The Notch signaling network in muscle stem cells during development, homeostasis, and disease

With the notable exception of blastocyst morphogenesis and the establishment of the three germ layers (ectoderm, endoderm, and mesoderm) [1], Notch signaling is involved in the formation of virtually every tissue studied to date and, not surprisingly, has emerged as a major regulator of stem cell functions. Notch is a highly conserved transmembrane plasma receptor that mediates cell-cell communication. Similar to the murine Notch receptors (Notch-1, -2, -3 -4 in mammals), its ligands (Delta-like (DLL) -1, -4 and Jagged (JAG)-1, -2 in mammals) are also transmembrane proteins, so physical cell-to-cell interaction is required for activation of the pathway (note that cis receptor/ligand interactions are predominantly inhibitory [2, 3] and secreted, cleaved ligands seem to have no activity in vivo [4, 5]). Notch is one of a handful of plasma membrane receptors that acts both as a cell surface receptor and a transcription factor, together with the leukocyte-common antigen-related receptor tyrosine phosphatase (LAR), the amyloid precursor protein, and the receptor tyrosine kinase receptor ERBB4 [6, 7]. Following ligand-triggered intramembrane proteolysis, the intracellular domain of Notch (NICD) is cleaved and translocates into the nucleus, where it acts as a transcriptional coactivator [8, 9]. There NICD forms a complex with CSL (RBPJ in vertebrates, Su(H) in flies, Lag-1 in roundworms) and stabilizes its binding to DNA to induce gene expression [10,11,12] (Fig. 1A).

Fig. 1figure 1

The Notch signaling pathway during myogenic progression and self-renewal. A Basic scheme of the Notch signaling pathway in murine muscle cells. The receptor is more highly expressed on the stem/progenitor cell (signal receiving cell), whereas the DLL ligands on the committed myoblasts and the mature myofibers (signal sending cells). Ligand-receptor interaction triggers intramembrane proteolysis and release of the intracellular domain of Notch (NICD). NICD then translocates into the nucleus where it forms a complex with its main downstream effector and DNA binding transcription factor RBPJ, and members of the coactivator Mastermind-like (MAML) family. The triprotein NICD transcriptional complex recruits additional coactivators and histone modifiers to activate transcription, not illustrated here for simplicity. B During MuSC activation and myogenic commitment, Notch signaling activity is downregulated. Quiescent MuSCs have high Notch activity (dark blue in color key), which maintains Pax7 and inhibits Myod and Myogenin expression. Immediately after MuSC activation, Notch activity rapidly declines and the cells express MYOD, which accelerates S-phase entry. During the proliferation phase, high Notch activity is restricted to some cells, which remain undifferentiated and self-renew to replenish the satellite cell pool (dotted arrow). Notch activation is principally triggered by ligand-bearing differentiating myoblasts (block arrow indicates direction of Notch signaling). Mature myofibers, in which Notch activity is insignificant (green color in color key), are the main source of ligand in the resting muscle and maintain MuSC quiescence by direct cell-cell interaction (block arrow)

Muscle stem cells (MuSCs) depend on Notch signaling activity from their emergence in the embryo to their residence in adult tissue, both in resting and regenerating conditions. Embryonic and adult MuSCs share several common features, including the expression of the paired-box homeodomain transcription factor PAX7, and execute an almost indistinguishable myogenic program, involving the sequential expression of specific muscle regulatory factors [13]. A prominent difference, however, between adult and embryonic MuSCs is the anatomy of their cellular niche, which is strictly defined by the myofiber and the basal lamina in adult MuSCs but is highly disorganized in the growing muscle. Another distinct difference is the proliferation of Pax7+ cells during murine muscle growth, whereas in adult tissue, MuSCs are mitotically quiescent. Regardless of the developmental stage of MuSCs, Notch signaling has been shown to be indispensable for both of these contrasting MuSC states. Both embryonic and adult MuSCs with abrogated Notch signaling ectopically differentiate, leading to a depletion of MuSCs and progenitors whereas in the context of growing tissue, Notch signaling mutations lead to sarcopenia [14,15,16,17,18,19,20,21]. In reciprocal experiments, overexpression of constitutively activated NICD, terminal myogenic differentiation was blocked, leading to a loss of muscle mass; a phenotype akin to the abrogation of the pathway [22,23,24,25]. In Table 1 we summarize the Notch signaling mouse models that have been reported with a muscle phenotype. Of note, the role of Notch signaling on muscle stem cell homeostasis and regeneration is conserved in other vertebrates, including the zebrafish [41] and the chick [42, 43].

