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Identification and expression of Smads associated with TGF-β/activin/nodal signaling pathways in the rainbow trout (Oncorhynchus mykiss)

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Abstract

The Smad proteins are essential components of the TGF-β/activin/nodal family signaling pathway. We report the identification and expression of transcripts representing three receptor Smads (Smad2a, Smad2b, and Smad3), two common Smads (Smad4a and Smad4b), and one inhibitory Smad (Smad7). Phylogenetic analysis suggests this gene family evolved through the combination of ancient and more recent salmonid genome duplication events. Tissue distribution, embryonic expression, and expression in growth hormone (GH) treated fish were assessed by reverse transcription PCR or qPCR. All six Smad transcripts were ubiquitously expressed in adult tissues. We observed the highest expression of the receptor Smads in unfertilized eggs, generally decreasing during early embryonic development and slightly increasing around 11 days post-fertilization (dpf). Smad7 expression was low for most of embryonic development, with a dramatic increase at the onset of muscle development (6 dpf), and at hatch (24 dpf). Smad4 expression was low during early embryonic development and increased after 14 dpf. The increased expression of Smad4 and Smad7 during late embryonic development may indicate modulation of gene expression by GH axis, which initiates activity during late embryonic development. These data were supported by the modulation of these Smads in the gill filament, stomach, and muscle following a GH treatment. Additionally, these changes are concurrent with the modulation of expression of TGF-β family members. Most significantly, the increased expression of Smad7 in the muscle is simultaneous with increased expression of MSTN1A and not MSTN1B during both embryonic development and following GH treatment. These data indicate a promyogenic role for Smad7 as previously identified in other non-fish species.

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References

  • Amthor H, Nicholas G, McKinnell I, Kemp CF, Sharma M, Kambadur R, Patel K (2004) Follistatin complexes myostatin and antagonizes myostatin-mediated inhibition of myogenesis. Dev Biol 270:19–30

    Article  PubMed  CAS  Google Scholar 

  • Andersson O, Korach-Andre M, Reissmann E, Ibanez CF, Bertolino P (2006) Growth/differentiation factor 3 signals through ALK7 and regulates the accumulation of adipose tissue and diet-induced obesity. Proc Natl Acad Sci USA 105:7252–7256

    Article  Google Scholar 

  • Biga PR, Cain KD, Hardy RW, Schelling GT, Overturf K, Roberts SB, Goetz FW, Ott TL (2004) Growth hormone differentially regulates muscle myostatin1 and -2 and increases circulating cortisol in rainbow trout (Oncorhynchus mykiss). Gen Comp Endocrinol 138:32–41

    Article  PubMed  CAS  Google Scholar 

  • Bilezikjian LM, Blount AL, Leal AMO, Donaldson CJ, Fischer WH, Vale WW (2004) Autocrine/paracrine regulation of pituitary function by activin, inhibin and follistatin. Mol Cell Endocrinol 225:29–36

    Article  PubMed  CAS  Google Scholar 

  • Bobe J, Nguyen T, Jalabert B (2004) Targeted gene expression profiling in the rainbow trout (Oncorhynchus mykiss) ovary during maturational competence acquisition and oocyte maturation. Biol Reprod 71:73–82

    Article  PubMed  CAS  Google Scholar 

  • Brunelli JP, Thorgaard GH (1999) Sequence, expression and genetic mapping of a rainbow trout retinoblastoma cDNA. Gene 226:175–180

    Article  PubMed  CAS  Google Scholar 

  • Brunelli JP, Robinson BD, Thorgaard GH (2001) Ancient and recent duplications of the rainbow trout Wilms’ tumor gene. Genome 44:455–462

    Article  PubMed  CAS  Google Scholar 

  • Cai Z, Gao C, Li L, Xing K (2010) Bipolar properties of red seabream (Pagrus major) transforming growth factor-beta in induction of the leucocytes migration. Fish Shellfish Immunol 28:695–700

    Article  PubMed  CAS  Google Scholar 

  • Chang C, Brivanlou AH, Harland RM (2006) Function of the two Xenopus Smad4s in early frog development. J Biol Chem 281:30794–30803

