© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev167080. doi:10.1242/dev.167080

RESEARCH ARTICLE β-Catenin is essential for differentiation of primary myoblasts via cooperation with MyoD and α-catenin Shuang Cui1,2, Liang Li3, Ruth T. Yu4, Michael Downes4, Ronald M. Evans4,5, Julie-Ann Hulin1, Helen P. Makarenkova6 and Robyn Meech1,*

ABSTRACT a hierarchy of myogenic regulatory factors (MRFs), such as MyoD, Canonical Wnts promote myoblast differentiation; however, the role of which is involved in both proliferation and differentiation, and β-catenin in adult myogenesis has been contentious, and its myogenin, which drives and modulates the differentiation program mechanism(s) unclear. Using CRISPR-generated β-catenin-null (Schultz and Jaryszak, 1985; Wozniak et al., 2005). Differentiation primary adult mouse myoblasts, we found that β-catenin was essential involves multiple highly coordinated processes, including exit from for morphological differentiation and timely deployment of the myogenic the cell cycle, migration, adhesion, fusion and formation of the gene program. Alignment, elongation and fusion were grossly impaired contractile apparatus that defines functional myofibers. in null cells, and myogenic gene expression was not coordinated with Several studies have defined crucial functions for the canonical β cytoskeletal and membrane remodeling events. Rescue studies and Wnt/ -catenin signaling pathway in muscle development and also in genome-wide analyses extended previous findings that a β-catenin- adult muscle regeneration. Wnt ligands bind to membrane receptor TCF/LEF interaction is not required for differentiation, and that β-catenin co-complexes involving the Frizzled and low-density lipoprotein enhances MyoD binding to myogenic loci. We mapped cellular receptor protein (LRP) families that associate with the so-called pathways controlled by β-catenin and defined novel targets in destruction complex (Stamos and Weis, 2013). Wnt binding elicits a myoblasts, including the fusogenic genes myomaker and myomixer. change in the destruction complex that suppresses ubiquitylation β We also showed that interaction of β-catenin with α-catenin was and degradation of -catenin (Li et al., 2012) As the core β important for efficient differentiation. Overall the study suggests dual transcriptional effector of the Wnt pathway, -catenin does not roles for β-catenin: a TCF/LEF-independent nuclear function that bind DNA directly but instead interacts with partner proteins to coordinates an extensive network of myogenic genes in cooperation regulate target genes. The classical transcriptional partners for β with MyoD; and an α-catenin-dependent membrane function that -catenin are the TCF/LEF family factors. In the absence of Wnt helps control cell-cell interactions. β-Catenin-TCF/LEF complexes may signals, TCF/LEF proteins bind and repress Wnt target genes via function primarily in feedback regulation to control levels of β-catenin interaction with Groucho/TLE co-repressors (Arce et al., 2009; and thus prevent precocious/excessive myoblast fusion. Daniels and Weis, 2005) and HDAC1 (Arce et al., 2009; Billin et al., 2000). Upon binding of Wnt ligand, stabilized β-catenin binds to KEY WORDS: Homeobox genes, Muscle stem cells, Myogenesis, TCF/LEF proteins and recruits histone acetyltransferases (HATs) Skeletal muscle, Transcription factors, Epigenetics, Cell signaling, (Ogryzko et al., 1996; Parker et al., 2008; Takemaru and Moon, Differentiation 2000; Hecht et al., 2000) to activate these targets (Hecht et al., 2000; Wodarz and Nusse, 1998; Posokhova et al., 2015). INTRODUCTION Canonical Wnt signals have been shown to play crucial roles in Skeletal muscle owes its considerable regenerative capacity to a muscle development; in particular, canonical Wnts induce expression population of resident muscle stem cells called satellite cells of Myf5 and MyoD in the somite/myotome and thus embryonic (Lepper et al., 2011; McCarthy et al., 2011; Murphy et al., 2011; myogenesis (see von Maltzahn et al., 2012 for a review). Less Sambasivan et al., 2011). These cells, which are situated between well understood is the role of canonical Wnt signals in adult the basal lamina and myofiber, are largely quiescent in uninjured (regenerative) myogenesis, although insights in this regard come adult muscle (Mauro, 1961; Rudnicki et al., 2008; Buckingham from recent studies both in vitro and in vivo. Canonical Wnt ligands et al., 2003). Soluble factors produced after muscle injury trigger have been found to promote myoblast differentiation in vitro (Otto satellite cells to re-enter the cell cycle, giving rise to a et al., 2008; Bernardi et al., 2011; Zhuang et al., 2014; Pansters et al., transit-amplifying population called myoblasts. Myoblasts express 2011; Tanaka et al., 2011; Brack et al., 2008), and can increase expression or activity of MyoD and/or myogenin (Ridgeway et al., 2000; Petropoulos and Skerjanc, 2002; Jones et al., 2015). Moreover, 1Department of Clinical Pharmacology, Flinders University, Bedford Park, SA 5042, studies by Kim et al. (2008) using C2C12 cells showed that β-catenin Australia. 2Department of Physiology, Shandong University School of Medicine, can interact with MyoD and enhance the ability of MyoD to bind and Jinan, Shandong 250012, China. 3Department of Biochemistry, Flinders University, β Bedford Park, SA 5042 and Department of Biochemistry, University of Adelaide, activate target genes. This study also found that -catenin knockdown North Tce, Adelaide, SA 5005, Australia. 4Gene Expression Laboratory, Salk impaired C2C12 differentiation; however, dominant-negative (DN) Institute, La Jolla, CA 92037, USA. 5Howard Hughes Medical Institute, Salk Institute, forms of TCF/LEF did not. This finding led to the suggestion that La Jolla, CA 92037, USA. 6Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. β-catenin function in myogenesis is TCF/LEF independent (Kim et al., 2008). However, a more recent study reported that expression *Author for correspondence ([email protected]) of DN-TCF4 in human adult primary myoblasts did impair R.M., 0000-0002-6831-0801 differentiation, and concluded that TCF4 is required for this process (Agley et al., 2017). The reason for these discrepant results

Received 2 May 2018; Accepted 16 January 2019 remains unclear, but may relate to the use of different cell models and DEVELOPMENT

1 RESEARCH ARTICLE Development (2019) 146, dev167080. doi:10.1242/dev.167080 assays. We have recently reported that Wnt treatment of adult primary Interestingly, even though key myogenic genes such as myogenin myoblasts leads to acquisition of active chromatin marks at myogenic were expressed in null cells in a delayed fashion, the cells still loci that are enriched for E-boxes but not for TCF/LEF elements displayed grossly impaired morphological differentiation, suggesting (Hulin et al., 2016). These data suggest that Wnt activates a gene that these molecular and morphological events are uncoupled by loss expression program that is TCF/LEF independent in adult myoblasts; of β-catenin. Our rescue studies supported the previous suggestion this conclusion is consistent with the findings of Kim et al. (2008), that β-catenin-TCF/LEF interactions are not required for but not those of Agley et al. (2017). differentiation. However, in contrast to a previous report, we also Conditional deletion studies in mice have raised uncertainty about found that interactions of β-catenin with α-catenin at membrane whether β-catenin is actually required for adult myogenesis in vivo, junction complexes are important for efficient differentiation. regardless of mechanism. In particular, Murphy et al. (2014) performed inducible Cre-mediated ablation of β-catenin in satellite RESULTS cells and reported no defect in adult muscle regeneration (Murphy Phenotypic characterization of β-catenin-null primary et al., 2014). They suggested that, in contrast to the embryonic state, β- adult myoblasts catenin may be redundant in adult myoblasts. Subsequently however, Low passage primary myoblasts derived from adult mice were Rudolf et al. (2016) performed very similar conditional deletion transduced with a viral vector targeting exon 5 of β-catenin (Fig. 1A). experiments using a different Cre-driver line, and found β-catenin to Loss of β-catenin mRNA was observed in several clones (<5% of be essential for efficient adult muscle repair. They also reported that parental cell levels), likely as a result of nonsense-mediated decay explanted myoblasts lacking β-catenin showed delayed differentiation (NMD) (Fig. 1B). All null clones had a similar phenotype (Fig. S1A), in vitro. The reason for the discrepancy between the two in vivo studies which included a rounded morphology and impaired differentiation, remains unresolved, but may relate to the presence of undetected as discussed further below. Loss of β-catenin protein was confirmed ‘recombination-escaper’ cells, which are known to have confounded at the protein level by immunostaining and immunoblotting for previous conditional ablation studies in satellite cells (Brack, 2014). selected lines (e.g. Fig. 1C,E). Consistent with the loss of β-catenin, To address the ongoing uncertainty about the cell-autonomous Axin2 failed to be induced in null cells after Wnt3a treatment requirement for β-catenin and its mechanism(s) of action, we used (Fig. 1D). Sequencing analysis of selected clones revealed frameshift CRISPR to generate adult primary mouse myoblast-derived β-catenin- deletions generating premature stop codons in both alleles; the null models. We then investigated the functions of β-catenin via resulting predicted proteins would be truncated to fewer than 40 rescue studies with mutant forms of the protein. We confirmed that amino acids (see Fig. S2A,B), although these are unlikely to be β-catenin is essential for timely activation of the myogenic gene produced because of NMD-mediated loss of the transcript. expression program by Wnt in primary myoblasts, and that β-catenin A representative null line is shown in Fig. 2 in comparison with is required for Wnt to enhance genome-wide MyoD binding. control cells that were transduced with empty lentiCRISPRv2 vector

