We have investigated the accumulation of mRNA transcripts of the atrial (or embryonic) myosin light chain MLC1A (MLCLmb), and the two adult fast muscle myosin light chains (MLC1F and MLC3F) during fetal skeletal muscle development in the mouse. In 15-day fetal muscle, MLC1A is the predominant mRNA detectable, by 18 days MLC1F has become the major transcript and MLC3F mRNA is detectable for the first time. By 12 days after birth, MLC1A transcripts are undetectable and MLC1F and MLC3F are similar in abundance. In fetuses treated with β-bungarotoxin and which therefore develop in the absence of functional nerve, MLC1A and MLC1F undergo normal transitions but MLC3F mRNA accumulation is significantly retarded. This demonstrates that these myosin light chain mRNAs accumulate with differing kinetics, and that MLC3F mRNA accumulation is nerve-dependent during fetal development. The results are discussed in terms of secondary muscle fibre formation, and in relation to the independent regulation of MLCIF and MLC3F mRNAs which are transcribed from the same gene.

Myosin alkali light chains (MLC) are encoded by a multigene family and show both tissue specificity and developmental regulation (for review, see Barton and Buckingham, 1985). In mammalian striated muscle, there are three principal MLC genes expressed. In adult fast skeletal muscle, the two fast isoforms MLC1F and MLC3F are encoded by a single gene (Robert et al. 1984; Periasamy et al. 1984; Nabeshima et al. 1984) and differ only in their N-terminal sequence. This gene uses two independent promoters (Strehler et al. 1985; Daubas et al. 1988) to generate primary transcripts that, following splicing, generate MLC1F- and MLC3F-specific mRNA, which are identical over most of their length but which differ in their 5’ coding and noncoding sequences. Slow skeletal muscle contains an isoform (MLC1S) which is the same (and is encoded by the same gene) as the cardiac isoform present in ventricular muscle in mammals (Barton et al. 1985a). In birds, this isoform is expressed in both atrial and ventricular muscle (Nakamura et al. 1988). During mammalian development there is expression of an embryonic isoform MLClemb (Whalen et al. 1978) in all striated muscles. This isoform persists in the atrial muscle where it is the major alkali light chain also known as MLC1A (Barton et al. 1985ft, 1988). The situation in birds is different, here an embryonic isoform (L23) is expressed transiently and at low levels in early striated muscle, but is a major isoform of developing smooth muscle and is expressed in brain at all stages (Takano-Ohmuro et al. 1985; Kawashima et al. 1987). There is no evidence for the expression of the mammalian isoform MLClemb in either smooth muscle or brain.

The influence of nerve stimulus on muscle fibre type and isoform composition in neonatal and adult muscle is well documented (for review see Pette and Vbrova, 1985). In particular the influence of nerve on myosin light chain expression has been examined in denervated (Matsuda et al. 1984), cross reinnervated (Weeds et al. 1974) and electrically stimulated muscle (Brown et al. 1983; Kirschbaum et al. 1989). In these experiments, it has been shown that, if fast skeletal muscle is denervated or stimulated with slow muscle type frequencies, it will gain characteristics of slow muscle and undergo isoform switching. For the myosin light chains this is typified by the re-expression of the slow (MLCls) isoform and a significant reduction in the accumulation of MLC3F protein and mRNA (Kirschbaum et al. 1989). In this situation, there is a transition in muscle fibre type from fast to slow resulting in mixed fibre populations with isoform content typical of mixed fibre muscles, i.e. predominantly MLC1S and MLC1F.

Transitions in the myosin light chain protein content of developing avian muscle have been well documented (Bandman et al. 1982; Crow et al. 1983; Takano-Ohmuro et al. 1985; Kawashima et al. 1988) and the influence of nerve contact examined in both neonatal muscle (Matsuda et al. 1984) and during early development (Merrifield and Konigsberg, 1987). In mammals, the pattern of myosin light chain gene expression during prenatal development is poorly documented, and the influence of prenatal denervation is unknown. In this paper, we describe the transitions in MLC1F, MLC3F and MLC1A (MLClemb) mRNA accumulation in mouse skeletal muscle taken at 15 days and 18 days in utero and 12 days after birth. mRNA accumulation was measured by the use of cloned probes and Sl-nuclease protection assay. In order to examine the influence of nerve on fetal development we have made use of bungarotoxin, a neurotoxin which binds to and destroys peripheral nerves (McCaig et al. 1987). Injection of β-bungarotoxin in utero destroys nerve fibres and subsequent muscle development therefore occurs in an aneural environment. Secondary muscle fibre formation is dependent on nerve activity, and fetal muscle that has been denervated by β-bungarotoxin injection, contains drastically reduced numbers of secondary fibres (Harris, 1981; Ross et al. 1987a, 1987b). In this study, denervation was effected at day 15 in utero and muscle analysed at day 18.

