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Fusogenic Micropeptide Myomixer Is Essential for Satellite Cell Fusion and Muscle Regeneration

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Fusogenic Micropeptide Myomixer Is Essential for Satellite Cell Fusion and Muscle Regeneration Fusogenic micropeptide Myomixer is essential for satellite cell fusion and muscle regeneration Pengpeng Bia,b,c, John R. McAnallya,b,c, John M. Sheltond, Efrain Sánchez-Ortiza,b,c, Rhonda Bassel-Dubya,b,c, and Eric N. Olsona,b,c,1 aDepartment of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; bHamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; cSenator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390; and dDepartment of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390 Contributed by Eric N. Olson, March 1, 2018 (sent for review January 3, 2018; reviewed by Bradley B. Olwin and Nadia A. Rosenthal) Regeneration of skeletal muscle in response to injury occurs through fusion and muscle formation during embryogenesis in mice and fusion of a population of stem cells, known as satellite cells, with zebrafish (14, 16–20). Myomaker is also required for fusion of injured myofibers. Myomixer, a muscle-specific membrane micro- SCs during adult muscle regeneration (21). peptide, cooperates with the transmembrane protein Myomaker Myomaker and Myomixer are up-regulated in SCs during to regulate embryonic myoblast fusion and muscle formation. To muscle regeneration (14, 16, 17, 21). However, the perinatal investigate the role of Myomixer in muscle regeneration, we used lethality of Myomixer null mice has precluded an analysis of its CRISPR/Cas9-mediated genome editing to generate conditional potential involvement in myofiber regeneration in response to knockout Myomixer alleles in mice. We show that genetic deletion injury (14). To directly assess the potential involvement of of Myomixer in satellite cells using a tamoxifen-regulated Cre Myomixer in SC-mediated muscle regeneration, we generated recombinase transgene under control of the Pax7 promoter abol- mice with conditional Myomixer null alleles, which were deleted ishes satellite cell fusion and prevents muscle regeneration, result- in SCs of adult mice with a tamoxifen (TMX)-inducible Cre ing in severe muscle degeneration after injury. Satellite cells devoid recombinase controlled by the Pax7 promoter (22). We show of Myomixer maintain expression of Myomaker, demonstrating that Myomixer is essential for the fusion of regenerative SCs and that Myomaker alone is insufficient to drive myoblast fusion. These the formation of new myofibers after injury of adult muscle. The findings, together with prior studies demonstrating the essentiality absence of Myomixer in SCs results in dramatic muscle de- of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation generation following injury, despite the expression of Myomaker throughout embryogenesis and adulthood. in these cells. Thus, at every stage of vertebrate myogenesis ex- aminedthusfar,MyomixerandMyomakerfunctioninanobliga- skeletal muscle | fusion | satellite cells | Myomaker | CRISPR/Cas9 tory partnership to control myoblast fusion and myofiber formation. Results keletal muscle is the largest tissue in the body, accounting for Myomixer ∼ Conditional Deletion of Myomixer in Mice. Germline null S 40% of human body mass. Skeletal muscle formation in- mice display perinatal lethality due to an absence of multinu- volves the differentiation and fusion of myoblasts to form mul- cleated myofibers (14), precluding an analysis of Myomixer tinucleated myofibers (1, 2). In adult skeletal muscle, resident function in adulthood. To circumvent the lethality of Myomixer muscle stem cells, called satellite cells (SCs), are required for null mice, we generated mice with conditional null alleles of the muscle growth, maintenance, and regeneration (3–5). SCs are characterized by their unique anatomical location between the basal lamina and plasma membrane of myofibers (6). Under Significance normal conditions, SCs are quiescent, whereas in response to injury they become activated and enter the cell cycle before their Skeletal muscle damaged by injury or disease can regenerate terminal differentiation and fusion into myofibers (7, 8). SCs are new muscle fibers. The regenerative properties of skeletal muscle marked by the expression of the paired-box transcription factor, involve fusion of activated muscle stem cells (satellite cells). We Pax7, and deletion of Pax7 from SCs prevents muscle re- recently discovered Myomixer, a conserved micropeptide that is generation (9). specifically expressed during muscle formation. Myomixer, The formation of myofibers through cell–cell fusion involves a together with its partner Myomaker, another muscle-specific