Fig. 1. Characterization of β-catenin-null primary myoblasts. (A) Strategy for Cas9/gRNA-mediated mutation of β-catenin. The blue nucleotides represent the gRNA target sequence; the highlighted nucleotides represent the protospacer adjacent motif (PAM). (B) β-Catenin mRNA expression was measured by RT-PCR in multiple independent CRISPR-generated myoblast lines and compared with that in wild-type myoblasts transfected with the empty vector (LentiCRISPRv2). Dataare normalized to the housekeeping gene RPS26 and show the average of two experiments performed in duplicate. Statistical analysis: one-way ANOVA (*P<0.05). (C) Western blot analysis using β-catenin antibody shows that β-catenin-null primary myoblasts are completely devoid of β-catenin protein expression. β-Actin was used as a loading control. (D) β-Catenin-null myoblasts show no induction of Axin2 mRNA after treatment with Wnt3a for 24 h. Statistical analysis: t-test (****P<0.05).

(E) Immunofluorescence labeling of β-catenin (red) in cultured wild-type and β-catenin-null primary myoblasts; nuclei are stained with DAPI (blue). Scale bars: 25 μm. DEVELOPMENT

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Fig. 2. Differentiation capacity of wild-type and β-catenin-null primary myoblasts. (A) Phenotype of wild-type and β-catenin-null primary myoblasts cultured in growth media (GM) showing lack of spontaneous differentiation. (B) Phenotype of wild-type and β-catenin-null primary myoblasts after 5 days of culture in Wnt3a-containing media showing greatly impaired differentiation. Scale bars: 100 μm.

and passaged a similar number of times. When cultured in growth expression of myogenic markers (Fig. S3). Overall, these data suggest media, the β-catenin-null cells appeared rounded relative to wild- that β-catenin is important for coupling of fusion and cytoskeletal type cells, and failed to undergo spontaneous differentiation upon remodeling with acquisition of molecular markers of differentiation. reaching confluence (Fig. 2A). Treatment with Wnt3a induced differentiation of wild-type cells, leading to formation of aligned Molecular characterization of β-catenin-null primary fibers within 24 h. After 5 days in culture, cells began to beat, adult myoblasts indicating spontaneous contractility (Fig. 2B and Movies 1-3). In To understand the molecular changes underlying the phenotypic contrast, β-catenin-null cells continued to proliferate after Wnt3a effects of loss of β-catenin, we performed transcriptomic analysis of treatment (Fig. S1B) and fused slowly and infrequently, giving rise wild-type myoblasts and two of the β-catenin-null lines after 24 h of to only very short fibers that were poorly aligned and did not beat, Wnt3a treatment. Comparative enrichment clustering of wild-type even after 5 days in culture (Fig. 2B and Movies 4-6). Upon long- myoblast RNAseq data using Toppcluster (Kaimal et al., 2010) term culture, some β-catenin-null cells fused into syncytia that identified the most significantly enriched Gene Ontology (GO) became detached from the substrate and formed spherical balls terms associated with the Wnt-induced transcriptome as muscle (Fig. 2B), indicating that loss of β-catenin impaired substrate contraction and striated muscle formation (Fig. 4A,C). Equivalent attachment. Cultures were immunostained with myogenin and analysis of β-catenin-null cells found no muscle-associated terms MyHC antibodies at different time points after treating cells with among the top 10 enriched terms GO categories (Fig. 4B,D). Wnt3a. In wild-type myoblasts, myogenin and MyHC were first Moreover, comparison of Fig. 4A with B shows that wild-type cells detected on day 1, whereas in β-catenin-null myoblasts, these (A) activated a very large number of genes (red nodes) that were markers were first detected on days 2-3 after induction of tightly linked within a small number of functional pathways (green differentiation (Fig. 3A-D). Interestingly, MyHC was highly nodes), which is indicative of a highly coordinated response to Wnt, expressed in unfused cells in both wild-type and null cultures, whereas null cells (B) activated very few genes within a large and in syncytia that did not elongate in the latter. Despite the number of pathways, suggesting a nonspecific response. The top 25 eventual acquisition of these myogenic markers in β-catenin-null pathways associated with the Wnt-induced transcriptome in wild- cells (Fig. 3A-D), morphological myogenesis (formation of type cells were further assessed using REACTOME: muscle multinucleated elongated myofibers) was essentially prevented. contraction (general, striated and smooth) were the top 3 enriched To confirm that the phenotype of the null cells was due to loss of pathways, followed by ECM proteoglycans/ECM organization, β-catenin, we transfected them with a constitutively active β-catenin Cdo, Cdkn1a/p21, calcitonin receptors, circadian clock, Igf-binding expression plasmid together with a GFP-expression plasmid to assess proteins, leptin, Tfap2, growth hormone signaling, Runx factors and efficiency. Notably, the null myoblasts transfected much more NrCAM (see Table S2). Interestingly, of the top 25 enriched efficiently than did wild-type myoblasts (∼80% of null cells were pathways associated with Wnt-downregulated genes in wild-type GFP-positive relative to 50-60% of wild-type myoblasts), possibly cells, 12 relate to Notch signaling and eight involve DNA repair (see owing to their rounded/poorly adherent phenotype. Null cells transfected Table S3). REACTOME analysis of the Wnt-induced transcriptome with stable β-catenin spontaneously elongated and fused, and increased in null cells suggested a weak enrichment of lipid-associated DEVELOPMENT

