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Endocrinology Vol. 148, No. 5 2106-2115
Copyright © 2007 by The Endocrine Society

Characterization of Rainbow Trout Myostatin-2 Genes (rtMSTN-2a and -2b): Genomic Organization, Differential Expression, and Pseudogenization

Dilip K. Garikipati, Scott A. Gahr, Eric H. Roalson and Buel D. Rodgers

School of Molecular Biosciences (D.K.G., B.D.R.), School of Biological Sciences (E.H.R.) and Department of Animal Sciences (B.D.R.), Washington State University, Pullman, Washington 99164; and United States Department of Agriculture/Agricultural Research Service (S.A.G.), National Center for Cool and Cold Water Aquaculture, Kearneysville, West Virginia 25430

Address all correspondence and requests for reprints to: Buel D. Rodgers, Ph.D., Department of Animal Sciences, 124 ASLB, Washington State University, Pullman, Washington 99164-6351. E-mail: danrodgers{at}wsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Myostatin is an extremely potent negative regulator of vertebrate skeletal muscle development. A phylogenetic analysis suggests that salmonids should possess four distinct genes, although only MSTN-1 orthologs have been characterized. Described herein are the rainbow trout (rt) MSTN-2a and -2b genes and subsequence analysis of their promoters and their quantitative expression profiles. Both genes are similarly organized, contain several putative myogenic response elements, and are legitimate MSTN-2 orthologs based on Bayesian analyses. However, rtMSTN-2b contains two in-frame stop codons within the first exon and unspliced variants of both transcripts were expressed in a tissue-specific manner. Complete splicing of rtMSTN-2a occurred only in brain, where expression is highest, whereas rtMSTN-2b transcripts were mostly present in unspliced forms. The presence of stop codons in the rtMSTN-2b open reading frame and the expression of mostly unspliced transcripts indicate that this particular homolog is a pseudogene. These results confirm our previous phylogenetic analysis and suggest that all salmonids likely possess four distinct myostatin genes. The tissue-specific expression and differential processing of both rtMSTN-2 transcripts as well the pseudogenization of rtMSTN-2b may reflect compensatory and adaptive responses to tetraploidization and may help limit rtMSTN-2a’s influences primarily to neural tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MYOSTATIN IS A MEMBER of the TGF-ß superfamily and a potent negative regulator of skeletal muscle growth in mammals (1). Myostatin null phenotypes in rodents and cattle are characterized by extreme gains in muscle mass commonly referred to as double muscling (2, 3, 4). Indeed, extraordinary musculature has also been reported in a child with a 5' splice site mutation in the myostatin gene’s first intron (5), indicating that myostatin’s function is widely conserved among mammals. Unlike mammals where myostatin expression is restricted primarily to skeletal muscle (4, 6) and to a much lesser extent in heart (7) and mammary glands (8), it is widely expressed in many different fish tissues (6, 9, 10, 11, 12, 13). Nevertheless, tissue-specific functions of myostatin have yet to be described and the existence of multiple myostatin genes (9, 12, 14, 15, 16) further complicates our understanding of myostatin biology in fish. Many salmonid species are highly prized for their commercial importance, and rainbow trout in particular are an emerging and alternative animal model for basic and biomedical research. Thus, improving muscle growth by controlling myostatin expression or bioactivity in these species could significantly impact aquaculture and academic communities alike.

An early genome duplication in the bony fish, before the teleost radiation, resulted in multiple copies of many genes (17, 18). A second duplication event occurred in the salmonids approximately 50–100 million years ago (19), although many paralogs may have been lost. Some of these genes have been characterized in rainbow trout and include homologs for Wilms’ tumor gene (19), glutamate synthetase (20), the inhibitors of DNA binding/differentiation (ID) genes (21), and major histocompatibility genes (22). Our previous phylogenetic analysis of the myostatin gene family (9) identified two distinct fish clades, MSTN-1 and MSTN-2, and determined that all of the salmonid myostatin genes previously identified were actually MSTN-1 orthologs. These genes were therefore reclassified as either MSTN-1a or MSTN-1b. This study also predicted the existence of two additional myostatin paralogs, namely MSTN-2a and -2b, in most if not all salmonid species, although none of these genes have been identified to date.

