691Journal of Cell Science 111, 691-700 (1998)Printed in Great Britain © The Company of Biologists Limited 1998JCS9708

Myogenic conversion of NIH3T3 cells by exogenous MyoD family members:

dissociation of terminal differentiation from myotube formation

Simona Russo 1,*, Daniela Tomatis 2,*, Ginetta Collo 3, Guido Tarone 4 and Franco Tatò 1,†

1Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Roma ‘LaSapienza’, Italy2Dipartimento di Genetica, Biologia e Chimica Medica, Università di Torino, Italy3Glaxo Institute for Molecular Biology, Geneva, Switzerland4Dipartimento di Psicologia, Università di Roma ‘La Sapienza’, Italy*The first two authors equally contributed to the work†Author for correspondence (e-mail: tato@axcasp.caspur.it)

Accepted 7 January 1998: published on WWW 23 February 1998

Myogenic regulatory factors (MRF) of the MyoD familyregulate the skeletal muscle differentiation program. Non-muscle cells transfected with exogenous MRF either areconverted to the myogenic lineage or fail to express themuscle phenotype, depending on the cell type analysed. Wereport here that MRF-induced myogenic conversion ofNIH3T3 cells results in an incomplete reprogramming ofthese cells. Transfected cells withdrew from the cell cycleand underwent biochemical differentiation but,surprisingly, terminally differentiated myocytes absolutelyfailed to fuse into multinucleated myotubes. Analysis ofmuscle regulatory and structural gene expression failed to

provide an explanation for the fusion defectiveness.However, myogenic derivatives of NIH3T3 cells were shownto be unable to accumulate the transcripts encodingmuscle-specific isoforms of the integrin subunit β1D andthe transcription factor MEF2D1b2, that depend onmuscle-specific alternative splicing. Our results suggestthat the fusion into myotubes is under a distinct geneticcontrol that might depend, at least partially, on differentialsplicing.

Key words: Myogenesis, Fusion, Differential splicing

SUMMARY

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INTRODUCTION

In recent years, the use of established cell lines has allowedmolecular understanding of how muscle specific genes turned on by the concerted action of muscle transcriptifactors belonging to the MyoD and the MEF2 families. Thfour known muscle regulatory factors (MRF) of the MyoDfamily (MyoD, myogenin, MRF4 and Myf5) are basic helixloop-helix (bHLH) transcription factors thought to activatmuscle gene expression by binding to a specific DNsequence, called E-box, after heterodimerization wubiquitously expressed E2A gene products such as E12/E4(Weintraub et al., 1991; Li and Olson, 1992). Moreover, whexpression vectors encoding a single MRF are introduced ia variety of non-muscle cell types, any MRF can lead to tactivation of the muscle differentiation program (Davis et a1987; Choi et al., 1990; Auradè et al., 1994). The MEF(myocyte-specific enhancer binding factor 2) proteins, anothclass of transcription factors that belongs to the MADsuperfamily (reviewed by Olson et al., 1995), are thought toimportant in transactivating muscle promoters not containiE-boxes as well as in cooperating with MyoD family membe(Molkentin et al., 1995). During myogenesis, sequentiactivation of individual myogenic factor genes suggests th

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these genes form a regulatory hierarchy and that particuproteins perform distinct functions (reviewed by Lyons anBuckingam, 1992). Knock-out experiments in micedemonstrated that MyoD and Myf5 play partially overlappinroles in the determination of muscle precursor cells, wheremyogenin appears to be essential for muscle cell differentiati(reviewed by Ludolph and Konieczny, 1995). In addition totranscriptional activation, alternative RNA splicing alsocontributes to developmentally regulated gene expressi(Smith et al., 1989). Alternative splicing of muscle-specifiexons is associated with several transcripts expresseddifferentiating muscle cells (Nadal-Ginard, 1990).

The skeletal muscle differentiation program usually involvepermanent withdrawal from the cell cycle (also referred to acommitment), biochemical differentiation and fusion intomyotubes (Okazaki and Holtzer, 1966; Nadal-Ginard, 1978Two cell systems are known where myoblast fusion genetically dissociated from biochemical differentiation: thBC3H1 cell line, capable of differentiating and expressinmany skeletal muscle-specific proteins, but not of forminmultinucleated myotubes (Shubert et al., 1974) and C2Ccells transformed by the v-myc oncogene, where fusion intomyotubes is severely inhibited, although the mononucleatmyocytes are indistinguishable from control myotubes in term

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of muscle-specific gene expression (Crescenzi et al., 19BC3H1 cells do, however, recover the ability to fuse whexogenous MyoD or MRF4 are forcibly expressed in thecells (Brennan et al., 1990; Block and Miller, 1992) and v-myc-transformed C2C12 cells can undergo phenotypic reversiomixed culture with normal fibroblasts, recovering the ability fuse into multinucleated myotubes (Crescenzi et al., 1994)previously shown for v-myc-transformed quail myoblasts (LaRocca et al., 1989).

Formation of multinucleated myotubes is thought to procethrough the induction of differentiation in myoblast cellfollowed by the aggregation and fusion of their plasmmembranes. Four major classes of molecules seem to mecell-cell and cell-matrix interactions during myogenesicadherins, N-CAM, integrins (reviewed by McDonald et a1995) and disintegrins (Yagami-Hiromasa et al., 199However, the molecular mechanisms of muscle cell fusion still poorly understood.

In this paper we show that myogenic conversion of NIH3Tcells by MyoD family members is incompletely achieved, afusion into myotubes is dissociated from commitment abiochemical differentiation. In spite of this defectivdifferentiation phenotype, transcriptional activation oendogenous muscle regulatory and structural genes was similar to that of fusion-competent myogenic cell lines. Wreport here that the inability to fuse was accompanied byextremely low competence for the differential splicing requirto generate the muscle-specific transcripts for the β1D integrinsubunit (Belkin et al., 1996) and for the transcription factMEF2-D (Martin et al., 1994). Thus, MRF-converted NIH3Tcells appear to represent an attractive system to study speaspects of differential splicing regulation as well as hodifferentiated muscle cells fuse with each other.

MATERIALS AND METHODS

Cell culture and transfectionsNIH3T3 cells were kindly provided by S. Aaronson, NIH, BethesdUSA; C3H10T1/2 cells were obtained from V. Sorrentino, DIBITMilano, Italy; C2C12 cells were provided by S. Alemà, IBC, RomItaly; L8 cells were a generous gift from D. Yaffe, Weizmann InstituRehovot, Israel.