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Fig. 3. Immunostaining analysis of myogenic markers in wild-type and β-catenin-null primary myoblasts after Wnt3a treatment. (A,C) Immunofluorescence staining for myogenin (A) or MyHC (C), both green. Nuclei were stained with DAPI (blue). Images were captured at 0, 24, 48, 72 and 120h post-treatment. (B,D) Quantification of myogenin- (B) or MyHC- (D) positive cells at each time point. Data are mean±s.e.m. Statistical analysis: one-way ANOVA (****P<0.0001). pathways, although significance was low and no pathway was two genome-wide analyses: the first was of activating-histone represented by more than two gene members (see Table S4). modifications in wild-type and β-catenin-null cells, the second was To validate the RNA-seq data, the β-catenin-dependence of of MyoD binding. In wild-type cells, activating modifications selected targets was tested by RT-PCR (Fig. 4E). Myogenin, (H3KAc and H3K4me3) were enriched at myogenic gene follistatin, and myomaker (see further details below) were induced promoters 24 h after Wnt3a treatment. These activated promoters by Wnt treatment of wild-type but not null cells, consistent with were enriched in MRF- and MEF-binding motifs, but not in TCF/ RNA-seq analysis. MyoD was not induced by Wnt3a in either cell LEF-binding motifs (Fig. 5A), consistent with our earlier study (Hulin type, again consistent with RNA-seq. et al., 2016). In contrast, β-catenin-null cells showed no enrichment of activating histone modifications at myogenic promoters, and there Essential fusion pore components myomaker and myomixer were no significantly enriched motifs. MyoD-ChIP-seq analysis in are targets of Wnt/β-catenin signaling in primary myoblasts wild-type cells 24 h after Wnt3a treatment revealed increased binding Among the novel Wnt/β-catenin-induced targets revealed by RNA-seq of endogenous MyoD at genes associated with myogenic were two genes essential for formation of the myoblast fusion pore: differentiation (Fig. 5B, left), and these loci were enriched in MRF- myomaker (Mymk; previously called Tmem8c) and myomixer (Mymx; and MEF-binding motifs as expected (Fig. 5C). In contrast, in null previously called myomerger, minion and GM7325) (Millay et al., cells, there was very little increase in MyoD binding after Wnt3a 2014, 2016; Quinn et al., 2017; Takei et al., 2017). Both genes were treatment, and the loci showing enhanced binding were not associated induced fivefold by Wnt3a in wild-type cells but not in null cells based with myogenic genes (Fig. 5B, right), and contained no statistically on RNA-seq data, and were induced by β-catenin transfection, as enriched motifs, suggesting that the enrichment was non-specific. assessed by RT-PCR (see Fig. S4). In addition, RNA-seq showed that Immunoblotting analysis of myoblasts showed no obvious four other factors recently identified within the myomaker-myomixer difference in MyoD or ID1 protein levels after Wnt treatment complex (Takei et al., 2017) were induced at least twofold by Wnt3a in (Fig. S5). Thus, increased MyoD binding to target genes likely wild-type cells but not null cells. These genes were dysferlin (Dysf), involves interaction of MyoD with β-catenin, as previously suggested synaptopodin-2-like (Synpo2l), junctional sarcoplasmic reticulum (Kim et al., 2008). To confirm this, recruitment of β-catenin and protein 1 (Jsrp1) and tripartite motif 72 (Trim72). Overall, these data MyoD was tested using ChIP-qPCR with the myogenin and suggest that Wnt might function in coordinating fusion pore assembly. myomaker promoters as exemplars. Wnt3a treatment of wild-type myoblasts increased binding of endogenous β-catenin and MyoD, at Wnt induces MyoD binding to myogenic loci in a β-catenin- the same proximal promoter location, for both genes (Fig. 5D,E). The dependent manner myogenin findings are consistent with previous work showing that β- Previous work using C2C12 cells showed that β-catenin could catenin knockdown reduces MyoD binding to the myogenin enhance the ability of MyoD to bind and activate specific target genes promoter (Kim et al., 2008). However, the binding of β-catenin and (Kim et al., 2008). Here, we had the opportunity to examine whether MyoD to the mouse myomaker promoter is a novel finding. We also loss of β-catenin altered the global landscape of MyoD binding and further confirmed the binding of MyoD to this promoter using gene activation in primary myoblasts. To achieve this, we performed heterologous MyoD expression (Fig. S6). DEVELOPMENT

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Fig. 4. Transcriptomic analysis of wild-type and β-catenin-null cells after Wnt3a treatment identifies the Wnt-induced gene set. (A,B) RNA-seq was performed on wild-type (A) and β-catenin-null (B) myoblasts treated with Wnt3a or control (L-cell) medium for 24 h. Genes upregulated by Wnt3a treatment (Wnt3a/L cell≥twofold, red nodes) were analyzed using Toppcluster to define associated pathways (green nodes). Network analysis of global gene expression changes in wild type shows many genes upregulated by Wnt3a in wild type but not in β-catenin-null myoblasts. (C,D) Gene Ontology (GO) terms significantly enriched in the differentially expressed gene set (Wnt3a/L cell≥twofold) identified in wild-type (C) and β-catenin-null (D) myoblasts. (E) Wnt3a induces expression of myogenic genes myomaker (Tmem8c), Fst and myogenin in a β-catenin-dependent manner. Quantitative RT-PCR was performed on wild-type and β- catenin-null myoblasts treated with Wnt3a-containing or control (L-cell) medium for 24 h. Data are normalized to the housekeeping gene RPS26 and are the average of three experiments performed in duplicate. Data are mean±s.e.m. Statistical analysis: t-test (*P<0.05, **P<0.01).

TCF/LEF-β-catenin interaction is not required for The mutant form of stable β-catenin (referred to here as the TCF- Wnt-mediated differentiation of primary myoblasts binding site mutant) was unable to induce Axin2 expression or Although a previous study indicated that β-catenin controls C2C12 TOPflash activity (Fig. S7), confirming functional ablation of TCF/ differentiation in a TCF/LEF-independent manner (Kim et al., 2008), LEF interaction. When this TCF-binding site mutant was transfected a more recent report concluded that adult primary myoblasts require into β-catenin null myoblasts, morphological differentiation was TCF/LEF for differentiation (Agley et al., 2017). To clarify the role of indistinguishable from that of cells transfected with wild-type β- TCF/LEF family members in differentiation, we attempted to rescue catenin (Fig. 6A). The full rescue of differentiation by the TCF- the phenotype of the β-catenin-null cells using a variant of β-catenin binding site mutant was confirmed by quantifying the number of that does not interact with TCF/LEF. We mutated two positively nuclei in MyHC-positive fibers (Fig. 6B,C). Overall, these data charged residues (‘charged buttons’)inβ-catenin that are known to be support the model (Kim et al., 2008) that β-catenin-mediated essential for interaction with TCF/LEF proteins (Graham et al., 2000). differentiation does not require TCF/LEF. DEVELOPMENT

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Fig. 5. Epigenomic analyses indicate that β-catenin promotes MyoD association with chromatin. (A) H3Kac and H3K4me3 ChIP-seq was performed using chromatin from wild-type myoblasts treated with control or Wnt3a-containing media for 24 h. Homer analysis was used to identify the top 10 motifs that are enriched in promoter regions that show increased levels of H3Kac and H3K4me3 (Wnt/L cell≥threefold) after Wnt3a induction (represented as target sequences with motif/total sequences with motif). (B) MyoD ChIP-seq analyses of wild-type and β-catenin-null primary myoblasts treated with Wnt3a medium for 24 h. Gene ontology (GO) analysis was performed on the set of genes showing increased MyoD binding after Wnt3a treatment (Wnt/L cell≥twofold) in wild-type (left) and β-catenin CRISPR-null (right) myoblasts. Wild-type myoblasts show significant enrichment of muscle-associated GO categories; β-catenin-null myoblasts do not show enrichment in any muscle-specific categories. (C) The top 15 motifs (Homer analysis) seen in promoter regions showing increased MyoD binding (Wnt/L cell≥twofold) after Wnt3a treatment of wild-type myoblasts (target sequences with motif/total sequences with motifs). Statistical analysis: multiple t-tests with P<0.05 considered significant. (D,E) Wnt3a increases binding of β-catenin and MyoD to the proximal promoters of the myomaker and myogenin genes, as shown by ChIP-qPCR analysis. Chromatin from wild-type primary myoblasts treated with control or Wnt3a-containing medium for 48 h was used for ChIP with β-catenin (D) or MyoD (E) antibodies. qPCR was used to assess enrichment of the myomaker promoter in both β-catenin- and MyoD-ChIP samples; the myogenin promoter was used as a control for MyoD-ChIP and the Axin2 enhancer was used as a control for β-catenin-ChIP. Data were first normalized to a control non-target locus (rDNA1) and then to the mock ChIP with preimmune IgG, set to a value of 1. n=3. Data are mean±s.e.m. *P<0.05, **P<0.001.

