ARTICLE

Expression and Neural Control of Follistatin VersusMyostatin Genes During Regeneration ofMouse SoleusAnne-Sophie Armand,1 Bruno Della Gaspera,1 Thierry Launay,1,2 Frederic Charbonnier,1,3 Claude L. Gallien,1

and Christophe Chanoine1*

Follistatin and myostatin are two secreted proteins involved in the control of muscle mass during development. Thesetwo proteins have opposite effects on muscle growth, as documented by genetic models. The aims of this work wereto analyze in mouse, by using in situ hybridization, the spatial and temporal expression patterns of follistatin andmyostatin mRNAs during soleus regeneration after cardiotoxin injury, and to investigate the influence of innervationon the accumulation of these two transcripts. Follistatin transcripts could be detected in activated satellite cells asearly as the first stages of regeneration and were transiently expressed in forming myotubes. In contrast, myostatinmRNAs accumulated persistently throughout the regeneration process as well as in adult control soleus. Denervationsignificantly affected both follistatin and myostatin transcript accumulation, but in opposite ways. Muscle denervationpersistently reduced the levels of myostatin transcripts as early as the young myotube stage, whereas the levels offollistatin mRNA were strongly increased in the small myotubes in the late stages of regeneration. These results arediscussed with regard to the potential functions of both follistatin, as a positive regulator of muscle differentiation,and myostatin, as a negative regulator of skeletal muscle growth. We suggest that the belated up-regulation of thefollistatin mRNA level in the small myotubes of the regenerating soleus as well as the down-regulation of themyostatin transcript level after denervation contribute to the differentiation process in denervated regeneratingmuscle. Developmental Dynamics 227:256–265, 2003. © 2003 Wiley-Liss, Inc.

Key words: follistatin; myostatin; muscle regeneration; denervation

Received 11 July 2002; Accepted 17 February 2003

INTRODUCTION

Follistatin, originally known as an ac-tivin-binding protein, and myostatin(or GDF-8), a member of the trans-forming growth factor beta (TGF-�)family of growth factors, have beenshown to be expressed in the devel-oping mouse myotome (Currie andIngham, 1998). Follistatin was shownto inhibit all aspects of bone mor-phogenetic protein (BMP) growth

factor activity in early Xenopus em-bryos. Follistatin can directly interact,at significantly high affinity, with mul-tiple BMPs, which are members ofthe TGF-� family (Iemura et al.,1998). During somitogenesis, BMP4 ishighly expressed in chick and mouselateral plate mesoderm (Pourquie etal., 1996). It is now established thatBMP4 represents the lateral plateactivity required to specify the lat-

eral somites and repress myogenesisin lateral dermomyotome cells. Fol-listatin-deficient mice develop anoverall reduction in skeletal musclemass, indicating that follistatin mayplay a role in the morphogenesis ofthe myotome (Matzuk et al., 1995).Amthor et al. (1996) suggested amodel whereby follistatin antago-nizes the BMP4-induced repressionof muscle cell fates in the develop-

1Biologie du Developpement et de la Differenciation Neuromusculaire, LNRS ESA 7060 CNRS, Universite Rene Descartes, Paris, France2UFR STAPS, Universite Rene Descartes, Paris, France3Departement STAPS, Universite d’Evry, Evry, France*Correspondence to: Christophe Chanoine, Laboratoire de Biologie du Developpement et de la Differenciation Neuromusculaire, CentreUniversitaire des Saints-Peres, Universite Rene Descartes, 45 rue des Saints-Peres, F-75720 Paris Cedex 06, France.E-mail: chanoine@biomedicale.univ-paris5.fr

DOI 10.1002/dvdy.10306

DEVELOPMENTAL DYNAMICS 227:256–265, 2003

© 2003 Wiley-Liss, Inc.

ing myotome. Myostatin acts to reg-ulate the morphogenesis of the skel-etal muscle mass (McPherron et al.,1997). Mice that are homozygous fora targeted disruption of the myosta-tin gene develop muscles that aretwo to three times the size of those oftheir wild-type littermates. This find-ing is essentially the opposite pheno-type to that seen in specific musclesof follistatin mutant mice (Matzuk etal., 1995). It has been suggested thatfollistatin may also regulate the ac-tivity of myostatin and control thelevel of division of forming myoblastsin specific locations in the myotome(Currie and Ingham, 1998). Thomaset al. (2000) showed that myostatinacts by inhibiting myoblast prolifera-tion. More recently, Lee andMcPherron (2001) clearly indicatedthat myostatin is negatively regu-lated by follistatin in vitro andstrongly suggested that follistatinblocks myostatin activity in vivo topromote muscle growth.

