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Skeletal muscle stem cells
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Skeletal muscle stem cells Margaret Buckingham and Didier Montarras In this review we shall discuss recent publications on the heterogeneity of muscle stem cells, signaling pathways that affect their behaviour and regulatory mechanisms that underlie their myogenic fate, with reference to insights provided by work on skeletal muscle formation in the embryo as well as the adult, with the mouse as a model of reference. Addresses Pasteur Institute, CNRS URA 2578, 28 rue du Dr Roux, 75015 Paris, France Corresponding author: Buckingham, Margaret ([email protected]) and Montarras, Didier ([email protected])
Current Opinion in Genetics & Development 2008, 18:330–336 This review comes from a themed issue on Pattern formation and developmental mechanisms Edited by Ottoline Leyser and Olivier Pourquie´ Available online 30th July 2008 0959-437X/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2008.06.005
Introduction The notion that satellite cells represent the major cell type responsible for post-natal skeletal muscle growth and regeneration has recently received further support [1–3]. These quiescent cells, located under the basal lamina of muscle fibres, become activated upon injury, proliferate and differentiate into new muscle fibres. During regeneration, the satellite cell pool is also reconstituted. Thus, satellite cells display two hallmarks of stem cells: lineage-specific differentiation and selfrenewal.
Heterogeneity between muscle progenitor cells Skeletal muscle formation [4] (Figure 1) depends on the myogenic regulatory factors, of which MyoD and Myf5/ Mrf4 determine muscle cell fate, whereas Myogenin, as well as MyoD and Mrf4, controls differentiation. At the onset of myogenesis in the embryo, Myf5/Mrf4 and then MyoD are directly activated in the dorsal somite by signals from adjacent tissues. Although the initial activation of MyoD depends on Myf5/Mrf4, it is subsequently expressed in the absence of these factors. Selective ablation of Myf5 expressing cells indicates that the MyoD positive population is distinct, suggesting a two cell lineage model [5,6]. However, a Myf5nlacZ allele is tranCurrent Opinion in Genetics & Development 2008, 18:330–336
scribed in MyoD positive cells [7], but this may be insufficient to generate enough Myf5 dependent Cre recombinase required for ablation. After the first wave of myogenesis that results in the formation of the early myotome, Pax3 and Pax7 act upstream of Myf5 and MyoD and thus control the entry of cells into the myogenic programme [8]. Pax3/Pax7 positive stem cells from the somite are subsequently present in all developing skeletal muscle masses and give rise to the satellite cells of post-natal muscle [4]. Myoblasts (Myf5/MyoD positive), isolated from late embryonic or foetal muscle have different properties, further demonstrated by transcriptome analysis [9]. This may reflect a contribution from the first wave of myogenesis to the embryonic population, but is also probably due to the onset of innervation in the foetal period. Spatial as well as temporal parameters lead to muscle stem cell heterogeneity. As development proceeds, Pax3 expression is downregulated and Pax7 marks satellite cells [10]; however, Pax3 continues to be transcribed in some muscles [11,12]. This is the case in the diaphragm, also distinguished by more severe MyoD [13] and FgfrL1 [14] mutant phenotypes, indicative of heterogeneity between different sites of myogenesis. Lbx1 deficient mouse embryos display a lack of limb muscles attributed to migration defects. The finding that Lbx1 marks activated satellite cells after injury [15] may be of importance in understanding satellite cell mobility, required for adult muscle repair. The Pax3/Pax7 positive cells in skeletal muscle before birth either proliferate or activate Myf5 and MyoD, followed by muscle differentiation. However, by contrast, the majority of quiescent Pax7 positive satellite cells (80%) transcribe Myf5, as indicated by b-galactosidase activity from a Myf5nlacZ allele [16] (Figure 2). This heterogeneity has been investigated using a Myf5Cre/Rosa26-YFP genetic approach [17], which shows that 10% of Pax7 positive satellite cells have never expressed significant levels of Myf5. Further heterogeneity, suggested by YFP (Myf5) positive cells that are Pax7 negative, may reflect the surface markers used for cell selection and the fact that Myf5 and therefore the Myf5Cre allele, is expressed in presomitic mesoderm [18], leading to labeling of other mesodermal lineages, see also [5]. With the available antibody tools, MyoD is not detected in quiescent satellite cells and expression of the gene is thought to be a hallmark of activation. www.sciencedirect.com
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Figure 1
division (parallel to the fibre), which represents 90% of all divisions scored, leads to symmetrical Pax7+/Myf5+ daughter cell fates [17]. This suggests that signaling from the fibre promotes myogenesis. Asymmetric segregation of Numb has also been documented in dividing satellite cells [21], although it is not clear how this correlates with Pax7 versus Pax7/Myf5 expression and indeed it was also detected in cultured satellite cells [22]. A further indicator of ‘stemness’, although the underlying mechanism is controversial [23], is non-random template DNA segregation. Pulse labeling with BrdU demonstrated asymmetric strand segregation in about 7% of satellite cells, which also showed asymmetric segregation of Numb [22]. Interestingly this can occur in satellite cells that have activated Myf5. Using pulses of halogenated thymidine analogues during injury induced regeneration, a strikingly higher figure (50%) of satellite cells undergoing asymmetric segregation of template DNA was reported [24]. These authors suggest that this is accompanied by cell fate decisions, based on desmin, as a marker of myogenic commitment and Scal for ‘stemness’, although the robustness of the latter may be questioned.
