Satellite cells as adult skeletal muscle stem cells

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Skeletal muscle structure and function

Skeletal muscle as a contractile unit

Skeletal muscle is one of the largest tissues in mammals. It allows voluntary movement and plays a key role in regulating metabolism and homeostasis of the organism. Throughout evolution, skeletal muscle is essentially defined by the succession of motor units which consists of a motoneuron and all of the muscle fibres innervated by that motoneuron (Figure 1). Myofibres are multinucleated cells and compose the cellular units of mature skeletal muscles. The structure of myofibres is strikingly repetitive at all sites in the organism, and the basic principles that govern the development of myofibres are conserved from Drosophila to humans. This structure is illustrated by the linear and repetitive arrangement of sarcomeres composed by an actin and myosin network together with associated proteins that enable muscle contraction (Figure 1). Different fibre types have been described, and these can be classified as slow-oxidative, fast-oxidative-glycolytic, and fast-glycolytic (Peter et al., 1972). The inherent contractile speed of each fibre-type cluster is determined essentially by the myosin motor protein isoform that is expressed predominantly. For example, the slow-oxidative unit expresses primarily a slow myosin heavy chain (MyHC) gene designated as slow or type I. The fast-oxidative unit expresses a combination of the fast type IIa and IIx MyHC genes, whereas the fast-glycolytic unit expresses both the fast IIb and IIx MyHC genes (Larsson et al., 1991). The accessibility of the hind limb Tibialis anterior muscle (below the knee), a mix of slow and fast fibres, has made it one the major sites for experimentation in studies on muscle homeostasis and regeneration. Finally, skeletal muscle allows the study of plasticity at the tissue and cellular level in different conditions such as overload (exercise), sarcopenia (muscle loss), ageing, and disease (myopathies).
The resident stem cells of skeletal muscle, historically called satellite cells, are located between the basement membrane containing a basal lamina, and the plasmalemma of the muscle fibre (Mauro, 1961) (Figure 1). Importantly, ≈90% of Muscle stem cells (MuSCs) are located in tight proximity with vessels (within 21μm) (Christov et al., 2007) (Figure 1), suggesting a communication between the vasculature and the MuSCs.
Figure 1. Scheme of skeletal muscle and associated structures. Skeletal muscles in general are attached at each end to the bone via tendons. Three connective tissue layers can be distinguished in skeletal muscle. The epimysium is the deep facia component that encloses the entire muscle and it is contiguous with the tendon and endosteum (facia surrounding bone). The perimysium encloses individual muscle fibers into fascicules (bundles). The endomysium is located between fibers and it encloses individual muscle fibers. Within the muscle cell (myofibre) the major intracellular source of calcium needed for muscle contraction is the sarcoplasmic reticulum, which connects to the transverse (T) tubules, and these surround the sarcomeres. Satellite cells are located between the basement membrane and the plasmalemma of the myofibre. Note the close proximity of the vessel, stained with India ink on the muscle section, and satellite cell from adult Myf5nlacZ/+ mouse stained with X-gal (upper image), or immunostained with GFP from a Myf5GPF-P/+ adult mouse. (Tajbakhsh, 2009)