An important feature of matureskeletal muscles is their ability to re-generate after injury. Satellite cells,closely associated with muscle fi-bers, are myoblast-like cells respon-sible for the regenerative capacityof muscles (Mauro, 1961; Plaghki,1985). Normally, these adult musclestem cells are mitotically quiescent,but they are activated in responseto injury. The regenerative process ischaracterized by the proliferation ofthe descendants of the activatedsatellite cells, before fusing to formnew myotubes. As myostatin is a keynegative regulator of skeletal mus-cle growth, its expression during mus-cle regeneration has been analyzedin many studies (Kirk et al., 2000; Ya-manouchi et al., 2000). However,considering that follistatin could an-tagonize myostatin function, it is sur-prising that no results have been re-ported about the accumulation offollistatin mRNA during mammalianmuscle regeneration.

To remediate this deficiency, theaim of this work was to characterizeclearly, by using in situ hybridization,the spatial and temporal expressionpatterns of follistatin vs. myostatintranscripts during regeneration ofthe mouse soleus after cardiotoxininjury. Because of the potential in-volvement of these two secreted

molecules in the control of the levelof division of forming myoblasts, wealso analyzed the effects of dener-vation on the accumulation of bothfollistatin and myostatin transcriptsduring the regeneration process. In-deed, it is well known that innerva-tion plays a crucial role in myogen-esis and that denervation activatesthe satellite cells in adult muscles(Weis et al., 2000).

RESULTS

In this study, we performed cardio-toxin injury-induced regenerationexperiments on soleus muscle ofadult mice to investigate the influ-ence of innervation on the expres-sion of both follistatin and myostatinduring muscle regeneration. Animalswere divided into two separategroups: whereas in all animals, thesoleus muscle was injured by cardio-toxin injection, in half of them, thesoleus was also subjected to dener-vation before toxin injection and inthe other half innervation was leftintact. The accumulation of follista-tin and myostatin mRNA in bothgroups was then analyzed at differ-ent days postinjection (P-I) by in situhybridization.

The sequence of histologicchanges observed in regeneratingmouse muscle after snake toxin in-jury has been described previously(Couteaux et al., 1988; Launay et al.,2001). In our present experiments,from 2–3 days P-I many mononucle-ated cells were observed located atthe edges and between necroticmyofibers (Fig. 1). They probably cor-respond, at least in part, to prolifer-ating myoblasts arising from satellitecells, in accordance with previousreports (Grounds et al., 1992; Kami etal., 1995). At 5 days P-I, young myo-tubes with central nuclei were pre-dominantly observed (Fig. 2). Be-cause all studies were done on serialsections, there was no problem inverifying the multinuclear nature ofnewly formed myotubes. One-month P-I, the majority of the nucleiremained in a central position (Fig.3C), which is characteristic of regen-erating mouse muscles, as previouslydescribed (Couteaux et al., 1988).

Analysis of follistatin transcript ac-cumulation revealed a hybridization

signal in the first stage of regenera-tion (2 days P-I; Fig. 1A). At highmagnification, it appeared that fol-listatin transcripts accumulated insome mononucleated cells locatedeither at the edges of some necroticmyofibers or between them (Fig.1B,D). No positive signal was de-tected in the uninjured contralateralmuscle (data not shown). The major-ity of these follistatin-positive mono-nucleated cells also expressedMyoD transcripts (Fig. 1C), as shownin Table 1. As MyoD is known to be agood marker of myogenic cells (Lau-nay et al., 2001), we can assumethat these follistatin-positive mono-nucleated cells corresponded inpart to activated satellite cells andtheir descendant proliferating myo-blasts.

