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1 School of Molecular Biosciences and
2 Department of Animal Sciences, Washington State University, 124 ASLB, Pullman, Washington 99164-6351, USA
3 United States Department of Agriculture, National Center for Cool and Cold Water Aquaculture, Kearneysville, West Virginia 25430, USA
(Requests for offprints should be addressed to B D Rodgers; Email: danrodgers{at}wsu.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A recent phylogenetic analysis of the entire myosta-tin/GDF-11 subfamily (Kerr et al. 2005) indicates that bony fish possess multiple myostatin genes and that a gene duplication event during early fish radiation (Amores et al. 1998, Postlethwait et al. 1998) produced two distinct myostatin clades: MSTN-1 and MSTN-2. A second duplication event within salmonids, likely resulting from tetraploidization, produced two subsequent divisions, one in each clade. This suggests that most, if not all, salmonids possess four distinct myostatin genes: two within the first clade (1a and 1b) and two in the second (2a and 2b). Both the previously identified rainbow trout cDNA clones, formerly named Tmyostatin-1 and Tmyostatin-2, are actually MSTN-1 orthologs. They were, therefore, renamed rtMSTN-1a and rtMSTN-1b respectively, which reflects their true evolutionary relationship to other myostatin genes (Kerr et al. 2005).
Myostatin genes have been characterized in mice (McPherron et al. 1997), humans (Gonzalez-Cadavid et al. 1998), cattle (Jeanplong et al. 2001), and pigs (Stratil & Kopecny 1999). Although cDNA clones have been characterized in many diverse fish species (Ostbye et al. 2001, Roberts & Goetz 2001, Rodgers & Weber 2001, Rodgers et al. 2001, Rescan et al. 2001, Maccatrozzo et al. 2001a, Kocabas et al. 2002, Kerr et al. 2005), very few genes have been completely characterized. This is particularly disconcerting since bony fish, especially teleosts, represent the largest group of extant vertebrates and many of these species are commercially important. Therefore, a better understanding of the genomic sequence and organization of different fish myostatin genes, as well as species-specific expression patterns will significantly interest comparative and agricultural biologists alike. This information will be particularly important in the identification and cloning of MSTN-2 genes from different salmonids and will help in distinguishing paralogs from orthologs.
The isolation and characterization of the rtMSTN-1a and rtMSTN-1b genes, including their respective promoter regions, are reported herein. We additionally report the quantitative assessment of the expression of each gene using detailed RNA panels generated from multiple stages of embryonic/larval development and different adult tissues. These studies indicate strong sequence conservation among all vertebrate myostatin genes. However, the expression patterns and putative promoter elements suggest that the function of myostatin in fish may not be as limited as in mammals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genomic DNA was extracted from rainbow trout (Oncorhynchus mykiss) fin clips. Briefly, 3 ml lysis buffer (30 mM Tris, 8 M urea, 4% w/v Chaps (pH 8.0) was added to 50 mg tissue and incubated overnight with proteinase K (20 mg/ml) at 60 °C. Three consecutive phenol:chloroform:isoamyl alcohol extractions were then performed and DNA quality was verified on a 1% agarose gel. Promoter regions were cloned using the Universal Genome walker kit (BD Biosciences, www.bdbiosciences.com) and the manufacturer’s protocol. Briefly, genomic DNA was digested with the blunt-end restriction enzymes DraI, EcoRV, PvuII, and StuI, and subsequently ligated to the provided adaptor linkers. Nested PCR with 94 °C initial denaturation was then performed using gene-specific primers homologous to the known 5'-coding region of each gene (Table 1
) and adaptor primers with the Advantage 2 PCR kit (BD Biosciences). The cycle parameters were as follows and used as default unless otherwise specified: an initial denaturation at 94 °C for 1 min, seven cycles of 94 °C for 30 s, and 72 °C for 3 min followed by 30 cycles of 94 °C for 30 s, and 67 °C for 3 min and a final extension at 67 °C for 4 min. The PCR products were subcloned into the Topo TA vector (Invitrogen, www.invitrogen.com) and sequenced in the university’s genomic core facility. Putative-regulatory elements were identified by subsequence analysis using Matinspector software (Genomatrix, Inc., www.genomatix.de), which searches consensus sequences of known cis-regulatory sequences. The intron sequences were obtained by nested PCR using gene-specific primers for rtMSTN-1a or rtMSTN-1b coding regions and adaptor primers (Table 1) as follows: initial denaturation at 94 °C for 1 min followed by 30 cycles of 94 °C for 30 s, 65 °C for 30 s, 72 °C for 3 min, and a final extension at 72 °C for 3 min. The resulting amplicons were then cloned as described and sequenced. Flanking primers, specific to each myostatin gene, were then used to amplify and clone the complete genes using Pfu polymerase (Stratagene, www.stratagene.com) and the pCR4-blunt Topo vector (Invitrogen). Intron/exon boundaries were determined by aligning the cDNA sequences to their respective gDNA sequences using ALIGN X (VectorNTI, www.invitrogen.com).
