REPORT◥

MUSCLE DEVELOPMENT

Control of muscle formation by thefusogenic micropeptide myomixerPengpeng Bi,1,2 Andres Ramirez-Martinez,1,2 Hui Li,1,2 Jessica Cannavino,1,2

John R. McAnally,1,2 John M. Shelton,3 Efrain Sánchez-Ortiz,1,2

Rhonda Bassel-Duby,1,2 Eric N. Olson1,2*

Skeletal muscle formation occurs through fusion of myoblasts to form multinucleatedmyofibers. From a genome-wide clustered regularly interspaced short palindromicrepeats (CRISPR) loss-of-function screen for genes required for myoblast fusion andmyogenesis, we discovered an 84–amino acid muscle-specific peptide that we callMyomixer. Myomixer expression coincides with myoblast differentiation and is essentialfor fusion and skeletal muscle formation during embryogenesis. Myomixer localizesto the plasma membrane, where it promotes myoblast fusion and associates withMyomaker, a fusogenic membrane protein. Myomixer together with Myomaker canalso induce fibroblast-fibroblast fusion and fibroblast-myoblast fusion. We conclude thatthe Myomixer-Myomaker pair controls the critical step in myofiber formation duringmuscle development.

Skeletal muscle is the largest tissue in thebody, accounting for ~40% of human bodymass. The formation of skeletal muscle be-gins with the specification of muscle cellfate by the myogenic transcription factors

Pax7 and MyoD, followed by the expression of avast number of genes that establishmuscle struc-ture and function (1, 2). A fundamental step inthis process is the fusion of mononucleated myo-blasts to form multinucleated myofibers (3–7).Similarly, in response to injury, myogenic pro-genitor cells within the adult musculature areactivated and fuse to generate new myofibers(8–10). Whereas many of the initial steps of myo-blast fusion are similar to those of other fuso-genic cell types (11), the components andmolecularbasis of fusion of specific cell types, such asmyoblasts, have not been fully defined.To identify new regulators of myogenesis, we

performed a genome-wide clustered regularlyinterspaced short palindromic repeats (CRISPR)loss-of-function screen for genes required for dif-ferentiation and fusion of C2C12 myoblasts, amouse muscle cell line (fig. S1A). We infected60 million C2C12 myoblasts with a lentiviral lib-rary comprising a pool of 130,209 single-guideRNAs (sgRNAs) and CRISPR-associated protein9 (Cas9) for CRISPR gene editing (12). Lentivirusinfection was performed at a multiplicity of in-fection in order to retain a 460-fold representa-

tion of the library. After puromycin selection for2 days,myoblast cultures were switched to differ-entiationmedium(DM) for 1week so as topromotemyotube formation. The cultures were subjectedto a brief exposure to low trypsin (0.00625%),which promoted the detachment of myotubes,leaving mononucleated myoblasts attached tothe dishes. Subsequent treatment with 0.25%trypsin allowed release of myoblasts. As a con-trol for separation of the myotube and myoblastpopulations, we detected myosin heavy chainby means of Western blot analysis, which washighly enriched in the myotube population(fig. S1B).We enumerated sgRNA representation inmyo-

blast andmyotube populations bymeans of high-throughput sequencing. Genes targeted bymultiplemyoblast-enriched sgRNAs were scored on thebasis of their relative abundance. This analysisrevealed numerous genes that were targeted bymultiple independent CRISPR sgRNAs that wereenriched inmyoblasts. Because we sought to iden-tify genes specifically required for myoblast dif-ferentiation or fusion, we narrowed down thislist by comparing these genes with transcripts thatare up-regulated during differentiation of C2C12myoblasts (13), as well as Pax7+ and Twist2+ myo-genic progenitors (fig. S1C) (14). Five genes fulfilledthese criteria (fig. S1C). One previously uniden-tified gene on the list, annotated as Gm7325, wastargetedby three independent sgRNAs in the initialscreen and stood out as the most up-regulatedgene of unknown function during differentiationof C2C12myoblasts andPax7+ andTwist2+muscleprogenitors (fig. S1C). This gene was also asso-ciated with conserved peaks for genomic bindingof the myogenic transcription factors MyoD andMyogenin (fig. S1D) (15) and was identified as aputative target of MyoD (16).

