SUPPLEMENTARY INFORMATION

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SUPPLEMENTARY INFORMATION oi:1.138/nture12343 Myogenin M-Cherin E9.5 E11.

tissue Tongue Lim Musle Hert Kiney Brin Liver Lung Bler Supplementry Figure 1. is skeletl musle-speifi in the emryo., E9.5 n 11.

5 n in the entire myotome of E11.5 emryos. is expresse t reltively lower levels t E9.5 ompre to E11.5. The top of the emryos piture is rostrl n the ottom ul.

1 SUPPLEMENTARY INFORMATION oi:1.138/nture12343 Myogenin M-Cherin E9.5 E11.5 Thymus Spleen White ipose tissue Tongue Lim Musle Hert Kiney Brin Liver Lung Bler Stomh 28s Fol expression (Reltive to Thymus) Thymus Spleen White ipose tissue Tongue Lim Musle Hert Kiney Brin Liver Lung Bler Supplementry Figure 1. is skeletl musle-speifi in the emryo., E9.5 n 11.5 emryos were setione trnsversely n rioisotopi in situ hyriiztions for, Myogenin, n M-herin were performe. Eh trnsript exhiite expression in the somites t E9.5 n in the entire myotome of E11.5 emryos. is expresse t reltively lower levels t E9.5 ompre to E11.5. The top of the emryos piture is rostrl n the ottom ul. Imges were pture using rkfiel mirosopy, onverte to re pseuo olor, n overlye on rightfiel imge., RNA ws isolte from multiple tissues of E19 wil-type emryos n ssesse for the presene of trnsripts y Northern lot., Quntittive rel-time PCR for on the sme tissues s in (). Sle rs: 2 μm. Stomh 1

RESEARCH SUPPLEMENTARY INFORMATION Kpn I Lous Trgeting Vetor X I 5 Proe 3 Proe 1 1s 2 3 4 5 FRT FRT 5 Arm E2SA

k) Trgete Lous Kpn I X I Kpn I X I Kpn I FRT FRT 1 E2SA IRES βgl β-t neo 1s 2 3 4 5 loxp loxp +/- +/- +/- +/-

+/- +/- +/- Qurieps P6 P6 P26 e Fol expression (Reltive to y ) 15 1 5 E17.

5 P7 Totl pups 5 25 57 Oserve Expete Oserve Expete Oserve Expete 13 (26%) 13 (25%) 8 (32%) 6 (25%) 24 (42%) 14

(25%) Supplementry Figure 2. The -LZ llele repitultes expression of RNA n loss of is lethl.

The strong splie eptor site (E2SA) results in -LZ fusion n interruption of ownstrem trnsription, generting

, Tissues hrveste from P6 +/- mie were stine with X-gl.

, X-gl stining on forelims from +/- emryos emonstrtes expression in musle fiers. The enotes ones.