Cell fate decisions and signaling pathways Cells that form skeletal muscle in the embryo. This takes place in waves; (1) Myf5+/Mrf4+ cells delaminate from the edges of the dermomyotome to form the early myotome. These cells also express Pax3. (2) Pax3+ cells delaminate and migrate away from the somites to form more distant muscle masses such as those in the limbs. (3) A third wave of MyoD+ cells from the edges of the dermomyotome contributes to the myotome. (4) The central epithelial structure of the dermomyotome breaks down releasing Pax3+/Pax7+ cells into the myotome, subsequently extending to all trunk muscles.
Stem cell behaviour in the satellite cell population The implication of the observed Myf5/Pax7 heterogeneity is that most satellite cells at some stage of muscle development had engaged the myogenic programme and then reverted to a quiescent satellite cell state. Indeed, even in cell culture, activated satellite cells can revert to a Pax7+/MyoD state [19,20]. The question is whether such cells have less capacity for self-renewal. In the Myf5Cre/Rosa26-YFP experiment, Pax7+/YFP (Myf5) cells were more efficient in reconstituting the satellite cell compartment, in grafting experiments, and furthermore constituted a reservoir for both Pax7+/Myf5 and Pax7+/Myf5+ cells [17] (Figure 2). Thus, Pax7+ cells, which have never engaged the myogenic programme, have more stem cell like properties. Interestingly, maintenance of this status correlates with the apical/basal polarity of cell division on the muscle fibre, such that the apical cell in contact with the basal lamina, maintains a Pax7+/Myf5 phenotype, in contrast to the basal cell that adopts a Pax7+/Myf5+ committed phenotype. Planar www.sciencedirect.com
Numb/Notch antagonism is thought to be involved in stem cell behaviour in many systems [25]. Delta1 stimulated Notch signaling has been implicated in satellite cell mobilisation in adult muscles, and forced activation of this pathway leads to increased regeneration in muscles of ageing mice [26]. Delta1 appears to be produced not only by activated satellite cells but also by injured fibres. This is further illustrated by the muscle regeneration defects displayed by Stra13-deficient mice [27]. Stra13 encodes a factor that antagonises the interaction between intracellular-activated Notch and its transcriptional effector, RBPJ. In the absence of Stra13, Notch signaling is enhanced, resulting in increased satellite cell proliferation and the failure of muscle differentiation. A potential link between Notch signaling and the transmembrane protein Megf10, a novel marker of quiescent and activated satellite cells, has also been suggested in the control of satellite cell proliferation [28]. During skeletal muscle development, conditional invalidation of genes encoding the Notch ligand, Delta1 [29] or the transcriptional mediator of Notch signaling, RBP-J [30], using a Pax3-Cre, promoted myogenic differentiation, with depletion of the Pax3/ Pax7 positive progenitor cell population, leading to a failure of skeletal muscle growth. In these examples, Notch signaling is regulating the balance between progenitor cell self-renewal and myogenic differentiation. However, Notch signaling has also been shown to influence cell fate decisions in the multipotent Pax3/Pax7 positive cells of the dermomyotome (dorsal somite). In mouse [31] and avian [32] embryos, a common Pax positive progenitor will give rise to the mural smooth Current Opinion in Genetics & Development 2008, 18:330–336
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Figure 2
Schematic representation of satellite cells at different stages from quiescence, activation/division, expansion to differentiation. Signaling pathways that promote these different satellite cell states are indicated.
muscle cells of blood vessels, such as the dorsal aorta, and to the skeletal muscle cells of the myotome. Components of the Notch signaling pathway are present in the Pax positive cells, and the overactivation of Notch signaling prematurely activates the smooth muscle cell fate at the expense of muscle cells, whereas inhibition of Notch signaling biases the cell fate choice in favour of skeletal muscle [32].
tiation [36]. Conversion of muscle progenitor cells to a fibrogenic lineage, observed upon increased Wnt signaling in muscle of aged mice, suggests that this pathway may also be involved in muscle homeostasis during ageing [37]. Interestingly, increased Wnt signaling was observed in the Klotho mutant mouse model of accelerated ageing, where Klotho is thought to act as a secreted Wnt antagonist [38].
