1 WNT/β-catenin signaling regulates multiple steps of myogenesis ...

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WNT/β-catenin signaling regulates multiple steps of myogenesis by regulating step-specific 1

targets 2

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Akiko Suzuki1,2, Richard C. Pelikan2, and Junichi Iwata1,2,3,* 4

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1Department of Diagnostic & Biomedical Sciences, and 2Center for Craniofacial Research, the 6

University of Texas Health Science Center at Houston School of Dentistry; 3The University of 7

Texas Graduate School of Biomedical Sciences at Houston, USA 8

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Competing financial interests 10

The authors declare no competing financial interests 11

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*Address correspondence to Junichi Iwata, [email protected]. 13

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Running title: WNT/β-catenin signaling regulates muscle development 15

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Keywords: WNT/β-catenin signaling, cell cycle, myoblast fusion, muscle homeostasis 17

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The word count for the Materials and Methods section: 1,177 words (7,328 characters excluding 19

spaces) 20

The combined word count for the Introduction, Results, and Discussion sections: 4,156 words 21

(24,976 characters excluding spaces) 22

MCB Accepted Manuscript Posted Online 9 March 2015Mol. Cell. Biol. doi:10.1128/MCB.01180-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 23

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Molecules involved in WNT/β-catenin signaling show specific spatiotemporal expression and 25

play vital roles in myogenesis; however, it is still largely unknown how WNT/β-catenin 26

signaling regulates each step of myogenesis. Here, we show that WNT/β-catenin signaling can 27

control diverse biological processes of myogenesis by regulating step-specific molecules. In 28

order to identify the temporally specific roles of WNT/β-catenin signaling molecules in muscle 29

development and homeostasis, we used in vitro culture systems for both primary mouse 30

myoblasts and C2C12 cells, which can differentiate into myofibers. We found that a blockade of 31

WNT/β-catenin signaling in the proliferating cells decreases proliferation activity, but does not 32

induce cell death, through the regulation of genes cyclin A2 (Ccna2) and cell division cycle 25C 33

(Cdc25c). During muscle differentiation, the inhibition of WNT/β-catenin signaling blocks 34

myoblast fusion through the inhibition of the Fermitin family homolog 2 (Fermt2) gene. 35

Blocking WNT/β-catenin signaling in the well-differentiated myofibers results in the failure of 36

maintenance of their structure by disruption of cadherin/β-catenin/actin complex formation, 37

which plays a crucial role in connecting a myofiber’s cytoskeleton to the surrounding 38

extracellular matrix. Thus, our results indicate that WNT/β-catenin signaling can regulate 39

multiple steps of myogenesis, including cell proliferation, myoblast fusion, and homeostasis, by 40

targeting step-specific molecules. 41


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INTRODUCTION 42

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Skeletal muscle plays highly specialized roles in the generation of force and is capable of 44

extensive metabolic and functional plasticity. Skeletal muscle also exhibits robust regenerative 45

capacity, and its formation is composed of complex and highly regulated processes among 46

embryonic development and regeneration in mature skeletal musculature (1). Muscle precursor 47

cells (aka satellite cells in the adult) lie under, or are embedded in, the basal lamina of the 48

myofiber in skeletal muscles and are the major source of regeneration and growth of skeletal 49

muscles (2). During development and regeneration in response to injury or recovery from 50

atrophy, muscle precursor cells start to proliferate, at which stage they are referred to as 51

myoblasts, and subsequently differentiate to form new myofibers or fuse with existing myofibers 52

(2). These processes require fine-tuned regulations of proliferation and differentiation that are 53

likely regulated by signal transduction pathways. 54

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The WNT family (wingless-type MMTV integration site family) of signaling molecules 56

consists of 21 secreted glycoprotein ligands. WNT ligands are essential to activate downstream 57

pathways (canonical β-catenin-dependent and/or non-canonical β-catenin independent pathways) 58

in physiological and pathological conditions. WNT/β-catenin signaling can regulate a variety of 59

genes in a specific spatiotemporal manner (3). In the absence of WNT ligands, β-catenin is 60

incorporated into a protein complex containing AXIN, adenomatous polyposis coli (APC) and 61

the serine-threonine kinase glycogen synthase kinase-3 (GSK3β), which is hereafter termed the 62

“destruction complex”. The destruction complex phosphorylates β-catenin and leads it to a 63


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degradation process by the ubiquitin-proteasome (4). In the presence of WNT ligands, WNT 64

ligands bind to a Frizzled receptor (FZD) and the low-density lipoprotein receptor-related protein 65

5/6 (LRP5/6), followed by the recruitment of AXIN to LRP5/6 (5). Subsequently, the destruction 66

complex is inactivated, and β-catenin can escape from its incorporation into the destruction 67

complex, resulting in its stabilization and translocation into the nucleus (4). Increased nuclear 68

concentrations of β-catenin result in induction of target genes by interacting with transcriptional 69

co-activators such as members of the T-cell factor/Lymphocyte-enhancement factor-1 70

(TCF/LEF-1) family (6). In addition, cytoplasmic accumulated β-catenin is involved in cell-cell 71

interactions in combination with cadherin and actin (7). 72

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WNT signaling is essential for a variety of developmental and regenerative processes 74

including embryonic muscle development and maintenance of skeletal muscle homeostasis in the 75

adult (8-10). For example, WNT ligands regulate the specification of skeletal myoblasts in the 76

paraxial mesoderm and induce location-specific expression of muscle regulatory factors (11). 77

Treatment with secreted Frizzled-related protein 3 (sFRP3), a soluble WNT antagonist, reduces 78

skeletal myogenesis in a dose-dependent fashion (12). Indeed, mice with deficiency of WNT/β-79

catenin signaling (Ctnnb1-/- mice) are embryonic lethal by E8.5 with increased cell death (13). 80

Mice with conditional depletion of β-catenin in the muscle precursor Pax7+ cell lineage 81

(Ctnnb1F/F;Pax7-Cre mice) show reduced muscle mass and slow myofibers (14). Mice with 82

expression of a constitutively active stabilized form of β-catenin in the Pax7+ lineage 83

(CtnnbΔex3;Pax7-Cre mice) exhibit reduced myogenesis but increased slow myofibers (14, 15). 84

Thus, dysregulation of WNT/β-catenin signaling can lead to severe developmental defects and 85


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perturbation of muscle homeostasis. Nevertheless, the temporally specific roles of WNT/β-86

catenin signaling during myogenesis remain unknown. 87

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The multiple steps of muscle development and regeneration, beginning with muscle 89

progenitor cell activation and ending with myofiber formation, are all subject to separate levels 90

of regulation and are affected by a variety of muscle disorders and atrophy (2, 14). In this study, 91

we investigated the role of WNT/β-catenin signaling in muscle biology, including cell 92

proliferation, differentiation, and homeostasis of muscle cells. We used both primary myoblasts 93

and C2C12 cells (a myoblast cell line) that have the ability to differentiate into myofibers in the 94

culture with differentiation inducers. This process provides an opportunity to investigate the 95

temporally specific role of WNT/β-catenin signaling during myogenesis. We found that WNT/β-96

catenin signaling can regulate multiple steps of muscle development, ranging from cell 97

proliferation to homeostasis, through the regulation of step-specific targets. Knowledge of the 98

temporally specific regulatory mechanism may be applied to therapeutic approaches to stimulate 99

effective skeletal muscle regeneration following muscle trauma or atrophy. 100

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MATERIALS AND METHODS 103

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Cell culture. C2C12 cells, a murine skeletal muscle cell line, were obtained from the American 105

Type Culture Collection (ATCC; CRL-1772). Primary myoblasts were isolated from the limb 106

and tongue of C57B6/J mice, as described previously (16). Briefly, for preparation of primary 107


