DEVELOPMENTAL DYNAMICS 201:6048 ( 1 M ) Analysis of MyoD, Myogenin, and Muscle-Specific Gene mRNAs in Regenerating Xenopus Skeletal Muscle NATHALIE NICOLAS, CLAUDE L. GALLIEN, AND CHRISTOPHE CHANOINE Laboratoire de Biologie du Dlveloppemnt, URA 1188 CNRS, Centre Uniuersitaire aks Saints-P&es, Uniuersitd Rene Rescartes, 75270 Paris C e b 06, France ABSTRACT We have analyzed in adult Xenopuo Zaeuis, using in situ hybridization, the spatial and temporal expression patterns of MyoD, myogenin, and or-skeletal actin and fast myosin heavy chain mRNAs during muscle regeneration following cardiotoxin injury. MyoD transcripts could be detected in the satellite cells as early as the first stage of regeneration and were expressed persistently throughout the regeneration process. Myogenin mRNAs were transiently expressed in forming myotubes. or-Skeletal actin and fast myosin heavy chain mRNAs were detected precociously, before the young myotube stage. This work has shown, for the first time, the presence of myogenin transcripts during Xenopus myogenesis. 0 1896 Wiley-Liss, Inc. Key words: MyoD, Myogenin, Regeneration,Skeletal muscle, Xenopus INTRODUCTION Degeneration and then regeneration of the muscle fibers occur following muscle injury (Allbrook, 1981; Plaghki, 1985). The satellite cells, quiescent myogenic precursor cells located between the plasma membrane and the basal lamina in normal muscle, are activated during muscle regeneration. They proliferate, fuse to form new plurinucleate myotubes, and finally mature into myofibers (Campion, 1984; Saadi et al., 1994). Some studies have suggested that muscle regeneration recapitulates the developmental process. In mammals and amphibians, the same myosin isoforms, i.e., embryonic, neonatal/larval, and adult, are synthesized during regeneration as are observed during normal development. Nevertheless, the appearance of adult isomyosins was more precocious in regenerating compared to developing muscles (d'Albis et al., 1987; Saadi et al., 1993, 1994). It has been suggested that satellite cells and embryonic myoblasts might be endowed with specific developmental programs and represent nonequivalent myogenic cells (Chevallier et al., 1987). The discovery of a family of skeletal muscle-specific genes, the MyoD family, involved in the early stages of determination and differentiation of muscle cells, provided a powerful approach for investigating important aspects of the de novo myogenesis that occurs during muscle regeneration. Members of the MyoD gene family, including MyoD itself (Davis et al., 19871, myoge0 1996 WILEY-LISS,INC. nin (Wright et al., 19891, MRF4 (Rhodes and Koniecmy, 1989; Braun et al., 1990; Miner and Wold, 1990)) and myfi (Braun et al., 1989)) encode for basic helixloop-helix (bHLH) proteins, which are DNA binding transcription factors able to convert nonmuscle cells to a muscle phenotype in culture (Schafer et al., 1990; Choi et al., 1990). These myogenic factors can promote the transcription of a number of muscle-specific genes such as the creatine kinase (Buskin and Hauschka, 1989) and myosin light chain 1/3 (Wentworth et al., 1991) genes. In mammals, myf5 and MyoD seem to intervene at the level of the precursor muscle-cell population, whereas myogenin and probably MRF4 intervene a t the level of muscle differentiation (rev. by Buckingham, 1994). The temporal and spatial appearance of myogenic factors mRNAs has been analyzed in mammalian, avian, and amphibian development (rev. by Gurdon et al., 1992; Sassoon, 1993; Lyons, 19941, but little is known about the expression of the MyoD gene family in regenerating muscle. To our knowledge, the only studies addressing this subject analyzed the localization of MyoD and myogenin mRNAs during rat (Kami et al., 1995; Koishi et al., 1995; Rantanen et al., 1995) and mouse (Fuchtbauer and Westphal, 1992; Grounds et al., 1992) muscle regeneration. During development of the anuran amphibian Xenopus laevis, the pattern of myogenic factors expression is markedly different from the murine and avian patterns during early embryogenesis. The key difference lies in the observation that XMyoD and Xmfl transcripts can be detected in presomitic mesoderm, which is in contrast to both avian and murine systems (Hopwood et al., 1989, 1991; Scales et al., 1990). Moreover, although a partial genomic myogenin clone has been isolated in Xenopus, no myogenin transcript was detected during