Muscle regeneration

The remarkable regenerative ability of skeletal muscle was shown several decades ago in rats that had received weekly injections of bupivacaine (anaesthetic drug that blocks sodium channels (see, (Gayraud-Morel et al., 2009)) for 6 months, and did not show reduction or exhaustion of muscle fibres repair capacity (Sadeh et al., 1985). Similarly in mouse, after 50 bupivacaine injections into the TA muscle mice regenerated their muscle without loss of myofibres or gain of fibrotic areas (Luz et al., 2002). In human, skeletal muscle injuries resulting from direct trauma (contusions), partial tears, fatigue, following surgical procedures or myopathies are common and present a challenge in traumatology, as therapy and recuperation are not well supported. The most commonly used acute murine injury models involve intramuscular injection of myotoxins (cardiotoxin and notexin), BaCl2, and mechanical injury (freeze, needle or crush injuries) (Gayraud-Morel et al., 2009; Hardy et al., 2016) (see also Annex 1). For the purpose of our study, we will focus on the injury following the injection of myotoxins. Cardiotoxin (CTX, protein kinase C inhibitor) and Notexin (NTX, phospholipaseA2) are isolated from snake venom, and they trigger an increase in Ca2+ influx followed by fibre depolarization and consequently myofibre hypercontraction and necrosis (Gayraud-Morel et al., 2009; Hardy et al., 2016). After trauma, skeletal muscle regeneration follows three distinguishable and overlapping phases (Figure 2). The first phase of degeneration following severe injury is characterized by necrosis and significant inflammation (0 to 5 days post-injury (dpi)). After clearance of cellular debris, new fibres form and they transiently express embryonic and neonatal Myosin Heavy Chain (MyHC) from 3-14 dpi. The remodelling phase is characterized by hyperplasia and hypertrophy regulated in part by the IGF-1/Akt and TGFβ /Smad pathways. IGF-1 affects the balance between protein synthesis and protein degradation thus inducing muscle hypertrophy, whereas TGFβ negatively controls muscle growth (Schiaffino et al., 2013).
Although satellite cells play a crucial role in restoring myofibres following injury, it is clear that other cells types impact on the regeneration process (Figure 3) (see Annex 1). For example, fibro-adipogenic progenitors (FAPs) reside in the muscle interstitium and they play a significant myogenic and trophic role in muscle physiology during regeneration (Fiore et al., 2016; Joe et al., 2010; Lemos et al., 2015; Uezumi et al., 2010). Similarly, macrophages play a critical role during the initial stages following tissue damage as they are required for phagocytosis and cytokines release. The first wave of macrophages (peak at 3dpi) promotes myoblast proliferation via the secretion of pro-inflammatory molecules such as TNFα (Tumor Necrosis Factor α), INFα (Interferon α) and IL6 (Interleukin 6) (Lu et al., 2011a). Subsequently, macrophages undergo a phenotypical and functional switch toward an anti-inflammatory fate characterized by the production of IL4 and IL10, for example (Arnold et al., 2007). As mentioned previously, this anti-inflammatory response stimulates FAPs, mesoangioblasts, and also directly myoblasts to promote differentiation and fusion (Chazaud et al., 2003; Saclier et al., 2013). In addition, pericytes, located peripheral to the endothelium of microvessels, are known to be involved in blood vessel growth, remodelling, homeostasis, and permeability (Armulik et al., 2011) (Figure 3). The integrity of vessels is essential for muscle repair and homeostasis and it has been proposed that microvascular insufficiency could be responsible for the local inflammation and necrosis observed in both dystrophin-deficient mouse and human (Cazzato, 1968). Moreover, pericytes in skeletal muscles are constituents of the satellite cell niche where they secrete molecules such as IGF1 (insulin growth factor-1) or ANGPT1 (angiopoetin-1) to modulate postnatal myofibres growth and satellite cell entry in quiescence, respectively (Kostallari et al., 2015).
Although the generation of new fibres is dependent on MuSCs, other cell types such as macrophages, monocytes, mesenchymal stromal cells (including FAPs, mesoangioblasts and PICs), pericytes and fibroblasts are also critical for the regeneration process. (Baghdadi and Tajbakhsh, Annex 1).