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myoblasts, hind limb muscle and tongue were dissected out from E15.5 C57B6/J mouse embryos 108

and digested by a 1.8 U/ml dispase solution (Gibco) for one hour at 37 °C and 5 % CO2. 109

Digested tissues were then suspended with growth medium [Dulbecco’s Modified Eagle’s 110

Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin, streptomycin, 2 mM 111

L-glutamate, 1 mM sodium pyruvate, and nonessential amino acids], and cells were collected by 112

centrifugation. Resuspended cells in growth medium were placed into a cell culture dish coated 113

with human fibronectin (BD BiocoatTM, BD Falcon) and cultured at 37 °C and 5 % CO2 in a 114

humidified incubator. Cell proliferation assays were performed using a cell counting kit (Dojindo 115

Molecular Technologies). Cells were treated with IWR1-endo (Tocris Bioscience, Bristol, UK) 116

at indicated concentration (0–80 µM) for 24 to 48 hours. BrdU incorporation assays (BrdU 117

incorporation for the last 1 hour) were performed using cells treated with vehicle or 80 µM 118

IWR1-endo for 36 hours. Incorporated BrdU was stained with a rat polyclonal antibody against 119

BrdU (Abcam), as described previously (17, 18). Myogenic differentiation was induced under 120

muscle differentiation medium [DMEM supplemented with 2% horse serum, 2 mM L-glutamate, 121

penicillin, streptomycin, and insulin (100 ng/ml)] for indicated days. To examine the effect of 122

IWR1-endo on myogenic differentiation, cells were treated with IWR1-endo for three to five 123

days at indicated concentration (0–10 µM). To investigate the effect of IWR1-endo on 124

maintenance of myofibers, well-differentiated C2C12 cells were cultured with vehicle or 1 μM 125

IWR1-endo under differentiation medium for another three to five days. The siRNA knockdown 126

for Ctnnb1 (Invitrogen), Ccna2, Cdc25c, and Fermt2 (Sigma-Aldrich) was performed as 127

described previously (17, 19). The overexpression of Fermt2 (OriGene Technologies, Inc., 128

Rockville, MD) was also performed as described previously (17). 129


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Tongue organ culture. Timed-pregnant C57B6/J mice were sacrificed at E14.5. Each 131

tongue was micro-dissected out and cultured in serum-free chemically defined BGJb medium 132

supplemented with penicillin, streptomycin, and 100 µg/ml ascorbic acid (16, 17). Tongue 133

explants were treated with vehicle or 80 µM IWR1-endo for 24 hours for BrdU incorporation 134

assay or for 72 hours for differentiation assay and then harvested, fixed in 4% 135

paraformaldehyde/0.1 M phosphate buffer (pH 7.4), and processed to analyze histology. BrdU 136

staining was performed as described previously (17). Immunohistological staining for myosin 137

heavy chain (MYH) was also performed as described previously (16, 20). 138

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Evaluation of myoblast fusion. After a five-day culture under the differentiation 140

medium with vehicle or IWR1-endo (1 µM), C2C12 cells were stained with MYH and 141

counterstained with DAPI for nuclei. The extent of fusion was calculated using the fusion index 142

(21, 22): Fusion % = (the number of nuclei in multiple nuclei myotubes) per (total number of 143

nuclei in MYH positive cells and myotubes) x 100. 144

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Immunoprecipitation. Extracts in cell lysis buffer containing 10 mM Tris (pH 7.5), 100 146

mM NaCl, 1% Triton X-100, and CompleteTM protease inhibitor (Roche Diagnostics) were lysed. 147

Immunoprecipitation assays were performed as described previously (17). A mouse monoclonal 148

antibody against pan-cadherin (Abcam) and a rabbit polyclonal antibody against actin (Abcam) 149

were used for immunoprecipitation assays. 150

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Immunoblotting. Immunoblots were performed, as described previously (18), using 152

rabbit polyclonal antibodies against cleaved caspase 3, active β-catenin, phosphorylated β-153

catenin (Cell Signaling Technology), and actin (Abcam) and mouse monoclonal antibodies 154

against pan-cadherin (Abcam) and GAPDH (Millipore). 155

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Immunofluorescent analysis. Immunofluorescent analysis was performed, as described 157

previously (20), using antibodies as follows: a rabbit polyclonal antibody against active β-catenin 158

(Cell Signaling Technology) and mouse monoclonal antibodies against MYH (Sigma-Aldrich) 159

and pan-cadherin (Abcam). DyLightTM 554-conjugated Phalloidin was used for staining F-actin 160

(Cell signaling Technology). Fluorescent images were taken by an inverted fluorescent 161

microscope (IX71, Olympus). Confocal images were taken by a laser confocal scanning 162

microscope (IX81 Fluoveiw FV1000, Olympus). 163

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Quantitative RT-PCR. For cell proliferation assays, total RNAs isolated from C2C12 165

cells treated with 80 µM IWR1-endo, or control vehicle, for 36 hours (n=6 per group), were 166

dissected with a QIAshredder and RNeasy mini extraction kit (Qiagen), as described previously 167

(19, 23). Gene expression profiling for cell cycle regulators was performed by PCR arrays 168

(PrimePCR assay, BioRad laboratories). The PCR primers were purchased from BioRad. For 169

myoblast fusion assays, total RNAs isolated from C2C12 cells cultured under differentiation 170

medium with 1 µM IWR1-endo or control vehicle for 12 hours to five days (n=6 per group) were 171

dissected with the QIAshredder and RNeasy mini extraction kit (Qiagen), as described 172

previously (16). For time-course experiments, C2C12 cells were treated with 0.1 μg/ml WNT3A 173


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for the indicated time with 80 µM IWR1-endo or control vehicle for cell proliferation and with 1 174

µM IWR1-endo or control vehicle for differentiation. The following PCR primers were used for 175

further specific analysis: Ccna2, 5’-TTGCTCCTCTTAAGGACCTTCC-3’ and 5’-176

CATTTAACCTCCATTTCCCTAAGGT-3’; Cdc25c, 5’-TGACGTCTATAGCCCCACC-3’ and 177

5’-TAAGCGGAGAGGCAGACATC-3’; Fermt2, 5’-GATCACTTTGGAAGGCGGGA-3’ and 178

5’-GCGCGTACTGCTTCTCGTTA-3’; and Gapdh, 5’-AACTTTGGCATTTGGAAGG-3’ and 179

5’-ACACATTGGGGGTAGGAACA-3’. The PCR primers for Ctnnb1 were purchased from 180

Santa Cruz Biotechnology, Inc. 181

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Reporter assay. Cignal TCF/LEF reporter construct (Qiagen) was transfected in C2C12 183

cells using Lipofectamine 3000 (Invitrogen). C12C12 cells were cultured under growth medium 184

or differentiation medium with/without 0.1 μg/ml WNT3A (R&D) and IWR1-endo (80 µM for 185

cell proliferation and 1 µM for differentiation) for 24 hours following the manufacturer’s 186

protocol. Luciferase assays were performed by the Dual-Glo® Luciferase assay system 187

(Promega). 188

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Comparative analysis of transcription factor binding site. The UCSC genome 190

browser was used to obtain the genomic sequences of the following murine genes including 5 191

kbp upstream of the respective transcription start site: Ccna2 (NM_009828.2), Ccnb1 192

(NM_172301.3), Cdc25c (NM_009860.2), Ccnd2 (NM_009829.3), Cdk1 (NM_007659.3), Cdk4 193

(NM_009870.3), Cdkn1a (NM_007669.4), Dmd (NM_007868.5), E2f5 (NM_007892.2), and 194

Fermt2 (NM_146054.2). These sequences were then mapped to seven additional mammalian 195


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genomes [human (Build 38), chimpanzee (Build 2.1.4), orangutan (Build 2.0.2), rhesus macaque 196

