Identification of a muscle factor related to MyoD in a fish species

Abstract

We have isolated the cDNA encoding a myogenic factor expressed in embryonic trout muscle by hybridization with a Xenopus MyoD cDNA. Nucleotide sequence analysis and amino acid comparison showed that this cDNA called TMyoD encodes a polypeptide of 276 amino acids with 70% identity to the entire Xenopus MyoD protein and 92% identity within the basic and myc-like region. Results from Northern blotting showed that the corresponding transcript is expressed both in adult and embryonic skeletal musculature and in an in vitro myogenesis system, but is undetectable in cardiac and smooth muscles and in non muscle tissues.Key words: MyoD; Myogenesis; Teleost; (Satellite cell)The MyoD gene identified by substract cloning for myoblast specific RNA is the prototype of a family of master regulators of skeletal myogenesis which includes in vertebrates three other members namely myogenin, Myf5 and MRF4 identified subsequently [1][2][3][4][5]. All these genes encode proteins that share a highly conserved central region termed the basic/helix-loop-helix(B-HLH) domain related to the c-myc superfamily and contain sequences essential for both dimerisation and DNA binding [6]. In multipotential 10T1/2 cells, transfection experiments have shown that forced expression of these exogenous myogenic factors is sufficient to drive them down the muscle differentiation pathway suggesting their functions in myogenic lineage determination [1][2][3][4][5]. In contrast to vertebrates whose genome encodes multiple members of the MyoD family, invertebrates, including Sea urchin [7], C. elegans [8] and Drosophila [9], appear to contain only a single myogenic factor encoding gene. However, the myogenic factor for Sea urchin and C. elegans activates myogenesis in 10T1/2 cells indicating a highly conserved mechanism for muscle genes activation. Myogenic factors have been studied in mammals, amphibians and birds [10], but nothing is known to date in fish. To analyse early developmental events leading to muscle formation in fish, we set out to isolate myogenic regulatory factors from Rainbow trout (Oncorhynchus mykiss) embryos (398 degree days) using a probe which spanned functional domains of the Xenopus MyoD cDNA. For this purpose, a Agtl0 cDNA library was constructed from poly(A) ÷ RNA of the trunk of rainbow trout embryos. The double strands cDNAs synthetized by the method of Gubler et al. [11] were size fractionated by gel filtration on a sepharose 4B column (Pharmacia) and the largest fractions were pooled, inserted into Agtl0 vector (Stratagene) and encapsided using an in vitro packaging kit (Amersham). After amplification of the cDNA library, approximately 5 • 105 plaques were screened at low stringency with a fragment from the Xenopus MyoD cDNA encompassing the B-HLH domain [12]. From 9 positive clones, we identified a single cDNA of 1.5 kb which had two EcoRI fragments of aproximately 1.2 and 0.3 kb. Restriction analysis and sequencing showed that this internal EcoRI site was not situated near a Notl site which is contained in the linker...

A gene with homology to myogenin is expressed in developing myotomal musculature of the rainbow trout and in vitro during the conversion of myosatellite cells to myotubes

“…This in vitro observation indicates that teleost myosatellite cells retain their potential to differentiate without expressing the myogenin gene. Therefore, the early detection of TMyoD transcripts in proliferating undifferentiated myosatellite cells [14] is consistent with the notion that in teleosteans, Tmyogenin functions downstream of TMyoD in a regulatory pathway. This sequential activation of myogenic regulatory genes is close to mammalian models, but contrasts with avian system in which the myogenin gene has been shown to be activated in both proliferative primary myoblasts and differentiated myofibre cultures [21].…”

“…In this study, we show that the red and white fibres display different levels of myogenin mRNA accumulation: Tmyogenin mRNA did not accumulate in the fast glycolytic white muscle fibres, but concentrated in the slow oxydative red fibres. Interestingly, this selective accumulation contrasts with the distribution of TMyoD which is found to be present in equal amounts in both fibre types of fish myotomal musculature [14]. This observation suggests that Tmyogenin takes part in the control of fibre type-specific gene expression.…”

“…This novel myogenic factor represents in teleosts the second member of a family of muscle regulatory factors that include the previously described TmyoD [14]. Northern analyses indicate that this novel myogenic factor is expressed in developing myotomal musculature, in red adult muscle layer and in vitro during the conversion of satellite cells to myotubes.…”

“…In this respect, we do not know whether there is a differential expression of myogenic regulators in white and red muscles. We have previously identified a teleost fish homologue of MyoD [14]. In this study, we report the cloning of a teleost cDNA related to myogenin.…”

“…The embryonic cDNA library was probed at low stringency with the previously described cDNA TMyoD [14]. After hybridization at 42°C for 16 h in 40% formamide, 6 x SSPE, 5 x Denhart's solution, 0.5% SDS and 0,1 mg/ml sheared denatured calf thymus DNA, filters were washed twice in 2 × SSPE, 0.5% SDS for 30 min at 45°C.…”