Two myostatin genes are differentially expressed in myotomal muscles of the trout (Oncorhynchus mykiss)
SCRIBE-INRA, Campus de Beaulieu, 35042 Rennes, France
*e-mail: rescan{at}beaulieu.rennes.inra.fr
Accepted July 27, 2001
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Summary |
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Key words: myostatin, gene expression, myotomal muscle, rainbow trout, Oncorhynchus mykiss, sexual maturation, development.
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Introduction |
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In fish, the myotomal musculature presents an interesting problem of differentiation and development because of the presence of two major types of fibre (slow-oxidative versus fast-glycolytic) that are grouped into physically distinct areas: the slow muscle occurs as a thin continuous strip that lies external to the fast muscle, which constitutes the major part of the myotomal musculature. In addition, fish myotomal muscle grows by both fibre hyperplasia and hypertrophy (Koumans and Akster, 1995), whereas increases in the number of fibres in mammals and birds stop shortly after embryonic development (Goldspink, 1972). Fibre hyperplasia refers to the increase in muscle fibre number due to the formation of new fibres resulting from a continuous proliferation of satellite cells and their fusion into new myofibres (Rowlerson and Veggetti, 2001). This peculiar muscle growth pattern is associated with the continuing expression in muscle of the mitogen fibroblast growth factor 6 (FGF6) far into adulthood (Rescan, 1998). Thomas et al. (Thomas et al., 2000) have shown that the downstream function of myostatin is to prevent the progression of myogenic cells into the cell division cycle. So, we wondered in this study whether the muscle hyperplastic growth in fast-growing fish species that involves continuous division of myogenic cells for fibre recruitment correlates with an absence of myostatin gene expression in muscle.
During the sexual cycle of salmonids, a dramatic loss of muscle mass accompanies the maturation of the gonads and the release of ova or spermatozoa. This muscle atrophy results from the decreasing contents of fat and proteins that are used for energy metabolism and gonadal growth (Aksnes et al., 1986). In the present study, we demonstrate that two distinct myostatin genes exist in trout and that these are differentially expressed in various tissues. Taking advantage of the anatomical separation of slow and fast muscle fibres in fish, we report the expression of the two myostatin genes in these two kinds of fibres in muscle of immature animals and in wasting muscle of maturing animals.
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Materials and methods |
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Cloning and sequencing of cDNAs
An embryonic trout cDNA library was screened at low stringency with a probe containing the coding region of bovine myostatin. After hybridisation for 16 h at 42°C in 40 % formamide, 6x SSPE (0.9 mol l1 NaCl, 6 mmol l1 EDTA, 60 mmol l1 NaH2PO4), 5x Denhardts solution, 0.5 % sodium dodecylsulphate (SDS) and 0.1 mg ml1 denatured calf thymus DNA, filters were washed twice in 2x SSPE, 0.5 % SDS at 50°C for 30 min. One positive clone was purified, subcloned in Bluescript and sequenced using an automatic sequencing system (ABI Prism 310, PE Biosystems). Using primers designed from the sequence of the positive clone and RNA extracted from fry muscle, two distinct cDNAs were obtained by reverse transcriptase/polymerase chain reaction (RT-PCR). These two cDNAS were subsequently completed at their 5' end by a rapid amplification of cDNA ends (RACE) protocol using the Gibco BRL kit (Version 2.0). Briefly, the first-strand cDNA was synthethised using the reverse primer 5'-CTTGTTCACCAGGTGGGTGTG-3' (nucleotides 10781098 in Tmyostatin 1 and 10721092 in Tmyostatin 2). After tailing of the cDNA using dCTP (final concentration 200 mmol l1) and terminal deoxynucleotidyl transferase, a PCR reaction was carried out using the reverse primer 5'-AGTCCCAGCCAAAGTCTTC-3' (nucleotides 9821000 in Tmyostatin 1 and 976994 in Tmyostatin 2) and a poly(G)-containing primer. The 5'Race products were then subcloned in a pCRII vector (InVitrogen) and sequenced. To ensure that no mutation had been introduced during amplification, additional RT-PCR and sequencing were carried out using specific primers flanking the open reading frame of Tmyostatin 1 and 2.
Isolation of genomic clones encompassing the first intron
To further identify structural differences within the two trout myostatin genes, we generated genomic clones encompassing the first intron. For this, PCR experiments with trout genomic DNA were carried out using two sets of primers designed from the position of the intron/exon junction in the human myostatin gene (Gonzales-Cadavid et al., 1998). The primer sets used were 5'-CTTCACATATGCCAATACATATTA-3' (nucleotides 5174, see Fig. 1) and 5'-GCGGTTCACCTGAATCTTCGAAC-3' (nucleotides 545567), to generate the Tmyostatin 1 genomic fragment, and 5'-TTCACGCAAATACGTATTCAC-3' (nucleotides 5070) and 5'-GTGCACCCATAGCTGTGCCCG-3' (nucleotides 568588) to generate the Tmyostatin 2 genomic fragment. The genomic fragments obtained were subcloned in a pCRII vector (inVitrogen) and sequenced.