Interaction of β-catenin and α-catenin is required for efficient 2005; Xu and Kimelman, 2007). We examined whether expression of differentiation of primary myoblasts amutantformofβ-catenin that cannot interact with α-catenin could Although these studies have focused on nuclear functions, β-catenin induce differentiation of β-catenin-null myoblasts. The residues that is also crucial at adherens junctions where cadherin/β-catenin/ control interaction of β-catenin with α-catenin have been previously α-catenin complexes link to the cytoskeleton (Huveneers and de defined (Aberle et al., 1996; Nieset et al., 1997); we mutated these

Rooij, 2013) and control local filament assembly (Yamada et al., residues (threonine 120 and valine 122) to alanine, generating a DEVELOPMENT

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Fig. 6. Comparison of the ability of wild-type and mutant forms of β-catenin to rescue differentiation of β-catenin-null myoblasts. (A) β-Catenin-null myoblasts were co-transfected with GFP and one of the following expression plasmids: empty pcDNA3, an α-catenin-binding site (BS) mutant form of β-catenin (which cannot interact with α-catenin), a TCF-binding site (BS) mutant form of β-catenin (which cannot interact with TCF/LEF) and wild-type β-catenin (constitutively active/stable). Scale bars: 50 μm. (B,C) Quantitation of differentiation was achieved by immunostaining with MyHC (B) and counting the percentage of nuclei (blue, DAPI) in MyHC-positive myofibers (C). Data are mean±s.e.m. Statistical analysis: one-way ANOVA (*P<0.05, ****P<0.0001).

variant of stable β-catenin that does not bind to α-catenin. This recently, Wnt was reported to mediate intercellular coupling of the mutant (referred to here as the α-catenin-binding site mutant) was circadian clock to the cell cycle in organoids (Matsu-Ura et al., 2016); able to induce Axin2 expression and TOPflash activity as effectively thus, it is possible that Wnt-mediated regulation of Bhlhe40 helps to as wild-type β-catenin (Fig. S7). The α-catenin-binding site mutant coordinate the synchronous cell cycle exit of neighboring myoblasts. only partially rescued differentiation (Fig. 6A-C), suggesting that, in Among the novel targets of Wnt/β-catenin identified were the addition to the nuclear functions of β-catenin, interactions between central fusion pore factors myomaker and myomixer. Myomaker was β-catenin and α-catenin at membrane junction complexes are also recently found to be essential for C2C12 cell fusion. Moreover, its required for efficient differentiation. ectopic expression in fibroblasts could induce them to fuse with C2C12 cells, but not with each other (Millay et al., 2014, 2016), DISCUSSION suggesting the existence of a myoblast-specific fusion partner. The overall goal of this study was to understand the cell-autonomous Myomixer was very recently identified as this protein (Leikina et al., requirement for β-catenin in myoblast differentiation. This was 2018). Interestingly, we found that transfection of myomaker prompted in part by conflicting findings from previous studies of into β-catenin-null myoblasts did not rescue their fusion and β-catenin disruption both in vitro and in vivo. Our morphological and instead caused toxicity (not shown). This may have been due to gene expression data using the β-catenin-null primary myoblast insufficient levels of myomixer and suggests that the levels of these model are consistent with, but extend beyond, those of Kim et al. two proteins must be carefully titrated. A set of potential fusion pore (2008) and Rudolf et al. (2016). Pathway analysis of transcriptomic components were recently identified via a proteomic analysis of data indicates that Wnt/β-catenin Wnt targets multiple myogenic myomixer-interacting proteins (Takei et al., 2017). Of these, four pathways: many involved in forming contractile apparatus, but others were Wnt/β-catenin induced based on RNA-seq data; this finding related to cell cycle control (e.g. Cdkn1a/p21 that was previously prompts further analysis of the role of Wnt in controlling fusion implicated in myoblast cell cycle exit; Zhang et al., 1999), ECM pore assembly. modulation, adhesion and fusion, and Igf signaling. Wnt-mediated Overall, the dysregulation of multiple classes of myogenic regulation of clock-related genes is intriguing and broadly consistent regulators and effectors is consistent with the observation that with previous studies showing that circadian regulators control longer-term null myoblast cultures ultimately expressed myogenin differentiation in muscle (Chatterjee and Ma, 2016). The most highly and MyHC, yet still failed to align, and any fusion that occurred Wnt-induced of the circadian genes, Bhlhe40 (SHARP-2, Dec1, appeared uncoupled from the assembly of the myofibrils that maintain Stra13), is a bHLH factor that functions in negative-feedback loop myofiber architecture (particularly evident in the formation of ball- with CLOCK/BMAL (Honma et al., 2002). Interestingly, Bhlhe40 shaped syncytia). Overall, we conclude that β-catenin coordinates was also reported to antagonize Notch in satellite cells (Sun et al., multiple processes, including cell cycle exit, matrix remodeling,

2007), preventing Notch-imposed inhibition of differentiation. Very fusion and cytoskeletal rearrangement/myofibril assembly. DEVELOPMENT