At 5 days P-I, a positive signal forfollistatin mRNA was detected in thesmallest multinucleated myotubes(Fig. 2A). At high magnification, itcould be more specifically seen thatfollistatin transcripts strongly accu-mulated in both the forming and thesmall newly formed myotubes,whereas the hybridization signalstrength drastically fell in the largermyotubes (Fig. 2B). This hybridizationsignal progressively decreased in thefollowing stages (Fig. 3A) and wasno longer detected in myofibers at30 days P-I (Fig. 3C).

At the beginning of the regenera-tion process (2 days P-I), a positivesignal for myostatin mRNA was de-tected in some mononucleate cellslocated between the necrotic myo-fibers (Fig. 4B,C). Low levels of myo-statin mRNA were also detected inthe necrotic myofibers (Fig. 4A,B),whereas a high level of myostatintranscripts was observed in the unin-jured contralateral soleus (Fig. 5A).From 5 days P-I, myostatin transcriptswere strongly expressed in the newlyformed multinucleated myotubes(Fig. 5B). Myostatin mRNAs were stillstrongly detected in subsequentstages of regeneration up to 30 daysP-I (Fig. 5C).

Denervation significantly regu-lated the follistatin and myostatintranscripts but in an opposite man-ner. Globally, follistatin mRNA accu-mulation was up-regulated by de-nervation during soleus regenerationand that of myostatin was down-

FOLLISTATIN VERSUS MYOSTATIN DURING REGENERATION 257

regulated. For myostatin, the effectof denervation was clearly observedat 5 days P-I, with a strong in situhybridization signal visible in theyoung myotubes of innervated mus-cle, whereas only a weak hybridiza-tion signal was seen in the smallmyotubes of denervated contralat-eral muscle (Fig. 5B,D). In the follow-ing stages of regeneration, at 14and 30 days P-I, the down-regulationof myostatin transcript accumula-tion by muscle denervation was stillobserved, but it was less drastic thanat 5 days P-I (Fig. 6). Quantificationof myostatin mRNA levels indicatedthat at 5 days P-I the level of myo-statin transcripts transiently de-

creased approximately threefoldcompared with innervated muscle(Fig. 6).

The response of muscle to dener-vation is more belated for follistatinthan for myostatin. No effect of mus-cle denervation was observed be-fore 15 days P-I, when the level offollistatin transcripts was up-regu-lated (Fig. 3A,B) more than threefoldcompared with innervated muscle(Fig. 6). Indeed, at this stage of re-generation, as well as at 30 days P-I,a strong hybridization signal for fol-listatin was detected in the smallmyotubes of denervated musclesbut not in innervated contralateralmuscles, where follistatin mRNAs

were not yet detected at 30 days P-I(Fig. 3C,D). It should be noted that,in these denervated soleus muscles,the follistatin transcripts accumu-lated more strongly in the smallestmyotubes compared with the largemyotubes, where the follistatin signalwas lower (Fig. 3D).

In the uninjured adult soleus, de-nervation also differentially regu-lated the accumulation of follistatinand myostatin transcripts, as previ-ously observed in regenerating mus-cle (Fig. 6). In these experiments, theadult soleus muscles were dener-vated and examined after 8 or 15days. Follistatin mRNA levels wereprogressively induced to approxi-

Fig. 1. In situ hybridization by using antisense riboprobes to follistatin (A,B,D) and MyoD (C) on transverse sections of regenerating soleusat 2 days postinjection. By using sense riboprobes, we did not detect hybridization signals (data not shown). Low-magnification (A) andhigh-magnification (B–D) brightfield photomicrographs are shown. Arrows in A indicate, at low-magnification, cells showing a positivesignal for follistatin transcripts. At high magnification (B,D), follistatin transcripts can clearly be seen to accumulate in some mononucle-ated cells expressing MyoD transcripts, located either at the edges of some necrotic myofibers (arrowheads) or between these myofibers(arrows). B and C show adjacent sections. Scale bar in D � 8 �m in A, 5 �m in B,C, 3 �m in D.

258 ARMAND ET AL.

mately two- to threefold in dener-vated muscle in comparison to con-tralateral muscle. Myostatin mRNAlevels, by contrast, were reducedapproximately twofold (Fig. 6).