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) and universal adaptor primers (UAP) provided in the kit. The PCR conditions were as follows: an initial denaturation at 94 °C for 1 min followed by 30 cycles of 94 °C for 30 s, 65 °C for 30 s, 72 °C for 2 min, and a final extension at 72 °C for 2 min. Nested PCR was subsequently performed with the 3'UTR2 forward primer and the abridged universal adaptor primer (AUAP) provided in the kit. The resulting amplicons were subcloned and sequenced as described. Embryonic and tissue collections
Rainbow trout were reared at the National Center for Cool and Cold Water Aquaculture, Kearneysville, WV, USA, according to the guidelines approved by the institutional animal care and use committee. An RNA panel was generated from 5000 pooled eggs from multiple females (Trout lodge, October 2004) that were fertilized by milt from two males. Following fertilization, eggs were incubated at approximately 13 °C throughout embryonic development. In addition to the unfertilized eggs (day 0), developing embryos were collected as whole egg samples daily for the first 14 days, every other day until hatch (day 24), and every third day, thereafter. Each sample contained 18 eggs or embryos or 9 post-hatched larvae that were pooled and several samples were collected at each time-point. Tissues were also removed from 2-year-old adult fish weighing approximately 2 kg. All samples were flash-frozen in liquid nitrogen and stored at –.80 °C until RNA isolation. Samples were first powdered using a liquid nitrogen-cooled Bessman Tissue Pulverizer (Spectrum Laboratories, www.spectropor.com) and total RNA was extracted using TRI-reagent with the high-salt solution modification to remove excess glycosylated proteins. The RNA was reconstituted in 20–50 µl nuclease-free water and treated with DNase (DNAse RQ-1; Promega, www.promega.com) to remove contaminating genomic DNA. Samples were then re-extracted with TRI-reagent and RNA quality was assessed by agarose gel electrophoresis.
RNA quantification using ‘real-time’ reverse transcriptase (RT)-PCR
The total RNA (2 µg) was reverse transcribed with 1 µg random primers (Promega) and 200 U Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT, Promega) in 40 µl. Subsequent real-time RT-PCR assays were conducted using the ABI Prism 7900HT Sequence Detection System (www.appliedbiosystems.com) and gene-specific primers (MSTN-1a F, MSTN-1a R, MSTN-1b F, and MSTN-1b R, Table 1). For each sample, 1 µl cDNA was combined with 7.5 µl of 2.SYBR Green PCR master mix (Applied Biosystems). For each reaction, 6 µl of this mixture was added to 9 µl primer mix containing 500 nM of each primer. The reactions were carried out as follows: 50 °C for 2 min, 95 °C for 10 min, then 40 cycles consisting of 95 °C for 15 s and 60 °C for 1 min. The cycling reaction was followed by a dissociation curve to verify amplification of a single product and amplicons were also verified by DNA sequencing.
