eae in delayed differentiation. myoblast vivo activate activate and In another Nfix one and of Matn2 expression whereby the loop positive-feedback a repressing of onset by part in least TGF- of genes. and p21 myogenic of the expression the and differentiation Matn2 restored of Rescue expression differentiation. myoblast and exit cycle cell retarded fBohmsr n oeua ilg,Fclyo aua cecsand Hungary. Sciences Szeged, Natural H-6720 of Szeged, Faculty of University Biology, Informatics, Molecular and Biochemistry of nvriy -84 untr Germany. Muenster, Szeged, D-48149 H-6701 University, Szeged, Sciences, of Hungary. Academy Hungarian Centre, Research cecs zgd -71See,Hungary. Szeged, H-6701 Szeged, Sciences, eecDea Ferenc the regeneration of muscle timing during the program controls myogenic matrilin-2 of deposition Extracellular ARTICLE RESEARCH 3240 2014 May 8 Accepted 2013; August 25 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This ` early The 1 2013). al., injured the et of necrosis Yin inflammation, sequential 2005; involves phase the Conboy, degenerative and by multistep Wagers (Charge architecture pathways a 2004; signaling tissue is multiple the of injury activation restores following that regeneration process muscle Skeletal INTRODUCTION shRNA, Matn2 TGF- Myogenesis, regeneration, Muscle WORDS: KEY genes myogenic the of and regeneration p21 muscle during and culture vivo in differentiating differentiation. myoblasts proliferating, fusing myogenic around and of deposition stages Matn2 early observed We the matrix extracellular controlling (Matn2) in matrilin-2 the protein for role a identify we Here, ABSTRACT zgd Hungary. Szeged, eea eiie nvriyo zgd -70See,Hungary. Szeged, H-6720 Szeged, of University Medicine, General Teeatoscnrbtdeulyt hswork this to equally contributed E authors and *These Lausanne of University Fe the of Biotechnology for of rca opnn fagntcsic htmdltsteostof onset the modulates that switch repair. genetic tissue a initiates a as that of Matn2 cascade identify component thus regulatory crucial data the Our of differentiation. myogenic modulator terminal establishing key cultures, a myoblast as primary Matn2 differentiating in and limbs ioa Mermod Nicolas uaMendler Luca uhr o orsodne([email protected] [email protected]) ([email protected]; correspondence for Authors nttt fBohmsr,Booia eerhCnr,HnainAaeyof Academy Hungarian Centre, Research Biological Biochemistry, of Institute ´ de 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,34–26doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. Trf3 ´ iecn of Silencing . aed asne H11 asne Switzerland. Lausanne, CH-1015 Lausanne, de rale b inln,BPsgaig F,Trf3 NFI, signaling, BMP signaling, ybatcl yl retadmsl eeeainwas regeneration muscle and arrest cycle cell myoblast , n ygncgnswr outyrdcdin reduced robustly were genes myogenic and 3 nttt fPyilgclCeityadPtoiceity Muenster Pathobiochemistry, and Chemistry Physiological of Institute Matn2 ´k 7 1, nttt fBoehooy nvriyo asne n Center and Lausanne, of University Biotechnology, of Institute 2 4 Matn2 ,LjsMa Lajos *, / Aniko , 2 eaiet idtp ie h xrsinlevels expression The mice. wild-type to relative 7 ,La eae h xrsino h d inhibitor Cdk the of expression the delayed ´szlo Keller-Pinte ´ b Matn2 niie ygncdfeetainat differentiation myogenic inhibited 1 .Puska G. ´ ´te Nfix ´s 4 , nttt fBohmsr,Fclyof Faculty Biochemistry, of Institute xrsin hc niie the inhibited which expression, 1,2, MyoD 2 * nttt fGntc,Biological Genetics, of Institute , ` ,E ´r and ´s ´ 4 ´ aKorpos va ,Be oePolytechnique cole 2,6 Myog l O ´la n blaKiss Ibolya and 6 ´ vdnLd,H-6726 Ltd., Avidin xliigthe explaining , n Rudnicki, and Matn2 ´ zsva 5 Department 1,3, 2 ´ri / 2 ,A *, 6 fetal anlaJuha Hajnalka , ´ in nsZvara gnes 1,6, ` euaoypormdrn mroi n ea eeomn and development (Charge fetal regeneration and muscle embryonic during program regulatory steps. stages, differentiation maturation is embryonic and This the myoblasts. recapitulating differentiation proliferating proliferation, of of by pool sarcolemma a followed the which create and cells, to regeneration membrane satellite fibers, to muscle basement of four the activation in leading between the repaired reside with endplates, starts then Repair motor is stages. muscle of The degeneration denervation. and myofibers 06.Tepoieaigmolssepestemyogenic the express myoblasts proliferating The 2006). elcdb aii-1-otiigbsmn membrane basement laminin-211-containing is ECM a fibronectin-rich differentiation 2002). by the Brandan, myogenic differentiation, replaced and (Osses with myoblast inhibition expression During interferes MRF the of assembly independently Importantly, ECM regeneration. of and differentiation 2010). al., fetal et activates (Messina Nfix genes whereas genes, muscle embryonic of induction the for muscle responsible al., in being Nfia the factors et 2000), transcription (Gronostajski, of morphogenesis of Winbanks members family 2013; (NFI/Nfi) specific I implicated al., factor al., also nuclear et et have Sartori data (Gardner 2009; Recent growth 2013). and al., muscle et Smad5 of Sartori regulator Smad1, 2011; positive Smad3 through a and is signaling Smad2 Smad8 the BMP by and whereas signals binding intracellular proteins, specific receptor of following initiation growth TGF- and 2005). differentiation Conboy, and (Charge (ECM) (e.g. matrix TGF- extracellular factors growth and of IGF signals morphogeneticFGF, inductive to by response regeneration in regulation muscle during and development merge or myotubes myofibers. multinucleated and cells existing primitive the with (Walsh form and to exit proteins, fuse contractile either cycle muscle-specific cell the of with starts expression irreversible differentiation and overt to Subsequently, Myog 1997). Cdk Perlman, and of the of expression p21 upregulation The the inhibitor to 2008). leads MyoD- myoblasts al., committed in et the Mrf4 and Deato (Deato a as complex 2007, 2010; TRF3–TAF3 Tjian, the by machinery, ( with gene TFIID al., accompanied transcription myogenin of replacement the is core et of activation This the dependent (Aziz 2011). of committing differentiate Asahara, switch factors and to transcription also Yokoyama other that precursors of program a myogenic network initiates expression a MyoD gene involves MyoD. muscle-specific and Myf5 complex (MRFs) factors regulatory ucedfeetaini ietdb osre myogenic conserved a by directed is differentiation Muscle h C a ait fdrc n nietefcso muscle on effects indirect and direct of variety a has ECM The fetal and embryonic during fine-tuned is program myogenic The 2, ,TbrSze Tibor *, ´sz 1 yi Sorokin Lydia , b ´na ,ctknsadvrossiuifo the from stimuli various and cytokines ), ´si ´ 2 b n unci 04 h n Garry, and Shi 2004; Rudnicki, and ,Mo aiymmesihbtmuscle inhibit members family 3 iaKiricsi nika ´ ,La ´ n unci 04 Wagers 2004; Rudnicki, and ´szlo Dux ´ Myog 4,5 4 , , eursthe requires )

Journal of Cell Science ihdlydmsl eeeainin regeneration muscle delayed with ybatclue.W eotdlydmoei differentiation myogenic delayed report from We cultures. myoblast nue uceregeneration muscle induced euainb h M7Sa inln aha a modulate 2008). al., can et pathway (Ichikawa signaling healing skin-wound BMP7–Smad that the and 2009), by al., regulation et (Malin for regeneration required nerve is peripheral Matn2 that al., demonstrated et have are and studies (Piecha Recent homo-oligomers 1999). skeletal membranes are basement they heart, Matn2 subepithelial and tissues, uterus, with connective 2011). associated dense the and loose al., in in muscle, amounts et smooth varying Klatt in 1999; deposited (Dea proteoglycans al., filamentous and form fibrils et collagen to connecting proteins by ECM networks other with interact oligomers isedfeetainadrepair. to of interactions and onset ECM–cell differentiation the linking tissue controlling thereby network program, myogenic signaling the a in Matn2 implicate decreasing an stelretmme ftemtii aiyo multidomain of (Dea family regeneration, matrilin proteins the muscle adaptor of ( member of largest muscle the gene model is Matn2 mouse matrilin-2 skeletal a the in including genes to ECM several contribute unknown. largely remains might of regeneration and stages differentiation components all at ECM interactions for (Thorsteinsdo role cell–ECM development significant muscle and a components revealed (Sanes, evidence ECM respectively ample (MTJ), Although junctions junctions 2003). neuromuscular myotendinous at and contacts (NMJ) muscle–tendon nerve– mediates and ECM muscle specialized functionally and structurally A of TGF- that establish irto n ifrnito,moie omto and formation cell myofiber myogenic Thorsteinsdo 1998; influences differentiation, al., et which (Gullberg and innervation myotubes, migration by deposited ARTICLE RESEARCH inlitniyi h nrae oto,weesmil Matn2 mainly whereas control, untreated the in intensity signal maturation. myofiber during and downregulated is proliferation it whereas myofibers, myoblast and myotubes early involves myocytes, to differentiation which that phase, concluded regeneration Henry, We and (Goody target integrin 2010). common a Ptk2), as known (also The 1C). of (Fig. expression of peak the and (Bgn) of Myog biglycan analysis Matn2 (QRT-PCR) as and RT-PCR such Quantitative components, (Sdc4). MyoD syndecan-4 ECM induction other the the of from of kinetics differed but profiles markers, differentiation expression myogenic partially the accumulation mRNA overlapped transient The marked its 1B). in (Fig. decline showed a expression by followed treatment, analysis after days time 4 Northern until upregulation over 1A). (Fig. weight repair soleus processes and necrosis soleus inflammation, injected of progress rat expected the the reflected notexin-induced in of Changes model regeneration. regeneration well-established muscle in the Matn2 for using role potential a assessed first We regeneration muscle Transient RESULTS ee erva transient reveal we Here, iraryaayi a hw nicesdepeso of expression increased an shown has analysis Microarray muolt eeldpeoiatyMt2oioeso low of oligomers Matn2 predominantly revealed Immunoblots Trf3 Matn2 RArvae .-odtasetatvto htfollowed that activation transient 6.5-fold a revealed mRNA , Nfix Matn2 Matn2 slne ybatclue,adaml dystrophy mild a and cultures, myoblast -silenced Matn2 n R eeepeso.Oeal u data our Overall, expression. gene MRF and peuaindrn skeletal during upregulation xrsin n ako an easteonset the delays Matn2 of lack and expression, b ´ RAlvlicesdsmlryt hto Fak of that to similarly increased level mRNA ta. 97 Ma 1997; al., et k a nii 2mols ifrnito by differentiation myoblast C2 inhibit can MyoD Matn2 Matn2 n lsl aalldta of that paralleled closely and nvivo in Matn2 ´ tre l,21) o distinct how 2011), al., et ttir peuaindrn notexin- during upregulation surgltddrn the during upregulated is Matn2 ´ Geshe l,2003). al., et (Goetsch ) te ´ ta. 02.Matrilin 2002). al., et s n ndifferentiating in and 2 / 2 ´ tre l,2011). al., et ttir ie ealso We mice. Matn2 Matn2 Myog ´ k omlzdt hs fteutetdcnrl( control untreated levels the expression of relative those the QRT-PCR, to using For normalized QRT-PCR (C). and protocol ( (B) green samples hybridization SYBR regenerating northern and to 2A). control subjected (Fig. from was sections derived Garry, H&E-stained RNA and of total (Shi analysis Pooled regeneration histological of on stage based the 2006), with aligned were expression A egtcagsdrn oei-nue eeeain aaso the show Data regeneration. regeneration. notexin-induced mean soleus during rat changes during Weight expression (A) gene Marker 1. Fig. D muoltaayi fMt2epeso uigrgnrto.t trimer; t, regeneration. monomer. during m, expression dimer; Matn2 d, of analysis Immunoblot (D) 6 ...( s.e.m. ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal n § ) ** 3); P , .1 BC iei hne nmre gene marker in changes Kinetic (B,C) 0.01. DD C t r shown. are ) n 5 3241 3)

