ARTICLE

Expression and Neural Control of Myogenic Regulatory Factor Genes During Regeneration of Mouse Soleus

Correspondence to: Christophe Chanoine, Laboratoire de Biologie du Développement et de la Différenciation Musculaire (EA 2507), Centre Universitaire des Saints-Pères, Université René Descartes, 45 rue des Saints-Pères, F-75720 Paris Cedex 06, France. E-mail: chanoine@biomedicale.univ-paris5.fr

	Summary

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Given the importance of the myogenic regulatory factors (MRFs) for myoblast differentiation during development, the aims of this work were to clarify the spatial and temporal expression pattern of the four MRF mRNAs during soleus regeneration in mouse after cardiotoxin injury, using in situ hybridization, and to investigate the influence of innervation on the expression of each MRF during a complete degeneration/regeneration process. For this, we performed cardiotoxin injury-induced regeneration experiments on denervated soleus muscle. Myf-5, MyoD, and MRF4 mRNAs were detected in satellite cell-derived myoblasts in the first stages of muscle regeneration analyzed (2–3 days P-I). The Myf-5 transcript level dramatically decreased in young multinucleated myotubes, whereas MyoD and MRF4 transcripts were expressed persistently throughout the regeneration process. Myogenin mRNA was transiently expressed in forming myotubes. These results are discussed with regard to the potential relationships between MyoD and MRF4 in the satellite cell differentiation pathway. Muscle denervation precociously (at 8 days P-I) upregulated both the Myf-5 and the MRF4 mRNA levels, whereas the increase of both MyoD and myogenin mRNA levels was observed later, in the late stages of regeneration (30 days P-I). This significant accumulation of each differentially upregulated MRF during soleus regeneration after denervation suggests that each myogenic factor might have a distinct role in the regulatory control of muscle gene expression. This role is discussed in relation to the expression of the nerve-regulated genes, such as the nAChR subunit gene family. (J Histochem Cytochem 49:887–899, 2001)

Key Words: myogenic regulatory factors, MRF4, muscle regeneration, denervation

	Introduction

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The four myogenic regulatory factors (MRFs) MyoD, Myf-5, myogenin, and MRF4 are basic helix–loop–helix transcription factors whose ectopic expression is able to convert a wide range of cultured cells to a muscle phenotype and which can promote the transcription of a number of muscle-specific genes. The functions of the MRFs in vivo have been investigated by determining their pattern of expression and by gene targeting. During development, the order of expression of MRF genes varies according to muscle origin and among species (Ludolph and Konieczny 1995 ; Yun and Wold 1996 ), and there is strong evidence that MRFs regulate contractile protein genes during embryonic/fetal differentiation. Experiments using knockout mice progressively elucidated the hierarchical relationships between the MRFs and established that functional redundancy is a feature of the MRF regulatory network. Previous studies showed that MyoD and Myf-5 play overlapping roles in myoblast specification, whereas myogenin and either MyoD or MRF4 are required for differentiation (Tajbakhsh and Buckingham 2000 ).

An important feature of mature skeletal muscles is their ability to regenerate after injury. Satellite cells, closely associated with muscle fibers, are myoblast-like cells responsible for the regenerative capacity of muscles (Plaghki 1985 ). These adult muscle stem cells are normally mitotically quiescent but are activated in response to injury. The regenerative process is characterized by proliferation of the descendants of the activated satellite cells before they fuse to form new myotubes. Given the importance of the MRFs for myoblast differentiation during development, the pattern of expression of the MRFs has been analyzed during muscle regeneration of mammals after different types of injury and using different methods of detection, including in situ hybridization (ISH) (Grounds et al. 1992 ; Fuchtbauer and Westphal 1992 ; Kami et al. 1995 ; Rantanen et al. 1995 ), Northern blotting (Marsh et al. 1997 ), RT-PCR (Mendler et al. 1998 ), immunohistology (Fuchtbauer and Westphal 1992 ), and Western blotting (Sakuma et al. 1999 ). Surprisingly, the only studies using ISH to address this subject analyzed the localization of MyoD and myogenin, but no results were reported about the accumulation of Myf-5 and MRF4 mRNA during mammalian muscle regeneration. Moreover, the previous findings appeared contradictory and have been discussed and attributed to the different methods used to induce muscle degeneration (Koishi et al. 1995 ; Nicolas et al. 1996 ). On the other hand, we point out the fact that some discrepancies among previous reports analyzing MRF expression after muscle injury by Northern blotting (Marsh et al. 1997 ) and RT-PCR (Mendler et al. 1998 ) could be due to difficulty in obtaining complete degeneration of the muscle.