The relative standard curve method was employed to quantify gene expression. For each primer set, a serial dilution of a mixed tissue cDNA was used to construct a standard curve for each assay plate. The standard curve was constructed by plotting the threshold cycle (CT) versus the natural log of input RNA (ng). This curve was then used to calculate the relative abundance of each transcript in each sample. Myostatin values were then normalized to those of 18s to control differences in RNA and cDNA loading. Each sample was run in triplicate on a single plate and each plate was run in duplicate. Assays were repeated with different samples and all data are presented as normalized gene expression.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Complete genomic clones for both rtMSTN-1a and rtMSTN-1b genes were isolated and sequenced (Figs 1 and 2). This includes, approximately, 2 kb upstream of each initiator. The annotated gene and promoter sequences were then deposited with GenBank and assigned the accession numbers DQ136028
[GenBank]
and DQ138300
[GenBank]
respectively. Each gene is organized into three exons of similar size with appropriate intron/exon splice sites, a pattern that is conserved with other fish species (Maccatrozzo et al. 2001b, Xu et al. 2003, Kerr et al. 2005) and mammals (McPherron et al. 1997, Gonzalez-Cadavid et al. 1998, Stratil & Kopecny 1999, Jeanplong et al. 2001). The three rtMSTN-1a exons are 490, 368, and 1600 bp in size respectively, and are separated by 1072 and 992 bp introns (Fig. 1A). The rtMSTN-1b gene is similarly organized with three exons of 486, 386, and 1419 bp with two, 564 and 778 bp introns intervening (Fig. 2A). The 3' UTRs were also cloned by 3' RACE using RNA from adult skeletal muscle and determined to be 1.2 kb in the rtMSTN-1a transcript and 1.1 kb in rtMSTN-1b. This is in contrast with the significantly shorter 3' UTRs reported previously (Rescan et al. 2001). In addition, two polyadenylation signal sequences (AATAAA) were detected in each 3'UTR at –17 to –11 bp and –83 to –76 bp from the poly A site in rtMSTN-1a and –19 to –13 bp and –86 to –80 bp in rtMSTN-1b (Figs 1D and 2D).
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). Comparative mapping of coding regions revealed a common organization between species, including conserved pre-mRNA splice sites. Codons flanking the first splice sites (3' end of exon 1 and 5' end of exon 2) are highly conserved among the fishes, while the second site (3' of exon 2 and 5' of exon 3) is conserved in fish and mammals (Fig. 3). Amino acid consensus sequences were, therefore, identified by comparative sequence analysis of intron/exoncoding junctions. These include MAT(E/K)|PXXI for the first junction and (G/E)(E/D)GL|XPF
for the second (X = any amino acid; = hydrophobic, likely L, I, or M). Indeed, multiple sequence alignments with myostatin proteins from different vertebrates, both previously published (Rodgers & Weber 2001) and repeated with newly discovered clones (data not shown), indicate that these motifs are highly conserved, the first in fishes and the second in all vertebrates.
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Subsequent analysis of the 2 kb promoter regions upstream of each gene using MatInspector software identified several putative muscle-specific transcription factor binding sites or cis-regulatory elements. These include Comp1 (cooperates with myogenic proteins 1), HAND2 (heart, autonomic nervous system, neural crest derivative 2), MEF 2 (myocyte enhancer factor 2), MusIn (muscle initiator), SRF (serum response factor), and TEF-1 (transcriptional enhancer factor 1) binding sites in the rtMSTN-1a promoter (Fig. 1). The rtMSTN-1b promoter contained all these putative elements except for the MusIn site and additionally contained a MEF 3 site (Fig. 2). Each promoter also contained an appropriately placed TATA box and several putative E-boxes, while a muscle TATA box was also found in the rtMSTN-1b promoter. A comparative analysis of all cloned myostatin gene promoters from fish revealed features common to all or most promoters and some features unique to a particular promoter as well (Fig. 4). Every promoter contained several E-box motifs and all, but the brook trout (bt) MSTN-1b promoter contained multiple Comp 1 sites and TATA boxes in close proximity to the transcription start sites. Studies with mammalian promoters indicate that MEF2, GRE (glucocorticoid-response element), and MyoD (myogenic differentiation factor) binding sites regulate myostatin promoter transactivation (Ma et al. 2001, Spiller et al. 2002, Forbes et al. 2006). These sites were also identified in the fish promoters. All, but the btMSTN-1b promoter contained multiple MEF2 sites and many were located within the first 500 bp. A GRE was only identified in the btMSTN-1b and zebrafish (zf) MSTN-2 promoters, while the latter additionally contained the only MyoD-binding site as well as two myogenin-binding sites. Roberts & Goetz (2003) previously identified a MyoD-binding site in the btMSTN-1b promoter, although this site was not identified in our analysis using the same, yet updated software package.