Reported herein is the discovery of MSTN-2a and -2b genes in the rainbow trout, the genomic organization of both alleles, including their respective promoter regions, and the quantitative assessment of rtMSTN-2a gene expression using comprehensive RNA panels derived from multiple developmental stages and various adult tissues. These results are the first to confirm the existence of four myostatin genes in any salmonid species and reveal a strong conservation of genomic organization among all vertebrate homologs. In addition, we identified rtMSTN-2b as a pseudogene as well as unspliced transcripts of both rtMTSN-2a and -2b, which has never been described in any vertebrate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Rainbow trout (Oncorhynchus mykiss) were obtained from the Washington State University hatchery, a Center of Reproductive Biology core facility. Fish were reared and used according to protocols preapproved by the Institutional Animal Care and Use Committee. The RNA panels were generated from embryos and adult tissues of rainbow trout obtained from the National Center for Cool and Cold Water Aquaculture according to the guidelines approved by a separate Institutional Animal Care and Use Committee.

Cloning and characterizing rtMSTN-2a and -2b genes
Genomic DNA (gDNA) was isolated from fin clips (~50 mg) after incubating in 3 ml lysis buffer (30 mM Tris, 8 M urea, 4% wt/vol CHAPS, pH 8.0) containing 20 mg/ml proteinase K at 60 C. Three consecutive phenol/chloroform/isoamyl alcohol extractions were then performed, and gDNA quality was verified on a 1% agarose gel.

Degenerate primers (DegP-1 and DegP-2, Table 1Go) were designed to recognize conserved regions within the C-terminal domain of different fish MSTN-2 genes (shi drum, seabream, fugu, and zebrafish) and were used to amplify custom rainbow trout genomic libraries. Briefly, gDNA was digested with the blunt end restriction enzymes DraI, EcoRV, PvuII, and StuI and subsequently ligated to adaptor linkers according to the kit manufacturer’s protocol (BD GenomeWalker Universal kit; BD Biosciences, Franklin Lakes, NJ). Nested PCR was performed using degenerate primers and the adaptor primers (Table 1Go) with the Advantage 2 PCR kit (BD Biosciences). Cycling parameters were as follows and were used as default unless otherwise specified: an initial denaturation at 94 C for 1 min, 32 cycles of 94 C for 30 sec, 65 C for 3 min, and a final extension period of 4 min at 67 C. The PCR products were subcloned into the Topo TA vector (Invitrogen, Carlsbad, CA) and sequenced in the university’s genomic core facility. This resulted in partial sequences of two new myostatin genes. Complete genomic clones were isolated using the gene-specific primers based on the partial sequences previously cloned. Whole gene sequences were then isolated by PCR using a high-fidelity polymerase (Pfu) (Stratagene, La Jolla, CA) and resequenced.


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  		TABLE 1. Primer sequences and annealing temperatures

 
The 3' untranslated region (UTR) of rainbow trout (rt)MSTN-2a was isolated using a 3'-RACE (rapid amplification of cDNA ends) kit (Invitrogen). Total RNA from juvenile skeletal muscle was extracted using Trizol and reverse transcribed using superscript reverse transcriptase, both according to the manufacturer’s protocols (Invitrogen). cDNA was then amplified by PCR using a gene-specific forward primer (PS-I F/PS-II F; Table 1Go) and the universal adaptor primers 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 sec, 60 C for 30 sec, and 72 C for 2 min and a final extension at 72 C for 3 min. Nested PCR was subsequently performed with the 3'-UTR2a primer and the abridged universal adaptor primer provided in the kit. The resulting PCR product was subcloned and sequenced as described.

Computational analysis
Putative cDNA sequences for each novel myostatin gene were constructed in silico from the predicted open reading frames using Genescan (www.genes.mit.edu/GENESCAN.html) and were confirmed by sequencing RT-PCR amplicons. The 2.4- and 1.5-kb regions upstream of the rtMSTN-2a and rtMSTN-2b coding regions, respectively, were also cloned and sequenced using similar protocols. Putative regulatory elements in the promoters were identified by subsequence analysis using Matinspector software (Genomatix Inc., www.genomatix.de), which searches for known consensus sequences of cis regulatory elements. Alignments of the putative rtMSTN proteins were performed using Vector NTI Align X (Invitrogen).