Cells for stable transfections were seeded at 1.5×105 on 60 mmdishes and transfected using the Transfectam Reagent (Promega7 hours with 5 µg of DNA. The cDNAs for rat MRF4 (Rhodes andKonieczny, 1989), mouse MyoD (Davis et al., 1987), rat myogen(Wright et al., 1989) and human Myf5 (Braun et al., 1989a) wedriven by the Moloney murine sarcoma virus long terminal rep(MoMSV-LTR) in the expression vector pEMSV-scribe. For selectiocells were cotransfected with the pSV2-Hygro expression veccarrying the gene for hygromycin resistance. 24-48 hours lahygromycin was added to a final concentration of 0.3 mg/ml astably transfected myogenic clonal populations were obtained wittwo weeks the selection procedure.

Growth medium (GM) for NIH(MRF) cells was Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 10% caserum; C3H(MRF4) and C2C12 cells were cultivated in DMEsupplemented with 20% fetal bovine serum; L8 cells were cultivain DMEM supplemented with 10% fetal bovine serum.

To assay myogenic differentiation, 2×105 cells were plated on 35mm dishes in GM. 24 hours later, cultures were fed wdifferentiation medium (DM) represented by DMEM supplementwith 10 µg/ml insulin and 5 µg/ml transferrin for three days.

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The proliferation rate of growing and differentiated NIH(MRFcells was assayed by measuring the percentage of nuclei whincorporated bromo-deoxyuridine (BrdU). 1.5×105 cells of each celltype were plated in duplicate on 35 mm dishes in GM and 24 holater were fed with GM or shifted in DM for 48 hours; cells were thefed with GM or DM containing 20 µM BrdU and allowed toincorporate the analogue for 24 hours.

Mixed cultures of proliferating NIH(MRF) and L8 cells were seup by co-seeding the two cell types in different ratios, ranging fro10:1 to 1:100. 24 hours later, GM was replaced with DM and fodays later cultures were stained for fluorescent detection of tropoT (TNT) and DNA as described below. All experiments werperformed on collagen-coated dishes.

ImmunofluorescenceA mouse monoclonal antibody that recognizes the skeletal mustroponin T (TNT) (Amersham) was used to assess termindifferentiation. Cells were fixed in 3% paraformaldehyde for 1minutes and permeabilized with 0.25% Triton X-100. The primaantibody anti-TNT diluted 1:500, was added for 30 minutes at 37°After washing with PBS, a rhodamine-conjugated goat anti-mouantibody (Cappel) was added for 30 minutes at 37°C.

Cultures labelled with BrdU were fixed for 20 minutes with 95%ethanol/5% acetic acid, treated for 10 minutes with 1.5 M HCl, rinsthree times with PBS/Triton and stained with anti-BrdU mA(Amersham) for 30 minutes at room temperature (RT). After washiwith PBS, a rhodamine-conjugated goat anti-mouse antibody (Capwas added for 30 minutes at RT. When the cells were double staifor BrdU and myosin heavy chain (MHC), we used a polyclonantiserum that recognizes the sketetal muscle MHC (kindly providby G. Salvatori, Istituto di Istologia, Roma, Italy) and positive celwere visualized with a fluorescein-conjugated goat anti-rabantibody (Cappel). Nuclei were stained with 1 µg/ml Hoechst 33258dye (Sigma).

mRNA analysisTotal RNA was prepared by lysing the cells with 1% SDS, 10 mTris-Cl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA. Followingcentrifugation for 1 hour at 100,000 g at 10°C, the supernatant wasextensively extracted with acidic phenol and cloroform and ethanprecipitated. For northern blot analysis 25 µg of RNA were resolvedon a 0.8% agarose-2.2 M formaldeyde gel and transferred nitrocellulose membrane. High-stringency hybridization anwashings were carried out according to standard procedures. detection of muscle-specific and constitutive transcripts, inserts wexcised with the appropriate restriction enzymes from the followiplasmids: pEMSV-MRF4, containing a 1.2 kb cDNA fragmenencoding the rat MRF4 (obtained by V. Sorrentino); pEMC11containing a 1.2 kb cDNA fragment encoding the murine Myo(kindly provided by the late H. Weintraub); pEMSV-myogenincontaining a 1.5 kb cDNA fragment encoding the rat myogen(obtained by V. Sorrentino); pBS/KS-Myf5, containing a 1.2 kcDNA fragment of the murine Myf5 (kindly provided by E. BoberHamburg University, Germany); pV2-E12, containing a 1.4 kb cDNfragment encoding the murine E12 (obtained by V. SorrentinopMCK-m36, containing a 1.3 kb cDNA fragment of the murine MCK(obtained from M. Bouché, Istituto di Istologia, Roma, Italy); pMHC25, containing a cDNA fragment of rat skeletal muscle MHC (kindprovided by D. A. Fischman, Cornell University, New York, USA)pBSI-M-cadherin, containing a 800 bp cDNA fragment of the murinM-cadherin (kindly provided by A. Starzinski-Powitz, KolnUniversity, Cologne, Germany); pBATMNC, containing a 2.2 kcDNA fragment of the N-cadherin coding region (kindly providefrom M. Takeichi, Kyoto University, Japan); pM1.3, containing a 60bp cDNA fragment of the murine N-CAM (obtained from ASalvatori, IBC, Roma, Italy); a plasmid containing a 1.2 kb cDN

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For analysis of meltrins α, β and γ expression, 2 µg of total RNAwere reverse-transcribed for 1 hour at 42°C using specific 3′-primers,named, respectively, α1, β1 and γ1. For analysis of β1 integrin,MEF2-D and β-TM splicing, 2 µg of total RNA were reverse-transcribed for 1 hour at 37°C using 18-base poly (dT) oligomerprimer and 100 units of M-MLV reverse trancriptase (BRL). cDNwas analysed by polymerase chain reaction (PCR) using the followprimers:

α1 5′-ATTTTCCCACACTTGGCATCTCTCAG-3′ (nt: 1,962-1,937);α2 5′-CACATTTGGCGAAGGCGCTCG-3′ (nt: 1,914-1,935);α3 5′-GAGTGTGACTGCGGAGAACCGGAGGAATG-3′ (nt:

1,523-1,551);β1 5′-GGGACTGCACTTCCTCTATTGGCCATTC-3′ (nt: 1,764-

1,737);β2 5′-AGTTTCCATAGGTGTCACCAG-3′ (nt: 1,705-1,726);β3 5′-GAGGAGGAATGTAAGAACCCTTGC-3′ (nt: 1,368-1,391);γ1 5′-TCCCTGAATGTCACAGTCATAATTC-3′ (nt: 1,988-2,012);γ2 5′-GACAGAAGCATTTACACACTG-3′ (nt: 1,965-1,986);γ3 5′-GTGGACCCATGCTGTGAAGGAAGC-3′ (nt: 1,380-1,403);B73 5′-TTGCCTTGCTGCTGATTTTGG-3′;B72 5′-AAAATCCGCCTGAGTAGG-3′; B98 5′-AAACTCAGAGACCAGCTTTAC-3′; T10 5′-TGAGAGCGAGAGAGGAATG-3′;T12 5′-TGTCATTGAGTGCGTTGTC-3′;M1 5′-GGACTCACTGGAGCAGAGC-3′;M2 5′-ACGACAGGGAGTGTGAGAG-3′;M3 5′-AAGGGATGATGTCACCAGG-3′;A1 5′-TCCTGCGTCTGGACCTGG-3′;A2 5′-CCATCTCTTGCTCGAAGT-3′;R5 5′-CTGAAGAGAGAGCCGAGGTGG-3′;R8 5′-GCAAACTCTGCTCGGGTCTCAGC-3′.Primers α2 and α3 generate a 420 bp cDNA fragment of meltri

α; primers β2 and β3 amplify a 370 bp cDNA fragment of meltrin βand primers γ2 and γ3 generate a 610 bp cDNA fragment of meltriγ. Amplification conditions for meltrins were as follows: denaturatioat 95°C for 1 minute, annealing at 55°C for 2 minutes and extensat 72°C for 3 minutes (30 cycles). Primers B73 and B72 are locaon the sixth exon and on the 3′ non coding region of the mouse β1integrin gene that are common to the β1A and β1D transcripts (Belkinet al., 1996). These primers generate a fragment of 188 bp on theβ1AcDNA and a fragment of 269 bp on the β1D cDNA. Primer B98 islocated on the β1D specific exon and when used with B73 generaa β1D specific cDNA fragment of 172 bp. Primers T10 and T12 alocated on exon 3 and on exon 9a, respectively, of the mouse β-TMgene (Wang and Rubenstein, 1992). Primer M3 is located on region (codon 167 to 173) immediately 3′ of the alternative exons 1aor 1b of the mouse MEF2-D gene (Martin et al., 1994). Primer M1located on exon 1a and generates with M3 a specific fragment ofbp (MEF2D1a2). Primer M2 is located on the muscle-specific ex1b and generates with M3 a fragment of 246 bp (MEF2D1bAmplification conditions were as follows: the annealing temperatof the reaction was decreased by 2°C every second cycle from 6to a touchdown at 50°C, at which temperature 20 cycles were carout. Denaturation was carried out at 90°C, extension at 72°C. PrimA1 and A2 are located on the mouse β-actin cDNA. Amplificationconditions for β-actin were 90°C for 1 minute followed by 2 cycleat 65°C for 30 seconds, 72°C for 30 seconds and by 18 cycles at 5for 30 seconds and 72°C for 30 seconds.

Primers R5 and R8 are located on exon 5 and exon 8, respectiof the mouse β-TM gene. Amplification conditions were as followsthe annealing temperature of the reaction was decreased by 2°C second cycle from 75°C to a touchdown at 65°C, at which tempera20 cycles were carried out. Denaturation was carried out at 90extension at 72°C. R5 and R8 amplify a fragment of 189 bp containeither exon 6a or exon 6b. A PvuII site is located only on exon 6a

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Western blottingCells were lysed on ice in RIPA buffer (150 mM NaCl, 1% NP400.5% DOC, 0.1% SDS, 50 mM Tris-Cl, pH 7.5) containing freshladded protease inhibitors (1 mM PMSF, 10 µg/ml leupeptin, 4 µg/mlpepstatin and 5 µg/ml aprotinin). Lysates were clarified by 10 minutescentrifugation at 12,000 g at 4°C and protein concentration wasdetermined by Bio-Rad protein assay reagent (Bio-Rad Laboratorie

For Immunoblot analysis of MEF2-A and MEF2-D proteins 20 µgof total cell lysates were resolved on 8% SDS-PAGE and transferonto a nitrocellulose filter. After blocking non-specific reactivity with5% dry skimmed milk dissolved in TBS-T (20 mM Tris-Cl, pH 7.5150 mM NaCl, 0.02% Tween-20), filters were probed overnight at 4°with polyclonal antibodies against MEF2-A or MEF2-D (Han anPrywes, 1995) diluted 1/1,000 in 5% dry skimmed milk in TBS-T. Foimmunoblot analysis of α7 isoforms 40 µg of total extracts wereresolved on 6% SDS-PAGE. After blocking non-specific binding wit5% BSA dissolved in TBS-T, filters were probed overnight at 4°with polyclonal antibodies against α7B and α7A diluted 1/1,000 in1% BSA in TBS-T. Rabbit polyclonal antisera for α7A and α7Bisoforms were raised against the following synthetic peptidecorresponding, respectively, to the last 21 or 23 amino acids of mouse α7A and α7B proteins:

CGTVGWDSSSGRSTPRPPCPST; CHPILAADWHPELGPDGHPVPATA.A cysteine residue was added at the NH2 for coupling to maleimi

activated keyhole limpet hemocyanin according to the manufactureinstructions (Pierce, Rockford, Illinois) and rabbits were immunizewith the conjugate. The α7B-specific antiserum was previouslycharacterized (Velling et al., 1996). The specificity of the anti-α7Aserum was proved by western blotting and immunoprecipitation extracts from CHO cells transfected with α7A cDNAs and on C2C12cells at different stages of differentiation:

After extensive washing, immune complexes were detected whorseradish peroxidase-conjugated goat anti-rabbit secondantiserum (Bio-Rad Laboratories) followed by enhancechemiluminescence reaction (Amersham).