(Build 1.0), rat (Build 5), dog (Build 3.1), and horse (Build equCab2)] through the BLAST tool, 197

as described previously (16, 19). Multiple alignments for these sequences were obtained using 198

the Clustal Omega tool with default parameters and settings (24). Binding motifs of LEF1 199

(minimal core sites: 5’-CTTTG-3’or 5’-CAAAG-3’; optimal sites: 5’-CTTTGWW-3’ or 5’-200

WWCAAAG-3’, W=A/T) (25-27) were searched in the aligned DNA sequences. 201

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Chromatin immunoprecipitation (ChIP) assay. Cell extracts were incubated with β-203

catenin antibody (Abcam) overnight at 4°C followed by precipitation with magnetic beads. 204

Washing and elution of the immune complexes, as well as precipitation of DNA, were performed 205

according to standard procedures, as described previously (16, 19). The putative LEF1 target 206

sites of the genes in the immune complexes were detected by PCR using the following primers: 207

Ccna2 gene, 5’-TGGTGTTGCAGATCTACCGT-3’ and 5’-208

TCTGCTAACAAAATGGCAATGC-3’ (-2364 to -2154); Cdc25c gene, 5’-209

TTCATCGGTCTCAGCTTCCC-3’ and 5’-AGTCACCACTGAGCCTTGTC-3’ (-3164 to -210

3029); and Fermt2 gene, 5’-TAGAGGTTCTAGCGGGGGTT-3’ and 5’-211

TTCAGGCCTTGGCTTTGAGT-3’ (-3138 to -2949). Positions of PCR fragments correspond to 212

NCBI mouse genome Build 38 (mm10). 213

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Statistical analysis. Two-tailed student’s t tests were applied for statistical analysis. A p 215

value ≤ 0.05 was considered statistically significant. For all graphs, data are represented as mean 216

± standard deviation (SD). 217


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RESULTS 219

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WNT/β-catenin signaling regulates cell proliferation activity through Ccna2 and Cdc25c 221

expression. 222

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We have recently reported that upregulation of Dkk1/4, WNT/β-catenin antagonists, 224

inhibits muscle development (20), suggesting that WNT/β-catenin signaling plays a role in 225

muscle development. To explore how WNT/β-catenin signaling regulates myogenesis, we used a 226

cell culture system of C2C12 cells, which are an immortalized mouse myoblast cell line 227

originally established from normal adult C3H mouse hind limb muscle. C2C12 cells express 228

WNT ligands during cell proliferation and differentiation (28). We performed cell proliferation 229

assays using C2C12 cells treated with or without WNT/β-catenin inhibitor IWR1-endo (0-80 230

μM) that can promote AXIN stabilization and thereby inhibit WNT signaling (29). Cell 231

proliferation activity was decreased by IWR1-endo treatment in the dose-dependent manner (Fig. 232

1A and B). To validate cell proliferation defects after treatment with IWR1-endo (80 μM) for 36 233

hours, we performed BrdU incorporation assay. Cells incorporated BrdU were reduced by the 234

treatment with IWR1-endo (Fig. 1C and D). We further confirmed that IWR1-endo had no 235

toxicity on C2C12 cells at the concentrations we tested. We detected no apoptosis with treatment 236

of 80 μM IWR1-endo (Fig. 1E). IWR1-endo specifically inhibits WNT/β-catenin signaling by 237

induction of the β-catenin destruction complex formation (29). As expected, non-phosphorylated 238

(active) β-catenin was decreased, and phosphorylated (inactive) β-catenin was increased at 12 239


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and 24 hours after the treatment with 80 μM IWR1-endo (Fig. 1F). To test whether IWR1-endo 240

specifically blocks WNT/β-catenin signaling cascade through transcriptional response elements 241

of LEF/TCF, which is a well-known β-catenin co-transcription factor, we performed LEF/TCF 242

luciferase reporter assays after treatment with WNT3A (0.1 μg/ml) and IWR1-endo (80 μM) for 243

24 hours using proliferating cells. WNT3A induced LEF/TCF-mediated promoter activity 244

approximately six times, and IWR1-endo (80 μM) completely inhibited the induction of 245

LEF/TCF-mediated promoter activity (Fig. 1G). These results indicate that IWR1-endo inhibits 246

canonical WNT/β-catenin signaling. 247

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Next, to identify downstream targets of WNT/β-catenin signaling, we performed PCR 249

array analyses of C2C12 cells treated with or without 80 μM IWR1-endo for 36 hours (n=5 per 250

group). In this analysis, we uncovered 7 genes that were differentially expressed (≥1.5-fold, 251

p<0.05), 2 genes that were more abundant in the treated group, and 5 genes that were more 252

abundant in the control group (Table 1). The genes altered in the treated group were consistent 253

with cell proliferation defects, with significant reductions in the expression levels of transcripts 254

related to cell cycle transition. We analyzed the promoter sequence (including 5-kbp upstream of 255

the transcription start site) of the candidate target genes. We found that both Ccna2 and Cdc25c 256

genomic regions have a phylogenetically conserved LEF1 recognition sequence (Tables 2 and 3). 257

To test whether β-catenin could bind to the promoter regions of Ccna2 and Cdc25c, we 258

performed chromatin immunoprecipitation (ChIP) assays. The binding site for LEF1 in each 259

gene was immunoprecipitated with β-catenin antibody but not with control IgG. Furthermore, the 260

immunoprecipitated amounts were decreased after the treatment with 80 μM IWR1-endo for 24 261


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hours in both Ccna2 and Cdc25c, indicating that the binding site for LEF1 in each Ccna2 and 262

Cdc25c gene is functional and responsive to WNT/β-catenin signaling activity (Fig. 1H and I). 263

Importantly, gene expression of Ccna2 and Cdc25c was induced by WNT3A ligands (0.1 μg/ml) 264

in the time dependent manner (Fig. 1J and K). Additionally, we analyzed cell proliferation 265

activity after treatment with siRNA knockdown for Ccna2, Cdc25c, and Ccna2/Cdc25c 266

combination. Gene expression of Ccna2 and Cdc25c was significantly reduced after the siRNA 267

knockdown by 80 % and 60 % reduction, respectively (Fig. 1L). Cell proliferation activity was 268

significantly reduced after the knockdown of Ccna2, Cdc25c, or Ccna2/Cdc25c combination 269

(Fig. 1M). Furthermore, we analyzed gene expression of Ccna2 and Cdc25c after siRNA 270

knockdown for Ctnnb1 (Fig. 1N and O). Cell proliferation activity was significantly reduced 271

after the knockdown for Ctnnb1 (Fig. 1P and Q). Taken together, our results indicate that gene 272

expression of Ccna2 and Cdc25c is regulated by the WNT/β-catenin pathway, and CCNA2 and 273

CDC25C play a crucial role during myoblast proliferation. 274

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WNT/β-catenin signaling regulates muscle differentiation through Fermt2 expression. 276

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Next, we investigated myogenic differentiation of C2C12 cells under a differentiation 278

medium with (at both 1 and 10 μM) and without IWR1-endo. Without the IWR1-endo treatment, 279

we could detect myofibers after a three-day culture in the differentiation medium. After a five-280

day culture in the differentiation medium without IWR1-endo, C2C12 cells were well-developed 281

into multinucleated myofibers. In contrast, C2C12 cells cultured in the differentiation medium 282

with 1 μM IWR1-endo were mostly mononucleated and failed to differentiate into myotubes. 283


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This suggests that WNT/β-catenin signaling is crucial for myoblast fusion, a critical process for 284

the terminal differentiation of skeletal muscle. We further tested higher concentrations of IWR1-285

endo. We found that treatment with IWR1-endo at 10 μM and higher blocked myoblast 286

differentiation of C2C12 cells (Fig. 2A). To quantify the fusion defect with treatment of 1 μM 287