Journal of Cell Science ooesdcesdatrdy4 hra h oligomer of the muscles amount regenerating in whereas The Matn2 of Immunolocalization 1D). 4, (Fig. day control after restored. the was formation the in decreased from antibody monomers resulted recognized the likely poorly elevated be post-injury might by that The day oligomers of 1 regeneration. cleavage proteolytic at during signal observed monomer were monomers ARTICLE RESEARCH ta. 07 aeye l,19;Msiae l,21;Montarras 2010; al., 3242 et Messina 1995; al., et Halevy 2007; al., et (Biressi differentiation muscle fetal mimic faithfully to reported model was a line cell this deposit monitored as medium, differentiation can in next cultured myoblasts myoblasts we that ECM, evidence Matn2-rich independent provide To Transient steps early reinnervation. the and in differentiation Matn2 for myogenic role of formed a suggested newly NMJs myotubes, and myoblasts, myofibers observed around cap The Matn2 2I). the of (Fig. around (TSCs) deposition area cells Schwann presynaptic terminal the by to formed and extended the membrane signal in Matn2 synaptic The receptors the 2H). acetylcholine formed (Fig. to membrane proximity recently postsynaptic Double re- close 2B,G). NMJs, in the (Fig. were re-established at specifically 4–7 deposition that between days Matn2 intense at showed NMJs number staining terminals nerve the also large and was and myofibers al., in deposition nerves Matn2 et around established 1991). (Grubb elevated Cullen, signals highly and nerve proliferation, Sesodia on myoblast 1991; depends Unlike maturation endplates. myofiber motor their destroys (supplementary and process fibronectin S1D). regeneration 2B–D). Fig. of material (Fig. the those control overlapped throughout the partially in collagen-1 than signal 14 Matn2 day The on endomysium myofiber tissue the interstitial in during broader and and decreased intense 14 more day was expression for it on but Matn2 maturation, nuclei stained myoblast central 2A). containing strongly Fig. still (arrow, myofibers assists but basement 2B–D) formed laminin-containing (Fig. that the desmin recently in of can not assembly membrane but strong proteins endomysium, relatively ECM the also was both in signal Matn2 the fusion. that and differentiation in implying participate formed 2B–D,F), (Fig. newly with fusing laminin merging myofibers desmin-positive with myoblasts surrounding and colocalized myotubes membrane Matn2 myoblasts, basement shown). the not in data S1B,C; Fig. material Lama2-positive supplementary 2B–F; around (Fig. and not macrophages and but granulocytes 2, Myog- day on MyoD-, myoblasts differentiating and proliferating around observed deposition myogenic, we thereby markers, Using Matn2 endomysium, cell antibody. from inflammatory the old and epitopes membrane by the basement recognition Matn2 in better of in linkages resulting release ECM proteolytic and oligomers a implied This the S1A,D). further Fig. at material 1 the supplementary day observed 2A–D; at in tissue (Fig. was interstitial post-injury the vessels signal in and blood myofibers Matn2 necrotic and of intense periphery nerves an to muscle, relative untreated staining endomysium by Matn2 weak determined the the were of 2; Unlike (Fig. S1). which sections Fig. muscle 2006), material of supplementary Garry, staining (H&E) and eosin and Shi hematoxylin 2005; al., known et during (Charge markers regeneration specific muscle of of stages that with Matn2 express of might localization types cell which reveal To hl epn h ucenre nat oei treatment notexin intact, nerves muscle the keeping While Matn2 ciaini ifrnitn 2myoblasts C2 differentiating in activation ´ n unci 04 Gorbe 2004; Rudnicki, and Matn2 Matn2 ecmae the compared we , xrsini C2 in expression aiu ndy – ndfeetainmdu Fg 3G), (Fig. medium differentiation in thus and 2–4 dimers days 2, to day trimers, on transiently increased Matn2 maximum at also medium of a the peak amount into secreted of The a monomers 3F). accumulation with (Fig. transient mRNAs increase the transient overlapping a exhibited uigteeryseso 2mols differentiation. myoblast C2 of steps and early myoblasts the proliferating thus by during data Matn2 of These deposition myotubes. the of confirmed appearance the with concomitantly iecn nefrdwt h rnin ciainof activation transient the with Thus, shown). interfered not silencing data experiments S2C; differentiation Fig. repeated material sh4 in (supplementary the lines and cell line silenced control sh3 the and changes between robust expression gene most myogenic the in revealed which QRT-PCR, comparative silencing cultures Thus, Matn2 myoblasts. the S2B). in Fig. of reduction material Matn2 expression supplementary the 4A,C; mRNA with differentiation (Fig. and correlation and in index immunofluorescence fusion decreased frequency, index was The S2A,B). myotube myotubes Fig. material primary (supplementary multinucleated cultures sarcomeric control to of that of in in fusion reduced frequency culturing their highly Upon the 2– and shown). by myocytes and not delayed was (data days formation 3 4B) data myotube (Fig. count medium, activity mitochondrial differentiation cell their on on based and at proliferated as and rates, shown) not similar (data myoblasts control to identical n once otelmnncnann aeetmembrane 3D). (Fig. basement 6 laminin-containing day by the myotubes multinucleated be around to to connected an fibronectin appeared into with and incorporated associated filaments being closely network Matn2 3E), filamentous (Fig. extensive The attachment cell 3D,E). myotubes (Fig. in primary involved 2 fibronectin-connected and day and myoblasts laminin from fusing formed surrounding it 0, filaments day on granules Long 3C). 3B,D). (Fig. (Fig. 2 6 day day from culture by the sarcomeric for staining staining laminin cells the spindle-shaped and by with followed desmin structures 2, myotube-like high day multinucleated aligned at of signal formation fibronectin and laminin desmin, Matn2, intense exhibiting myotubes and myocytes spindle-shaped to differentiated myoblasts medium, differentiation growth In 3A–D). in (Fig. proliferating myoblasts deposition, fibronectin butonlytracesofdesminandno and laminin C2 Matn2, showed Polygonal 0) (day medium 1996). al., et iial splmnaymtra i.SB.Pirto Prior S2B). Fig. by determined material as the decreased line, protein differentiation, (supplementary control Matn2 from the the ranging of of similarly levels expression that 4A); expression (Fig. of with QRT-PCR sh3, 17.6% and to were sh4 of 52.2% Ctrl) sh7, expression (line lines the vector in and control differentiation genes. the for marker or tested sh7) and and established sh4 either sh3, stable-knockdown expressing (lines Independent 2005). constitutively myogenic al., lines for et expression (sh)RNA (Kaufman needed using short-hairpin system myoblasts be transposon-based C2 in efficient might expression an its Matn2 silenced we whether differentiation, differentiation address myoblast C2 To for required is Matn2 eae ybatdfeetainwsfrhrspotdby supported further was differentiation myoblast Delayed The fine in myoblasts proliferating by deposited was Matn2 Whereas xrsindlydbt h ifrnito n uinof fusion and differentiation the both delayed expression ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal Matn2 Matn2 RAlvli ifrnitn ybat also myoblasts differentiating in level mRNA Matn2 Matn2 RAlvl eescesul reduced successfully were levels mRNA slne ybatclue eaieto relative cultures myoblast -silenced slne el eemorphologically were cells -silenced a atnnimmunofluorescence -actinin Matn2 a atnnapae in appeared -actinin MyoD treigshRNA -targeting a -actinin-positive MyoD and Matn2 Myog and

Journal of Cell Science EERHARTICLE RESEARCH h aniae n,cnrlyncetdmoie;e,edmsu;m,mols;m,moie;m,moue ,nre m,ncoi yfbr pm, myofiber; necrotic ben nmf, shown are nerve; (G) n, area myotube; outlined mt, the myofiber; or mf, (C,D,F) image myoblast; newly entire mb, the the endomysium; of at em, images vessel. signal myofiber; Single-channel blood Matn2 nucleated (A,B). v, strong magnification centrally perimysium; the higher cnf, Note at image. image 4. main main day the the w on of membrane, NMJs section basement to a the Matn2 show within of fusing and Localization and Rhodamine-labeled d Matn2 (G–I) differentiating by at post-injury. between (arrowhead) myoblasts visualized days differentiating myoblasts as 2 and around NMJs, at proliferating and established arrow), Myog-expressing (star) (Lam, and tissue laminin-111 laminin MyoD- interstitial for and around the stained (B–D) Matn2 in Matn2 of deposition Deposition for Matn2 (E) regeneration. cryosections (F) (B). soleus serial tissue rat of interstitial during Immunofluorescence indicates deposition (B–D) star Matn2 cells. and inflammatory changes indicates morphological star of Progress 2. Fig. a bnaooi inl tNJ H ros.TC iulzdb FPsann r oee yMt2rc C tteNJ() Insets (I). NMJ the at ECM Matn2-rich by covered are staining GFAP by visualized TSCs arrows). (H, NMJs at signals -bungarotoxin a bnaooi BX tiigo ctlhln eetr G roha) hr spriloverlap partial is There arrowhead). (G, receptors acetylcholine of staining (BTX) -bungarotoxin ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal A & tiigo rnvreprfi etos The sections. paraffin transverse of staining H&E (A) a Lm2 C rdsi Ds D.The (D). (Des) desmin or (C) (Lama2) 2 y2post-injury. 2 ay ihi also is hich 3243 eath

Journal of Cell Science EERHARTICLE RESEARCH elln a etr h rprtmn fmyogenic of timing proper 3244 targeted sequence the the mouse mutated the in restore we shRNA the end, by this can To differentiation. line cell genes. differentiation-related and Matn2 and p53 control unrelated (e.g. the genes ECM of Matn2 expression the in Col6a1 rise transient the the the of explaining differentiation of and readily exit lines. myoblasts, cell cycle silenced cell control commitment, in t delayed medium. differentiation observed the of into level Comparison daily (F) secreted image. marker main was other the that below for Myog Matn2 magnification or higher of at alone analysis shown Matn2 is Immunoblot for E (G) in images hybridization. white monomer. Separate in northern m, 4. outlined ce by dimer; area day upon MRFs The d, on behind E. and trimer; fibron left and (arrowhead) Matn2 D adhesions and adhesions B, for focal (Lam) focal in laminin-111 levels vinculin-positive images paxillin-positive with mRNA main the at the Matn2 mark cells above and Arrows of shown the (B) 0). colocalization are to Desmin DAPI (day Partial linked medium. myoblasts (D) are differentiation proliferating myotube. filaments by in mt, Matn2 (asterisk) (mmt) myoblast; deposition myotubes mb, Matn2 multinucleated differentiation. Granular to of (E) differentiating progress cultures the myoblast demonstrates of (C) (B–E) markers other and 3. Fig. et etse hte ecigMt2snhssi silenced a in synthesis Matn2 rescuing whether tested we Next, Fak swl swt h rgesv nraei h 2 mRNA p21 the in increase progressive the with as well as , Matn2 eeecdn ouao fcl–C otcs and contacts, cell–ECM of modulator a encoding gene a , iecn xrsaseii feto h xrsino MRF of expression the on effect specific a exerts silencing ntecnrlln.A omre leainwsse for seen was alteration marked no As line. control the in and xrsini rlfrtn n ifrnitn 2myoblasts. C2 differentiating and proliferating in expression Sdc4 ,teepeso fwihprlee htof that paralleled which of expression the ), Matn2 iecn lohmee h activation the hampered also silencing Matn2 Col1a1 DAi h hr oiin of positions third the in cDNA RA,ti niae that indicated this mRNAs, Lama2 , AE hs-otatiae A n obeimnfursec o Matn2 for immunofluorescence double and (A) images Phase-contrast (A–E) dt o hw) m inln,wihcnrl ucegrowth muscle controls which control signaling, similar the Bmp were shown). of rates proliferation not that myoblast (data to the in and similar 4G,H), 6 frequency (Fig. day line a at at culture appeared sh3-res2 the the myotubes the of differentiation, in During noted use both medium. not differentiation the was filamentous in decrease level given a Matn2 expression, the expected, Matn2 in line drive As to in promoter control CMV 4E,F). constitutive and (Fig. the sh3-res2 extracts medium complemented ECM of differentiation the those of conditioned levels reached Matn2 myoblasts analyses The further 4E–I). for (Fig. (sh3-res2) clones stable puromycin-resistant into cDNA modified a while the Matn2- introduced we harboring capacity Then, vector 4D). expression protein-coding (Fig. constitutive site target its shRNA the preserving disrupting thereby codons, iecds3molssadslce o ol of pools for selected and myoblasts sh3 silenced ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal a a -actinin-positive atnnstaining -actinin lmovement. ll ci (Fn). ectin and s ,