Denervation and preparation of RNA

C3H pregnant mice, dated from the time of conception, were sacrificed either at day 15 or day 18 for the preparation of fetal skeletal muscle RNA. β-bungarotoxin-treated fetuses were prepared as previously described (Weydert et al. 1987). Briefly, pregnant mice were anaesthetised and individual fetuses injected intraperitoneally with 1 μg of β-bungarotoxin (Boehringer) in utero. At day 18 mice were sacrificed and RNA extracted from both treated and untreated litter mates. β-bungarotoxin-treated fetuses were clearly paralysed and had markedly reduced muscle mass. Skeletal muscle was dissected from the hind limbs or ribs and frozen in liquid nitrogen. For the preparation of RNA, tissue was homogenized in 4 M-LiCl/8 M-urea and RNA selectively precipitated and re-extracted in LiCl/urea as in Barton et al. (1985a). RNA was also prepared from normal newborn (12 day C3H) mice. RNA from myotubes formed by the myogenic cell line T984-C110 (Jakob et al. 1978) was prepared as previously described (Barton et al. 1985a).

Probes used in SI protection assay

In order to detect MLC1F and MLC3F mRNA transcripts, we have used genomic DNA fragments cloned into M13 which encompass either exon 1 or exon 2 of the MLC1F/MLC3F gene (see Robert et al. 1984). These exons are specific to MLC1F or MLC3F mRNAs, respectively. Clone LC101 contains a genomic DNA fragment encompassing the whole of exon 1 of the MLC1F/MLC3F gene inserted into the SmaI site of M13 mp8. Synthesis from the universal primer and sub-sequent restriction at the HpaI site located within the 5’ noncoding sequence generates an antisense strand of about 320 nucleotides of which 130 are complementary to exon 1 (see Fig. 1). Clone LC40 contains the exon 2 of the MLC1F/ MLC3F gene inserted into the SmaI site of M13 mp8. This is the 5’ noncoding exon of MLC3F mRNA. Synthesis from the M13 universal primer and restriction with DdeI generates an antisense strand of 180 nucleotides, 70 of which are complementary to exon sequence. Clone PS6 contains the cDNA insert of the MLC1A clone pC110.4 (see Barton et al. 1988) subcloned in M13 mpl9. Synthesis from the M13 universal primer and restriction with PvuII generates an antisense strand of 320 nucleotides of which 227 are complementary to MLC1A mRNA.

Fig. 1.

Myosin alkali light chain specific probes used in SI analysis. In each case, the DNA fragment used for probe preparation is shown together with the restriction site used, the size and position of the antisense probe generated, and the size and position of the resulting protected fragment. In the case of MLC1F and MLC3F, these are cloned genomic DNA fragments containing exons 1 and 2 of the mouse MLC1F/MLC3F gene which are specific for MLC1F and MLC3F, respectively. In the case of MLC1A (MLClemb), this is a cDNA clone covering 3’ coding and noncoding sequence (see text). Exon sequences are indicated as boxes with hatched boxes representing 5’-non-coding sequence. Dotted lines represent M13 vector sequences.

Fig. 1.

Myosin alkali light chain specific probes used in SI analysis. In each case, the DNA fragment used for probe preparation is shown together with the restriction site used, the size and position of the antisense probe generated, and the size and position of the resulting protected fragment. In the case of MLC1F and MLC3F, these are cloned genomic DNA fragments containing exons 1 and 2 of the mouse MLC1F/MLC3F gene which are specific for MLC1F and MLC3F, respectively. In the case of MLC1A (MLClemb), this is a cDNA clone covering 3’ coding and noncoding sequence (see text). Exon sequences are indicated as boxes with hatched boxes representing 5’-non-coding sequence. Dotted lines represent M13 vector sequences.