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A quantitative analysis indicated that both rtMSTN-1a and rtMSTN-1b were similarly expressed at low levels during the early stages of development. However, levels of both transcripts rose substantially after eyeing with rtMSTN-1a mRNA levels always greater than those of rtMSTN-1b (Fig. 5A). Expression of both, peaked and dropped immediately before hatching and then continued to rise thereafter. A similar peak in rtMSTN-1a expression also occurred just before yolk sac absorption, although this was not observed with rtMSTN-1b (Fig. 5C). A closer analysis of early developmental stages indicated that neither gene was significantly expressed during gastrulation, although expression of both steadily rose during somitogenesis peaking and then subsided at its end (Fig. 5B).
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Expression of both rtMSTN-1a and rtMSTN-1b was detected in every tissue sampled, including brain, pituitary gland, heart, ovary, testis, kidney, stomach, pyloric ceca, intestine, liver, pancreas, peripheral blood leukocytes, erythrocytes, spleen, gills, branchial arches, fins, skin, eyes, white and red skeletal muscles, and fat (Fig. 6). This includes, some tissues not known to express either myostatin (pituitary, stomach, pyloric ceca, pancreas, leukocytes, erythrocytes, spleen, brachial arches, fins, skin, eyes, and fat) and several others previously thought not to express rtMSTN-1b in particular, which has only been identified in brain and skeletal muscle (Rescan et al. 2001). Expression of both genes was highest (note log scale) in brain, testes, eyes, muscle, and surprisingly spleen. Individual tissue levels of both transcripts were similar in many tissues, but not all. Those of rtMSTN-1a were approximately 50-fold higher than rtMSTN-1b levels in fins and expression of the latter was almost 100-fold greater in leukocytes and gills, and 1000-fold in heart.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2) is highly similar to that of other homologs previously characterized in mammals and nearly identical to those in other fish. Indeed, exon boundaries and pre-mRNA splice sites are even conserved (Fig. 3), especially the second, which separates the coding region of the latency-associated peptide from the bioactive domain of mature myostatin. The amino acid identity of the mature bioactive domains of most fish and mammalian species is 88% (Rodgers & Weber 2001), indicating that both primary sequence and gene organization are highly conserved among vertebrates. Although a more comprehensive analysis of genes from divergent fish species and other vertebrate classes is needed to determine the degree of conservation across taxa, these data suggest that strong selective pressures are likely to be responsible and particularly important in preserving fidelity of the third exon. Teleosts commonly possess multiple copies of individual genes. This is a result of an early genome duplication event prior to the teleost radiation, but after the divergence of ray- and lobe-finned fishes (Amores et al. 1998, Postlethwait et al. 1998). A second duplication event specifically within the salmonids (Phillips & Rab 2001) gave rise to additional (‘a’ and ‘b’) myostatin paralogs within each MSTN-1 and MSTN-2 sister clade (Kerr et al. 2005), although none of these genes has been identified to date. Nevertheless, the high degree of genomic and sequence conservation shared among all myostatin genes and fish homologs (Fig. 3) should aid in their isolation and characterization.
