Myogenesis is a complex process in which committed myogenic cells differentiate and fuse into myotubes that mature into the muscle fibres of adult organisms. This process is initiated by a cascade of myogenic regulatory factors expressed upon entry of the cells into the myogenic differentiation programme. However, external signals such as those provided by the extracellular matrix (ECM) are also important in regulating muscle differentiation and morphogenesis. In the present work, we have addressed the role of various ECM substrata on C2C12 myoblast behaviour in vitro. Cells grown on fibronectin align and fuse earlier than cells on laminin or gelatine. Live imaging of C2C12 myoblasts on fibronectin versus gelatine has revealed that fibronectin promotes a directional collective migratory behaviour favouring cell-cell alignment and fusion. We further demonstrate that this effect of fibronectin is mediated by RGD-binding integrins expressed on myoblasts, that N-cadherin contributes to this behaviour, and that it does not involve enhanced myogenic differentiation. Therefore, we suggest that the collective migration and alignment of cells seen on fibronectin leads to a more predictable movement and a positioning that facilitates subsequent fusion of myoblasts. This study highlights the importance of addressing the role of fibronectin, an abundant component of the interstitial ECM during embryogenesis and tissue repair, in the context of myogenesis and muscle regeneration.
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Boettiger D, Enomoto-Iwamoto M, Yoon HY, Hofer U, Menko AS, Chiquet-Ehrismann R (1995) Regulation of integrin α5β1 affinity during myogenic differentiation. Dev Biol 169:261–272
Brand-Saberi B, Krenn V, Grim M, Christ B (1993) Differences in the fibronectin-dependence of migrating cell populations. Anat Embryol (Berl) 187:17–26
Brand-Saberi B, Gamel AJ, Krenn V, Müller TS, Wilting J, Christ B (1996) N-Cadherin is involved in myoblast migration and muscle differentiation in the avian limb bud. Dev Biol 178:160–173
Buckingham M (2006) Myogenic progenitor cells and skeletal myogenesis in vertebrates. Curr Opin Genet Dev 16:525–532
Burattini S, Ferri P, Battistelli M, Curci R, Luchetti F, Falcieri E (2004) C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. Eur J Histochem 48:223–233
Cachaço AS, Pereira CS, Pardal RG, Bajanca F, Thorsteinsdóttir S (2005) Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse. Dev Dyn 232:1069–1078
Danen EHJ, Sonnenberg A (2003) Integrins in regulation of tissue development and function. J Pathol 200:471–480
Dhawan J, Rando TA (2005) Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. Trends Cell Biol 15:666–673
Disatnik MH, Rando TA (1999) Integrin-mediated muscle cell spreading. The role of protein kinase C in outside-in and inside-out signaling and evidence of integrin cross-talk. J Biol Chem 274:32486–32492
García AJ, Vega MD, Boettiger D (1999) Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation. Mol Biol Cell 10:785–798
Goody MF, Henry CA (2010) Dynamic interactions between cells and their extracellular matrix mediate embryonic development. Mol Reprod Dev 77:475–488
Huttenlocher A, Lakonishok M, Kinder M, Wu S, Truong T, Knudsen KA, Horwitz AF (1998) Integrin and cadherin synergy regulates contact inhibition of migration and motile activity. J Cell Biol 141:515–526
Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11–25
Jansen KM, Pavlath GK (2008) Molecular control of mammalian myoblast fusion. Methods Mol Biol 475:115–133
Knudsen KA, Myers L, McElwee SA (1990) A role for the Ca2+-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis. Exp Cell Res 188:175–184
Lan MA, Gersbach CA, Michael KE, Keselowsky BG, García AJ (2005) Myoblast proliferation and differentiation on fibronectin-coated self assembled monolayers presenting different surface chemistries. Biomaterials 26:4523–4531
Larsen M, Artym VV, Green JA, Yamada KM (2006) The matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr Opin Cell Biol 18:463–471
Linask KK, Ludwig C, Han MD, Liu X, Radice GL, Knudsen KA (1998) N-cadherin/catenin-mediated morphoregulation of somite formation. Dev Biol 202:85–102
Martins GG, Rifes P, Amândio R, Rodrigues G, Palmeirim I, Thorsteinsdóttir S (2009) Dynamic 3D cell rearrangements guided by a fibronectin matrix underlie somitogenesis. PLoS One 4:e7429
May MJ, Entwistle G, Humphries MJ, Ager A (1993) VCAM-1 is a CS1 peptide-inhibitable adhesion molecule expressed by lymph node high endothelium. J Cell Sci 106:109–119