2 RESEARCH SUPPLEMENTARY INFORMATION Kpn I Lous Trgeting Vetor X I 5 Proe 3 Proe 1 1s FRT FRT 5 Arm E2SA IRES βgl β-t neo 1s 2 3 Arm t (4.9 k) loxp loxp (6. k) Trgete Lous Kpn I X I Kpn I X I Kpn I FRT FRT 1 E2SA IRES βgl β-t neo 1s loxp loxp +/- +/- +/- +/- +/- Tongue Hert Bler Brin White ipose tissue Forelim. +/- +/- +/- Qurieps P6 P6 P26 e Fol expression (Reltive to y ) E17.5 +/- 3 7 Criotoxin injury (Dys) f Fol expression 1..5 (Reltive to ) 1.5 E13.5 E14 g Age E17.5 P7 Totl pups Oserve Expete Oserve Expete Oserve Expete 13 (26%) 13 (25%) 8 (32%) 6 (25%) 24 (42%) 14 (25%) myomker +/- 24 (48%) 25 (5%) 11 (44%) 13 (5%) 33 (58%) 29 (5%) 13 (26%) 13 (25%) 6 (24%) 6 (25%) 14 (25%) Supplementry Figure 2. The -LZ llele repitultes expression of RNA n loss of is lethl., An IRES-β-gltosise n neomyin ssette ws inserte in intron 1. The strong splie eptor site (E2SA) results in -LZ fusion n interruption of ownstrem trnsription, generting null llele. These trgete ES ells were otine from KOMP s esrie in Mterils n Methos., Tissues hrveste from P6 +/- mie were stine with X-gl. -LZ expression ws not etete in ny non-skeletl musle tissues., X-gl stining on forelims from +/- emryos emonstrtes expression in musle fiers. The enotes ones., Qurieps from P6 WT, n P6 n P26 +/- mie were stine with X-gl to visulize postntl own-regultion of the -LZ llele. e, WT tiilis nterior (TA) musle ws injure with riotoxin n nlyze for trnsripts y qpcr t y, 3, n 7 fter injury. f, RNA ws sent from the tongue musle of E13.5, E14, n emryos. Dt re presente s men ± SEM. P <.5 ompre to. g, null emryos were otine t expete Menelin frequenies t n E17.5. However, we i not oserve knokout t P7 ue to lethlity. Sle rs:,, 2 mm;, 2 μm. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Diphrgm Interostl E12 MyoD Fol expression (Reltive to ) E17.5 Msseter Myogenin e E14 Forelim E13.5 E14 f Desmin 4 Diphrgm TUNEL DAPI g Supplementry Figure 3. is neessry for proper musle formtion espite norml speifition., Prffin setioning n H&E stining on iphrgm n interostl musles revel lk of musle fiers in E17.5 emryos. The epits ones. The enotes iphrgm., Msseter musle from n E17.5 WT n emryos ws prffin setione n H&E stine to emonstrte the neessity of for formtion of skeletl musles of the he., MyoD in situ hyriiztion shows omprle expression in E12 WT n emryos., qpcr for myogenin revele no ifferenes t the inite ges in the tongues of WT (n=3) n (n=3) null mie. Dt re presente s men ± SEM. e, E14 forelims n iphrgm were setione n stine with routine H&E. musle is present t this stge of evelopment in the forelims n iphrgm. The enotes ones of the lim. The shows the iphrgm. Sle rs, 2 μm for forelim n 1 μm for iphrgm. f, Desmin stining on WT n forelims emonstrtes the presene of musle ells. The enotes ones. g, forelims were stine for DNA frgmenttion with the TUNEL retion n o-stine with DAPI. Apoptoti nulei were more reily etete in musle ompre to WT musle. Sle rs:,, f, g 2 μm;, 2 mm W W W. N A T U R E. C O M / N A T U R E 3

RESEARCH SUPPLEMENTARY INFORMATION, 3 Fusion Inex, 5 Hoehst Myosin % nulei in

expression (Reltive to ) 5 4 3 2 1 Myogenin Ckm Myh4 f y 1 y 2 y 3 y 4 Myosin

>1 1-3 4-1 >1 # of nulei h % nulei in myotues Fusion Inex 8 6 4 2 Supplementry

, null myolsts were ifferentite n we oserve smll numer of i-nulete ells (rrow).

ell with t lest two nulei)., myolsts file to fuse even fter 5 ys of ifferentition.

lter levels of ifferenttion (n=3 for n n=3 for ).