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Results |
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Discussion |
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In fish, and this is particularly true of salmonid species, genes often occur in pairs as the result of ancient genome duplication. The presence of two prolactin-encoding genes (Yasuda et al., 1986), two somatostatin-encoding genes (Moore et al., 1999) and two MyoD-encoding genes (Rescan and Gauvry, 1996) has been reported in salmonids. This genetic redundancy is believed to have created new structural proteins and/or to have modified their expression pattern (Meyer and Schartl, 1999; Shimeld, 1999). In the present study, we show that, after the duplication that led to their emergence, the two trout myostatin genes also evolved separately, acquiring distinct expression patterns. While Tmyostatin 1 is ubiquitously expressed, suggesting a role in growth regulation of many tissues in the trout, Tmyostatin 2 is preferentially expressed in muscle and brain.
It would be of great interest to determine whether the additional insertion of a 509-nucleotide fragment in the first intron of Tmyostatin 1 contributes to the differences observed in the expression pattern of the two myostatin genes. Surprisingly, in addition to the differential expression of the two trout myostatin genes in non-muscle tissues, major differences are also observed in their expression in muscles: while the synthesis of Tmyostatin 1 mRNA appears to be constitutive, that of Tmyostatin 2 mRNA is subject to regulation depending on the fibre phenotype (slow versus fast) and on the physiological status of the animal (immature versus mature). The constitutive expression of Tmyostatin 1 raises the question of its role in the homeostasis of muscles. In this regard, further studies are needed to ascertain whether the two proteins are processed in a similar manner and with the same kinetics in muscles, and it would be of interest to define the relative contribution of each of the two proteins to global myostatin activity.
In mammalian muscle, hyperplasia is restricted to the pre- and perinatal periods. In contrast, in some teleosts, including salmonids, hyperplasia continues, together with hypertrophic growth, far into adulthood (Rowlerson and Veggetti, 2001; Stickland, 1983). In this peculiar context, we expected to correlate the continuous proliferation of myogenic cells that leads to the formation of new myofibres in teleosts with an absence of myostatin expression. Surprisingly, we clearly demonstrated the presence of Tmyostatin 1 transcript in both slow and fast muscle and of Tmyostatin 2 transcript in slow muscle. Therefore, hyperplastic growth of muscle in trout cannot be attributed to the absence of myostatin gene transcription. However, the sequencing analysis showed that there is no obvious mutation within the coding region of the myostatin gene that would disrupt myostatin function.
The targeted inactivation of the myostatin gene in mice and the identification of mutations in the myostatin gene in double-muscled cattle provide strong evidence that myostatin is a negative regulator of muscle mass (MacPherron et al., 1997; Grobet et al., 1997; MacPherron and Lee, 1997; Kambadur et al., 1997; Grobet et al., 1998). In accordance with such a regulatory function, wasting muscle in HIV-infected men has been shown to express higher levels of myostatin than non-pathological muscle (Gonzales-Cadavid et al., 1998). Since dramatic muscle atrophy occurs during the sexual cycle of salmonids, we examined whether the increase in myostatin expression might mediate the loss of muscle mass in maturing fish. We observed that the amount of Tmyostatin 1 transcript was similar in muscles from immature and mature trout and that the amount of Tmyostatin 2 mRNA decreased dramatically in muscles from spawning and spermiating trout. These observations, which preclude an association between an upregulation of myostatin and muscle atrophy in maturing trout, are reminiscent of previous studies that have either reported the lack of a strong correlation between mouse muscle atrophy induced by unloading and over-expression of myostatin (Carlson et al., 1999) or shown that, in unloaded muscle subjected to intermittent loading, over-expression of myostatin may occur without causing muscle mass loss (Wehling et al., 2000). Therefore, we believe that myostatin is not a mediator of muscle atrophy in vertebrates.
Recently, with the intention of elucidating the molecular mechanisms by which myostatin exerts its activity, Thomas et al. (Thomas et al., 2000) have shown that myostatin specifically upregulates p21, a cyclin-dependent kinase inhibitor, and decreases the level of Cdk2 proteins, thus preventing the progression of myoblasts into the cell cycle. Although these data do not totally exclude additional roles for myostatin in the homeostasis of differentiated cells, they reinforce the idea that myostatin exerts its effect on myogenic mononucleated cells rather than on already existing muscle fibres.
In summary, rainbow trout possess two myostatin-encoding genes that have evolved separately. One (Tmyostatin 1) is ubiquitously expressed in trout tissues and appears to be constitutively expressed in muscle. The other (Tmyostatin 2) is preferentially expressed in muscle and is subject to considerable regulation, depending on the fibre phenotype and the physiological status of the animal. Finally, neither Tmyostatin 1 nor Tmyostatin 2 is upregulated during the muscle wasting that accompanies sexual maturation.
During the reviewing of this paper, Roberts and Goetz (Roberts and Goetz, 2001) reported the isolation of two cDNAs encoding myostatin homologues in another species of trout (Salvelinus fontinalis). These authors have isolated one cDNA from muscle and brain and a second cDNA from ovarian tissue. The isoform they isolated from muscle and brain is very probably homologous to the Tmyostatin 2 described here. Unfortunately, Roberts and Goetz (Roberts and Goetz, 2001) did not give the full sequence of the ovarian myostatin cDNA or the expression pattern of the ovarian isoform in various tissues, particularly in muscle. So, the homology between the ovarian isoform and the Tmyostatin 1 gene remains to be established.
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Acknowledgments |
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References |
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