Aquaculture 280 (2008) 247–255

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The isolation and characterization of myostatin gene in Japanese flounder(Paralichthys olivaceus): Ubiquitous tissue expression anddevelopmental specific regulation

Qiwang Zhong, Quanqi Zhang ⁎, Yanjie Chen, Yeying Sun, Jie Qi, Zhigang Wang, Shuo Li, Chunmei Li, Xun LanLaboratory of Marine Genetics and Breeding, College of Marine Life Science, Ocean University of China, No.5 Yushan Road, Qingdao, 266003, PR China

⁎ Corresponding author. Tel./fax: +86 532 8203 1931.E-mail address: qzhang@ouc.edu.cn (Q. Zhang).

0044-8486/$ – see front matter © 2008 Elsevier B.V. Adoi:10.1016/j.aquaculture.2008.04.015

a b s t r a c t

a r t i c l e i n f o

Article history:

Myostatin (MSTN) is a nega Received 6 September 2007Received in revised form 31 March 2008Accepted 9 April 2008

Keywords:Paralichthys olivaceusMyostatinGDF 11Expression profilesSNPMicrosatellite

Paralichthys olivaceus

tive regulator of skeletal muscle growth and development. In the current study,the MSTN gene was cloned from one of flatfish species, Japanese flounder (Paralichthys olivaceus). In the5520-bp genomic sequence, three exons, two introns, and 5' and 3' flanking sequences were identified. Theputative amino acid sequence was 377 residues long, including a signal peptide, nine conserved cysteineresidues, and a RXXR proteolytic processing site. The overall amino acid sequence of the P. olivaceus MSTN(pMSTN) was highly conserved with that of other organisms. Phylogenetic analysis suggested theevolutionary relationships of pMSTN with other known MSTN and GDF-11 genes. There were fourmicrosatellite sequences identified in noncoding regions and 19 SNPs identified in coding sequences.Expression analysis revealed that pMSTN gene was expressed not only in the skeletal muscle, but also inother tissues, including cartilage, which has not been reported in fish previously. Although pMSTN wasexpressed in gill, no transcript could be detected in FG-9307, a continuous gill cell line from P. olivaceus.Quantitative RT-PCR analysis indicated that the expression of pMSTN appeared to be developmentallyregulated. The pMSTN mRNA detected in unfertilized eggs indicated the maternal deposit. Considering of theubiquitous tissue distribution and the continuous expression of pMSTN during the whole embryonicdevelopment in Japanese flounder, MSTN gene in fish may play different roles in comparison with that inmammals.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Myostatin (MSTN, also known as GDF-8) is a member of thetransforming growth factor-β superfamily. In mammals, MSTN mRNAis expressed almost exclusively in the cells of skeletal muscle lineagein developing and adult animals and functions as a negative regulatorof muscle growth by inhibiting the hypertrophy and hyperplasia ofskeletal muscle cells (McPherron et al., 1997; Lee and McPherron,1999). MSTN deficient mice showed a 2- to 3-fold increase in skeletalmuscle size and mass in comparison with their wild-type littermates(McPherron et al., 1997). Similarly, two domestic breeds of cattle withnatural MSTN mutation, the Belgian Blue and the Piedmontese,showed abnormally increased muscular mass (Kambadur et al., 1997;Grobet et al., 1997).

Because of its role in the regulation of muscle growth, potentialapplications in aquaculture have been considered, and many MSTNsequences have been isolated from diverse fish species. Unlikemammals, the expression of fish MSTN occurs in many different

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tissues in addition to skeletal muscle, indicating that the role of fishMSTN might not be restricted in muscle growth control. Interestingly,discrepant expression distributions were observed in some fishspecies. For example, MSTN mRNA was found in both intestine andliver of channel catfish (Ictalurus punctatus) (Kocabas et al., 2002).However, in the same tissues of shi drum (Umbrina cirrosa) noexpression of MSTN was observed (Maccatrozzo et al., 2002), and intilapia (Oreochromis mossambicus) MSTN gene was expressed inintestine but not in liver (Rodgers et al., 2001). The differential tissuedistribution was especially significant in some salmonid species,which had two isoforms of MSTN-1 gene in the genome and eachisoform exhibited different expression patterns (Rescan et al., 2001;Ostbye et al., 2001; Roberts and Goetz, 2001). When the develop-mental expression profiles of MSTNs in different fish species wereexamined, discrepant results were also reported (Rodgers et al., 2001;Kocabas et al., 2002; Roberts and Goetz, 2003). Recent phylogeneticanalysis have identified a novel type of fish MSTN, MSTN-2, inzebrafish (Biga et al., 2005; Kerr et al., 2005) and salmonids species(Garikipati et al., 2007; Ostbye et al., 2007). Two distinct fish clades,MSTN-1 and MSTN-2, existing in some fish genomes suggested anearly genome duplication event in the teleost lineage (Postlethwaitet al., 1998; Taylor et al., 2003). A second duplication event specifically

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within the salmonids produced additional MSTN paralogs within eachMSTN-1 and MSTN-2 clade. These different MSTN genes exhibiteddifferential tissue and developmental expression patterns (Biga et al.,2005; Kerr et al., 2005; Garikipati et al., 2007; Ostbye et al., 2007),although they shared high identity in amino acid sequence. Thus,despite the similarities in sequence and structure, diverse actions ofMSTN in different fish species may exist. Isolation and analysis ofMSTN genes from other fish species will help to understand thesefunctional variances.