Table 1 Notch signaling mouse mutants with MuSC phenotype

Effectively, a major function of Notch signaling in skeletal muscle is to sustain an upstream population of founder cells irrespective of their cycling status by safeguarding their undifferentiated state (Fig. 1B). More recent studies have elucidated the modus operandi of Notch pathway members, providing mechanistic explanations of their antimyogenic activity, their crosstalk with other signaling molecules and the interactions between heterologous cell types. In this review, we will provide an updated view on the ways by which Notch signaling factors and other modifying partners are operating during the establishment, maintenance, and self-renewal of MuSCs.

The core antimyogenic activity of notch decoded

The inhibition of differentiation by Notch signaling is principally driven by the well-characterized and highly conserved direct targets of the pathway, the basic-helix-loop-helix transcriptional repressors of the Hes/Hey family. In mouse skeletal muscle cells, the strongest responders to Notch activation amongst them (in order of transcriptional fold-induction) are HeyL, Hey1, and Hes1 [16, 23, 44], whereas in human myoblasts HES1 seems to be the most highly induced [11, 45]. Overexpression, though, of HeyL or Hes1 alone in the immortalized myogenic cell line C2C12 is not sufficient to block myogenesis [46]. It is the HEY1 repressor that does so by being recruited to the promoters of Myogenin and myocyte enhancer factor 2C (Mef2C) genes, whose products are critical for muscle differentiation [44]. Surprisingly, Hey1 germline knockout mice do not show MuSCs abnormalities [16]. Similarly, the number of adult MuSCs is mildly decreased or not affected at embryonic day (E) 17.5 in the hindlimb muscle of HeyL germline knockout mice [16]. Double-knock out Hey1/HeyL mice on the other hand, exhibit severe regenerative defects due to a reduction in MuSC number, resulting from increased Myod and Myogenin expression [16]. More recently, it was shown that HEYL forms heterodimeric complexes with HES1 and acts synergistically to bind with higher affinity to the Myogenin promoter [33] (Fig. 2A). Consistently, the removal of HeyL in Hes1 null cells (Pax3Cre; Hes1flox/flox; HeyL−/−) exacerbates the MuSC deficiency observed in Pax3Cre; Hes1flox/flox single mutant muscles [34].

Fig. 2figure 2

The Notch signaling network in murine muscle stem cells. A In the proliferating cells, enhancer competition and negative autoregulation establish an oscillatory system (pendulum sign) comprising transcription factors and ligands that regulate each other. Competition is also occurring for the transcriptional co-activator Mastermind-like 1 between the NICD activating complex and the differentiation factor Mef2c, while Mef2c is under the control of the Notch-controlled Dusp1 kinase that targets p38. Notch signaling also safeguards cells from spontaneous fusing by repressing the expression of the membrane activator of myoblast fusion Myomaker (Mymk). Ligand presentation on the growing fibers is stimulated by extrinsic cues, including mechanical stress (fetal chick fibers) and circulating sex hormones (puberty and muscle regeneration). The factors that maintain quiescence (Fig. 2B) are reiterated for self-renewal. Double-headed arrow indicates protein interaction; pendulum sign indicates oscillation; dashed arrow line indicates self-renewal; *the YAP/Jag2 link has been demonstrated in chick embryos. B Notch signaling maintains both quiescent and activated MuSCs by engaging different targets and interactors. Quiescent MuSC express Notch-1, -2, and -3 and, in the mouse, the principal ligand is Dll4 from the myofibers. Diverse direct NICD/RBPJ transcriptional targets execute different functions: the Hes/Hey repressors prevent the expression of differentiation factors, collagen V chain encoding genes directly contribute to the build-up of the quiescent niche, and micro-RNA mir-708 anchors MuSCs by targeting molecules involved in cell migration

The redundancy observed between the different HES/HEY proteins is not conserved in the context of muscle hypertrophy. Following overload-induced hypertrophy, the majority of MuSCs gets activated but shows some distinct features compared to the classical activation that is described in regenerating muscle and HeyL, unlike Hey1, is not downregulated upon activation. In fact, HeyL is required for the proliferation of MuSCs during induced hypertrophy, contrary to muscle regeneration [47].

