tenascin-C?
André B. Gonçalvesa,b, Marianne Deriesa and SólveigThorsteinsdóttira,b.
a Centro de Ecologia, Evolução e Alterações Ambientais, Departamento de Biologia Animal,
Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.
b Instituto Gulbenkian de Ciência, 2781-901 Oeiras, Portugal
Contribution for the publication:
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- ... non-applicable O ... no intervention I ... minor contribution II ... moderate contribution
III .... major contribution/full execution
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Abstract
Developing and adult skeletal muscle is surrounded by an extracellular matrix (ECM) including that of its connective tissue such as tendons. However, little is known about how the ECM affects the behaviour and maintenance of muscle stem cells (MuSCs) during development. Thus, we studied the spatial relationship between laminin, fibronectin and tenascin-C matrices and MuSCs, from their origin in the dermomyotome until they invade the first embryonic skeletal muscle, the epaxial myotome. For this we used 3D analysis of images obtained from whole mount immunohistochemistry of wild type mouse embryos. We demonstrate that each type of ECM exhibits a distinct distribution pattern which suggests they may have specific roles in MuSC delamination and colonisation of the myotome. Our data set the stage for studying how MuSC-ECM interactions contribute towards proper muscle development.
Keywords: Extracellular matrix; Muscle stem cells; Myotome; Laminin, Fibronectin,
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Introduction
The extracellular matrix (ECM) is a non-cellular three-dimensional (3D) macromolecular network composed of proteoglycans and fibrous proteins that can bind to each other (Järveläinen et al., 2009; Schaefer and Schaefer, 2010). Fibrous proteins are predominantly glycoproteins, including laminins, fibronectin, collagens, elastins and tenascins (Alberts et al., 2007), which provide physical adhesive scaffolds for cells. Cell-ECM communications via cell surface receptors can trigger a variety of intracellular signal transduction pathways and cytoskeletal reorganisation (Harburger and Calderwood, 2009; Humphries et al., 2006). ECMs have been shown to affect not only differentiated cells in a variety of tissues, but also provide an environment regulating stem cell behaviour (Gattazzo et al., 2014). In this study, we focus on determining the distribution patterns of laminin, fibronectin and tenascin matrices during epaxial (deep back) muscle development and in particular their relationship with skeletal muscle progenitor cells, also called muscle stem cells (MuSCs).
All muscles of the body derive from the epithelial somites of the paraxial mesoderm which mature into several compartments originating different tissues. Ventrally, each somite forms the mesenchymal sclerotome, which gives rise to the axial skeleton, while the dorsal part of the somite stays epithelial and forms the dermomyotome (Christ et al., 2007). The dermomyotome contains progenitors for several mesodermal cell types, including embryonic muscle stem cells (MuSCs) (Ben-Yair and Kalcheim, 2005; Seale et al., 2008). Dermomyotomal MuSCs are the progenitor pool of trunk and limb skeletal muscles and are characterised by the expression of the transcription factors Pax3 and/or Pax7 (Buckingham, 2006). During early stages of myogenesis in the somites, MuSCs delaminate from the dermomyotome in synchronous waves to form the myotome, the third somitic compartment and first muscle to develop in the embryo. The last compartment to differentiate is the syndetome, the anlagen of axial tendon. This compartment is localised between the myotome and the sclerotome and its development depends on specific signals produced by the myotomal myocytes (Brent et al., 2003).