Japanese flounder (Paralichthys olivaceus) is a commerciallyimportant fish species and the breeding of fast-growing and anti-disease varieties has been carried out in our laboratory. As a part ofthis research, many growth and anti-disease related genes have beenidentified. Here we reported the isolation, characterization andexpression analysis of the MSTN gene from P. olivaceus (pMSTN).Our results showed that pMSTN gene was closely related to fishMSTN-1 genes and expressed in many tissues. Meanwhile, pMSTNmRNA could be detected in unfertilized eggs and appeared to bedevelopmentally regulated.

2. Materials and methods

2.1. Isolation of RNA and genomic DNA

All experimental fish were collected from the fish hatchery ofShandong Huaxin-Haida MariBio Co., Ltd., PR China. The fish wereover anesthetized with MS222, and heart, liver, spleen, gills, kidney,brain, intestine, eyes, ovary, testis, cartilage, blood and skeletal musclewere dissected, immediately frozen in liquid nitrogen and stored at−80 °C. Fertilized eggs of P. olivaceus were obtained by artificialfertilization and maintained at 16 °C in clean sea water with aeration.Embryos at different developmental stages were determined undermicroscope, collected and preserved in liquid nitrogen until used.Total RNA was isolated separately from the sampled tissues usingTrizol reagent (Invitrogen), DNA contamination was removed byDNase I treatment. The absence of genomic DNA contamination wasverified by PCR using 18S rRNA gene specific primers and RNAtemplate lacking a reverse-transcription step. Genomic DNA wasisolated from the blood. Simply, 0.1 ml blood was collected fromcaudal vein using 1 ml syringes, dissolved in 5 ml DNA extractionbuffer containing 10 mM Tris–HCl pH 8.0 125 mM NaCl 10 mM EDTApH 8.0 0.5% SDS and 4 M Urea (TNES) and stored at room temperatureuntil used.15 μl of preserved samplewas diluted by 600 μl TNES buffer.After overnight digestion with proteinase K at the final concentrationof 100 μg/ml at 50 °C, and extraction with phenol and chloroform/isoamyl alcohol, DNAwas precipitated with isopropanol, washed with70% ethanol and resuspended in ddH2O.

2.2. Cloning and sequencing of MSTN cDNA

Total RNA used for obtaining MSTN cDNA was extracted fromskeletal muscle. The first strand cDNA was generated with M-MLVreverse transcriptase (Promega) and oligo(dT)17 primers (Tiangen).Three degenerate primers were designed based on conserved re-gions of MSTN in other fish species. Initially, a partial fragmentwas obtained using two degenerate primers Fw1 (5'-TCKTGGCAR-AGYATMGACG-3') and Rv1 (5'-ATARYCCAGTCCCAGCCRAA-3'). A largercDNA region was obtained using the degenerate primers Fw2 (5'-CARATTYTBAGYAARCTBCG) and Rv1 in the first PCR followed by Fw2and a nested primer Rv2 (5'-AAGTCAACTGTGAGCGGATAGC-3'), aspecific primer designed on the base of the sequenced fragment

Fig. 1. Genomic sequences of P. olivaceus MSTN locus (accession number DQ997779). CDS5' untranslated region, the introns and the 3' untranslated region are shown in small letters. Tthe CDS. An arrow indicates the transcriptional start site obtained by 5'-RACE. Simple sequnderlined and labeled, and E boxes are shown in boldface and labeled. Poly-A signal is no

above, in the second PCR. The SMART RACE cDNA Amplification kit(Clontech) was used to isolate the 5' and 3' unknown regionsaccording to the manufacturer's instruction using the specific primersbased on the obtained sequence information. The resulting full-lengthcoding sequence was confirmed by PCR using two gene specificprimers (Fw3: 5'-CAA ACCTCCCACCAGAGAAAATG-3' and Rv3: 5'-AGACGCTCTCCGTCCCAACTC-3') flanking the entire open readingframe of pMSTN. All the PCR products were separated on agarosegels, purified, cloned into the pMD-18T vector (TaKaRa) andsequenced.