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In agreement with Kim et al. (2008), our studies indicated that cells may be distinct from its MyoD-dependent functions in β-catenin enhances MyoD binding to target genes. Moreover, using committed myoblasts. ChIP-seq we were able to generalize this model, showing that Wnt We found that interaction of β-catenin with α-catenin is important enhances MyoD binding to myogenic loci across the genome. We for efficient differentiation, which is in contrast to the findings of also confirmed that β-catenin and MyoD co-bind to common loci Kim et al. (2008) in C2C12 cells. However, Kim et al. expressed using analytical ChIP; however, we were unable to gain a genome- mutant β-catenin in the background of wild-type β-catenin, and the wide picture of colocalization because β-catenin ChIP-seq results extent to which the mutant can act in a dominant-negative manner is were inconsistent (not shown). This is possibly because β-catenin unclear. Thus, our rescue studies in null cells might appear to be a associates in large complexes in myoblasts that are inconsistently more-reliable approach, although with the caveat that any represented during shearing/library construction; a technical overexpression of β-catenin could cause artefacts by altering limitation to be addressed in future work. How β-catenin stoichiometry. It is also possible that primary myoblasts and promotes MyoD binding is not yet clear, but might involve post- C2C12 cells have different requirements for these interactions. transcriptional changes. Given that Wnt/β-catenin drives histone Adherens junctions might be more important for interaction, modification, it may also increase the stability of MyoD binding alignment and fusion of primary myoblasts than C2C12 cells, within chromatin. β-Catenin can associate with p300 and CBP, and which are less migratory and more adhesive. The exact role of these complexes are known to target different gene programs β-catenin at the myoblast membrane remains to be determined, (Sasaki et al., 2013). MyoD also interacts with p300 and CBP; although it likely relates to coupling cadherin-mediated adhesion to however, only p300 appears to be required for embryonic cytoskeletal remodeling (Wrobel et al., 2007; Ozawa, 2015). It is myogenesis (Polesskaya et al., 2001; Roth et al., 2003). We found also interesting to consider the relationship between fusion pores that chemical inhibition of the β-catenin-p300 interaction can impair and adherens junctions. In Drosophila muscle, electron-dense myoblast differentiation, whereas disruption of β-catenin-CBP fusion plaques overlap with N-cadherin-rich junctions (Hamp et al., interaction does not (Fig. S8). Whether these co-activators are 2016). Thus, it is possible that myomaker/myomixer and β-catenin differentially involved in the functions of β-catenin-MyoD and membrane functions overlap; future work should examine their β-catenin-TCF/LEF complexes, is being further examined. localization and interaction during differentiation. Interestingly, Kim et al. (2008) used dominant-negative (DN)-TCF/LEF recent work has shown that a β-catenin-α-catenin interaction may expression to conclude that TCF/LEF proteins were redundant for also be involved in the pro-proliferative functions of Wnt/β-catenin differentiation. We came to the same conclusion via a different in satellite cells; in particular, loss of this interaction after deletion of approach in which null cells were transfected with a form of cadherins induced a break in quiescence by increasing nuclear β-catenin that cannot interact with TCF/LEF. However, a very recent β-catenin/TCF complexes (Goel et al., 2017). Thus, it appears that study by Agley et al. (2017) found that overexpression of dominant- membrane-linked β-catenin functions (like its nuclear functions) negative (DN)-TCF4 in human myoblasts impaired differentiation. may also be involved in multiple steps of myogenesis. The reasons for these discrepant results may relate to different cell Although this study focused on in vitro models to define β-catenin models and assays; however, in the study by Agley et al., it is function, it is relevant to discuss the recent in vivo studies of satellite notable that DN-TCF4 expression dramatically reduced levels of cell-specific β-catenin ablation. As mentioned previously, Murphy active β-catenin (Agley et al., 2017); this would be expected to et al. (2014) found that loss of β-catenin had no effect on impair differentiation regardless of the mechanism of β-catenin regeneration, whereas Rudolf et al. (2016) reported significantly action. Overexpression of active β-catenin also led to formation of impaired regeneration. The reason for the discrepancy remains thinner fibers (Agley et al., 2017), which is presumably due to unclear, although the possibility that the earlier study was precocious differentiation of myoblasts at the expense of compromised by the presence of unrecognized ‘recombination proliferation (Brack et al., 2008; Murphy et al., 2014; Rudolf escapers’ has been put forward (Rudolf et al., 2016). Among the et al., 2016). A parsimonious model that agrees with all of these Wnt/β-catenin targets that we have identified are miRNAs that studies is that β-catenin-TCF interactions are not required to activate repress Pax7 by binding the 3′UTR (Cui et al., 2019). Murphy et al. the differentiation program of committed myoblasts, but are (2014) used a Pax7-Cre-driver allele that contained the Pax7 3′UTR required for appropriate feedback to the Wnt pathway (via targets (Murphy et al., 2011); it is possible that endogenous β-catenin (via its such as Axin2) to prevent precocious/excessive fusion. There is also miRNA targets) inhibited cre expression via this 3′UTR, increasing evidence that Wnt/β-catenin induces proliferation of uncommitted the likelihood that a subset of cells may escape recombination. Given (e.g. MyoD-negative) satellite cells (Otto et al., 2008), and this is the apparently pro-proliferative role of β-catenin (via TCF/LEF) in likely to involve TCF/LEF-mediated targeting of genes such as satellite cells (Suzuki et al., 2015), any such escapers might have a Ccna2 and Cdc25c (Suzuki et al., 2015). Consistent with this, when selective advantage post-activation. In contrast, Rudolf et al. (2016) Murphy et al. (2014), examined Wnt/β-catenin activity during used a Pax7-cre driver allele that did not contain this 3′UTR (Lepper muscle regeneration using a TCF/LEF-GFP reporter, they found et al., 2009) and hence would not have been subject to repression, that 6% of cells were GFP+ before injury, 23% at 1 day post-injury possibly allowing more efficient recombination. Although this (dpi) and only 0.6% at 3 dpi. This suggested that β-catenin/TCF/ hypothesis obviously requires formal testing, it is reasonable to state LEF activity is high in activated/proliferating satellite cells, but very that any regulation of a cre driver allele (in this case Pax7) by its low in differentiating myoblasts where we would argue that intended target (in this case β-catenin) has potential to generate β-catenin/MyoD activity predominates instead (MyoD+ cells were confounding effects. also highest at 3 dpi in Murphy et al., 2014). Of note, during In summary, we report that differentiation of primary adult embryogenesis, TCF/LEF is also involved in Wnt-mediated myoblasts requires interaction of β-catenin with MyoD, but not induction of somitic Myf5 expression in proliferating progenitor TCF/LEF, in agreement with previous findings in C2C12 cells (Kim cells (Borello et al., 2006). In general, it is clear that Wnt/β-catenin et al., 2008), but also involves the interaction of β-catenin with regulates multiple steps of myogenesis (Suzuki et al., 2015) and α -catenin at adherens junction complexes. We also identify a novel

TCF/LEF-dependent functions of Wnt in progenitor and satellite role for Wnt/β-catenin in regulation of fusion pore assembly. Further DEVELOPMENT