RESULTS

MRF-converted NIH3T3 cells display a fusion-defective phenotypeWe studied the influence of the myogenic regulatory facto(MRF) MRF4, MyoD, myogenin and Myf5 on thedifferentiation potential of the murine cell line NIH3T3. Cellswere cotransfected with a vector conferring hygromyciresistance and a vector carrying the cDNAs encoding eithMRF4 (Rhodes and Konieczny, 1989), MyoD (Davis et al1987), myogenin (Wright et al., 1989) and Myf5 (Braun et a1989a), under the control of the MoMSV-LTR. High myogeniconversion frequency was observed and stably transfecmyogenic clonal populations were obtained within two weekfollowing the selection procedure. We chose for furtheanalysis a representative clone for each MRF and wcollectively refer to these myogenic populations as NIH(MRFcells or, specifically, NIH(MRF4) cells for the NIH3T3 cellstransfected with the MRF4 cDNA and similarly NIH(MyoD),NIH(myogenin) and NIH(Myf5) cells. We also generatedmyogenic derivatives of the murine cell line C3H10T1/2 fo

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Table 1. Competence for terminal differentiation, fusion-index and rate of entry into S phase of myogenic

derivatives of NIH3T3 or C3H10T1/2 cells in proliferatingand differentiating conditions

% TNT+ % Fusion % BrdU+

indexGM DM DM GM DM

C3H(MRF4) 4 55 60 82 30NIH(MRF4) 0.4 50 <0.1 90 29NIH(MyoD) <0.1 30 <0.1 85 32NIH(mgn) <0.1 32 <0.1 88 30NIH(Myf5) 0.5 60 <0.1 89 25

Fusion index refers to the percentage of nuclei in MHC+ cells that were inmyotubes containing three or more nuclei. The experiment was repeatedseveral times, using different cell plating densities. The values represent datafrom a representative experiment that included the northern blot analysisshown in Fig. 2.

GM, growing medium; DM, differentiation medium; mgn, myogenin.

comparison with NIH(MRF) cells; the MRF4-converted clonapopulation of C3H10T1/2 cells is referred to as C3H(MRFcells.

NIH(MRF) cells were characterized in terms odifferentiative potential: terminal differentiation was induceby feeding the cells with a serum-free medium (referred toDM) for three days and biochemical differentiation wadetermined by immunofluorescence staining for troponin(TNT), a muscle-specific marker. NIH(MRF) cells underwebiochemical differentiation with high efficiency (see Table 1but, surprisingly, myotube formation could not be observed athe differentiated cells remained strictly mononucleated (sFig. 1A and Table 1); the inability to fuse into myotubeappeared to be independent from the efficiency to terminadifferentiate. The fusion-defective phenotype appeared tovery stable and was independent from the type of substrdifferentiation medium formulation and cell density. Myotubewere observed neither in the primary clones on the origitransfection dishes nor in the 12 clones expanded into lines, from which the clonal strains here described weselected. So far, we never detected myotubes containing th

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Fig. 1. Fusion-defective phenotype of NIH(MRF4) cells and itscorrection by cocultivation of NIH(MRF4) and L8 cells. NIH(MRF4cells [2×105 ] cultivated alone (A) or seeded together with L8 ratmyoblasts (B) [105 L8 cells+104 NIH(MRF4) cells] were induced todifferentiate in DM for four days. Cells were then stained byimmunofluorescence for TNT detection and with Hoechst 33258 dto distinguish mouse (speckled) from rat (homogenous) nuclei. Noin A the absence of fusion in differentiated (TNT+) NIH(MRF4) cellsand in B the presence of chimeric myotubes containing mouse anrat nuclei. Arrowheads show mouse nuclei in hybrid myotubes. Ba50 µm.

cellrereeor more nuclei in cultures of differentiated NIH(MRF) celland we estimate that the frequency of myotube formationthese cells is in the order of 10−6 or less.

Biological properties of NIH(MRF) cellsWe asked whether NIH(MRF) cells could fully undergcommitment to irreversible withdrawal from the cell cycleNIH(MRF4) or NIH(Myf5) cells were induced to differentiatein DM for three days and then were fed with growth mediu(GM) containing 20 µM bromo-deoxyuridine (BrdU). Cellswere fixed after 12, 24 and 48 hours and double stained skeletal muscle myosin heavy chain (MHC) and BrdU, identify terminally differentiated, MHC+ cells and BrdU+,proliferating cells. No double-positive cells were found at antime. Thus, despite their impaired ability to fuse, NIH(MRFcells fully undergo myogenic commitment and, unlike BC3Hcells (Lathrop et al., 1985), irreversibly withdraw from the cecycle.

We checked whether NIH(MRF) cells were able to cofuwith other myoblasts competent for fusion, by cocultivatinNIH(MRF4) or NIH(Myf5) cells together with rat L8myoblasts. As described in Materials and Methods, varyinumbers of the two cell types were seeded together and shito DM; after differentiation had taken place, the cells wefixed and stained to visualize TNT expression and nuclemorphology. As shown in Fig. 1B, many myotubes containboth rat and mouse nuclei, demonstrating that the NIH(MRcells were not impaired in fusing with other myoblascompetent for myotube formation and about 40% of coculturdifferentiated NIH(MRF4) cells were recruited into chimerimyotubes. Recruitment of NIH(MRF) cells into L8 myotubewas best observed in the presence of a large excess (5- tofold) of the rat L8 myoblasts. No fusion was obtainecultivating the fusion-defective NIH(MRF) cells in conditionedmedium from normal differentiating and fusing myoblasts ococultivating NIH(MRF) cells with normal fibroblasts.

It is well known that fusion into myotubes can be preventin biochemically differentiated myocytes by manipulatinenviromental conditions such as lowering calcium levels in tmedium (Paterson and Strohman, 1972). In NIH(MRF) celhowever, addition of the calcium ionophore ionomycin durinor after differentiation did not rescue fusion into myotubes.

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Fig. 2. Northern blot analysis of muscle-specific regulatory andstructural gene expression. Total RNA was extracted fromNIH(MRF), C3H(MRF4) and C2C12 cells cultivated in either GM orDM. The same filter was sequentially hybridized with the indicatedprobes, as described in Materials and Methods. Parallel cultures wereanalysed by immunofluorescence for TNT expression and DNAsynthesis (see Table 1). Note the presence of high levels of myogtranscripts in NIH(MRF4), NIH(Myf5) and C3H(MRF4) cellscultivated in GM and the variable pattern of endogenous MRF croactivation by different exogenous MRF; mgn, myogenin.