Phylogenetic analyses was performed using Bayesian inference as implemented in MrBayes version 3.1 (23) and the newly discovered myostatin genes, rtMSTN-2a and -2b, along with previously characterized myostatin genes from the following species: human (GenBank accession no. AF019627), chicken (AY448007), seabream (AF258448 and AY046314), shi drum (AF316881 and AY059386), fugu (AY445322 and AY445321), zebrafish (AY258034 and AY687474), striped bass (AF290910), white perch (AF290910), white bass (AF197194), tilapia (AF197193), king mackerel (AF317667), little tunny (AF317666), Atlantic salmon (AJ344158 and AJ297267), brook trout (AF313912 and AF247650), rainbow trout (AF273035 and AF273036), coho salmon (AY434465 and AF394687), blue catfish (AY540992) and channel catfish (AF396747). Amino acid alignments were created using Clustal X 1.83 (24) using default parameters. Bayesian analyses used a mixed amino acid model and 10 million generations were performed with four chains (Markov Chain Monte Carlo) sampling every 1000 generations, which resulted in a distribution of 10,000 trees. To test for the occurrence of stationarity, convergence, and mixing within the 10 million generations, multiple analyses were started from different random locations in tree space. The posterior probability distributions from these separate replicates were compared for convergence to the same posterior probabilities across branches. Majority-rule consensus trees of those sampled in Bayesian inference analyses yielded probabilities that the clades are monophyletic (25). Trees sampled during the burn-in of the chain, the first two million generations, were discarded to assure that only trees sampled after stationarity was established were included and the remaining trees were loaded into PAUP version 4.0 (26). Consensus trees were then created to display branches with posterior probabilities greater than 50%.

Qualitative analysis of rtMSTN-2a and -2b expression in adult tissues
Brain, skin, gill filaments, gill arches, heart, kidney, spleen, intestine, stomach, liver, eyes, and red and white skeletal muscle were collected from 2-yr-old male and female rainbow trout, snap frozen in liquid nitrogen, and stored at –80 C. Samples were powdered, and total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol and treated with DNase (DNase RQ-1; Promega, Madison, WI) to remove contaminating gDNA. Samples were then reextracted with TRIzol reagent, and RNA quality was assessed by agarose gel electrophoresis. Assays were also performed using the RNA panel described below. Three different primer sets (PS), each spanning an intron, were used to amplify specifically rtMSTN-2a and -2b transcripts (see Fig. 5AGo). The PS-I reverse (R) primer (Table 1Go) is specific for rtMSTN-2a and primers PS-II R and PS-III forward (F) are specific for rtMSTN-2b gene transcripts. However, the other primers cross-hybridize to both rtMSTN-2 transcripts. Nevertheless, primer specificity was confirmed by sequencing amplicons. The PCR conditions were as follows: an initial denaturation at 94 C for 1 min followed by 30 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 90 sec and a final extension at 72 C for 3 min. After the initial 30 cycles, an additional 30 cycles of PCR was performed using 5 µl of the PCR product as template. To confirm that the unspliced amplicons were not from gDNA contamination, equal quantities of RNA from gills were treated with or without RNase A following the manufacturer’s protocol (Fermentas, Hanover, MD) and reextracted with TRIzol reagent. cDNA was then synthesized and amplified with primers specific to rtMSTN-2a (PS-I), rtMSTN-2b (PS-II/PS-III), or both rtMSTN-2 genes (PS-I F and PS-III R). The PCR protocol included an initial denaturation at 94 C for 1 min followed by 35 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 2 min and a final extension at 72 C for 3 min. An aliquot of 5 µl was then reamplified using the same protocol. Qualitative analysis of rtMSTN-1a, -1b, and -2a was performed in different sections of the brain with gene-specific primers (Table 1Go). The PCR protocol included an initial denaturation at 94 C for 1 min followed by 30 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 90 sec and a final extension at 72 C for 2 min.


Figure 5
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  		FIG. 5. Expression of rtMSTN-2 transcripts. A, Primer annealing locations (triangles) on rtMSTN-2 gene maps used for expression analysis. Exons are boxed and amplicon sizes (lines connecting primers), resulting from the amplification of gDNA or unprocessed transcripts are indicated. B, Expression analysis of rtMSTN-2a and -2b and ß-actin in different male rainbow trout tissues using the primer sets shown above. Initial amplification for 30 cycles was performed, and 5 µl of the product was then used for another 30 cycles of PCR. Exact amplicon sizes are indicated and were verified by sequencing. C, Gene expression in different female tissues. r. sk. mus., Red skeletal muscle; w. sk. mus., white skeletal muscle.