8 RESEARCH ARTICLE Development (2019) 146, dev167080. doi:10.1242/dev.167080 work will be required to understand exactly how β-catenin Lipofectamine LTX (Thermo Fisher Scientific). Quantitative-RT-PCRs coordinates activation of myogenic gene expression with cell were performed 48 h post-transfection. Transfections were performed in alignment and fusion, and the extent to which the membrane triplicate and repeated two to six times; significance was assessed using ’ t functions of β-catenin are important for this. In addition, defining Student s -test. Transfection with TOPflash used Lipofectamine LTX, as well differential co-factor requirements for β-catenin/MyoD and as pRL-Null renilla-luciferase reporter (Promega) as an internal reference. β Luciferase assays were performed 48 h post-transfection using a Dual -catenin/TCF/LEF complexes might ultimately allow differential Luciferase assay kit (Promega). TOPFlash luciferase assays were performed targeting of desirable pro-myogenic versus other potentially in HEK293T cells according to our previous studies (Zhuang et al., 2014). deleterious (e.g. pro-fibrotic) functions of β-catenin in muscle. Chromatin immunoprecipitation MATERIALS AND METHODS ChIP was performed using a modified MicroChIP protocol (Dahl and Primary myoblast cultures Collas, 2008) in primary myoblasts as described previously (Zhuang et al., Primary myoblasts were isolated from postnatal day 21 TP1-Venus mice by 2014) using MyoD antibody (M-318, Santa Cruz Biotechnology), rabbit immuno-FACS, as previously described (Zhuang et al., 2014); details are β-catenin antibody (9562, Cell Signaling Technology) or an equivalent provided in the supplementary Materials and Methods. TP1-Venus are amount of appropriate pre-immune IgG (Santa Cruz Biotechnology or Cell an ICR transgenic strain [RIKEN BioResource Center (RBRC06137)] Signaling Technology). In some ChIP experiments, wild-type myoblasts (Kohyama et al., 2005; Sasaki et al., 2011). Imaging of myoblast cultures and β-catenin-null CRISPR myoblasts were plated at the same density and was carried out using an Olympus CKX41 inverted microscope and DP22 treated for 24 h with Wnt3a medium or L cell control medium. In other ChIP camera. experiments, wild-type myoblasts were transfected with MyoD1/pcDNA3 using Lipofectamine LTX (Thermo Fisher Scientific). After ChIP, purified Plasmids genomic DNA was analyzed by quantitative PCR (qPCR) with primers that LentiCRISPR v2 and TOPflash plasmids were from Addgene (plasmids amplify the target regions or a control non-target locus (rDNA1). Details are 52961 and 12456) (Sanjana et al., 2014). The constitutively active β-catenin provided in the supplementary Materials and Methods, and sequences of cDNA was obtained from Dr Victor Korinek and subcloned into pcDNA3 primer used for genomic PCR are in Table S1. vector. The MyoD1/pcDNA3 vector was generated in the laboratory previously. pMM043 (GFP-expression) plasmid was obtained from RNA isolation and quantitative reverse transcription-PCR Dr Michael Michael (Flinders University, Australia). TOPFlash plasmid RNA was prepared from cells using TRIzol (Life Technologies); after DNase was obtained from Addgene (plasmid 12456). treatment, cDNA was synthesized using NxGen M-MuLV reverse transcriptase (Lucigen) and random primers (New England Biolabs). Quantitative RT-PCR Site-directed mutagenesis was performed using a Corbett Rotorgene (Qiagen) and GoTaq SYBR green The TCF/LEF interaction sites in the β-catenin/pcDNA3 were mutated by (Promega). Significance was assessed using Student’s t-test. Sequences of the replacement K312 and K435 with glutamic acid: the β-catenin TCF- primersusedforqRT-PCRareshowninTableS1. mutant (K312E/K435E). The α-catenin interaction sites in the β-catenin/ pcDNA3 were mutated by the replacement T120 and V122 with alanines: Immunofluorescence labeling β-catenin α-catenin-mutant (T120A/ V122A). Site-directed mutagenesis Wild-type and β-catenin-null primary myoblasts were plated at 1.5×104 per (SDM) was then performed using the QuikChange site directed mutagenesis well in collagen-coated 96-well plates before treatments with Wnt3a or protocol (Agilent). All mutant plasmids were confirmed by sequencing. L-cell control medium. Wells were fixed at 0 h, 24 h, 48 h, 72 h and 120 h post-treatment with 3.7% formaldehyde in PBS for 10 min at room CRISPR temperature, rinsed with PBS and permeabilized with 0.5% Triton-X for gRNAs were inserted into the LentiCRISPRv2 vector according to the 5 min. Primary myoblasts were then blocked with 1% BSA in PBS for recommended protocols (Sanjana et al., 2014). Briefly, oligonucleotides 30 min before addition of antibodies. Rabbit anti-β-catenin (9562, Cell corresponding to sgRNA targeting exon 5 of β-catenin were annealed Signaling Technology), mouse anti-myogenin (F5D-c, DSHB) or anti- and ligated into LentiCRISPRv2. After confirmation by sequencing, the MyHC (MF-20-c, DSHB) antibodies were applied at 4 µg/ml overnight at LentiCRISPRv2-β-catenin vector was packaged as described below and 4°C; fluorescently labeled secondary antibodies (Dylight-594 or Dylight- used to transduce primary adult mouse myoblasts at low passage. 488; Vector Labs) were applied at 10 µg/ml for 2 h. Nuclei were Transduced myoblasts were subjected to puromycin selection and counterstained with 1 µg/ml DAPI for 5 min. Cells were imaged with an fluorescence-activated cell sorting or limiting dilution plating to obtain Olympus IX71 microscope. To quantify the percentage of myogenin- or clonal lines. Multiple mutant cell lines were expanded and characterized by MyHC-positive cells at each time point, the number of fluorescently labeled sequencing and gene expression analysis. cells was divided by the total number of DAPI stained nuclei using ImageJ in 20 fields per condition. Viral packaging and transduction HEK293T cells were plated at a density of 4×105 cells/well in a 6-well plate Proliferation assay 24 h before transfection. Transfection complexes consisted of plasmid DNA A dye-retention assay was used to assess proliferation. Briefly, 1×106 wild-type (1.5 µg viral plasmid, 1.5 µg gag-pol plasmid, 1 µg VSV-G plasmid), 8 µl of or β-catenin-null myoblasts were washed and incubated with 1 µl of Vibrant DiI Lipofectamine 2000 (Thermo Fisher Scientific) and 200 µl of serum-free cell-labeling solution (Thermo Fisher Scientific) in 1 ml serum-free RPMI at media per well. Cells were cultured for 24 h before collecting viral 37⁰C for 10 min. Labeling efficiency was assessed by flow cytometry using the supernatant. Viral supernatant was harvested and cleared by centrifugation, Accuri C6 (BD Biosciences) and was always more than 95%. The cells were and polybrene (Sigma) was added at 4 µg/ml final concentration. plated in a 24-well plate at 5×104 cells/well. After various times in culture, the Spinfection was carried out by spinning plates for 3 h at 1150 g at 30°C. cells were harvested using trypsin and the mean DiI fluorescence level of the The viral supernatant was replaced with fresh media and cells were grown population was measured by flow cytometry. The change (decrement) in mean for a further 48 h before analysis. Lentiviral methods were approved by the DiI fluorescence at each time point was calculated as a fold over the initial (time Flinders Institutional Biosafety Committee (IBC2008-14). 0) fluorescence level. The experiment was repeated twice in duplicate with similar results and a representative experiment is shown. Transfections Primary myoblasts seeded in 12-well plates at 1×105 cells/well were RNA-seq co-transfected with pcDNA3, β-catenin/pcDNA3, MyoD1/pcDNA3, β-Cat Wild-type and β-catenin-null CRISPR myoblasts were plated at 3.3×105 K312E/K435E or β-Cat T120A/V122A and GFP expression plasmids cells per well in collagen-coated six-well plates before treatment with Wnt3a pMM043 (0.66 µg each plasmid unless otherwise stated) using or control conditioned L-cell medium. Primary myoblasts were harvested at DEVELOPMENT