In vitro and in vivo experiments have shown that the expression of myogenic factors depends on different types of regulation, including those by thyroid hormone (Carnac et al. 1992 ; Hughes et al. 1993 ; Muscat et al. 1994 ), retinoic acid (Arnold et al. 1992 ; Carnac et al. 1993 ), and innervation (Hughes et al. 1993 ). In adult muscle, denervation positively regulates the expression of the transcripts encoding myogenic factors. In mouse, Duclert et al. 1991 showed that the rate of myogenin transcripts is rapidly upregulated (about 30-fold) in denervated muscle compared to innervated muscle. Denervation also increases the rate of MyoD and MRF4 transcripts (about 10-fold) in muscle (Duclert et al. 1991 ; Eftimie et al. 1991 ). Adams et al. 1995 showed that long-term denervation induces a long-term upregulation of the expression of MyoD and Myf5 transcripts, whereas the overexpression of MRF4 and myogenin is transitory. Little is known, to our knowledge, about the effect of denervation on MRF expression during muscle regeneration apart from the work of Koishi et al. 1995 showing that denervation upregulated the accumulation of MyoD protein in regenerating muscle of adult rat, although it is well known that innervation has a critical role in determining adult muscle phenotype. It is also well established that embryonic myoblasts and satellite cells are not equivalent cells (Stockdale 1992 ) that are subjected to distinct neural and hormonal environments. In particular, in the new myogenesis, which takes place after muscle injury, innervation is continuously present. Therefore, it appears important to analyze the modulating influence of innervation on MRF expression during the course of the regenerating process.

For these reasons, the aims of this work were (a) to clearly characterize the spatial and temporal expression pattern of the four MRF transcripts during regeneration of the mouse soleus after cardiotoxin injury, using ISH. It is well established that this snake toxin offers the advantage of inducing a complete degeneration of the myofibers without affecting the satellite cells, blood vessels, or muscle innervation (Nicolas et al. 1996 ) and (b) to analyze the effect of denervation on the accumulation of the four MRF mRNAs during the regeneration process. It seems particularly important to know if the accumulation of each MRF is differently altered by denervation, which would suggest the existence of a distinct role for each MRF in the regulation of muscle gene expression.

	Materials and Methods

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Animals and Muscle Injury
Studies were carried out with adult female mice Mus musculus Swiss (about 30 g) originating from the breeding center R. Janvier (Le Genest Saint-Isle, France). Animals were anesthetized by IP injection of 3.5% chloral hydrate. The skin was cut and pure cardiotoxin from Naja mossambica nigricollis venom (Latoxan; Valence, France) (10^-5 M in 0.9% NaCl) was injected into the soleus muscle. Five animals were analyzed for each stage of muscle regeneration except for 4 days post injection (P-I) when only three animals were analyzed.

Denervation
Before cardiotoxin injury, the soleus muscle was denervated as follows. A double proximal ligature and a double distal ligature with silk thread, separated by 3 mm, were placed on the tibial nerve that innervates several muscles including the soleus muscle. The nerve was then cut between these ligatures. Five animals were analyzed for each stage of muscle regeneration except for 4 days P-I, when only three animals were analyzed.

Preparation and Prehybridization of Tissue Sections
The procedure for fixing, embedding, and sectioning tissues 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. Then 7-µm-thick serial sections were mounted on TESPA-coated RNase-free glass slides. Sections were deparaffinized in xylene, digested with proteinase K, postfixed, treated with dithiothreitol/iodoacetamine/N-ethylmaleimide (to reduce nonspecific ³⁵S binding; Zeller and Rogers 1989 ), treated with triethanolamine/acetic anhydride, washed, and dehydrated.