Wnt signaling also plays important roles in promoting myogenesis in the embryo. Canonical Wnt signaling directly activates the early transcription of Myf5 in the somite [33] and non-canonical Wnt signaling, in a PKCdependent pathway, promotes the transcriptional activity of Pax3, leading to expression of MyoD and myogenesis [34]. During adult myogenesis, it is proposed that a precise temporal regulation of Wnt and Notch signaling is required for efficient muscle repair, with Notch signaling promoting proliferation of satellite cells and Wnt signaling subsequently ensuring commitment to terminal differentiation. In this scheme, the control of GSK3-b, an intracellular signaling intermediate, plays a crucial role in the switch from one pathway to another [35]. Contrasting results, however, have been obtained in ex vivo studies where b-catenin (Wnt)-dependent signaling leads satellite cells to undergo self-renewal rather than differen-
Other signaling pathways are also implicated in myogenesis. FGF signaling promotes muscle differentiation [39] and manipulation of the antagonist, Sprouty, modulates the balance between progenitor cell self-renewal and muscle differentiation in Pax3/7 positive progenitor cells, expressing both Fgfr4 and Sprouty1 [40]. Quiescent satellite cells express both genes, with downregulation of Sprouty1 on activation in culture [41]. In Fgfr4 mutant mice, muscle regeneration is compromised [42]. Extracellular heparan sulfatases are required for muscle regeneration [43] and may affect FGF signaling during the transition from satellite cell proliferation to differentiation. At an earlier stage in regeneration, sphingolipid signaling is involved in the transition of satellite cells from quiescence to proliferation [44]. Among new markers of satellite cells, found as a result of microarray analyses, the calcitonin receptor is exclusively expressed on quiescent
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satellite cells and its stimulation delays their activation, pointing to a role for this signaling pathway in the regulation of quiescence [41].
complex that modifies chromatin adjacent to the site where Pax7 binds, permitting transcriptional activation [56].
TGFb signaling also modulates myogenesis, and mice with mutations in myostatin show extensive muscle growth, accompanied by impaired force generation in the adult [45]. Recent results in the chick embryo [46] now show that this member of the TGFb family affects the balance between proliferation and differentiation of myogenic precursor cells (Pax7+/Myf5+) by promoting cell cycle withdrawal and, in the embryonic context, muscle differentiation, although in the adult it may promote satellite cell quiescence.
In order to understand how Pax3/7 regulate muscle stem cells, it is essential to identify their targets. Fgfr4 has now been identified as a direct target, via a 30 regulatory sequence that drives expression of the gene at sites of myogenesis [40]. Sprouty1 also lies genetically downstream of Pax3/7, providing a mechanism for Pax-dependent modulation of self-renewal versus differentiation. Pax3/7 also directly activates a 57.5 kb Myf5 regulatory element, which is necessary for transcription at sites of myogenesis in the embryo, notably in the limbs [57]. This element is also regulated by Six1/4 in the embryo [58], further illustrating the upstream role of this family of transcription factors in myogenesis [4]. Myf5 transcription in satellite cells depends on other regulatory regions [59,60]; however, the Pax site in the 57.5 kb sequence was shown to bind the Pax7/histone methyltransferase complex in satellite cells [56], perhaps because the locus is open. Observations on a requirement for Pax7 activation of Myf5, leading to myogenic commitment in this study, would be expected to apply to the small fraction of Pax7+/ Myf5 satellite cells, since Pax-independent myogenesis takes place in the presence of Myf5 [11].
Within muscles, satellite cells are often located close to blood vessels, with reciprocal paracrine effects on myogenesis and angiogenesis [47]. Blood vessel associated cells with myogenic potential [48] [49] may reflect this juxtaposition.
