The muscles of the trunk and limbs derive from somites (Figure 1A), which are transient paraxial mesodermal structures that form pairwise on either side of the neural tube, following an anterior-posterior developmental gradient. The somite is initially a spherical unit of polarized epitheloid cells that soon after subdivides into two compartments, the ventral mesenchymal sclerotome and the dorsal epithelial dermomyotome. Shortly afterwards, myogenic precursor cells from the epaxial and hypaxial lips of the dermomyotome undergo an epithelial-mesenchymal transition (EMT) and accumulate underneath, where they differentiate and elongate to form the myocytes of the myotome, the first myogenic structure to develop in the body (Buckingham and Relaix, 2015; Deries and Thorsteinsdóttir, 2016). The epaxial region of the myotome gives rise to the deep back muscles, whereas the hypaxial myotome is the source of body wall muscles and most other trunk muscles (Buckingham and Relaix, 2015; Deries and Thorsteinsdóttir, 2016). In segments adjacent to the limb-region cells of the hypaxial dermomyotome undergo an EMT, leave the epithelial structure, and migrate toward the fore and hind limbs to form dorsal and ventral muscle masses in the limb-bud mesenchyme, where they begin to differentiate and express muscle-specific genes (Biressi et al., 2007; Deries and Thorsteinsdóttir, 2016).

Cell commitment in the somite is highly dependent on a number of transcription factors which act in a hierarchical molecular cascade to orchestrate the specification, determination, and differentiation of myogenic precursors. In the genetic hierarchy that regulates the onset of trunk myogenesis, Pax3 and Myf5 play a dominant role (Buckingham and Relaix, 2015). Pax3 is already transcribed in pre-somitic mesoderm adjacent to the first somite (Schubert et al., 2001) and then throughout the newly formed somites. As somites mature Pax3 expression becomes confined to the dermomyotome (Goulding et al., 1991) and persists in myogenic progenitor cells that delaminate and migrate from the somite to more distant sites of myogenesis such as the limb (Buckingham and Relaix, 2015). Myogenic cells that have activated the myogenic determination genes Myf5/Myf6 and MyoD downregulate Pax3 and delaminate from the edges of the dermomyotome (Buckingham and Relaix, 2015). The epaxial myotome then start to form. This depends on the early epaxial activation of Myf5, which is driven by Wnt and Shh signaling, without any Pax3 and/or Pax7 requirement (Borello et al., 2006; Buckingham and Relaix, 2015). These cells do not activate MyoD but rather Myog and differentiate (Kablar et al., 2003). On the other hand, the activation of Myf5 in the hypaxial somite as well as in the limb depends on PAX3 (Bajard et al., 2006; Buckingham and Relaix, 2015). At this stage MYF6 also acts as a myogenic determination factor (Kassar-Duchossoy et al., 2004). The Myod gene is activated after the onset of Myf5 expression in the rest of the dermomyotome and limbs (Hu et al., 2008). Finally, the transcription factor MYOG is required for the onset of the expression of terminal differentiation genes needed for the fusion of myocytes and the formation of myotubes (Bentzinger et al., 2012).

Although, all skeletal muscle throughout the body originates within paraxial mesoderm, in the head, identifiable compartments such as the somites in the trunk are not evident histologically or by most molecular criteria. This unsegmented head mesoderm is remodeled at the early stages of embryonic development (Figures 1B,C). The unsegmented head mesoderm gives rise to all craniofacial skeletal muscles, which can be cataloged as four distinct populations: extra-ocular (EOMs), branchial, laryngoglossal, and axial neck muscles (Noden and Francis-West, 2006; Tzahor, 2015). EOMs are formed by cells from the cranial paraxial mesoderm that migrate through the first branchial arch (FBA) as well as from the prechordal mesoderm (Jacob et al., 1984; Evans and Noden, 2006; Tzahor, 2015; Figure 1D). Branchial arch muscles are formed mainly by migrating cells from the cranial paraxial mesoderm and the lateral splanchnic mesoderm (Harel et al., 2009; Sambasivan et al., 2009; Tzahor, 2015). Laryngoglossal muscles develop from migratory myoblasts arising from occipital somites that form a condensed mesenchymal band, the hypoglossal cord, which elongates and similarly brings myoblasts ventral to pharynx (Hammond, 1965; Hazelton, 1970; Tzahor, 2015). Finally, in the transition zone between the head and the trunk are the axial neck muscles. They arise from medio-dorsal and latero-ventral domains of occipital and cervical somites (Noden, 1983; Couly et al., 1992; Matsuoka et al., 2005).