9 RESEARCH ARTICLE Development (2019) 146, dev167080. doi:10.1242/dev.167080

24 h post-treatment, and RNA was prepared as previously described. RNA Buckingham, M., Bajard, L., Chang, T., Daubas, P., Hadchouel, J., Meilhac, S., quality was confirmed using the Agilent 2100 Bioanalyzer and RNA-Seq Montarras, D., Rocancourt, D. and Relaix, F. (2003). The formation of skeletal libraries prepared using the TruSeq RNA Sample Preparation Kit v2 muscle: from somite to limb. J. Anat. 202, 59-68. Chatterjee, S. and Ma, K. (2016). Circadian clock regulation of skeletal muscle according to the Illumina protocol. Multiplexed libraries were validated using growth and repair. F1000Research 5, 1549-1549. the Agilent BioAnalyzer, normalized and pooled for sequencing. High- Cui, S., Li, L., Mubarokah, S. N. and Meech, R. (2019). Wnt/β-catenin signalling throughput sequencing was performed on the HiSeq 2500 system (Illumina) induces the MyomiRs miR-133b and miR-206 to suppress Pax7 and induce the with a 100 bp read length. Image analysis and base calling were carried out myogenic differentiation program. J. Cell. Biochem. (in press). using Illumina HiSeq Analysis Software. Short read sequences were mapped Dahl, J. A. and Collas, P. (2008). A rapid micro chromatin immunoprecipitation to a UCSC mm10 reference sequence using the RNA-Seq aligner STAR assay (ChIP). Nat. Protocols 3, 1032-1045. (Dobin et al., 2013). Known splice junctions from mm10 were supplied to the Daniels, D. L. and Weis, W. I. (2005). Beta-catenin directly displaces Groucho/TLE de novo repressors from Tcf/Lef in Wnt-mediated transcription activation. Nat. Struct. Mol. aligner and junction discovery was also permitted. Differential gene Biol. 12, 364-371. expression analysis, statistical testing and annotation were performed using Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, Cuffdiff 2 (Trapnell et al., 2013). Transcript expression was calculated as P., Chaisson, M. and Gingeras, T. R. (2013). STAR: ultrafast universal RNA-seq gene-level relative abundance in fragments per kilobase of exon model per aligner. Bioinformatics 29, 15-21. million mapped fragments and employed correction for transcript abundance Goel, A. J., Rieder, M. K., Arnold, H. H., Radice, G. L. and Krauss, R. S. (2017). bias (Roberts et al., 2011). Results for genes of interest were also explored Niche cadherins control the quiescence-to-activation transition in muscle stem visually using the UCSC Genome Browser. cells. Cell Rep 21, 2236-2250. Graham, T. A., Weaver, C., Mao, F., Kimelman, D. and Xu, W. (2000). Crystal structure of a β-catenin/Tcf complex. Cell 103, 885-896. Statistical analyses Hamp, J., Löwer, A., Dottermusch-Heidel, C., Beck, L., Moussian, B., ̈ GraphPad Prism was used for statistical analysis. One-way ANOVA or Flötenmeyer, M. and Onel, S.-F. (2016). Drosophila Kette coordinates t-tests were used as specified in the figure legends. myoblast junction dissolution and the ratio of Scar-to-WASp during myoblast fusion. J. Cell Sci. 129, 3426-3436. Hecht, A., Vleminckx, K., Stemmler, M. P., van Roy, F. and Kemler, R. (2000). Competing interests The p300/CBP acetyltransferases function as transcriptional coactivators of The authors declare no competing or financial interests. [beta]-catenin in vertebrates. EMBO J. 19, 1839-1850. Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y. Author contribution and Honma, K. (2002). Dec1 and Dec2 are regulators of the mammalian Conceptualization: H.P.M., R.M.; Methodology: S.C.; Investigation: S.C., L.L., M.D., molecular clock. Nature 419, 841-844. R.T.Y., J.-A.H., R.M.; Resources: M.D., R.T.Y., R.M.E.; Data curation: M.D., R.T.Y.; Hulin, J. A., Nguyen, T. D., Cui, S., Marri, S., Yu, R. T., Downes, M., Evans, R. M., Writing - original draft: S.C., H.P.M., R.M.; Writing - review & editing: R.M.; Makarenkova, H. and Meech, R. (2016). Barx2 and Pax7 regulate Axin2 Supervision: M.D., R.T.Y., R.M.; Project administration: R.M.; Funding acquisition: expression in myoblasts by interaction with beta-catenin and chromatin R.M.E., R.M. remodelling. Stem Cells 34, 2169-2182. Huveneers, S. and de Rooij, J. (2013). Mechanosensitive systems at the cadherin– Funding F-actin interface. J. Cell Sci. 126, 403-413. Jones, A. E., Price, F. D., Le Grand, F., Soleimani, V. D., Dick, S. A., Megeney, This work was supported by National Institutes of Health (1R01EY026202 and L. A. and Rudnicki, M. A. (2015). Wnt/beta-catenin controls follistatin signalling to 1R01EY028983 to H.P.M.), by an Australian Research Council Fellowship regulate satellite cell myogenic potential. Skelet Muscle 5, 14. (FT110100573 to R.M.) and by a Flinders Foundation grant (to R.M.). Deposited in Kaimal, V., Bardes, E. E., Tabar, S. C., Jegga, A. G. and Aronow, B. J. (2010). PMC for release after 12 months. ToppCluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems. Nucleic Acids Data availability Res. 38, W96-W102. RNA-seq and ChIP-seq data reported in this paper have been deposited in the Kim, C.-H., Neiswender, H., Baik, E. J., Xiong, W. C. and Mei, L. (2008). {beta}- National Center for Biotechnology Information (NCBI) Sequence Read Archive Catenin interacts with MyoD and regulates its transcription activity. Mol. Cell. Biol. (SRA) database under accession number SRP185730. 28, 2941-2951. Kohyama, J., Tokunaga, A., Fujita, Y., Miyoshi, H., Nagai, T., Miyawaki, A., Supplementary information Nakao, K., Matsuzaki, Y. and Okano, H. (2005). Visualization of spatiotemporal Supplementary information available online at activation of Notch signaling: live monitoring and significance in neural http://dev.biologists.org/lookup/doi/10.1242/dev.167080.supplemental development. Dev. Biol. 286, 311-325. Leikina, E., Gamage, D. G., Prasad, V., Goykhberg, J., Crowe, M., Diao, J., References Kozlov, M. M., Chernomordik, L. V. and Millay, D. P. (2018). Myomaker and Aberle, H., Schwartz, H., Hoschuetzky, H. and Kemler, R. (1996). Single amino myomerger work independently to control distinct steps of membrane remodeling acid substitutions in proteins of the armadillo gene family abolish their binding to during myoblast fusion. Dev. Cell 46, 767-780.e7. Lepper, C., Conway, S. J. and Fan, C.-M. (2009). Adult satellite cells and -catenin. J. Biol. Chem. 271, 1520-1526. embryonic muscle progenitors have distinct genetic requirements. Nature 460, Agley, C., Lewis, F., Jaka, O., Lazarus, N., Velloso, C., Francis-West, P., Ellison- 627-631. Hughes, G. M. and Harridge, S. D. R. (2017). Active GSK3β and an intact β- Lepper, C., Partridge, T. A. and Fan, C.-M. (2011). An absolute requirement for catenin TCF complex are essential for the differentiation of human myogenic Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. progenitor cells. Sci. Rep. 7, 13189. Development 138, 3639-3646. Arce, L., Pate, K. T. and Waterman, M. L. (2009). Groucho binds two conserved Li, V. S. W., Ng, S. S., Boersema, P. J., Low, T. Y., Karthaus, W. R., Gerlach, J. P., regions of LEF-1 for HDAC-dependent repression. BMC Cancer 9, 159. Mohammed, S., Heck, A. J. R., Maurice, M. M., Mahmoudi, T. et al. (2012). Wnt Bernardi, H., Gay, S., Fedon, Y., Vernus, B., Bonnieu, A. and Bacou, F. (2011). signaling through Inhibition of β-catenin degradation in an intact Axin1 complex. Wnt4 activates the canonical β-catenin pathway and regulates negatively Cell 149, 1245-1256. myostatin: functional implication in myogenesis. Am. J. Physiol. Cell Physiol. Matsu-Ura, T., Dovzhenok, A., Aihara, E., Rood, J., Le, H., Ren, Y., Rosselot, 300, C1122-C1138. A. E., Zhang, T., Lee, C., Obrietan, K. et al. (2016). Intercellular coupling of the Billin, A. N., Thirlwell, H. and Ayer, D. E. (2000). Beta-catenin-histone deacetylase cell cycle and circadian clock in adult stem cell culture. Mol. Cell 64, 900-912. interactions regulate the transition of LEF1 from a transcriptional repressor to an Mauro, A. (1961). Satellite cell of skeletal muscle fibers. J. Cell Biol. 9, 493-495. activator. Mol. Cell. Biol. 20, 6882-6890. McCarthy, J. J., Mula, J., Miyazaki, M., Erfani, R., Garrison, K., Farooqui, A. B., Borello, U., Berarducci, B., Murphy, P., Bajard, L., Buffa, V., Piccolo, S., Srikuea, R., Lawson, B. A., Grimes, B., Keller, C. et al. (2011). Effective fiber Buckingham, M. and Cossu, G. (2006). The Wnt/β-catenin pathway regulates hypertrophy in satellite cell-depleted skeletal muscle. Development 138, Gli-mediated Myf5 expression during somitogenesis. Development 133, 3657-3666. 3723-3732. Millay, D. P., Sutherland, L. B., Bassel-Duby, R. and Olson, E. N. (2014). Brack, A. S. (2014). Pax7 is back. Skeletal Muscle 4, 24-24. Myomaker is essential for muscle regeneration. Genes Dev. 28, 1641-1646. Brack, A. S., Conboy, I. M., Conboy, M. J., Shen, J. and Rando, T. A. (2008). A Millay, D. P., Gamage, D. G., Quinn, M. E., Min, Y.-L., Mitani, Y., Bassel-Duby, R. temporal switch from notch to Wnt signaling in muscle stem cells is necessary for and Olson, E. N. (2016). Structure-function analysis of myomaker domains

normal adult myogenesis. Cell Stem Cell 2, 50-59. required for myoblast fusion. Proc. Natl. Acad. Sci. USA 113, 2116-2121. DEVELOPMENT