Fig. 3. Western blot detection of MEF2-A and MEF2-D proteins.Total extracts were prepared from NIH(MRF4), C3H(MRF4) andC2C12 cells cultivated in either GM or DM. Antibodies againstMEF2-A showed an heterogeneity of MEF2 polypeptides due tophosphorylation with two different mobilities, respectively, of 69 and58 kDa (Dodou et al., 1995); moreover, it has to be mentioned thatantibodies against MEF2-A partially cross-react with MEF2-C (Hanand Prywes, 1995). Antibodies against MEF2-D showed anhomogenous pattern with a single band migrating as a 58 kDaprotein. Note that MEF2-A and MEF2-D protein levels werecomparable among the three cell types, with a slight up-regulationupon terminal differentiation.

Muscle-specific gene expression in NIH(MRF) cellsIn an attempt to understand the nature/cause of the fusdefective phenotype, NIH(MRF) cells were analysed to asswhether regulatory and coregulatory factors of myogenewere correctly expressed as compared to fusion compeC3H(MRF4) and C2C12 cells.

Northern blot analysis (Fig. 2) showed that: (i) NIH(MRF4NIH(Myf5) cells and, to a lesser extent, C3H(MRF4) celaccumulated transcripts for MyoD and myogenin both proliferative (GM) and in differentiative (DM) conditions, avariance with previously reported data (Miner and Wold, 199Auradè et al., 1994); (ii) NIH(MyoD) cells activated myogeniexpression only in differentiating conditions anNIH(myogenin) cells could express endogenous MyoD, bonly upon shift to DM; (iii) none of the exogenous MRF coulactivate endogenous MRF4 or Myf5 expression in aexperimental conditions and, surprisingly, NIH(Myf5) cellfailed to accumulate detectable levels of transcripts for texogenous Myf5, at least by northern blot analysis (data shown; see also Auradè et al., 1994). It has to be stressedexpression of myogenin in proliferating NIH(MRF4)NIH(Myf5) or C3H(MRF4) cells cultivated in GM cannot bequantitatively accounted for by contaminating differentiatecells. At least 86% of NIH(MRF4), NIH(Myf5) orC3H(MRF4) cells in GM, from which RNA was extracted ananalysed in Fig. 2, were still competent to enter the S phasthe subsequent 24 hours (see Table 1). E2A expressappeared to be constitutive and showed no substandifference between myogenic derivatives of NIH3T3 oC3H10T1/2 cells and C2C12 myoblasts (not shown). We a

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checked the expression of some structural genes such asmuscle-specific isoforms of MHC and muscle creatine kina(MCK) that appeared normally upregulated upon termindifferentiation (Fig. 2).

Given the striking similarity among the different NIH(MRF)cells, further investigations were limited, unless otherwisindicated, to the analysis of NIH(MRF4) cells, chosen as thexpress exogenous MRF4 as well as endogenous MyoD amyogenin.

Although MEF2 proteins are widely expressed in nonmyogenic cells (Dodou et al., 1995), the role of MEF2 factoin activating muscle-specific genes is of great importan(Molkentin et al., 1995). Western blot analysis was performeon total extracts from growing or differentiated NIH(MRF4)C3H(MRF4) and C2C12 cells to assay the expression of tMEF2 family of transcription factors. As shown in Fig. 3MEF2-A and MEF2-D protein levels were comparable amonthe three cell types analysed with a slight up-regulation upmyogenic terminal differentiation. Altogether these datindicate that, independently of which MRF initiated themyogenic conversion, regulatory and structural musclspecific genes are correctly expressed in the fusion-defectNIH(MRF) cells, without obvious differences with myogeniccells competent for myotube formation.

Expression of adhesion molecules and muscle-specific alternative splicing events in fusion-defective NIH(MRF) cellsVarious membrane proteins mediating both cell-cell and cematrix interactions, including cadherins, N-CAM, integrins(reviewed by McDonald et al., 1995) and members of thmetalloprotease/disintegrin protein family (Yagami-Hiromaset al., 1995) have been suggested to participate in myotuformation. In an attempt to understand why the myogenderivatives of NIH3T3 cells are unable to fuse into

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Fig. 5. RT-PCR analysis of meltrin α, β and γ expression. RT-PCRreactions were performed on total RNA from differentiatedNIH(MRF4), C3H(MRF4) and C2C12 cells. Note that there were nosignificant differences in terms of meltrin α and β expressionbetween fusion-defective and fusion-competent cells, althoughMRF4-converted NIH3T3 and C3H10T/2 cells express a loweramount of meltrin α compared to C2C12 cells. Surprisingly, parentalNIH3T3 and C3H10T1/2 fibroblasts could express meltrins α and β.Meltrin γ expression is ubiquitous and served as a positive control.

multinucleated myotubes, we also analysed the expressioseveral surface molecules. Northern blot analysis wperformed in NIH(MRF4), C3H(MRF4) and C2C12 cells, boin proliferating and differentiating conditions, to check thexpression of M-cadherin, a muscle-specific cadhe(Donalies et al., 1990), and of N-cadherin (Miyatani et a1989) and N-CAM (Goridis et al., 1985), two widely expresscell-adhesion molecules. Fig. 4 shows that: (i) N-CAexpression strongly increased upon terminal differentiati(ii) M-cadherin mRNA levels were up-regulated idifferentiated NIH(MRF4) cells but remained stable C3H(MRF4) and in C2C12 cells; (iii) N-cadherin expressiowas stable in NIH(MRF4) and C2C12 cells and slighincreased in differentiated C3H(MRF4) cells. Howevesignificant differences between the fusion-defective ce[NIH(MRF4)] and the fusion-competent cell[C3H(MRF4)/C2C12] were not detected.

Expression of meltrin-α, a member of themetalloprotease/disintegrin protein family, has been suggeto be required for myotube formation (Yagami-Hiromasa et 1995). Accordingly, we investigated whether defective meltexpression might explain the fusion defect in NIH(MRF) celHowever, reverse transcriptase-polymerase chain reaction PCR) analysis shown in Fig. 5 indicated that meltrin-α wasexpressed in parental NIH3T3 or C3H10T1/2 cells and tthere were no significant differences between their myogederivatives, thus suggesting that meltrin-α neither shows clearmuscle-specificity in vitro nor is likely to be involved in thfusion-defective phenotype of NIH(MRF) cells.

