ARTICLE |
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
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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 (23 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 49:887899, 2001)
Key Words: myogenic regulatory factors, MRF4, muscle regeneration, denervation
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Introduction |
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The four myogenic regulatory factors (MRFs) MyoD, Myf-5, myogenin, and MRF4 are basic helixloophelix 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 (
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 (
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 (
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 (
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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
Probe Preparation
The following probes were used to generate antisense cRNAs: MRF4 template is a fragment (BmsAI-XbaI; positions 853990) of rat MRF4 (, linearized using MLUI, and transcribed using T3 RNA polymerase. Myogenin is a 695-nt 3' fragment cloned in pBluescript M13-65-7 (
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 100150 nucleotides in length by limited alkaline hydrolysis according to
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.
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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 (
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At high magnification, analysis of Myf-5 transcript accumulation revealed a strong hybridization signal in the first stage of regeneration (23 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 1A1D). 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 23 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 312 initial hours after injury (
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 23 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.
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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).
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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 5D5G). 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).
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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.
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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 (
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.
It has been shown that newborn mice deficient for both MyoD and Myf-5 are totally devoid of skeletal myoblasts and muscle (
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 (
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 (
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 (-subunit is replaced by an adult
-subunit (
-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 (
-subunit gene (
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 (
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Acknowledgments |
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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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