Journal of Cell Science oprdwt oto ( control with compared EERHARTICLE RESEARCH utrsuigTqa rbs akrmN eesaegvna odvle eaiet the to relative values fold as given are levels mRNA silenced Marker the probes. TaqMan from using (F) Cytoskeletal cultures shown. lysates is cell Matn2 t and of of form (E) trimeric index medium differentiation a the differentiation Only and conditioned cultures. the index myoblast the for Ctrl fusion on frequency, and depicted sh3-res2 performed myotube are rescued was Primary sequences Immunoblotting (C) Coding interference. medium. (D) shRNA growth medium. to differentiation in resistant in assay 5 MTS day the using on lines by determined was lines expressing cell stably silenced lines cell C2 proliferating in QRT-PCR of effect The 4. Fig. atnnpstv el nteclue.()Dfeetainidxa eemndo a .()QTPRaayi frltv eeepeso Rl ex (Rel. expression gene relative of analysis QRT-PCR (I) 5. day on determined as index Differentiation (H) cultures. the in cells -actinin-positive Matn2 n 5 –) o – n ,rpeettv aafo he needn xeiet r shown. are experiments independent three from data representative I, and E–G For 3–5). iecn n eceo ifrnito n akrgn xrsino 2myoblasts. C2 of expression gene marker and differentiation on rescue and silencing Matn2 hN s7 h n h)o h oto etr(tl.()Tepoieainrt ftecnrland control the of rate proliferation The (B) (Ctrl). vector control the or sh3) and sh4 (sh7, shRNA Matn2 Hprt hN,teendogenous the shRNA, ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal RAlvl aaso h mean the show Data level. mRNA b atnsre salaigcnrl G muoloecnefor Immunofluorescence (G) control. loading a as served -actin Matn2 (A) Matn2 n h ececN ln,wihis which clone, cDNA rescue the and RAlvl eemaue by measured were levels mRNA 6 ...(–,H.*** H). (A–C, s.e.m. . ntesame the in p.) sh3, P ecell he , 0.001, 3245

Journal of Cell Science fxhsbe hw odieatasrpinlsic from switch transcriptional a drive myogenesis to fetal shown to embryonic been of connection has in regeneration Nfix regulation tissue of and TGF- cells onset the with stem the adult in of assess control involved possibly to are Matn2 that us factors transcription prompted differentiation ( chain heavy myosin ybat,weesMt2epeso ecersoe the sh3 of restored differentiation rescue the expression TGF- on 5A). Matn2 (Fig. effect inhibition delaying whereas hampered small and myoblasts, TGF- line a However, control only 5A,B). the (Fig. TGF- phosphorylation of Smad1/5/8 and differentiation TGF- the Matn2 of inhibited addition whether the myoblasts. differentiating expected, assessed in As another next one regulate al., might et we signaling Winbanks 2009; Thus, al., et Sartori 2012). 2011; muscle al., and et differentiation (Gardner myogenic growth inhibit to known is signaling h oto,bta nerirtm-on.Dsi and it Desmin and time-point. culture, earlier rescued an of that the at to similar but in expression of control, 2 level day could the a reach expression at to p21 steadily cells, detected increased sh3 p21 in of be level expression low the already its in not Unlike seen was 4F). were (Fig. expression differences large Smad4 but S3B). altered, Smad1/5/8 Fig. of comparable phosphorylation material similar yielded (supplementary the 2013), from al., judged et as Winbanks effects, 2013; al., et (Sartori ARTICLE RESEARCH an nteEMfo h oto n h-e2cultures, sh3-res2 and control the 3246 from ECM the in Matn2 Matn2 TGF- and 2008), al., et (Ichikawa types cell Matn2 differentiation myoblast an TGF- driving switch genetic a of response. part differentiation be might all-or-none Nfix implying S3A), and Fig. Matn2 material that supplementary and 4I Fig. (compare genes muscle monitoring fetal When activating 2010). and al., et Nfia (Messina of effect the suppressing of myoblasts the expression in Matn2-rescued increased increase the the differentiating muscle-specific and 4I), in of (Fig. restoration expression the the in deposition gene confirmed its and QRT-PCR expression ECM. Matn2 the restoring the by Thus, of reversed p21 expression of increased pathways. induction the TGF- despite the signaling is differentiation, hampered overt it Matn2 several and S3B,C), of modulate expression Fig. decreased material to (supplementary likely Matn2 Erk1/2) (also p42/44 As and as Fak cultures. of known sh3-res2 phosphorylation the the altered in also silencing rescued also was expression rtis neetnl,ars nMt2epeso bv a above expression of expression Matn2 downstream the the on Nfi control in turn of to and of needed rise might likely expression was level a Matn2 threshold the Interestingly, regulating that proteins. by suggested differentiation This only myogenic myogenic expression. preceded or expression The gene their 4I). silent and (Fig. cells, cells Matn2-expressing silenced either the in were Nfia levels low but at expressed myoblasts, Matn2-rescued that Nfia observed we myogenesis, during h idn htMt2epeso ih euaemyoblast regulate might expression Matn2 that finding The b eeide ciae nbt h ifrnitn oto and control differentiating the both in activated indeed were and b inln represses signaling RAlvl,adi ossetyrdcdtedpsto of deposition the reduced consistently it and levels, mRNA a ensont ergltdb mdsgaigi other in signaling Smad by regulated be to shown been has xrsin f rtishv enipiae nthe in implicated been have proteins Nfi expression. inln nemdaeSa4 n hsefc ol be could effect this and Smad4, intermediate signaling Matn2 b inln Paaie l,20;Paaie l,2010). al., et Plasari 2009; al., et (Plasari signaling RA cuuae ihsmlrkntc in kinetics similar with accumulated mRNAs Myh1 Myh1 b mRNAs. ) Matn2 outyihbtdteices in increase the inhibited robustly 1 MyoD and nvivo in Mstn Matn2 b xrsinand expression and odfeetainmedium differentiation to 1 Nfix ee ns3rs cultures sh3-res2 in genes Myog ee a aalldby paralleled was level Nfix n nC utr by culture C2 in and n,t esrdegree, lesser to and, or n fteadult the of and b Nfia n Smad2/3 and expression b a had 1 -actin Nfix Nfix b , ybatdfeetain(i.6–) rncito rmP from Transcription 6A–C). and (Fig. regeneration differentiation muscle myoblast during activation gene transient the direct yDepeso erae hra fxepeso increased expression myoblasts, P Nfix C2 of whereas In the binding activity. decreased the inhibitory, promoter expression eliminated the MyoD strongly increased that was proteins mutation sites both NfMdm1 MyoD the and whereas NFI in (Nfm1) overlapping mutation the NFI-contact-point An 6E,G). (Fig. myoblasts hnemrel nete ae ycnrs,teP the contrast, By case. either in markedly change Fg B ro) ossety rncito rmP from transcription days Consistently, 4 arrow). formation and myofiber during 6B, h decline a (Fig. 6 by followed between injury, soleus upregulated after reproducibly was transcript n ja,20;Daoe l,20) n both and 2008), al., et Deato (Deato 2007; myogenesis Tjian, and during and machinery induction is transcription Myog Trf3 basal MyoD-dependent As the for levels. of Smad4 transition expression the highest for the p21 needed and levels in Matn2 TGF- expression lowest decrease the the in prominent Smad4 obtained most induced was The strongly 5C,D). (Fig. medium differentiation decreased the h, (12–36 differentiation of TGF- stages Similarly, early 5C,D). Fig. the during especially O- el ofre hti csa natvtro h P the of activator an as acts it that confirmed cells COS-7 rvosfnigta fxcnpooemoeei,w next we myogenesis, promote the can Matn2 on regulate Nfix directly and might Nfix whether that myogenesis assessed early finding during previous kinetics comparable with that observation the on Based Matn2 h d1mtto oeaeyicesdteP sites. the overlapping increased distal moderately the mutation to two Mdm1 binding the The for that indicating compete binding, can the NFI proteins inhibited Low to MyoD interaction. weakly of this amounts abolished bound (NfMdm1) motifs isoform consensus NFI both 6D,F). was CTF1 (Fig. binding (Mdm1) the Pd motif this and MyoD and MyoD the site, Both in start mutation Pd by transcription the abolished shift the to mobility near binding Electrophoretic MyoD element 6D). showed (Fig. (EMSA) proteins assay both for sites u esddrn h omto fmliulae myotubes P multinucleated the in motifs of formation the arrow). 6C, during differentiation,(Fig. myoblast ceased C2 of stages but early the during transiently n h TGF- the can Matn2 that concluded and we MRFs Overall, two 5D). the activate (Fig. of cells expression sh3 the abrogated from fully and level and Matn2 control TGF- Matn2-expressing whereas myoblasts, of sh3-res2 differentiation upon and induction TGF- Matn2 of extracellular Accordingly, induction low differentiation. that myoblast suggest data TGF- our medium, TGF- and decrease p21, the Interestingly, the myoblasts. in sh3 differentiating from Matn2 ifrnito ih tmi atfo h niiinof inhibition the from part Trf3 in stem might differentiation etdwiho h ptem(P upstream the of which tested euneaayi eeldpttv F-adMyoD-binding and NFI- putative revealed analysis Sequence 2 2 n ehp also perhaps and p21 , 135/ b d die uieaeatvt Fg H.Ni xrsinin expression Nfix 6H). (Fig. activity luciferase -driven inln c ncnett nii 2 xrsinand expression p21 inhibit to concert in act signaling 1 stasrbdfo w rmtr (Ma promoters two from transcribed is satvtdb h ygncNi n yDregulators MyoD and Nfix myogenic the by activated is ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal RAado ihmlclrms an complexes Matn2 high-molecular-mass of and mRNA Trf3 2 b 0 ptemeeet n on uain disrupting mutations point and element, upstream 107 compromised 1 Trf3 n 2 xrsinadmoei differentiation, myogenic and expression p21 and d Trf3 Matn2 ein nldn oetal vrapn binding overlapping potentially including region, b , n usqetyreduced subsequently and –mdptwymdae niiinof inhibition 1–Smad-pathway-mediated Nfix xrsin hra ohTGF- both whereas expression, Nfix b and -rae h el,wihdisplayed which cells, sh3 1-treated b Matn2 ute eue h o eesof levels low the reduced further 1 Trf3 u rdwsra rmtr(P promoter downstream or ) expression. Nfia b ute eue h silenced the reduced further 1 and nuto ndifferentiation in induction RAlvl paralleled levels mRNA Nfix Matn2 RA accumulated mRNAs ´ te ´ b ta. 02,we 2002), al., et s MyoD blse the abolished 1 Matn2 d 2 xrsin As expression. ciiyi C2 in activity 2+5DNA 12/+15 d and d increased silencing u -specific d b sdid as , Matn2 i not did and 1 d Myog )can ,

Journal of Cell Science EERHARTICLE RESEARCH Fg IJ.Cepeso fMo n fxddntrelieve not did Nfix repressor and P a the as MyoD of acted repression of MyoD MyoD-mediated Coexpression whereas species, 6I,J). Nfib (Fig. and Nfia the TGF- of effect The 5. Fig. salaigcnrl ,tie;d ie;m ooe.()QTPRo he oldprle utrsuigteSB re protocol. green SYBR the using cultures parallel Cytoskeletal pooled expression. three p21 of and QRT-PCR Smad4 (D) Matn2, monomer. for m, samples dimer; pooled d, three trimer; of t, analysis control. blot loading Western a (C) as immunoblotting. by assessed was b .()Imnfursetsann for staining Immunofluorescent (A) 1. b ntedfeetaino tl h n h-e2myoblasts. sh3-res2 and sh3 Ctrl, of differentiation the on 1 a atnno a fdfeetain B md// hshrlto uigtedfeetaino h oto elline cell control the of differentiation the during phosphorylation Smad1/5/8 (B) differentiation. of 3 day on -actinin d niaigta yDcan MyoD that indicating , fiiybnigt ieoverlapping high- their site MyoD with a with agreement and to in assays binding Nfix, EMSA by affinity in activation binding the competitive block fully ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal el eeclue ndfeetainmdu ihadwtotTGF- without and with medium differentiation in cultured were Cells Matn2 b atnserved -actin transcription 3247

Journal of Cell Science EERHARTICLE RESEARCH 3248 activation the assessed also we the 2008), of al., activate et (Ichikawa to cells reported other Smad was TGF- putative BMP7 the harbors As mediate also might promoter that Matn2 sites The site. initiation 6. Fig. e etpg o legend. for page next See Matn2 rmtrb M7 efudta M7ldt a to led BMP7 that found We BMP7. by promoter b niioyefc Fg 6D). (Fig. effect inhibitory 1 MATN2 n t rmtrin promoter its and ylwdsso fcadNi Fg 6K). (Fig. Nfix and Nfic of doses low by ygnss hra h xrsino yDa ae tgsof early stages in later at species MyoD NFI of of expression upregulation the whereas the myogenesis, from results activity hefl nraei P in increase threefold vrl,w ocueta rnin nraein increase transient a that conclude we Overall, ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal d ciiyadicesdistransactivation its increased and activity Matn2 P d