Several lines of evidence suggest a role for extracellumatrix (ECM) molecules and their integrin receptors in skele

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Fig. 4.Northern blot analysis of genes encoding adhesion molecuinvolved in myotube formation. Total RNA was extracted fromNIH(MRF4), C3H(MRF4) and C2C12 cells cultivated in either GMor DM. The same filter was sequentially hybridized with theindicated probes, as described in Materials and Methods. N-CAMand N-cadherin mRNAs showed an heterogenous pattern withmultiple transcripts, indicated in the figure, as previously reported(Hamshere et al., 1991; Miyatani et al., 1989). Note that there weno significant differences in N-CAM, N-cadherin and M-cadherinexpression between the fusion-defective and the fusion-competenmyogenic cells.

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muscle differentiation (reviewed by McDonald et al., 1995). Iskeletal muscle, α7β1 integrin is the major laminin receptor(Song et al., 1992; Von der Mark et al., 1991) and speciisoforms of the α7 and β1 subunits are generated by alternativsplicing during muscle differentiation (Collo et al., 1993Belkin et al., 1996). The integrin α7 is present in proliferatingmyoblasts and its expression increases upon termidifferentiation. Two isoforms of the α7 integrin subunit, namedα7A and α7B, can be generated by alternative splicing of thcytoplasmic domain (Collo et al., 1993; Ziober et al., 199Song et al., 1993). The undifferentiated myoblasts of tmyogenic cell line C2C12 express only α7B, whereas theexpression of α7A correlates in these cells with terminadifferentiation, fusion and formation of myotubes. Analternative splicing event in the β1 integrin transcriptsgenerates a new isoform of this molecule, named β1D, that isobserved only in skeletal and cardiac muscle. The expressof β1D is developmentally regulated, replacing the commoβ1A isoform during muscle differentiation either in vivo or invitro (Belkin et al., 1996).

We asked whether NIH(MRF) cells could express themuscle specific alternative-spliced isoforms of the laminreceptor. To control the expression and differential splicing the β1 integrin subunit, RT-PCR was performed on total RNof NIH(MRF4), C3H(MRF4) and C2C12 cells grown for threedays in either GM or DM. As shown in Fig. 6A-B, the β1subunit transcript was clearly spliced to the β1D form both inC3H(MRF4) and C2C12 cells, even though the β1A is still thepredominant isoform. On the contrary, in NIH(MRF4) cellsthe β1A mRNA isoform, expressed at the myoblast stag(GM), remained the only detectable β1 species and there wasno muscle-specific alternative splicing to the β1D isoformupon terminal differentiation. While fusion-competent cell

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Fig. 6.RT-PCR analysis of muscle-specific differential splicing of β1integrin, MEF2-D and β-TM mRNAs. RT-PCR reactions wereperformed with RNA from NIH(MRF4), C3H(MRF4) and C2C12cells cultivated in either GM or DM. (A) RT-PCR reaction wasperformed using primers B72 and B73, annealing with both β1A andβ1D transcripts. The 188 bp product corresponding to the β1Atranscript is present in all samples, the 269 bp product representsβ1D isoform which is expressed only upon terminal differentiation C3H(MRF4) and C2C12 cells, but not in NIH(MRF4) cells. (B) Lacof β1D specific transcript in NIH(MRF4) cells was confirmed by RTPCR using β1D specific primers B73 and B98 (see Materials andMethods). (C) RT-PCR reaction was performed using primers T10and T12 that can selectively amplify from exon 3 only if exon 9a,diagnostic of the skeletal muscle-specific isoform, is present in theβ-TM transcript. C3H10T1/2 and NIH3T3 fibroblasts acquire theability to perform this muscle-specific differential splicing only upoMRF4-induced myogenic conversion. Note that β-TM muscle-specific expression does not depend on terminal differentiation.(D) RT-PCR reaction was performed using primers M1 and M3 thaamplify, in all cell types, the MEF2D1a2 transcript containing thewidely expressed exon 1a. (E) Primers M2 and M3 amplify theMEF2D1b2 transcript containing exon 1b. This muscle-specificisoform is expressed, like the β1D integrin isoform, upondifferentiation only in C3H(MRF4) and in C2C12 cells, but not inNIH(MRF4) cells. (F) Amplification of the β-actin cDNA confirmequivalent amounts of cDNA in each amplification.

Fig. 7.Western blot detection of the α7B or α7A integrin isoforms.Total extracts were prepared from NIH(MRF4), C3H(MRF4) andC2C12 cells cultivated either in GM or DM for three days.(A) Undifferentiated myoblasts already express α7B. In C3H(MRF4)and NIH(MRF4) cells α7B increases significantly duringdifferentiation, while it remained stable in C2C12 cells. (B) Theexpression of the muscle-specific isoform α7A is concomitant withterminal differentiation in all three cell types, although C3H(MRF4)and NIH(MRF4) cells express lower levels compared to C2C12 cells.

displayed increased levels of the β1D isoform upon longercultivation times, NIH(MRF4) cells were β1D negative evenafter six days in DM (data not shown). Preliminary data extethe β1D differential splicing defective phenotype also tNIH(MyoD) and NIH(myogenin) cells.

To assess whether also regulation of α7 subunit expressionwas abnormal in NIH(MRF4) cells, western blot analysis wperformed on total extracts from growing or differentiateNIH(MRF4), C3H(MRF4) and C2C12 cells. C2C12 celleither in GM or DM expressed α7B (Fig. 7A); C3H10T/2 andNIH3T3 cells did not express α7B, consistent with their

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fibroblastic phenotype (not shown; see also Song et al., 19Von der Mark et al., 1991; Collo et al., 1993), whereas themyogenic derivatives C3H(MRF4) and NIH(MRF4) cellsdisplayed the α7B isoform. The muscle-specific integrinisoform α7A was not detectable in all cell types analysed (Fi7B) in GM, but upon differentiation this integrin subuniappeared not only in C2C12 and C3H(MRF4) cells but alsoNIH(MRF4) cells. The α7A levels in C3H(MRF4) and inNIH(MRF4) cells were lower, compared to C2C12 cells, whilα7B levels were comparable among the three cell types in DThese differences, however, are not apparently linked to tcompetence for myotube formation.

