Muscle contraction activates YAP and NOTCH signalling and thus regulates the pool of muscle progenitor cells during foetal myogenesis Synopsis 

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Limb muscle formation

Limb muscles derive from waves of migrating progenitor cells that reside in the hypaxial lips of the dermomyotomes (Chevallier et al., 1977; Christ et al., 1974). In mouse embryos, the migrating muscle progenitors express only Pax3 and activation of Pax7 occurs once the cells reach the limbs. In chick embryos, migrating muscle progenitors co-express Pax3 and Pax7 (reviewed by Duprez, 2002; reviewed by Biressi et al., 2007a). The first muscle progenitors start to be observed in forelimb buds at embryonic day 2.5 (E2.5) in the chick (Fig. 2) and at E10.5 in the mouse (reviewed by Duprez 2002; reviewed by Biressi et al., 2007a; Tozer et al., 2007).

Embryonic, foetal and adult myogenesis

Skeletal muscle formation occurs in successive and overlapping phases. During the embryonic phase, a first wave of myogenesis that relies on Pax3+ cells takes place and Pax3+ cells give rise to embryonic myoblasts. The first fusion events occur between embryonic myoblasts and give rise to embryonic fibres that function as scaffold for subsequent muscle growth. Embryonic myogenesis takes place at E2.5 in the chick and at E11.5 in the mouse (Fig. 4) (reviewed by Duprez, 2002; reviewed by Biressi et al., 2007a). During foetal myogenesis, a second wave of progenitors differentiates. Foetal progenitors express Pax7 and give rise to foetal myoblasts. Foetal myoblasts fuse with themselves and with pre-existing embryonic fibres allowing the muscle growth. Foetal myogenesis starts at E6 in the chick and at E14.5 in mice (Fig. 4) (reviewed by Duprez 2002; reviewed by Biressi et al., 2007a). By the end of foetal myogenesis, the pool of satellite cells (Pax7+ cells) that is required for the peri-natal muscle growth and adult homeostasis is established. Foetal muscle progenitors adopt a satellite cell-like position under the basal lamina at E17 and E16 in the chick and mouse, respectively (Fig. 4) (reviewed by Biressi et al., 2007a).

The balance between proliferation and differentiation

The mechanisms involved in muscle formation and growth rely on a tight control of the cell cycle. I define here exit or withdrawal from the cell cycle as an irreversible process, that is accompanied by terminal differentiation and distinguish it from a reversible stop in proliferation that is observed in quiescence. Proteins that play a central role in cell cycle regulation are required to switch between myoblast proliferation and differentiation. In addition, MRFs and cell cycle-associated factors control each other to ensure appropriate muscle development. Most studies on cell cycle regulation during myogenesis were performed in vitro and rely frequently on the use of the C2C12 cell line. Very little is known about cell cycle regulation during myogenesis in vivo.
During development, proliferating myoblasts are characterized by the presence of Pax3 and/or Pax7 and the early MRF Myf5 (Fig. 6). MyoD starts to be expressed in proliferating myoblasts, and strong expression of MyoD forces cells into cell cycle exit and myogenic differentiation (Fig. 6, 7). The activity of MyoD in proliferating myoblasts was proposed to be inhibited to allow the progression through the cell cycle (Fig. 7) (reviewed by Kitzmann and Fernandez, 2001). In proliferating myoblasts, MyoD is associated with histone deacetylases (HDCA), which inhibit trans-activation of muscle-related genes (reviewed by Ciemerych et al., 2011). In addition, the inhibitor of differentiation proteins (Id) are Figure 6 – Progression of myogenic cells towards differentiation. Different molecular markers characterising the distinct stages of myogenic differentiation are illustrated. Muscle progenitors and satellite cells can be considered “quiescent cells”. Brackets around Myf5 indicate that a subset of satellite cells have expressed Myf5 in their developmental history.

Regulation of the muscle progenitor pool

The balance between proliferation and differentiation of the muscle progenitors during the different myogenesis steps is critical to achieve muscle growth and maintain the undifferentiated pool of muscle progenitors. Several signalling pathways have been identified to play a role in the maintenance of the muscle progenitor pool in vivo. Notch signalling is associated with the maintenance of muscle progenitors, inhibition of terminal differentiation and formation and positioning of satellite cells (Bröhl et al., 2012; Delfini et al., 2000; Hirsinger et al., 2001; Mourikis et al., 2012a; Schuster-Gossler et al., 2007; Vasyutina et al., 2007a). Studies on the role of BMP signalling pathway in myogenesis have revealed two distinct functions in embryonic and foetal myogenesis. In embryonic myogenesis, activation of BMP pathway increases the muscle progenitor pool and blocks differentiation whereas in foetal development, activation of BMP leads to muscle growth with increased numbers of muscle progenitors and fibres (Amthor et al., 1998, 1999; Wang et al., 2010). Fibroblast growth factor (FGF) signalling activates proliferation and blocks myogenic differentiation in vitro (reviewed by Olson, 1992). In addition, in vivo activation of FGF signalling by FGF4 in chick limbs decreases Pax3 and MyoD expression levels and results in less muscle fibres (Edom-Vovard et al., 2001). The role of Wnt signalling pathway in myogenesis is quite controversial, and in vitro studies demonstrate either a role in promoting or blocking myogenesis (Gavard et al., 2004; Goichberg et al., 2001; Perez-Ruiz et al., 2008). However, in vivo analyses suggest a role of Wnt in the maintenance of foetal myoblasts (Hutcheson et al., 2009) and satellite cell expansion (Le Grand et al., 2009).