2.3. Identification of single nucleotide polymorphisms (SNPs)

For SNPs identification, 30 individuals were assessed in this study.RNA from different muscle samples was isolated with Trizol reagentand treated with DNase I as above. Then, cDNA was generated witholigo(dT)17 primers (Tiangen) using M-MLV reverse transcriptase(Promega). The entire open reading frame of pMSTN were amplifiedusing Taq Platinum DNA Polymerase (Tiangen) and primers Fw3 andRv3. PCR products were separated on agarose gels, purified and clonedinto the pMD-18T vector (TaKaRa) and four clones of each individualwere sequenced.

2.4. Isolation of genomic sequences of MSTN

The promoter sequence was isolated using the strategy of thegenome walking method with some modifications. Simply, 20 μggenomic DNA was digested with restriction enzyme EcoRI and thenligated to a special adaptor (obtained by annealing of two oligonu-cleotides: 5'-CTCGTAGACTGCGTACC-3' and 5'-AATTGGTACGCAGTC-3'). A semi-nested PCR was conducted using an outside primer (5'-CTCGTAGACTGCGTACCAATTC-3') on the adaptor and two gene specificprimers (Rw4: 5'-GACCACATCCCTGTTGTCATCTC-3' and Rv5: 5'-TCCCAGCACGTCGTACTGGTC-3') which were designed on the 5'endof MSTN cDNA. The intron sequences were obtained using two sets ofprimers encompassing the presumed introns (Fw4: 5'-GGGAGATGA-CAACAGGGATG-3' and Rv6: 5'-TTAATCTCGATGCCCCAGTTG-3' forintron 1; Fw5: 5'-AGACCAACTGGGGCATCG-3' and Rv2: 5'-AAGT-CAACTGTGAGCGGATAGC-3' for intron 2).

2.5. Sequence analysis

The putative MSTN and GDF-11 protein sequence were alignedwith ClustalX (Thompson et al., 1997). Phylogenetic analysis wasconducted using MEGA3.0 (Kumar et al., 2004). Unreliable regions(e.g. signal sequences) and gap-containing positions were excludedfrom the analysis. A phylogenetic tree was constructed using theneighbour-joining method (Saitou and Nei, 1987) based on thePoisson-corrected distances. Node robustness was evaluated by thebootstrap method (N=1000 replications). Bay scallop (Argopectenirradians) MSTN was included as an outgroup.

2.6. The culture of the continuous cell line FG-9307

The continuous cell line FG-9307 was established from the gills ofthe flounder P. olivaceus (Tong et al., 1997). 1×105 cells were plated in25 cm2

flask and cultured at 20 °C in minimal essential medium(MEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mlpenicillin and 100 μg/ml streptomycin. Upon reaching 80% confluency,the cells were harvested and the total RNA was isolated using Trizolreagent and treated with DNase I as above.

(Coding sequence) are shown in capital letters, whereas the 5' regulatory region, thehe deduced amino acid sequences are shown by single letter code of amino acids belowuence repeats (microsatellites) are underlined. TATA box and CAAT box are doubledted in 3' untranslated region with gray highlighting.

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2.7. Tissue distribution expression of MSTN

Total RNA was isolated from different tissues of adult flounder. Foreach sample, the same amount of RNA (2.0 μg) was reverse-transcribedusing M-MLV reverse transcriptase (Promega) and oligo(dT)17 primers(Tiangen). Equal amounts of the RT products were amplified for 40cycles with two specific primers (Fw5: 5'-AGACCAACTGGGGCATCG-3'andRv7: 5'-CCAACTCAAGAGCATCCGCAAC-3') encompassing the secondintron. PCR productswere analyzed on a 1.5% agarose gel and confirmedby sequencing. The quality of extractedRNA and the efficiencyof reversetranscription were tested using a pair of β-actin primers (ActinFw: 5'-GAGATGAAGCCCAGAGCAAGAG-3' and ActinRv: 5'-CAGCTGTGGTGGT-GAAGGAGTAG-3').

2.8. Developmental expression profile of MSTN

Total RNA was extracted from each pool of oocytes/embryos (n=3pools of 50 oocytes/embryos per developmental stage) using theTrizol reagent (Invitrogen) according to the manufacturer's instruc-tions. As for larva after hatching, 15 larvae were pooled for RNAextraction. The cDNA was generated with M-MLV reverse transcrip-tase, random hexamers and 2.0 μg of RNA from samples at differentdevelopmental stages. Each amplification was performed in a 15 μlfinal volume containing 1× SYBR Green Realtime PCR MasterMix (TOYOBO), 0.2 μM each of specific forward and reverse primer,and 1.2-μl diluted cDNA. The reaction conditions were as follows: aninitial 1 min activation and denaturation step at 95 °C, followed by 45cycles of 5 s at 95 C, 10 s at 60 °C, 20 s at 72 °C. Fluorescent detectionwas performed after each extension step. A dissociation protocol wasadded after thermocycling to verify that only a single product wasamplified. A serial dilution of plasmids containing either 18S rRNA orpMSTN gene fragments was used to compute the amounts oftemplate. All samples were amplified in duplicate and means wereused for further analyses. Primers used for Quantitative PCR weredesigned specially for pMSTN (Fw6: 5'-CTCCGAACATCAGCCGAGAC-3';Rv8: 5'-GACCACATCCCTGTTGTCATCTC-3') or 18S rRNA (18SFw: 5'-CCTGAGAAACGGCTACCACATC-3'; 18SRv: 5'-CCAATTACAGGGCCTC-GAAAG-3').