There is a single publication describingGm7325as a gene expressed in embryonic stem (ES) cellsand germ cells, but the function was not eluci-dated (17). Here, we show that Gm7325 is re-quired for myoblast fusion and promotes themixing of cell membranes; thus, we named thisprotein Myomixer. In the experiments that fol-low, we demonstrate the key role of this proteinin the control of myoblast fusion and muscleformation.The mouseMyomixer gene spans three exons

with the open reading frame (ORF) in exon 3 (fig.S1E). To confirm that Myomixer is essential formyoblast fusion, we disrupted the gene in C2C12cells and mouse primary myoblasts with lentivi-ruses that expressed Cas9 and a pair of sgRNAsthat were separated by 122 base pairs (bp) in theMyomixer ORF (fig. S1E). Sequencing of genomicpolymerase chain reaction (PCR) fragments gen-erated with primers flanking the sgRNA targetsequences from these puromycin stably selectedcell cultures revealed numerous indel mutationsthat disrupted theMyomixerORF (fig. S1F).Westernblot analysiswith an antibody against amino acids24 to 84 confirmed the absence of Myomixerprotein in the knockout (KO) cultures (Fig. 1Aand fig. S1I).Disruption of the Myomixer gene prevented

fusion of C2C12 cells and primary mouse myo-blasts but did not affect expression of myosinheavy chain, a marker for differentiation (Fig.1, A and B, and fig. S1, G to I). Quantification ofthe percentage of myonuclei in mono- versusmultinucleated cells showed that ~89% of myo-blasts targeted with the Myomixer sgRNAs weremononucleated after transfer to DM for 7 days(Fig. 1C). We never observedmyotubes with morethan five nuclei in the Myomixer KO cultures,whereas ~82% of myonuclei in wild-type (WT)cultures were contained in myotubes with morethan fivenuclei (Fig. 1C).MyoDandmyogeninwereexpressed normally in differentiatedMyomixerKO cells, as was Myh8 (myosin heavy chain 8)(fig. S1J), indicating a selective blockade to myo-blast fusion independent ofmuscle differentiation.The ORF of mouse Myomixer encodes an 84–

amino acid micropeptide (Fig. 1D). Orthologs ofMyomixer are conserved across diverse vertebratespecies (Fig. 1D). In humans, a transcript encod-ing Myomixer is annotated as uncharacterizedLOC101929726. Myomixer amino acid sequenceis not annotated in fish or amphibians, presumablybecause ORFs smaller than 100 amino acids arenot typically annotated. However, we identifiedputative Myomixer orthologs in turtle, frog, andfish genomeswith conservation of several residues,especially many arginine, leucine, and alanineresidues (Fig. 1D). The proteins from the variousspecies contain an N-terminal hydrophobic seg-ment, followed by a positively charged helix andan adjacent hydrophobic helix.Myomixer proteinsfrom mammals and marsupials also contain adistinct C-terminal helix that is missing fromother organisms (Fig. 1D).Because the N terminus contains an extended

hydrophobic segment, we postulated that itmight serve as a membrane anchor. Indeed,

RESEARCH

Bi et al., Science 356, 323–327 (2017) 21 April 2017 1 of 5

1Department of Molecular Biology, Hamon Center forRegenerative Science and Medicine, University of TexasSouthwestern Medical Center, Dallas, TX 75390, USA. 2Sen.Paul D. Wellstone Muscular Dystrophy Cooperative ResearchCenter, University of Texas Southwestern Medical Center,Dallas, TX 75390, USA. 3Department of Internal Medicine,University of Texas Southwestern Medical Center, Dallas,TX 75390, USA.*Corresponding author. Email: eric.olson@utsouthwestern.edu

