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Cloning, expression profiling and promoter functional analysis of Bone morphogenetic protein 6 and 7 in tongue sole (Cynoglossus semilaevis)

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Abstract

Bone morphogenetic proteins (BMPs) play crucial roles in vertebrate developmental process and are associated with the mechanisms which drive early skeletal development. As a first approach to elucidating the role of BMPs in regulating fish bone formation and growth, we describe the cloning, expression profiling and promoter functional analysis of bmp6 and bmp7 in tongue sole (Cynoglossus semilaevis). The full length of bmp6 and bmp7 cDNA sequences is 1939 and 1836 bp, which encodes a protein of 428 and 427 amino acids, respectively. Tissue expression distribution of bmp6 and bmp7 was examined in 14 tissues of mature individuals by quantitative real-time PCR (qRT-PCR). The results revealed that bmp6 was predominantly expressed in the gonad, and bmp7 exhibited the highest expression level in the dorsal fin. Further comparison of bmp6 expression levels between female and male gonads showed that the expression in the ovary was significantly higher than in the testis. Moreover, bmp6 and bmp7 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp6 was observed in the egg stage (multi-cell and gastrula stage); while bmp7 exhibited the highest expression in the larva stage (1–4 days old). The high expression levels of BMP6 in the ovary as well as at early embryonic stages indicated that the maternally stored transcripts of bmp6 might play a role in early embryonic development. Whole-mount in situ hybridization showed that bmp6 and bmp7 exhibited similar spatial expression patterns. Both bmp6 and bmp7 signals were first detected in the head and anterior regions in newly hatched larvae, and then, the mRNAs appeared in the crown-like larval fin, jaw, operculum and fins (pectoral, dorsal, pelvic and anal) along with early development. Subsequently, we characterized the 5′-flanking regions of bmp6 and bmp7 by testing the promoter activity by luciferase reporter assays. Positive regulatory regions were, respectively, detected at the location of −272 to +28 and −740 to −396 in bmp6 and bmp7 gene. The predicted transcription factor binding sites (CREB, AP1 and methyl-CpG-binding protein) in the regions might participate in the transcriptional regulation of these two genes.

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References

  • Balemans W, Hul WV (2002) Extracellular regulation of BMP signaling in vertebrates: a cocktail of modulators. Dev Biol 250:231–250

    Article  CAS  PubMed  Google Scholar 

  • Baron D, Houlgatte R, Fostier A, Guiguen Y (2005) Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow trout. Biol Reprod 73:959–966

    Article  CAS  PubMed  Google Scholar 

  • Bragdon B, Moseychuk O, Saldanha S, King D, Julian J et al (2011) Bone morphogenetic proteins: a critical review. Cell Signal 23:609–620

    Article  CAS  PubMed  Google Scholar 

  • Celeste AJ, Iannazzi JA, Taylor RC, Hewick RM, Rosen V et al (1990) Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci 87:9843–9847

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y et al (2003) Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg (Am Vol) 85:1544–1552

    Article  Google Scholar 

  • Chubinskaya S, Hurtig M, Rueger DC (2007) OP-1/BMP-7 in cartilage repair. Int Orthop 31:773–781

    Article  PubMed  PubMed Central  Google Scholar 

  • de Castro E, Sigrist CJ, Gattiker A, Bulliard V, Langendijk-Genevaux PS et al (2006) ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucl Acids Res 34:362–365

    Article  Google Scholar 

  • Ducy P, Karsenty G (2000) The family of bone morphogenetic proteins. Kidney Int 57:2207–2214

    Article  CAS  PubMed  Google Scholar 

  • Erickson GF, Shimasaki S (2003) The spatiotemporal expression pattern of the bone morphogenetic protein family in rat ovary cell types during the estrous cycle. Reprod Biol Endocrinol 1:1–20

    Article  Google Scholar 

  • Faustino M, Power D (1998) Development of osteological structures in the sea bream: vertebral column and caudal fin complex. J Fish Biol 52:11–22

