Andreia M. Nunes1, Inês Antunes1, André Gonçalves1, Patrícia Ybot-Gonzalez2, Marianne Deries1, and
Sólveig Thorsteinsdóttir1,3
1Centro de Ecologia, Evolução e Alterações Ambientais, Faculdade de Ciências, Universidade de Lisboa,
1749-016 Lisbon, Portugal; 2Departamento de Pediatria, Hospital Infantil Virgen del Rocio, Sevilla, Spain; 3Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal
Abstract
Skeletal muscle fibers are surrounded by a laminin-containing basement membrane essential for skeletal muscle health. During embryonic and fetal development, several laminin isoforms are in contact with myogenic cells, but it is still unclear which cells within the muscle masses produce these different laminins. Here we characterized the laminin expression dynamics throughout mouse myogenesis in utero to determine which cells are responsible for producing the laminins. The dermomyotome, the source of skeletal muscle cells, produces laminins 111 and 511 which are deposited in the dermomyotomal basement membrane as well as in the myotomal basement membrane during early stages of myotome myogenesis. Our data also revealed that the myotome synthesizes laminin 211 and 221 which are deposited in its basement membrane. Later on, as the dermomyotome dissociates and muscle progenitor cells enter the myotome, these cells and/or the differentiated myocytes express laminins 111 and 511. The dermomyotomal and myotomal laminin matrices are disassembled and/or degraded during primary myogenesis, but the expression of Lama2, Lama5 and Lamb1 is maintained within the muscle masses. During fetal myogenesis, both mononucleated cells and myofibers appear to synthesize laminins. Interestingly, our results suggest that the muscle stem cell population in early fetal muscle masses is heterogeneous with regard to its laminin repertoire in that some Pax7-positive cells have no assembled laminin around
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them while others have laminins 211, 511 and/or 411. Our results reveal that laminin synthesis and assembly involve cells at multiple stages of myogenic development as well as several different laminin isoforms. This work highlights the need to thoroughly study how this complexity contributes to the development of healthy skeletal muscle.
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Introduction
Skeletal muscles form through a highly coordinated process termed myogenesis. Myogenesis in the body starts at E8.5 in the mouse, when some cells from the dermomyotome are induced by neighboring tissues to activate myogenic regulatory factors (MRFs), transcription factors that initiate the myogenic program, and delaminate from the dermomyotome into the underlying space to constitute the myotome (Venters et al., 1999; Gros et al., 2004; Hollway and Currie, 2005; Buckingham, 2006). In the myotome, these cells divide once or twice before differentiating into myocytes which align and elongate along the rostral-caudal axis. As development proceeds, more Pax3- and/or Pax7- (Pax3/Pax7) positive dermomyotomal cells commit to myogenesis and are progressively added to the myotome where they differentiate contributing to myotomal growth (Venters et al., 1999; Gros et al., 2004; Hollway and Currie, 2005). Pax3/Pax7- positive cells are initially epithelial in the dermomyotome and lined by a laminin- containing basement membrane, but by the end of myotomal myogenesis, the dermomyotome dissociates and proliferating Pax3/Pax7-positive muscle progenitor cells (hereafter designated muscle stem cells) invade the myotomal space (Ben-Yair and Kalcheim, 2005; Kassar-Duchossoy et al., 2005; Relaix et al., 2005; Gros et al., 2005; Thorsteinsdóttir et al., 2011). Between E11.5 and E14.5 some of those muscle stem cells differentiate into myoblasts that fuse with myocytes and/or each other to generate multinucleated primary myotubes (Kelly and Zacks, 1969; Ross et al., 1987; Deries et al., 2010; Thorsteinsdóttir et al., 2011), while others remain as muscle stem cells (Kassar- Duchossoy et al., 2005; Relaix et al., 2005). Then from E14.5 until birth, many Pax7- positive muscle stem cells differentiate into secondary myoblasts which fuse with each other to form the secondary myotubes, and with the primary myotubes and recently formed secondary myotubes to increase their size (Duxson and Usson, 1989; Harris et al., 1989; Biressi et al., 2007; Thorsteinsdóttir et al., 2011). Pax7-positive muscle stem cells
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that do not differentiate during fetal myogenesis become the satellite cells of post-natal and adult muscle (Kassar-Duchossoy et al., 2005; Relaix et al., 2005; Tajbakhsh, 2009).
Laminins are major components of basement membranes which surround epithelial, endothelial, muscle, nerve, and fat cells (Thorsteinsdóttir et al., 2011). Laminins are secreted to the extracellular environment as trimers produced by cells which synthesize the three (α, β, γ) chains (Kumagai et al., 1997; Yurchenco et al., 1997; Yurchenco, 2015). Different combinations of 5 α chains, 3 β chains and 3 γ chains generate 16 different laminins, which are named according to their chain composition, e.g. laminin 111 is a trimer of α1, β1 and γ 1 while laminin 211 is composed of α2, β1 and γ1 chains (Aumailley et al., 2005). Up to 8 laminins, namely laminins 111, 121, 211, 221, 411, 421, 511 and 521, are present in skeletal muscle either during development or in adulthood (Durbeej, 2010; Thorsteinsdóttir et al., 2011).
During myotomal myogenesis, two laminin basement membranes are present: the dermomyotome basement membrane that lines the basal side of the dermomyotome; and the myotome basement membrane which separates the myotome from the sclerotome (Bajanca et al., 2006; Deries et al., 2012). The dermomyotome and myotome basement membranes display a similar laminin composition since laminins 111 and 511 are assembled in both (Bajanca et al., 2006). In addition, laminin 211 is assembled among myotomal myocytes and is also incorporated in the myotomal basement membrane (Cachaço et al., 2005; Nunes et al., 2017). Laminins are not assembled during primary myogenesis (Cachaço et al., 2005; Nunes et al., 2017) but during secondary myogenesis, primary and secondary myotubes are progressively surrounded by a basement membrane containing laminins 211, 411 and 511 (Patton et al., 1997; Nunes et al., 2017). Muscle stem cells initially intermingle with myotubes in the muscle masses, but enter their niche under the myotube basement membrane around E16.5 (Ontell and Kozeka, 1984; Kassar- Duchossoy et al., 2005). Muscle stem cells, myoblasts and myotubes are known to interact
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Here, we characterized the laminin expression dynamics during myogenesis. We demonstrate that dermomyotomal cells produce their own laminin matrix and provide laminins for the assembly of the myotomal basement membrane during early stages of myotome development. We also show that myotome maturation correlates with laminin synthesis by myotomal myocytes and possibly by that of dermomyotome-derived muscle stem cells invading the myotome as the dermomyotome dissociates. During primary myogenesis, laminin genes are expressed in the muscle masses although no assembled laminins are detected. Finally, we show that mononucleated cells and myotubes both contribute to the laminin matrix within the fetal muscle masses.