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BMP signalling pathway

BMPs are growth factors of the TGFβ superfamily, which also includes Activins, Nodals and Growth/Differentiation factors (GDFs). TGFβ family members play a role in cell growth, differentiation and morphogenesis. BMPs act as morphogens, i.e. they are secreted and can induce distinct responses depending on their concentration. Dimerization of BMPs is required for activation of the BMP receptors that possess intracellular serine/threonine kinase domains. Two type II and two type I receptors are assembled in the receptor complex (reviewed by Massagué, 2012). In the presence of the ligand, the type II receptor phosphorylates the type I receptor (Fig. 9A). The kinase activity of type I receptors depends on this phosphorylation in a domain rich in glycine and serine (GS domain). The type II receptors that mediate BMP signals are mainly the BMP receptor II (BMPRII) but also the Activin receptor type IIa and IIb (ActRIIa/IIb). The type I subunits of the BMP receptor complexes are mainly the BMP receptors Ia and Ib (BMPRIa/Ib) (also named Activin-like receptor 3 (Alk3) and Alk6, respectively) but also in certain contexts the receptors Alk1 and Alk2 (reviewed by Moustakas and Heldin, 2009). The activated type I receptors phosphorylate and activate the regulatory Smad proteins (R-Smads), the effectors of the pathway, that translocate to the nucleus and function as transcription factors (Fig. 9A). The BMP type I receptors (Alk3/6/1/2) signal via Smad1/5/8 (Fig.9 A), while the type I receptors that bind TGFβ signal via Smad2/3 (reviewed by Moustakas and Heldin, 2009). The Smad proteins function in trimers, where two phosphorylated R-Smads bind to the common mediator (co-Smad) Smad4. Smad4 is required for trimerization of Smad1/5/8 and Smad2/3 (reviewed by Massagué et al., 2012). The third class of Smads, the Inhibitory-Smads (I-Smads) Smad6 and Smad7, bind to the type I receptor and interfere with phosphorylation and activation of R-Smads. Smad7 blocks the activity of the different TGFβ type I receptors whereas Smad6 has higher affinity for BMPRIa/Ib (Fig. 9A) (reviewed by Moustakas and Heldin, 2009).

Crosstalk between Notch and TGFβ/BMP signalling pathways

The Notch and BMP signalling pathways play similar roles in the maintenance of the muscle progenitor pool during foetal myogenesis. Mechanisms of crosstalk between these two pathways started to be explored in different developmental processes, including myogenesis.
Notch and TGBβ/BMP inhibit myogenic differentiation of C2C12 cells and the two factors can cooperate (Fig.10) (Blokzijl, 2003; Dahlqvist, 2003). While exogenous BMP4 inhibited myogenic differentiation of C2C12 cells, blocking Notch signalling by a pharmacological agent (L-685,458) or a dominant-negative RBP-J interfered with this result (Dahlqvist et al., 2003). This indicates that BMP4-mediated inhibition of myogenic differentiation in vitro requires active Notch signalling. In addition, exogenous BMP4 increases the expression of Notch target genes Hes1 and Hey1, which suggests a synergistic effect of the two pathways in the inhibition of differentiation (Fig. 10) (Dahlqvist et al., 2003). Exogenous TGFβ in C2C12 cells also increases the expression of Hes1, effect that is no longer observed in the presence of a dominant-negative RBP-J (Fig. 10) (Blokzijl et al., 2003). In addition, in vivo experiments in the chick embryo showed that activation of TGFβ signalling by overexpression of the constitutively active Alk5 also up-regulates Hes1 (Fig. 10) (Blokzijl et al., 2003).
In these studies, direct interactions between NICD and Smad3 (the effector of the TGFβ signalling pathway) or Smad1 (the effector of the BMP signalling pathway) were observed (Fig. 10) (Blokzijl et al., 2003; Dahlqvist et al., 2003). In addition, NICD, Smad1 and Smad3 can be recruited to Notch/RBP-J binding sites on DNA, which potentiates the activation of Notch target genes. In particular, Smad1/NICD complexes bind to the Hey1 promoter (Fig. 10) (Dahlqvist et al., 2003). In accordance, the activation of Notch reporter constructs, which respond to transfection with NICD, is potentiated by BMP4 (Dahlqvist et al., 2003). Furthermore, Smad3/NICD but not Smad3 activated a reporter construct carrying RBP-J binding sites (Blokzijl et al., 2003). These results indicate that BMP/TGFβ and Notch signalling pathways synergistically interact to potentiate the inhibition of myogenic differentiation (Fig. 10).

Table of contents :

Table of Contents
Gene Nomenclature and Abbreviations
State of the ar
1 – Skeletal muscle development
1.1 – Skeletal muscle specification in the head, trunk and limbs
1.2 – Limb muscle formation
2 – Embryonic, foetal and adult myogenesis 
3 – Regulation of the muscle progenitor pool
3.1 – The balance between proliferation and differentiation
3.2 – Signalling pathways
3.2.1 – Notch signalling pathway
3.2.2 – BMP signalling pathway
3.2.3 – Crosstalk between Notch and TGFβ/BMP signalling pathways
4 – Muscle as mechanical tissue
4.1 – Importance of muscle contraction during development
4.2 – The mechano-sensitive gene Yap and its role in myogenesis
1 – The interplay between NOTCH and BMP signalling pathways is different during proliferation and differentiation during foetal myogenesis Synopsis 
Materials and Methods
2 – Muscle contraction activates YAP and NOTCH signalling and thus regulates the pool of muscle progenitor cells during foetal myogenesis Synopsis 
Materials and Methods
Additional Information
Discussion and Perspectives


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