IWR1-endo, we counted the number of nuclei in the cells stained with a myosin heavy chain 288

(MYH), a differentiation marker for myofibers. In the control, almost 100% of muscle cells had 289

more than 3 nuclei [as shown by fusion index (ratio of muscle cells with multiple nuclei per all 290

muscle cells)]. In contrast, approximately 90% of the cells treated with 1 μM IWR1-endo had 291

single nuclei in each cell (Fig. 2B and C). Consistent with the IWR1-endo induced fusion defect, 292

the length of muscle fibers in the treated group was significantly shorter than those in the control 293

group (approximately 1.4 mm in control vs. approximately 0.1 mm in the treated group) (Fig. 294

2D). Furthermore, we confirmed that IWR1-endo treatment decreased non-phosphorylated 295

(active) β-catenin and increased phosphorylated (inactive) β-catenin at 1 and 10 μM (Fig. 2E). 296

Importantly, the treatment with either 1 or 10 μM IWR1-endo induced no apoptosis in the 297

process of muscle differentiation (Fig. 2F). To validate further that IWR1-endo treatment had no 298

toxic effects on myoblasts, we cultured C2C12 cells with 1 μM IWR1-endo under the 299

differentiation medium for five days (fusion defects at Day 5 treated with IWR1-endo) and then 300

switched the medium with or without 1 μM IWR1-endo for another five days (Fig. 2G). The 301

inhibited myoblast fusion at Day 5 was completely restored at Day 10 without IWR1-endo 302

(IWR1-endo was removed from the culture medium after five days of the culture), indicating that 303

WNT/β-catenin signaling specifically inhibits myoblast fusion but does not induce apoptosis. 304

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After this, we explored target molecules which were involved in myoblast fusion and 306

altered by the 1 μM IWR1-endo treatment at Day 5. Among 11 candidate molecules (Nphs1, 307

Ptk2, Fermt2, Adam12, Itga3, Itgb1, Cdh2, Cdh15, Myof, Dysf, and Tmem8c), gene expression of 308

Fermitin family homolog 2 (Fermt2, aka Kindlin2) was significantly decreased by the treatment 309

of 1 μM IWR1-endo (1.8-fold reduction, p<0.01) (Fig. 3A). Decreased Fermt2 expression at Day 310

2 was restored at Day 7 after the depletion of 1 μM IWR1-endo, indicating that Fermt2 311

expression is regulated by WNT/β-catenin signaling (Fig. 3B). To test whether IWR1-endo 312

specifically blocks WNT/β-catenin signaling cascade through transcriptional response elements 313

of LEF/TCF, we performed LEF/TCF luciferase reporter assays after treatment with WNT3A 314

(0.1 μg/ml) and IWR1-endo (1 μM) at the muscle differentiation stage. WNT3A induced 315

LEF/TCF-mediated promoter activity approximately six times, and IWR1-endo (1 μM) 316

completely inhibited the induction of LEF/TCF-mediated promoter activity (Fig. 3C). These 317

results indicate that IWR1-endo inhibits canonical WNT/β-catenin signaling. FERMT2, a focal 318

adhesion protein, can regulate integrin activation and mediate cell-cell or cell-matrix adhesion 319

(30, 31). We analyzed the promoter sequence of the Fermt2 gene (including 5-kbp upstream of 320

the transcription start site). We found that the murine Fermt2 genomic region has a conserved 321

LEF1 recognition sequence (Tables 1 and 2). To test whether β-catenin could bind to the 322

promoter region of Fermt2, we performed ChIP assays. The binding site for LEF1 in the 323

promoter region of Fermt2 was immunoprecipitated with β-catenin antibody but not with control 324

IgG. In addition, the immunoprecipitated amounts were decreased by treatment with 1 μM 325

IWR1-endo for 48 hours, indicating that the binding site for LEF1 in the Fermt2 promoter region 326

is functional and responsible for WNT/β-catenin signaling activity (Fig. 3D and E). Gene 327


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expression of Fermt2 was induced by WNT3A ligands (0.1 μg/ml) in the time dependent manner 328

(Fig. 3F). Furthermore, gene expression of Fermt2 was significantly reduced after siRNA 329

knockdown for Ctnnb1 (Fig. 3G and H). Myoblast fusion was disrupted by the knockdown of 330

Ctnnb1 (Fig. 3I). Taken together, our results indicate that gene expression of Fermt2 is regulated 331

by the WNT/β-catenin pathway. 332

333

To validate the function of Fermt2 during muscle differentiation, we cultured C2C12 334

cells under the differentiation medium with treatment of siRNA knockdown for Fermt2. Gene 335

expression of Fermt2 was significantly reduced after the siRNA knockdown at 80 % reduction 336

(Fig. 3J). Myoblast fusion was disrupted by the knockdown of Fermt2 (Fig. 3K). In addition, 337

overexpression of Fermt2 in the condition of 1 μM IWR1-endo treatment restored the myofiber 338

fusion defect (Fig. 3L and M). Altogether, our results indicate that gene expression of Fermt2 is 339

regulated by WNT/β-catenin pathway, and Fermt2 plays a crucial role in myoblast fusion. 340

341

The cadherin/β-catenin/actin complex formation mediated by WNT/β-catenin signaling is 342

crucial for muscle homeostasis. 343

344

To test if WNT/β-catenin signaling is involved in muscle homeostasis, we treated well-345

differentiated C2C12 cells with or without 1 μM IWR1-endo (Fig. 4A and B). The long and 346

straightened myofibers were disrupted and changed their form to a round, multinucleated shape 347

after the IWR1-endo treatment (Fig. 4B). Next, we analyzed gene expression of the molecules 348

related to cytoskeletal structure formation (MYH1/4, caveolin 3, and desmin); however, we 349


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failed to identify genes altered by the treatment with 1 μM IWR1-endo (Fig. 4C), which suggests 350

that the WNT/β-catenin pathway may regulate cytoskeletal structure formation at the 351

posttranscriptional level. Previous studies have demonstrated that β-catenin can bind to both 352

cadherin and actin (32, 33). An extracellular matrix niche is crucial for regulating the movement 353

and morphology of skeletal muscle cells (34). We hypothesized that the cadherin/β-catenin/actin 354

complex plays a crucial role in myofiber morphogenesis by anchoring skeletal structure 355

molecules to the connective tissue (niche) composed of an extracellular matrix. To test the 356

molecular interaction among them, we performed immunoprecipitation for actin, cadherin, and 357

β-catenin (Fig. 4D). Our results indicate that cadherin/β-catenin/actin complex existed in 358

myofibers and that it was disrupted by the treatment with 1 μM IWR1-endo. Thus, β-catenin is 359

crucial for forming the cadherin/β-catenin/actin complex. In addition, we confirmed that 1 μM 360

IWR1-endo induced no apoptosis at the maturation/maintenance stage of muscle cells (Fig. 4E). 361

To further validate our findings of the complex formation regulated by WNT/β-catenin signaling, 362

we performed immunofluorescent analyses of these complex molecules with or without 363

treatment of 1 μM IWR1-endo. Actin stained with phalloidin was straightened and paralleled 364

each other in control. In contrast, actin formation was disrupted and accumulated after the 365

treatment with 1 μM IWR1-endo (Fig. 4F). Without IWR1-endo treatment, cadherin and β-366

catenin were co-localized in the myofibers. In contrast, there was poor co-localization of 367

cadherin and β-catenin after the treatment with 1 μM IWR1-endo (Fig. 4G and H). Thus, our 368

data indicate that β-catenin is an essential binding partner for both the cytoplasmic tail of 369

cadherin and actin and is crucial for the maintenance of myofiber morphology. 370

