Characterization of Rainbow Trout Myostatin-2 Genes (rtMSTN-2a and -2b): Genomic Organization, Differential Expression, and Pseudogenization (original) (raw)
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Gene, 2007
Whereas the negative muscle regulator myostatin (MSTN) in mammals is almost exclusively expressed in the muscle by a single encoding gene, teleost fish possess at least two MSTN genes which are differentially expressed in both muscular and non-muscular tissues. Duplicated MSTN-1 genes have previously been identified in the tetraploid salmonid genome. From Atlantic salmon we succeeded in isolating the paralogous genes of MSTN-2, which shared about 70% identity with MSTN-1a and -1b. The salmon MSTN-2a cDNA encoded a predicted protein of 363 residues and included the conserved C-terminal bioactive domain. MSTN-2a seemed to be primarily expressed in the brain, and a functional role of teleost MSTN-2 in the neurogenesis similar to the inhibitory action of the closely related GDF-11 in the mammalian brain was proposed. In contrast, a frame-shift mutation in exon 1 of salmon MSTN-2b would lead to the synthesis of a putatively non-functional truncated protein. The absence of processed MSTN-2b mRNA in the examined tissues indicated that this gene has become a non-functional pseudogene. The differential, but partially overlapping, expression patterns of salmon MSTN-2a, -1a and -1b in muscular and non-muscular tissues are probably due to the different arrangement of the potential cis-acting regulatory elements identified in their putative promoter regions. Single and paired E-boxes in the MSTN-1b promoter were shown to bind both homo-and hetero-dimers of the myogenic regulatory factor MyoD and E47 in vitro of importance for initiating the myogenic program. Analyses of nucleotide substitution patterns indicated that the teleost MSTNs essentially have evolved under purifying selection, but a subset of amino acid sites under positive selective pressure were identified within the MSTN1 branch. The results may reflect the evolutionary forces related to adoption of the different functional roles proposed for the teleost MSTN isoforms. The phylogenetic analysis of multiple vertebrate MSTNs suggested at least two separate gene duplication events in the fish lineage. Linkage analysis of polymorphic microsatellites within intron 2 of salmon MSTN-1a and -1b mapped the two genes to different linkage groups in agreement with the tetraploid origin of the duplicated salmonid MSTN-1 and MSTN-2 genes.
BMC Evolutionary Biology, 2012
Background: Most fishes possess two paralogs for myostatin, a muscle growth inhibitor, while salmonids are presumed to have four: mstn1a, mstn1b, mstn2a and mstn2b, a pseudogene. The mechanisms responsible for preserving these duplicates as well as the depth of mstn2b nonfunctionalization within the family remain unknown. We therefore characterized several genomic clones in order to better define species and gene phylogenies. Results: Gene organization and sequence conservation was particularly evident among paralog groupings and within salmonid subfamilies. All mstn2b sequences included in-frame stop codons, confirming its nonfunctionalization across taxa, although the indels and polymorphisms responsible often differed. For example, the specific indels within the Onchorhynchus tshawytscha and O. nerka genes were remarkably similar and differed equally from other mstn2b orthologs. A phylogenetic analysis weakly established a mstn2b clade including only these species, which coupled with a shared 51 base pair deletion might suggest a history involving hybridization or a shared phylogenetic history. Furthermore, mstn2 introns all lacked conserved splice site motifs, suggesting that the tissue-specific processing of mstn2a transcripts, but not those of mstn2b, is due to alternative cis regulation and is likely a common feature in salmonids. It also suggests that limited transcript processing may have contributed to mstn2b nonfunctionalization. Conclusions: Previous studies revealed divergence within gene promoters while the current studies provide evidence for relaxed or positive selection in some coding sequence lineages. These results together suggest that the salmonid myostatin gene family is a novel resource for investigating mechanisms that regulate duplicate gene fate as paralog specific differences in gene expression, transcript processing and protein structure are all suggestive of active divergence.