 
Quantitative analysis of gene expression in developing embryos and in adult tissues
An RNA panel was generated from 5000 pooled eggs from multiple females (Trout Lodge, October 2004) that were fertilized by milt from two males. After fertilization, eggs were incubated at 13 C throughout embryonic development. Unfertilized eggs (d 0) and developing embryos were collected as whole egg samples daily for the first 14 d, every other day until hatch (d 24), and every third day thereafter. Each sample pool contained 18 eggs/embryos or nine post-hatched larvae that were pooled, and several samples were collected at each time point. Frozen samples were first powdered using a liquid nitrogen cooled Bessman tissue pulverizer (Spectrum Laboratories, Rancho Dominguez, CA) and total RNA was extracted using TRI reagent (Sigma, St. Louis, MO) with the high-salt-solution modification to remove the excess glycosylated proteins and treated with DNase as described. Samples were then reextracted with TRI reagent, and RNA quality was assessed by agarose gel electrophoresis. The adult tissues were collected, and mRNA was extracted as previously described.

Total RNA (2 µg) was reverse transcribed with 1 µg oligo-dT primers (Promega) and 200 U Maloney murine leukemia virus reverse transcriptase (Promega). Subsequent real-time RT-PCR assays were conducted using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) and gene-specific primers (QMSTN-2a F and QMSTN-2b R, Table 1Go). For each sample, 1 µl cDNA was combined with 5 µl of 2x SYBR Green PCR master mix (Applied Biosystems). For each reaction, 6 µl of this mixture was added to 9 µl of the primer mix containing 500 µM of each primer. The reactions were performed as follows: 50 C for 2 min, 95 C for 10 min, and then 40 cycles of 95 C for 15 sec 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. 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 vs. the natural log of input RNA. This curve was then used to calculate the abundance of each transcript in each sample. Myostatin values were then normalized to those of 18s to control for differences in RNA and cDNA loading. Each sample was run in triplicate on a single plate, each plate was run in duplicate, and data are presented as normalized gene expression values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genomic organization of rtMSTN-2a and -2b gene
Complete genomic clones for both rtMSTN-2a and -2b genes were isolated and sequenced (Fig. 1Go, A and B). This includes approximately 2.4 kb upstream from the translational start site for rtMSTN-2a and 1.5 kb for rtMSTN-2b. The annotated gene and promoter sequences of rtMSTN-2a and -2b were deposited into GenBank and assigned the accession numbers DQ138301 and DQ177320, respectively. The genomic organization of both genes was similar to all myostatin genes characterized to date (4, 9, 13, 27, 28, 29, 30). The three exons of rtMSTN-2a are 340, 371, and 1063 bp in size, respectively, and separated by 158- and 255-bp introns (Fig. 1AGo). Similarly, the rtMSTN-2b is also organized into three exons with 346, 320, and 381 bp (partial), respectively, and are separated by 158- and 239-bp introns (Fig. 1AGo). The 3'-UTR of rtMSTN-2a was also cloned by 3' RACE using RNA and was determined to be 782 bp, which is considerably shorter than the 3'-UTRs of the rtMSTN-1 genes (13). A partial 3'-UTR of rtMSTN-2b was also cloned and found to be indistinguishable from that of rtMSTN-2a. Therefore, the exact size of rtMSTN-2b’s 3'-UTR could not be determined.


Figure 1
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  		FIG. 1. Genomic structure and organization of the rtMSTN-2a and -2b genes. A and B, Maps of rtMSTN-2a and -2b genes and putative myogenic cis regulatory elements within the promoter regions starting from the initiator. Exons are boxed with the open reading frame (ORF) in white, the UTR of rtMSTN-2a in gray, and the putative 3'-UTR of rtMSTN-2b hatched. Each individual cis element is placed relative to its position within the promoter and is color-coded as indicated in E. Putative E-boxes are numbered. The 51-bp cassette missing from the second exon of rtMSTN-2b is indicated by red lines. The asterisks below the first exon in B indicate in-frame stop codons. C and D, Sequence of the promoter regions for rtMSTN-2a and -2b, respectively, with color-coded cis elements. Boxed are consensus sequences [CAN(T/A)TG] for E-boxes. Nucleotide positions correspond to the start codon. E, Key to the color-coded promoter elements in A–D and their corresponding nucleotide positions.

 
In silico analysis of rtMSTN-2a and -2b promoters
Subsequence analysis of the upstream 2.4- and 1.5-kb sequence for rtMSTN-2a and -2b, respectively, identified several putative muscle-specific transcriptional factor binding sites or cis regulatory elements. These regulatory elements include COMP1 (cooperates with myogenic proteins), MEF2 (myocyte enhancer factor), SRF (serum response factor), TEF-1 (transcriptional enhancer factor 1), MyoD (myogenic differentiation), and Muscle sp Mt site (Fig. 1Go). Each promoter also contained a muscle-specific TATA box and several putative E-boxes. Most importantly, a MyoD binding site and several MEF2 sites were identified, although the MyoD site was found only in the rtMSTN-2a promoter (–1658 and –1644). The presence of these putative sites is particularly intriguing because both regulate myostatin expression in different mammalian systems (31, 32, 33).