Myogenic regulatory mechanisms Artificially high levels of Pax7 in satellite cells are not compatible with differentiation and Myogenin appears to be crucial for Pax7 downregulation [50]. Rapid Pax protein degradation, essential for myogenic progression, depends on post-transcriptional mechanisms that differ for Pax3 and Pax7 [51]. Interestingly Pax3, but not Pax7, is regulated by ubiquitination dependent proteosomal degradation, involving a novel role for protein monoubiquitination. Pax3 and Pax7 play an important role in muscle stem cell survival, both in the embryo [8] and post-natally when muscle growth and regeneration are compromised in Pax7 mutant mice, because of progressive loss of activated satellite cells due to cell death [11]. Satellite cells that lack MyoD, a marker of activation and myogenic commitment, display increased survival when grafted [52], also seen with freshly isolated satellite cells that are not yet activated [3]. Pax3/7 are required for MyoD expression [11], so this will not contribute to their anti-apoptotic function. However, other mechanisms affect satellite cell survival. In vivo manipulation of Necdin, expressed in activated satellite cells, shows that it protects against apoptosis and accelerates differentiation, possibly through its interaction with proteins involved in cell cycle progression, such as p53 [53]. An interesting factor found in satellite cells is PW1 that acts through the p53 pathway to regulate satellite cell expansion and muscle homeostasis [54]. Neuregulin receptors of the ErbB family, present in activated satellite cells, are also associated with cell survival [55]. Pax3/7 function, like that of other Pax factors, probably depends on co-factors [4]. Recently it has been shown that Pax7 is present in a histone methyltransferase www.sciencedirect.com
Mrf4 and Myf5 genes are linked in the same locus, with regulatory elements that direct the distinct spatio-temporal patterns of these two genes lying within the intragenic region and, notably, also 50 of Mrf4-Myf5. Manipulation of the promoter/enhancer composition of the locus reveals a delicate equilibrium between promoter/enhancer interactions, regulated by transcriptional balancing sequences that can also act as cryptic promoters [61]. Myf5 and MyoD have largely overlapping functions as determination factors during development. However, in the adult, MyoD mutant mice regenerate less well, apparently owing to impaired differentiation of satellite cells [62], although survival is also affected [52]. Viable Myf5 mutant mice, in which the neighbouring Mrf4 gene is not affected, have now also been shown to have a regeneration defect, revealed in response to certain types of injury and to chronic muscle regeneration [63,64]. Although satellite cell number in vivo is not detectably altered, effects on proliferation in culture are observed, also seen with late foetal myoblasts in the absence of Myf5 [65]. Another player in the regulation of satellite cells is Sox15, which activates Foxk1 and is required for proliferation of the satellite cell population. In Sox15 mutant mice regeneration is impaired [66]. The regulatory cascade that leads from Pax3/7 to Myf5/ MyoD and then to myogenic differentiation or to the Current Opinion in Genetics & Development 2008, 18:330–336
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renewal of the upstream stem cell population is beginning to emerge. In this context recent results in Drosophila on transcriptional networks governing the role of Twist in mesoderm formation [67] and Ladybird in muscle and heart cell specification [68] are conceptually important. Contrary to what was originally concluded from mutant analyses, these key regulators act at multiple levels, in a feed-forward model of the cascade that leads from a multipotent stem cell to a differentiated muscle.
Acknowledgements MB and DM thank Didier Rocancourt for the illustrations. Work on myogenesis in the Buckingham laboratory is supported by the Pasteur Institute and the Centre National de la Recherche Scientifique, with grants from the Association Franc¸aise contre les Myopathies, the EU Integrated Project ‘EuroSyStem’ and EU Networks of Excellence, ‘Cells into Organs’ and ‘MYORES’.
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This paper addresses the complex regulation of the Mrf4-Myf5 locus that encodes these two myogenic regulatory factors and shows that transcriptional balancing elements determine promoter/enhancer interactions leading to the distinct expression profiles of the two genes.
66. Meeson AP, Shi X, Alexander MS, Williams RS, Allen RE, Jiang N, Adham IM, Goetsch SC, Hammer RE, Garry DJ: Sox15 and Fhl3 transcriptionally coactivate Foxk1 and regulate myogenic progenitor cells. EMBO J 2007, 26:1902-1912.
62. Megeney LA, Kablar B, Garrett K, Anderson JE, Rudnicki MA: MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev 1996, 10:1173-1183.
67. Sandmann T, Girardot C, Brehme M, Tongprasit W, Stolc V, Furlong EE: A core transcriptional network for early mesoderm development in Drosophila melanogaster. Genes Dev 2007, 21:436-449.
63. Gayraud-Morel B, Chretien F, Flamant P, Gomes D, Zammit PS, Tajbakhsh S: A role for the myogenic determination gene Myf5 in adult regenerative myogenesis. Dev Biol 2007, 312:13-28. 64. Ustanina S, Carvajal J, Rigby P, Braun T: The myogenic factor Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification. Stem Cells 2007, 25:2006-2016. 65. Montarras D, Lindon C, Pinset C, Domeyne P: Cultured myf5 null and myoD null muscle precursor cells display distinct growth defects. Biol Cell 2000, 92:565-572.