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immunostaining of intact C2C12 myoblasts ex-pressing Myomixer with FLAG tag at the C ter-minus revealed the presence of the protein onthe cell surface (Fig. 1E), suggesting that the Nterminus is embedded in the membrane. Consis-tent with these findings, fractionation of C2C12myotubes intomembrane and cytosolic fractionsshowed the association of Myomixer with mem-branes (Fig. 1F). Similarly, in human kidney 293cells infected with a Myomixer-expressing retro-virus, we found that the proteinwas preferentiallylocalized to the membrane fraction, althoughweak staining was also seen in the cytosolicfraction (Fig. 1F).Myomixer protein andmRNA expressionwere

up-regulated during differentiation of C2C12myo-blasts and declined after myoblast fusion (Fig. 2Aand fig. S2A). Similarly, Myomixer was stronglyup-regulated during differentiation of isolated

Pax7+ and Twist2+ adult muscle progenitor cells,which contribute to muscle regeneration (fig.S2B) (14). During pre- and postnatal muscle de-velopment, Myomixer transcripts showed a peakof expression at embryonic day 14.5 (E14.5) anddeclined thereafter (fig. S2C). Western blot anal-ysis revealed the presence of the Myomixer pro-tein in limb tissue from mouse embryos at E13.5and in skeletal muscle at postnatal day 2, whereasthe protein was undetectable in adult muscleof normalmice or in nonmuscle tissues (Fig. 2B).In situ hybridizations of mouse embryo sectionsat E12.5 and E15.5 showed intense expressionof Myomixer exclusively in developing skeletalmuscles throughout the limbs and body wall(Fig. 2C). In response to cardiotoxin (CTX) injuryof adult muscle, Myomixer expression was ra-pidly up-regulated, reaching a peak at day 3 afterinjury and declining thereafter (Fig. 2D). Im-

munostaining of CTX-treatedmuscle also showedrobust expression of Myomixer in the myosin-positive muscle cells in the region of regeneration(fig. S2D). Consistent with the micropeptide’spotential role in muscle regeneration, MyomixermRNA expression was up-regulated in muscleof mdx mice, a model of muscular dystrophythat exhibits muscle degeneration and regen-eration during disease progression (Fig. 2E).To assess the potential involvement of

Myomixer in muscle formation in vivo, we inacti-vated the gene during mouse embryogenesisthrough CRISPR-Cas9mutagenesis using the samepair of sgRNAs that was shown to effectively tar-get the gene in C2C12 cells and primary myoblasts.Fertilized zygotes were injected with Cas9 mRNAand sgRNAs, followed by implantation into pseudo-pregnant female mice. Embryos were harvested atE17.5 and analyzed. From 65 embryos analyzed,

Bi et al., Science 356, 323–327 (2017) 21 April 2017 2 of 5

Fig. 1. Myomixer is a small-membrane protein that is essential for myo-blast fusion. (A) Western blot showing Myomixer, Myosin, and Gapdh ex-pression in WTor Myomixer KO primary myoblast cultures after 4 days in DM.(B) WT and Myomixer KO primary myoblasts differentiated for 1 week andstained with MY32 antibody (myosin) and Hoechst show the requirement ofMyomixer for fusion. Scale bar, 50 mm. (C) Quantification of fusion in WTandMyomixer KO primary myoblast cultures (n = 3 pairs). (D) Amino acid se-quence ofMyomixer and cross-species homology. Basic residues are blue, acidicresidues are red, cysteines are green, and leucines are yellow. H, helix; C,

coil. (E) Live-cell staining of Myomixer-transfected C2C12 myoblasts show-ing cell-surface localization of Myomixer with a C-terminal FLAG tag. Lamininwas stained after FLAG staining and permeabilization. (F) Western blotanalysis of cytosolic (C) and membrane (M) fractions of retrovirus-Myomixer–infected 293 cells and WT C2C12 cells (day 4 after differentiation). Gapdhblot was used as a positive control of cytosolic proteins. N-Cadherin andepidermal growth factor (EGF) receptor blots were used as positive con-trols of membrane proteins. *P < 0.05, ***P < 0.001, Student’s t test. Dataare mean ± SEM.