Sequence analysis
The coding sequences of both genes are similar (Figs. 2Go and 3Go), although rtMSTN-2b’s is smaller and lacks 51 bp from the second exon (Fig. 1Go), which corresponds to 17 amino acids present in the other rtMSTN proteins (Fig. 3Go). The putative rtMSTN-2 proteins possess all of the elements conserved in other myostatin proteins (Fig. 2Go). However, two in-frame stop codons were detected within the rtMSTN-2b ORF (Fig. 2BGo), which were coded by the first exon (Fig. 1BGo). A multiple sequence alignment of the four rtMSTN amino acid sequences, including the stop codons in rtMSTN-2b, revealed several unique regions including a nine-residue epitope that is missing from the rtMSTN-2 sequences (Fig. 3Go). Individual comparisons indicate that rtMSTN-2a and -2b are 81% identical overall and 94% in the conserved C-terminal domain, whereas rtMSTN-2a is 66 and 91%, respectively, to the rtMSTN-1 proteins. However, the C-terminal domains of the rtMSTN-2 sequences contained two notable differences: a methionine substitution for lysine 354 (rtMSTN-1a) and a histidine substitution for arginine 369 (Fig. 3Go). This particular arginine is replaced by either histidine (seabream, shi drum, and fugu) or leucine (zebrafish) in other MSTN-2 proteins.


Figure 2
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  		FIG. 2. Annotated cDNA sequences of rtMSTN-2a and -2b. Nucleotide positions are numbered, conserved cysteine residues in the C-terminal regions are circled, and the proteolytic processing sites are boxed. The in-frame stop codons in rtMSTN-2b are highlighted by gray boxes.

 

Figure 3
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  		FIG. 3. Alignment of rtMSTN-1a, -1b, -2a, and -2b amino acid sequences. Alignments were performed using VECTOR NTI Align X. Identities between proteins are shown in the consensus whereas gaps (. . .) and stop codons (*) are indicated in the individual sequences. The in-frame stop codons in rtMSTN-2b are also circled. The conserved proteolytic RXXR site is boxed, and the residues highlighted in gray indicate notable differences in the conserved carboxy-terminal bioactive domain.

 
Phylogenetic analyses and comparative exon mapping
The current Bayesian inference analysis of different fish myostatin sequences produced only minor differences from the previous maximum likelihood analysis of the entire myostatin gene family (9). Multiple independent analyses converged on the same posterior probability values for branches, suggesting that convergence and mixing are occurring. Most of the major internal branches are well supported, and the consensus tree clearly supports the placement of the newly discovered proteins within the MSTN-2 clade. This not only confirms the identity of rtMSTN-2a and -2b as legitimate MSTN-2 orthologs but also validates our previous phylogenetic analysis and suggests that other salmonid MSTN-2 genes likely exist as well. Comparative mapping of coding regions of different myostatin genes reveals a similar organization in all species. Codons flanking the first and the second exon are highly conserved among the fishes despite minor differences in rtMSTN-2a and -2b, whereas the codons flanking the second and third exon are highly conserved among all vertebrate species (Fig. 4BGo). The previously identified consensus sequence (13) for the codons flanking the first and second exon, MAT(E/K) | PXXI (X = any amino acid), was slightly different in the rtMSTN-2 proteins [MAT(E/K) vs. MA(T/K)], whereas the consensus between the second and third exons, (G/E)(E/D)GL | XPF{phi} ({phi} + hydrophobic amino acid), was found in both rtMSTN-2 coding frames. This further indicates a high level of conservation of myostatin genes as reflected in both gene sequences and organization.


Figure 4
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  		FIG. 4. Phylogenetic analysis and comparative exon mapping of the myostatin proteins. A, Bayesian inference analysis was performed on a single matrix composed of homologous myostatin sequences from the indicated species. Two independent analyses were performed and produced identical results. Bayesian posterior probability values are shown above each branch when greater than 50%. The individual clades are shaded and labeled. B, Comparative exon mapping of the myostatin proteins. All the vertebrate myostatin genes characterized to date are organized into three exons. The first amino acid coded by each exon and the last amino acid of each protein (sequential white boxes) are shown above. Amino acids coded by adjacent regions to each exonic boundary are shown inside the boxes. In all the fish genes, the codon of the proline residue located at the first exonic boundary is partially coded by nucleotides in the first and second exons as shown. The in-frame stop codons in rtMSTN-2b are indicated with asterisks.