Xenopus development (Jennings, 1992). Nothing is known about the pattern of expression of the myogenic factor genes in regenerating muscles of Xenopus . In this work, using in situ hybridization, we have characterized the spatial and temporal expression pattern of MyoD and myogenin transcripts during the re- Received September 27, 1995; accepted March 25, 1996. Address reprint requestdcorrespondenceto Christophe Chanoine, Laboratoire de Biologie du DBveloppement, URA 1188 CNRS, Centre Universitaire des Saints-Phres, UniversitB Renee Descartes, 45 rue des Saints-PQres,75270 Paris cedex 06, France. MyoD, MYOGENIN, AND MUSCLE-SPECIFIC GENE mRNAs Fig. 1. MHCf, MyoD, myogenin, and a-skeletal actin rnRNA localization in regenerating muscles. In situ hybridization using antisense riboprobes to MHCf ( A X ) , MyoD (SF), myogenin (G-I), and a-skeletal actin (CL) was performed on transverse sections at different stages of muscle regeneration: mononucleate cell/young rnyotube (A,D,G,J), large 61 rnyotube (B,E,H,K), and mature myofiber (C,F,I,L) stages. Using sense riboprobes, we did not detect hybridization signals (data not shown). Low-magnification darkfield photomicrographs are shown. Scale bar = 100 pm. 62 NICOLAS ET AL. Fig. 2. Brightfield photomicrographs at high magnification showing that signals for a-skeletal actin and MHCf are detected in mononucleate cells during the first stage of regeneration. Arrowheads indicate some mononucleate cells expressing both a-skeletal actin (A) and MHCf (B). Scale bar = 20 pm. generation of Xenopus muscles following cardiotoxin injury. We also examined the accumulation of mRNAs coding for two muscle structural proteins, a-skeletal actin and a fast myosin heavy chain. RESULTS "he sequence of histological changes observed in Xenopus regenerating muscle following cardiotoxin injury has been previously described (Saadi et al., 1994). A single injection of cardiotoxin caused an almost complete degeneration of the myofibers within 24 hr. The degenerated muscle retained the appearance of continuous tubes owing to the persistence of the basal lamina sheaths of the myofibers. The regeneration process began to appear from the fifteenth day postinjection. In the present study, regenerative stages corresponded to the following days: mononucleate celldyoung myotubes, 15 days postinjection; large myotubes, 20 days postinjection; and mature myofibers, 1 month postinjection. At low magnification, analysis of MyoD and myogenin transcript accumulation revealed, in both cases, a strong hybridization signal in the first stage of regeneration analyzed (Fig. lD,G). For MyoD, this strong hybridization signal decreased progressively thereafter until the mature myofiber stage, at which only a weak positive signal for MyoD mRNA was observed (Fig. lE,F). For myogenin, from mononucleate cells/young myotubes to large myotubes, there is a significant decrease of the positive signal for myogenin mRNAs (Fig. lH),which was not yet detected in mature myofibers (Fig 11). a-Skeletal actin and fast myosin heavy chain (MHCf) mRNAs were detected as early as the first stage of regeneration (Fig. lA,J), and the hybridization signal strength increased progressively during later stages of regeneration (Fig. lB,C,K,L). At high magnification, it appeared that the a-actin and myosin heavy chain mRNAs began to be expressed before the formation of the young myotubes and were detected in mono- nucleate cells between the necrotic myofibers (Fig. 2A,B). MyoD transcripts began to be detected in mononucleate cells, which were probably satellite cells in concordance with Grounds et al. (1992) and Kami et al. (19951, located, on the one hand, a t the edges of some myofibers that were not totally degenerated following cardiotoxin treatment and, on the other hand, between these myofibers (Fig. 3A,B). No positive signal was detected in the uninjured contralateral muscle (data not shown). On the same sections, the new formed plurinucleate myotubes strongly expressed MyoD mRNAs (Fig. 3C,D). A low-positive signal was still observed a t 30 days postinjection in the large myotubes, which are characterized by the migration of the nuclei at the periphery (Fig. 3E,F). For myogenin transcripts, a strong positive signal was observed in the first stage of regeneration until the young myotube stage (Figs. 4, 5). More precisely, no positive signal was detected in the satellite cells located a t the