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68. Junion G, Bataille L, Jagla T, Da Ponte JP, Tapin R, Jagla K: Genome-wide view of cell fate specification: Ladybird acts at multiple levels during diversification of muscle and heart precursors. Genes Dev 2007, 21:3163-3180. A systems biology approach to the genetic network that is regulated by the Ladybird gene during myogenesis in Drosophila reveals a feedforward mechanism where Ladybird intervenes at multiple levels in the myogenic cascade. This is an example of the mode of regulation observed during mesoderm induction, reported in [67].
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Skeletal muscle stem cells Margaret Buckingham and Didier Montarras In this review we shall discuss recent publications on the heterogeneity of muscle stem cells, signaling pathways that affect their behaviour and regulatory mechanisms that underlie their myogenic fate, with reference to insights provided by work on skeletal muscle formation in the embryo as well as the adult, with the mouse as a model of reference. Addresses Pasteur Institute, CNRS URA 2578, 28 rue du Dr Roux, 75015 Paris, France Corresponding author: Buckingham, Margaret ([email protected]) and Montarras, Didier ([email protected])
Current Opinion in Genetics & Development 2008, 18:330–336 This review comes from a themed issue on Pattern formation and developmental mechanisms Edited by Ottoline Leyser and Olivier Pourquie´ Available online 30th July 2008 0959-437X/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2008.06.005
Introduction The notion that satellite cells represent the major cell type responsible for post-natal skeletal muscle growth and regeneration has recently received further support [1–3]. These quiescent cells, located under the basal lamina of muscle fibres, become activated upon injury, proliferate and differentiate into new muscle fibres. During regeneration, the satellite cell pool is also reconstituted. Thus, satellite cells display two hallmarks of stem cells: lineage-specific differentiation and selfrenewal.
Heterogeneity between muscle progenitor cells Skeletal muscle formation [4] (Figure 1) depends on the myogenic regulatory factors, of which MyoD and Myf5/ Mrf4 determine muscle cell fate, whereas Myogenin, as well as MyoD and Mrf4, controls differentiation. At the onset of myogenesis in the embryo, Myf5/Mrf4 and then MyoD are directly activated in the dorsal somite by signals from adjacent tissues. Although the initial activation of MyoD depends on Myf5/Mrf4, it is subsequently expressed in the absence of these factors. Selective ablation of Myf5 expressing cells indicates that the MyoD positive population is distinct, suggesting a two cell lineage model [5,6]. However, a Myf5nlacZ allele is tranCurrent Opinion in Genetics & Development 2008, 18:330–336
scribed in MyoD positive cells [7], but this may be insufficient to generate enough Myf5 dependent Cre recombinase required for ablation. After the first wave of myogenesis that results in the formation of the early myotome, Pax3 and Pax7 act upstream of Myf5 and MyoD and thus control the entry of cells into the myogenic programme [8]. Pax3/Pax7 positive stem cells from the somite are subsequently present in all developing skeletal muscle masses and give rise to the satellite cells of post-natal muscle [4]. Myoblasts (Myf5/MyoD positive), isolated from late embryonic or foetal muscle have different properties, further demonstrated by transcriptome analysis [9]. This may reflect a contribution from the first wave of myogenesis to the embryonic population, but is also probably due to the onset of innervation in the foetal period. Spatial as well as temporal parameters lead to muscle stem cell heterogeneity. As development proceeds, Pax3 expression is downregulated and Pax7 marks satellite cells [10]; however, Pax3 continues to be transcribed in some muscles [11,12]. This is the case in the diaphragm, also distinguished by more severe MyoD [13] and FgfrL1 [14] mutant phenotypes, indicative of heterogeneity between different sites of myogenesis. Lbx1 deficient mouse embryos display a lack of limb muscles attributed to migration defects. The finding that Lbx1 marks activated satellite cells after injury [15] may be of importance in understanding satellite cell mobility, required for adult muscle repair. The Pax3/Pax7 positive cells in skeletal muscle before birth either proliferate or activate Myf5 and MyoD, followed by muscle differentiation. However, by contrast, the majority of quiescent Pax7 positive satellite cells (80%) transcribe Myf5, as indicated by b-galactosidase activity from a Myf5nlacZ allele [16] (Figure 2). This heterogeneity has been investigated using a Myf5Cre/Rosa26-YFP genetic approach [17], which shows that 10% of Pax7 positive satellite cells have never expressed significant levels of Myf5. Further heterogeneity, suggested by YFP (Myf5) positive cells that are Pax7 negative, may reflect the surface markers used for cell selection and the fact that Myf5 and therefore the Myf5Cre allele, is expressed in presomitic mesoderm [18], leading to labeling of other mesodermal lineages, see also [5]. With the available antibody tools, MyoD is not detected in quiescent satellite cells and expression of the gene is thought to be a hallmark of activation. www.sciencedirect.com
Skeletal muscle stem cells Buckingham and Montarras 331
Figure 1
division (parallel to the fibre), which represents 90% of all divisions scored, leads to symmetrical Pax7+/Myf5+ daughter cell fates [17]. This suggests that signaling from the fibre promotes myogenesis. Asymmetric segregation of Numb has also been documented in dividing satellite cells [21], although it is not clear how this correlates with Pax7 versus Pax7/Myf5 expression and indeed it was also detected in cultured satellite cells [22]. A further indicator of ‘stemness’, although the underlying mechanism is controversial [23], is non-random template DNA segregation. Pulse labeling with BrdU demonstrated asymmetric strand segregation in about 7% of satellite cells, which also showed asymmetric segregation of Numb [22]. Interestingly this can occur in satellite cells that have activated Myf5. Using pulses of halogenated thymidine analogues during injury induced regeneration, a strikingly higher figure (50%) of satellite cells undergoing asymmetric segregation of template DNA was reported [24]. These authors suggest that this is accompanied by cell fate decisions, based on desmin, as a marker of myogenic commitment and Scal for ‘stemness’, although the robustness of the latter may be questioned.