DISCUSSION

This study provides a detailed spatialand temporal analysis of gene ex-pression for follistatin and myostatinduring a complete degenerationand regeneration process of mousesoleus. The opportunity is also ex-ploited to analyze the influence ofdenervation on the accumulation oftwo transcripts encoding proteinsthat have opposite effects on mus-cle growth. Indeed, as it well estab-lished that denervation involvesmuscle atrophy, it seems importantto better understand the expressionof follistatin and myostatin mRNAs indenervated regenerating muscleswith regard to potential functions offollistatin in muscle morphogenesisand of myostatin as a negative reg-ulator of skeletal muscle growth.

Expression of Follistatin vs.Myostatin During Regenerationof Soleus Muscle

Previous work analyzed the accu-mulation of myostatin mRNA (Ya-manouchi et al., 2000; Mendler etal., 2000) and protein (Sakuma et al.,2000; Kirk et al., 2000; Mendler et al.,2000) during muscle regeneration,but no data are available for follista-tin during the regeneration processin mammals. In birds, the in vitro workof Kocamis et al. (2001) analyzedthe expression of follistatin mRNA insatellite cells derived from chickenmuscles in the stages from initiationof proliferation to fusion.

In the present study, we foundthat, after cardiotoxin injection, themyostatin mRNA levels fell in the ne-crotic fibers before being up-regu-lated in the subsequent stages ofmuscle regeneration, whereas fol-listatin mRNA was transiently ex-pressed in forming myotubes. Wecannot assume that, at 2 days P-I, allmononucleate cells expressing fol-

listatin and/or myostatin transcriptswere myogenic cells. Yamanouchiet al. (2000) reported that myostatintranscripts are expressed during ratmuscle regeneration both in myo-genic and nonmyogenic mono-nucleate cells. It is also well estab-lished that follistatin is not muscle-specific and is widespread inmammalian tissues (Kogawa et al.,1991).

The general pattern of myostatinmRNA accumulation is in accor-dance with its role as an inhibitor ofmyoblast proliferation. A similar con-clusion was drawn by Yamanouchi etal. (2000) and by Mendler et al. (2000),in studies whose findings are globallyin line with ours. In the latter report,after notexin injection, analysis of themyostatin mRNA levels by reversetranscriptase-polymerase chain reac-tion (PCR) showed that they droppedbelow the detection limit on day 1 inrat soleus and then progressively in-creased on the following days (Men-dler et al., 2000). The same study re-ported that myoblasts proliferating in

Fig. 2. In situ hybridization using antisense riboprobes to follistatin on transverse sections of regenerating soleus at 5 days postinjection.Low-magnification (A) and high-magnification (B) brightfield photomicrographs are shown. Arrowhead in A indicates a small myotubeexpressing follistatin mRNA. Scale bar in B � 8 �m in A, 5 �m in B.

FOLLISTATIN VERSUS MYOSTATIN DURING REGENERATION 259

vitro produced a low level of myosta-tin mRNA, which increased upon in-duction of differentiation. Yamanou-chi et al. (2000), by using in situ

hybridization, observed mononucle-ated cells positive for myostatin mes-sage in the regenerating area of ratmuscle, 48 hr after bupivacaine injec-

tion. However, in contrast to other re-sults, including ours here, they did notdetect a positive signal for myostatinmRNA in intact skeletal muscle. By us-

Fig. 3. In situ hybridization using antisense riboprobes to follistatin on transverse sections of control (A,C) and denervated (B,D)regenerating soleus at 15 (A,B) and 30 (C,D) days postinjection. Note in D that the small myotubes of denervated muscle stronglyaccumulate follistatin mRNA. A and B are darkfield photomicrographs. C and D are brightfield. Scale bar � 8 �m in D (applies to A–D).

260 ARMAND ET AL.

ing Western blot analysis, Sakuma etal. (2000) indicated that, in the rat, themyostatin protein level rapidly de-creased in regenerating tibialis ante-rior muscle subjected to bupivacaineinjection and was restored by 28 daysP-I. After notexin injection, Kirk et al.(2000), by using immunohistochemis-try, obtained similar results that regen-erating myotubes of rat contained nomyostatin at the time of the fusionand, during subsequent myotube en-largement, low levels of myostatinwere observed.