Journal of Cell Science E tutr fteP the of Structure (E) a eemndi MA ,fe rb.()Teefc fteNIadMyoD and NFI the of P the effect on The D (G) in shown probe. mutations free motif F, EMSA. in determined was tiigada nrae ubro Ki67 of 7E,F; number (Fig. increased dystrophy an muscular and desmin mild staining decreased Consistently, S4H). a Fig. material indicating supplementary mice, type rncit.Pie ar pcfcfrP the for and specific promoters pairs alternative Primer two transcripts. the E1–E3, exons the illustrating the of Regulation 6. Fig. ARTICLE RESEARCH eeldadlydrgnrto in Immunofluorescence regeneration of 5 delayed 7G,H). and (Fig. a 3 days revealed at respectively mice Matn2-deficient regeneration, in observed were cells notexin-induced in following injury muscle anterior tibialis of establish regeneration myogenesis further in Matn2 To of newborn S4A–G). function than the was Fig. adults in material signal in lower (supplementary intensity was Matn2 mice signal endomysium and the high peri- and epi-, MTJs, 2A,G), the and (Fig. tendons nerves, muscles in observed rat to regeneration Similarly muscle proper impairs deficiency Matn2 positive-feedback This of expression high expression. ensure would Matn2 loop regulatory increase further differentiation of myoblast upregulation early transient the the during activate explains each other 4–6), Nfix (Figs the and Matn2 of that expression finding the with together involving repress might differentiation # ynce a losgiiatyhge nbt h nrae and untreated of the both muscles in regenerating higher significantly also was myonuclei el,NIpoen a nraeP increase can proteins NFI cells, the on the pFMyoD) from and of MyoD activity (pFNfi luciferase and plasmids proteins expression NFI of of amounts expression indicated forced of effect The (H–K) iewsmr eeoeeu n ie pitn curdmore occurred splitting fiber and in heterogeneous myofiber frequently cross- the more myofiber but significantly, was average differ The body size not 7A). did to (Fig. (CSA) weight area 3–5 muscle sectional days the at in ratio weight seen were differences Significant niec fcnrlyncetdmoiesicesdfivefold increased myofibers untreated nucleated the in centrally of incidence Pd oxrsinwt fxcnrpesi J.Bp a nraeP increase can Bmp7 (J). it repress can Nfix with coexpression C.()Ncetd eune hwn h uaiefco-idn ie nthe in sites factor-binding putative P differentiation the myoblast showing sequences C2 Nucleotide and (D) (B) (C). regeneration soleus during pairs primer otewl-yeadmtn Pd mutant and wild-type isoform) the (NFIC to GST–CTF1 and GST–MyoD S-transferase), (glutathione GST ybat tl eetdisd h aeetmmrn fthe of membrane basement the regenerating of inside and number necrotized large detected the and still staining myoblasts desmin delayed the by indicated of downregulation the differentiation. myogenic to contribute Matn2 would expression MyoD Matn2 initial The in regeneration. increase muscle in and increase differentiation myoblast oedpnetatvto yNi n fx() aarpeetthe represent Data (K). Nfix mean and Nfic by activation dose-dependent eiqatttv TPRaayi sn P using (Ma analysis previously RT-PCR described Semi-quantitative been have and (arrows), depicted P d , 2 einadi h idtp W)admtn esoso N elements DNA of versions mutant and (WT) wild-type the in and region 0.05, 2+5adPd and 12/+15 6 ... * s.e.m.; terydfeetainsae,weesteltrices in increase later the whereas stages, differentiation early at xrsini eaiefebc opdrn terminal during loop negative-feedback a in expression ### Matn2 Matn2 P Matn2 , Nfix P Matn2 .0 a indicated). (as 0.001 , ncot( knockout 2 .5 ** 0.05, Matn2 Pd( ciaina h ne ftemoei program, myogenic the of onset the at activation 135/ d and lcfrs uincntut F h idn fpurified of binding The (F) construct. fusion -luciferase xrsina a sepce ola oan to lead to expected is 1 day at expression 2 2 342)Luc / 2 2 Matn2 P 2 0.Mttdncetdsaesoni oe case. lower in shown are nucleotides Mutated 107. Nfia , / hni idtp ie(i.7–) The 7B–D). (Fig. mice wild-type in than 2 Matn2 .1 *** 0.01, 2 Matn2 uce,adtepooto fcentral of proportion the and muscles, 2+5adPd and 12/+15 Matn2 rmtrb yDadNfi. and MyoD by promoter d nC ybat n O- el.I COS-7 In cells. COS-7 and myoblasts C2 in xrsin(i.4) hc would which 4I), (Fig. expression d ciiy() hra yDaoeo upon or alone MyoD whereas (I), activity Matn2 ciiyof activity u 2 P 2 n P and / 2 , 2 / 2 .0 rltv to [relative 0.001 / nvivo in 2 n idtp ie(i.7). (Fig. mice wild-type and ) ieta ntoeo wild- of those in than mice u n P and - d ucefbr tdy and 5 days at fibers muscle xrsin mechanism A expression. Matn2 Pd( n o nrnccnrl(C are (IC) control intronic for and 2 135/ 2 enx oprdthe compared next we , 342)Luc d + seii n control and -specific dfcetmc,as mice, -deficient 2 n Ki67 and 0 N elements DNA 107 ´te Pd( Nfix nC myoblasts. C2 in Matn2 se l,2002). al., et ´s d A Schematic (A) 2 ciiyadits and activity , 342)Luc Nfia + Matn2 MyoD nvivo in during and ], + oneuaini h xrsinof expression the in downregulation xrsinof expression ale ciaino 2 and p21 2, of day activation on earlier of activation 50–100-fold levels a the after Interestingly, 8B). (Fig. uclrdsrpywr enin seen mild were of signs regeneration, dystrophy of stages late muscular At 7I). (Fig. post-injury 7 eiinycnas fetitgi inln Fg CD.p42 in 8C,D). inhibited (Fig. also signaling was integrin Matn2 phosphorylation affect that (Erk2) also indicated can respectively, deficiency differentiation, their during mroi a 65(1.)(al ) an eiinyalso deficiency Matn2 the 1). of (Table expression the (E16.5) decreased significantly 16.5 day embryonic ifrnito akr ( markers differentiation in derived a myoblasts drop revealed and analysis QRT-PCR limbs 361-fold Remarkably, fetal mice. newborn in from expression gene specific S4I,J). Fig. material (supplementary animals wild-type neato a otiuet ygncdfeetainhas role differentiation crucial a myogenic uncovers study to This understood. contribute poorly have this remained how and muscles can However, development interactions. myopathic interaction muscle cell–ECM proper of and upon dependence function normal crucial the of underlined studies Numerous DISCUSSION versus h al tgso ucedifferentiation. muscle might of integrin MyoD stages and and early BMP7 Nfix fetal the timely with of and forms regulator proper the it drive early and loops we activation and regulatory transient Thus, key its feedback genes. a that muscle and of as differentiation, fetal acts myogenic cascade of Matn2 the activator that the conclude on Nfix, for turning and MRFs selectively needed thereby differentiation, is Matn2 S3C). Fig. material (supplementary fpiaymols ifrnito eeasse in assessed were induction transient 28–50-fold a of differentiation by of accompanied activation was 160-fold expression myoblast transient a medium, differentiation primary of yfbrsltigas curdmr rqety whereas older frequently, in and pronounced more myonuclei more occurred not Central was also S4H). fibrosis 8A; splitting Fig. Fig. 7I; myofiber (Fig. material fibrosis increased supplementary and inflammation necrosis, of aaipyta an eiinyipistegoa switch as global such preventing regulators, the robustly myogenic by of impairs induction differentiation deficiency the muscle Matn2 terminal our that towards TFIID 2007), the Tjian, imply of and switch (Deato and data the complex E14.5 with Trf3–Taf3-containing along a between 2010), to place al., from et takes switch (Messina myogenesis E17.5 Nfix-directed fetal to an embryonic Because regulators. myogenesis ucegns n tas rvne h nuto fthe of induction the the prevented Nfic also with it and interfered genes, muscle deficiency Matn2 of activation Thus, transient 8B). (Fig. induced Taf3 ybat hmevs steepeso of expression the as themselves, myoblasts MyoD erae ta n a xrsinin expression Fak and Itga5 Decreased et etse h feto an eiinyo h muscle- the on deficiency Matn2 of effect the tested we Next, ae oehr u aaidct httemyoblast-secreted the that indicate data our together, Taken ycnrs,i h bec fMt2 h xrsinof expression the Matn2, of absence the in contrast, By h euaoycsaeidcdb an seiie ythe by elicited is Matn2 by induced cascade regulatory The Trf3 Tfr3 , nemdae fteBMP/TGF- the of intermediates , MyoD Matn2 , Fak ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal , Taf3 Taf3 , , Nfic +/+ and Itga Myog MyoD , Nfix , utrdmolss(al ) hntekinetics the When 1). (Table myoblasts cultured Nfix Nfi and n te ygncmre ee a not was genes marker myogenic other and , . and Nfix Trf3 Fak and Nfic Eno Myog and , Itga5 ndifferentiating in Trf3 Myh1 , xrsin olwdb robust by followed expression, Nfia Myh1 Nfia ekda a ,floigthe following 7, day at peaked eealdcesdin decreased all were and ihakntc iia othose to similar kinetics a with euaigftladembryonic and fetal regulating and )in Matn2 Nfix Matn2 Taf3 Bmp7 Matn2 b , 2 Nfia inln pathways. signaling MyoD Matn2 a / 2 2 Trf3 Matn2 inln during signaling 5 nuto during induction Trf3 2 n yelevated by and / 2 , / ie including mice, Matn2 2 Bmp7 , , ybat and myoblasts +/+ ea ib at limbs fetal Fak and Taf3 2 / 2 Matn2 myoblasts Bmp7 2 and Nfix / n late and , 2 hnin than Matn2 Bmp7 3249 Trf3 mice . Taf3 2 and / 2 , ,

Journal of Cell Science EERHARTICLE RESEARCH n uigmolssi eurdfrtml myoblast timely for required is 3250 myoblasts fusing differentiating of proliferating, and upregulation around transient deposition Matn2 a by differentiation that followed muscle find skeletal We in regeneration. Matn2 and component ECM the for 7. Fig. e etpg o legend. for page next See Matn2 hscnlso sspotdb h idn htslnigor silencing that finding the by supported of knockout is regeneration conclusion muscle during This and culture in differentiation ybat yihbtn h ciaino Rs p21, MRFs, of activation the inhibiting by myoblasts ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal Matn2 easmoei ifrnito rmcultured from differentiation myogenic delays Nfix n other and nvivo in .