Satellite cells as adult skeletal muscle stem cells

A brief history

The regenerative potential of muscle was first shown in the 1860s, but almost a century elapsed before the satellite cell was discovered. Using electron microscopy, Alexander Mauro observed a group of mononucleated cells located at the periphery of the adult skeletal muscle fibres from the Tibialis anticus of the Xenopus and rat (Mauro, 1961). These cells were named satellite cells due to their localisation on the periphery of the myofibres (Figure 4).
Figure 4. Electron micrograph of a typical myonucleus (A) and satellite cells (B) in mouse.
Muscle satellite cell (S) is inside the basal lamina (arrowheads) and outside the sarcolemma (arrows) with an independent cytoplasm. In contrast, a myonucleus (M) is located inside the sarcolemma of the muscle fibre. Bar: 1μm. (Sinha-Hikim et al., 2003)
The absence of satellite cells in cardiac muscle prompted him to speculate a role for these cells as skeletal-muscle specific precursor cells: « satellite cells are merely dormant myoblasts that failed to fuse with other myoblasts and are ready to recapitulate the embryonic development of skeletal muscle fibre when the main multinucleate cell is damaged » (Mauro, 1961). Interestingly, the position of this cell adjacent to the myofibre appears to be highly conserved in evolution, and similar satellite cells have been observed in multiple species, from the arthropods to mammals (see Baghdadi and Tajbakhsh, Annex 1). Electron microscopy also revealed other morphological characteristics of satellite cells: large nuclear-to-cytoplasmic ratio, few organelles, small nucleus, and condensed interphase chromatin.
The role of satellite cells in regeneration was first assessed after crush injury to the small web muscles of the East African fruit bat, Eidolon helvum (Church and Noronha, 1965). This study reported that satellite cells disappear from the highly injured area at the same time as the emergence of mitotic myoblasts, then reappear on myotubes after repair. Authors provided evidence that satellite cells were skeletal muscle “reserve cells”, capable of generating new fibres upon injury and replenishing the initial pool of cells. Additional [3H]-Thymidine tracing experiments combined with electron microscopy demonstrated that satellite cells are mitotically quiescent in adult muscle contribute to myofibre nuclei upon injury (Moss and Leblond, 1970; Reznik, 1969). The same studies also demonstrated that satellite cells give rise to proliferating myoblasts (myogenic progenitors cells), which were previously shown to form multinucleated myotubes in vitro (Konigsberg, 1963; Snow, 1977; Yaffe, 1969). Moreover, in vivo [3H]-Thymidine donor satellite cells specific labelling after free grafting of the muscle showed the presence of labelled nuclei on the periphery of regenerated myofibres in the host (Gutmann et al., 1976).