The expression pattern of follistatinmRNA during soleus regeneration isconsistent with its observed accu-mulation during myogenesis of satel-lite cells in vitro (Kocamis et al.,2001). In both cases, a transient ex-pression was observed, character-ized by a peak of follistatin mRNAlevels in forming myotubes. Follistatincan directly interact with multiplemembers of the TGF-� family, at sig-nificantly high affinity (Iemura et al.,1998). Activins inhibited pectoralmuscle cell differentiation in culture,whereas follistatin stimulated thisprocess (Link and Nishi, 1998). It hasbeen suggested that the overall ac-tivin-b and follistatin mRNA expres-sion patterns found in satellite cells(i.e., both increase when fusionstarts) may contribute to the inhibi-tory functions of follistatin on activins(Michel et al., 1993). Considering thenegative regulation of myostatin byfollistatin in vitro (Lee and McPher-ron, 2001) and the expression pat-tern of both myostatin and follistatinmRNA during soleus regeneration,we suggest that follistatin may inter-act with myostatin or other TGF-� su-perfamily members, such as bone

morphogenetic proteins or activins,to participate in myotube enlarge-ment. It should be noted that follista-tin is not only an antagonist but isable, at certain concentrations, toact as a cofactor promoting bindingof TGF-� members, such as BMP-7and BMP-2, to their receptor(Amthor et al., 2002). This finding sug-gests that follistatin also acts topresent BMPs to myogenic cells at aconcentration that permits stimula-tion of embryonic muscle growth.

Denervation OppositelyRegulates the Accumulation ofFollistatin and MyostatinTranscripts During SoleusRegeneration

It is well established that denervationof adult muscle alters within days theexpression levels of numerous struc-tural proteins, transcription factors,and trophic factors (d’Albis et al.,1988; Hughes et al., 1993; Adams etal., 1995), resulting in muscle fibersthat show many characteristics ofembryonic fibers. Morphologic alter-ations such as atrophy of muscle fi-bers are also observed (Couteaux etal., 1988), as well as the proliferationof satellite cells (Weis et al., 2000).

Our findings showed that dener-vation significantly regulated bothfollistatin and myostatin transcripts,but in an opposite manner, duringmuscle regeneration as well as inadult soleus. To our knowledge, nodata have been published on theeffect of muscle denervation on fol-listatin expression, but one report an-alyzed the influence of innervationon myostatin expression during mus-cle regeneration (Sakuma et al.,2000). The down-regulation of myo-statin in regenerating mouse soleusby denervation observed in our workis consistent with the findings of Sa-kuma et al. (2000), who observed agradual diminution of myostatin pro-tein in denervated soleus of adultrat. However, the basis for the rela-tively large variation in the effect ofdenervation on the accumulation ofmyostatin mRNA observed depend-ing on the stage of regeneration(see Fig. 6) remains unclear andshould be clarified in further experi-ments. Mendler et al. (2000) showed

that the increase of myostatin tran-script levels during soleus regenera-tion coincided with the time of rein-nervation, and it decreased whenthe new endplates were estab-lished. These findings are consistentwith the fact that myostatin geneexpression is under neuronal control.However, the relationship betweenthe myostatin mRNA level and inner-vation is not likely to be causative,because in cell cultures in vitro themRNA levels also changed despitethe absence of neural influence(Mendler et al., 2000).

Myostatin-null mice show a dra-matic and widespread increase inskeletal muscle mass due to an in-crease in the number of muscle fi-bers (hyperplasia) and their thick-ness (hypertrophy) (McPherron etal., 1997). Denervation of skeletalmuscle induces activation and pro-liferation of satellite cells (Weis et al.,2000) as well as hyperplasia phe-nomena (Sola et al., 1973; Jimena etal., 1993). Taking into account therole of myostatin in the control ofmuscle mass based on the geneticmodel, we suggest that the de-crease in myostatin transcripts in de-nervated regenerating soleus couldregulate the hyperplasia associatedwith muscle denervation. We couldconsider the expression pattern ofboth follistatin and myostatin tran-scripts induced by denervation as agene dosage compensation oppos-ing muscle atrophy and as being in-volved in the acquisition and/ormaintenance of differentiated mus-cle phenotype. It is well establishedthat in denervated regeneratingmuscle, the expression of structuralmuscle genes occurs, giving rise todifferentiated atrophied myofibers(d’Albis et al., 1988). Recently, Rioset al. (2002) proposed that myostatinnegatively regulates muscle massnot only by decreasing the prolifer-ation rate of myoblasts but also byinhibiting terminal muscle differenti-ation. However, in the absence of acomplete analysis of follistatin andmyostatin proteins in denervated re-generating muscle, it appears illu-sive to speculate about specificroles for each of them.