10 RESEARCH ARTICLE Development (2019) 146, dev167080. doi:10.1242/dev.167080

Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A. and Kardon, G. Sambasivan, R., Yao, R., Kissenpfennig, A., Van Wittenberghe, L., Paldi, A., (2011). Satellite cells, connective tissue fibroblasts and their interactions are Gayraud-Morel, B., Guenou, H., Malissen, B., Tajbakhsh, S. and Galy, A. crucial for muscle regeneration. Development 138, 3625-3637. (2011). Pax7-expressing satellite cells are indispensable for adult skeletal muscle Murphy, M. M., Keefe, A. C., Lawson, J. A., Flygare, S. D., Yandell, M. and regeneration. Development 138, 3647-3656. Kardon, G. (2014). Transiently active Wnt/beta-catenin signaling is not required Sanjana, N. E., Shalem, O. and Zhang, F. (2014). Improved vectors and genome- but must be silenced for stem cell function during muscle regeneration. Stem Cell wide libraries for CRISPR screening. Nat. Methods 11, 783-784. Reports 3, 475-488. Sasaki, N., Kiso, M., Kitagawa, M. and Saga, Y. (2011). The repression of Notch Nieset, J. E., Redfield, A. R., Jin, F., Knudsen, K. A., Johnson, K. R. and signaling occurs via the destabilization of mastermind-like 1 by Mesp2 and is Wheelock, M. J. (1997). Characterization of the interactions of alpha-catenin with essential for somitogenesis. Development 138, 55-64. alpha-actinin and beta-catenin/plakoglobin. J. Cell Sci. 110, 1013-1022. Sasaki, T., Hwang, H., Nguyen, C., Kloner, R. A. and Kahn, M. (2013). The small Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H. and Nakatani, Y. molecule Wnt signaling modulator ICG-001 improves contractile function in (1996). The transcriptional coactivators p300 and CBP are histone chronically infarcted rat myocardium. PLoS ONE 8, e75010. acetyltransferases. Cell 87, 953-959. Schultz, E. and Jaryszak, D. L. (1985). Effects of skeletal muscle regeneration on Otto, A., Schmidt, C., Luke, G., Allen, S., Valasek, P., Muntoni, F., Lawrence- the proliferation potential of satellite cells. Mech. Ageing Dev. 30, 63-72. Watt, D. and Patel, K. (2008). Canonical Wnt signalling induces satellite-cell Stamos, J. L. and Weis, W. I. (2013). The beta-catenin destruction complex. Cold proliferation during adult skeletal muscle regeneration. J. Cell Sci. 121, Spring Harb. Perspect Biol. 5, a007898. 2939-2950. Sun, H., Li, L., Vercherat, C., Gulbagci, N. T., Acharjee, S., Li, J., Chung, T.-K., Ozawa, M. (2015). E-cadherin cytoplasmic domain inhibits cell surface localization Thin, T. H. and Taneja, R. (2007). Stra13 regulates satellite cell activation by of endogenous cadherins and fusion of C2C12 myoblasts. Biol. Open 4, antagonizing Notch signaling. J. Cell Biol. 177, 647-657. 1427-1435. Suzuki, A., Pelikan, R. C. and Iwata, J. (2015). WNT/β-catenin signaling regulates Pansters, N., van der Velden, J., Kelders, M., Laeremans, H., Schols, A. and multiple steps of myogenesis by regulating step-specific targets. Mol. Cell. Biol. Langen, R. (2011). Segregation of myoblast fusion and muscle-specific gene 35, 1763-1776. β expression by distinct ligand-dependent inactivation of GSK-3 . Cell. Mol. Life Sci. Takei, D., Nishi, M., Fukada, S., Doi, M., Okamura, H., Uezumi, A., Zhang, L., 68, 523-535. Yoshida, M., Miyazato, M., Ichimura, A. et al. (2017). Gm7325 is MyoD- Parker, D. S., Ni, Y. Y., Chang, J. L., Li, J. and Cadigan, K. M. (2008). Wingless dependently expressed in activated muscle satellite cells. Biomed. Res. 38, signaling induces widespread chromatin remodeling of target loci. Mol. Cell. Biol. 215-219. 28, 1815-1828. Takemaru, K. I. and Moon, R. T. (2000). The transcriptional coactivator CBP Petropoulos, H. and Skerjanc, I. S. (2002). Beta-catenin is essential and sufficient interacts with beta-catenin to activate gene expression. J. Cell Biol. 149, 249-254. for skeletal myogenesis in P19 cells. J. Biol. Chem. 277, 15393-15399. Tanaka, S., Terada, K. and Nohno, T. (2011). Canonical Wnt signaling is involved Polesskaya, A., Naguibneva, I., Fritsch, L., Duquet, A., Ait-Si-Ali, S., Robin, P., in switching from cell proliferation to myogenic differentiation of mouse myoblast Vervisch, A., Pritchard, L. L., Cole, P. and Harel-Bellan, A. (2001). CBP/p300 cells. J. Mol. Signal. 6, 12. and muscle differentiation: no HAT, no muscle. EMBO J. 20, 6816-6825. Trapnell, C., Hendrickson, D. G., Sauvageau, M., Goff, L., Rinn, J. L. and Posokhova, E., Shukla, A., Seaman, S., Volate, S., Hilton, M. B., Wu, B., Morris, H., Swing, D. A., Zhou, M., Zudaire, E. et al. (2015). GPR124 functions as a Pachter, L. (2013). Differential analysis of gene regulation at transcript resolution WNT7-specific coactivator of canonical beta-catenin signaling. Cell Rep 10, with RNA-seq. Nat. Biotechnol. 31, 10.1038/nbt.2450. 123-130. von Maltzahn, J., Chang, N. C., Bentzinger, C. F. and Rudnicki, M. A. (2012). Wnt Quinn, M. E., Goh, Q., Kurosaka, M., Gamage, D. G., Petrany, M. J., Prasad, V. signaling in myogenesis. Trends Cell Biol. 22, 602-609. and Millay, D. P. (2017). Myomerger induces fusion of non-fusogenic cells and is Wodarz, A. and Nusse, R. (1998). Mechanisms of Wnt signaling in development. required for skeletal muscle development. Nat. Commun. 8, 15665. Annu. Rev. Cell Dev. Biol. 14, 59-88. Ridgeway, A. G., Petropoulos, H., Wilton, S. and Skerjanc, I. S. (2000). Wnt Wozniak, A. C., Kong, J., Bock, E., Pilipowicz, O. and Anderson, J. E. (2005). signaling regulates the function of MyoD and myogenin. J. Biol. Chem. 275, Signaling satellite-cell activation in skeletal muscle: markers, models, stretch, and 32398-32405. potential alternate pathways. Muscle Nerve 31, 283-300. Roberts, A., Pimentel, H., Trapnell, C. and Pachter, L. (2011). Identification of Wrobel, E., Brzoska, E. and Moraczewski, J. (2007). M-cadherin and beta-catenin novel transcripts in annotated genomes using RNA-Seq. Bioinformatics 27, participate in differentiation of rat satellite cells. Eur. J. Cell Biol. 86, 99-109. 2325-2329. Xu, W. and Kimelman, D. (2007). Mechanistic insights from structural studies of β- Roth, J. F., Shikama, N., Henzen, C., Desbaillets, I., Lutz, W., Marino, S., catenin and its binding partners. J. Cell Sci. 120, 3337-3344. Wittwer, J., Schorle, H., Gassmann, M. and Eckner, R. (2003). Differential role Yamada, S., Pokutta, S., Drees, F., Weis, W. I. and Nelson, W. J. (2005). of p300 and CBP acetyltransferase during myogenesis: p300 acts upstream of Deconstructing the cadherin-catenin-actin complex. Cell 123, 889-901. MyoD and Myf5. EMBO J. 22, 5186-5196. Zhang, P., Wong, C., Liu, D., Finegold, M., Harper, J. W. and Elledge, S. J. Rudnicki, M. A., Le Grand, F., McKinnell, I. and Kuang, S. (2008). The molecular (1999). p21(CIP1) and p57(KIP2) control muscle differentiation at the myogenin regulation of muscle stem cell function. Cold Spring Harb. Symp. Quant. Biol. 73, step. Genes Dev. 13, 213-224. 323-331. Zhuang, L., Hulin, J. A., Gromova, A., Tran Nguyen, T. D., Yu, R. T., Liddle, C., Rudolf, A., Schirwis, E., Giordani, L., Parisi, A., Lepper, C., Taketo, M. M. and Le Downes, M., Evans, R. M., Makarenkova, H. P. and Meech, R. (2014). Barx2 Grand, F. (2016). beta-catenin activation in muscle progenitor cells regulates and Pax7 have antagonistic functions in regulation of wnt signaling and satellite tissue repair. Cell Reports 15, 1277-1290. cell differentiation. Stem Cells 32, 1661-1673. DEVELOPMENT

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