    Article  Google Scholar 

  • Fraser M, Anderson T, de Nys R (2004) Ontogenic development of the spine and spinal deformities in larval barramundi (Lates calcarifer) culture. Aquaculture 242:697–711

    Article  Google Scholar 

  • Frota IMA, Leitão CCF, Costa JJN, Hurk RVD, Saraiva MVA et al (2013) Levels of BMP-6 mRNA in goat ovarian follicles and in vitro effects of BMP-6 on secondary follicle development. Zygote 21:270–278

    Article  CAS  PubMed  Google Scholar 

  • Gitelman SE, Kobrin M, Lee A, Fet V, Lyons K et al (1997) Structure and sequence of the mouse Bmp6 gene. Mamm Genome 8:212–214

    Article  CAS  PubMed  Google Scholar 

  • Hochheimer A, Tjian R (2003) Diversified transcription initiation complexes expand promoter selectivity and tissue-specific gene expression. Genes Dev 17:1309–1320

    Article  CAS  PubMed  Google Scholar 

  • Hogan BL (1996) Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev 10:1580–1594

    Article  CAS  PubMed  Google Scholar 

  • Holland LZ, Holland PW, Holland ND, Ferraris J, Palumbi S (1996) Revealing homologies between body parts of distantly related animals by in situ hybridization to developmental genes: amphioxus versus vertebrates. Mol Zoolog Adv Strategies Protoc 267:473–483

    Google Scholar 

  • Juengel J, McNatty K (2005) The role of proteins of the transforming growth factor-β superfamily in the intraovarian regulation of follicular development. Hum Reprod 11:144–161

    Article  Google Scholar 

  • Juengel JL, Reader KL, Bibby AH, Stan L, Ian R et al (2006) The role of bone morphogenetic proteins 2, 4, 6 and 7 during ovarian follicular development in sheep: contrast to rat. Reproduction 131:501–513

    Article  CAS  PubMed  Google Scholar 

  • Kang Q, Sun M, Cheng H, Peng Y, Montag A et al (2004) Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther 11:1312–1320

    Article  CAS  PubMed  Google Scholar 

  • Kawai S, Sugiura T (2001) Characterization of human bone morphogenetic protein (BMP)-4 and -7 gene promoters: activation of BMP promoters by Gli, a sonic hedgehog mediator. Bone 29:54–61

    Article  CAS  PubMed  Google Scholar 

  • Kingsley DM (1994) The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8:133–146

    Article  CAS  PubMed  Google Scholar 

  • Krause C, Kloen P, Dijke PT (2011) Elevated transforming growth factor β and mitogen-activated protein kinase pathways mediate fibrotic traits of Dupuytren’s disease fibroblasts. Fibrogenesis Tissue Repair 4:14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kugimiya F, Kawaguchi H, Kamekura S, Chikuda H, Ohba S et al (2005) Involvement of endogenous bone morphogenetic protein (BMP) 2 and BMP6 in bone formation. J Biol Chem 280:35704–35712

    Article  CAS  PubMed  Google Scholar 

  • Lankford SE, Weber GM (2010) Temporal mRNA expression of transforming growth factor-beta superfamily members and inhibitors in the developing rainbow trout ovary. Gen Comp Endocrinol 166:250–258

    Article  CAS  PubMed  Google Scholar 

  • Lee WS, Yoon SJ, Yoon TK, Cha KY, Lee SH et al (2004) Effects of bone morphogenetic protein-7 (BMP-7) on primordial follicular growth in the mouse ovary. Mol Reprod Dev 69:159–163

    Article  CAS  PubMed  Google Scholar 

  • Li CW, Ge W (2011) Spatiotemporal expression of bone morphogenetic protein family ligands and receptors in the zebrafish ovary: a potential paracrine-signaling mechanism for oocyte-follicle cell communication. Biol Reprod 85:977–986