    Article  PubMed  CAS  Google Scholar 

  • Chomczynski P, Mackey K (1995) Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 19:942–945

    PubMed  CAS  Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 162:156–159

    Article  PubMed  CAS  Google Scholar 

  • Coulibaly I, Gahr S, Yao J, Rexroad C (2006) Embryonic expression of UCP2 in rainbow trout (Oncorhynchus mykiss). Fish Physiol Biochem 32:249–253

    Article  CAS  Google Scholar 

  • Delalande JM, Rescan P-Y (1999) Differential expression of two non-allelic MyoD genes in developing and adult myotomal musculature of the trout (Oncorhynchus mykiss). Dev Genes Evol 209:432–437

    Article  PubMed  CAS  Google Scholar 

  • Dick A, Mayr T, Bauer H, Meier A, Hammerschmidt M (2000) Cloning and characterization of zebrafish smad2, smad3 and smad4. Gene 246:69–80

    Article  PubMed  CAS  Google Scholar 

  • Feng XH, Derynck R (2005) Specificity and versatility in TGF-beta signaling through Smads. Annu Rev Cell Dev Biol 21:659–693

    Article  PubMed  CAS  Google Scholar 

  • Gabillard J-C, Duval H, Cauty C, Rescan P-Y, Weil C, Le Bail P-Y (2003) Differential expression of the two GH genes during embryonic development of rainbow trout (Oncorhynchus mykiss) in relation with the IGFs system. Mol Reprod Dev 64:32–40

    Article  PubMed  CAS  Google Scholar 

  • Gahr SA, Palti Y, Rexroad CE III (2004) Genomic characterization of a novel pair of ID genes in the rainbow trout (Oncorhynchus mykiss). Anim Genet 35:317–320

    Article  PubMed  CAS  Google Scholar 

  • Gahr SA, Rodriguez MF, Rexroad CE III (2005) Identification and expression profile of the ID gene family in the rainbow trout (Oncorhynchus mykiss). Biochim Biophys Acta 1729:64–73

    Article  PubMed  CAS  Google Scholar 

  • Gahr SA, Weber GM, Rexroad CE III (2006) Fasting and refeeding affect the expression of the inhibitor of DNA binding (ID) genes in rainbow trout (Oncorhynchus mykiss) muscle. Comp Biochem Physiol B Biochem Mol Biol 144:472–477

    Article  PubMed  Google Scholar 

  • Gahr SA, Vallejo RL, Weber GM, Shepherd BS, Silverstein JT, Rexroad CE III (2008) Effects of short-term growth hormone treatment on liver and muscle transcriptomes in rainbow trout (Oncorhynchus mykiss). Physiol Genomics 32:380–392

    PubMed  CAS  Google Scholar 

  • Garber MJ, DeYonge KG, Byatt JC, Lellis WA, Honeyfield DC, Bull RC, Schelling GT, Roeder RA (1995) Dose–response effects of recombinant bovine somatotropin (Posilac) on growth performance and body composition of two-year-old rainbow trout (Oncorhynchus mykiss). J Anim Sci 73:3216–3222

    PubMed  CAS  Google Scholar 

  • Garikipati DK, Gahr SA, Rodgers BD (2006) Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes. J Endocrinol 190:879–888

    Article  PubMed  CAS  Google Scholar 

  • Garikipati DK, Gahr SA, Roalson EH, Rodgers BD (2007) Characterization of rainbow trout myostatin-2 genes (rtMSTN-2a and -2b): genomic organization, differential expression, and pseudogenization. Endocrinology 148:2106–2115

    Article  PubMed  CAS  Google Scholar 

  • Hill JA, Kiessling A, Devlin RH (2000) Coho salmon (Oncorhynchus kisutch) transgenic for a growth hormone gene construct exhibit increased rates of muscle hyperplasia and detectable levels of differential gene expression. Can J Fish Aquat Sci 57:939–950