Subsequent analysis of the rtMSTN-1a and rtMSTN-1b promoter regions identified several putative cis-regulatory elements that could contribute to the myogenic process. Some of these elements were also identified in the comparable promoters of brook trout (Salvelinus fontinalis) and zebrafish myostatin genes, including multiple MEF2 sites in each (Fig. 4). A putative MyoD site was also identified in the brook trout MSTN-1b promoter by Roberts & Goetz (2003), although this particular site was not identified in our analysis using the same, yet updated software. Among these fish genes, however, MyoD sites were identified in the zebrafish MSTN-2 promoter, which also contained far more putative myogenic elements than its counterpart (Kerr et al. 2005). Mammalian myostatin promoters contain E-boxes and other elements critical to the differentiation and maturation of skeletal muscle, including both MyoD- and MEF2-binding sites. Indeed, both these sites have been implicated in the regulation of myostatin-gene expression in different animal and cellular systems (Spiller et al. 2002, Salerno et al. 2004, Shyu et al. 2005). Expression of both rtMSTN-1a and rtMSTN-1b genes increases as somitogenesis progresses and rapidly decreases as it ends (Fig. 5B). This is consistent with increased transactivational activity of these and other myogenic-regulatory factors and myostatin’s developmental expression profile in mouse embryos (McPherron et al. 1997). A functional assessment of promoter activity is needed to definitively determine whether these transcription factors regulate either rtMSTN-1a or rtMSTN-1b gene expression in developing skeletal muscle. The ubiquitous nature of MSTN-1 expression in fish, however, suggests that additional elements unrelated to myogenesis altogether may be active as well.
Former attempts to define the developmental and tissue-specific expression profiles of fish MSTN-1 genes revealed a far more diverse expression pattern than, which occurs in mammals (Ostbye et al. 2001, Rescan et al. 2001, Rodgers et al. 2001, Maccatrozzo et al. 2001b, Kocabas et al. 2002, Roberts & Goetz 2003, Rodgers et al. 2003, Vianello et al. 2003, Johansen & Overturf 2005). These studies were still somewhat limited and mostly qualitative assessments. Rescan et al. reported that rtMSTN-1a mRNA levels were substantially higher than those of rtMSTN-1b in most adult tissues and at the three stages of development (eyeing, hatching, and free-swimming larvae). The one exception was adult brain where expression appeared equal for both genes. This study also indicated a very limited distribution of rtMSTN-1b expression, which was restricted to the brain and the skeletal muscle. By contrast, Ostbye et al. reported a much wider tissue distribution and apparently higher levels, in some tissues, of Atlantic salmon MSTN-1b expression. Both these studies used qualitative RT-PCR assays that do not account for primer efficiency and other aspects of non-quantitative PCR amplification and could have easily underestimated rtMSTN-1b expression. By contrast, our use of comprehensive RNA panels and a quantitative ‘real-time’ assay suggest that both rtMSTN-1a and rtMSTN-1b genes are expressed much earlier embryologically, specifically during somitogenesis, and in more adult tissues. Expression of both genes was detected in all tissues sampled and surprisingly high in spleen and eyes, which possibly indicates novel functional roles for myostatin in the growth and/or differentiation of immune and proliferative cells of the eye (Reh & Levine 1998). Johansen and Overturf also analyzed developmental expression of rtMSTN-1a and rtMSTN-1b using a quantitative RT-PCR assay. Although only a few developmental stages were sampled (eyed, hatched/sac present, and swim-up fry), their results also indicate that the expression of both genes rises substantially after eyeing and rtMSTN-1b mRNA levels are significantly higher than previously reported. Myostatin expression in mammals is first detected within the developing myotome (Kambadur et al. 1997, McPherron & Lee 1997), although former attempts to localize myostatin message in fish somites have produced mixed results (Xu et al. 2003, Amali et al. 2004, Kerr et al. 2005). Nevertheless, our results are the first to identify a temporal expression pattern in fish that is consistent with a functional role during the early stages of muscle development as levels of both rtMSTN-1a and rtMSTN-1b rise substantially throughout somitogenesis and begin to subside just before this developmental period ends.
The expression patterns described and the subsequence analysis of the different promoters further support a role for both the MSTN-1 genes during fish myogenesis, although the ubiquitous expression pattern in different adult tissues suggests that the functional role of cytokine is far more diverse than that in mammals. The presence of multiple fish genes that are differentially expressed throughout development and adult tissues also suggests that the precise role of a particular gene may vary between tissues. A better understanding of physiological factors that influence the expression of each gene and the transcriptional machinery involved will, therefore, help distinguish the potential divergent actions of myostatin in fish and mammals.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Received 15 March 2006
Received in final form 9 June 2006
Accepted 21 June 2006
Made available online as an Accepted Preprint 30 June 2006
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