Journal of Cell Science htMt2cnmdf nernadohr(..Fk Erk2) Fak, (e.g. other that and and integrin modify pathways Matn2-dependent can signaling the show data Matn2 Our for 8E). (Fig. that hypothesis differentiation myogenic a of onset timely propose we the literature, in involved genes of Matn2 integrin activation and as the Bmp7 pathways, impairs signaling also various deficiency al., expression coordinating et gene Mourikis by muscle-specific 2007; induce changes al., thus et might Biressi Matn2 2005; 2012). al., et (Blais transition of aa n i7Mo muoloecne rohaspitto point Arrowheads Ki67 immunofluorescence. Ki67/MyoD Ki67/ and Lama2/desmin, by Lama2 revealed was muscles regenerating Matn2-deficient i.7 idmsua ytoh n eae iilsatro muscle anterior tibialis delayed and in dystrophy regeneration muscular Mild 7. Fig. ARTICLE RESEARCH n etrdteidcinof induction the restored and in expression Matn2 rescuing Conversely, genes. myogenesis-related rwhfcosuo aae(i.8) an ih otiueto releasing contribute might reservoir, Matn2 polynucleated 8E). a (Fig. of provides damage upon ECM rather factors growth organized myofibers and highly myotubes the signaling, 8E). factor of (Fig. myoblasts upregulation in this genes Matn2-dependent 2008), muscle-specific the al., and MRF et mediate Deato might 2007; Tjian, complex and (Deato differentiation to with known is formed complex Trf3–Taf3 activate complexes the As Smad factors. transcription multiprotein Matn2-elicited other of on the composition turn to Thus, the needed is program. signaling differentiation ECM muscle of the activation the impairs previous the with well correlates This 2010). by activation the with differentiation interferes 2008; of also al., muscle deficiency et Matn2 Deato while 2007; 2007). terminal genes Tjian, Jones, and marker (Deato in myogenic complex others many of Trf3–Taf3 inhibiting subset role a the activating pivotal that specifically proposal a the these plays support differentiation, myogenic further of data modulator ( global upstream MRF an of ( activation promoter genes muscle-specific core the the and of transition complex of global the lack compromising thereby a by accompanied ynce futetd(,rgt n eeeaig()auttbai anterior tibialis adult all (F) myonuclei/animal, versus regenerating (2000 non-peripheral and shown of right) are ratio (E, of the untreated myofibers and of all left) versus myonuclei (E, nucleated centrally anterior of tibialis ratio untreated The post-injury. 28 day on myofibers/muscle, (300–400 (C) ( regeneration uniida h niae asatrntxnijcini idtp (WT, wild-type in injection notexin and after bars) days closed indicated the at quantified otergto h aniae l uniaiedt ersn the represent data quantitative All image. main images the single-channel mean as of 28 shown right and is the 14 area to days outlined The on dystrophy. the myofibers mild whereas regenerating shows regeneration, and delayed degenerating on indicate of myofibers post-injury enrichment regenerating 7 of and membrane 5 basement myoblasts days the numerous inside the mice, detected Matn2-deficient still desmin-positive In around myotubes. 3–7 and days myoblasts between upregulation Matn2 transient group, nuclei/animal Ki67 ae ntepeetwr n aaaalbefo the from available data and work present the on Based hra h os C urudn ybat aosgrowth favors myoblasts surrounding ECM loose the Whereas h ucergnrto ea in delay regeneration muscle The Matn2 Nfix Matn2 + + MyoD MyoD 6 ... * s.e.m.; hc eie h ea ygnccl ae(esn tal., et (Messina fate cell myogenic fetal the defines which , slne ybat teutdtedlyi differentiation in delay the attenuated myoblasts -silenced MyoD + + ea-pcfcepeso uigtemyoblast–myotube the during expression fetal-specific uliwti h MyoD the within Ki67 nuclei of Quantification (H) cells. n 5 P Matn2 Matn2 –) h S rqec itiuin()adaeaeCSA average and (B) distribution frequency CSA The 3–6). , n ocueagoa wthtwrsterminal towards switch global a cause to and .5 ** 0.05, n 5 a 2 2 ) I obeimnfursec indicates immunofluorescence Double (I) 3). inln ahasdrn differentiation. during pathways signaling 5 / / 2 2 P Myh1 K,oe as ie A egtcagsduring changes Weight (A) mice. bars) open (KO, mice. , .1 *** 0.01, Trf3 n .I diint dniyn an as Matn2 identifying to addition In ). Nfix AF iilsatro eeeainwas regeneration anterior Tibialis (A–F) 5 Trf3 + ) D uniiaino pi myofibers split of Quantification (D) 3). n xrsinadmrel delays markedly and expression 5 ulipplto (10,000–30,000 population nuclei Matn2 P n ygncmre genes. marker myogenic and ) G rlne rlfrto in proliferation Prolonged (G) 3). , and 0.01. Trf3 + essalnce and nuclei all versus MyoD Taf3 eiinydramatically deficiency osbyb modifying by possibly , Matn2 nvivo in induction , Myog 2 / 2 observation ,p1and p21 ), iewas mice nvivo in , ygnss uha yg esssutlltrsae,when stages, of later regulators until downstream persists Myog, and as targets such MyoD myogenesis, of expression pcfcgn xrsinpormdie yfrhrpositive (Halevy muscle- p21 further the and of Myog by MyoD, initiation driven by the formed program loops such promote expression autoregulatory reached, turn, are gene in Nfix specific and can, Matn2 Nfix ensure both might that of loop levels positive-feedback sufficient This that of 8E). expression (Fig. the another activate Nfix one and Matn2 whereby loop feedback It 2009). TGF- al., and that et finding (Schabort the incorporates differentiation also myoblast inhibit are to acts signals Matn2-elicited nucleus. cell extracellular the to the transduced fully to how required are (Knight studies understand fusion further However, myoblast 2011). in Kothary, and implicated was that phosphorylation euaoycsaeft elwt ro bevtoso the ( of observations genes prior muscle This ( with 2010). genes fetal al., MRF well et of induction Aziz fits concomitant 1997; Perlman, cascade and regulatory Walsh 1995; al., et nkeigwt hs idns eosre eeewound-healing severe in observed defects we findings, Matn2 healing. these skin-wound and with of BMP7 keeping timing of the In control and to 2010) 2008) al., al., et et (Ichikawa Plasari 2009; al., of the et observations TGF- (Plasari prior in between with al., Nfix line relationships et in (Nebl of regulatory also types are role cell conclusions other Our described in is 1994). previously pathways p21, mitogenic regulatory as a of such a regulation by inhibitors, of cycle supported interpretation cell also Our and Nfix 2010). between al., interplay et Wright, Messina and (Funk 1992; transcription can muscle-specific species in Nfi cofactor that a finding as the act with and 2006), al., et Ouellet 1999; tgso ifrnito Fg E.Ti sacmaidb the by accompanied is This of 8E). MyoD, a repression (Fig. whereby concomitant Nfix, differentiation of of established block will stages also Nfix, be of effect possibly the can overcoming and loop Matn2 negative-feedback of expression increased TGF- Matn2 whereby mechanism molecular a Dea ciainrqie nernbnigadcnrcieforces contractile and binding their and integrin ECM, (Massague the requires in structures activation fibrillin-bound and or fibronectin fibronectin- with TGF- interaction instance, For TGF- its fibrillin. might through al., modulate Matn2 pathways et ECM, indirectly signaling Klatt the and in 2007; or protein al., adaptor complex directly et an Mann as laminin–nidogen 2002; Working 2011). al., fibrillins, et proteins, (Piecha collagens, ECM fibronectin and as integrins with interaction such its by adhesion cell f-idn ie nthe in of reports sites prior Nfi-binding with be consistent be to would remains this intermediates, However, established. regulatory these between control occurs directly also can Nfix Whether 2010). the gene al., muscle-specific et MyoD-dependent (Aziz expression increase can (Biressi Nfix cells genes, 2010). C2 al., et differentiating Messina 2007; in al., and et myotubes embryonic ifrnito.As differentiation. of activation transient u oe si iewt rvososrain htTGF- that observations previous with line in is model Our ycnrligteepeso frgltr n C target ECM and regulatory of expression the controlling By ial,ormdlpoie ehnsi xlnto o the for explanation mechanistic a provides model our Finally, ´ MyoD n .Ks,dt o hw) u eut rvd vdnefor evidence provide results Our shown). not data Kiss, I. and k Matn2 ih ocmtnl oto F expression. NFI control concomitantly might ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal ´ , Matn2 02.Mt2dfcec loipie 4 (Erk2) p42 impaired also deficiency Matn2 2012). , Myog xrsin hspeetn h ne fapositive- a of onset the preventing thus expression, 2 n/rp1gns n hte edakloop feedback a whether and genes, p21 and/or / 2 MyoD ieo 5B/ eei akrud(F. background genetic C57BL/6 a on mice Matn2 Myog Myh7 xrsini undo nrsos to response in on turned is expression Matn2 b , yforced by ) aiymmesaesoe nlatent in stored are members family n 2 rmtr Jhno tal., et (Johanson promoters p21 and MyoD , Nfix and b MyoD b b b poe M signaling BMP opposes 1 -eitdrglto of regulation 1-mediated and BPSa n other and /BMP/Smad Matn2 Myog n te f species Nfi other and Nfix , MyoD Myog uigmyogenic during xrsina late at expression xrsinin expression hra the whereas , , Mef2a and ) 3251 b 1

Journal of Cell Science EERHARTICLE RESEARCH 3252 differentiation. myoblast during expression gene marker on Matn2 of effect The 8. Fig. e rosidct ciainpoessafce ietyo nietyb an inln.B,bsmn ebae n fibronectin. Fn, membrane; basement BM, illus signaling. are Matn2 differentiation by myoblast indirectly to or expression directly Matn2 affected linking processes interactions activation Regulatory indicate differentiation. arrows myoblast Red of model A (E) densitometry. by efre sn h yrre rtcl eut hwtemean Matn2 the show Results protocol. SybrGreen the using performed yfbr cuuae hc trcsnmru D5pstv nlmaoycls(ro) edn oicesdfboetnadclae- deposi collagen-3 in and expression fibronectin gene increased marker to of leading analysis (arrow), QRT-PCR cells (B) inflammatory insets). CD45-positive numerous attracts which accumulate, myofibers 2 / 2 K)piaymols utrs D A eeto yimnbotn nmols utrsdifferentiating cultures myoblast in immunoblotting by detection FAK (D) cultures. myoblast primary (KO) xvivo ex 6 ...()Imnbo nlsso integrin of analysis Immunoblot (C) s.e.m. ifrnitn ybatclue eie from derived cultures myoblast differentiating ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal A mardrgnrto tdy2 in 28 day at regeneration Impaired (A) a nlmso 1. mro n nwl-ye(T and (WT) wild-type in and embryos E16.5 of limbs in 5 xvivo ex Matn2 o n ,tepoen eequantified were proteins the D, and C For . 2 / 2 and Matn2 Matn2 +/+ 2 rtdschematically. trated ebr mice, newborn / 2 ie Necrotic mice. in(magnified tion