BMC Genetics
Myostatin (MSTN) belongs to the transforming growth factor-beta superfamily and is a potent negative regulator of skeletal muscle development and growth in mammals. Most teleost fish possess two MSTN paralogues. However, as a consequence of a recent whole genome-duplication event, salmonids have four: MSTN-1 (-1a and -1b) and MSTN-2 (-2a and -2b). Evidence suggests that teleost MSTN plays a role in the regulation of muscle growth. In the current study, the MSTN-1b gene was re-sequenced and screened for SNP markers in a commercial population of Atlantic salmon. After genotyping 4,800 progeny for the discovered SNPs, we investigated their association with eight harvest traits - four body-weight traits, two ratios of weight traits, flesh colour and fat percentage - using a mixed model association analysis. Three novel SNPs were discovered in the MSTN-1b gene of Atlantic salmon. One of the SNPs, located within the 5[prime] flanking region (g.1086C > T), had a significant association ...
Evolution & Development, 2005
The myostatin (MSTN)-null phenotype in mammals is characterized by extreme gains in skeletal muscle mass or ''double muscling'' as the cytokine negatively regulates skeletal muscle growth. Recent attempts, however, to reproduce a comparable phenotype in zebrafish have failed. Several aspects of MSTN biology in the fishes differ significantly from those in mammals and at least two distinct paralogs have been identified in some species, which possibly suggests functional divergence between the different vertebrate classes or between fish paralogs. We therefore conducted a phylogenetic analysis of the entire MSTN gene sub-family. Maximum likelihood, Bayesian inference, and bootstrap analyses indicated a monophyletic distribution of all MSTN genes with two distinct fish clades: MSTN-1 and -2. These analyses further indicated that all Salmonid genes described are actually MSTN-1 orthologs and that additional MSTN-2 paralogs may be present in most, if not all, teleosts. An additional zebrafish homolog was identified by BLAST searches of the zebrafish Hierarchical Tets Generation System database and was subsequently cloned. Comparative sequence analysis of both genes (zebrafish MSTN (zfMSTN)-1 and -2) revealed many differences, primarily within the latency-associated peptide regions, but also within the bioactive domains. The 2-kb promoter region of zfMSTN-2 contained many putative cis regulatory elements that are active during myogenesis, but are lacking in the zfMSTN-1 promoter. In fact, zfMSTN-2 expression was limited to the early stages of somitogenesis, whereas zfMSTN-1 was expressed throughout embryogenesis. These data suggest that zfMSTN-2 may be more closely associated with skeletal muscle growth and development. They also resolve the previous ambiguity in classification of fish MSTN genes.
2006
The distinct growth capacities exhibited by slow-and fast-growing rainbow trout strains were associated with the potential to form new muscle fibres. The structural variability of myostatin genes (myostatin 1 and myostatin 2), negative regulator of muscle growth, was then investigated in trout red muscle. Myostatin cDNAs did not show any missense mutations in its coding sequence, in any of the analysed strains. However, several silent single nucleotide polymorphisms (SNP) were found in both myostatin genes. Other allelic polymorphisms, probably related to the existence of heterozygosity within strains, were also detected in myostatin 1. No sequence variability was found in the myostatin genes, in either of the two strains, which would obviously modify the functional properties of the corresponding proteins.
Development Genes and Evolution, 1999
Previously we identified two nonallelic MyoD encoding genes in the rainbow trout. These two MyoD genes (TMyoD and TMyoD2) were duplicated during the tetraploidization of the salmonid genome. In this study we show that TMyoD and TMyoD2 exhibit a distinct spatiotemporal pattern of expression that defines discrete cell populations in the developing somite. TMyoD expression is first detected in the mid-gastrula on either side of the elongating embryonic shield. During the anterior-to-posterior wave of somite formation the TMyoD transcript is initially present in adaxial cells of both the presomitic mesoderm and the forming somites. A lateral extension of TMyoD expression occurs only when the myotomes acquire their characteristic chevron shape pointing rostrally. By contrast, the initial expression of TMyoD2 occurs in somites that have already formed and is limited to the posterior compartment of somites. Further, in postlarval trout we observed a differential expression of TMyoD and TMyoD2 genes in muscle fibers with differing phenotype. Collectively, these data provide evidence that the two trout MyoD encoding genes have evolved to become functionally different. A comparison of the expression patterns of the two trout MyoD genes with that of myogenin allowed us to position them in the regulatory pathway leading to muscle differentiation.