Cell fate decisions and signaling pathways Cells that form skeletal muscle in the embryo. This takes place in waves; (1) Myf5+/Mrf4+ cells delaminate from the edges of the dermomyotome to form the early myotome. These cells also express Pax3. (2) Pax3+ cells delaminate and migrate away from the somites to form more distant muscle masses such as those in the limbs. (3) A third wave of MyoD+ cells from the edges of the dermomyotome contributes to the myotome. (4) The central epithelial structure of the dermomyotome breaks down releasing Pax3+/Pax7+ cells into the myotome, subsequently extending to all trunk muscles.
Stem cell behaviour in the satellite cell population The implication of the observed Myf5/Pax7 heterogeneity is that most satellite cells at some stage of muscle development had engaged the myogenic programme and then reverted to a quiescent satellite cell state. Indeed, even in cell culture, activated satellite cells can revert to a Pax7+/MyoD state [19,20]. The question is whether such cells have less capacity for self-renewal. In the Myf5Cre/Rosa26-YFP experiment, Pax7+/YFP (Myf5) cells were more efficient in reconstituting the satellite cell compartment, in grafting experiments, and furthermore constituted a reservoir for both Pax7+/Myf5 and Pax7+/Myf5+ cells [17] (Figure 2). Thus, Pax7+ cells, which have never engaged the myogenic programme, have more stem cell like properties. Interestingly, maintenance of this status correlates with the apical/basal polarity of cell division on the muscle fibre, such that the apical cell in contact with the basal lamina, maintains a Pax7+/Myf5 phenotype, in contrast to the basal cell that adopts a Pax7+/Myf5+ committed phenotype. Planar www.sciencedirect.com
Numb/Notch antagonism is thought to be involved in stem cell behaviour in many systems [25]. Delta1 stimulated Notch signaling has been implicated in satellite cell mobilisation in adult muscles, and forced activation of this pathway leads to increased regeneration in muscles of ageing mice [26]. Delta1 appears to be produced not only by activated satellite cells but also by injured fibres. This is further illustrated by the muscle regeneration defects displayed by Stra13-deficient mice [27]. Stra13 encodes a factor that antagonises the interaction between intracellular-activated Notch and its transcriptional effector, RBPJ. In the absence of Stra13, Notch signaling is enhanced, resulting in increased satellite cell proliferation and the failure of muscle differentiation. A potential link between Notch signaling and the transmembrane protein Megf10, a novel marker of quiescent and activated satellite cells, has also been suggested in the control of satellite cell proliferation [28]. During skeletal muscle development, conditional invalidation of genes encoding the Notch ligand, Delta1 [29] or the transcriptional mediator of Notch signaling, RBP-J [30], using a Pax3-Cre, promoted myogenic differentiation, with depletion of the Pax3/ Pax7 positive progenitor cell population, leading to a failure of skeletal muscle growth. In these examples, Notch signaling is regulating the balance between progenitor cell self-renewal and myogenic differentiation. However, Notch signaling has also been shown to influence cell fate decisions in the multipotent Pax3/Pax7 positive cells of the dermomyotome (dorsal somite). In mouse [31] and avian [32] embryos, a common Pax positive progenitor will give rise to the mural smooth Current Opinion in Genetics & Development 2008, 18:330–336
332 Pattern formation and developmental mechanisms
Figure 2
Schematic representation of satellite cells at different stages from quiescence, activation/division, expansion to differentiation. Signaling pathways that promote these different satellite cell states are indicated.