edge of the necrotic myofibers, in contrast to that observed for MyoD (Fig. 4A). A strong positive signal began to be observed in the satellite cells that lined up (Fig. 4A,B) and fused (Fig. 5A-C) to give young myotubes with central nuclei (Fig. 5D).During this sequence of myogenic events, the cytoplasm of the spindle-shaped fusing cells progressively increased and strongly expressed the myogenin mRNAs in forming myotubes (Fig. 5B-D). Transcripts of MyoD and myogenin were not detected in mononucleate cells, such as red blood cells and macrophages, leukocytes, or fibroblasts, or in the widened extracellular spaces around the plurinucleate myotubes (Figs. 3C, 5D). All these results are summarized in Figure 6. DISCUSSION The earlier findings on the expression pattern of MyoD and myogenin in muscle regeneration of mammals seemed contradictory. Grounds et al. (1992) detected significant levels of MyoD and myogenin MyoD,MYOGENIN, AND MUSCLE-SPECIFIC GENE mRNAs 63 Fig. 3. Photomicrographs at high magnification showing signals for MyoD mRNA during myotube formation. A,B: Mononucleate cells. C,D: Young rnyotubes. E,F: Large myotubes. A, C, and E are brightfield micrographs; B, D, and F are darkfield micrographs. Arrowheads indicate the nuclei of some mononucleate cells (A,B) and myotubes (C-F) expressing MyoD mRNA. Arrows show that the mononucleate cells (here, some red blood cells) around the myotubes do not show any signal for MyoD mRNA. Scale bar = 20 km. mRNAs in mononuclear cells during the regeneration process but not in the newly formed myotubes. In the same way, Koishi et al. (1995)showed an accumulation of MyoD protein in satellite cells but not in myotubes. MyoD transcripts and proteins were detected in myotubes by others (Fuchtbauer and Westphal, 1992; Kami et al., 1995). These different results have been attrib- uted to the methods used to induce muscle degeneration (Koishi et al., 1995). On the one hand, Fuchtbauer and Westphal (1992) induced degeneration by transplanting muscles and in doing so would have denervated and tenotomized the muscles. On the other hand, in the studies of Grounds et al. (1992) and Koishi et al. (19951,the muscles were injured by crushing and freez- 64 NICOLAS ET AL. Fig. 4. Photomicrographsat high magnification showing signals for myogenin mRNA during the first stage of regeneration. A,B: Positive signal in satellite cells that lined up and fused. Note that the mononucleate cells located at the edge of the necrotic fibers do not exwess myogenin mRNA (arrows). Scale bars = 10 km in A, 5 pm in B. ing, respectively. These two techniques induce muscle necrosis, with minimal damage to tendons and innervation. The snake toxin, cardiotoxin, used in our experiment offered the advantage of a selective action. It induces a complete degeneration of the myofibers but does not affect the satellite cells, blood vessels, or muscle innervation (Couteaux and Mira, 1985). We showed that, during muscle regeneration of X . Zaeuis, MyoD mRNAs are expressed persistently throughout the regeneration process, whereas myogenin mRNAs are transiently expressed during the first stages of regeneration. a-Skeletal actin and adult MHC mRNAs appear also to be expressed precociously as early as the beginning of the muscle regeneration. These observations clearly show that development and regeneration of X . laevis muscles are characterized by different patterns of expression for the different myogenic factors and muscle structural gene transcripts. During Xenopus muscle development, whereas Mohun et al. (1984) observed the accumulation of a-skeletal actin mRNAs in embryos that had just completed gastrulation, Radice and Malacinski (1989) showed that fast MHC mRNAs begin to accumulate during terminal differentiation of muscle a t the time of anatomical metamorphosis. We have shown (Saadi et al., 1994) a t the protein level that larval muscles of Xenopus contained only the larval myosin isoforms. The precocious accumulation of fast MHC mRNAs during the first stages of the regeneration process agrees well with previous studies that showed the accumulation of the fast MHC protein as early as the first stages of regeneration in Xenopus (Saadi et al., 1994), urodelan amphibian (Saadi et al., 19931, and rat (d’Albis et al., 1987, 1988) muscles. In Xenopus, MyoD mRNAs have been detected prior to mesoderm induction (Rupp and Weintraub, 1991; Frank and Harland, 1991) but