SI protection assay

Labelled probes were generated from clones LC101 (MLC1F), LC40 (MLC3F) and PS6 (MLC1A) by synthesis from the universal primer in the presence of α32P dCTP and α32P-dTl’P and unlabelled dGTP and dATP. Following synthesis and digestion with the appropriate restriction enzyme (see above), the desired fragment was isolated on 6% acrylamide gel. SI protection was carried out as described in Barton et al. (1988) using 200 ng equivalent of pqly(A)+ RNA (in total RNA) per reaction and >80000ctsmin−1 of probe (Sp.Act. 400Ci mmole−1). The products of SI digests were analysed on 6 % acrylamide gels with appropriate size markers and exposed to X-ray film at –80°C. Quantification of signal intensity was achieved using various exposure times and scanning densitometry (Bio-Rad).

Detection of MLC1A, MLCIF and MLC3F mRNAs

In order to quantify mRNAs encoding MLC1A (MLClcmb). MLC1F and MLC3F we have used SI protection analysis. This technique avoids complications due to cross hybridisation between mRNAs of similar sequence such as the myosin light chains and allows the simultaneous analysis of two or more probes thereby reducing experimental variation to a minimum. Probes specific for MLC1A, MLC1F and MLC3F were derived as shown in Fig. 1, and were tested using RNA from myotubes formed by the cell line T984-C110 (Jakob et al. 1978) which expresses all three isoforms. When hybridised to RNA from this cell line the MLC1F and MLC1A probes both show a single protected fragment of the predicted size (Fig. 2). In the case of MLC3F, a series of bands of 70±2 nucleotides is seen. This probe extends past the site of initiation of transcription of MLC3F mRNA (see Robert et al. 1984) and these bands probably result from multiple transcription start sites, as has also been noted in the case of the rat MLC1F/MLC3F gene (Strehler et al. 1985). Multiple bands around 70 nucleotides in length are seen with all RNA samples analysed here and are therefore not specific to the T984-C110 cell line.

Fig. 2.

Detection of MLC1F, MLC1A and MLC3F mRNAs by SI protection. RNA from the myogenic cell line T984-C110 was hybridised with SI probes derived as shown in Fig. 1. Following hybridisation and digestion with SI nuclease, protected products were analysed on acrylamide gels (track 3) and compared with both undigested probe (track 1) and probe hybridised with ribosomal RNA (track 2). The size of fragments was determined by reference to a labelled pBR322 HpaII digest (M).

Fig. 2.

Detection of MLC1F, MLC1A and MLC3F mRNAs by SI protection. RNA from the myogenic cell line T984-C110 was hybridised with SI probes derived as shown in Fig. 1. Following hybridisation and digestion with SI nuclease, protected products were analysed on acrylamide gels (track 3) and compared with both undigested probe (track 1) and probe hybridised with ribosomal RNA (track 2). The size of fragments was determined by reference to a labelled pBR322 HpaII digest (M).

Myosin light chain mRNA accumulation in normal and denervated muscle

The relative abundance of mRNAs encoding MLC1A (MLClemb), MLC1F and MLC3F was determined in RNA from normal 15 day fetal, 18 day fetal, newborn (12-day-old) skeletal muscle, and from β-bungarotoxin-treated fetuses. Fig. 3 shows the results of a typical experiment showing the accumulation of these mRNA. As the probes are labelled along their length, and as different experiments required differing exposure times to allow optimal analysis by scanning densitometry, we have pooled data from a number of experiments and have corrected in each case for length of protected fragment and for exposure times. The linear relationship of optical density to mRNA concentration was confirmed using two different probes and serial RNA dilutions (data not shown). The pooled data from three independent experiments and two batches of RNA are represented in Figs 4 and 5. Firstly, we quantified the relative accumulation of each mRNA during normal development as shown in Fig. 4. These data are derived from three determinations each of which gave similar (±<10 %) values. At 15 days in utero, MLC1A is the most abundant MLC mRNA present; MLC1F mRNA is detectable at a lower level (ratio MLC1A: MLC1F 3:1) but MLC3F mRNA was not detected at this stage. By 18 days, MLC1F becomes the predominant transcript although MLC1A is also abundant. MLC3F mRNA is detectable in 18 day muscle although at a significantly lower abundance than MLC1F (ratio MLC3F:MLC1F 1:2.2). By 12 days after birth only MLC1F and MLC3F mRNA are detectable and by this stage MLC3F has become a major transcript which was, in this analysis, in fact more abundant than MLC1F (MLC3F: MLC1F 1:0.8).