Probe Preparation
The following probes were used to generate antisense cRNAs: MRF4 template is a fragment (BmsAI-XbaI; positions 853–990) of rat MRF4 (Rhodes and Konieczny 1989 ) subcloned in pGEM-T (Promega; Madison, WI), cut with SalI, and transcribed with T7 RNA polymerase. MyoD template is a fragment (positions 750–1785) of MyoD1 (Davis et al. 1987 ), cloned in pVZCII, linearized using MLUI, and transcribed using T3 RNA polymerase. Myogenin is a 695-nt 3' fragment cloned in pBluescript M13-65-7 (Wright et al. 1989 ), cut with HindIII, and transcribed with T7 RNA polymerase. Myf-5 template is a 5' fragment (BalI-ApaLI; position 15–326) of mouse Myf-5 subcloned in pBluescript (Ott et al. 1991 ), cut with HindIII, and transcribed with T7 RNA polymerase.

cRNA probes were made by in vitro transcription in the presence of 50 µCi [³⁵S]-UTP at 1200 Ci/mmol (NEN; Boston, MA) according to the manufacturer's instructions (Promega). However, unlabeled UTP was omitted from the reaction medium to achieve synthesis of RNA probes with a specific activity of 10⁹ cpm/µg.

Probes were hydrolyzed to an average of 100–150 nucleotides in length by limited alkaline hydrolysis according to Cox et al. 1984 for efficient hybridization and were used at 50,000 cpm/µl hybridization solution.

Hybridization and Washing Procedures
High-stringency conditions for hybridization and post-hybridization were followed. Sections were hybridized overnight at 53C with post-hybridization washing in 2 x SSC, 50% formamide, 50 mM DTT at 65C for 30 min. Autoradiography was carried out with Kodak NTB-2 track emulsion, developed in Kodak D19 developer, and stained lightly with Giemsa.

Quantitative Evaluation of the Hybridization Signal
Hybridization signals were analyzed using a specific minicomputerized densitometric program developed for use with the Visilog 4.15 image analysis software (Noesis; Saclay, France). Briefly, images were converted to a gray scale and quantification of staining was carried out by recording the value of light intensity, measured by the program, in each considered cell. Results were reported as light intensity by surface area. One section of each stage originating from four distinct experimental muscles was analyzed. Each quantification was repeated three times for the same section with comparable results.

	Results

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In this study we performed cardiotoxin injury-induced regeneration experiments on soleus muscle of adult mice to investigate the influence of innervation on the expression of the MRFs during muscle regeneration. Animals were divided into two distinct groups: in all animals the soleus muscle was injured by cardiotoxin injection, and in half of them the soleus was also subjected to denervation before toxin injection. The accumulation of MyoD, Myf-5, myogenin, and MRF4 mRNA was then analyzed at different days P-I using ISH.

The sequence of histological changes observed in regenerating mouse muscle after snake toxin injury has been previously described (Couteaux et al. 1988 ). In our experiments, from 2–3 days P-I many mononucleated cells located between necrotic myofibers were observed (Fig 1A and Fig 2A). They probably corresponded, at least in part, to proliferating myoblasts arising from activated satellite cells in accordance with previous reports (Grounds et al. 1992 ; Kami et al. 1995 ). At this stage and on the following day (4 days P-I) (Fig 2E), myoblasts that lined up and fused were observed. Some isolated young myotubes with central nuclei also began to be identified at 4 days P-I (Fig 2F). Because all studies were done on serial sections, there was no problem in verifying the multinuclear nature of newly formed myotubes. In contrast to that, we classified cells as mononucleated if they appeared only on one or two sections. Young myotubes with central nuclei were predominantly observed at 5 days P-I (Fig 3B). One month P-I, the majority of the nuclei remained in a central position, which is a characteristic of regenerating mouse muscles previously described (Couteaux et al. 1988 ) (Fig 4D).

View larger version (129K): [in this window] [in a new window]   Figure 1. In situ hybridization using antisense riboprobes to Myf-5 on transverse sections of control (A–E) and denervated (F) regenerating soleus at 3 (A–D) and 8 (E,F) days P-I. Using sense riboprobes, we did not detect hybridization signals (data not shown). (A,C,E,F) Brightfield photomicrographs; (B,D) darkfield photomicrographs of the same sections shown in A and C, respectively. Arrowhead indicates an activated satellite cell strongly expressing Myf-5 mRNA. Arrow points to a Myf-5-positive myoblast located between the necrotic myofibers. Bar = 5 µm.