Considering the role of follistatin inmuscle differentiation demonstratedby in vitro studies (Link and Nishi,

TABLE 1. Percentage ofMononucleated Cells

Accumulating Follistatin and/orMyoD Transcripts in Regenerating

Soleus 2 Days Postinjectiona

Follistatin 17.7MyoD 5.3Follistatin and MyoD 59.7

aThe number of cells counted was1000, using serial sections (seeExperimental Procedures section).

FOLLISTATIN VERSUS MYOSTATIN DURING REGENERATION 261

1998) and analysis of follistatin (-/-)mice, it could be suggested that thebelated up-regulation of follistatinmRNA level in the small myotubes ofthe regenerating soleus as well asthe down-regulation of myostatintranscript levels regulate the differ-entiation process in denervated re-generating muscle.

EXPERIMENTAL PROCEDURES

Animals and Muscle Injury

Studies were carried out with adultfemale mice Mus musculus Swiss(approximately 30 g) originating

from the breeding center R. Janvier(France). Animals were anesthetizedby intraperitoneal injection of 3.5%chloral hydrate. The skin was cut andpure cardiotoxin from Naja Mossam-bica nigricollis venom (Latoxan,France) (10�5 M in 0.9% NaCl) wasinjected into soleus muscle. Four an-imals were analyzed for each stageof muscle regeneration.

Denervation

Before cardiotoxin injury, the soleusmuscle was denervated as follows.A double proximal ligature and adouble distal ligature with silk thread,

separated by 3 mm, were per-formed on the tibial nerve, innervat-ing several muscles including the so-leus muscle, which was then cutbetween these ligatures. Five ani-mals were analyzed for each stageof muscle regeneration. We alsoperformed denervation of adult so-leus without cardiotoxin injury.

Preparation and Prehybridationof Tissue Sections

The procedure for fixing, embed-ding, and sectioning tissues was es-sentially the same as that describedby Wilkinson et al. (1987). Briefly, tis-sues were fixed in 4% paraformalde-hyde in PBS, dehydrated, and infil-trated with paraffin. Then 7-�m-thickserial sections were mounted onTESPA-coated RNase-free glassslides. Sections were deparaffinizedin xylene, digested with proteinaseK, post-fixed, treated with dithiothre-itol/iodoacetamine/N-ethylmaleim-ide (to reduce nonspecific 35S bind-ing; Zeller and Rogers, 1989), treatedwith triethanolamine/acetic anhy-dride, washed, and dehydrated.

Probe Preparation

The following probes were used togenerate antisense cRNAs: totalRNA from muscle tissue was pre-pared by using the RNeasy kit (QIA-GEN, Inc., Valencia, CA) and re-verse transcription was performedusing the Omniscript RT kit (QIAGEN).

Myostatin was amplified by PCR us-ing the following primers: F (Forward)5�-TAGCAGATTCAATAGTGGTC-3�and R (Reverse) 5�-ATTGAAATTT-GACTGGGAGC-3�, producing a143-bp product (positions 2278-2420)from the 3� end of the myostatincDNA. This fragment was cloned inpGEM-T (Promega Biotec, Madison,WI), cut with SalI, and transcribedusing T7 RNA polymerase.

Follistatin was amplified by PCR us-ing the following primers: F (Forward)5�-AGTTCATGGAGGACCGCAGC-3�and R (Reverse) 5�-AATCCGATTACA-GGTCACAC-3�, producing a 528-bpproduct (positions 59-585) from the 5�-end of the follistatin cDNA. This frag-ment was cloned in pGEM-T (Pro-mega Biotec), cut with NcoI, andtranscribed by using Sp6 RNA poly-merase.