    Article  CAS  PubMed  Google Scholar 

  • Ling F, Wang T, Wei L, Zhu X, Chen Y et al (2009) Cloning and characterization of the 5’-flanking region of the pig AgRP gene. Biochemi Biophys Res Commun 381:236–240

    Article  CAS  Google Scholar 

  • Liu XZ, Zhuang ZM, Ma AJ, Chen SQ, Sun ZZ et al (2005) Reproductive biology and breeding technology of Cynoglossus semilaevis. Mar Fish Res 26:7–14

    CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Ma Q, Zhuang Z, Feng W, Liu S, Tang Q (2015) Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole (Cynoglossus semilaevis). Acta Oceanologica Sinica 34:90–97

    Article  CAS  Google Scholar 

  • Massague J (1990) The transforming growth factor-beta family. Annu Rev Cell Biol 6:597–641

    Article  CAS  PubMed  Google Scholar 

  • Mehler MF, Mabie PC, Zhang D, Kessler JA (1997) Bone morphogenetic proteins in the nervous system. Trends Neurosci 20:309–317

    Article  CAS  PubMed  Google Scholar 

  • Messeguer X, Escudero R, Farré D, Núñez O, Martínez J et al (2002) PROMO: detection of known transcription regulatory elements using species-tailored searches. Bioinformatics 18:333–334

    Article  CAS  PubMed  Google Scholar 

  • Mizrahi O, Sheyn D, Tawackoli W, Kallai I, Oh A et al (2012) BMP-6 is more efficient in bone formation than BMP-2 when overexpressed in mesenchymal stem cells. Gene Ther 20:370–377

    Article  PubMed  Google Scholar 

  • Moghaddam A, Elleser C, Biglari B, Wentzensen A, Zimmermann G (2010) Clinical application of BMP 7 in long bone non-unions. Arch Orthop Trauma Surg 130:71–76

    Article  PubMed  Google Scholar 

  • Morrissey J, Hruska K, Guo G, Wang S, Chen Q et al (2002) Bone morphogenetic protein-7 improves renal fibrosis and accelerates the return of renal function. J Am Soc Nephrol 13:14–21

    Article  Google Scholar 

  • Otsuka F, Moore RK, Shimasaki S (2001) Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary. J Biol Chem 276:32889–32895

    Article  CAS  PubMed  Google Scholar 

  • Perron JC, Dodd J (2012) Structural distinctions in BMPs underlie divergent signaling in spinal neurons. Neural Dev 7:16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Riko N, Kenji H, Fumiyo I, Fumitaka I, Atsuko S et al (2008) Signal transduction and transcriptional regulation during mesenchymal cell differentiation. J Bone Miner Metab 26:203–212

    Article  Google Scholar 

  • Sampath TK, Coughlin JE, Whetstone RM, Banach D, Corbett C et al (1990) Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily. J Biol Chem 265:13198–13205

    CAS  PubMed  Google Scholar 

  • Sandelin A, Carninci P, Lenhard B, Ponjavic J, Hayashizaki Y et al (2007) Mammalian RNA polymerase II core promoters: insights from genome-wide studies. Nat Rev Genet 8:424–436

    Article  CAS  PubMed  Google Scholar 

  • Schlunegger MP, Grütter MG (1992) An unusual feature revealed by the crystal structure at 2.2 Å resolution of human transforming growth fact or-β2. Nature 358:430–434

    Article  CAS  PubMed  Google Scholar 

  • Schmid B, Fürthauer M, Connors SA, Trout J, Thisse B et al (2000) Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation. Development 127:957–967

    CAS  PubMed  Google Scholar 

  • Shi Y, Massague J (2003) Mechanisms of tgf-Beta signaling from cell membrane to the nucleus. Cell 113:685–700