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we obtained nine motionless embryos that ap-peared to lack skeletal muscle and were nearlytransparent so that internal organs and boneswere apparent (Fig. 3A).Histological sections through limb, body wall,

and diaphragmmusculature revealed an absenceof multinucleated myofibers in Myomixer KO

embryos (Fig. 3B). Instead, presumptive muscle-forming regionswere populatedbymononucleatedcells that stained for myosin expression (Fig. 3C).Tissues other than muscle appeared normal inthese embryos. The cutting sites of the twosgRNAs were 122 base pairs apart in the thirdexon of the Myomixer gene. PCR with primers

amplifying this region showed mutant micewith deletions that ranged from 163 to 470 bp,reflecting different indels (fig. S3, A and B).Western blot of hindlimbs confirmed the absenceofMyomixer protein in the KO embryos (Fig. 3D).The muscle phenotype of Myomixer mutant

mice is reminiscent of that seen in mice lacking

Bi et al., Science 356, 323–327 (2017) 21 April 2017 3 of 5

Fig. 2. Myomixer expression during muscle developmentand regeneration inmice. (A) Western blot showing Myomixer,Myosin, and Gapdh expression during C2C12 differentiation.(B) Western blot showing Myomixer and Gapdh expression inthe indicated tissues. SKM, skeletal muscle. (C) In situ hybridiza-tion showing Myomixer transcript expression in transversesections of mouse embryos at E12.5 and E15.5. Scale bar,250 mm (a), 500 mm (b), and 200 mm (c). Image c is a mag-nification of the boxed area shown in image b. H, heart; R,radius. (D) Myomixer mRNA expression in CTX-injured skeletalmuscle from 1-month-old WT mice, as determined with quantitative PCR. (n = 3 mice/time point). (E) Up-regulation of Myomixer mRNA expression in mdxcompared with WTmuscle, as detected with quantitative PCR (n = 4 pairs). Data are mean ± SEM.

Fig. 3. Myomixer is essential for muscle development in mice. (A) WTandMyomixer KO embryos at E17.5 dissected and skinned to reveal the lack ofmuscle in Myomixer KO limbs. (B) Hematoxylin and eosin (H&E) staining ofE17.5 limb muscles shows lack of muscle fibers in Myomixer KO embryos.Scale bar, 50 mm. (C) Immunohistochemistry of E17.5 limb muscles usingMY32 (myosin) antibody. WT myofibers show multinucleation, which isabsent in Myomixer KO sections. Scale bar, 50 mm. (D) Western blot analysisof Myomixer and Gapdh in forelimb tissues of E17.5 WT and Myomixer KOembryos.

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Myomaker, a fusogenic transmembrane muscleprotein (18, 19), suggesting a functional relation-ship between these two regulators of myoblastfusion. To assess the functional relationship be-tween Myomixer and Myomaker in myoblastfusion, we retrovirally expressed each protein inC2C12 myoblasts and monitored myotube for-mation. As shown in Fig. 4A, Myomixer markedlyenhanced the fusion of C2C12 cells. Moreover,when Myomixer and Myomaker were overex-pressed together in C2C12 cells, they promotedthe formation of massive multinucleated myo-tubes, which is indicative of their synergisticactivity (Fig. 4A and fig. S4).Myomaker can induce fusion of 10T1/2 mouse

fibroblasts with myoblasts (18, 19). In contrast,Myomixer expressing 10T1/2 cells did not fusewith C2C12 cells (Fig. 4B). To investigate whetherMyomixer can synergize with Myomaker to pro-mote heterologous cell fusion, we infected greenfluorescent protein (GFP)–labeled 10T1/2 cellswith Myomaker and Myomixer retroviruses andmixed them with mCherry-labeled C2C12 cells(fig. S5A).We found that expression ofMyomixerand Myomaker together in 10T1/2 cells induceddramatic fusion to C2C12myotubes so that only afew mononucleated GFP-labeled fibroblasts re-mained in the cultures (Fig. 4B). Moreover, whenMyomaker and Myomixer were coexpressed intwo populations of 10T1/2 fibroblasts labeledwith GFP or mCherry and the cells were mixed,fibroblast-fibroblast fusion was observed (Fig. 4Cand fig. S5B). Multinucleated fibroblasts coex-pressing GFP and mCherry, which appearedyellow, often resembled bird nests filled witheggs (Fig. 4C).Western blot confirmed expressionof Myomaker andMyomixer in the appropriatecells (fig. S5C). We observed no effect of eitherprotein on the level of expression of the other,indicating that their synergy does not reflect aneffect on stability of either protein. A summary ofthe effects ofMyomixer andMyomaker on fusionis shown in fig. S5D.To further test the functional dependency of