muscle cells of blood vessels, such as the dorsal aorta, and to the skeletal muscle cells of the myotome. Components of the Notch signaling pathway are present in the Pax positive cells, and the overactivation of Notch signaling prematurely activates the smooth muscle cell fate at the expense of muscle cells, whereas inhibition of Notch signaling biases the cell fate choice in favour of skeletal muscle [32].
tiation [36]. Conversion of muscle progenitor cells to a fibrogenic lineage, observed upon increased Wnt signaling in muscle of aged mice, suggests that this pathway may also be involved in muscle homeostasis during ageing [37]. Interestingly, increased Wnt signaling was observed in the Klotho mutant mouse model of accelerated ageing, where Klotho is thought to act as a secreted Wnt antagonist [38].
Wnt signaling also plays important roles in promoting myogenesis in the embryo. Canonical Wnt signaling directly activates the early transcription of Myf5 in the somite [33] and non-canonical Wnt signaling, in a PKCdependent pathway, promotes the transcriptional activity of Pax3, leading to expression of MyoD and myogenesis [34]. During adult myogenesis, it is proposed that a precise temporal regulation of Wnt and Notch signaling is required for efficient muscle repair, with Notch signaling promoting proliferation of satellite cells and Wnt signaling subsequently ensuring commitment to terminal differentiation. In this scheme, the control of GSK3-b, an intracellular signaling intermediate, plays a crucial role in the switch from one pathway to another [35]. Contrasting results, however, have been obtained in ex vivo studies where b-catenin (Wnt)-dependent signaling leads satellite cells to undergo self-renewal rather than differen-
Other signaling pathways are also implicated in myogenesis. FGF signaling promotes muscle differentiation [39] and manipulation of the antagonist, Sprouty, modulates the balance between progenitor cell self-renewal and muscle differentiation in Pax3/7 positive progenitor cells, expressing both Fgfr4 and Sprouty1 [40]. Quiescent satellite cells express both genes, with downregulation of Sprouty1 on activation in culture [41]. In Fgfr4 mutant mice, muscle regeneration is compromised [42]. Extracellular heparan sulfatases are required for muscle regeneration [43] and may affect FGF signaling during the transition from satellite cell proliferation to differentiation. At an earlier stage in regeneration, sphingolipid signaling is involved in the transition of satellite cells from quiescence to proliferation [44]. Among new markers of satellite cells, found as a result of microarray analyses, the calcitonin receptor is exclusively expressed on quiescent
Current Opinion in Genetics & Development 2008, 18:330–336
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Skeletal muscle stem cells Buckingham and Montarras 333
satellite cells and its stimulation delays their activation, pointing to a role for this signaling pathway in the regulation of quiescence [41].
complex that modifies chromatin adjacent to the site where Pax7 binds, permitting transcriptional activation [56].
TGFb signaling also modulates myogenesis, and mice with mutations in myostatin show extensive muscle growth, accompanied by impaired force generation in the adult [45]. Recent results in the chick embryo [46] now show that this member of the TGFb family affects the balance between proliferation and differentiation of myogenic precursor cells (Pax7+/Myf5+) by promoting cell cycle withdrawal and, in the embryonic context, muscle differentiation, although in the adult it may promote satellite cell quiescence.
In order to understand how Pax3/7 regulate muscle stem cells, it is essential to identify their targets. Fgfr4 has now been identified as a direct target, via a 30 regulatory sequence that drives expression of the gene at sites of myogenesis [40]. Sprouty1 also lies genetically downstream of Pax3/7, providing a mechanism for Pax-dependent modulation of self-renewal versus differentiation. Pax3/7 also directly activates a 57.5 kb Myf5 regulatory element, which is necessary for transcription at sites of myogenesis in the embryo, notably in the limbs [57]. This element is also regulated by Six1/4 in the embryo [58], further illustrating the upstream role of this family of transcription factors in myogenesis [4]. Myf5 transcription in satellite cells depends on other regulatory regions [59,60]; however, the Pax site in the 57.5 kb sequence was shown to bind the Pax7/histone methyltransferase complex in satellite cells [56], perhaps because the locus is open. Observations on a requirement for Pax7 activation of Myf5, leading to myogenic commitment in this study, would be expected to apply to the small fraction of Pax7+/ Myf5 satellite cells, since Pax-independent myogenesis takes place in the presence of Myf5 [11].
Within muscles, satellite cells are often located close to blood vessels, with reciprocal paracrine effects on myogenesis and angiogenesis [47]. Blood vessel associated cells with myogenic potential [48] [49] may reflect this juxtaposition.