    Article  CAS  PubMed  Google Scholar 

  • Shimasaki S, Moore RK, Otsuka F, Erickson GF (2004) The bone morphogenetic protein system in mammalian reproduction. Endocr Rev 25:72–101

    Article  CAS  PubMed  Google Scholar 

  • Simon M, Feliers D, Arar M, Bhandari B, Abboud HE (2002) Cloning of the 5’-flanking region of the murine bone morphogenetic protein-7 gene. Mol Cell Biochem 233:31–37

    Article  CAS  PubMed  Google Scholar 

  • Smith A, Avaron F, Guay D, Padhi B, Akimenko M (2006) Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblast differentiation and function. Dev Biol 299:438–454

    Article  CAS  PubMed  Google Scholar 

  • Sugiura K, Su YQ, Eppig JJ (2010) Does bone morphogenetic protein 6 (BMP6) affect female fertility in the mouse? Biol Reprod 83:997–1004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sun D, Piez KA, Ogawa Y, Davies DR (1992) Crystal structure of transforming growth factor-beta 2: an unusual fold for the superfamily. Science 257:369–373

    Article  CAS  Google Scholar 

  • Tamada H, Kitazawa R, Gohji K, Kamidono S, Maeda S et al (1998) Molecular cloning and analysis of the 5’-flanking region of the human bone morphogenetic protein-6 (BMP-6). Biochim Biophys Acta 1395:247–251

    Article  CAS  PubMed  Google Scholar 

  • Tong X (2010) Studies on early development and related physiological traits in Turbot. Institute of Oceanology, Qingdao

    Google Scholar 

  • Urist MR, Mikulski A, Lietze A (1979) Proceedings of the national academy of science of the United States of America. Proc Natl Acad Sci 76:1828–1832

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wagemans F, Vandewalle P (1999) Development of the cartilaginous skull in solea solea: trends in pleuronectiforms. Annales des Sciences Naturelles-Zoologie et Biologie Animale 20:39-52

    Article  Google Scholar 

  • Wan RJ, Jiang YW (2004) Morphological and developmental characters at the early stages of the tonguefish Cynoglossus semilaevis. Acta Zoologica Sinica 50:91–102

    Google Scholar 

  • Wise SB, Stock DW (2006) Conservation and divergence of Bmp2a, Bmp2b, and Bmp4 expression patterns within and between dentitions of teleost fishes. Evol Dev 8:511–523

    Article  CAS  PubMed  Google Scholar 

  • Wozney JM (1998) The bone morphogenetic protein family: multifunctional cellular regulators in the embryo and adult. Eur J Oral Sci 106:160–166

    Article  CAS  PubMed  Google Scholar 

  • Yukiko O, Ikumi H, Keiji I, Eri S, Shinichi M et al (2014) Bmp7 and Lef1 are the downstream effectors of androgen signaling in androgen-induced sex characteristics development in medaka. Endocrinology 155:449–462

    Article  Google Scholar 

  • Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL, Mcmullen JR et al (2007) Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med 13:952–961

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors are very grateful to Haiyang hatchery station (Qingdao, Shandong Province) for their support in fish culture and sampling. The research was financially supported by the National Natural Science Foundation of China (Grant No.: 31201981), Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2016RC-LX02) and the Applied Basic Research Programs of Qingdao (14-2-4-15-jch).

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Authors and Affiliations

  1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, China

    Qian Ma, Wenrong Feng, Zhimeng Zhuang & Shufang Liu

  2. Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266200, China

    Qian Ma & Shufang Liu

  3. Function Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266200, China

    Zhimeng Zhuang

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  1. Qian Ma
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Correspondence to Zhimeng Zhuang.

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Ma, Q., Feng, W., Zhuang, Z. et al. Cloning, expression profiling and promoter functional analysis of Bone morphogenetic protein 6 and 7 in tongue sole (Cynoglossus semilaevis). Fish Physiol Biochem 43, 435–454 (2017). https://doi.org/10.1007/s10695-016-0298-z

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