3. Results

3.1. Identification of pMSTN gene

Initially, a partial fragment of approximately 300 bp was obtainedfrom flounder skeletal muscle cDNA using two degenerate primers(Fw1–Rv1), designed on the basis of highly conserved regions of MSTNsequences in other species. A larger cDNA region (692 bp) was thenisolated by semi-nested PCR with two degenerate primers Fw2, Rv1and a specific primer derived from the sequenced fragment above. Theremaining unknown regions of the cDNA were obtained by 5' and 3'RACE reactions. Overlapping sequences of all fragments yielded a full-length cDNA with 117 bp of 5' UTR, 1304 bp of 3' UTR and 1173 bp ofthe entire open reading frame (accession number: DQ997779) (Fig. 1).The predicted amino acid sequence of pMSTN is 377 residues long andhas an estimated Mr of 42.8 kD. As with the previously identifiedMSTN orthologs, the predicted pMSTN contained a putative signalpeptide sequence (the putative cleavage site was at amino acid 22–23), nine conserved cysteine residues found in all TGF-β familymembers, and a RXXR proteolytic processing site.

Fig. 2. Multiple alignment of different MSTN and GDF11 proteins. The accession numbers forU. cirrosa MSTN (AAL26886), S. salar MSTN 1a (CAC51427), S. salar MSTN 1b (CAC19541), Dcompiled from alignment with the A. anser (AAL35276), M. gallopavo (O42221) and G. gal(AAO46885) and R. norvegicus (AAB86691) for mammalian MSTN proteins. The mammalia(AAF21633), and R. norvegicus (XP_343149) GDF11 sequence. Consensus sequence is shadeindicated with asterisks.

A BLASTP analysis revealed that the putative amino acid sequenceshared high similarity with other MSTNs, especially with fish MSTNs.It was nearly 65% identity withmammalian or avianMSTNs, while 93%and 92% with shi drum (AAL26886) and striped bass (AAK67983)MSTN, respectively. However, high scores were also obtained when itwas compared with zebrafish and mammalian GDF-11, 67.4% and 64%similarity, respectively. The alignment of the pMSTN, GDF-11 andMSTN sequences from other species showed that most of the identicalamino acids lied within the highly conserved C-terminal region(Fig. 2). To further understand the identity of the pMSTN, a phy-logenetic tree was constructed with vertebrate MSTN and GDF-11sequences. As shown in Fig. 3, the mammalian and avian MSTNs weregrouped together (100% bootstrap support), while all of the fishMSTNs (MSTN-1 and MSTN-2) were grouped together and thenclustered with GDF-11 with 91% bootstrap support. The putativepMSTN sequence was located within the fish MSTN-1 group (99%bootstrap support), confirming that it was one of the teleost MSTNs,but not GDF-11.

3.2. Genomic MSTN analysis

Sequence analysis of the pMSTN gene identified a putative TATAbox site and a putative CCAAT box site. In addition, twelve putativemuscle-specific MyoD binding sites (CAxxTG), known as E boxes, wereidentified (Fig. 1). Among them, two sites which located at position916 bp and 933 bp upstream from the transcriptional start site,respectively, were closer than any other two sites. Closely linked Eboxes also exist in zebrafish MSTN-1 (zfMSTN-1) (Xu et al., 2003) andbovine MSTN promoter (Spiller et al., 2002), and were consideredcritical for its muscle-specific expression (Spiller et al., 2002).

Two introns were identified by comparing the pMSTN cDNAsequence with the corresponding genomic DNA fragments. Introns 1and 2 of pMSTNwere 362 bp and 762 bp in length, respectively (Fig.1).The sizes of the two introns were similar to seabream and shi drum's.However, they were very different from that of channel catfish(1758 bp/intron 1 and 300 bp/intron 2) and considerably smaller thantheir mammalian counterparts (1808 bp/intron 1, 1977 bp/intron 2 forporcine MSTN; 1789 bp/intron 1, 2400 bp/intron 2 for human MSTN).In accordance with other MSTN genes, pMSTN gene showed theconserved intron–exon boundaries conforming to the GT-AG rule.