A novel second myostatin gene is present in teleost fish
FEBS Letters, 2001
We report on the isolation and characterisation of the complete cDNA sequence encoding a novel bone morphogenetic protein-like protein (sbMSTN-b) in the teleost fish Sparus aurata. The encoded protein is 68% identical to S. aurata MSTN at the amino acid level, and homologues were also found in Umbrina cirrosa and Tetraodon nigroviridis. Phylogenetic analysis suggests that the MSTN-b gene may be present in most, perhaps all, teleost fish species. RT-PCR on different tissues/stages indicates that MSTN-b is expressed almost exclusively in the central nervous system, starting from late larval stages. Quantitative analyses indicate an increase of sbMSTN-b expression in the brain associated with metamorphosis, at the same time as completion of nervous system differentiation. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
BMC Genetics, 2013
Background: Myostatin (MSTN) belongs to the transforming growth factor-β superfamily and is a potent negative regulator of skeletal muscle development and growth in mammals. Most teleost fish possess two MSTN paralogues. However, as a consequence of a recent whole genome-duplication event, salmonids have four: MSTN-1 (−1a and -1b) and MSTN-2 (−2a and -2b). Evidence suggests that teleost MSTN plays a role in the regulation of muscle growth. In the current study, the MSTN-1b gene was re-sequenced and screened for SNP markers in a commercial population of Atlantic salmon. After genotyping 4,800 progeny for the discovered SNPs, we investigated their association with eight harvest traits -four body-weight traits, two ratios of weight traits, flesh colour and fat percentage -using a mixed model association analysis. Results: Three novel SNPs were discovered in the MSTN-1b gene of Atlantic salmon. One of the SNPs, located within the 5′ flanking region (g.1086C > T), had a significant association with harvest traits (p < 0.05), specifically for: Harvest Weight (kg), Gutted Weight (kg), Deheaded Weight (kg) and Fillet Weight (kg). The haplotype-based association analysis was consistent with this result because the two haplotypes that showed a significant association with body-weight traits, hap4 and hap5 (p < 0.05 and p < 0.01, respectively), differ by a single substitution at the g.1086C > T locus. The alleles at g.1086C > T act in an additive manner and explain a small percentage of the genetic variation of these phenotypes. Conclusions: The association analysis revealed that g.1086C > T had a significant association with all body-weight traits under study. Although the SNP explains a small percentage of the variance, our results indicate that a variation in the 5′ flanking region of the myostatin gene is associated with the genetic regulation of growth in Atlantic salmon.
Journal of Fish Biology, 1999
In the trout Oncorhynchus mykiss genome, two distinct MyoD genes (TMyoD and TMyoD2) and a single myogenin gene (Tmyogenin) have been identified. The two MyoD genes are believed to arise from a recent tetraploidization of the salmonid genome. During the anterior-to-posterior wave of somite formation, the TMyoD transcript is present initially in adaxial cells of both the presomitic mesoderm and the forming somites. A lateral extension of TMyoD labelling is observed in maturing somites when progressively they acquire the characteristic chevron shape. In contrast, the initial expression of TMyoD2 takes place in somites which have been formed already and is limited to cells of the posterior domain of the somites. Later, when all the myotomes have acquired their chevron shape, TMyoD2 transcript disappears progressively from the inner part of the myotomes until it is present only in the superficial part where slow oxidative fibres differentiate. The expression of Tmyogenin is detected first in adaxial cells of forming somites. Shortly after the formation of the somite, Tmyogenin expression extends from the adaxial cells to the posterior lateral regions of the somites and then progresses towards the anterior region. TMyoD, TMyoD2 and Tmyogenin are dynamically and differentially expressed in the developing somite suggesting that they are playing distinct roles in the early myogenesis of trout. 1999 The Fisheries Society of the British Isles