Molecular regulation of muscle stem cell emergence

During early development, muscle stem/progenitor cells migrate underneath the dorsal part of the somites called the dermomyotome (DM) and differentiate into mononucleated myocytes to form the myotome. In response to key transcription factors, committed myocytes align and fuse to generate small multinucleated myofibres during primary myogenesis in the embryo (from E11-E14.5), then myofibres containing a few hundred myonuclei during secondary myogenesis (from E14.5-to birth). During the early and late perinatal period that lasts about 4 weeks, continued myoblast fusion, or hyperplasia, is followed by muscle hypertrophy (Sambasivan and Tajbakhsh, 2007; Tajbakhsh, 2009; White et al., 2010) (Figure 5).
The developmental origin of satellite cells was first shown in a chick-quail chimera study: satellite cells of quail origin were found after replacement of chick somitic mesoderm by one from quail. In addition, electroporation of the central dermomyotome (the dorsal somite) in the trunk with a molecular marker showed that marked cells gave rise to Pax7+ satellite cells after hatching, thereby establishing the dermomyotome origin of satellite cells, in chick (Armand et al., 1983; Gros et al., 2005). Further evidences that satellite cells also originate from Pax3/7+ cells coming from the somites have been reported in the mouse (Kassar-Duchossoy et al., 2005; Relaix et al., 2005). Emerging satellite cells are found underneath a basement membrane from about 2 days before birth in mice and they further proliferate until the mid-perinatal stage (Kassar-Duchossoy et al., 2005). The majority of quiescent MuSCs are established from about 2-4 weeks after birth (Tajbakhsh, 2009; White et al., 2010). During prenatal and postnatal myogenesis, stem cell self-renewal and commitment are governed by a gene regulatory network that includes the paired⁄homeodomain transcription factors Pax3 and Pax7, and basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs), Myf5, Mrf4, Myod and Myogenin (Figure 5). Pax3 plays a critical role in establishing MuSCs during embryonic development (except in cranial-derived muscles) and Pax7 during late foetal and perinatal growth. Indeed, Pax3:Pax7 double mutant mice exhibit severe hypoplasia due to a loss of stem and progenitor cells from mid embryonic stages, and these Pax genes appear to regulate apoptosis (Relaix et al., 2006; Relaix et al., 2005; Sambasivan et al., 2009). During perinatal growth, Pax7 null mice are deficient in the number of MuSCs and fail to regenerate muscle after injury in adult mice (Lepper et al., 2009; Oustanina et al., 2004; Seale et al., 2000; von Maltzahn et al., 2013).
Experiments using simple or double knockout mice have shown the temporal and functional roles of these different factors during myogenesis. Myf5, Mrf4 and Myod assign myogenic cell fate of muscle progenitor cells to give rise to myoblasts (Kassar-Duchossoy et al., 2004; Rudnicki et al., 1993; Tajbakhsh et al., 1996) whereas Myogenin plays a crucial role in myoblast differentiation prenatally (Hasty et al., 1993; Nabeshima et al., 1993) but not postnatally as the conditional mutation of Myogenin in the adult has a relatively mild phenotype (Knapp et al., 2006; Meadows et al., 2008; Venuti et al., 1995). In the adult, Myod deficient mice that survive have increased precursor cell numbers accompanied by a delay in regeneration (Megeney et al., 1996; White et al., 2000); whereas Myf5 null mice display a slight delay in repair (Gayraud-Morel et al., 2007). These studies suggested that Myf5, Mrf4 and Myod could in some cases compensate for each other’s function. Whereas Mrf4 plays a role in embryonic progenitors, Myf5 and Myod continue to regulate muscle progenitor cell fate throughout foetal and postnatal life. Interestingly, additional transcription factors have been shown to interact with MYOD to regulate myogenesis. For instance, ChiP-seq data demonstrated that KLF5 (Kruppel-like factor, member of a subfamily of zinc-finger transcription factors) (Hayashi et al., 2016) as well as RUNX1 (Umansky et al., 2015) binding to Myod-regulated enhancers is necessary to activate a set of myogenic differentiation genes.
The MRFs form heterodimers with members of the E-protein bHLH family (E2A, E2-2 and HEB) and bind to a consensus E-box sequence (CANNTG) to activate muscle-specific gene expression. Although there are millions of consensus E-boxes in the genome that can bind of the myogenic bHLH factors, the productivity of this occupancy and the specifity of binding is determined by flanking nucleotides in the E-box, thereby effectively reducing the number of sites that are functional (Cao et al., 2010).
It is likely that MRFs combined with other transcription factors fine-tune the myogenesis process and it would be important to further explore the set of co-activators/repressors required for each step of muscle repair.
Pax3 and Pax7 expressions decline in the foetus. Myf5, Myod and Mrf4 expressing instruct to the progenitors cell a myogenic program. Desmin is an intermediate filament protein express in the muscle and Myosin is a component of the contractile apparatus. Around E16.5 Pax7+ cells appear in satellite cell position (see also Fig. 6).
(Sambasivan and Tajbakhsh, 2007)

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Heterogeneity in the muscle stem cell population