Further analysis revealed several microsatellite sites in pMSTNgene. Two microsatellite sequences existed in the 5' upstream region,(TA)8 and (AC)6. One (AC)13 motif was found in the second intron andan imperfect (AC)12 motif in 3'-UTR (Fig. 1). All these microsatellitesites showed the polymorphism to some extent by sequencing (datanot shown). The single nucleotide polymorphism (SNP) within theCDS (coding sequence) of pMSTN genewas also identified (Table 1). Allthree exons contained the SNPs, most of which distributed in thesecond and the third exon. Eight of these SNPs were neutral withoutcausing changes in amino acid sequence; however, the other 11 SNPswere missense mutations resulting in amino acid changes. These 11missensemutations sites distributed in all three functional domains ofpMSTN protein with one SNP site in signal peptide, six in propeptideand four in mature peptide.

3.3. Tissue distribution and developmental profile of pMSTN gene

The tissue distribution of pMSTN was examined by RT-PCR usingthe primers designed on either side of the second intron. The

these sequences are as follows: P. olivaceus (DQ997779),M. saxatilisMSTN (AAK67983),. rerio GDF11 (AAN03678). The non-fish MSTN sequences represented the consensuslus (AAB86688) for avian MSTN proteins and the H. sapiens (AAC96327), M. musculusn GDF11 sequence obtained from a consensus of H. sapiens (NP_005802), M. musculusd. Solid bar indicates RXXR proteolytic cleavage domain and conserved cysteines are

Fig. 3. Phylogenetic analysis of MSTN and GDF11 amino acid sequences in representative vertebrates. Bay scallop (A. irradians) MSTN was included as an outgroup. Numbers at treenodes refer to percent bootstrap values after 1000 replicates; the scale bar refers to a phylogenetic distance of 0.1 amino acid substitutions per site. The accession numbers formyostatin and GDF11 sequences are as follows: M. americana MSTN (AAK67984), M. saxatilis MSTN (AAK67983), M. chrysops MSTN (AAK28707), U. cirrosa MSTN (AAL26886),S. aurata MSTN (AAK53545), P. olivaceus (DQ997779), S. salar MSTN 1a (CAC51427), S. salar MSTN 1b (CAC19541), S. salar MSTN 2a (ABN72587), S. salar MSTN 2b (EF392864),O. mykiss MSTN 1a (AAK71707), O. mykiss MSTN 1b (AAK71708), O. mykiss MSTN 2a (ABA42587), O. mykiss MSTN 2b (DQ177320), S. fontinalis MSTN b/m (AAK08152), I. punctatusMSTN (AAK84666), D. rerio MSTN 1(AAB86693), D. rerio MSTN 2 (NP_001019991), M. gallopavo (O42221), G. gallus MSTN (AAB86688), A. anser MSTN (AAL35276), O. aries MSTN(AAB86689), B. taurus MSTN(BAB79498,) R. norvegicus MSTN (AAB86691,) M. musculus MSTN (AAO46885), H. sapiens MSTN (AAC96327), P. hamadryas MSTN (AAB86686),A. irradians MSTN (AAT36326), D. rerio GDF11 (AAN03678), M. musculus GDF11 (AAF21633), R. norvegicus GDF11 (XP 343149) and H. sapiens GDF11 (NP 005802).

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specificity of the PCR products of the expected size was verified bysequencing. The pMSTN mRNA was detected in all 13 tissues tested,including skeletal muscle, eyes, ovary, brain and kidney with highexpression level, and heart, liver, testis, spleen, gills and intestine withrelatively low expression level (Fig. 4A). Also, expressionwas detectedin cartilage and blood, which were not reported in other fishpreviously. Moreover, when the brain was dissected into five distinctregions, differential expression was found. The transcription levelswere high in cerebrum and mesocephalon, moderate in diencephalonand myelencephalon, and low in cerebellum (Fig. 4B). Althoughtissue-specific expression was found in gill filaments, no signal couldbe detected in FG-9307, a continuous gill cell line from P. olivaceus(Fig. 4C).

Quantitative RT-PCR analysis indicated that the expression ofpMSTN appeared to be developmentally regulated (Fig. 5). The pMSTNmRNA could be detected in unfertilized eggs, indicating the maternaldeposit, whichwas in accordancewith the result obtained in zebrafish(Vianello et al., 2003). After fertilization, the expression level ofpMSTN remained low and stable until 40%-epiboly stage. Then theexpression of pMSTN increased significantly at neurula stage, andelevated continually except for a slight decrease at heart-beatingstage. Interestingly, after hatching a second sharp elevation wasobserved at the stage of 1-day larva, more than two fold higher thanthat at hatching stage, and maintained relatively high until three daysafter hatching, the last stage we studied. This intricate expressionprofile suggests a varied role of pMSTN in embryonic development.

Fig. 4. Tissue distribution of pMSTN. A: RT-PCR analysis of pMSTN expression in 2-year-old Japanese flounder tissues. B: RT-PCR expression profile in different part of brain.C: Results of amplication of a pMSTNmRNA fragment with gill cDNA (positive), FG-9307cDNA and ddH2O (negative). A fragment of β-actin mRNA was amplified in all samplesby means of RT-PCR.

