© 2020. Published by The Company of Biologists Ltd | Development (2020) 147, dev184507. doi:10.1242/dev.184507 RESEARCH ARTICLE Myosin heavy chain-embryonic regulates skeletal muscle differentiation during mammalian development Megha Agarwal1,2,‡, Akashi Sharma1,3,‡, Pankaj Kumar1,2,‡, Amit Kumar1,*,‡, Anushree Bharadwaj1, Masum Saini1, Gabrielle Kardon4 and Sam J. Mathew1,2,3,§ ABSTRACT and myogenin. Once committed, the progenitors become myoblasts Myosin heavy chain-embryonic (MyHC-emb) is a skeletal muscle- that fuse with each other and with progenitors to generate myofibers, specific contractile protein expressed during muscle development. which are tubular multinucleate fibers that contain the contractile Mutations in MYH3, the gene encoding MyHC-emb, lead to Freeman– machinery essential for muscle contraction. Myofibers are composed Sheldon and Sheldon–Hall congenital contracture syndromes. Here, we of sarcomeres, the functional contractile unit of the skeletal muscle, characterize the role of MyHC-emb during mammalian development made up of alternating arrays of thick and thin filaments. MRFs are using targeted mouse alleles. Germline loss of MyHC-emb leads to crucial for the transcriptional regulation of the structural and neonatal and postnatal alterations in muscle fiber size, fiber number, contractile proteins that form the mature muscle fibers. fiber type and misregulation of genes involved in muscle differentiation. During mammalian development, myofibers are generated in two Deletion of Myh3 during embryonic myogenesis leads to the depletion of distinct stages, namely the embryonic and fetal phases of myogenesis. the myogenic progenitor cell pool and an increase in the myoblast pool, During the embryonic phase [embryonic day (E) 9.5-13.5 in mice], a whereas fetal myogenesis-specific deletion of Myh3 causes the small proportion of the embryonic myogenic progenitors fuse with depletion of both myogenic progenitor and myoblast pools. We reveal each other to give rise to primary myofibers, whereas in the fetal that the non-cell-autonomous effect of MyHC-emb on myogenic phase (E14.5-17.5), fetal myogenic progenitors fuse with each other progenitors and myoblasts is mediated by the fibroblast growth factor and with primary myofibers to give rise to secondary myofibers (FGF) signaling pathway, and exogenous FGF rescues the myogenic (Biressi et al., 2007a,b; Zammit et al., 2008). Although embryonic differentiation defects upon loss of MyHC-emb function in vitro. Adult and fetal myogenic progenitors differ from each other with respect to Myh3 null mice exhibit scoliosis, a characteristic phenotype exhibited their morphology, proliferation rates and response to growth factors, by individuals with Freeman–Sheldon and Sheldon–Hall congenital the myofibers derived from them, namely primary and secondary contracture syndrome. Thus, we have identified MyHC-emb as a myofibers, respectively, also exhibit distinct characteristics (Biressi crucial myogenic regulator during development, performing dual et al., 2007b; Stockdale, 1992; Zammit et al., 2008). cell-autonomous and non-cell-autonomous functions. Thesarcomericthickfilamentisprimarily composed of myosin, a heterohexamer comprising a pair each of myosin heavy chains This article has an associated ‘The people behind the papers’ interview. (MyHCs), regulatory light chains and essential light chains. MyHCs have an N-terminal globular head region with actin-binding and KEY WORDS: Skeletal muscle, Myosin heavy chain-embryonic, ATPase activity, a neck region where the light chains bind, and a C- Myogenesis, Development, Mice, Muscle progenitors, FGF, Signaling terminal coiled-coil tail region (Schiaffino and Reggiani, 1996). The contractile velocity of muscles is directly correlated with their myosin INTRODUCTION ATPase activity, varying from muscle to muscle and classified broadly The vertebrate skeletal muscle develops through a complex series of as fast or slow (Barany, 1967). Of the seven mammalian skeletal steps involving cell fate determination, cell migration and muscle MyHCs, five are expressed during adult life: MyHC-IIa differentiation. The early myogenic progenitors originate in the (encoded by Myh2), MyHC-IIx (encoded by Myh1) and MyHC-IIb somites, expressing Pax3 followed by Pax7 paired box transcription (encoded by Myh4) are adult ‘fast’ isoforms; MyHC-slow (encoded by factors, and migrate to their target tissues such as limbs, the Myh7)isthe‘slow’ isoform; and MyHC-extraocular (encoded by diaphragm or the tongue. Commitment to the myogenic lineage Myh13) is a unique ‘fast’ isoform expressed in the extraocular and occurs as a result of four basic helix-loop-helix transcription factors laryngeal muscles (Narusawa et al., 1987; Parker-Thornburg et al., called myogenic regulatory factors (MRFs) – Myf5, MyoD, MRF4 1992; Weydert et al., 1983; Wieczorek et al., 1985). Mice lacking MyHC-IIb or -IIx are significantly smaller, weigh less than the 1Developmental Genetics Laboratory, Regional Centre for Biotechnology (RCB), controls, and exhibit reduced grip strength, muscle weakness and NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, increased interstitial fibrosis (Acakpo-Satchivi et al., 1997). In Faridabad, 121001 Haryana, India. 