Compelling evidence from several studies has demonstrated that the satellite cell population is heterogeneous regarding their gene set of expression, proliferation rate, differentiation potential, stemness and even survival.
One remarkable example is demonstrated by the heterogeneity in satellite cells derived from skeletal muscle arising from different developmental origins: head (non-segmented paraxial mesoderm) versus limb (somites) that showed distinct molecular signatures. Cranial mesoderm derived muscles (except extraoculars) are Tbx1-dependent, whereas somite-derived muscles are Pax3-dependent (Sambasivan et al., 2011a). Furthermore, Alx4, Pitx1/2 are specifically expressed in the cranial mesoderm-derived extraoccular muscles (EOM) (Sambasivan et al., 2009). In addition, EOM-derived satellite cells showed greater ex vivo growth, self-renewal capacities and in vivo transplantation efficiency (Stuelsatz et al., 2015).
Similarly, single fibre transplantation experiments suggested that heterogeneity exists in muscles with the same developmental origin, but different anatomical location: MuSCs isolated from EDL (Extensor digitorium longus) or soleus muscles have superior engraftment potential compared to MuSCs from TA (Tibialis anterior) (Collins et al., 2005). Given that the MuSCs were grafted with their adjacent fibre in those experiments, this result could also be explained by the heterogeneity in the stem cell niche rather than cell-autonomous properties of the satellite cells.
Strikingly, even within a single muscle cell population, heterogeneity has been reported. Continuous in vivo labelling with the thymidine analogue BrdU (5′-bromo-2′-deoxyuridine) in 4weeks-old rats revealed two populations: about ≈80% of satellite cells readily marked over the first 5 days and a slow cycling minority of cells not fully saturated upon 2 weeks of treatment. This second population named “reserve cells” was proposed to maintain quiescence during muscle growth/homeostasis and enter cell-cycle only upon trauma (Schultz, 1996). Furthermore, freshly isolated single myofibres from Myf5nlacZ and Myf5Cre;R26RYFP mice showed ≈13% of MuSCs that never express Myf5 (Pax7+/β-gal—; Pax7+/YFP—, respectively), suggesting a more stem-like fate (Kuang et al., 2007). This Myf5— population is capable of asymmetric cell division and replenish the stem cell pool upon engraftment, whereas the Myf5+ undergo differentiation. These results suggest a hierarchical organisation of quiescent MuSCs: with a more stem population that will give rise to the more committed cells upon activation while self-renew to repopulate the quiescent niche. However, this phenotype is less pronounced with another Myf5Cre allele, and eventually all satellite cells experience Myf5 expression, therefore it is unclear how the genetically modified mice reflect stem-like behaviour over time (Sambasivan et al., 2013). Indeed, the presence/absence of labelling relies on the efficiency of the Cre-recombinase that has been shown to not faithfully represent Myf5 expression in every condition, a phenomenon that has been reported also for other tissues (Comai et al., 2014).
To address some of these issues, a Tg:Pax7-nGFP mouse has been used to fractionate the satellite cell population in both quiescent and injured muscles based on the nGFP intensity. Interestingly, fractionation of the Pax7-nGFP population by FACS into Pax7High (Top 10%) and Pax7Low (Bottom 10%) revealed that the Pax7High population displays more stem-like features such as lower metabolic activity, longer time to enter cell cycle compared to Pax7Low that express more activation/differentiation genes (e.g: Myod, Myogenin, see below section 3.1.2), and higher expression of stem cell markers. Notably, Pax7High cells were considered to be in a more dormant cell state (deeper quiescence), however serial transplantation of these subpopulations did not show dramatic differences in contribution to the niche (Rocheteau et al., 2012).
Recent technological advancements in single cell RNAseq, methylome analysis and mass cytometry now permit investigations of cellular heterogeneity within specific cell populations (Angermueller et al., 2016; Grun et al., 2016; Spitzer and Nolan, 2016). For example, analysis of single cells by multiparameter sequencing-based analysis, specifically RNAseq and bisulfite based methylome analysis, allows the investigation of epigenetic, genomic and transcriptional heterogeneities. Although powerful, some limitations include sequence depth and coverage of the genome. On the other hand, CyTOF based mass cytometry is based on a combination of markers conjugated to metal isotopes, and this led to the identification and classification of subpopulations of myogenic cells following muscle injury (Porpiglia et al., 2017). These emerging technologies can be used to assess the relative potential and role of a whole population at the single cell level and promise to give further insights into understanding MuSC heterogeneities.