begin to accumulate strongly in the prospective somite region of early gastrulae (Hopwood et al., 1989); MyoD mRNAs are still detected in mature myofibers. These observations are consistent with the expression pattern of MyoD mRNAs during Xenopus muscle regeneration reported herein. We have shown that MyoD mRNAs accumulate prior to myogenin mRNAs during muscle regeneration, which is Consistent with the expression of myogenic factors observed in cultured muscle cells lines (Montarras et al., 1991). The transient accumulation of myogenin mRNAs in the first stages of Xenopus muscle regeneration is consistent with the transient expression of myogenin in mammalian development (Wright et al., 1989) but differs from the results of Jennings (1992) in Xenopus development, who used RNAse protection assay and did not detect myogenin mRNAs. The strong signal observed in fusing cells during myotube formation accounts for the potential involvement of myogenin during muscle differentiation, in the myoblast to myotube transition (Wright et al., 1989; Buckingham, 1994). However, the accumulation of myogenin mRNA is not necessarily correlated to the detection of myogenin protein. Cusella-de Angelis et al. (1992) showed that, in developing somites of mouse, myogenin protein was detectable 2 days after myogenin mRNA. The problem is to know whether the transient accumulation of myogenin mRNA during Xenopus myogenesis is specific for the regeneration process or if the nondetection of myogenin transcripts by RNAse protection assay during development is due to a low and/or transient expression of the myogenin gene. This is a crucial question; the failure to observe detectable levels of myogenin in Xenopus development has suggested the hypothesis that MyoD could play the role of myogenin in amphibian myogenesis (Sassoon, 1993). The persistence of MyoD mRNAs in regenerated myofibers and in adult myofibers (Hopwood et al., 1989) poses the problem of its functional significance. One hypothesis, suggested by Hopwood et al. (19891, is that a persisting low concentration of MyoD mRNA in ma- MyoD, MYOGENIN, AND MUSCLE-SPECIFIC GENE mRNAs 65 ture myofibers may be required for the maintenance of muscle gene expression. Because, a t least in the Xenopus embryo, the nuclear localization of XMyoD protein appears to be a highly regulated event (Rupp et al., 1994), analysis of the intracellular location of XMyoD in regenerated myofibers could give more information on its function. Nevertheless, it should be noted that the distribution of XMyoD protein during muscle development of Xenopus closely follows that of XMyoD mRNA and is characterized by a continuous nuclear localization; this is consistent with the idea of its potential involvement in the maintenance of muscle differentiation (Hopwood et al., 1992). A better understanding of Xenopus regeneration now will involve the study of transcripts of the two other myogenic factors, myf5 and MRF4. In particular, the timing of accumulation of myE mRNAs could give more information on the determination of the precursor muscle cell population. Rudnicki et al. (1993) showed, in double myf51MyoD knock-out mice, a total absence of skeletal muscle. Nevertheless, injection of MyoD orland myf5 RNA into fertilized Xenopus eggs produced a transient wave of ectopic a-cardiac actin (the first striated muscle-specifictranscript during vertebrate development) expression, although ectopic muscle formation was not induced (Hopwood and Gurdon, 1990; Hopwood et al., 1991), suggesting that additional factors are required for complete muscle differentiation. The precocious expression of MyoD, myogenin, and fast MHC mRNAs in satellite cells and young myotubes of regenerating muscles support the idea that these two HLH proteins could regulate the expression of fast MHC and be involved in muscle differentiation. We currently do not have any information on the structure of the MHC promoter in Xenopus, but Takeda et al. (1992)showed in the mouse that the promoter of the fast IIB MHC has a sequence corresponding to the motif called MEF1, which has been shown in other muscle genes to bind the myogenic regulatory factors (Olson, 1990; Weintraub et al., 1991). Understanding the potential role of MyoD and myogenin in the expression of the fast MHC gene involves analysis of their expression during metamorphosis, a t which time many new muscles are formed and the full range of adult fiber types arises (Rev. in Radice et al., 1989). It is known that the peak of circulating thyroid hormone during metamorphosis controls the larval to fast MHC transition (Chanoine et al., 1987, 1989, 1990). Muscat et al. (1995) suggested a putative regulatory cascade involved in the direct regulation of myogenesis by thyFig. 