Journal of Cell Science Bmp7 Itga5 MyoG MyoD 2 +0.43 p21 Ckm Nfix Nfic Matn2 Eno iddsrpy u osntbokmsl ifrnito or differentiation as muscle mice, block transgenic not in does regeneration but dystrophy, why explain mild can This arrows). black 8E, Matn2 (Fig. Matn2 of presence nebynco ea ucedvlpetwsosre ylight that by observed observed recently was we development However, muscle microscopy. fetal or embryonic in rmtrocpnyb ygi 2myocytes. C2 in Myog by occupancy promoter fiinl n oesol hnupon less than but slowly program, also more regulatory might myogenic and proteins the efficiently ECM and other MyoD by activate signaling with combination ihu biu bomlte (Ma abnormalities obvious without Myh1 Nfib al .Cmaio fteepeso eeso akrgnsi ea ib n ybatclue from cultures myoblast and limbs fetal in genes marker of levels expression the of Comparison 1. Table ARTICLE RESEARCH aafo h NOEcnotu htrva osbebnigof the binding possible to a reveal MyoD that consortium ENCODE the from data reports is online regulation recent Matn2 by in supported MRFs of involvement The expressed. become differ terminally of components a earpoess si sepesdi ait fcommitted of variety a (Szabo in of liver processes regenerative expressed the in functions is it and types it the cell proliferating facilitates as of also range Matn2-rich processes, broad possibly the a repair to NMJs the 1996), contributes likely at al., Matn2 guide process. TSCs et reinnervation and (Son by and NMJs produced induce differentiation at matrix TSCs nerves myogenic As of timely sprouting regeneration. and muscle proper skeletal for 2013). required al., et (Sartori atrophy dramatic a caused Fak eet ihvral eeiydpnigo h genetic the on depending severity Smad Dea variable (F. background with defects (Fulla diethylnitrosamine-induced to hepatocarcinogenesis susceptibility increased an display eesmo vrg ratio Average symbol Gene ieb QRT-PCR by mice Taf3 codnl,ohrmmeso h F aiyhv been have the in NFI family tissue adult NFI with of TGF- the processes concert regenerative of other in in and members implicated tissues, other factors various Accordingly, of transcription and processes differentiation cell of steps repair early the regulate thereby can and it communication, that ECM–cell support controls together Matn2 data that These hypothesis 2009). the al., et (Malin nerves peripheral Nfia Trf3 Mean nteasneo an,BPsgaigaoeo in or alone signaling BMP Matn2, of absence the In vrl,ti okietfe an sa motn C protein ECM important an as Matn2 identifies work this Overall, 2 6 e1x)n rdc in product no (ex1ex2) / ... n s.e.m., eiinyol easmoei ifrnito,causing differentiation, myogenic delays only deficiency 2 ieas hwdaml ypty u denervation but myopathy, mild a showed also mice 5 Matn2 ´ .C 9. ta. 07,si Ihkw ta. 08 and 2008) al., et (Ichikawa skin 2007), al., et ´ 2 2 2 2 2 2 2 2 1. ea ib ybatcultures Myoblast limbs fetal E16.5 2 2 2 t n .Ks,dt o hw) uce of Muscles shown). not data Kiss, I. and k +0.24 +0.46 +0.01 auswr omlzdt h C the to normalized were values 1.87 1.59 0.53 1.35 2.40 1.69 6.05 0.93 0.64 3.55 8.50 rmtri 2molssadincreased and myoblasts C2 in promoter 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 .67.66E 0.46 .90.0006*** 0.29 .30.006** 0.33 .80.001*** 0.68 .40.0007*** 0.54 .50.03* 0.55 .93.73E 2.09 .90.01** 0.69 .60.04* 0.56 .17.2E 0.61 .93.87E 1.19 .401 +1.17 0.18 0.34 .302+1.30 0.2 0.33 .204+1.37 0.4 0.12 .409 +0.99 0.94 0.34 ´ ta. 04 n wound-healing and 2014) al., et r nitdmoies uha Myh1, as such myofibers, entiated a Matn2 fcrmtnimmunoprecipitation chromatin of o2( log2 ´ te ´ ta. 04.N deficiency No 2004). al., et s 2 D b / 2 C Matn2 t mro opoutin product no embryos ) Trf3 inln pathways. signaling 2 / 2 nuto nthe in induction t Matn2 P au fteinvariant the of value iedevelop mice au odcag vrg ratio Average change Fold value 2 2 2 2 09*** 2 07*** 05*** 10*** / 2 mice 2 2 2 360.79 2 2 2 2 2 2 2 2 66.26 11.71 3.20 1.44 2.55 1.90 3.64 5.27 3.20 1.56 Rps18 sdsrbdpeiul (Za previously described as ifrnito eimo a n a hne daily. changed was and 0 day times. The three least 0. at day on repeated In were as microscope. experiments TGF- medium Differentiation defined R medium Cell ng/ml was 5 differentiation Olympus differentiation this an experiments, to and with by some photographed cultures induced serum, were was horse confluent cultures Differentiation 2% 80% 1995). containing al., serum the bovine et thigh fetal switching (Halevy the 20% 1997), containing al., Gibco) from et medium (FBS, (Springer prepared growth previously in al., were described propagated et as were Montarras which mice 1977; neonatal myoblasts, Saxel, of and muscle primary (Yaffe and 7 clone 1996) line cell myoblast the C2 Csongra under Institute, and culture Cell Control Animals and Laboratory Care of Health Health of Use Hungary. Animal County, Institutes and the National Care of the approval the with treatments for accordance animal Guide All in muscle. conducted 0.3 anterior were injecting tibialis the by into littermates (Sigma-Aldrich) wild-type and mice at stored and by weighed control removed, of were muscles legs soleus anesthetized notexin-treated entire The or (10 pentobarbital. 20 were sodium of of hydrate overdose an injection rats chloral intramuscular by Wistar 4% induced was of 3-month-old necrosis injection study, intraperitoneal this For treatment Animal METHODS AND MATERIALS ta. 04.Msl eeeainwsidcdi 10-week-old in induced was regeneration Muscle 2004). al., et reigi spnaecoe ylqi nitrogen. liquid by cooled isopentane in freezing otx fTGF- of context rtis uha an,t h euaino h ne fa of onset the target of ECM regulation processes. the regeneration and tissue to factors adult Matn2, of transcription variety as NFI these such Collectively, the 1997). proteins, al., link et al., Iozzo et 1996; findings (Alevizopoulos al., genes et ECM Alonso other 1995; TGF- NFIC several in An as reported 2010). TGF- acting been al., a et sites harbor (Lee Plasari to NFI-binding regeneration 2009; shown hair al., and was to et isoform leading Plasari healing cycle 2009; al., follicle skin-wound et the of morphogenesis, onset tooth the in implicated h eeainof generation The .* P , ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal .5 ** 0.05, 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0.90 1.42 1.39 0.57 0.82 0.88 1.21 0.97 0.40 1.27 0.69 1.32 0.55 1.84 2.38 P 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 , .00.01** 0.30 1 0.00001*** 018 .10.0004*** 0.21 .50.02* 0.15 .30.0014** 0.13 .60.006** 0.16 .60.002** 0.16 .40.004** 0.24 .70.34 0.37 .20.02* 0.42 .50.05* 0.25 .30.03* 0.43 .70.02* 0.07 .10.002** 0.71 .60.0009*** 0.46 Matn2 .1 *** 0.01, b inln.Frisac,Ni a been has Nfic instance, For signaling. a Matn2 o2( log2 ´ 2 o ta. 96.Teaiaswr ildby killed were animals The 1996). al., et dor / P 2 , 2 iehsbe eotdpeiul (Ma previously reported been has mice D .0 (two-sample 0.001 / 2 C t culture au odchange Fold value P ) b Imntos a de to added was (Immunotools) 1 b rsosv lmnshave elements -responsive Matn2 b rsosv oan and domain, -responsive 2 m t / -test). 2 (60 g esswild-type versus m m m 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 fnotexin, of g )o notexin of l) /) Muscle l/g). 1.87 2.67 2.63 1.48 1.76 1.84 2.31 1.95 1.31 2.42 1.62 2.49 1.46 3.59 5.21 2 80 Matn2 ˚ after C 3253 ´ 2 te ´ / ´ 2 d s

Journal of Cell Science rmr n R-ojgtdscnayatsr r itdin the listed at with repeated are quantified were Experiments times. were antisera three (BioRad). Images 1999). least program al., S1. secondary One-4.2.3. et Table Quantity (Piecha HRP-conjugated material previously supplementary and described as Primary developed and blotted omlsrm uliwr tie ihDP.NJ eedetected for were NMJs proper used DAPI. 10% S1. with with antibodies Table stained blocked were material was secondary Nuclei supplementary antibodies serum. in the normal and listed of binding are Nonspecific coverslips Primary 10- on acetone-fixed grown 1999). of Belgium). (Piecha immunofluorescence al., previously Mariakerke, described using Software, as determined et performed (MedCalc were was distributions software Immunohistochemistry fiber-size Digimizer and the CSA average The immunofluorescence and immunohistochemistry Histology, ARTICLE RESEARCH 3254 following 2005), derivative al., the generate et to 1 of (Kaufman system total pSUPER A vector KD2639. the for pFP/Neo-H1 inserted recommendations and the phosphorylated explorer RNAi annealed, into program were the Oligonucleotides using 4D). Matn2 (Fig. targeting shRNA designed We rescuing and Silencing Mix Master at min Expression 15 program: Gene following TaqMan the 2011). with al., 95 the used et were and (Nagy Biosystems) previously (Applied sets described probe with as performed TaqMan protocol, was of grown QRT-PCR green limbs. cultures SYBR cultures fetal differentiating the E16.5 three myoblast or from also primary medium from and medium three growth isolated in lines, was either cell RNA C2 For total S2). differentiating Table pooled material QRT-PCR, (supplementary (Dea fragments previously ( cDNA muscles described gene-specific soleus as rat analysis from northern isolated for was RNA total Pooled analysis RNA 1% 4 EGTA, containing mM 10,000 at 1 buffer at EDTA, centrifugation min mM ice-cold After 10 1 inhibitors. in NaCl, protease M and extracted 0.15 X-100 7.5, Triton was pH Tris-HCl layer mM 20 cell myoblast analysis The immunoblot and extraction Protein with equipped softwares. 8.0 Photoshop X4 Adobe microscope CorelDraw The with and created camera. Windows; were for E600 Slider Figures 4.0.9 (version Instruments). RT software Diagnostic Eclipse SPOT Spot using a processed were Nikon with images (Dako), photographed medium a and mounting epifluorescence with fluorescent with viewed mounted were specimens tetramethylrhodamine with eaiegn xrsinlvl r rsne slog as presented are levels expression gene Relative 4hatrpaig h el eesee no1-mdse o 2-week a for At dishes (Fermentas). 10-cm 500 into 500 containing seeded ExGene medium were growth using cells in the vector DNA selection plating, control helper after pFP/Neo-H1 pFvFP h or of 24 KD2639 ng purified 50 of and ng 500 with medium rmr n aMnpoest r itdi upeetr material supplementary in listed (C cycle are threshold sets Individual S2. probe Table TaqMan and primers values. CycloAb oteaeaeC average the to tbytransfected stably xrsini tlattreidpnetexperiments. (sh3- gene independent rescued marker three and and least differentiation lines at myogenic cell in for expression sh7) tested and were sh4, cultures res2) (sh3, 1 silenced to (Ctrl), for recombinant silenced control resistant the line and cell were cDNA sh3 vector the that modified expression into The introduced pCEP-Pu was 4D). the plasmid (Fig. into capacity inserted but coding was shRNA the protein by targeted its sequence preserving the mutating by Matn2 mouse length colonies. single resistant orescue To ˚ ;4 ylso 95 of cycles 45 C; SB re)o oteC the to or green) (SYBR ) ˚ ,spraatpoen eesprtdb SDS-PAGE, by separated were proteins supernatant C, Matn2 t auso he nenlcnrlgns( genes control internal three of values Matn2 iecn,w oiidtecN noigfull- encoding cDNA the modified we silencing, 6 ˚ 10 o 5sad60 and s 15 for C slne ellnswr salse rmG418- from established were lines cell -silenced 5 m Matn2 /lprmcnwr old Independent pooled. were puromycin g/ml 2molsswr otasetdi growth in co-transfected were myoblasts C2 a bnaooi Ivtoe,120.The 1:200). (Invitrogen, -bungarotoxin expression t ausof values t auswr omlzdeither normalized were values ) ˚ m m o i.Gene-specific min. 1 for C roetosadcells and cryosections m /lG1 Cmrx,and (Cambrex), G418 g/ml xvivo ex Hprt ´ ta. 97,using 1997), al., et k ndifferentiation in Tqa probe). (TaqMan Hprt 2 n Matn2 aiso fold or ratios 5 )adused and 3) , Rps18 Clones . g and for experiments. u %plarlmd gel. polyacrylamide 5% run (Szabo previously 1.25 with or odvle eaiet htfor as the that expressed to were to vectors activities relative empty Luciferase mean DNA. values appropriate total the fold of Opti-MEM with amount BMP7 in adjusted same ng/ml 50 were tested of effect mixtures The was 2011). al., (ImmunoTools) et (Nagy previously described h rga aprwsue osac o uaieNIadMyoD and NFI double- synthesize putative to for and promoter search Matn2 to the used in sequences was recognition Jaspar program The EMSA (Ma previously described as promoter- 6A), using (Fig. PCR 2002). oligo(dT)- pairs by to primer followed synthesis, specific subjected cDNA were first-strand Biochemicals) primed Molecular (Roche DNase 1- nuclei. analysis promoter of Alternative number the total as the mean defined by was the divided index represent nuclei differentiation as myotube Data defined The harboring was of cells. index myotubes number of the fusion The number of as field. total microscope number calculated given was a the in fields frequency cells of microscope containing myotube myotubes chosen of of nuclei, Primary number sarcomeric randomly 3000–4000 evaluation counted. of for In were the total (DAPI). stained a For nuclei were containing protocol. and (Promega), cultures the kit actinin manufacturer’s myoblast with proliferation the performed cell differentiation, was to solution function One according mitochondrial AQueous for CellTiter96 assay MTS assays The differentiation and proliferation Cell eemnduigaoewyaayi fvrac AOA ihKyPlot * with 15; (ANOVA) beta mean variance 2.0 of the version analysis one-way as a using presented determined are Data analysis Statistical Pd( assay expression Transient 1.25–2.5 with incubated were probes the binding, NFI of 0.6– fmol dC) with 20–30 incubated purified 2011). were were probes al., 2.5 DNA proteins et mutant the and (Nagy wild-type and previously end-labeled expressed, described were as 1999) GST–MyoD essentially and al., 1995) al., et et (Alevizopoulos (Biesiada NFIC an human the GST–CTF1, of encoding isoform Constructs 6D). (Fig. oligonucleotides stranded fmueNIgns(huhye l,19)i pcDNA5 pFNfib, in 1998) (pFNfia, sequences al., coding et proteins the (Chaudhry harbour NFI genes Forced plasmids NFI mouse mouse latter of 2011). The gift or a pFNfix). and (pFMyoD, al., 3–5 Sartorelli) pFNfic MyoD for with et Vittorio plasmids phosphate from expression performed (Nagy of were calcium amounts were previously increasing activities the assays described luciferase or expression and as (Fermentas) respectively, measured, 500 method, ExGene coprecipitation using plates 3–5 with transfected (Stratagene). kit Mutagenesis Directed arigpitmttosi h yDmtfna h tr ie(Mdm1, site start the near motif MyoD the Pd in mutations point carrying the into osnu eune NMm)o h f/yDcmoieelement composite Nfi/MyoD the of Pd (NfMdm1) sequences consensus m 2molss(utrdi rwhmdu)adCS7clswere cells COS-7 and medium) growth in (cultured myoblasts C2 2 2 lqoso oa N ape rae ihRNase-free with treated samples RNA total of aliquots l 2 N m oyd-C,a ecie rvosy(isaae l,19) otest To 1999). al., et (Biesiada previously described as poly(dI-dC), 2+5 nteNImtf(f1 ri ohteNIadMyoD and NFI the both in or (Nfm1) motif NFI the in 12/+15) 135/ fGTo S–yDi h rsneo 0 go poly(dI- of ng 300 of presence the in GST–MyoD or GST of g 6 342)Luc ...fo tlattreidpnetexperiments. independent three least at from s.e.m. Pvu ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal 2 0 eemd sn h C ae QuikChange based PCR the using made were 107 II/ m Pst a osrce yisrigthe inserting by constructed was aho S-ue T1adMo,a described as MyoD, and CTF1 GST-fused of each g ´ ieo h G3BscVco Poea.Derivatives (Promega). Vector pGL3-Basic the of site I ta. 95,adsprtdb lcrpoei napre- a on electrophoresis by separated and 1995), al., et m P fwl-yeadmtn eotr nsix-well in reporters mutant and wild-type of g , .5 ** 0.05, 6 ...fo he ofv independent five to three from s.e.m. , P 6 ulidvddb h oa number total the by divided nuclei 3 , ...Saitclsgiiac was significance Statistical s.e.m. .1 *** 0.01, Pd( § 2 a 342)Luc ulidvddb the by divided nuclei 3 atnnpstv myoblasts -actinin-positive H P , 2 Gbo.Transfection (Gibco). m 0.001. 4/6 P 342/+60 frpresand reporters of g eut r the are Results . m fGST–CTF1 of g 9 TFA,as UT-FLAG, d ´ te fragment ´ TM setal., Site- a -