series of events, including cell–cell recognition and adhesion, membrane protein, is necessary for muscle formation during cytoskeletal reorganization, and, ultimately, membrane merger embryogenesis. Here, we show the absolute requirement of Myomixer for the fusion of satellite cells and regeneration of (10). Although a variety of proteins have been shown to partic- adult muscle in response to injury. Our findings provide in- ipate in myoblast fusion (11, 12), much remains to be learned sights into the mechanisms of muscle formation and suggest about the underlying mechanisms and the interplay between opportunities for enhancing muscle regeneration through muscle-specific and ubiquitous components of the fusion pro- manipulation of Myomixer and Myomaker. cess. Recently, we discovered two muscle-specific membrane proteins, Myomaker and Myomixer, which are essential for Author contributions: P.B., R.B.-D., and E.N.O. designed research; P.B., J.R.M., J.M.S., and myoblast fusion during embryogenesis (13, 14). Myomaker is a E.S.-O. performed research; P.B., J.R.M., J.M.S., E.S.-O., and E.N.O. analyzed data; and P.B., seven-pass transmembrane protein capable of promoting fusion R.B.-D., and E.N.O. wrote the paper. of fibroblasts with myoblasts (13, 15). Myomixer, also referred to Reviewers: B.B.O., University of Colorado; and N.A.R., The Jackson Laboratory. as Myomerger (16) and Minion (17), is a micropeptide which The authors declare no conflict of interest. lacks autonomous fusogenic activity, but stimulates the fusogenic Published under the PNAS license. activity of Myomaker (14, 16, 17). Moreover, coexpression of this 1To whom correspondence should be addressed. Email: [email protected]. pair of membrane proteins is sufficient to confer fusogenic po- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tential to cell types that otherwise cannot fuse (14, 16, 17). Ge- 1073/pnas.1800052115/-/DCSupplemental. netic deletion of Myomaker or Myomixer abolishes myoblast Published online March 26, 2018. 3864–3869 | PNAS | April 10, 2018 | vol. 115 | no. 15 www.pnas.org/cgi/doi/10.1073/pnas.1800052115 Downloaded by guest on September 23, 2021 Myomixer gene using clustered regularly interspaced short pal- Cas9 cut site with 36 base pairs (bp) on the protospacer-adjacent indromic repeats (CRISPR) and Cas9 endonuclease-mediated motif (PAM)-distal side, and a 91-bp extension on the PAM- homology-directed repair (HDR) to insert loxP sites flanking proximal side of the cleavage (24). The ssODNAs of this design Myomixer exons. Of note, the Myomixer gene encodes two iso- were named ssODNA1–4, allowing HDR-mediated insertion of forms: a short isoform of 84 amino acids translated from an ORF loxP sites 1–4 after cutting by sgRNA1–4(Fig.1A and E). in exon 3, and a longer isoform of 108 amino acids translated Genotyping of filial 0 (F0) MymxloxP1,2 mice with two pairs of from an extended ORF in exons 2 and 3. Through examination primers flanking loxP1 (Fig. 1B) or loxP2 sites (Fig. 1C) and of RNA-seq data of C2C12 myoblasts (23), we found that the another pair of primers flanking both loxP sites revealed 10% short isoform is the predominantly expressed isoform during (2 out of 20 mice) correct targeting efficiency, although large myoblast differentiation (Fig. S1A). Nevertheless, to discern any deletions and the insertion of a single loxP site were also ob- potential function of the 24-amino acid N-terminal region of the served. We confirmed the simultaneous insertions of the long isoform of Myomixer, we generated two floxed Myomixer loxP1 and loxP2 sites from two founder F0 mice after cloning alleles, termed MymxloxP1,2 and MymxloxP3,4, allowing deletion of and sequencing a mixture of PCR products (Fig. S1 B and C). In the short and long isoforms by inserting two loxP sites flanking parallel, a targeting efficiency of 14% (4 out of 28 mice) was exon 3 (Fig. 1A) or exons 1–3 (Fig. 1E), respectively. achieved for the generation of F0 MymxloxP3,4 mice. The simul- Mouse zygotes were injected with five components for each taneous insertions of the loxP3 (Fig. 1F) and loxP4 (Fig. 1G) targeting scheme: two single guide RNAs (sgRNAs) targeted to sites from these founders were also confirmed by cloning and sequences flanking Myomixer exons; two single-stranded oligo- sequencing of genomic PCR products (Fig. S1 E and F). + deoxynucleotide DNA (ssODNA) donors, each containing a We isolated intramuscular fibroblasts from F1 MymxloxP1,2/ + loxP site flanked by short arms with homology to the desired and MymxloxP3,4/ mice to validate the functionality of the loxP sites insertion site; and Cas9 mRNA (Fig. 1 A and E). Cas9 nuclease in allowing Cre-mediated DNA recombination of the Myomixer asymmetrically releases
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