371


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Molecular mechanisms of myogenesis mediated by WNT/β-catenin signaling are conserved 372

in primary muscle cells. 373

374

We tested whether the mechanisms we found in C2C12 cells were conserved in primary 375

muscle cells from C57B6/J mice. We isolated primary muscle cells from the limb and tongue of 376

mice. We performed BrdU incorporation assays to test cell proliferation activity with or without 377

treatment of 80 μM IWR1-endo. In primary myoblasts from both the limb and tongue, IWR1-378

endo reduced cell proliferation activity (Fig. 5A-C). Gene expression of Ccna2 and Cdc25c was 379

significantly decreased by treatment of IWR1-endo in both primary limb and tongue myoblasts 380

(Fig. 5D). Then, we investigated muscle differentiation of primary myoblasts from both the limb 381

and tongue. The treatment with 1 μM IWR1-endo strikingly inhibited myoblast fusion in both 382

primary limb and tongue myoblasts. Gene expression of Fermt2 was significantly reduced by the 383

treatment with IWR1-endo (Fig. 5F). Myoblasts differentiated into myofibers within seven days 384

in a culture under the differentiation medium in the control. In contrast, treatment with 1 μM 385

IWR1-endo blocked myotube formation by inhibiting myoblast fusion (Fig. 5G). Next, we 386

investigated the maintenance ability of muscle cells. The well-differentiated and straightened 387

myofibers changed their shape after treatment with 1 μM IWR1-endo, as seen in C2C12 cells 388

(Fig. 5H). These results indicate that WNT/β-catenin-mediated signaling mechanisms are well 389

conserved in primary muscle cells. Furthermore, to test if the mechanisms were conserved in a 390

muscular tissue, we cultured tongue explants from C57B6/J mice with or without IWR1-endo. In 391

the tongue explants, IWR1-endo treatment (80 μM, 24 hours) resulted in decreased cell 392

proliferation, as seen in our cell culture system (Fig. 6A and B). In addition, IWR1-endo 393


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treatment (80 μM, 72 hours) resulted in defects in the elongation of muscle fibers (Fig. 6C). 394

Collectively, these results suggest that WNT/β-catenin signaling can regulate multiple steps of 395

muscle development, including proliferation, differentiation, and structural homeostasis, by 396

regulating step-specific targets (Fig. 6D). 397

398

DISCUSSION 399

400

Mice with deficiency in WNT ligands and their receptors FZDs show early embryonic lethality, 401

often affecting multiple tissues (3, 10). WNT/β-catenin signaling is active within myogenic cells, 402

as shown by BAT-gal mice, in which seven TCF/LEF-binding sites drive nuclear LacZ 403

expression when bound by transcriptionally active TCF/LEFs (35). During axial myogenesis, 404

WNT/β-catenin signaling is crucial for dermomyotome and myotome formation (14, 36-38). 405

WNT ligands can positively regulate the number of dermomyotomal Pax3+ and Pax7+ 406

progenitors (39-41). During fetal myogenesis, β-catenin affects myofiber number and positively 407

regulates the number of slow fibers in mice with conditional deletion of Ctnnb1 in muscle 408

progenitor cells (Pax7iCre/+;Ctnnb1Δ/fl2-6 mice) (14). Subsequently, WNT ligands are necessary 409

and sufficient to promote normal Myf5 and MyoD expression (42-45). WNT/β-catenin activation 410

also induces satellite cell proliferation during skeletal muscle regeneration (46). Compared with 411

studies on early muscle specification as described above, relatively little is known about prenatal 412

and postnatal myogenesis. 413

414


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In this study, we investigated how WNT/β-catenin signaling regulates muscle biology 415

using both primary myoblasts and C2C12 cells. We found that WNT/β-catenin signaling can 416

regulate specific targets crucial for each myoblast proliferation, fusion, and homeostasis of 417

myofibers. We also found that during myoblast proliferation, gene expression of Ccna2 and 418

Cdc25c was specifically downregulated by the inhibition of WNT/β-catenin signaling by IWR1-419

endo. Ccna2 is ubiquitously expressed and is essential for cell-cycle progression of embryonic 420

stem cells (47). Mice with loss of Ccna2 exhibit early embryonic lethality (48). In cancer, Ccna2 421

is often aberrantly expressed and impacts cell proliferation (49). The CDC25 protein 422

phosphatases (CDC25A, -B, and -C) drive cell cycle transitions by activating key components of 423

the cell cycle engine (50). Mice with loss of Cdc25c exhibit normal development, suggesting that 424

the other CDC25 protein phosphatases can compensate for its function during embryogenesis 425

(51). Indeed, mice lacking Cdc25a or all three Cdc25 genes are early embryonic lethal with 426

growth retardation by E7.5 (50). Although WNT/β-catenin signaling targets gene expression of 427

both Ccna2 and Cdc25c, gene expression of Ccna2 mediated by WNT/β-catenin signaling may 428

be more important for myoblast proliferation in vivo. 429

430

A recent study suggests a potential role of WNT/β-catenin signaling in myoblast fusion 431

(52). Members of the R-spondin family (RSPO) of secreted cysteine-rich proteins (RSPO1, -2, -3, 432

and -4) activate the WNT/β-catenin signaling pathway at the receptor level (53, 54). RSPO2 433

promotes myogenic differentiation and myocyte fusion via activation of WNT/β-catenin 434

signaling in C2C12 cells (55). RSPOs can regulate WNT receptor expression and control 435

myogenesis through gene expression of the TGFβ antagonist Fst (56). Thus, WNT signaling is 436


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activated through WNT ligands, RSPO, and non-canonical (β-catenin-independent) WNT 437

signaling activators during muscle development. During myoblast fusion, the cell signaling 438

pathway is tightly regulated and controls downstream target molecules involved in membrane 439

fusion and cytoskeletal remodeling (57). For example, a recent study indicates that Tmem8c (aka 440

Myomaker) specifically expresses in skeletal muscles and plays a crucial role in myoblast fusion 441

during muscle development (58). In our study, the bioinformatics analysis of Tmem8c promoter 442

region (up to 5-kb upstream of transcription start site of Tmem8c gene) detected no TCF/LEF 443

optimal binding site (WNT/ β-catenin response element). In addition, gene expression of 444

Tmem8c was not altered by IWR1-endo treatment. These data suggest that gene expression of 445

Tmem8c is regulated by the other signaling pathways such as transforming growth factor beta 446

(TGFβ), fibroblast growth factor (FGF), and hedgehog (HH). Among 11 candidate molecules, 447

only Fermt2 expression was significantly (> 1.5-fold change, p < 0.05) decreased by IWR1-endo 448

treatment. The other molecules involved in myoblast fusion tested in this study (Nphs1, Ptk2, 449

Adam12, Itga3, Itgb1, Cdh2, Cdh15, Myof, and Dysf) may be also controlled by the other 450

signaling pathways. 451

452

A previous study shows that RNAi knockdown for Fermt2 in C2C12 cells results in 453

significant abnormalities during the early stages of myogenesis (31). In addition, targeted 454

disruption of the murine Fermt2 gene results in early embryonic lethality with musculature 455

developmental defects (59). These findings, coupled with our results, indicate that WNT/β-456

catenin-mediated Fermt2 expression is crucial for myogenic fusion. Although Fermt2 null mice 457

are lethal by the stage of muscle fusion, the future study using mice with conditional deletion of 458


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Fermt2 will allow us to investigate the role of Fermt2 during muscle fusion. Moreover, Fermt2 459

may play additional roles aside from myoblast fusion during muscle development. Interestingly, 460

different steps of muscle differentiation are also regulated by WNT/β-catenin signaling. For 461

example, inhibition of GSK3β results in activated WNT/β-catenin signaling and leads to 462

enhanced differentiation of C2 myogenic cells (60). The RSPO family of proteins, WNT/β-463

catenin signaling agonists, can promote myogenic differentiation in cell culture (55). 464