On y 3, myotues with >2 nulei were pprent in C2C12 ells with over-expresse,

g, Quntittion of the perentge of myosin + ells tht ontine the inite numer of

4 RESEARCH SUPPLEMENTARY INFORMATION, 3 Fusion Inex, 5 Hoehst Myosin % nulei in myotues Hoehst Myosin y: Myosin Myogenin GAPDH e Fol expression (Reltive to ) Myogenin Ckm Myh4 f y 1 y 2 y 3 y 4 Myosin Hoehst g 1 % myosin + ells y 1 y 2 y 3 y > > > >1 # of nulei h % nulei in myotues Fusion Inex Supplementry Figure 4. governs fusion n not the levels of myogeni proteins., null myolsts were ifferentite n we oserve smll numer of i-nulete ells (rrow)., The fusion inex ws lulte s the perentge of nulei ontine in myotues ( myosin + ell with t lest two nulei)., myolsts file to fuse even fter 5 ys of ifferentition., Western lots for myosin n myogenin were performe on C2C12 ells on the inite y of ifferentition fter or infetion. e, qpcr for myogeni ifferentition genes revele tht meite fusion enhnement i not lter levels of ifferenttion (n=3 for n n=3 for ). f, C2C12 myolsts were immunostine with myosin n Hoehst (nulei) to evlute the kinetis of fusion fter or infetion. -infete C2C12s exhiite n inrese rte of pperne of myotues with multiple nulei. On y 3, myotues with >2 nulei were pprent in C2C12 ells with over-expresse, wheres these myotues were not etete in ontrol ultures on this y. g, Quntittion of the perentge of myosin + ells tht ontine the inite numer of nulei on y 1 y 4 of ifferentition. Dt re presente s men ± SEM. P <.5 ompre to -infete ells on tht y of ifferentition. h, The fusion inex on y 4 of ifferentition ws quntifie s in () n showe n inrese in myotue nulei fter infetion. Sle rs:,, 1 μm, f, 2 μm. 4

SUPPLEMENTARY INFORMATION RESEARCH -Flg Kyte-Doolittle Hyropthy Plot 4. Hyrophoiity. Hyrophiliity Flg -4. 2 4 6 8 1 12 14 16 18 2 22 Amino i numer Supplementry Figure 5.

5 SUPPLEMENTARY INFORMATION RESEARCH -Flg Kyte-Doolittle Hyropthy Plot 4. Hyrophoiity. Hyrophiliity Flg Amino i numer Supplementry Figure 5. mino i onservtion n hyrophoiity., Amino i lignment of proteins from Humn, Dog, Pig Mouse, Oppossum, n Zerfish shows strong onservtion., Kyte-Doolittle plot for -Flg shows its highly hyrophoi nture. Regions ove the horizontl line re onsiere hyrophoi. Flg, enote y the green line, ws engineere fter mino i 61 of. W W W. N A T U R E. C O M / N A T U R E 5

Empty -Flg Hoehst Myosin 3 3 3 -Flg RESEARCH SUPPLEMENTARY

-Flg Hoehst GM13 Merge -Flg Hoehst Cylophilin D Merge -Flg Hoehst

Supplementry Figure 6. -Flg lolizes to memrne omprtments.

lot nlysis using Flg ntioy etete expression of -Flg in whole ell

, -Flg ws over-expresse in C2C12 ells to test for its ility to

Cells were stine 4 ys fter ifferentition.

memrne (M) frtions were otine using ifferentil entrifugtion.

frtion ws use to show effiient seprtion of the ellulr omprtments.