 
Qualitative analysis of rtMSTN-2a and -2b expression
Amplification of rtMSTN-2 transcripts in different adult tissues was performed using three primer sets. A schematic of hybridization sites and amplicon sizes are shown in Fig. 5AGo. In most tissues, both transcripts were expressed at low or undetectable levels except for brain where rtMSTN-2a was readily detected (Fig. 5Go, B and C). However, amplicons corresponding to both spliced and unspliced transcripts were detected for both rtMSTN-2a and -2b and were confirmed by sequencing. PS-I, which is specific for rtMSTN-2a, amplified both spliced and unspliced transcripts in most tissues, although the amplified variant appeared to be more abundant. However, only the spliced variant was detected in brain. By contrast, only unspliced rtMSTN-2b transcripts were detected regardless of the tissue. This was confirmed using two gene-specific primer sets that spanned either the first (PS-II) or second (PS-III) intron. To rule out the possibility of gDNA contamination of our cDNA, we amplified ß-actin using gDNA or gill cDNA as template and intron spanning primers (Fig. 6AGo). However, the larger amplicons produced with gDNA template were not produced with cDNA even after 60 cycles. Amplifying gill cDNA with primers that recognize both rtMSTN-2 transcripts produced unspliced amplicons for both genes and spliced for rtMSTN-2a (verified by sequencing). To further illustrate that these amplicons originated from RNA, samples were treated with or without RNase before cDNA synthesis and were eventually amplified with primers specific to rtMSTN-2a or -2b or both transcripts (Fig. 6BGo). Regardless of primers, all of the amplicons produced in the control samples either disappeared or were significantly reduced with RNase treatment. Taken together, these results indicate that the detection of unspliced transcripts for both rtMSTN-2 genes in different tissues is not due to gDNA contamination. The expression pattern shown represents a single male fish; however, it was additionally confirmed in a female fish and by using the RNA panel described herein. Only unspliced transcripts were present in ovaries (Fig. 5CGo) and testes (data not shown), and no evidence of sexually dimorphic mRNA processing was detected in any tissue. Nevertheless, this is the first report of myostatin splice variants for any species and possibly reflects a novel mechanism for limiting rtMSTN-2a influences to the brain and for further silencing rtMSTN-2b.


Figure 6
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  		FIG. 6. Pre-mRNA processing of rtMSTN-2 transcripts. A, Amplification of ß-actin and rtMSTN-2 genes from gill cDNA and gDNA. Total RNA was treated twice with DNase, reextracted, and used to synthesize cDNA. Primers for ß-actin and those that recognize both rtMSTN-2 genes (forward primer of PS-I and reverse primer of PS-III) were then used for 30 and 60 cycles of PCR. The same primers were also used to amplify gDNA. B, Expression of MSTN-2 transcripts at 35 and 70 cycles using cDNA synthesized from RNase-treated and nontreated gill RNA. Separate but equal RNA pools were treated with RNase and nuclease-free water, reextracted, and used for cDNA synthesis before PCR (2, all MSTN-2 primers; 2a and 2b, gene-specific primers).

 
Embryonic and adult tissue expression
Embryonic expression of rtMSTN-2a remained very low throughout development and did not differ significantly between sample times (data not shown). In adults, expression of rtMSTN-2a was detected in all tissues sampled and was highest in brain, which was 10-fold higher than in testis, spleen, and red and white skeletal muscle and at least 100-fold higher than all other tissues (Fig. 7AGo). Qualitative analysis of rtMSTN-1a, -1b, and -2a expression in different brain regions revealed gene-specific expression patterns (Fig. 7BGo). Although rtMSTN-1b was similarly expressed in all regions (myelencephalon, cerebellum, proencephalon, and optic lobe), rtMSTN-1a and -2a expression was barely detected in the proencephalon and appeared highest in the cerebellum, albeit using a qualitative assay. Comparing the normalized expression of all the three genes in selected tissues revealed that rtMSTN-1b expression was consistently highest (Fig. 7CGo). In the brain, it was 12-fold higher than rtMSTN-2a expression, which in turn was 8-fold higher than that of rtMSTN-1a. In red and white muscle, however, rtMSTN-2a expression was 6- to 10-fold lower than rtMSTN-1a expression and over 100-fold lower in eye.