Myomaker onMyomixer for cell fusion, wemixedMyomaker-expressing 10T1/2 cells with WT orMyomixer-KOC2C12myoblasts and switched themtoDM for 1 week, after whichwe immunostainedformyosin. AlthoughMyomaker-expressing 10T1/2cells fusedwithWTC2C12 cells, theywere unableto fuse with Myomixer KO C2C12 cells (fig. S5E).This implies that Myomaker relies on Myomixerfor cell fusion in trans. Indeed, Myomaker wasexpressednormally inMyomixerKOmyoblasts andembryos, suggesting a dependency of MyomakeronMyomixer for normalmuscle fusion anddevel-opment (fig. S6, A and B).To begin to define the mechanistic basis of the

cooperativity between Myomixer and Myomaker,we examined whether the two proteins physicallyinteract. Indeed, we found that in coimmuno-precipitation assays, FLAG-taggedMyomaker co-immunoprecipitated with Myomixer in 10T1/2fibroblasts and differentiated C2C12 cells (Fig. 4D).Replacement of arginines at amino acid position34, 38, or 46 with glutamic acid residues withinthe charged segment of Myomixer diminished

Bi et al., Science 356, 323–327 (2017) 21 April 2017 4 of 5

Fig. 4. Myomixer binds Myomaker and synergizes to induce cell fusion. (A) MY32 (myosin) immuno-stainingof C2C12 cells infectedwith retroviruses expressingMyomaker,Myomixer, or both anddifferentiatedfor 4 days. Nuclei were counterstained with Hoechst and pseudocolored green. Scale bar, 50 mm. (B andC)Fluorescence images of GFP,mCherry, and Hoechst to counterstain nucleus. Scale bars, 100 mm (B), 50 mm(C). Arrows point to multinucleated GFP+/mCherry+ cells. (D and E) Coimmunoprecipitation assays wereperformedby using 10T1/2 cells or C2C12myoblasts infectedwith retroviruses expressingMyomixer and/orFLAG-Myomaker. IP, immunoprecipitation; IB, immunoblot. (F) MY32 (myosin) immunostaining of C2C12cells infected with retroviruses expressing Myomaker, together with WTor mutated versions of Myomixer,differentiated for 1 week. Nuclei were counterstained with Hoechst and pseudocolored green. Scale bar, 50mm. R, arginine; E, glutamic acid; C, cysteine; L, leucine; A, alanine; EEEAA, R34E-R38E-R46E-C52A-L54A.

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association with Myomaker but did not affectmembrane association (Fig. 4E and fig. S7A). TheEEEAA mutant failed to synergize with Myo-maker to stimulate fusion of C2C12 myoblasts(Fig. 4F and fig. S7B), as well as heterologousfusion of C2C12 myoblasts with 10T1/2 fibro-blasts (fig. S7C), indicating a correlation betweenthe association of Myomixer with Myomaker andtheir synergistic fusogenic activity. Althoughmutation of cysteine 52 to alanine in the secondhydrophobic region disrupted the fusogenic ac-tivity of Myomixer (Fig. 4F and fig. S7B), thismutation did not prevent membrane localiza-tion (fig. S7A) or interaction with Myomaker incoimmunoprecipitation experiments (Fig. 4E).This suggests that the second hydrophobic do-main of Myomixer may mediate the fusogenicfunction after its binding withMyomaker. A sum-mary of the effects of Myomixer mutations isshown in fig. S7D. Given the cross-species hom-ology of Myomixer orthologs, we tested wheth-er the ortholog from zebrafish (Danio rerio)could also promote heterologous fusion of cells.As shown in fig. S7E, this zebrafish peptide ofonly 75 amino acids induced cell fusion whenoverexpressed with mouse Myomaker. Consist-ent with this finding, deletion of the C terminusof mouse Myomixer (MyomixerDC) did not abol-ish its fusogenic function (fig. S7E). Thus, weconclude thatMyomixer is an evolutionarily con-served regulator of myoblast fusion and that theC-terminal region that is specific to higher ver-tebrates is dispensable for function, at least inthis assay.The requirement of Myomixer for fusion of