5 . Photomicrographs at high magnification showing signals for myogenin mRNA during myotube formation. Forming myotubes ( A X ) and newly formed myotubes (D) with central nuclei strongly express myogenin mRNA. B and C are the same view in brightfield (B) and darkfield (C) photomicrographs.Arrowheads indicate the nuclei of the fusing satellite cells. Arrows show that the mononucleate cells around the rnyotubes do not show any signal for myogenin mRNA. Scale bar = 20 pm. 66 NICOLAS ET AL. I I I MyoD M yogentn MHCf a skeletal actin Fig. 6. Appearance and modulation of transcript accumulation for the myogenic factors (myogenin, MyoD) and the contractile protein transcripts (for a-skeletal actin and fast myosin heavy chain) in regenerating Xenopus skeletal muscle. The level of intensity of the signal resulting from hybridization is reflected in the thickness of the lines. roid hormone: At the level of the muscle cells, T3 activates one or several members of the MyoD gene family, which can activate contractile protein genes. Several in vitro and in vivo studies have shown that MyoD, but not myogenin, is regulated by thyroid hormone (Carnac et al., 1992; Hughes et al., 1993; Muscat et al., 1994). Insofar as thyroid hormone was not detected in the serum of adult Xenopus (Leloup and Buscaglia, 1977), it now seems important to analyze the influence of thyroid hormone on the accumulation of MyoD and myogenin mRNAs during muscle regeneration in comparison to normal development to understand better the factors regulating Xenopus myogenesis. EXPERIMENTAL PROCEDURES Muscle Injury Injury was performed on adult Xenopus laevis. Animals were anesthetized with tricaine methane sulfate (MS222), and pure cardiotoxin from Nuja mossarnbica M in 0.9% nigricollis venom (Latoxan, France) NaC1) was injected into the right anterior brachial muscle of the forelimb (Saadi et al., 1994). Probes RNA probes were made by in vitro transcription in the presence of 50 pCi [35SlUTP at 1,200 Ci/mmol (NEN research product) according to the manufacturer’s instructions (Promega Biotec, Madison, WI). However, unlabeled UTP was omitted from the reaction medium in order to achieve synthesis of RNA probes with a specific activity of -10’ cpmlpg. XmyoD template is a 3’ fragment (BamHI-EcoRI; positions 8721469) of XmyoD2-24 (Hopwood et al., 1989) subcloned in pGEM 42 (Promega Biotec), cut with BamHI, and transcribed with SP6 RNA polymerase. In agreement with the work of Scales et al. (19901, the two XmyoD transcripts, Xlmfl and Xlmfl5, are detected by this probe. Xmyogenin, pCJMG2 (Jennings, 19921, was linearized using SphI and transcribed using SP6 RNA polymerase. a-Skeletal actin, pSP73-M32 (3’-UTR fragment subcloned into EcoRI-Hind11 sites of pSP73; Mohun et al., 1984), was linearized using EcoRI and transcribed using T7 RNA polymerase. Adult myosin heavy chain is a 330 nt 3‘ fragment (PstI-EcoRI)of A7 clone (Radice and Malacinski, 1989) cut with ApaI and transcribed with SP6 polymerase. A7 transcripts are expressed in fast-type fibers (Nicolas, personal communication). Probes were hydrolyzed to an average of 100-150 nucleotides by limited alkaline hydrolysis according to Cox et al. (1984) and used at 50,000 cpdF1 hybridization solution. In Situ Hybridization The procedure for fixing, embedding, and sectioning tissues was as for mouse embryos and, as was the procedure for in situ hybridization, was essentially the same as that described by Wilkinson et al. (1987). Briefly, tissues were fixed in 4% paraformaldehyde in PBS, dehydrated, and infiltrated with paraffin. Serial sections, 6 pm thick, were mounted on TESPA-coated RNase-free glass slides. Sections were deparafinized in xylene, treated with dithiothreitol (DTT)/iodoacetamine/N-ethylmaleimide (to reduce nonspecific 35S binding; Zeller and Rogers, 1989), treated with triethanolaminelacetic anhydride, washed, and dehydrated. High-stringency conditions for hybridization were followed, with posthybridization washing in 2 x SSC, 50% formamide, 50 mM DTT at 65°C for 30 min. 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