Figure 7
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  		FIG. 7. Comparative expression analysis of different rainbow trout myostatin genes in different adult tissues. A, Real-time RT-PCR was used to quantify rtMSTN-2a expression in different adult tissues. Assays were repeated twice and run in triplicate from pooled tissue samples. Ant kid, Anterior/head kidney; PBL, peripheral blood lymphocytes; Pit, pituitary; Trk, trunk; Intest, whole intestine; RBS, red blood cells; Wt, white. B, Qualitative analysis of rtMSTN-1a, -1b, and -2a expression in different brain sections. Cereb, Cerebellum; Myelen, myelencephalon; O.L., optic lobe; Proen, proencephalon. C, Real time RT-PCR was used to quantify and compare normalized expression of rtMSTN-1a, -1b, and -2a in brain, eye, and red and white skeletal muscle using the pooled tissue samples in A.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An early genome duplication event before the teleost radiation, but after the divergence of ray- and lobe-finned fishes, resulted in multiple copies of many genes (17, 18). A second event specifically in the salmonids produced additional copies of some genes (19) in this family. To date, two myostatin genes have been identified in zebrafish (9, 27), brook trout (34), rainbow trout (15), Atlantic salmon (12), coho salmon (35), seabream (16, 36), shi drum (37), and fugu (Fernandas J. M., J. R. Kinghorn, and I. A. Johnston, GenBank accession nos. AY445321 and AY445322). However, all the salmonid genes are actually MSTN-1 paralogs because no MSTN-2 genes have been identified in these species until now. Indeed, a phylogenetic analysis (Fig. 4AGo) and sequence alignment (Fig. 3Go) clearly indicate that rtMSTN-2a and -2b are legitimate MSTN-2 paralogs. Organization of the newly discovered rtMSTN-2 genes (Fig. 1Go, A and B) is highly conserved with three exons, identical to rtMSTN-1 genes and other vertebrate genes characterized to date. The second exon/intron boundary, which separates the coding regions for the latency-associated peptide from that of the bioactive domain of both rtMSTN-2 genes, is also highly conserved (Fig. 4BGo). However, comparable introns in both rtMSTN-2 genes have similar sequences but are significantly smaller than their rtMSTN-1 counterparts (13), which is further indicative of their orthologous relationship. These data validate our previous phylogenetic analysis (9) and strongly suggest the presence of additional myostatin genes in other salmonid species. Whether or not all genes are functionally active, similarly expressed, or lost/silenced to pseudogenization in all salmonids remains to be determined. These studies are dependent upon the characterization of genes from additional salmonid species and possibly from other basal fish groups. Thus, comparative mapping of different myostatin genes (Fig. 4BGo) and the identification of consensus sequences for exon boundaries should prove invaluable.

Subsequence analysis of rtMSTN-2a and -2b promoter regions identified several putative cis regulatory elements that could contribute to the myogenic process. Some of these promoter elements were also found in other fish myostatin genes including brook trout (14) and zebrafish (9). However, a putative MyoD binding site was found only in promoters for zebrafish MSTN-2 and rtMSTN-2a. In mammals, MyoD and MEF2 are critical to the differentiation of skeletal muscle and induce myostatin expression (31, 32, 33). However, rtMSTN-2a expression was relatively low in skeletal muscle when compared with brain expression (Fig. 7Go), which is consistent with tissue expression levels of other MSTN-2 genes (16, 38). MyoD is also expressed in brain and helps in the early differentiation of neurons (39). Nevertheless, MyoD binding sites alone cannot explain the possible preferential expression of MSTN-2 genes in brain because MSTN-1 gene expression also occurs in the tissue. Indeed, MSTN-1a and -1b are both differentially expressed in brook trout (34) and in rainbow trout brains (Fig. 7BGo). A recent computational analysis of the structural changes that occurred throughout the evolution of the myostatin gene family failed to identify markers of positive selection in any taxonomic group (40). Thus, diversity in muscle mass among vertebrates may have evolved from changes in the cis regulation of myostatin gene expression rather than from changes in the protein structure per se (40).