Journal of Cell Science epciey.Dpstdi M o meit release. immediate for BO/00349/03, PMC and in BO_00781/11/8 Deposited numbers respectively]. [grant Sciences of Academy h artwork. the Aniko assistance, expression MyoD the Aniko Emo for thank CA) also Angeles, We Sartorelli, Los plasmids. Vittorio California, and of Southern antiserum (University of Wagener Matn2 (University Raimund for vector, Germany) pFP/Neo-H1 Cologne, of Cologne, gift the for Germany) Zolta thank We Acknowledgements ARTICLE RESEARCH uk .D n rgt .E. W. Wright, and D. W. Funk, Roncaglia, E., Tenedini, S., Monteverde, G., Lamorte, E., Tagliafico, S., Biressi, V. Sartorelli, and L. Kedes, Y., Hamamori, E., Biesiada, J. F. Dilworth, and C. Q. Liu, A., Aziz, R. A. Kornblihtt, and G. C. Pesce, R., C. Alonso, Wahli, T., Weid, der von M., Tsai-Pflugfelder, Y., Dusserre, A., Alevizopoulos, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.141556/-/DC1 online available material Supplementary material Supplementary A the Avidin by to co-financed GOP-1.3.1-11/B-2011-0002 TA Union, numbers Ltd., European [grant the Fund by Regional number supplied European to [grant also 304077] OTKA was FP7-PEOPLE-2011-CIG from Funding number grants [grant L.M. by Curie and Marie Hungary I.K.; and Council, to PD-101421]; Research 008/2006] Medical number Fund the Research [grant Scientific and (ETT) Hungarian TO49608]; the number from [grant grants (OTKA) by supported was work This Funding manuscript. the I.K. wrote revisions; and financed research and the experiments L.M. conceived manuscript; evaluated the and edited designed and L.G.P. data and interpreted evaluated and materials, reagents provided contributed N.M. L.D. results; and L.S. data; analyzed and experiments E L.M., F.D., contributions Author interests. competing no declare authors The interests Competing et,M . ar .T,Steo . nue . u .adTin R. Tjian, and P. Hu, C., Inouye, T., Sottero, T., M. Marr, D., M. Deato, R. Tjian, and D. M. Deato, Dea Dea Dynlacht, and Y. Kluger, R., Sharan, D., Acosta-Alvear, M., Tsikitis, A., Blais, Fulla M. R. Gronostajski, and D. A. Vitullo, Z., A. Chaudhry, Charge ´ Z n .e eespotdb h Ja the by supported were L.Me. and .Z. idn ie o ai ei-ophlx ula atr1 myocyte-specific 1, factor nuclear recognizing helix-loop-helix, factors basic with for interacts sites myogenin binding complexes: multicomponent for by revealed is myoblasts fetal cells. purified and on analysis embryonic expression gene genome-wide of diversity al. et phenotypic S. Tajbakhsh, G., Intrinsic M. Angelis, Cusella-De S., multiprotein alpha-actin Ferrari, S., Ferrari, cardiac E., a human of the components of activity as for interact promoter. required Sp1 complex and transcriptional proteins helix-loop-helix muscle. skeletal in 691-695. expression gene tissue-specific establishing H3. histone with interacts activator N. Mermod, and W. yDtresTF/R3t ciaemoei transcription. myogenin activate to TAF3/TRF3 targets MyoD myogenesis. during proteins. matrix extracellular oligomeric of family matrix superfamily. cartilage module of A-like relative type factor closest Willebrand Chem. the von matrilin-2, the within of protein expression and structure D. B. the of transcription the in ATF-2 with cooperate NF-I gene. and fibronectin CP1 proteins NI sfrsdfeetal ciaesml esscmlxNFI-responsive complex versus simple activate differentially promoters. isoforms (NFI) regeneration. muscle 553-569. 105. uo eeomn i r12adGSK-3 and Erk1/2 e93469. via development tumor Duda k . aee,R,Ks,I n alsn M. Paulsson, and I. Kiss, R., Wagener, F., ´k, I. Kiss, and M. Paulsson, C., Bachrati, D., Piecha, F., ´k, k Horva ˝ke r . ah,K,Dea K., Baghy, A., ´r, ´ ,S .adRdik,M A. M. Rudnicki, and B. S. ´, s . is .adKvlzy I. Kovalszky, and I. Kiss, J., ´s, O-..--0801 n TA and MOP-4.2.2-1-2008-0013 20) niiilbupitfrmoei differentiation. myogenic for blueprint initial An (2005). 272 ´ o.Cl.Biol. Cell. Mol. K,A .K., t,KtlnHegedu Katalin ´th, 9268-9274. , .Bo.Chem. Biol. J. nIis(a Delbru (Max Ivics ´n ´Go ´ Z,TS,MK,LM. .. B.O A.K., L.Me., M.K., T.S., .Z., .Bo.Chem. Biol. J. readErno and ¨rbe ee Dev. Genes 19) rln-ihTFbt-epnietranscriptional TGF-beta-responsive proline-rich A (1995). hso.Rev. Physiol. k . Pe F., ´k, 19 20) wthn ftecr rncito machinery transcription core the of Switching (2007). 273 io,Erzse Simon, ´ 2577-2584. , 19) ylcapiiainadslcino targets of selection and amplification Cyclic (1992). sadGbilaLeho Gabriella and ˝s 18538-18546. , ˝Za c etrfrMlclrMdcn,Berlin, Medicine, Molecular for Center ¨ck 271 21 tri,B,Zsa B., ´terfia, drfrtcnclavc n Ma and advice technical for ´dor 20) ellradmlclrrglto of regulation molecular and Cellular (2004). ee Dev. Genes 2137-2149. , 84 22271-22279. , ´ MOP-4.2.2.A-11-1-KONV-2012-0035]. nsBla elwhpo h Hungarian the of fellowship Bolyai ´nos 209-238. , 21) euaigamse regulator: master a Regulating (2010). 21) ako arln2fvr liver favors Matrilin-2 of Lack (2014). btBala ´bet b ahasi vivo. in pathways ´ n ..performed H.J. and . 9 k . Ta Y., ´k, arxBiol. Matrix 19) h arln:anovel a matrilins: The (1999). 3051-3066. , c o xeln technical excellent for ˝cz ´sha 19) h CCAAT-binding The (1996). z,KadaKa Klaudia ´zy, e.Biol. Dev. 19) ygncbasic Myogenic (1999). 19) ula atrI factor Nuclear (1998). ta,P,Shf,Z., Schaff, P., ´trai, 18 o.Cell Mol. ee Dev. Genes 19) Primary (1997). 55-64. , Epigenetics LSONE PLoS 304 raTo ´ria ´vai, 633-651. , 32 (2008). (2007). .Biol. J. t for ´th 96- , 19 5 9 , , , afa,C . Izsva D., C. Kaufman, A. K. Jones, oasn . ens . ag,K,Bcbre,A,Anl,H .and H. H. Arnold, A., Buchberger, K., Ragge, H., Meents, M., Johanson, oz,R . ilrsti . hra . udc,A . ailo,K . Uitto, G., K. Danielson, D., A. Murdoch, B., Sharma, J., Pillarisetti, V., R. A. Iozzo, Nakagawara, and T. Ozaki, T., Koda, Y., Suenaga, T., J., Ichikawa, G. Hannon, J., Rhee, X., S. Skapek, B., D. Spicer, G., B. S. Novitch, O., F.Halevy, I. C. Tiger, and Schofield, L. Lohikangas, T., and Velling, D., B. Gullberg, J. Harris, D., B. Grubb, M. R. Gronostajski, ob,A,Bce,D . u,L,Sekvc,E,Keas . ad,E and E. Bagdi, L., Krenacs, E., Stelkovics, L., Dux, L., D. Becker, A., Gorbe, A. C. Henry, and F. M. Goody, an .H,Snl,G,Gbur .M,El,J . alsn .and M. Paulsson, A., J. Eble, M., Massague J. Gebauer, G., Sengle, H., H. Mann, Aszo R., Wagener, D., Guseva, E., Sonnenberg-Riethmacher, D., Malin, otc,S . ak,T . alro .D,Rcado,J .adGry .J. D. Garry, and A. J. Richardson, D., T. Gallardo, J., T. Hawke, C., S. Goetsch, P. Rotwein, and D. Kuninger, P., Knollman, D., Alzhanov, S., Gardner, el . emd .adCt,A C. A. Cato, and N. Mermod, G., Nebl, Ma M., R. Gronostajski, H., H. Son, S., J. Ko, M., H. Kim, T., J. Park, S., D. Lee, R. Kothary, and D. J. Knight, R. Wagener, and M. Paulsson, D., C. Neacsu, K., A. Becker, R., A. Klatt, Ma ay . Ke A., Nagy, C. Pinset, and F. Gros, J., IIan, T., Johnson, F., Aurade, D., Montarras, L., Perani, M., Cassano, A., Magli, S., Monteverde, S., Biressi, G., Messina, orks . oaarsnn . absvn .adTjahh S. Tajbakhsh, and R. Sambasivan, S., Gopalakrishnan, P., Mourikis, ´te ´te hknt h core. the to shaken rnpsnbsdRA etr eit fiin eekokoni human in knockdown gene efficient mediate cells. vectors RNAi transposon-based protein. E1A adenovirus by inhibited Commun. is Res. myf5 Biophys. Biochem. of recruitment MEF2-mediated Sandmo eavaancerfco -idn element. 1-binding factor nuclear a factor- growth via transforming beta by activation Transcriptional promoter. gene perlecan A. Mauviel, and J. wounding. to 994-1000. response keratinocyte in in involved migration is matrilin-2 of expression MyoD. DeltaNp63/BMP-7-dependent by p21 of induction with B. muscle A. skeletal Lassar, and D. Beach, regenerating dystrophies. muscular the in and development in muscle junctions rat. neuromuscular the of formed muscle newly soleus at transmission development. ycrnzdcl yl oto rcd ybatfso nrgnrtn skeletal regenerating in vivo. fusion in myoblast muscle precede control cycle cell synchronized T. Krenacs, development. embryonic mediate 77 matrix extracellular their oa deinformation. adhesion focal R. Wagener, regeneration. D. nerve peripheral in Riethmacher, participates 2 matrilin and A. Irintchev, 20) rncitoa rfln n euaino h xrclua arxduring matrix extracellular the of regeneration. regulation muscle and profiling Transcriptional (2003). IGF-II. by initiated TGF- 9484-9488. euaino xrsino rncito atrN1b arsoncogene. Ha-ras by NF1 factor Chem. transcription of expression of regulation Sox9. by L- activation tune promoter: finely TATA matrilin-1 Biol. near Cell. the bound factors of Nfi regulation and Sox5/Sox6 zone-specific plate growth conserved, 630. root tooth during differentiation odontoblast development. C. and J. proliferation Park, cell and odontogenic H. for P. Choung, I., M. Cho, myogenesis. skeletal mammalian to critical assembly. matrix extracellular Biol. of modulators matrilins: The nacrbnigfco ,adCM1factor. COMP1 and 2, factor enhancer-binding 20) oprtv nlsso h os n ua ee Mt2and (Matn2 genes filament-for human and a matrices. mouse matrilin-2, extracellular the for of MATN2) analysis Comparative (2002). euae ea-pcfctasrpini eeoigseea muscle. skeletal al. developing et E. in C. 554-566. transcription Campbell, L., fetal-specific Starnes, regulates E., Tagliafico, E., normal Roncaglia, display matrilin-2 protein development. adaptor adult matrix and extracellular embryonic the lacking Mice . omn .T,Bra . Puska E., Barta, T., S. Oommen, A., of cells. potential stem specification muscle temporal the skeletal maintains activity a Notch Cell-autonomous involves stage. C2, myoblast operating the line, MyoD, at and cell as II factor early myogenic growth as insulin-like mouse between control the positive mutual in differentiation Autonomous s . ops . De E., Korpos, L., ´s, s . ioa,C,Mo C., Nicolae, L., ´s, 475-488. , 43 b .RA eeSilencing Gene RNAi J. ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal niismsl ifrnito ybokn uorn inln pathways signaling autocrine blocking by differentiation muscle inhibits ,J. ´, 269 320-330. , lr A. ller, ¨ ns,E,Rnsnoj . Molna O., Rentsendorj, E., ´nesi, 31 7371-7378. , 21) TGF (2012). 20) rncito taeisi emnlydfeetae cells: differentiated terminally in strategies Transcription (2007). 686-699. , 20) rnin peuaino onxn3gpjntosand junctions gap connexin43 of upregulation Transient (2005). 20) arln eit ekcl tahetwtotpromoting without attachment cell weak mediate Matrilins (2007). Gene .Bo.Chem. Biol. J. itce.Cl Biol. Cell Histochem. 19) rncitoa ciaino h ygnngn by gene myogenin the of activation Transcriptional (1999). 20) oe fteNICFgn aiyi rncito and transcription in family gene NFI/CTF the of Roles (2000). 19) tutrladfntoa hrceiaino h human the of characterization functional and Structural (1997). o.Endocrinol. Mol. ee Dev. Genes 249 k . azr .adIis Z. Ivics, and A. Katzer, Z., ´k, hso.Genomics Physiol. arxBiol. Matrix a,F,Lu . ee,D . Aszo R., D. Beier, Z., Liu, F., ´ak, b 31-45. , gln . Dea M., rgelin, ¨ .Physiol. J. inligi context. in signalling arxBiol. Matrix Development 284 19) orlto ftria elccears of arrest cycle cell terminal of Correlation (1995). 1 21) yai neatosbtenclsand cells between interactions Dynamic (2010). 21) h ygncknm:poenkinases protein kinome: myogenic The (2011). 97-104. , 21 17293-17303. , 21 .Cl Sci. Cell J. arxBiol. Matrix 265 2113-2117. , 25 441 163-174. , 123 ice.Bohs e.Commun. Res. Biophys. Biochem. 26 128-137. , 20) h xrclua-arxprotein extracellular-matrix The (2009). 222-232. , s .G tal. et G. L. ´s, k . is .adAszo and I. Kiss, F., ´k, 405-421. , 167-174. , igpoenwdl itiue in distributed widely protein ming 573-583. , 14 139 20) ula atrICi essential is I-C factor Nuclear (2009). r . Sze A., ´r, 19) ottasrpinldown- Post-transcriptional (1994). .Bo.Chem. Biol. J. klt Muscle. Skelet. 261-271. , 4536-4548. , a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 109 rc al cd c.USA Sci. Acad. Natl. Proc. 23 Science rn.Biosci. Front. 195-204. , 551-560. , ´na .Cl Sci. Cell J. 19) Neuromuscular (1991). s,T,Sinko T., ´si, 19) nern during Integrins (1998). 267 21) Evolutionarily (2011). 20) rgPrince Frog (2005). 272 n.J ice.Cell Biochem. J. Int. i .adKs,I. Kiss, and A. di, ´ o.Rpo.Dev. Reprod. Mol. 1 1018-1021. , 29. , 5219-5228. , 3 D1039-D1050. , 122 i A. di, ´ 21) Nfix (2010). . Zvara, I., , ´ 995-1004. , Cell 13 i A., di, ´ (2008). (2004). (2012). (1996). (2011). (2011). .Biol. J. 3255 ,616- 369 140 Mol. 89 , , ,