In the mouse embryo, myogenesis is first triggered in the epaxial lip (the dorso-medial lip or DML) of the dermomyotome by signals from axial tissues (Buckingham, 2006; Deries and Thorsteinsdóttir, 2016; Tajbakhsh, 2009). MuSCs in the epaxial lip upregulate Myf5 at E8.0 (Ott et al., 1991), de-epithelialize and colonise the epaxial (dorsal) myotome (Venters et al.,
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1999). Subsequently, a second migratory wave from the hypaxial lip (the ventro-lateral lip or VLL) of the dermomyotome colonises the hypaxial (ventral) myotome and soon after, a third wave originating from the rostral and the caudal lips forms myocytes that increase the thickness of the myotome (Gros et al., 2004; Venters et al., 1999). In the myotome, MuSCs express the myogenic regulatory factors, Myf5, Mrf4, MyoD and Myogenin to differentiate into mononucleated, postmitotic myocytes that express muscle structural proteins such as desmin and myosin heavy chain (MHC). Near the end of myotome development (E11.0), myocytes start to fuse with each other and with myoblasts to form multinucleated myotubes (Ben-Yair and Kalcheim, 2005; Hollway and Currie, 2005). Between E10.5 and E11.5, the central portion of the dermomyotome de-epithelializes releasing a fourth wave of MuSCs into the myotome. These MuSCs can either proceed to myogenic differentiation or remain undifferentiated and proliferative, forming a muscle stem cell reservoir for embryonic, foetal and postnatal growth and which gives rise to the satellite cells of the adult (Ben-Yair and Kalcheim, 2005; Gros et al., 2005; Kassar-Duchossoy et al., 2005; Relaix et al., 2005). Between E11.5 and E12.5, the epaxial (dorsal) myotome transforms into four epaxial (deep back) muscle groups including the most dorsal, the transversospinalis muscle group (Deries et al., 2010), whereas the hypaxial myotome forms hypaxial (abdominal and intercoastal) muscles (Christ et al., 1983; Kalcheim et al., 1999). All these events are accompanied by dynamic changes in the surrounding ECM. Indeed, ECM containing laminin, fibronectin and tenascin-C exhibit specific dynamic distribution patterns during mouse epaxial myogenesis (Bajanca et al., 2006; Cachaço et al., 2005; Deries et al., 2012; Nunes et al., 2017) and some of these matrices have been shown to play specific roles during myogenesis in the somites (Bajanca et al., 2006).
Basement membranes of all tissues contain laminin matrices (Durbeej, 2010). The epithelial dermomyotome is surrounded dorsally by its laminin-containing basement membrane and ventrally the myotome assembles its own basement membrane which makes a physical border with the sclerotome (Anderson et al., 2009; Bajanca et al., 2006; Tajbakhsh et al.,1996). Then, from E11.5, dermomyotomal and myotomal laminin matrices are disassembled and are not assembled again before the end of primary myogenesis (E14.5) after which each myotube is progressively wrapped with a laminin basement membrane (Cachaço et al., 2005; Deries et al., 2012; Nunes et al., 2017).
Fibronectin, which can form both pericellular and interstitial matrices, is the most abundant ECM glycoprotein in the early embryo. It supports cell adhesion, migration and growth and interacts with collagens, tenascins and other molecules of the ECM (Hynes, 1992;
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Rozario and DeSimone, 2010; Zollinger and Smith, 2017). Fibronectin surrounds the somites and as somites mature, it is present at somitic boundaries and forms thick cables within the myotome (Deries et al., 2012).
Tenascins are modular hexadimeric proteins of the interstitial matrix that can associate with fibronectin (Hsia and Schwarzenbauer, 2005). Tenascin-C, the most abundant and well- studied tenascin isoform during development, has a restricted and often transient distribution pattern in the embryo (Jones and Jones, 2000; Riou et al., 1992). It is particularly enriched at intersegmental borders from which cables of tenascin-C matrix extend into the myotome, lying parallel to the myotomal myocytes (Deries et al., 2012). Later in development it becomes confined to tissues bearing tensile and mechanical stress, specifically in tendons, ligaments and smooth muscles (Chiquet-Ehrismann et al., 2014).
Although an increase knowledge exists underlying the importance of the ECM during skeletal muscle differentiation, little is known about its effect on the identity and behaviour of MuSCs. In this work, we carefully describe the spatial relationship between Pax3/Pax7-positive MuSCs and laminin, fibronectin and tenascin-C ECMs using wild type embryos during development of the epaxial myotome/muscles in the mouse embryo. We found that each of the studied ECM is in a position compatible with playing a role in the regulation of MuSCs.