The genetic hierarchy governing primary myogenesis in the trunk does not appear to operate for head-muscle formation. Activation of the myogenic program in the head therefore depends on different upstream factors, responds differently to signaling pathways and also displays site-dependent regulation. Branchial-arch-derived muscles depend on Myf5/Myf6/Myod, whereas extra-ocular muscle formation is initiated by Myf5/Myf6 and in their absence cannot be restored by Myod (Tajbakhsh and Buckingham, 1999).

During fetal myogenesis, secondary fibers in trunk, limbs, and head are generated by the fusion of fetal myoblasts. Secondary fibers form initially at the site of innervation of the primary fiber and are surrounded by the same basal lamina as the primary fiber on which they lie (Duxson et al., 1989). The secondary myotubes remain attached for a short period to primary fibers and subsequently elongate and become independent fibers, which can be distinguished from primary fibers by their relative small size (Kelly and Zacks, 1969). Although, the genetic networks that rule this second stage of prenatal myogenesis is less understood, it is known that the MRFs MYF5, MYOD, and MYOG are also crucial, since in Myog^−/− as well as Myf5^−/−:MyoD^−/− double-mutant secondary myogenesis is completely inhibited (Venuti et al., 1995; Kassar-Duchossoy et al., 2004).

The regulatory inputs that orchestrate myogenesis during prenatal myogenesis are partially reactivated in adult muscle repair. In adulthood, the maintenance as well as the repair of muscle tissue are both directed mainly by SCs. These cells, originally identified via electron microscopy in 1961 by Alexander Mauro, are located underneath the basal lamina and adjacent to the plasma membrane of the skeletal-muscle myofiber (Mauro, 1961; Figure 2). In their quiescent state, SC express the transcription factor Pax7 and represent a genuine stem-cell population indispensable for skeletal-muscle repair (Lepper et al., 2011; Murphy et al., 2011; Sambasivan et al., 2011; Miersch et al., 2017; Stuelsatz et al., 2017). It has been established that SCs in adult muscle represent a lineage continuum of the embryonic myogenic progenitor cells. Thus, while SCs of the body and limbs arise from somites, in common with the muscle that they are associated with (Armand et al., 1983; Gros et al., 2005; Relaix et al., 2005; Schienda et al., 2006), the SCs located in head muscles also originate from the cranial mesoderm (Harel et al., 2009). Within a context of physiological stimuli (physical exercise or pathological conditions) SCs become activated, proliferate, differentiate and fuse to form multinucleated myofibers in order to undergo proper myogenesis (Lepper et al., 2011; Murphy et al., 2011; Sambasivan et al., 2011; Miersch et al., 2017; Stuelsatz et al., 2017; Figure 2). In this regard, numerous studies have revealed a striking similarity between adult and embryonic myogenesis, where the core regulatory network composed of the MRFs MYF5, MYOD, MYOG, and MYF6 is mainly required (Bentzinger et al., 2012; Segalés et al., 2016; Figure 2).

Most of what is known about Pitx2 concerns early (embryonic) myogenesis. However, a new role for Pitx2 has recently been unraveled during fetal myogenesis. L'Honoré et al. by using Pitx2:Pitx3 double conditional mutants, have shown that Pitx2/3 control the expression of the antioxidant system through the regulation of Nrf1 and antioxidant enzymes during muscle differentiation (L'honoré et al., 2014a). Thus, Pitx2/3 depletion at the onset of differentiation induces an abnormal increase of reactive oxygen species (ROS) levels in differentiating myoblasts and leads to impaired myogenesis due to apoptosis of these cells. These results emphasize the role of Pitx2 controlling redox conditions during fetal myogenesis.

During adult life the maintenance and repair of skeletal-muscle tissue is directed by SCs. The regulation of SC function in adults requires the redeployment of many of the regulatory networks fundamental for developmental myogenesis. Although, several efforts have been made during the last few years to disentangle the role of Pitx2 in embryonic and fetal stages of myogenesis, studies linking Pitx2 to adult myogenesis have only recently emerged and are still controversial.