Notch signaling seems to have evolved several safety lock mechanisms in muscle cells to prevent ectopic differentiation from occurring and to enhance regulation of transition states. In addition to the direct transcriptional repression of Mef2C by HEY1, a fine balancing mechanism has been uncovered whereby the essential transcriptional NICD coactivator, Mastermind-like 1 (MAML-1), is bound and sequestered by the MEF2C protein [48]. In C2C12 cells, Notch induces the expression of Dusp1, a dual-specificity MAPK phosphatase that blocks the activity of the MAPK family member p38 [49]. This prevents p38 from phosphorylating and activating MEF2C and E47, which would usually drive myogenesis and promote MYOD/E47 association [49, 50]. An unexpected function of HEYL in myoblast fusion has more recently been uncovered, unravelling another safety lock of the Notch system. Since Notch inhibition drives early differentiation, the direct regulation on fusion had been overlooked. By analyzing chick embryonic myogenesis, Esteves and colleagues discovered that Notch inhibition increased the expression of TMEM8C, the chick orthologue of the fusion master gene Myomaker. Specifically, HEYL was found to bind and repress the regulatory regions of TMEM8C, thus hindering myoblast fusion [51] (Fig. 2A).

At the level of receptors, using tamoxifen-inducible MuSC-specific knockout mice for Notch1 and Notch2, it was shown that the size of the adult satellite cell population is slightly reduced in Notch2-cKO, not significantly affected in Notch1-cKO, but almost completely depleted in double Notch1/Notch2 knockout mice [28]. The functional redundancy between NOTCH 1 and 2 during muscle regeneration was later confirmed with neutralizing antibodies [52]. Germline Notch3 knockout mice however, had an abnormally high number of MuSCs, pointing to an antagonistic function when compared with the other Notch receptors [29]. Several reports have compared NOTCH1 and NOTCH3 functions in different contexts and concluded that these Notch paralogues display distinct roles in stem cells. In neural stem cells of the zebrafish pallium, Notch3 maintains quiescence, in striking contrast from the role of Notch1 (Notch1b in zebrafish) that prevents differentiation of activated progenitors [53]. A similar functional dichotomy is observed in murine MuSCs, whereby Notch3 knock out leads to an increase in quiescent MuSCs and accelerated proliferation of activated MuSCs on isolated muscle fibers [29]. It remains unclear and controversial on how NOTCH3 operates mechanistically to antagonize Notch signaling. In muscle cells, one hypothesis examines the possibility of NOTCH3 induction of Nrarp functioning as a negative feedback regulator of Notch signaling by attenuating NICD-mediated transcription [54]. The loss of NOTCH3 during muscle growth and regeneration could lead to over-stimulation of Notch signaling by Nrarp downregulation which could in turn partially explain the increased number of PAX7 cells, and thus the muscle hyperplasia in Notch3 KO mice. Despite these hypotheses, further studies using conditional MuSC-targeted deletion of Notch3 would be required to acquire more precise information on the role of this locus during MuSC growth and homeostasis.

Dynamics and source of notch ligands in growing and resting muscle

The origin of MuSCs in the mouse trunk can be traced back to mesodermal cells of the dermomyotome [55], a transient epithelial structure in the somites formed in the mouse around embryonic day (E) 9. The muscle founder cells are initially marked by the transcription factors PAX3 and later by both PAX3 and PAX7 [56,57,58]. Their emergence in the dermomyotome does not seem to require Notch signaling, since they are unaffected in Notch mutant embryos [19, 20]. Like the mammalian central nervous system, Notch signaling is essential for maintenance but not the generation of neural stem cells [59]. This is consistent with the expression pattern of the Notch receptors -1, -2 and -3, and the ligands Jag2 and Dll-1 in embryonic MuSCs, which are excluded from the dermomyotome [RNA in situ hybridization [19];)]. The expression of Notch ligands is instead confined to the underlying myotome, where differentiating muscle cells reside and where Notch activity is critical. In Dll-1 and Rbpj (Pax3Cre; Rbpjflox/flox and Pax7CreERT2; Rbpjflox/flox) mutant mouse embryos, severe muscle hypotrophy has been observed due to precocious differentiation of the self-renewing MuSC population in the myotome [19, 20]; our unpublished data). Notably, the observation that the muscle phenotype in embryos with globally reduced activity of DLL1 resembles the Rbpj null MuSCs, strongly suggests that the prevalent Notch ligand in embryonic myogenesis is DLL1.