Functions of muscle stem cells

Adult myogenesis

The absolute requirement for MuSCs was shown by genetic elimination of satellite cells postnatally using an inducible diphtheria toxin system that leads to an arrest in translation and subsequent cell death. This resulted in failed regeneration and replacement of the damaged muscle tissue with inflammatory and adipogenic cells (Lepper et al., 2011; Murphy et al., 2011; Sambasivan et al., 2011b). Nevertheless, some outstanding questions remain regarding the potential role of other interstitial cells in muscle repair (see Baghdadi and Tajbakhsh, Annex 1).
Examination of β-galactosidase activity in Myf5nlacZ mice indicated that the Myf5 locus is active in 90% of quiescent satellite cells, which suggests that most satellite cells are committed to the myogenic lineage (Beauchamp et al., 2000). Satellite cell physiology and progression throughout the myogenic program are tightly controlled by a hierarchy of transcription factors (Yablonka-Reuveni and Rivera, 1994) (Figure 6). At homeostasis, MuSCs remain quiescent and reside in G0-phase within their sublaminal niche contiguous to the myofibre (Schultz et al., 1978). It is thought that all adult quiescent satellite cells express the transcription factor Pax7 (Seale et al., 2000); its paralogue Pax3 is also expressed in a subset of satellite cells of certain muscles (Relaix et al., 2006). While Pax3 plays a critical role during embryonic myogenesis, most satellite cells, however, downregulate Pax3 before birth (Kassar-Duchossoy et al., 2005). As mentioned above, in myogenesis, Pax7 and Pax3 play overlapping but non-redundant roles. These functional differences can be explained by differential binding affinities for paired versus homeobox motifs, suggesting differences in DNA binding and chromatin status affinities (Soleimani et al., 2012). Upon injury, MuSCs activate, re-enter the cell cycle and undergo cellular division to give rise to myoblasts, a highly proliferative transient amplifying cell population (Figure 6). In the adult, MRFs are also responsible for both myogenic lineage specification as well as for the regulation differentiation. Although MYF5, but not MYOD protein is expressed in satellite cells, Myod and Myf5 genes are both rapidly upregulated upon activation (Cooper et al., 1999; Gayraud-Morel et al., 2012). Finally, terminal differentiation is initiated by the downregulation of Pax7 (Olguin and Olwin, 2004) and the upregulation of Myogenin and Mrf4 to generate elongated myocytes that will further fuse into myotubes (Cornelison et al., 2000; Cornelison and Wold, 1997) (Figure 6). Essentially, a subpopulation of activated satellite cells, exit the cell cycle and return to quiescence in order to maintain the stem cell pool for future regeneration (Figure 6).
Figure 6. Muscle regeneration following different forms of injury. Following mild or severe injury, quiescent muscle stem cells (MuSCs) activate, differentiate and fuse to repair the damaged fibre. The myogenic process is tightly regulated by the action of key transcription factors and regulators. (Baghdadi and Tajbakhsh, Annex 1)

Satellite cell activation and differentiation

Immediately following muscle injury, Myod expression is rapidly upregulated and MYOD protein is already detectable within satellite cells as early as 12 h after injury, before the first cell division that takes place from about 20h (Rocheteau et al., 2012; Smith et al., 1994). This early expression of Myod is proposed to be associated with a subpopulation of committed satellite cells, which are poised to differentiate without proliferation (Rantanen et al., 1995). In contrast, the majority of satellite cells express either Myod or Myf5 by 24h following injury and subsequently co-express both factors (Cornelison and Wold, 1997; Gayraud-Morel et al., 2012; Zammit et al., 2002) (Figure 6). Interestingly, ectopic expression of Myod in NIH-3T3 and C3H10T1/2 fibroblasts is sufficient to activate the complete myogenic program in these cells (Hollenberg et al., 1993); thus expression of Myod is an important determinant of myogenic commitment and differentiation, and its absence promotes proliferation and delayed differentiation (Myod—/—)(Sabourin et al., 1999). During satellite cell activation, Pax7 and Pax3 target genes to promote proliferation and commitment to the myogenic lineage, while repressing genes that induce terminal myogenic differentiation (Soleimani et al., 2012). For example, PAX7 and PAX3 induce the expression of Myf5 by direct binding to distal enhancer elements and Myod by binding to the proximal promoter (Bajard et al., 2006; Hu et al., 2008). Moreover, p38 kinase (p38γ) also negatively regulates the transcriptional potential of Myod by phosphorylation, which leads to a repressive Myod complex occupying the Myogenin promoter (Gillespie et al., 2009). This observation is supported by the premature expression of Myogenin and reduced proliferation of myoblasts in p38-decificent muscle (Gillespie et al., 2009).
Terminal differentiation is initiated by the expression of Myogenin and later Mrf4 (Smith et al., 1994; Yablonka-Reuveni and Rivera, 1994) (Figure 6). ChIP-on-chip experiments (Bergstrom et al., 2002; Cao et al., 2006) and ChIP-Seq analysis (Cao et al., 2010) revealed MYOD and MYOGENIN specific target genes. These studies suggested a hierarchical organization involved in satellite cell activation and differentiation with regard to MRFs. MYOD directly activates Myogenin and Mef2 transcription factors, a large portion of downstream targets are muscle-specific  structural and contractile genes, such as those encoding actins, myosins, and troponins, essential for proper myofibres function.
p38α/β kinase stimulates the binding of MYOD and MEF2s to the promoters of muscle-specific genes, leading to the recruitment of chromatin remodelling complexes promoting myogenesis (Cox et al., 2003; Wu et al., 2000).
Besides MRFs and their regulators, other post-transcriptional factors have been shown to be involved in myogenic differentiation such as micro-RNAs (see Chapter 3).