Fig. 3.

mRNA accumulation in normal and denervated fetal muscle: RNA from 15-day fetal muscle (track 1) normal 18-day fetal muscle (track 2), β-bungarotoxin-treated 18-day fetal muscle (track 3) and 12-day postnatal muscle (track 4) was analysed for the accumulation of MLC1A (MLClemb), MLC1F and MLC3F mRNA. Additional normal 18-day fetal muscle samples (tracks 5 and 7) and β-bungarotoxin-treated samples (tracks 6 and 8) are shown for MLC3F.

Fig. 3.

mRNA accumulation in normal and denervated fetal muscle: RNA from 15-day fetal muscle (track 1) normal 18-day fetal muscle (track 2), β-bungarotoxin-treated 18-day fetal muscle (track 3) and 12-day postnatal muscle (track 4) was analysed for the accumulation of MLC1A (MLClemb), MLC1F and MLC3F mRNA. Additional normal 18-day fetal muscle samples (tracks 5 and 7) and β-bungarotoxin-treated samples (tracks 6 and 8) are shown for MLC3F.

Fig. 4.

MLC1A, MLC1F and MLC3F mRNA accumulation during normal fetal muscle development. The results are corrected for both relative length of the protected fragment and for autoradiographic exposure times. The relative abundance is expressed in arbitrary units relative to the level of MLC3F mRNA in new born muscle (100).

Fig. 4.

MLC1A, MLC1F and MLC3F mRNA accumulation during normal fetal muscle development. The results are corrected for both relative length of the protected fragment and for autoradiographic exposure times. The relative abundance is expressed in arbitrary units relative to the level of MLC3F mRNA in new born muscle (100).

Fig. 5.

Effect of denervation of fetal muscle development: MLC1A, MLC1F and MLC3F mRNA accumulation was quantified in RNA from 18-day normal and 18-day β-bungarotoxin-treated litter mates. In each case, results are expressed as accumulation in toxin-treated fetuses as a % of that seen in their normal litter mates. Results are from three independent experiments and two independent series of RNA preparations. Bar lines indicate ±1 standard deviation.

Fig. 5.

Effect of denervation of fetal muscle development: MLC1A, MLC1F and MLC3F mRNA accumulation was quantified in RNA from 18-day normal and 18-day β-bungarotoxin-treated litter mates. In each case, results are expressed as accumulation in toxin-treated fetuses as a % of that seen in their normal litter mates. Results are from three independent experiments and two independent series of RNA preparations. Bar lines indicate ±1 standard deviation.

To examine the effect of denervation by j3-bungaro-toxin treatment, we have quantified the accumulation of MLC1A, MLC1F and MLC3F mRNAs in fetuses treated with toxin at day 15 and allowed to develop today 18. Both toxin-treated fetuses and their normal litter mates were examined. The results of a typical experiment are shown in Fig. 3 and the pooled (corrected) data from all experiments is shown in Fig. 5 where it is expressed as mRNA abundance in treated animals compared to their untreated litter mates. The results indicate that there is a slight (16%) increase in MLC1A mRNA accumulation in treated fetuses compared to normal, that there is no significant change for MLC1F but that there is a major decrease in MLC3F mRNA accumulation which is reduced to less than half that of normal.

We have used cloned probes corresponding to the mRNAs encoding the myosin alkali light chains MLC1A (MLClemb), MLC1F and MLC3F to examine the pattern of accumulation of their transcripts during development of normal and denervated fetal muscle. The results show a transition in the abundance of MLC1A and MLC1F between day 15 and day 18 in utero such that, at day 15, MLC1A is the major mRNA but, at day 18, MLC1F is predominant. Accumulation of MLC3F mRNA is not detected at day 15. By 18 days, MLC3F is present but remains significantly less abundant than MLC1F, by 12 days after birth, MLC3F is as abundant as MLC1F.