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4. Discussion

In this study, we isolated the sequence of MSTN gene fromJapanese flounder. The deduced amino acid sequence presented thetypical characteristic of TGF-b family members with nine conservedcysteine residues and a RXXR proteolytic processing site within theconserved carboxyl-terminal portion. When aligned with other MSTNgenes, the highest amino acid similarity was found in its fishorthologs, and some parts of the intron 2 of pMSTN shared morethan 80% identities with its teleost counterparts (e.g. largemouth bass,EF071854 and Sea Perch, AY965685). Phylogenetic analysis alsoshowed that the putative pMSTN clustered within the fish MSTNgroup with 99% bootstrap support. This evidence confirmed that thesequence we obtained was one of the teleost MSTNs.

Sequence analysis revealed the presence of four microsatellite siteswithin the pMSTN gene, 2 in the 3' UTR and 2 within introns.Analyzing the association between the polymorphism of thesemicrosatellite sites and the rate of muscle growth may facilitate theselection of advantageous mutants within this locus. Anotherinteresting feature was that a large number of single nucleotidepolymorphism (SNP) located within the CDS of pMSTN. In mammals,many SNPs of MSTN genes were identified, and some of them werebelieved to correlate with abnormally increased muscular mass(Grobet et al., 1998; Jiang et al., 2002; Clop et al., 2006). However, asfor fish MSTN, to date, only 11 SNP sites were reported in channelcatfish (Kocabas et al., 2002). Here we reported 19 SNP sites whichdistributed in all three exons and 11 of them caused changes in aminoacid sequences. Within the signal peptide region the missensemutations at 10 (coordinate was shown in Table 1) changed a serineresidue (S) to a proline (P). Although the substitution of S for P in thesignal peptide did not change the putative cleavage site, it is unclearwhether the efficiency of cleavage would be affected by this change ofamino acid. More expected substitutions occurred in the propeptideandmature peptide domains of pMSTN, where together 10 amino acidchanges were found. Prior research showed that the mature peptidedimer functioned as a negative regulator of muscle mass (McPherronet al., 1997; Zimmers et al., 2002), and the propeptide played animportant role in regulating the activity of the mature peptide dimer(Thies et al., 2001; Lee and McPherron, 2001; Wolfman et al., 2003).Thus, the amino acid substitutions in these regions could potentiallyaffect the activity of pMSTN, although the changes found in pMSTN didnot occur at the conserved cysteine sites and the RXXR motif. Forexample, the substitutions within the propeptide may strengthen its

Table 1Single nucleotide polymorphic (SNP) sites within the ORF of P. olivaceus MSTN gene

Nucleotidelocation

Coordinate (nucleotide/amino acid)

Basechange

Amino acidchange

Amino acidlocation

Exon I 10/4 TNC SNP Signal peptideExon I 98/33 CNT ANV PropeptideExon I 193/65 ANG SNG PropeptideExon II 427/143 TNC FNL PropeptideExon II 444/148 GNA No change PropeptideExon II 497/166 CNT ANV PropeptideExon II 626/209 ANG DNG PropeptideExon II 636/212 ANG No change PropeptideExon II 702/234 GNA No change PropeptideExon II 727/243 TNC SNP PropeptideExon II 747/249 ANG No change PropeptideExon III 777/259 CNA No change PropeptideExon III 783/261 CNT No change PropeptideExon III 808/270 GNA ANT Mature peptideExon III 827/276 ANT ENV Mature peptideExon III 860/287 CNT PNL Mature peptideExon III 861/287 GNA No change Mature peptideExon III 867/289 ANG No change Mature peptideExon III 1094/365 ANG KNR Mature peptide

The coordinate of base was determined from the first base of the initial translation site.

affinity with the mature peptide and make it difficult to dissociatefrom themature peptide and block the function of themature peptide.Thus, the amino acid changes within the conserved mature peptidemay weaken the activity on muscle growth inhibition, such as thewell-known double-muscled cattle, Piedmontese. TheMSTN sequenceof this contains a missense mutation in exon 3, resulting in a sub-stitution of tyrosine for an invariant cysteine in the mature region ofthe protein, resulting in a complete or almost complete loss offunction (Kambadur et al., 1997). Further research is required todetermine the relationship between these SNPs and the growth ofmuscle.

