80
Krasnov A, Teerijoki H, Gorodilov Y, Mölsä H (2003) Cloning of rainbow trout (Oncorhynchus mykiss) α-actin, myosin regulatory light chain genes and the 5′-flanking region of α-tropomyosin. Functional assessment of promoters. J Exp Biol 206:601–608
Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J, Lind K, Sindelka R, Sjöback R, Sjögreen B, Strömbom B, Ståhlberg A, Zoric N (2006) The real-time polymerase chain reaction. Mol Aspects Med 27:95–125
Moutou KA, Canario AVM, Mamuris Z, Power DM (2001) Molecular cloning and sequence of Sparus aurata skeletal myosin light chains expressed in white muscle: developmental expression and thyroid regulation. J Exp Biol 204:3009–3018
Mugue NS, Ozernyuk ND (2006) Comparative structural analysis of myosin light chains and gene duplication in fish. Biol Bull 33:30–34
Mugue NS, Tikhonov AV, Ozernyuk ND (2005) Ontogenetic and phylogenetic analysis of myosin light chain proteins from skeletal muscles of loach Misgurnus fossilis. Biol Bull 32:473–477
Nam YK, Noh CH, Kim DS (1999) Transmission and expression of an integrated reporter construct in three generations of transgenic mud loach (Misgurnus mizolepis). Aquaculture 172:229–245
Ozernyuk ND, Nareiko VG, Smirnova YA, Zinov’eva RD (2004) Pattern of skeletal muscle differentiation in fish: molecular biological approaches. Biol Bull 31:209–215
Pan X, Zhan H, Gong Z (2008) Ornamental expression of red fluorescent protein in transgenic founders of white skirt tetra (Gymnocorymbus ternetzi). Mar Biotechnol 10:497–501
Ravi V, Venkatesh B (2008) Rapidly evolving fish genomes and teleost diversity. Curr Opin Genet Dev 18:544–550
Song HY, Nam YK, Bang IC, Kim DS (2009) Embryogenesis and early ontogenesis of a marine medaka, Oryzias dancena. Korean J Ichthyol 21:227–238
Stewart CN Jr (2006) Go with the glow: fluorescent proteins to light transgenic organisms. Trends Biotechnol 24:155–162
Sweeney HL (1995) Function of the N terminus of the myosin essential light chain of vertebrate striated muscle. Biophys J 68:112S–118S
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739
Tang DD, Gunst SJ (2004) The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle. J Biol Chem 279:51722–51728
Thiébaud P, Rescan P-Y, Barillot W, Rallière C, Thézé N (2001) Developmental program expression of myosin alkali light chain and skeletal actin genes in the rainbow trout Oncorhynchus mykiss. Biochim Biophys Acta Gene Struct Expr 1519:139–142
Wan H, He J, Ju B, Yan T, Lam TJ, Gong Z (2002) Generation of two-color transgenic zebrafish using the green and red fluorescent protein reporter genes gfp and rfp. Mar Biotechnol 4:146–154
Watabe S (1999) Myogenic regulatory factors and muscle differentiation during ontogeny in fish. J Fish Biol 55:1–18
Williams DW, Muller F, Lavender FL, Orban L, Maclean N (1996) High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish (Danio rerio) embryos. Transgenic Res 5:433–442
Zeng Z, Liu X, Seebah S, Gong Z (2005) Faithful expression of living color reporter genes in transgenic medaka under two tissue-specific zebrafish promoters. Dev Dyn 234:387–392
Zhu H, Wang G, Li G, Han M, Xu T, Zhuang Y, Wu X (2005) Ubiquitous expression of mRFP1 in transgenic mice. Genesis 42:86–90
Acknowledgments
This study was supported by a research fund from the Ministry of Land, Transport and Maritime Affairs, Korea (Project # 20088033-1). Authors would like to express sincere thanks to Dr. Keun-Yong Kim for his invaluable comments and critical review on the analysis of molecular phylogeny in this study.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Lee, S.Y., Kim, D.S. & Nam, Y.K. Molecular characterization of fast skeletal muscle-specific myosin light chain 2 gene (mlc2f) in marine medaka Oryzias dancena . Genes Genom 35, 289–303 (2013). https://doi.org/10.1007/s13258-013-0071-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13258-013-0071-y