View larger version (141K): [in this window] [in a new window]   Figure 2. In situ hybridization using antisense (A,C–E) and sense (B,F) riboprobes to MRF4 on transverse sections of control regenerating soleus at 3 (A–D) and 4 (E,F) days P-I. C is a detail of A at high magnification, showing two activated satellite cells (arrowheads) strongly expressing MRF4 mRNA, closely associated with the periphery of the necrotic myofibers. In D, the arrow points to an MRF4-positive myoblast located between the necrotic myofibers. In E, open arrowheads indicate myoblasts that lined up and fused; closed arrowheads (E,F) indicate young myotubes with central nuclei. Bar: A,B = 5 µm; C,D = 2 µm; E,F = 3 µm.

View larger version (213K): [in this window] [in a new window]   Figure 3. In situ hybridization using antisense (A,C–E) and sense (B) riboprobes to MRF4 on transverse sections of control (A–C,E) and denervated (D) regenerating soleus at 5 (A,B), 8 (C,D), and 30 (E) days P-I. Bar = 8 µm.

View larger version (169K): [in this window] [in a new window]   Figure 4. In situ hybridization using antisense riboprobes to MyoD on transverse sections of control regenerating soleus at 3 (A,B), 5 (C), and 30 (D) days P-I. Arrow points to a MyoD-positive myoblast located between the necrotic myofibers. Using sense riboprobes, we did not detect hybridization signals (data not shown). A and B are the same view in brightfield (A) and in darkfield (B). Bars: A,B = 5 µm; C,D = 8 µm.

At high magnification, analysis of Myf-5 transcript accumulation revealed a strong hybridization signal in the first stage of regeneration (2–3 days P-I). More precisely, Myf-5 transcripts were detected in mononucleated cells located either at the edges of some necrotic myofibers or between these myofibers (Fig 1A–1D). Because no positive signal was detected in the uninjured contralateral muscle (data not shown), we can assume that these Myf-5-positive mononucleated cells corresponded to activated satellite cells and to their descendent proliferating myoblasts, respectively (see Fig 1C). However, at 2–3 days P-I, it appears difficult to affirm that the Myf-5-positive cells closely associated with the necrotic myofibers were activated satellite cells. Those were already detected within the 3–12 initial hours after injury (Grounds et al. 1992 ; Cooper et al. 1999 ). These cells might be already generating cuffs within the necrotic fibers as an intermediate step for forming early myotubes. From 4 days P-I in young multinucleated myotubes, there was a significant decrease of the positive signal for Myf-5 mRNAs, which then disappeared at 8 days P-I (Fig 1E). The fact that Myf-5 protein was detected in myotubes (Cooper et al. 1999 ), whereas we could not detect Myf-5 mRNA, is not contradictory and accounts for a precocious and transient expression of Myf-5 gene during muscle regeneration. At this stage the amount of Myf-5 mRNA is not enough to be detected by ISH.

In contrast to the pattern observed for Myf-5 and for MRF4 as well as MyoD transcripts, a positive signal was observed as early as the first stages of regeneration analyzed and was continuously detected to 30 days P-I (Fig 2 Fig 3 Fig 4). For these two MRFs, a strong hybridization signal was detected in both cells closely associated with the necrotic myofibers (Fig 2C) and in cells between the necrotic myofibers (Fig 2D, Fig 4A, and Fig 4B). As shown for MRF4, this strong positive signal was always detected in myoblasts that lined up (Fig 2E) and in the small newly formed myotubes at 4 days P-I (Fig 2E). From 5 to 30 days P-I, both MRF4 (Fig 3A, Fig 3C, and Fig 3E) and MyoD (Fig 4C and Fig 4D) mRNAs were still detected in multinucleated myotubes. During this period, the hybridization signal strength decreased progressively for MyoD mRNA, whereas the MRF4 gene showed a decreased expression up to 8 days P-I, followed by an increased expression at 30 days P-I.

At the beginning of the regeneration process, at 2–3 days P-I, no positive signal for myogenin mRNAs was detected in mononucleated cells located at the edge of the necrotic myofibers, but some myogenin-positive myoblasts located between the necrotic myofibers were clearly observed (data not shown). Nevertheless, a strong hybridization signal for myogenin was detected in the myoblasts that lined up and fused (Fig 5A and Fig 5B) before drastically decreasing in young multinucleated myotubes. At 5 days P-I, only a few mononucleated cells still expressed myogenin transcripts (Fig 5C). No positive signal was detected at 30 days P-I (Fig 5D and Fig 5F). All these results are summarized in Fig 6.