Osses N, Brandan E (2002) ECM is required for skeletal muscle differentiation independently of muscle regulatory factor expression. Am J Physiol Cell Physiol 282:C383–C394
Rieger S, Senghaas N, Walch A, Köster RW (2009) Cadherin-2 controls directional chain migration of cerebellar granule neurons. PLoS Biol 7:e1000240
Rochlin K, Yu S, Roy S, Baylies MK (2010) Myoblast fusion: when it takes more to make one. Dev Biol 341:66–83
Rosen GD, Sanes JR, LaChance R, Cunningham JM, Roman J, Dean DC (1992) Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis. Cell 69:1107–1119
Rozario T, DeSimone DW (2010) The extracellular matrix in development and morphogenesis: a dynamic view. Dev Biol 341:126–140
Ruoslahti E (1991) Integrins. J Clin Invest 87:1–5
Sanes JR (1982) Laminin, fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane. J Cell Biol 93:442–451
Sastry SK, Lakonishok M, Thomas DA, Muschler J, Horwitz AF (1996) Integrin α subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation. J Cell Biol 133:169–184
Siegel AL, Atchison K, Fisher KE, Davis GE, Cornelison DD (2009) 3D timelapse analysis of muscle satellite cell motility. Stem Cells 27:2527–2538
Takahashi S, Leiss M, Moser M, Ohashi T, Kitao T, Heckmann D, Pfeifer A, Kessler H, Takagi J, Erickson HP, Fässler R (2007) The RGD motif in fibronectin is essential for development but dispensable for fibril assembly. J Cell Biol 178:167–178
Theveneau E, Marchant L, Kuriyama S, Gull M, Moepps B, Parsons M, Mayor R (2010) Collective chemotaxis requires contact-dependent cell polarity. Dev Cell 19:39–53
Thorsteinsdóttir S, Deries M, Cachaço AS, Bajanca F (2011) The extracellular matrix dimension of skeletal muscle development. Dev Biol 354:191–207
Turner DC, Lawton J, Dollenmeier P, Ehrismann R, Chiquet M (1983) Guidance of myogenic cell migration by oriented deposits of fibronectin. Dev Biol 95:497–504
van der Flier A, Gaspar AC, Thorsteinsdóttir S, Baudoin C, Groeneveld E, Mummery CL, Sonnenberg A (1997) Spatial and temporal expression of the β1D integrin during mouse development. Dev Dyn 210:472–486
Vellón L, Royo F, Matthiesen R, Torres-Fuenzalida J, Lorenti A, Parada LA (2010) Functional blockade of α5β1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells. BMC Cell Biol 11:81
von der Mark K, Öcalan M (1989) Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype. Differentiation 40:150–157
Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725–727
Yao CC, Ziober BL, Sutherland AE, Mendrick DL, Kramer RH (1996) Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor. J Cell Sci 109:3139–3150
Yurchenco PD, Amenta PS, Patton BL (2004) Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol 22:521–538
Zammit PS, Partridge TA, Yablonka-Reuveni Z (2006) the skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem 54:1177–1191
Zeschnigk M, Kozian D, Kuch C, Schmoll M, Starzinski-Powitz A (1995) Involvement of M-cadherin in terminal differentiation of skeletal muscle cells. J Cell Sci 108:2973–2981
We are grateful to the members of our group for helpful discussions and to our laboratory rotation students, Márcio Madureira and Ana Rita Leitoguinho, for help with RT-PCR. The MF20, F5D and MNCD2 antibodies developed by D.A. Fishman, by W.E. Wright and M. Takeichi and by H. Matsunami, respectively, were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA52242.
R.V. and this work were supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) project PTDC/BIA-BCM/67437/2006.
Electronic supplementary material
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Time-lapse movie of C2C12 cells on gelatine with startingconfluence of 60%. Total film length: 10h50min. Colour scale corresponds to time. (AVI 7.32 mb)
Time-lapse movie of C2C12 cells on fibronectin with startingconfluence of 60%. Total film length: 10h50min. Colour scale corresponds to time. (AVI 7.60 mb)
Time-lapse movie of C2C12 cells on gelatine with startingconfluence of 90%. Total film length: 12h15min. Colour scale corresponds to time. (AVI 8.61 mb)
Time-lapse movie of C2C12 cells on fibronectin with startingconfluence of 90%. Total film length: 12h15min. Colour scale corresponds to time. (AVI 8.79 mb)
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Vaz, R., Martins, G.G., Thorsteinsdóttir, S. et al. Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model. Cell Tissue Res 348, 569–578 (2012). https://doi.org/10.1007/s00441-012-1364-1
- Myoblast behaviour
- Live imaging
- Extracellular matrix
- C2C12 cells