To investigate whether the fusion-defective NIH(MRF4cells might fail to synthesize other muscle-specific isoformgenerated by alternative splicing, we analysed by RT-PCR tadditional mRNAs, generated by a muscle-specific alternatisplicing event: theβ-tropomyosin (β-TM) mRNA and theMEF2D1b2 mRNA. The rodent β-ΤΜ gene (Helfman et al.,1986) codes for two isoforms referred to as β-ΤΜ in skeletalmuscle and TM-1 in fibroblasts and smooth muscle. TMmRNA contains exons 6a and 9b, whereas β-ΤΜ mRNAcontains exons 6b and 9a (Wang and Rubenstein, 1992). PCR analysis was performed to assess exon 9a or exonsplicing as markers for correct β-ΤΜ mRNA expression inNIH(MRF4), C3H(MRF4) and C2C12 cells cultivated both inGM and DM. As shown in Fig. 6C, while parental NIH3T3and C3H10T1/2 cells failed to splice exon 9a, all myogencells could perform the alternative splicing and generated tmuscle-specific β-TM mRNA; moreover, as previouslyreported (Wang and Rubenstein, 1992), the amount of β-TMtranscript increased upon terminal differentiation. In the caof exon 6b splicing, RT-PCR analysis was performed withdifferent approach in order to distinguish the non-specific ex6a from the muscle-specific exon 6b, because they cannotseparated by size. A PvuII site is located only in exon 6a andthe digestion of 6a amplified cDNA with the PvuII enzymegenerates two fragments of 39 bp and 150 bp, respectiv(Balvay et al., 1994). As shown in Fig. 8, under differentiatio

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Fig. 8.RT-PCR analysis of exon 6bsplicing in β-TM transcripts inNIH(MRF4), C3H(MRF4) and C2C12cells cultivated in either GM or DM. RT-PCR was performed using primers R5and R8 that amplify a fragment of therodent β-TM gene containing eitherexon 6a or exon 6b. Upondifferentiation, NIH(MRF4),C3H(MRF4) and C2C12 cells expressedthe muscle-specific exon 6b as provedby the absence of the PvuII site in thePCR products. The presence of the PvuIIsite, diagnostic of exon 6a, is proved bythe appearance of a 150 bp band afterdigestion with PvuII (Balvay et al., 1994). The 189 bp band contains exon 6b and undigestible PCR product. Although the incomplete PvuIIdigestion prevents a quantitative estimation of the 6a/6b ratio, the results suggest a shift from exon 6a to exon 6b splicing upon differentiation.Equal amounts (15 µl) of undigested (−) or digested (+) amplified cDNA were analysed on a 2% agarose gel.

conditions, NIH(MRF4), C3H(MRF4) and C2C12 cellexpressed the muscle-specific exon 6b as suggested byabsence of the PvuII site in the PCR products. Exon 6a, aexpected, was present in fibroblasts and proliferatinundifferentiated myogenic cells, and was almost absentterminally differentiated myocytes or myotubes. These resustrongly indicate that NIH(MRF4) cells are competent perform the muscle-specific splicing of exons 6b and 9a aexpress a bona fideβ-TM transcript.

As shown above (Fig. 3), the MEF2-A and MEF2-transcription factors were abundantly expressed in NIH(MRcells as well as in C3H(MRF4) and C2C12 cells. Transcrifor MEF2-D are widely expressed, but alternative splicinevents give rise to a muscle-specific isoform namMEF2D1b2, containing exon 1b instead of the most commexon 1a of the MEF2D1a2 isoform (Martin et al., 1994). RPCR analysis was performed on proliferating and differentiaNIH(MRF4), C3H(MRF4) and C2C12 cells. As shown in Fig6E, the muscle-specific isoform of MEF2-D, like the β1Dintegrin isoform, was not detected in NIH(MRF4) cells, whiit was correctly expressed by the fusion-competeC3H(MRF4) and C2C12 cells. As expected, the ubiquitoisoform MEF2D1a2 mRNA was present in all cell types (Fi6D).

The surprising inability of NIH(MRF) cells to generate thmuscle-specific isoforms of the β1D integrin subunit and theMEF2-D transcription factor might be, directly or indirectlylinked to the fusion-defective phenotype, although this does involve a generalized inability to perform muscle specifidifferential splicing: α7A integrin subunit and β-TMtranscripts, in fact, undergo muscle-specific alternative splicin NIH(MRF4) cells.

DISCUSSION

It is well established that forced expression of MyoD or othbasic-helix-loop-helix myogenic factors can convert differecell types into myogenic cells (Davis et al., 1987; Choi et a1990; Auradè et al., 1994). Myogenic conversion appears toan all or none phenomenon, influenced by cell history acharacteristics: if a given cell type is susceptible to conversithen terminal differentiation and myotube formation can

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easily observed (Davis et al., 1987; Choi et al., 1990; Auraet al., 1994); alternatively, particular cell types can brefractory to myogenic conversion, and these cells fail activate the myogenic program in spite of exogenous Myoexpression (Schafer et al., 1990).The data presented in paper illustrate some unusual properties of myogenderivatives of the NIH3T3 cell line, that pinpoint a thirdpossible phenotype elicited by forced expression of Myofamily members, namely induction of terminal differentiatiowithout fusion into multinucleated myotubes.

Independently of which MRF initiated myogenicconversion, NIH(MRF) cells were fully competent fopermanent withdrawal from the cell cycle and transcriptionactivation of several muscle-specific genes, but terminadifferentiated myocytes were not competent to fuse inmultinucleated myotubes. Such a fusion defective phenotywas not ostensibly linked to a failure in activating thexpression of muscle-specific transcription factors necessarmaintain the muscle phenotype (Braun et al., 1989b). Althouthere were differences in the pattern of endogenous Mcross-activation by different exogenous MRF, all thNIH(MRF) cells activated MyoD and myogenin expression DM. At variance with previous reports (Miner and Wold, 1990Auradè et al., 1994), our data indicate that in NIH(MRF4NIH(Myf5) and C3H(MRF4) cells myogenin expression iturned on before withdrawal from the cell cycle and termindifferentiation. The reason(s) for these discrepancies is not understood; however, myogenin expression in cyclinmyoblasts has been reported in C2C12 cells (Andrès aWalsh, 1996) and in quail myoblasts (Russo et al., 1997). Wregard to the MEF2 class of transcription factors, comparatanalysis of parental NIH3T3 cells, NIH(MRF) cells and fusioncompetent myogenic cells failed to indicate significadifferences in MEF2-A or MEF2-D expression, at least at tprotein level, hence supporting the conclusion that the fusdefective phenotype of NIH(MRF) cells cannot be ascribedan obvious defect in myogenic factor expression.