Table of contents :

Chapter 1. Skeletal muscle and its resident stem cells
1. Skeletal muscle structure and function
1.1. Skeletal muscle as a contractile unit
1.2. Muscle regeneration
2. Satellite cells as adult skeletal muscle stem cells
2.1. A brief history
2.2. Molecular regulation of muscle stem cell emergence
2.3. Heterogeneity in the muscle stem cell population
3. Functions of muscle stem cells
3.1. Adult myogenesis
3.1.1. Satellite cell activation and differentiation
3.1.2. Satellite cell self-renewal
Chapter 2. Stem cell niche is essential for quiescence
1. Stem cell quiescence
1.1. Identification of quiescent stem cells
1.2. Ex vivo induction of quiescence
1.3. Molecular signature of quiescence
1.3.1. Epigenetic control
1.3.2. Cell cycle regulators
2. Molecular signature of MuSCs
2.1.1. Calcitonin receptor
2.1.2. Teneurin-4 or Odz4
2. The stem cell niche
2.1. Extracellular matrix: powerful modulator of cell behaviour
2.2. ECM-cell interaction
2.3. Biophysical properties of ECM
2.4. Collagens constitute a major component of the ECM
2.4.1. Insights from Collagen V
3. The MuSCs niche
3.1. Extracellular matrix and associated factors
Chapter 3. Post-transcriptional regulation of myogenesis: a role for microRNAs
1. The discovery of microRNAs
2. MicroRNAs: Genomics, biogenesis, mechanism and function
2.1. Biogenesis of microRNAs
2.2. MicroRNAs arise from distinct genomic loci
2.3. MicroRNA prediction tools
3. MicroRNAs in cell and tissue regulation
4. Regulation of myogenesis by microRNAs
5. Inhibition of microRNAs using “Antagomirs”
Chapter 4. Notch signalling is a pleiotropic regulator of stem cells
1. An introduction to the world of Notch
2. Notch receptors, ligands and the cascade
3. Notch targets genes and their regulation
4. Notch signalling in the regulation of stem cell fate
5. Notch signalling in skeletal muscle and satellite cells
Part I: Notch-induced Collagen V maintains muscle stem cells by reciprocal activation of the Calcitonin Receptor
Part II: The Notch-induced microRNA-708 maintains quiescence and regulates migratory behavior of adult muscle stem cells
1. Context of this thesis project
2. Notch signalling regulates ECM niche components
3. Notch signalling positions MuSCs in their niche
4. Potential regulation of Notch signalling by microRNAs
ANNEX 1: Review Regulation and phylogeny of muscle regeneration
ANNEX 2: Resource paper Comparison of multiple transcriptomes using a new analytical pipeline Sherpa exposes unified and divergent features of quiescent and activated skeletal muscle stem cells
ANNEX 3: Small-RNA sequencing identifies dynamic microRNA deregulation during muscle lineage progression


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