myoblasts in vivo and in vitro, its ability to

synergistically promote fusion together withMyomaker, and the physical and functionalinteraction between these proteins indicate thatthey govern the critical step of multinucleationof skeletal muscle. We hypothesize that Myo-mixer activates the fusogenic activity ofMyomakerto drive membrane mixing, perhaps by estab-lishing a fusion pore.The small size of Myomixer places it in the

category of micropeptides, characterized by un-processed ORFs of less than 100 amino acids(20). A majority of micropeptides identified todate are embedded in membranes (21, 22). Thereare some striking similarities between Myomixerand the heart-specific micropeptide DWORF(Dwarf open reading frame), which localizes tothe sarcoplasmic reticulum of cardiomyocyteswhere it associates with the sarco/endoplasmicreticulum Ca2+-ATPase (SERCA) calcium adeno-sine triphosphatase (ATPase) and stimulates itsactivity (23). We speculate that the activities ofmany membrane proteins may be governed byassociation with micropeptides that are as yetunidentified.

REFERENCES AND NOTES

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(Shanghai) 37, 789–796 (2005).18. D. P. Millay et al., Proc. Natl. Acad. Sci. U.S.A. 113, 2116–2121 (2016).19. D. P. Millay et al., Nature 499, 301–305 (2013).20. B. R. Nelson, D. M. Anderson, E. N. Olson, Circ. Res. 114, 18–20

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ACKNOWLEDGMENTS

We are grateful to J. Cabrera for graphics, B. Chen forCRISPR library data processing, W. Ye for chromatinimmunoprecipitation sequencing data analysis, and N. Grishinand J. Pei for bioinformatic analysis of Myomixer orthologs. Wethank K. White in the Histopathology Core for technicalassistance. We also thank H. Zhou, L. Amoasii, A. Bookout,Z. Wang, and N. Liu for biological materials and other membersof the Olson laboratory for technical advice. This work wassupported by grants from the NIH (AR-067294, HL-130253,DK-099653, and HD-087351) and the Robert A. WelchFoundation (grant 1-0025 to E.N.O.).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/356/6335/323/suppl/DC1Materials and MethodsFigs. S1 to S7References (24–30)

7 February 2017; accepted 26 March 2017Published online 6 April 201710.1126/science.aam9361

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Control of muscle formation by the fusogenic micropeptide myomixer

Rhonda Bassel-Duby and Eric N. OlsonPengpeng Bi, Andres Ramirez-Martinez, Hui Li, Jessica Cannavino, John R. McAnally, John M. Shelton, Efrain Sánchez-Ortiz,

originally published online April 6, 2017DOI: 10.1126/science.aam9361 (6335), 323-327.356Science 

, this issue p. 323Sciencecells, such as fibroblasts.membrane protein called Myomaker. Notably, the Myomaker-Myomixer pair can also promote the fusion of nonmusclemyoblast fusion. This small peptide, called Myomixer, physically interacts with and stimulates the activity of a fusogenic

amino acid peptide that promotes− identified an 84et al.fully understood. Studying cell culture and mouse models, Bi muscle precursor cells, or myoblasts, differentiate and fuse together during embryogenesis. The fusion process is not

Adult skeletal muscles are characterized by long, multinucleated cells called myofibers. Myofibers form whenMicromanaging muscle cell fusion

ARTICLE TOOLS http://science.sciencemag.org/content/356/6335/323

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2017/04/05/science.aam9361.DC1

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