Pharmacological activations of WNT/β-catenin signaling can also facilitate the differentiation of 465

satellite cells and myoblasts (60-62). Thus, WNT/β-catenin signaling may play a pivotal role for 466

the volume and size of skeletal muscles by regulating various steps of myoblast differentiation 467

and fusion. 468

469

In this study, we found that during muscle maturation/maintenance, a blockade of 470

WNT/β-catenin signaling resulted in disrupted cell adhesion via loss of cadherin/β-catenin/actin 471

complex formation. Previous studies indicate that β-catenin plays dual roles in WNT/β-catenin 472

signaling and in cadherin-mediated cell adhesion (32, 63). Beta-catenin co-localized with 473

cadherin in membranes may be essential for proper cell structural formation. Cadherin family 474

molecules exhibit distinctly regulated tissue distribution and control morphogenesis. Skeletal 475

muscle cells express both N-cadherin (encoded by Cdh2) and M-cadherin (encoded by Cdh15) 476

throughout myogenesis, from progenitors to myofibers, in both development and regeneration 477

(64, 65). Mice with loss of Cdh2 are embryonic lethal by E10 with disorganized somites (66). 478

The in vitro studies of Cdh2-/- cells indicate that N-cadherin is not essential for myogenesis, but 479

other cadherins may compensate for the lack of N-cadherin (67). M-cadherin is a classical 480


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calcium-dependent cell adhesion molecule that is highly expressed in developing skeletal 481

muscle, satellite cells, and cerebellum (65). Mice with loss of M-cadherin exhibit no gross 482

developmental defects (68). These results strongly suggest that loss of the combination of N- and 483

M-cadherins or other cadherins, or both, is essential for myogenic defects. The actin family is 484

categorized into four muscle-specific isoforms: α-skeletal; α-cardiac; α-smooth; and γ-smooth. It 485

is further categorized into two ubiquitously expressed isoforms: β-cytoplasmic and γ-cytoplasmic 486

(69). Skeletal myoblasts almost exclusively express β- and γ-cytoplasmic actins, but on 487

differentiation, these cytoplasmic isoforms are drastically downregulated and sequentially 488

replaced by α-cardiac and α-smooth actins, and ultimately α-skeletal actin (69). Mice with 489

deficiency of α-skeletal actin are indistinguishable from their littermates at birth but die in the 490

early neonatal period (postnatal day 1 to 9) with skeletal muscle defects (70). Recent studies 491

indicate that γ-cytoplasmic actin is required for cytoskeletal maintenance but not development 492

(71, 72). Thus, actin isoforms may also largely compensate for the lack of an individual actin. 493

Compared with cadherin and actin, β-catenin may play the most critical role in the cadherin/β-494

catenin/actin complex formation. 495

496

Among skeletal muscles, tongue muscles are the most developed muscle at birth for 497

suckling, compared with the other muscles (73, 74). Interestingly, mice lacking Myf5 and Pax3 498

do not develop skeletal muscle in the trunk or limb, yet head muscles form normally (75). In 499

chick embryo, WNT signals, which promote trunk myogenesis, have been shown to block 500

myogenesis in branchial arches (76). These findings suggest that craniofacial muscle 501

development may be differently regulated compared to that of trunk and limb muscles. In this 502


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study, we investigated primary myoblasts from the tongue compared with that of the limb. We 503

found that requirement of WNT/β-catenin signaling was similar among tongue and limb 504

myogenesis. 505

506

Our findings may be relevant in human disease, given that altered WNT/β-catenin 507

signaling is implicated in multiple malformations and syndromes including muscle disorders in 508

humans (77-79). For example, in patients with muscular defects, such as myopathies and 509

atrophy, WNT/β-catenin signaling is most likely altered due to genetic and/or epigenetic cause(s) 510

(80). Our results highlight that WNT/β-catenin signaling is a widely used mechanism in skeletal 511

muscles which regulate step-specific targets during myogenesis. Our findings on the mechanisms 512

of action of WNT/β-catenin signaling offer several intriguing possibilities into the potential for 513

therapeutic interventions. 514

515

516

ACKOWLEDGMENTS 517

518

We thank Dr. Yoshihiro Komatsu for discussion. This study was supported by the UT Houston 519

School of Dentistry faculty start-up fund to Junichi Iwata. 520


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specification of resident CD45+ adult stem cells during muscle regeneration. Cell 697

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62. Brack AS, Conboy IM, Conboy MJ, Shen J, Rando TA. 2008. A temporal switch 699

from notch to Wnt signaling in muscle stem cells is necessary for normal adult 700

myogenesis. Cell stem cell 2:50-59. 701

63. Gates J, Peifer M. 2005. Can 1000 reviews be wrong? Actin, alpha-Catenin, and 702

adherens junctions. Cell 123:769-772. 703

64. Krauss RS, Cole F, Gaio U, Takaesu G, Zhang W, Kang JS. 2005. Close encounters: 704

regulation of vertebrate skeletal myogenesis by cell-cell contact. Journal of cell science 705

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68. Hollnagel A, Grund C, Franke WW, Arnold HH. 2002. The cell adhesion molecule 715

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72. Sonnemann KJ, Fitzsimons DP, Patel JR, Liu Y, Schneider MF, Moss RL, Ervasti 729

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its absence leads to a progressive myopathy. Developmental cell 11:387-397. 731


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73. Noden DM, Francis-West P. 2006. The differentiation and morphogenesis of 732

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Association of Anatomists 235:1194-1218. 734

74. Yamane A. 2005. Embryonic and postnatal development of masticatory and tongue 735

muscles. Cell and tissue research 322:183-189. 736

75. Tajbakhsh S, Rocancourt D, Cossu G, Buckingham M. 1997. Redefining the genetic 737

hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. 738

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76. Tzahor E, Kempf H, Mootoosamy RC, Poon AC, Abzhanov A, Tabin CJ, Dietrich S, 740

Lassar AB. 2003. Antagonists of Wnt and BMP signaling promote the formation of 741

vertebrate head muscle. Genes & development 17:3087-3099. 742

77. Al-Qattan MM. 2011. WNT pathways and upper limb anomalies. The Journal of hand 743

surgery, European volume 36:9-22. 744

78. Kim N, Vu TH. 2006. Parabronchial smooth muscle cells and alveolar myofibroblasts in 745

lung development. Birth defects research. Part C, Embryo today : reviews 78:80-89. 746

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biology 16:81-90. 748

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cell proliferation, and myogenic differentiation. Cell death and differentiation 20:1194-752

1208. 753


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Abbreviations 754

755

APC, adenomatous polyposis coli; ChIP, chromatin immunoprecipitation; FZD, Frizzled 756

receptor; GSK3β, serine-threonine kinase glycogen synthase kinase-3; H&E, Hematoxylin and 757

Eosin; IP, immunoprecipitation; LEF-1, Lymphocyte-enhancement factor-1; LRP5/6, low-758

density lipoprotein receptor-related protein 5/6; MyHC, myosin heavy chain; NCBI, National 759

Center for Biotechnology Information; sFRP3, secreted Frizzled-related protein 3; TCF, T-cell 760

factor. 761

762

763

FIGURE LEGENDS 764

765

FIG 1. WNT/β-catenin signaling regulates myoblast cell proliferation via Ccna2 and Cdc25c 766

expression. (A) Cell proliferation assay of C2C12 cells with IWR1-endo treatment for 0-48 hours 767

at each indicated concentration (0, 1, 5, 10, 20, 40, 60, and 80 μM). N=6 per group. ***, p<0.001. 768

