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Experimental Procedures
Author Contributions
Acknowledgments
Introduction Adult vertebrate muscle has an exceptional capacity for regeneration, mediated by a dedicated population of muscle stem cells. These muscle stem cells, termed satellite cells, were first identified by their unique anatomical position between the sarcolemma and basement membrane of myofibers (Mauro, 1961). Subsequently, satellite purchase Pyrrolidinedithiocarbamate ammonium were found to express the transcription factor Pax7 (Seale et al., 2000), and Pax7 is required for their maintenance in adult mice (Günther et al., 2013; Kuang et al., 2006; Oustanina et al., 2004; Relaix et al., 2006; von Maltzahn et al., 2013). Recent genetic labeling and ablation studies in mouse, using Pax7 mice, have definitively established that satellite cells are the endogenous stem cells necessary and sufficient for muscle regeneration (Lepper et al., 2009, 2011; Murphy et al., 2011; Sambasivan et al., 2011). During regeneration, satellite cells activate, proliferate, and give rise to transit-amplifying myoblasts, which differentiate into myocytes that fuse with one another to form multinucleate myofibers. In addition, like other stem cells, satellite cells self-renew. Canonical Wnt/β-catenin signaling is an important regulator of many adult stem cells (Holland et al., 2013) and has been proposed to be critical for satellite cells and muscle regeneration. Wnts are secreted glycoproteins that function as ligands, and β-catenin is the central mediator of canonical Wnt signaling (Niehrs, 2012). In the absence of Wnts, β-catenin is phosphorylated and targeted for degradation. The binding of Wnts to their receptors leads to the formation of stabilized, unphosphorylated β-catenin that translocates to the nucleus, where it binds to TCF/LEF proteins and activates transcription of Wnt-responsive genes. Many studies have identified Wnt pathway components as being active during muscle regeneration (Brack et al., 2008, 2009; Le Grand et al., 2009; Polesskaya et al., 2003; Zhao and Hoffman, 2004). Based largely on gain-of-function, primarily in vitro experiments, multiple labs have proposed that Wnt/β-catenin signaling is essential for muscle regeneration, although the conclusions of these papers are often contradictory (reviewed in von Maltzahn et al., 2012). However, no studies have explicitly examined in vivo whether Wnt/β-catenin signaling is necessary and sufficient specifically within satellite cells and their derivatives for muscle regeneration. In this study, we use a highly sensitive reporter of Wnt/β-catenin signaling (TCF/Lef:H2B-GFP; Ferrer-Vaquer et al., 2010), as well as a reagent (Pax7) that our lab has generated to genetically manipulate satellite cells with high specificity and efficiency (Murphy et al., 2011), to test the role of this signaling pathway specifically within satellite cells and their derivatives during muscle regeneration. We find that Wnt/β-catenin signaling is transiently active in myoblasts during regeneration. However, β-catenin is not required cell autonomously for muscle regeneration. Instead, downregulation of transiently activated β-catenin is critical for limiting the regenerative response, as continuous regeneration deleteriously leads to increased fibrosis and an increased number of small myofibers. Thus, surprisingly, we show that it is not activation of Wnt/β-catenin signaling but rather silencing of this activation that is important for muscle regeneration.
Results
Discussion Wnt/β-catenin signaling has been proposed to be critical for adult muscle regeneration (reviewed in von Maltzahn et al., 2012). Here, we explicitly test the role of this signaling pathway specifically within satellite cells and their derivatives during muscle regeneration in vivo. We find that Wnt/β-catenin signaling is transiently active in myoblasts during muscle regeneration. However, unlike previous studies, we find that β-catenin is not required in myogenic cells for regeneration, but instead downregulation of transiently activated β-catenin is critical for limiting the regenerative response. Thus, we show that it is not activation but rather silencing of Wnt/β-catenin signaling that is important for muscle regeneration (summarized in Figure 7).