Myogenic regulatory mechanisms Artificially high levels of Pax7 in satellite cells are not compatible with differentiation and Myogenin appears to be crucial for Pax7 downregulation [50]. Rapid Pax protein degradation, essential for myogenic progression, depends on post-transcriptional mechanisms that differ for Pax3 and Pax7 [51]. Interestingly Pax3, but not Pax7, is regulated by ubiquitination dependent proteosomal degradation, involving a novel role for protein monoubiquitination. Pax3 and Pax7 play an important role in muscle stem cell survival, both in the embryo [8] and post-natally when muscle growth and regeneration are compromised in Pax7 mutant mice, because of progressive loss of activated satellite cells due to cell death [11]. Satellite cells that lack MyoD, a marker of activation and myogenic commitment, display increased survival when grafted [52], also seen with freshly isolated satellite cells that are not yet activated [3]. Pax3/7 are required for MyoD expression [11], so this will not contribute to their anti-apoptotic function. However, other mechanisms affect satellite cell survival. In vivo manipulation of Necdin, expressed in activated satellite cells, shows that it protects against apoptosis and accelerates differentiation, possibly through its interaction with proteins involved in cell cycle progression, such as p53 [53]. An interesting factor found in satellite cells is PW1 that acts through the p53 pathway to regulate satellite cell expansion and muscle homeostasis [54]. Neuregulin receptors of the ErbB family, present in activated satellite cells, are also associated with cell survival [55]. Pax3/7 function, like that of other Pax factors, probably depends on co-factors [4]. Recently it has been shown that Pax7 is present in a histone methyltransferase www.sciencedirect.com
Mrf4 and Myf5 genes are linked in the same locus, with regulatory elements that direct the distinct spatio-temporal patterns of these two genes lying within the intragenic region and, notably, also 50 of Mrf4-Myf5. Manipulation of the promoter/enhancer composition of the locus reveals a delicate equilibrium between promoter/enhancer interactions, regulated by transcriptional balancing sequences that can also act as cryptic promoters [61]. Myf5 and MyoD have largely overlapping functions as determination factors during development. However, in the adult, MyoD mutant mice regenerate less well, apparently owing to impaired differentiation of satellite cells [62], although survival is also affected [52]. Viable Myf5 mutant mice, in which the neighbouring Mrf4 gene is not affected, have now also been shown to have a regeneration defect, revealed in response to certain types of injury and to chronic muscle regeneration [63,64]. Although satellite cell number in vivo is not detectably altered, effects on proliferation in culture are observed, also seen with late foetal myoblasts in the absence of Myf5 [65]. Another player in the regulation of satellite cells is Sox15, which activates Foxk1 and is required for proliferation of the satellite cell population. In Sox15 mutant mice regeneration is impaired [66]. The regulatory cascade that leads from Pax3/7 to Myf5/ MyoD and then to myogenic differentiation or to the Current Opinion in Genetics & Development 2008, 18:330–336
334 Pattern formation and developmental mechanisms
renewal of the upstream stem cell population is beginning to emerge. In this context recent results in Drosophila on transcriptional networks governing the role of Twist in mesoderm formation [67] and Ladybird in muscle and heart cell specification [68] are conceptually important. Contrary to what was originally concluded from mutant analyses, these key regulators act at multiple levels, in a feed-forward model of the cascade that leads from a multipotent stem cell to a differentiated muscle.
Acknowledgements MB and DM thank Didier Rocancourt for the illustrations. Work on myogenesis in the Buckingham laboratory is supported by the Pasteur Institute and the Centre National de la Recherche Scientifique, with grants from the Association Franc¸aise contre les Myopathies, the EU Integrated Project ‘EuroSyStem’ and EU Networks of Excellence, ‘Cells into Organs’ and ‘MYORES’.
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336 Pattern formation and developmental mechanisms
This paper addresses the complex regulation of the Mrf4-Myf5 locus that encodes these two myogenic regulatory factors and shows that transcriptional balancing elements determine promoter/enhancer interactions leading to the distinct expression profiles of the two genes.
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68. Junion G, Bataille L, Jagla T, Da Ponte JP, Tapin R, Jagla K: Genome-wide view of cell fate specification: Ladybird acts at multiple levels during diversification of muscle and heart precursors. Genes Dev 2007, 21:3163-3180. A systems biology approach to the genetic network that is regulated by the Ladybird gene during myogenesis in Drosophila reveals a feedforward mechanism where Ladybird intervenes at multiple levels in the myogenic cascade. This is an example of the mode of regulation observed during mesoderm induction, reported in [67].
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