In spite of the absence of fusion, NIH(MRF) cells closeresembled ‘bona fide’ myogenic cell lines and fusioncompetent myogenic derivatives of the C3H10T1/2 cell linSeveral muscle-specific structural genes were properly regulated upon terminal differentiation and myogenconversion was accompanied by the increased expressio

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many genes encoding surface molecules potentially involin the fusion process as M-cadherin (Zeschnigk et al., 199N-cadherin (Knudsen et al., 1990) and N-CAM (Peck aWalsh, 1993). Furthermore, although terminally differentiatNIH(MRF) cells failed to fuse with each other, they could brecruited into myotubes formed by coculture with fusiocompetent rat myoblasts, giving rise to chimeric myotubcontaining rat and mouse nuclei.

Genetic dissociation of terminal differentiation frommyotube formation has been previously observed in ttransformed myogenic cell lines: the brain tumour-derivBC3H1 cell line (Shubert et al., 1974) and the C2C12 cell ltransformed by the v-myc oncogene (Crescenzi et al., 1994NIH(MRF) cells represent a novel instance where fusion inmyotubes is genetically dissociated from the commitment abiochemical differentiation steps of the myogenic programnon-transformed cells. Furthermore, the fusion defectphenotype of the BC3H1 cell line was fully corrected bexogenous MyoD or MRF4 expression (Brennan et al., 19Block and Miller, 1992), and impaired myotube formation bv-myc-transformed C2C12 myoblasts could be reversedmixed cultures containing normal fibroblasts (Crescenzi et 1994). However, the first experimental approach is evidennot applicable to NIH(MyoD) or NIH(MRF4) cells and thsecond one was ineffective in rescuing myotube formationNIH(MRF) cells. Thus, the phenotype of NIH(MRF) cellswhile reinforcing our previous suggestion that myotuformation can be genetically dissociated from MRF-dependtranscriptional activation (Crescenzi et al., 1994), is likely depend on mechanism(s) different from those responsiblethe defectiveness of BC3H1 cells or v-myc-transformed C2C12myoblasts.

All the above discussed findings suggest that the fusdefect in NIH(MRF) cells might depend on a subtquantitative or qualitative deficiency. Analysis by RT-PCR β1D integrin subunit and MEF2D1b2 expression indicated tNIH(MRF) cells are unable to perform the differential splicinrequired to generate these muscle-specific transcriAlthough this finding represents the only defect so far detecin NIH(MRF) cells, the link between the absence of β1D andMEF2D1b2 and the fusion-defective phenotype is not yet cleThe absence of β1D and MEF2D1b2 transcripts does noappear to reflect a generalized deficiency in muscle-specdifferential splicing of NIH(MRF) cells. Expression of α7A,the muscle-specific isoform of the α7 integrin subunit, and β-TM muscle-specific transcripts also depend on muscle-spedifferential splicing, and both of them were easily detecteThe absence of relevant genomic sequence informations the still incomplete understanding of differential splicinregulation do not allow us to discriminate whether the obserdifferences reflect the existence of more than one classmuscle-specific differential splicing or depend on quantitative defect that preferentially causes interference whighly demanding alternative splicing events.

A possible role for β1D as a crucial effector involved inmyocyte fusion contrasts with the facts that NIH(MRFmyocytes can be recruited into myotubes formed by fusicompetent myoblasts in vitro (see Fig. 1B) and that β1 nullmuscle cells are recruited by wild-type cells into muscle fibin chimeric mouse embryos (Fassler and Meyer, 199Attempts to express exogenous β1D in NIH(MRF4) cells by

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transfection failed to rescue myotube formation (unpublisheobservations). The possibility remains that β1 null or β1Ddeficient cells can be recruited into myotubes formed bnormal myoblasts through a β1-independent mechanism. Theavailable data suggest that various MEF2 family members afunctionally equivalent (Molkentin et al., 1995). Attempts tooverexpress MEF2-C or MEF2D1b2 in NIH(MRF4) cellsfailed to provide evidence for a direct role of eithetranscription factor in regulating myocyte fusion (unpublisheobservations). Thus, we cannot formally exclude the possibilthat the defective differential splicing and the inability to fusinto myotubes are not causally linked in NIH(MRF4) cellsAlternatively, it is possible to envisage the inability to generathe β1D and the MEF2D1b2 specific mRNAs as a symptorather than the cause of the fusion-defective phenotype. Ittempting to speculate that functional expression of specieffector(s) involved in membrane fusion of differentiatemyocytes may display the same differential splicinrequirements as those involved in β1D and the MEF2D1b2expression.

In conclusion, we have shown here a novel example of hocell history and characteristics may influence the activation myogenesis by forced expression of MyoD family memberMyogenic derivatives of NIH3T3 cells appear to represent ainteresting model to unravel the mechanism of cell fusion myogenesis as well as to dissect subtle aspects of the activaof the muscle-specific differential splicing.

We thank the following persons for providing valuable reagenused in this study: S. Alemà, E. Bober, M. Bouché, D. A. FischmaE. Olson, R. Prywes, A. Salvatori, G. Salvatori, C. Schneider, Sorrentino, A. Starzinsky-Powitz, M. Takeichi and D. Yaffe. We thanA. Fujisawa-Sehara for providing sequence information to amplimeltrin-α. We thank our collegues who contributed a critical reviewof this manuscript. We also thank M. Migneco for the help in thpreparation of the figures. This work has been supported by grafrom Associazione Italiana per la Ricerca sul Cancro, TelethoConsiglio Nazionale delle Ricerche (Progetto FinalizzatApplicazioni Cliniche della Ricerca Oncologica) and FondazionCenci-Bolognetti.

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