2Manipal Academy of Higher Education, addition, two developmental isoforms, MyHC-embryonic (encoded Manipal, 576104, Karnataka, India. 3KIIT University, Patia, Bhubaneswar, 751024, Odisha, India. 4Department of Human Genetics, University of Utah, by Myh3) and MyHC-perinatal (encoded by Myh8), are expressed 15 N 2030 E, Salt Lake City, UT 84112, USA. during embryonic, fetal and neonatal development (Condon et al., *Present Address: Dept. of Pathology and Lab Medicine, David Geffen School of 1990; Periasamy et al., 1984, 1985; Schiaffino et al., 2015; Whalen Medicine at UCLA, 10833 Le Conte Ave, Los Angeles, CA 90095, USA. ‡These authors contributed equally to this work et al., 1981). Although MyHC-slow is expressed in the adult muscle, it is also expressed during developmental stages in the myofibers. § Author for correspondence ([email protected]) MyHC-embryonic and -perinatal (MyHC-emb and -peri) are not S.J.M., 0000-0001-7908-6094 normally expressed in adult muscle, except during skeletal muscle regeneration following injury or disease when they are transiently re- Received 3 September 2019; Accepted 12 February 2020 expressed (Sartore et al., 1982; Schiaffino et al., 1986). DEVELOPMENT 1 RESEARCH ARTICLE Development (2020) 147, dev184507. doi:10.1242/dev.184507 Although several transcriptional regulators and their functions in myofibers, mediates non-cell-autonomous effects on myogenic the distinct phases of myogenesis have been well studied, little is progenitors and myoblasts via secreted fibroblast growth factor known about the roles of downstream effectors such as MyHCs in (FGF) signals. Supplementation with FGF in the culture media myogenic differentiation. This is especially interesting with respect rescues the myogenic differentiation defects caused by loss of to developmental MyHCs because their expression correlates with MyHC-emb function in vitro.AdultMyh3 null mice exhibit scoliosis, specific developmental time points and myogenic phases, and one of the phenotypes exhibited by individuals with FSS and SHS. mutations in these MyHCs lead to congenital syndromes. Mutations Our results demonstrate that MyHC-emb is a crucial regulator of in the MyHC-emb encoding MYH3 gene lead to Freeman–Sheldon mammalian myogenesis. (FSS), Sheldon–Hall (SHS) and Multiple-Pterygium congenital contracture syndromes, indicating that developmental MyHCs have RESULTS vital, yet unidentified, roles during development (Toydemir et al., Loss of MyHC-embryonic leads to alterations in myofiber 2006b,c). number, area and fiber type In this study, we characterize the role of MyHC-emb in myogenic MyHC-emb is expressed during the embryonic and fetal stages of differentiation during embryonic, fetal, neonatal and postnatal muscle development, and mutations in the MYH3 gene coding for stages of development using targeted mouse alleles. We find that MyHC-emb protein cause congenital contracture syndromes MyHC-emb performs novel cell-autonomous and non-cell- (Chong et al., 2015; Tajsharghi et al., 2008; Toydemir et al., autonomous functions during myogenesis. At the cell-autonomous 2006c). Because few studies have been undertaken to investigate the level, MyHC-emb regulates muscle fiber size, fiber number and fiber molecular functions of MyHC-emb, we generated Myh3 targeted type, whereas at the non-cell-autonomous level it regulates myogenic mouse alleles. We generated a conditional Myh3fl3-7 allele, where progenitor and myoblast differentiation. MyHC-emb expressed in exons 3-7 of Myh3 were flanked by LoxP sites (Fig. 1A), and a Fig. 1. Loss of MyHC-embryonic leads to neonatal myogenic differentiation defects. (A,B) Schematics depicting the Myh3fl3-7 allele, where LoxP sites (red arrowheads) flank exons 3-7 (A) and the Myh3Δ allele lacking exons 3-7 of Myh3 (B). (C-D″) Cross-sections through the hind limbs of P0 Myh3+/+ (C,C′) and Myh3Δ/Δ (D,D′) mice labeled by immunofluorescence for MyHC-slow (red), laminin (green) and DAPI (blue). ‘E’ and ‘S’ denote EDL and soleus muscles, respectively, and ‘Tib’ and ‘Fib’ denote tibia and fibula bones, respectively, in C′ and D′;C″ and D″ are magnifications of boxed areas of the flexor digitorum longus (FDL) muscle from C′ and D′, respectively. (E,F) Laminin (green) labeling on sections through the soleus