View larger version (125K): [in this window] [in a new window]   Figure 5. In situ hybridization using antisense riboprobes to myogenin on transverse sections of control (A–D,F) and denervated (E,G) regenerating soleus at 3 (A,B), 5 (C), and 30 (D–G) days P-I. Using sense riboprobes, we did not detect hybridization signals (data not shown). A and B are the same view in brightfield (A) and in darkfield (B). (A) Arrows indicate myoblasts that lined up. (C) Arrow points to a mononucleated cell expressing myogenin transcripts. Arrowhead indicates a myotube in which myogenin transcripts were not still detected. Bars: A–E = 5 µm; F,G = 33 µm.

View larger version (9K): [in this window] [in a new window]   Figure 6. Appearance and modulation of transcript accumulation for the myogenic factors (Myf5, MyoD, myogenin, and MRF4) in regenerating soleus of mouse, depending on P-I day and stage of regeneration. The level of intensity of the signal resulting from hybridization is reflected in the thickness of the lines.

Denervation significantly upregulated the four MRF transcripts differentially. For Myf-5, the effect of denervation was observed at 8 days P-I, because at this stage ISH permitted detection of Myf-5 mRNA in the young myotubes of denervated muscle, whereas no hybridization signal was seen in myotubes of innervated contralateral muscle (Fig 1E and Fig 1F). This upregulation of the level of Myf-5 transcripts by denervation was transitory, because no effect of muscle denervation was visible on the previous or following days. Quantification of Myf-5 mRNA levels indicated that the level of Myf-5 transcripts transiently increased about 15-fold compared to innervated muscle (Fig 7C).

View larger version (26K): [in this window] [in a new window]   Figure 7. Quantitative analysis of MRF expression at different stages of regeneration (from 3 to 30 days P-I) in control () and denervated () soleus. (A) MRF4; (B) myogenin; (C) Myf-5; (D) MyoD. Error bars represent SD between each section of the four experimental muscles (see Materials and Methods).

As observed for Myf-5, MRF4 transcript levels were transiently upregulated by denervation at 8 days P-I (about fivefold; Fig 7A). A strong hybridization signal was detected in denervated muscles in comparison to innervated contralateral muscles, in which the hybridization signal strength for MRF4 was weaker (Fig 3C and Fig 3D).

The response of muscle to denervation is more belated for myogenin and MyoD compared to Myf-5 and MRF4. No effect of muscle denervation was observed before 30 days P-I, when the levels of both myogenin and MyoD transcripts were upregulated. Indeed, at this stage of regeneration a strong hybridization signal for myogenin was detected in myotubes of denervated muscles compared to that observed in innervated contralateral muscles where myogenin mRNA was not detected (Fig 5D–5G). The increase of MyoD mRNA levels was also seen at 30 days P-I (data not shown). The greatest increase was observed for myogenin mRNA (more than 40-fold) (Fig 7B), whereas MyoD transcript levels increase only about threefold (Fig 7D).

	Discussion

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This study provides a detailed spatial and temporal analysis of gene expression for the four myogenic regulatory factors (Myf-5, MyoD, myogenin, and MRF4) during a complete degeneration/regeneration process of mouse soleus. These results are compared in Table 1 with previous works analyzing MRF expression using both ISH and immunohistology in regenerating muscles of mammals. This compilation enables us to point out the discrepancies existing as a function of the type of muscle injury. This report also offers the opportunity to analyze the influence of denervation on the accumulation of each of the MRF transcripts during a complete regeneration process.

MRF4 Transcripts Are Strongly Expressed by Myoblasts of Regenerating Muscles
Expression of muscle structural genes (myosin isoforms, actins) has been extensively analyzed during muscle regeneration in mammals (d'Albis et al. 1988 ; Toyofuku et al. 1992 ), but it is surprising that no systematic ISH experiments have been performed on the expression of the four MRFs during muscle regeneration, given the importance of the MRFs for myogenesis. In particular, no data are published for Myf-5 or MRF4 during the regeneration process in mammals, apart from the work of Cooper et al. 1999 analyzing the expression of Myf-5 using heterozygous Myf-5- nlacZ mice after cardiotoxin injury.