There is an overall increase in MLC mRNA detected at 18 days compared to 15 days. For MLC1A, the increase (3-fold) parallels the increase in fibre number during this period (e.g. from 1500 to 4800 in the rat diaphragm, Harris, 1981), suggesting that the actual concentration of this mRNA per fibre remains constant over this period. In the case of MLC1F, the increase in mRNA accumulation (10-fold) indicates a net increase in amount per fibre as well as the formation of new (secondary) fibres. These results parallel our earlier study of myosin heavy chain transitions over this period where the embryonic MHC mRNA increased in proportion to fibre type, but the perinatal mRNA showed much more rapid accumulation to become the predominant isoform at 18 days (Weydert et al. 1987). The three MLC mRNAs therefore show different patterns of accumulation. MLC1A is predominant at 15 days, remains constant in concentration during fetal growth and is rapidly lost after birth. MLC1F is present at low abundance at 15 days and increases with age. MLC3F mRNA begins to accumulate after 15 days in utero but increases in abundance more rapidly than MLC1F until they are of similar concentration to the adult. The lag in accumulation of MLC3F mRNA compared to MLC1F is also seen at the level of protein accumulation where, at 18 days in utero, mouse skeletal muscle contains only trace amounts of MLC3F protein (Barton et al. 1985b), as is also the case in developing skeletal muscle in the rat (Whalen, 1978) and in chick (Crow et al. 1983).

The period between day 15 and day 18 in utero is a major phase of secondary muscle fibre formation in the mouse (Ontell and Kozeka, 1984a,b). At 15 days, primary muscle fibres predominate in skeletal muscle but, by 18 days, secondary muscle fibres form the major part of the muscle mass. Nerve contact is already established during this time period, and nerve electrical activity is essential for correct development and the formation of secondary muscle fibres (Harris, 1981; Ross et al. 1987a,b). In this study, we demonstrate that the principal effect of denervation by β-bungarotoxin treatment on myosin alkali light chain expression is a reduction of MLC3F mRNA. The fact that MLC1A and MLC1F mRNA accumulation proceed normally in these denervated muscles indicates that correct regulation of the transcripts is nerve-independent. In addition, as the denervated 18 day muscle contains predominantly primary muscle fibres this indicates that MLC1A and MLC1F are both expressed, and both correctly regulated, in primary muscle fibres. The switch from MLC1A to MLC1F is not therefore simply due to transition from predominantly primary to predominantly secondary fibres. These results parallel earlier studies showing that myosin heavy chain transitions occur in β-bungarotoxin denervated fetal muscle (Weydert et al. 1987).

In the case of MLC3F mRNA accumulation, there is an approximately 50 % reduction in denervated muscle compared to normal. Reduction in MLC3F protein accumulation has been documented in the case of chick limb bud grafts cultured on the chorioallantoic membrane of chick hosts using antibodies to detect MLC1F and MLC3F accumulation (Merrifield and Konigsberg, 1987). Here we show that mouse muscle denervated in situ by β-bungarotoxin treatment has significantly reduced MLC3F mRNA accumulation. This situation resembles that of the chick limb bud grafts in that nerve contacts were absent so that these experiments do not distinguish between the relative importance of nerve contact and nerve activity. Experiments comparing the effects of nerve paralysis and nerve destruction on muscle development have revealed few differences in the effects of these treatments (Harris, 1981), suggesting that nerve activity is the most important factor. This indicates that it is nerve activity itself that is important in MLC3F regulation, and we demonstrate that regulation is effected at the level of mRNA accumulation. Similar indications as to the importance of nerve activity have been provided by experiments using myogenic cell cultures where MLC3F accumulation is associated with external electrical stimulation (Srihari and Pette, 1981) and contraction (Moss et al. 1986), as well as in experiments comparing the effects of paralysis and denervation on MHC accumulation in fetal muscles (Harris et al. 1989).

It is of particular interest that MLC1F and MLC3F mRNAs accumulate with different kinetics, as they are generated from a single gene (Robert et al. 1984; Periasamy et al. 1984; Nabeshima et al. 1984) by the use of two independent promoters (Strehler et al. 1985; Daubas et al. 1988). Recent data from nuclear run-on assays carried out in our laboratory, have shown that mRNA accumulation is paralleled by transcriptional activity during normal development, for MLC1A, MLC1F and MLC3F (R. Cox, personal communication). The two promoters of the MLC1F/MLC3F gene are therefore sequentially activated during development and respond differently to denervation.

The authors wish to thank Benoit Robert for providing MLC1F and MLC3F clones, Arlette Cohen for assistance in SI analysis, and Professor M. Yacoub for his support during the preparation of this manuscript. The work was supported by the Muscular Dystrophy Association of America, the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Association pour la Recherche sur le Cancer and the New Zealand Medical Research Council.

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