(B) Cell proliferation ratio at 48 hours after treatment with IWR1-endo at indicated 769

concentrations (0-80 μM). N=6 per group. ***, p<0.001. (C) BrdU staining (brown) in C2C12 770

cells after treatment with vehicle control or 80 μM IWR1-endo for 24 hours. Nuclei are 771

counterstained with Hematoxylin. Arrows indicate BrdU positive cells. Scale bar, 50 μm. (D) 772

Quantitation of the percentage of BrdU-labeled nuclei in C2C12 cells treated with 80 μM IWR1-773

endo or vehicle control for 24 hours. N=12 per each group. ***, p<0.001. (E) Immunoblotting 774

analysis of caspase 3 in C2C12 cells treated with vehicle control (lane 1), 80 μM IWR1-endo 775


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(lane 2), or positive control (tissue sample from adult wild-type mouse small intestine, lane 3) for 776

24 hours. (F) Immunoblotting analysis of time course (0, 6, 12, and 24 hours) for activated β-777

catenin (ABC), phosphorylated β-catenin (p-β-catenin), and GAPDH in C2C12 cells after 778

treatment with vehicle control or 80 μM IWR1-endo for 24 hours. (G) Reporter assay for 779

WNT/β-catenin signaling activity after treatment with/without (+/-) WNT3A (0.1 μg/ml) and 780

IWR1-endo (80 μM) in C2C12 cells cultured in growth medium. (H) ChIP analysis of DNA 781

fragments immunoprecipitated with a β-catenin-specific antibody or with an isotype-specific 782

(IgG) control antibody after treatment with/without 80 μM IWR1-endo (IWR1) for 24 hours. 783

Immunoprecipitated products were PCR amplified with primers flanking the putative LEF1-784

binding region of indicated genes. 2% input lane shows PCR amplification of the sonicated 785

chromatin before immunoprecipitation. (I) ChIP-qPCR analysis of β-catenin binding on the 786

Ccna2 and Cdc25c promoter regions using β-catenin antibody following treatment with/without 787

80 μM IWR1-endo treatment for 24 hours. ChIP with IgG without IWR1-endo treatment 788

(orange), IgG with IWR1-endo treatment (green), α-β-catenin antibody without IWR1-endo 789

treatment (red), and α-β-catenin antibody with IWR1-endo treatment (blue). (J, K) Quantitative 790

RT-PCR analysis of gene expression of Ccna2 (J) and Cdc25c (K) after treatment with WNT3A 791

(0.1 μg/ml) for 0, 1, 2, and 3 hours (h) with/without 80 μM IWR1-endo. **, p<0.01; ***, 792

p<0.001. (L) Quantitative RT-PCR analysis after treatment with siRNA for control (orange), 793

Ccna2 (red), Ccna2/Cdc25c combination (green), and Cdc25c (blue). ***, p<0.001. (M) Cell 794

proliferation assay of C2C12 cells treated with siRNA for control (orange), Ccna2 (red), Cdc25c 795

(blue), and Ccna2/Cdc25c combination (green). ***, p<0.001. (N) Ctnnb1 expression after 796

treatment with siRNA for control (opened column) and Ctnnb1 (closed column). ***, p<0.001. 797


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(O) Quantitative RT-PCR analysis of Ccna2 and Cdc25c expression after treatment with siRNA 798

for control (opened column) and Ctnnb1 (closed column). ***, p<0.001. **, p<0.01. (P) BrdU 799

staining (brown) in C2C12 cells after treatment with control or Ctnnb1 siRNA knockdown for 24 800

hours. Nuclei are counterstained with Hematoxylin. Arrows indicate BrdU positive cells. Scale 801

bar, 50 μm. (Q) Quantitation of the percentage of BrdU-labeled nuclei in C2C12 cells after 802

treatment with control or Ctnnb1 siRNA knockdown for 24 hours. N=12 per each group. ***, 803

p<0.001. 804

805

FIG 2. WNT/β-catenin signaling regulates myoblast fusion during muscle development. (A) 806

Immunohistochemical analysis of myosin heavy chain (green) in C2C12 cells during muscle 807

differentiation (Day 3 and Day 5 after induction for muscle differentiation) with IWR1-endo at 0, 808

1, or 10 μM. Negative control was continued to culture C2C12 cells in growth medium. Cells are 809

counterstained with DAPI (blue). Scale bars 100 μm. (B) The number of nuclei (x-axis of graph) 810

was measured in MYH positive cells. Vehicle control (blue bars), 1 μM IWR1-endo treatment 811

(red bars). (C) Fusion index in cells treated with vehicle control (blue bar) or 1 μM IWR1-endo 812

(red bar). ***, p<0.001. (D) Length of muscle fibers. Vehicle control (blue bar), 1 μM IWR1-813

endo (red bar). ***, p<0.001. (E) Immunoblotting analysis of activated β-catenin (ABC) and 814

phosphorylated β-catenin (P-β-catenin) after treatment with vehicle (Control) or IWR1-endo (1 815

or 10 μM) for three days. (F) Immunoblotting analysis of caspase 3 after treatment with vehicle 816

(Control) or IWR1-endo (1 or 10 μM) for three days. PC, positive control (tissue sample from 817

adult wild-type mouse small intestine). (G) C2C12 cells were cultured under differentiation 818

medium with vehicle (control) or 1 μM IWR1-endo for five days (Day 5). The cells treated with 819


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1 μM IWR1-endo for 5 days (Day 5) were continued to culture with or without 1 μM IWR1-endo 820

for another five days (Day 10). The fusion defect by the treatment with 1 μM IWR1-endo at Day 821

5 was rescued by the removal of IWR1-endo at Day 10. 822

823

FIG 3. WNT/β-catenin signaling regulates Fermt2 expression during myoblast fusion. (A) PCR 824

array analysis after treatment with vehicle control (blue bars) or 1 μM IWR1-endo (red bars) for 825

48 hours. N=5 per treatment. **, p<0.01. (B) Quantitative RT-PCR analysis of Fermt2 after 826

treatment with vehicle control (blue) or 1 μM IWR1-endo (red) for two days (Day 2) or after 827

another two-days culture after the removal of 1 μM IWR1-endo at Day 5 of muscle 828

differentiation (Day 7, green). **, p<0.01. (C) Reporter assay for WNT/β-catenin signaling 829

activity after treatment with/without (+/-) WNT3A (0.1 μg/ml) and IWR1-endo (1 μM) in C2C12 830

cells cultured under differentiation medium. (D) ChIP analysis of DNA fragments 831

immunoprecipitated with a β-catenin-specific antibody or with an isotype-specific (IgG) control 832

antibody after treatment with/without 1 μM IWR1-endo (IWR1) for 48 hours under the 833

differentiation medium. Immunoprecipitated products were PCR amplified with primers flanking 834

the putative LEF1-binding region of Fermt2. 2% input lane shows PCR amplification of the 835

sonicated chromatin before immunoprecipitation. (E) ChIP-qPCR analysis of β-catenin binding 836

on the Fermt2 promoter region using β-catenin antibody following treatment with/without 1 μM 837

IWR1-endo treatment for 48 hours under the differentiation medium. ChIP with IgG without 838

IWR1-endo treatment (orange), IgG with 1 μM IWR1-endo treatment (green), α-β-catenin 839

antibody without IWR1-endo treatment (red), and α-β-catenin antibody with 1 μM IWR1-endo 840

treatment (blue). (F) Quantitative RT-PCR analysis of gene expression of Fermt2 after treatment 841


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with WNT3A (0.1 μg/ml) for 0, 1, 2, and 3 hours (h) with/without 1 μM IWR1-endo. ***, 842

p<0.001. (G) Ctnnb1 expression after treatment with siRNA for control (opened column) and 843