Our study shows that during soleus muscle regeneration three MRF mRNAs are concomitantly strongly expressed during the early stages of the regeneration process. Myf-5, MyoD, and MRF4 transcripts are detected in proliferating myoblasts and are not detectable in quiescent satellite cells, whereas myogenin mRNAs begin to be detected later in forming myotubes. Cooper et al. 1999 clearly showed that adult activated satellite cells initially express MyoD or Myf-5, or both MRF proteins. In a recent work, Weis et al. 2000 reported that MRF4 protein was detected in nuclei of activated satellite cells of rat muscles denervated for 24 hr. This reinforces our results and supports the fact that MRF4 transcripts accumulate more precociously (before 2–3 days P-I) in regenerating muscle. From analysis of the expression pattern of the MRFs in satellite cells isolated from rat muscle and cultured in vitro, it was found that MyoD was expressed before the first evidence of proliferation, Myf-5 and MRF4 were expressed at intermediate times, and myogenin was detected coincident with the first evidence of differentiation (Smith et al. 1994 ; Yablonka-Reuveni and Rivera 1994 ). Cornelison and Wold 1997 found that activated single satellite cells from fibers isolated and cultured in vitro first express Myf-5 or MyoD before co-expressing both Myf-5 and MyoD, and progress through their normal developmental program leading to terminal differentiation, characterized by the expression of both myogenin and of MRF4. In the developing forelimb of the mouse, the different MRFs are progressively expressed over a period of 3 days, MRF4 mRNA accumulating in later fetal stages in differentiated muscle fibers (reviewed in Sassoon 1993 ). In contrast to that, in somites MRF4 is characterized by a biphasic pattern of expression. MRF4 transcripts are transiently detected in somites during development from 9.5 to 12.5 days p.c. and are later upregulated in fetal muscle to reach relatively high levels in mature innervated muscle (Bober et al. 1991 ). For MRF4 mRNA, the same type of biphasic pattern of expression is observed during soleus regeneration. Indeed, we showed a transient downregulation of the accumulation of the MRF4 transcripts from 4 to 8 days P-I in regenerating muscle.

It has been shown that newborn mice deficient for both MyoD and Myf-5 are totally devoid of skeletal myoblasts and muscle (Rudnicki et al. 1993 ), whereas myogenin (-/-) mice form myoblasts that fail to form myotubes efficiently in vivo (Hasty et al. 1993 ). The transient accumulation of myogenin mRNAs in fusing myoblasts during soleus regeneration is consistent with the transient expression of myogenin in mammalian development (Wright et al. 1989 ). This accounts for the requirement of myogenin in the myoblast-to-myotube transition, including myoblast fusion and terminal differentiation (reviewed in Buckingham 1994 ).

Our results should be discussed in relation to recent studies using MRF4/MyoD double mutants and MyoD (-/-) mice, given new information on both the overlapping functions of MyoD and MRF4 and the specific MRF expression pathway detected in satellite cell myogenesis vs fetal or embryogenic myogenesis. MRF4/MyoD double mutants displayed a severe muscle deficiency similar to that in myogenin mutants (Rawls et al. 1998 ). Myogenin was expressed in these double mutants, indicating that myogenin is insufficient to support normal myogenesis in vivo and that there are overlapping functions for MRF4 and MyoD in the muscle differentiation pathway. Using a MyoD (-/-) mutant, Megeney et al. 1996 showed that MyoD plays a crucial role in satellite cell function, the transition from proliferation to differentiation being delayed in satellite cells from mice lacking MyoD even if Myf-5 was upregulated (Sabourin et al. 1999 ; Yablonka-Reuveni et al. 1999 ). In the recent report of Cornelison et al. 2000 , a central conclusion was that there is a strong epistasics relationship between MyoD and MRF4 in satellite cells, arguing for an absolute and nonredundant requirement for MyoD to support proper MRF4 expression in activated satellite cells. This suggested a specific involvement of this pair of MRFs in the satellite cell differentiation pathway clearly different from that observed during embryonic myogenesis because, at birth, the mice mutants lacking MyoD are phenotypically normal and show levels of expression of Myf-5, myogenin, and MRF4 transcripts that are indistinguishable from those of wild-type mice (Rudnicki et al. 1992 ).