The first evidence regarding Pitx2 expression in SCs was reported by Ono et al. (2010). These authors showed that all Pitx2 isoforms are expressed in proliferating SC-derived myoblasts. They analyzed SCs with a different ontology, comparing those of the extensor digitorum longus (EDL) of the limb with SCs from the masseter of the head (MAS). They found that Pitx2b and Pitx2c levels were higher in cells from the EDL than from the MAS, with Pitx2c being the main Pitx2 isoform expressed in proliferating limb SCs (Ono et al., 2010). Based on these distinct gene-expression profiles, the authors suggest that, even after activation and entry into the cell cycle, SCs retain an identity consistent with their ontogeny underlying their distinct properties. Subsequent studies have pointed out that Pitx2a, Pitx2b, and Pitx2c were expressed at very low levels in proliferating SCs, but increased during the early stages of myogenic differentiation. Meanwhile the constitutive expression of any Pitx2 isoform suppressed SC proliferation, with the cells undergoing greater myogenic differentiation (Knopp et al., 2013). However, additional evidence underlying the functional relevance of Pitx2 on SC proliferation has been reported. For example, Herbet et al. demonstrated that Pitx2 is crucial in maintaining the phenotype of myogenic precursor cells in the extraocular muscles (EOM; Hebert et al., 2013). In this analysis, the authors found that the higher levels of Pitx2 expression in EOM in comparison with limb muscles were concomitant with longer proliferative state in EOM-derived SCs as compared with limb cells. In addition, the knockdown of Pitx2 in SCs isolated from EOM slowed their proliferation rate, and a similar trend was seen for SCs isolated from tibialis anterioris muscle. These data led to the conclusion that Pitx2 helps maintain a proliferating pool of myogenic precursor cells. Finally, the authors highlight that this greater proliferative capacity may facilitate the repair of damaged EOM tissue, thereby contributing to the sparing of EOM in muscular dystrophies (Hebert et al., 2013).

More recently, a study conducted in our laboratory has provided additional information about the molecular mechanisms by which the Pitx2 transcription factor regulates cell proliferation in SCs (Lozano-Velasco et al., 2015). We have reported that Pitx2c expression is higher in early-activated SCs than in long-term activated ones, and our in vitro Pitx2c gain-of-function experiments have revealed that Pitx2c stimulates Ccnd1 and Ccnd2 expression, accelerating cell proliferation during early satellite-cell activation. Moreover, we have demonstrated that such Pitx2c effect on SCs proliferation is due to Pitx2c-mediated downregulation of the miRNAs miR-15b, miR-106b, miR-23b, and miR-503 (Figure 3B). The existence of the Pitx2-miRNA pathway controlling the expression of key regulatory cell-cycle genes in early-activated SCs revealed a role of Pitx2 in satellite-cell activation. Although, muscle SCs are promising targets for cell therapies, the paucity of SCs that can be isolated or expanded from adult muscle tissue is limiting; thus these findings provide new molecular tools to overcome such a bottleneck. It bears noting that our analyses also showed that Pitx2c can increase Myf5 expression by down-regulating miR-106b (Figure 3B), thus expanding the Myf5⁺ satellite-cell population and revealing a role for Pitx2c in promoting satellite-cell populations more primed for myogenic commitment (Lozano-Velasco et al., 2015). In this context it should be highlighted that in several muscular disorders such as muscular dystrophies, the progressive muscle wasting and weakness is often associated with exhaustion of muscle-regeneration potential. Therefore, the progressive loss of muscle mass has been attributed, at least partly, to the inability of muscle stem cells to efficiently regenerate tissue loss as the result of the disease (Berardi et al., 2014). Thus, critical for the development of effective strategies to treat muscle disorders is the optimization of approaches targeting muscle stem cells and capable of regenerating tissue loss as the result of the disease or as the result of normal muscle turnover (Bertoni, 2014). Notably, very recent reports have been pointed out that muscle stem cells should be considered as a therapeutic target for restoring muscle function in individuals with DMD (Chal et al., 2015; Dumont et al., 2015). Therefore, identification of new Pitx2 functions in the context of SC biology may significantly contribute to the clarification of the molecular and cellular mechanisms of skeletal-muscle regeneration and may help to develop therapeutic strategies for muscular disorders.

Notably, the analysis of adult single and double Pitx2:Pitx3 conditional mutant mouse lines targeted to the muscle stem-cell compartment revealed that double mutant SCs undergo senescence with impaired regeneration after injury, suggesting that Pitx2-mediated changes in ROS levels are required for differentiation of SCs (L'honoré et al., 2014b).

All these data provide new insight into the function of Pitx2 in the molecular mechanisms that control SC behavior and might thus have future application to enhance the regenerative capacity of these myogenic precursor cells. Further analysis using in vivo models could aid in understanding how the Pitx2-mediated effects on SCs can influence the kinetics of muscle regeneration.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.