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    • Using the highly sensitive TCF Lef H

    2018-10-24

    Using the highly sensitive TCF/Lef:H2B-GFP reporter, we demonstrate that Wnt/β-catenin signaling is transiently active during muscle regeneration, specifically in myoblasts 1 dpi. Our finding that myoblasts transiently transduce Wnt/β-catenin signals agrees with previous analyses of nuclear β-catenin and TOPGAL reporter expression (Brack et al., 2007, 2008). Results from others (Abiola et al., 2009; Le Grand et al., 2009; Polesskaya et al., 2003; Zhao and Hoffman, 2004) show that Wnts are upregulated during regeneration, although the cellular origin of these Wnts is unresolved. The Wnt antagonists, sFRPs 1, 2, and 4 are also strongly upregulated during regeneration (Le Grand et al., 2009; Zhao and Hoffman, 2004), and this likely is the endogenous molecular mechanism by which Wnt/β-catenin signaling, activated at 1 dpi, is subsequently silenced. We explicitly tested the requirement for β-catenin in satellite order olopatadine hcl and their derivatives for muscle regeneration. Despite efficient deletion of β-catenin, satellite cells were able to self-renew and regenerate muscle (although a subtle phenotype, undetectable in our assays, is possible). Interestingly, we did see that with β-catenin deletion myofibers shifted to larger cross-sectional areas at 28 dpi or with reinjury. Given that constitutive β-catenin activation prolonged regeneration and resulted in a shift to smaller, regenerating myofibers, the shift to larger myofibers with loss of β-catenin may indicate premature differentiation of myofibers. However, the finding that TAs regenerated from β-catenin satellite cells are GFP+ and do not differ in overall size from control TAs suggests that a potential requirement of β-catenin to inhibit premature differentiation is modest, at best. Consistent with the lack of a significant phenotype with β-catenin loss, previous studies have produced contradictory findings. Most experiments have been conducted in vitro and, using a variety of techniques to inhibit β-catenin signaling, have found decreased satellite cell proliferation (Otto et al., 2008), less differentiation (Brack et al., 2008; Descamps et al., 2008; Kim et al., 2008), or more differentiation (Gavard et al., 2004; Tanaka et al., 2011). Only Brack et al. (2008, 2009) inhibited Wnt/β-catenin signaling in vivo, via injection of sFRPs into regenerating TAs or genetic deletion of β-catenin coactivators BCL9 and BCL9-2 (via Myf5;Bcl9;Bcl9-2 mice) after BaCl2 or freeze injury. They concluded that Wnt/β-catenin is necessary to promote muscle differentiation, but addition of sFRPs blocks both canonical and noncanonical Wnt signaling (Li et al., 2008) and does not specifically target myogenic cells, and the genetic BCL9/BCL9-2 deletion potentially affects satellite cell development. Thus, previous phenotypes attributed to β-catenin necessity in satellite cells for regeneration may reflect in vitro conditions or in vivo reveal the role of canonical signaling in muscle progenitors during development or in other cell types involved in muscle regeneration or the function of noncanonical signaling in regeneration. The transient activation of Wnt/β-catenin signaling in myoblasts suggested the alternative hypothesis that not activation but silencing of signaling is critical for proper muscle regeneration. To test this, we examined the effects of constitutive β-catenin activation. Previous studies testing this have primarily been conducted in vitro, via Wnt3a or LiCl delivery to cultured satellite cells, and found constitutive Wnt/β-catenin signaling either prevents differentiation (Gavard et al., 2004; Kuroda et al., 2013; Tanaka et al., 2011) or promotes differentiation and fusion (Bernardi et al., 2011; Brack et al., 2008; Han et al., 2011; Pansters et al., 2011). Two papers (Brack et al., 2008; Le Grand et al., 2009) tested in vivo (using either BaCl2 or freeze injury) the effects of increased signaling and concluded that Wnt/β-catenin signaling promoted premature differentiation. However, these in vivo experiments activated, via ectopic Wnt3a, signaling in all cell types (including muscle connective tissue fibroblasts and endothelial cells) during regeneration. Our experiments constitutively activating β-catenin specifically in satellite cells revealed that satellite cells are largely insensitive to increased β-catenin, as we saw no effects on satellite cell expansion or proliferation after injury. However, constitutive β-catenin activation did alter the kinetics of the regenerative process, as myoblasts (which normally transiently express β-catenin) and subsequently myocytes and regenerating myofibers are present for an extended period. Thus, continued activation of β-catenin signaling prolongs the myoblast phase of regeneration, although it does not ultimately block differentiation. This prolonged regeneration negatively impacts muscle structure as it results in smaller myofibers and increased fibrosis.