Fig. 4. In situ hybridization by using sense (A) and antisense (B,C) riboprobes to myostatin ontransverse sections of regenerating soleus at 2 days postinjection. Low-magnification (A,B) andhigh-magnification (C) brightfield photomicrographs are shown. Arrowheads indicate mono-nucleate cells accumulating myostatin mRNA. Scale bar in C � 5 �m in A,B, 3 �m in C.

262 ARMAND ET AL.

Fig. 5. Myostatin mRNA localization in adult and regenerating soleus muscles. In situ hybridization using antisense riboprobes tomyostatin on transverse sections of control adult muscle (A) and of innervated (B,C) and denervated (D) regenerating soleus at 5 (B,D)and 30 (C) days postinjection. Scale bar � 8 �m in D (applies to A–D).

FOLLISTATIN VERSUS MYOSTATIN DURING REGENERATION 263

MyoD template is a fragment (po-sitions 750-1785) of MyoD1 (Davis etal., 1987), cloned in pVZCII�, linear-ized by using MLUI, and transcribedby using T3 RNA polymerase. All nu-cleotide sequences were checkedby using the dideoxynucleotidechain-termination method.

cRNA probes were made by invitro transcription in the presence of50 �Ci [35S]UTP at 1,200 Ci/mmol(NEN Research Products) accordingto the manufacturer’s instructions(Promega Biotec). Unlabeled UTPwas omitted from the reaction me-dium to obtain RNA probes with aspecific activity of 109 cpm/�g.Probes were hydrolyzed to an aver-age of 100–150 nucleotides in lengthby limited alkaline hydrolysis accord-ing to Cox et al. (1984) for efficienthybridization and used at 50,000cpm/�L hybridization solution.

Hybridization and WashingProcedures

High-stringency conditions for hy-bridization and posthybridizationwere used. Sections were hybridizedovernight at 53°C with posthybridiza-tion washing in 2� standard salinecitrate, 50% formamide, 50 mM di-thiothreitol at 65�C for 30 min. Auto-radiography was carried out withKodak NTB-2 track emulsion, devel-oped in Kodak D19 developer, andstained lightly with Giemsa.

Quantitative Analysis

Hybridization signals were analyzedby using a specific minicomputer-ized densitometric program devel-oped for use with the Visilog 4.15 im-age analysis software (Noesis,Saclay, France), consisting of anIMC 500 camera and an IMC 500digitizer. Briefly, images were con-verted to a gray scale, and quanti-fication of staining was carried outby recording the value of light inten-sity measured by the program ineach considered cell. Results werereported as light intensity per surfacearea. Four sections of each stageoriginating from three to five distinctexperimental muscles were ana-lyzed. Each quantification was re-peated three times for the samesection with comparable results.

The number of follistatin- and/orMyoD-positive mononucleated cellsdetected at 2 days P-I was alsocounted by using a computer-as-sisted image analysis system Visilog4.15 (Noesis; Les Ulis, France).Briefly, three serial sections wereanalyzed. One was probed withthe antisense follistatin RNA probe,the following was probed with theantisense MyoD RNA probe, andthe remaining section was probedwith the sense MyoD probe. Sec-tions were stained with Giemsaand 1,000 mononucleated cellswere counted. The identificationand counting of positive nuclei forfollistatin and/or for MyoD was car-ried out at high magnification andexpressed as percentage of the to-tal nuclei.

Statistical Analyses

The data are presented as means �SD. Four sections of each stage orig-inating from three to five distinct ex-perimental muscles were analyzed.Statistical comparisons betweengroups were assessed by using Stu-dent’s t-test, with probabilities of lessthan 0.01 considered as being signif-icant.

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Fig. 6. Quantitative analysis of myostatin and follistatin expression at different stages ofregeneration (from 2 to 30 days postinjection) in innervated and denervated soleus. C,Control adult muscle. Adult soleus were also denervated and examined after 8 days (D8)and 15 days (D15). Error bars represent standard deviation for the four sections of the threeto five experimental muscles (see Experimental Procedures section). Values are means �SD. *Significantly different from innervated regenerating muscle value (P � 0.01). **Signif-icantly different from innervated control adult muscle value (P � 0.01).

264 ARMAND ET AL.

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