    Article  CAS  Google Scholar 

  • Howell M, Itoh F, Pierreux CE, Valgeirsdottir S, Itoh S, ten Dijke P, Hill CS (1999) Xenopus Smad4beta is the co-Smad component of developmentally regulated transcription factor complexes responsible for induction of early mesodermal genes. Dev Biol 214:354–369

    Article  PubMed  CAS  Google Scholar 

  • Johnston IA (1999) Muscle development and growth: potential implications for flesh quality in fish. Aquaculture 177:99–115

    Article  Google Scholar 

  • Kalujnaia S, McWilliam IS, Zaguinaiko VA, Feilen AL, Nicholson J, Hazon N, Cutler CP, Cramb G (2007) Transcriptome approach to the study of osmoregulation in the European eel Anguilla anguilla. Physiol Genomics 31:385–401

    Article  PubMed  CAS  Google Scholar 

  • Knight PG, Glister C (2006) TGF-beta superfamily members and ovarian follicle development. Reproduction 132:191–206

    Article  PubMed  CAS  Google Scholar 

  • Kollias HD, Perry RLS, Miyake T, Aziz A, McDermott JC (2006) Smad7 promotes and enhances skeletal muscle differentiation. Mol Cell Biol 26:6248–6260

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245

    Article  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Lau M-T, Ge W (2005) Cloning of Smad2, Smad3, Smad4 and Smad7 from the goldfish pituitary and evidence for their involvement in activin regulation of goldfish FSHb promoter activity. Gen Comp Endocrinol 141:22–38

    Article  PubMed  CAS  Google Scholar 

  • Leder EH, Silverstein JT (2006) The pro-opiomelanocortin genes in rainbow trout (Oncorhynchus mykiss): duplications, splice variants, and differential expression. J Endocrinol 188:355–363

    Article  PubMed  CAS  Google Scholar 

  • Liu T, Feng XH (2010) Regulation of TGF-β signaling by protein phosphotases. Biochem J 430:191–198

    Article  PubMed  CAS  Google Scholar 

  • Massague J, Chen YG (2000) Controlling TGF-beta signaling. Genes Dev 14:627–644

    PubMed  CAS  Google Scholar 

  • McCormick SD (2001) Endocrine control of osmoregulation in teleost fish. Am Zool 41:781–794

    Article  CAS  Google Scholar 

  • Medeiros EF, Phelps MP, Fuentes FD, Bradley TM (2009) Overexpression of follistatin in trout stimulates increased muscling. Am J Physiol Regul Integr Comp Physiol 297:R235–R242

    Article  PubMed  CAS  Google Scholar 

  • Miyake T, Alli NS, McDermott JC (2010) Nuclear function of smad7 promotes myogenesis. Mol Cell Biol 30:722–735

    Article  PubMed  CAS  Google Scholar 

  • Miyazono K, Kusanagi K, Inoue H (2001) Divergence and convergence of TGF-β/BMP signaling. J Cell Physiol 187:265–276

    Article  PubMed  CAS  Google Scholar 

  • Moustakas A, Heldin CH (2009) The regulation of TGF-β signal transduction. Development 136:3699–3714

    Article  PubMed  CAS  Google Scholar 

  • Murani E, Muraniova M, Ponsuksili S, Schellander K, Wimmers K (2007) Identification of genes differentially expressed during prenatal development of skeletal muscle in two pig breeds differing in muscularity. BMC Dev Biol 7:109. doi:10.1186/1471-213X-7-109

    Article  PubMed  Google Scholar 

  • Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin N-E, Heldin C-H, ten Dijke P (1997) Identification of Smad7, a TGF-β-inducible antagonist of TGF-β signaling. Nature 389:631–635

    Article  PubMed  CAS  Google Scholar 

  • Pelegri F (2003) Maternal factors in zebrafish development. Dev Dyn 228:535–554

    Article  PubMed  CAS  Google Scholar 

  • Pollet N, Mazabraud A (2006) Insights from Xenopus genomes. Genome Dyn 2:138–153