The rtMSTN-2 genes are highly similar to each other, and their coding sequences are shorter than that of the rtMSTN-1 genes, which is not necessarily true in other species (9, 16, 37). Like other myostatin genes, the bioactive domains of both are entirely coded by the third exons, and each coding sequence contains the modular architecture common to other TGF-ß and myostatin gene family members. A prominent difference between the two genes is that rtMSTN-2b lacks a 51-bp cassette found in rtMSTN-2a (corresponding to 17 amino acids) and contains two in-frame stop codons within the first exon. Qualitative expression analysis of rtMSTN-2b indicates that neither intron is spliced from the primary transcript regardless of tissue (Fig. 5Go). Both introns contain in-frame stop codons, which prohibits the production of functional protein even in the presence of alternative translation start sites. The presence of stop codons in the coding sequence and in the unspliced intronic regions indicates that the rtMSTN-2b gene is a pseudogene. Both spliced and unspliced variants of rtMSTN-2a were also detected in all tissues surveyed except brain, which expressed only the correctly spliced transcript. The pre-mRNA splice sites for both rtMSTN-2 transcripts are identical; thus, the presence of correctly spliced rtMSTN-2a in some tissues is likely due to higher levels of rtMSTN-2a expression rather than differential processing. Whether or not MSTN-2b orthologs from other salmonid species are also pseudogenes remains to be determined. Furthermore, the exact timing of rtMSTN-2b pseudogenization or of any other salmonid MSTN cannot be estimated until other MSTN-2 genes are isolated from these species.

The bioactive C-terminal domain of myostatin is highly conserved in all species. In fact, the amino acid identity shared between most fish and mammalian species is 88% (6). These comparisons were made with MSTN-1 orthologs, and although the bioactive domains of MSTN-2 proteins are also well conserved, notable differences exist. These include methionine for lysine and histidine for arginine substitutions in the C terminus (Fig. 3Go). The latter conserved substitution is also seen in other fish MSTN-2 proteins (fugu, seabream, and shi drum) and may be unique to many MSTN-2 orthologs. The former, however, occurs in a region hypothesized to have contributed to enhanced musculature in domesticated and wild bovids (4, 41, 42). Myostatin’s actions are mediated by binding to the activin receptor ActRIIb (43, 44) because in fact any perturbation in ActRIIb signaling increases muscle mass (44, 45, 46). The carboxy terminus of activin is nearly identical to that of myostatin and is critical to ActRIIb signaling (47). Any change in myostatin’s structure could potentially impact the activation of ActRIIb. Future functional studies are therefore needed to determine whether MSTN-1 and -2 proteins bind ActRIIb equally or to other receptors.

Quantitative analysis of rtMSTN-2a expression in different stages of embryonic development revealed no significant differences during any stage (data not shown), suggesting that rtMSTN-2a may not play a significant role throughout development. This is consistent with the expression of MSTN-2 orthologs in other fish species including seabream (16) and zebrafish (9, 38), although MSTN-2 expression did appear to rise slightly during early somitogenesis in zebrafish. Nevertheless, this is in stark contrast to the developmental expression patterns of other fish MSTN-1 orthologs (13, 14, 38, 48), particularly rtMSTN-1a and -1b, which increased significantly during somitogenesis and after hatching and yolk sac absorption (13). In adult tissues, expression of rtMSTN-2a was comparable to that of MSTN-1a and -1b; however, it was at least 10-fold higher in brain than in any other tissue. In addition, brain was the sole tissue that expressed only the correctly spliced transcript. Teleost fish exhibit an enormous capacity to replace damaged neurons in the central nervous system, a process that involves a subdivision of the cerebellum (49, 50). Each myostatin gene was differentially expressed in different brain sections, although rtMSTN-2a levels were highest in the cerebellum (Fig. 7BGo). Qualitative analysis of MSTN-2 expression in seabream tissues indicates that it occurs primarily in brain (16), which is consistent with its expression in zebrafish, although more sensitive and quantitative assays detected low levels of MSTN-2 expression in other zebrafish tissues as well (51). These data together suggest that the biological actions of rtMSTN-2a, and possibly other MSTN-2 orthologs, may be limited primarily to the brain and may help regulate neurogenesis.

The rainbow trout is a unique model organism and representative of the salmonidae family. A better understanding of myostatin genes in this fish will add significantly to our understanding of the gene family’s evolution. Although similar to other myostatin genes in many ways, both subtle and obvious differences distinguish the trout genes. Whether or not pseudogenization or alternative splicing also occurs among MSTN-2 genes of other salmonids or whether their expression occurs primarily in neural tissue remains to be determined. These studies will nevertheless be aided by the characterization of MSTN-2 genes from other species and by additional computational and functional analysis.


   Acknowledgments
 
We acknowledge Dr. Caird Rexroad III, Kristy Anderson, and Roseanna Athey for their assistance with embryonic sampling and tissue collection.


   Footnotes
 
This work was supported by a grant from the United States Department of Agriculture (2004-34468-15199) to B.D.R.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 8, 2007

Abbreviations: F, Forward; gDNA, genomic DNA; PS, primer set; R, reverse; rt, rainbow trout; UTR, untranslated region.

Received September 22, 2006.

Accepted for publication January 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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