The previous studies suggested that the strong accumulation of both the MRF4 and MyoD transcripts in proliferating myoblasts observed in our in vivo analysis could account for a specific regenerating pathway in which MRF4 is upregulated by MyoD, in addition to the expression of other MRFs, Myf-5 and myogenin, which could reinforce the differentiation program. The fact that MRF4 protein was not detected in adult innervated muscle, whereas it transiently accumulated in both myofiber and satellite cell nuclei of denervated muscle, has also suggested that MRF4 may have important roles in the gene programs induced by activation after denervation and during muscle regeneration (Weis et al. 2000 ). However, both the function of MyoD and the relationships between MyoD and MRF4 during muscle regeneration seem more complex, probably depending in part of the model used to study the regeneration process. In a recent study, White et al. 2000 , using whole muscle grafts, showed that muscle regeneration was delayed but not impaired in MyoD (-/-) mice. Moreover, Sabourin et al. 1999 indicated that in primary MyoD -/- myogenic cells derived from adult muscle MRF4 expression was delayed but did occur.

MRF Gene Expression Is Differentially Upregulated by Denervation in Regenerating Mouse Soleus
Our findings showed that denervation positively regulates the accumulation of the four MRF transcripts during soleus regeneration. Nevertheless, the increase in the level of myogenic transcripts depended on both the MRF studied and the stage of regeneration. After denervation, Myf-5 and MRF4 mRNA were transiently upregulated at 8 days P-I whereas the increase in both MyoD and myogenin transcripts appeared much later, at 30 days P-I. These results emphasize the emerging idea that each MRF has evolved a specialized as well as a redundant role in skeletal muscle formation (Tajbakhsh and Buckingham 2000 ; Valdez et al. 2000 ), which suggests that each MRF may regulate a specific set of genes. On the basis of a detailed time-course analysis of MRFs and nAChR expression in denervated muscle (Eftimie et al. 1991 ) and of the differential regulatory control of MRFs on the different nAchR subunit gene expression, this hypothesis may be supported. During development, after the innervation of muscle, the embryonic -subunit is replaced by an adult -subunit (Schuetze and Role 1987 ). Durr et al. 1994 indicated that in the promoter of the -subunit gene, E-box elements enhance promoter activity in muscle and mediate transactivation by MRFs. Myogenin and Myf-5 were much more efficient than MRF4 or MyoD, which exerted only little transactivation. In contrast, MRF4 is the MRF which preferentially transactivates the -promoter (Sunyer and Merlie 1993 ). These data and the fact that MRF4 expression overlaps with the formation of neuromuscular connections and is strongly associated with that of the -subunit gene (Rohwedel et al. 1998 ), have suggested that MRF4 might be responsible for the synaptic regulation of nAChR genes in adult muscle. However, the fact that MRF4 protein could not be detected in sole plate nuclei of innervated muscle, nor did it accumulate preferentially at the junction after denervation (Weis et al. 2000 ), does not favor this idea.

In the absence of a complete analysis of MRF protein accumulation in denervated regenerating muscle, it appears illusive to speculate about specific role for each of them. It should be emphasized that both muscle denervation and muscle regeneration cause muscle to exhibit many properties of fetal myotubes, which results in the transcriptional activation of nAChR genes in extrasynaptic nuclei (Koltgen and Franke 1994 ) and the re-expression of embryonic/fetal isoforms of contractile protein (Buonanno and Rosenthal 1996 ). This could account for the precocious accumulation of MRFs in the early stages of muscle regeneration. The fact that denervation leads to a rapid increase of MRF4 protein before MyoD and myogenin (Weis 1994 ; Weis et al. 2000 ) is consistent with our findings showing that MRF4 mRNA accumulates strongly before MyoD and myogenin transcripts in denervated regenerating muscle. In agreement with Weis et al. 2000 , we suggest that MRF4 may mediate early responses to denervation such as the quick upregulation of both embryonic-type and adult-type nAChR detected in regenerating muscle after denervation (Brenner et al. 1992 ). In denervated regenerating muscles, MyoD and myogenin might induce other muscle-specific genes than the nAChR genes, as previously suggested by Koishi et al. 1995 in the case of MyoD.

	Acknowledgments

Anne-Sophie Armand held a doctoral fellowship from the Ministère de l'Education Nationale, de la Recherche et de la Technologie (MENRT).

We thank Drs S. Tajbakhsh from M. Buckingham's laboratory and D. Daegelen for the cDNAs. We also thank Dr R. Cassada for helpful advice.

Received for publication April 25, 2000; accepted March 1, 2001.

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