Journal of Cell Science ule,S,Vgeut . esr,M,Lcec . run .adGue and R. Drouin, S., Leclerc, M., Lessard, F., Vigneault, S., Ouellet, E. Brandan, and N. Osses, ARTICLE RESEARCH 3256 eoi,S n uln .J. M. Cullen, and S. Sesodia, U. M. C. L. Niesler, Sorokin, and and P. F. F. Schuler, Moore, B., Loos, M., Merwe, der van J., E. Schabort, Mo C., Wiberg, D., Piecha, Mo S., Muratoglu, D., Piecha, atr,R,Shri,E,Bau,B,Broaz,S,Za,J,Ez,E., Enzo, J., Zhao, S., Bortolanza, B., Blaauw, E., Schirwis, R., Sartori, and R. Abraham, B., Blaauw, C., Mammucari, M., Patron, G., Milan, R., Sartori, R. J. Sanes, M. Grounds, M., Davies, M., Maley, R., Hallmann, C., Roder, B., Ringelmann, Ho S., Edelmann, A., G., McNair, Plasari, M., R. Gronostajski, Y., Dusserre, A., Calabrese, G., Plasari, C394. expression. factor regulatory muscle of independently opooyo eeeaigrtslu muscles. soleus rat regenerating of morphology vivo. in and vitro an in cells in myogenic differentiation cell progenitor muscle manner. skeletal isoform-independent delay TGF-beta’s (2009). networks. fibrillar forms and cells of variety great a Chem. by expressed is Dea and M. Paulsson, cells. 1A human inhibitor proliferating in kinase NFI cyclin-dependent 6487. by the gene of (p21) regulation Transcriptional (2006). tnzu . oie,E,Tnoo . er,A tal. et A. Ferry, mass. L., muscle Toniolo, controls E., Mouisel, A., Stantzou, adulthood. M. Sandri, dystrophic of Chem. muscle Biol. J. skeletal in tenascin-C mice. and dy/dy 129ReJ fibronectin, chains, alpha5 L. Sorokin, and wound cycling. follicle skin to A. signaling Calabrese, beta1 factor growth transforming progression. healing and factor N. Mermod, growth and L. Michalik, proteins. matrix M. Paulsson, 274 13353-13361. , m .Physiol. J. Am. 20) h aeetmmrn/aa aiao kltlmuscle. skeletal of lamina membrane/basal basement The (2003). 20) md n rncito atr oto ucems in mass muscle control factors transcription 3 and Smad2 (2009). 20) arln2itrcswt tefadwt te extracellular other with and itself with interacts Matrilin-2 (2002). 278 .Bo.Chem. Biol. J. 21) ula atrICrgltsTFbt-eedn hair TGF-beta-dependent regulates I-C factor Nuclear (2010). ice.J. Biochem. 19) xrsino aii lh1 lh2 lh4 and alpha4, alpha2, alpha1, laminin of Expression (1999). 12601-12604. , x.Cl Res. Cell Exp. o.Cl.Biol. Cell. Mol. k F.´k, a.Genet. Nat. gln . enad,D . Dea P., D. Reinhardt, M., rgelin, ¨ 20) C srqie o kltlmsl differentiation muscle skeletal for required is ECM (2002). 296 19) arln2 ag,oioei arxprotein, matrix oligomeric large, a Matrilin-2, (1999). 367 x.Cl Res. Cell Exp. gln . asr . tdr . is I., Kiss, D., Studer, N., Hauser, M., rgelin, ¨ 19) xrsino aii sfrsi mouse in isoforms laminin of Expression (1995). 285 C1248-C1257. , 20) ula atrIClnsplatelet-derived links I-C factor Nuclear (2009). gr . usre . emd .and N. Mermod, Y., Dusserre, F., gger, ¨ 715-721. , 19) h feto eevto nthe on denervation of effect The (1991). 34115-34125. , .Cl Sci. Cell J. 45 246 29 1309-1318. , 6006-6017. , 165-182. , 315 108 caNeuropathol. Acta uli cd Res. Acids Nucleic 373-384. , 3795-3805. , m .Physiol. J. Am. 21) M signaling BMP (2013). k . arr .and P. Maurer, F., ´k, 82 282 rn .L. S. ´rin, 34 21-32. , ,C383- 6472- , .Biol. J. i,H,Pie .adRdik,M A. M. Rudnicki, and F. Price, H., Yin, Za H. Asahara, and S. Yokoyama, af,D n ae,O. Saxel, and D. Yaffe, Watt, C., Beyer, C., B. Bernardo, Y., Liu, H., Qian, L., J. Chen, E., C. C., Winbanks, Beyer, V., P. Sepulveda, E., R. Thomson, L., K. Weeks, E., C. Winbanks, H. Perlman, and K. Walsh, M. I. Conboy, and J. A. Wagers, Thorsteinsdo Szabo Szabo M H. Blau, and T. Rando, L., J. M. Springer, W. Thompson, and T. J. Trachtenberg, J., Y. Son, h,X n ar,D J. D. Garry, and X. Shi, dr . ede,L,VrHyn . u,L n utc,F. Wuytack, and L. Dux, M., Heyen, Ver L., Mendler, E., ´dor, elniche. cell sfrsi h a oesmsl eeeaigfo oei-nue necrosis. notexin-induced from Ca(2+)-ATPase regenerating sarcoplasmic/endoplasmic-reticulum muscle J. soleus the Biochem. rat the of in levels isoforms mRNA in Sci. Life Mol. Cell. el sltdfo ytohcmuemuscle. mass. mouse muscle dystrophic al. from skeletal isolated et of cells J. regulator B. positive Turner, a T., Biol. is Cell Connor, J. axis E., al. protein R. et morphogenetic Thomson, A. I., M. Febbraio, K. I., G. myostatin. Smad3 Lancaster, of by independently M., regulated is mTOR J. hypertrophy and Allen, muscle skeletal L., Follistatin-mediated J. (2012). Chen, H., Qian, Dev. Genet. Opin. Curr. myogenesis. adult in controversies and Cell concepts current regeneration: muscle 191-207. xrclua arxdmnino kltlmsl development. muscle skeletal of dimension matrix extracellular vlclsdrn a ie regeneration. liver rat during cells oval Toke I., ftecriaemti rti gene. region protein silencer the matrix in cartilage site the protein-binding family of factor-I nuclear a of Identification e ok Y onWly&Sons. & Wiley John NY: York, junctions. New neuromuscular In of muscle. reinnervation and sprouting Neurosci. guide Trends and induce n disease. and . Lo E., , ´ . ota . ecnoj . Ra A., Rencendorj, J., Moitra, P., , ´ 122 s .M,Lt,G,La G., Lotz, M., A. ´s, ora fCl cec 21)17 2035 doi:10.1242/jcs.141556 3240–3256 127, (2014) Science Cell of Journal 659-667. , i . ops . amnh . otnegr . Dea Z., Rottenberger, E., Batmunkh, E., Korpos, C., di, ´ hso.Rev. Physiol. tr . eis . Cachac M., Deries, S., ttir, ´ urn rtcl nHmnGenetics Human in Protocols Current 203 320 ee Dev. Genes 345-357. , 107-113. , 19 68 280-285. , 1843-1849. , 17) eilpsaigaddfeetaino myogenic of differentiation and passaging Serial (1977). 20) ucese el ndvlpet regeneration, development, in cells stem Muscle (2006). 7 93 597-602. , 19) elcceei pnmoei differentiation. myogenic upon exit cycle Cell (1997). 20 23-67. , ´szlo 1692-1708. , 21) h ygnctasrpinlnetwork. transcriptional myogenic The (2011). .e al. et V. , ´ 20) ellradmlclrsgaue of signatures molecular and Cellular (2005). 21) aelt el n h ucestem muscle the and cells Satellite (2013). .Bo.Chem. Biol. J. key . ac,T n is I. Kiss, and T. Rauch, G., ´khely, arxBiol. Matrix o .S n aac,F. Bajanca, and S. A. ¸o, 19,rvsd20) eedlvr to delivery Gene 2002). revised (1997, . .Cl Biol. Cell J. 20) xrsino arln2in matrilin-2 of Expression (2007). Nature e.A .Bye,Ui 13.4. Unit Boyle), L. A. (ed. 26 270 197 270 554-560. , 10212-10221. , 19) cwn cells Schwann (1996). 997-1008. , 725-727. , 21) h bone The (2013). 19) Changes (1996). e.Biol. Dev. 21) The (2011). k . Kiss, F., ´k, (1995). 354 ,

Journal of Cell Science