Ctnnb1 (closed column). ***, p<0.001. (H) Quantitative RT-PCR analysis of Fermt2 expression 844

after treatment with siRNA for control (opened column) and Ctnnb1 (closed column). ***, 845

p<0.001. (I) Immunofluorescent images for MHC in C2C12 cells treated with siRNA for control 846

and Ctnnb1. Cells are counterstained with DAPI (blue). Scale bars 100 μm. (J) Quantitative RT-847

PCR analysis after treatment with siRNA for control (opened column) and Fermt2 (closed 848

column). ***, p<0.001. (K) Immunofluorescent images for MHC in C2C12 cells treated with 849

siRNA for control and Fermt2. Cells are counterstained with DAPI (blue). Scale bars 100 μm. 850

(L) Quantitative RT-PCR analysis after treatment with overexpression vector for control (opened 851

column) and Fermt2 (closed column). ***, p<0.001. (M) Immunofluorescent images for MHC in 852

C2C12 cells treated with overexpression vector for control and Fermt2. Cells are counterstained 853

with DAPI (blue). Scale bars 100 μm. 854

855

FIG 4. WNT/β-catenin signaling regulates homeostasis. (A) Schematic diagram of time 856

schedule. After the completion of myofiber formation (Day 0), myofibers were cultured under 857

differentiation medium with vehicle (Control) or IWR1-endo for indicated time. (B) 858

Immunohistochemical analysis of MYH (green) in the differentiated C2C12 cells treated with 859

vehicle (Control) or 1 μM IWR1-endo for indicated days (Day 0, 3, and 5). Cells are 860

counterstained with DAPI (blue). Scale bars 100 μm. (C) Quantitative RT-PCR analysis of 861

indicated molecules in the differentiated C2C12 cells treated with vehicle (Control) or 1 μM 862

IWR1-endo at Day 3. NS, not significant. (D) Immunoblotting (IB) analysis with indicated 863


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antibodies after immunoprecipitation (IP) by beads with indicated antibody of extracts from 864

C2C12 cells treated with vehicle (Control) or 1 μM IWR1-endo at Day 5. Lane 1 and 2: 2% input 865

from vehicle control (lane 1) and IWR1-endo (lane 2) treatment groups. Lane 3 and 4: IgG 866

control for vehicle control (lane 3) and IWR1-endo (lane 4) treatment group. (E) Immunoblotting 867

analysis of caspase 3 treated with vehicle control or IWR1-endo. PC, positive control (tissue 868

sample from adult wild-type small intestine). (F) Immunofluorescent analysis of Phalloidin (red) 869

or cadherin (green)/β-catenin (red) in C2C12 cells at Day 5. Cells are counterstained with DAPI 870

(blue). Top images, scale bar 25 μm; lower images, scale bar 20 μm. (G) Confocal microscopic 871

images for cadherin (green) and β-catenin (red) in C2C12 cells at Day 5. Cells are counterstained 872

with DAPI (blue). Scale bar 25 μm. (H) Quantification of colocalized dots. Colocalized dots 873

(yellow) per total dots (green or red) on the plasma membrane. ***, p<0.001. 874

875

FIG 5. WNT/β-catenin signaling mechanism in primary myoblasts. (A) BrdU staining (brown) 876

in primary myoblasts from the limb after treatment with vehicle (control) or IWR1-endo for 24 877

hours. Nuclei are counterstained with Hematoxylin. Arrows indicate BrdU positive nuclei. Scale 878

bar, 50 μm. (B) BrdU staining (brown) in primary myoblasts from the tongue after treatment 879

with vehicle (control) or IWR1-endo for 24 hours. Nuclei are counterstained with Hematoxylin. 880

Arrows indicate BrdU positive nuclei. Scale bar, 50 μm. (C) Quantitation of the percentage of 881

BrdU-labeled nuclei in primary myoblasts derived from the limb or the tongue treated with 882

vehicle (blue bars) or IWR1-endo (red bars) for 24 hours. N=6 per each treatment. ***, p<0.001. 883

(D) Quantitative RT-PCR analysis of Ccna2 and Cdc25c in primary myoblasts from the limb (L) 884

or the tongue (T) after treatment with vehicle (blue bars) or IWR1-endo (red bars) for 24 hours. 885


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N=6 per each treatment. *, p<0.05; **, p<0.01. (E) Fusion index of primary muscle cells from 886

the limb or the tongue after myogenic induction with vehicle (blue bars) or IWR1-endo (red 887

bars) for three days. ***, p<0.001. (F) Quantitative RT-PCR analysis of Fermt2 in primary cells 888

from the limb or the tongue after treatment with vehicle (blue bars) or IWR1-endo (red bars) for 889

three days. ***, p<0.001. (G) Immunofluorescent analysis of MYH (green) in primary muscle 890

cells from the limb (upper panels) or the tongue (lower panels) cultured under the differentiation 891

medium with vehicle or IWR1-endo for five days. Cells are counterstained with DAPI (blue). 892

Scale bars 100 μm. (H) Immunofluorescent analysis of MYH (green) in primary muscle cells 893

from the tongue after well-differentiated into myofibers. The well-differentiated cells (Day 0) 894

were treated with vehicle (control) or 1 μM IWR1-endo for seven days (Day 7) under the 895

differentiation medium. Cells are counterstained with DAPI (blue). Scale bars 100 μm. 896

897

FIG 6. Conserved WNT/β-catenin signaling mechanisms in tongue explants. (A) BrdU staining 898

(brown) in tongue explants from C57B6/J mice after treatment with vehicle (control) or 1 μM 899

IWR1-endo for 24 hours. Nuclei are counterstained with Methylene Blue. Arrows indicate BrdU 900

positive nuclei. Scale bar, 50 μm. (B) Quantitation of the percentage of BrdU-labeled nuclei in 901

tongue explants treated with vehicle (blue bars) or 1 μM IWR1-endo (red bars) for 24 hours. 902

N=6 per each treatment. ***, p<0.001. (C) Immunohistological analysis of MYH (green) in 903

tongue explants cultured with vehicle or 10 μM IWR1-endo for five days. Cells are 904

counterstained with DAPI (blue). Scale bar, 50 μm. (D) Model of WNT/β-catenin signaling 905

during myogenesis. The schematic diagram depicts our model of the mechanism of WNT/β-906

catenin signaling pathway in muscle cells. WNT/β-catenin signaling regulates gene expression of 907


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Ccna2 and Cdc25c in the cell proliferation stage. WNT/β-catenin signaling regulates gene 908

expression of Fermt2 in the differentiation stage. Βeta-catenin forms a complex with cadherin 909

and actin to elongate the myofiber. Disruption of the complex formation results in disorganized 910

myofiber morphology. 911

912

Table 1. Differentially expressed genes related to cell proliferation based on PCR array analysis 913

in C2C12 cells treated with vehicle control or IWR1-endo for 24 hours. Table lists cell cycle 914

regulators altered with 1 μM IWR1-endo treatment compared to controls expressed as a ratio. *, 915

p<0.05; **, p<0.01; ***, p<0.001. Ratio based on the geometric means of expression scores 916

derived from control and treatment groups. All relevant transcripts with a ≥1.5-fold change with 917

p<0.05 are shown. 918

919

Table 2. Distribution of potential LEF1 recognition sequences in promoters of selected genes of 920

interest. Sequences 5-kbp upstream of transcription start sites of indicated genes were analyzed. 921

The number of minimal (CTTTG) and optimal (CTTTGWW) binding sites of indicated genes 922

are given. Note that the optimal binding sites are a subset of the minimal core sites. The number 923

of phylogenetically conserved sites in at least six mammals is given in the right column. 924

925

Table 3. Evolutionary conserved LEF1 recognition sites within the promoters of Ccna2, Cdc25c, 926

and Fermt2. Binding site motif is underlined in all cases. 927


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