A similar scenario, with the same actors, seems to be played during regeneration in adult muscles. Using paralogue-specific antagonizing antibodies, it was shown that blocking DLL1 4 days after cardiotoxin-induced injury led to severe self-renewal defects. Using anti-DLL4, -JAG1 or -JAG2 during regeneration, instead, gave no regeneration phenotype [52]. The data produced by those two studies is comprehensive and has established that during embryonic myogenesis and adult muscle regeneration the essential Notch ligand is DLL1, whose function is not compensated by either DLL4 or JAG-1/2. However, in both studies, cells were targeted indiscriminately, thus preventing a precise pinpoint to the actual signal-sending cell type. In fact, both embryonic myogenesis and adult muscle regeneration involve extensive cell migration and mixing of MuSCs with diverse cell types [60, 61]. Mesenchymal cells, connective tissue fibroblasts, endothelial cells (ECs), and inflammatory cells from regenerating tissue, all constitute potential Notch signaling activators. This complex cellular system was genetically dissected using Pax7CreERT2; Dll1flox/flox mice [31]. This mutation did not affect the quiescent MuSCs, and activation occurred accordingly as was observed using α-DLL1 neutralizing antibodies [52]. However, at the later stages of regeneration, the cells differentiated prematurely rendering decreased PAX7+ and increased MYOG+ cells at 4 dpi, and even fewer PAX7+ cells at 7 dpi and 21 dpi [31]. Importantly, this phenotype was recapitulated on isolated myofibers from Pax7CreERT2; Dll1flox/flox mice. Since Dll-1 was mutated specifically in the MuSCs and not in the myofibers, these results demonstrated definitively that DLL1 produced by committed myoblasts maintains the MuSCs in a classical lateral inhibition manner, as the authors comment [31]. Although the MuSC-myoblast interaction between receptor and ligand seems to be predominant, it is not the only one observed. A unique example of Notch/DLL1 interaction between heterologous cell types has been uncovered during embryonic myogenesis in the developing chick. In this system, Notch is transiently activated in a subpopulation of muscle cells at the epaxial lip of the dermomyotome, by DLL1+ progenitors migrating neural crest cells [62].

The cellular arrangement in adult, resting muscle, however, is strikingly different from the growing (embryonic and regenerating) muscle, and so are the Notch factors involved. In resting muscle, MuSCs are positioned between the basement membrane and the myofiber, the sole cell with which there is known physical contact. Hence, based on the nature of Notch signaling, the myofiber is the strongest candidate for the source of Notch ligands. Yet, ECs have drawn special attention as an alternative source of ligand, since MuSCs are closely associated to capillaries in human and mouse muscle [63, 64]. In a recent study, in silico analysis of gene expression datasets led to the hypothesis that DLL4 from ECs interact with NOTCH1 and NOTCH3 on MuSCs, through the basal lamina, to maintain the latter in a quiescent state [64]. However, in vivo experimental evidence would be needed to corroborate this intriguing theory. To date, no apparent physical contact has been demonstrated between MuSCs and ECs, a prerequisite for Notch signaling-across the basal membrane, and soluble forms of Notch ligands have not been shown to exist or be active in vivo.

Despite the suggestions of alternative sources, several studies point to the myofibers as the major and essential source of ligands. In fetal chick myogenesis, Notch activation in MuSCs is achieved by muscle contractions that force the myonuclear localization of the mechanosensory transcription factor YAP and thus the expression of the Notch ligand JAG2 [65]. In mice, an intriguing study showed that circulating sex hormones enhance the presentation of DLL1 and JAG1 ligands in the membrane of newly formed myofibers during puberty, by modulating the activity of the E3 ubiquitin ligase Mindbomb 1 (Mib1) [27] (Fig. 2A). Of interest, inhibition of sex hormones by orchiectomy significantly reduced the number of self-renewed quiescent PAX7+ cells during puberty and muscle regeneration but had no impact on the maintenance of the quiescent MuSC in homeostatic muscles. One possible explanation for this distinction is that sex hormones impact Mib1 only in fresh myotubes and not adult myofibers. Alternatively, sex hormones might regulate Notch ligand presentation in other cell types, which are in contact with the MuSC only during growth and regeneration, but not in resting muscles. A third explanation could be that other ligands are dominant to maintain quiescent MuSCs. In fact, it is now established that DLL4 is expressed in newly formed myotubes and mature myofibers [32, 66, 67] in an MIB1-dependant manner, and it is required for the maintenance of quiescent MuSCs. The original investigation using primary cell culture models [66] was later confirmed in vivo using conditional Dll-4 flox deletion in muscle fibers (tamoxifen-inducible HSACreMER; Dll4floxflox mice) [32]. Thirty days after injecting tamoxifen for seven consecutive days resulted in a 50% decrease in the total number of PAX7+ MuSCs in Dll-4

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