MSTN gene exhibited diverse expression patterns in differentvertebrates. In mammals, MSTN mRNA was mainly observed inskeletal muscle (McPherron et al., 1997), cardiomyocytes and Purkinjefibers of the heart (Sharma et al., 1999), and mammary gland (Ji et al.,1998). However, in teleost fish, MSTN gene was expressed in varioustissues. Also, in rainbow trout, unspliced variants of both rtMSTN-2transcripts were expressed in a tissue-specific manner. Completesplicing of rtMSTN-2a occurred only in brain, where expression ishighest, whereas rtMSTN-2b transcripts were mostly present inunspliced forms in all tested tissues (Garikipati et al., 2007). In thisstudy the pMSTN mRNAwas found in all 13 tissues tested, and for thefirst time we detected the expression of MSTN in cartilage. Thisubiquitous expression was in accordance with the results from other

Fig. 5. Quantitative analyses of the expression profiles of pMSTN gene in P. olivaceusembryos from unfertilized egg to 3-day old larva. Columns correspond to: unfertilizedegg (Unf), 64-cell (64C), lateblastula (Lab), 40% epiboly (40%-ep), neurula (Neu), eye-bud(Eyb), tail-bud forming (Tbf), tail-bud (Tab), heart-beating (Heb), hatching (Hat), 1-dayold larva (1-D) stage, 2-day old larva (2-D) stag e and 3-day old larva (3-D) stage. Therelative expression variance is showed as ratio between the amount of pMSTN mRNAnormalized to the corresponding 18S rRNA values in a particular developmental stageand that in unfertilized eggs. Data are shown as mean±SEM (n=3) (Pb0.05).

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fish species (Maccatrozzo et al., 2001; Kocabas et al., 2002; Garikipatiet al., 2006), and suggests different physiological functions thanskeletal muscle growth regulation in other tissues. For example, thepresence of pMSTN in cartilage suggests that it may participate in thecontrol of the growth of cartilage; its expression in gonads suggeststhat it may help to maintain reproductive tissues. Recent studies haveidentified myostatin expression (MSTN-1a and -1b) in peripheralblood lymphocytes as well as spleens of rainbow trout (Garikipatiet al., 2006). Theses results are consistent with the observation inP. olivaceus, where low abundance of MSTN mRNA was detected inblood and spleen tissue and suggests the possible role in immunereaction. Furthermore, in zebrafish, stocking stress greatly elevatedthe expression of two MSTN genes (zfMSTN-1 and zfMSTN-2) inspleens and suggests that myostatin may play an important role inmodulating immune system function during conditions of stress(Helterline et al., 2007). More detailed analysis showed that thedifferential expression levels even existed in the different regions ofthe Japanese flounder brain. Similar results was also obtained from thebrain of brook trout by Northern blot analysis, in which only opticlobes, hindbrain and hypothalamus showed the hybridization signal(Roberts and Goetz, 2001). This differential expression in brainindicated that pMSTN could anticipate the growth and differentiationof neural system. Tissue-specific expression in gill filaments in vivoprompted us to investigate whether pMSTN mRNA could express inFG-9307, an immortalized gill cell line cultured. However, no pMSTNexpression was detected. This expression change may be due to thedifferentiation status of the cells, just as observed in rat L6 myoblasts,an immortalized rat skeletal muscle cell line (Rodgers et al., 2001).

In mammals, the expression of MSTN was in accordance with thedevelopment of muscle. Inmouse, MSTNmRNA could first be detectedas early as day 9.5 post-coitum in developing somites (McPherronet al., 1997). However, in fish the developmental expression of MSTNgene seemed more intricate. In tilapia and gilthead seabream, MSTNmRNA was only present in posthatched larvae, none in embryos(Maccatrozzo et al., 2001; Rodgers et al., 2001); in zebrafish, twodiscrepant results occurredwhen the expression patterns of zfMSTN-1was explored. Xu showed that no MSTNmRNA could be detected by insitu hybridization until 4 days post-fertilization, and only the lowlevels of MSTN expression at 24 hpf using the more sensitive RT-PCRmethod (Xu et al., 2003). However, Vianello et al. (2003) demonstratedthe existence of MSTN mRNA in just-fertilized eggs, reflecting thematernal transcripts stored in the mature oocyte. The reason causingthese contrary results remain yet unknown. More recently someresearchers found that the transcripts of the second zebrafish MSTNgene (zfMSTN-2) could also be detected in unfertilized eggs, exhibitingthe maternal origin. In this study the pMSTN mRNAwas first detectedin unfertilized eggs, suggesting a maternal origin. Then it kept arelatively constant expression level with a little fluctuation beforeNeurula Stage. As there is no somite formation before Neurula Stage,this result indicated that the pMSTNmight participate in the formationand differentiation of three germ layers during the early embryodevelopment. FromNeurula Stage, the expression of pMSTN increasedsignificantly and elevated continually with a slight decrease at heart-beating stage, then increased at hatching stage and thereafter. Thisexpression pattern was consistent with somitogenesis in Japaneseflounder and similar to the expressionpattern of zfMSTN-1 (Vianello etal., 2003). After hatching, the activity of the skeletal muscles enhancedmarkedly, accompanied with the significant increase of pMSTNexpression. The elevation of the pMSTN level after hatching seemedto coincide with the post-embryonic development of the muscles infish, just like zebrafish, inwhich there is a large increase in the numberand size of both slow and fast muscle fibers during larval growth(Koumans andAkster,1995). The increase of the pMSTNmRNAperhapsreflects that MSTN acts as a negative regulator, antagonizing the fastgrowth of muscle during early larval development, so as to keep themuscle at a physiological normal rate of growth.