ifferentition to ssess o-loliztion with known proteins tht re

Speifilly, the ells were fixe n permeilize n stine with Flg ntioy

6 Empty -Flg Hoehst Myosin Flg RESEARCH SUPPLEMENTARY INFORMATION Tuulin C=ytosol M=memrne Gph -Flg Hoehst EEA1 Merge -Flg Hoehst GM13 Merge -Flg Hoehst Cylophilin D Merge -Flg Hoehst PDI Merge ER Mitohonri Golgi Enosomes -Flg C M Flg VDAC Flg Supplementry Figure 6. -Flg lolizes to memrne omprtments., C2C12 ells were infete with the inite retroviruses n Western lot nlysis using Flg ntioy etete expression of -Flg in whole ell lystes., -Flg ws over-expresse in C2C12 ells to test for its ility to inrese myolst fusion t levels similr to untgge. Cells were stine 4 ys fter ifferentition., C2C12 ells were lyse in hypotoni uffer n the ytosol (C) n memrne (M) frtions were otine using ifferentil entrifugtion. Western lot nlysis using Flg ntioy etete -Flg protein only in the memrne frtion. Detetion of VDAC for the memrne frtion n Tuulin for the ytosoli frtion ws use to show effiient seprtion of the ellulr omprtments., C2C12 ells were infete with -Flg n immunostine on y 2 of ifferentition to ssess o-loliztion with known proteins tht re speifi for ertin intrellulr omprtments. Speifilly, the ells were fixe n permeilize n stine with Flg ntioy n either Erly Enosome Antigen 1 (EEA1, enosomes), Golgi mtrix protein (GM13), Cylophilin D (Mitohonri), or protein isulfie isomerse (PDI, enoplsmi retiulum). Nulei were stine using Hoehst. -Flg exhiite prtil o-loliztion with enosomes n ER. Sle rs: 2 μm. 6 W W W. N A T U R E. C O M / N A T U R E

7 SUPPLEMENTARY INFORMATION RESEARCH Vehile + ytohlsin D + lntrunulin B Myosin Hoehst -Flg - ytohlsin D + ytohlsin D Hoehst Flg Live Supplementry Figure 7. A funtionl tin-ytoskeleton is neessry for funtion., C2C12 myolsts were infete with either - or -retrovirus n ifferentite in the presene of vehile (.1% EtOH), ytohlsin D, or lntrunulin. over-expression i not overome the eleterious effets of tin inhiition on myolst fusion., Live stining of C2C12 ells fter infetion with -Flg virus with n without ytohlsin D tretment emonstrtes tht tin ynmis oes not regulte lotion of on the ell surfe. Sle rs:, 2 μm;, 2 μm. 7

RESEARCH SUPPLEMENTARY INFORMATION 1T1/2 A sre C2C12s BrU Empty

1T1/2 A sre C2C12s BrU Differentite & BrU Stin Chimeri Myotue sre

Supplementry Figure 8. BrU++ firolsts fuse to C2C12 myolsts.

either Empty- or -retrovirus, followe y mixing with sre-infete

Anlysis of BrU inorportion in sre-myotues (rrows) revele ws

We oserve empty-infete BrU+ firolsts juxtpose to sre-myotues, ut

8 RESEARCH SUPPLEMENTARY INFORMATION 1T1/2 A sre C2C12s BrU Empty Differentite & BrU Stin Non-Chimeri Myotue Empty sre BrU Hoehst 1T1/2 A sre C2C12s BrU Differentite & BrU Stin Chimeri Myotue sre BrU Hoehst M Em yo pty m k er % BrU+ myotue nulei Supplementry Figure 8. BrU++ firolsts fuse to C2C12 myolsts., 1T1/2 firolsts were trete with BrU overnight n then infete with either Empty- or -retrovirus, followe y mixing with sre-infete C2C12 ells n inue to ifferentite for 4 ys. Anlysis of BrU inorportion in sre-myotues (rrows) revele ws suffiient to fuse firolsts to C2C12 ells. We oserve empty-infete BrU+ firolsts juxtpose to sre-myotues, ut negligile fusion., Quntittion of the perentge of myotue nulei tht were BrU+ in the inite ultures. Sle rs: 1 μm. 8 W W W. N A T U R E. C O M / N A T U R E

Supplementary figure 1

Supplementary figure 1 Supplementry figure 1 Igf 1 mrna 5 15 1 5 Arg1 mrna 16 1 8 Col11 mrna 5 3 1 Mmp 13 mrna 15 1 5 Tslp mrna 3 1 Il5 mrna 3 1-1 Il33 mrna 6 Figure 1s. Kinetis of woun heling ftors n erly Th-type response ytokines