    Article  PubMed  CAS  Google Scholar 

  • Rescan P-Y, Collet B, Ralliere C, Cauty C, Delalande J-M, Goldspink G, Fauconneau B (2001) Red and white muscle development in the trout (Oncorhynchus mykiss) as shown by in situ hybridisation of fast and slow myosin heavy chain transcripts. J Exp Biol 204:2097–2101

    PubMed  CAS  Google Scholar 

  • Rowlerson AM, Veggetti A (2001) Cellular mechanisms of post-embryonic muscle growth in aquaculture species. In: Johnston IA (ed) Fish physiology, vol 18. Muscle development and growth. Academic Press, San Diego, pp 103–140

    Google Scholar 

  • Schulte PM, Down NE, Donaldson EM, Souza LM (1989) Experimental administration of recombinant bovine growth hormone to juvenile rainbow trout (Salmo gairdneri) by injection or by immersion. Aquaculture 76:145

    Article  Google Scholar 

  • Steinbacher P, Haslett JR, Obermayer A, Marschallinger J, Bauer HC, Sanger AM, Stoiber W (2007) Myod and myogenin expression during myogenic phases in brown trout: a precocious onset of mosaic hyperplasia is a prerequisite for fast somatic growth. Dev Dyn 236:1106–1114

    Article  PubMed  CAS  Google Scholar 

  • Tan Q, Zagrodny A, Bernaudo S, Peng C (2009) Regulation of membrane progestin receptors in the zebrafish ovary by gonadotropin, activin, TGF-beta and BMP-15. Mol Cell Endocrinol 312:72–79

    Article  PubMed  CAS  Google Scholar 

  • Tian X, Halfhill AN, Diaz FJ (2010) Localization of phosphorylated SMAD proteins in granulosa cells, oocytes and oviducts of female mice. Gene Expr Patterns 10:05–112

    Article  Google Scholar 

  • Wrana JL (2000) Regulation of Smad activity. Cell 100:189–192

    Article  PubMed  CAS  Google Scholar 

  • Wrana JL, Attisano L (2000) The Smad pathway. Cytokine Growth Factor Rev 11:5–13

    Article  PubMed  CAS  Google Scholar 

  • Xu L (2006) Regulation of Smad activities. Biochim Biophys Acta 1759:503–513

    Article  PubMed  CAS  Google Scholar 

  • Yanagisawa K, Osada H, Masuda A, Kondo M, Saito T, Yatabe Y, Takagi K, Takahashi T (1998) Induction of apoptosis by Smad3 and down-regulation of Smad3 expression in reponse to TGF-β in human normal lung epithelial cells. Oncogene 17:1743–1747

    Article  PubMed  CAS  Google Scholar 

  • Zhu X, Topouzis S, Liang L-F, Stotish RL (2004) Myostatin signaling through Smad2, Smad3 and Smad4 is regulation by the inhibitory Smad7 by a negative feedback mechanism. Cytokine 26:262–272

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors acknowledge Jill Birkett and for technical assistance with the qPCR Assays, Drs. Issa Coulibaly and Fernanda Rodriguez for assistance in sample collection, Jim Everson, Josh Kretzer, and Jon Leasor for their assistance with animal care, and Roseanna Long and Kristy Shewbridge for general laboratory assistance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. Posilac® slow release rbST and vehicle were kindly provided by Dr. Gregg Bogosian (Monsanto, St. Louis, MO, USA).

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  1. Biology Department, St. Vincent College, 300 Fraser Purchase Rd., Latrobe, PA, 15650, USA

    Scott A. Gahr

  2. United States Department of Agriculture, Agriculture Research Service, National Center for Cool and Cold Water Aquaculture, Kearneysville, WV, 25430, USA

    Gregory M. Weber & Caird E. Rexroad III

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  1. Scott A. Gahr
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Correspondence to Scott A. Gahr.

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Gahr, S.A., Weber, G.M. & Rexroad, C.E. Identification and expression of Smads associated with TGF-β/activin/nodal signaling pathways in the rainbow trout (Oncorhynchus mykiss). Fish Physiol Biochem 38, 1233–1244 (2012). https://doi.org/10.1007/s10695-012-9611-7

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