Phylogenetic analysis revealed that the fish MSTN sequencesgrouped near GDF-11 sequences, but not mammalian and avian MSTNsequences. This location in the phylogenetic tree is consistent with theexpressionpatterns of GDF-11. Inmammals, GDF-11 genewas expressedin dental pulp, heart, kidney, brain and many other adult tissues(Nakashima et al., 1999). Further, immunolocalization studies showedthat the expression patterns of the fish MSTN, such as Atlantic salmonMSTN-1 genes, were highly consistent with the patterns of mammalianMSTN and GDF-11 together (Ostbye et al., 2001; Radaelli et al., 2003).These results, together with the sequence similarities between the fishMSTN and the mammalian MSTN and GDF-11, prompted a hypothesisthat fish MSTN gene might represent the ancestral gene whichduplicated and radiated during the evolution of vertebrates into thetwo mammalian TGFs: MSTN and GDF-11 (Ostbye et al., 2001), andhence fish should not express GDF-11. However, GDF-11 gene wasrecently discovered in zebrafish and this fact strongly suggested thatfishcould express both MSTN and GDF-11 (Biga et al., 2005). MSTN in fishmight retain the imprints of the common ancestral genewhich gave riseto ancestral GDF-11 and MSTN by means of gene duplication event(Radaelli et al., 2003). An early genome duplication event before theteleost radiation, but after the divergence of ray- and lobe-finned fishes,resulted in multiple copies of many genes (Postlethwait et al., 1998;Taylor et al., 2003). The discoveries of MSTN-2 genes in zebrafish (Bigaet al., 2005; Kerr et al., 2005) and salmonids species (Garikipati et al.,2007; Ostbye et al., 2007), together with the recent phylogeneticanalysis and the results presented here, strongly suggested that therewas an early duplication of theMSTNgene in the fish lineage (Kerr et al.,2005). A second duplication event specifically in the salmonidsproduced additional paralogs (‘a' and ‘b') within each MSTN-1 andMSTN-2 sister clade (Kerr et al., 2005), and then the divergence of theexpression pattern among these molecules occurred later in evolution.For example, the Atlantic salmonMSTN-1b expressed in brain, intestine,gonads, heart, kidney, spleen, tongue, eyes, and red and white muscles,while the Atlantic salmon MSTN-1a expressed in brain, intestine, gills,tongue, eyes, and white muscles (Ostbye et al., 2001). However, theexpression patterns of these two MSTN-1 genes together are highlysimilar to that of pMSTN. The similar phenomena could also be foundwhen compared the tissue distribution of MSTNs in rainbow trout orbrook troutwith that in Japaneseflounder. The location of the pMSTN inthe phylogenetic tree suggests that pMSTN is aMSTN-1 ortholog. Due tothe early genomeduplication event in ray-finned fishes, onemayexpecta paralog to the pMSTN gene that would be orthologous to MSTN-2;however, noMSTN-2 orthologwas identified in this study. The lack of anobserved MSTN-2 transcript may be due to technical reasons, but couldalso be due to chromosome loss or loss of function through geneinactivation or loss of individual regulatory elements due to mutationand random genetic drift after the genome duplication event (Forceet al., 1999). Thus, in P. olivaceus there might be only one MSTN gene,pMSTN, which evolved from the ancestral MSTN gene and still retainssome characteristics of the common ancestral gene which gave rise toancestral MSTN gene and ancestral GDF-11 gene.

In conclusion, the present study provided cDNA sequence and thegenomic organization of pMSTN gene. Expression analyses revealedthat pMSTN was developmentally regulated during embryogenesisand expressed ubiquitously in adult. These expression patternsindicated that fish MSTN might participate in many varied physiolo-gical processes other than skeletal muscle growth inhibition. Inaddition, we identified several microsatellite motifs and SNPs inpMSTNgene. In consideration of the function ofMSTN inmammals as anegative regulator of skeletal muscle growth, thesemolecularmarkersmight facilitate marker-assisted selection of advantageous mutants.

Acknowledgements

Wewould like to thank Dr. YuanminWu, from Funglyn BioTech Co.,Ltd., for technical assistance. This work was also supported by grants

255Q. Zhong et al. / Aquaculture 280 (2008) 247–255

from the National High Technology Research and DevelopmentProgram of China (No. 2006AA10A404), the National Basic ResearchProgram (No. 2004CB117401) and the National Natural ScienceFoundation of China (No. 30671624 and No. 30600455).

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