Wnt7a-induced cell migration requires Dvl2 and Rac1

Our previous work revealed a requirement of Rac1 for Wnt7a signaling in satellite cells (Bentzinger et al., 2013b). We observed that polarized peripheral Fzd7-tdTomato colocalized with GFP-Rac1 in C2C12 cells (Fig. 3 A). This suggested that the small GTPase Rac1 might also be involved in Wnt7a-mediated cell polarization and migration. In agreement with this hypothesis, overexpression of Wnt7a significantly increased the activation of Rac1 (Fig. 3 B and Fig. S3 A). Many forms of Wnt signaling require Disheveled (Dvl) proteins. At the protein level, Dvl2 is the most expressed Dvl in mouse primary myoblasts (unpublished data). siRNA SMARTpool-mediated knockdown of Dvl2 (siDvl2) from Wnt7a-stimulated cells significantly reduced the activation of Rac1 when compared with the scrambled siRNA (siSCR) control (Fig. 3 B and Fig. S3, A and B). Dvl has been suggested to serve as a scaffolding protein for downstream Wnt signaling effectors. Indeed, we observed an increased Rac1 association with Dvl2 in Wnt7a-stimulated cells (Fig. 3 C and Fig. S3 C).

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Figure 3.

Dvl2 and Rac1 are required for Wnt7a-induced cell migration. (A) Fzd7-tdTomato colocalizes with GFP-Rac1 (arrowhead) in the periphery of C2C12 cells but not in cytoplasmic vesicles. Bar, 5 µm. (B) Rac1 activation assay of mouse primary myoblasts transduced with Wnt7a-HA (Wnt7a) retrovirus or an empty control virus (EV). In addition, all cells were either treated with an siRNA SMARTpool targeting Dvl2 (siDvl2) or with a scrambled control (siSCR). Total Rac1 is shown as a loading control. (C) Coimmunoprecipitation of Rac1 with Dvl2 in primary myoblasts that were infected with Wnt7a or EV. More Rac1 associates with Dvl2 in Wnt7a-expressing cells. (D) Scratch assay with mouse primary myoblasts that were Wnt7a or vehicle treated. The cells were also transfected with either siSCR or siDvl2. Error bars represent means ± SEM; n = 3. *, P < 0.05. n.s., no significant difference. (E) Scratch assay with mouse primary myoblasts that overexpress EV or Fzd7-Flag and that were treated with siDvl2 or siSCR. Error bars represent means ± SEM; n = 3. *, P < 0.05. (F) Dominant-negative Rac1 (Rac1-DN) prevents Wnt7a-induced mouse primary myoblast migration in scratch assays. Error bars represent means ± SEM; n = 3. **, P < 0.01. (G) Rac1-DN prevents Fzd7-Flag–induced mouse primary myoblast migration in scratch assays. Error bars represent means ± SEM; n = 3. *, P < 0.05.

As predicted by these results, siDvl2 prevented both Wnt7a- and Fzd7-Flag overexpression–mediated migration of mouse primary myoblasts in scratch assays (Fig. 3, D and E). Moreover, the expression of dominant-negative Rac1-T17N (Rac1-DN) also antagonized Wnt7a- and Fzd7-mediated migration in primary mouse myoblasts (Fig. 3, F and G). Our biochemical analysis revealed no significant activation of other small GTPases such as Cdc42 and RhoA (unpublished data). Nevertheless, both dominant-negative Cdc42-T17N (Cdc42-DN) and RhoA-T19N (RhoA-DN) were able to prevent Wnt7a-induced cell migration (Fig. S3 D). Thus, we conclude that Dvl2 and the small GTPases Rac1, Cdc42, and RhoA are essential for the induction of cell motility by Wnt7a.

Wnt7a signaling in myoblasts is strictly noncanonical

Wnt7a at a concentration of 50 ng/ml does not activate β-catenin in satellite cells, primary myoblasts (Le Grand et al., 2009), or muscle myofibers (von Maltzahn et al., 2011). Moreover, ectopic expression of Wnt7a does not induce the TOP-flash luciferase reporter (Molenaar et al., 1996) in C2C12 myoblasts (Kuroda et al., 2013). To further clarify the signaling mechanisms downstream of Wnt7a and to exclude a dose-dependent effect on β-catenin, we tested different concentrations of this factor for its ability to induce TOP-flash activity in C2C12 cells. Although the canonical Wnt3a showed a dose-dependent response (Fig. 4 A), increasing concentrations of Wnt7a had no effect on TOP-flash reporter activity (Fig. 4 B). Collectively, these data support the notion that Wnt7a signaling in all cells of the adult muscle lineage is strictly noncanonical.

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Figure 4.

The Fzd7/Wnt7a signal is strictly noncanonical. (A) Wnt3a induces a dose-dependent increase in TOP-flash luciferase reporter activity in C2C12 cells. Error bars represent means ± SEM; n = 3. *, P < 0.05. n.s., no significant difference. (B) Wnt7a does not activate the TOP-flash reporter at any tested concentration. Error bars represent means ± SEM; n = 3. (C) Wnt7a treatment decreases the abundance of peripheral Fzd7-YFP and leads to its accumulation in intracellular clusters (arrowheads). Bar, 5 µm. (D) Inhibition of clathrin-dependent endocytosis with monodansylcadaverine (MDC) prevents Wnt7a-induced migration of primary myoblasts in scratch assays. Error bars represent means ± SEM; n = 3. ***, P < 0.001. (E) Substantial amounts of Wnt7a-HA are present in intracellular stores (arrowhead) 72 h after a 3-h exposure to conditioned supernatants produced in COS-1 cells (top). In primary myoblasts from Fzd7^−/− mice, Wnt7a does not show such intracellular accumulation (bottom). Bars, 2 µm.

Endocytosis is required for Wnt7a-induced cell migration

The activation of Rac1 by noncanonical Wnt signals has been shown to depend on clathrin-mediated endocytosis (Sato et al., 2010). Therefore, we set out to test whether Wnt7a treatment could alter the subcellular distribution of Fzd7. Indeed, Wnt7a-stimulated primary myoblasts displayed a higher abundance of large intracellular aggregates and reduced peripheral Fzd7-YFP (Fig. 4 C). In agreement with this observation, treatment with monodansylcadaverine, an inhibitor of clathrin-mediated endocytosis (Schlegel et al., 1982), prevented Wnt7a-mediated migration in scratch assays (Fig. 4 D). Importantly, we observed that myoblasts that were exposed to conditioned medium containing HA epitope-tagged Wnt7a (Wnt7a-HA) quickly endocytosed this factor. After 3 h of incubation and several subsequent washing steps, endocytosed Wnt7a-HA was detectable for ≥72 h in intracellular structures in primary myoblasts (Fig. 4 E) and C2C12 cells (Fig. S3 E). No intracellular Wnt7a-HA could be observed in Fzd7^−/− myoblasts exposed to the conditioned medium (Fig. 4 E). These observations demonstrate that both Fzd7 and Wnt7a endocytosis are interdependent and require activation of noncanonical signaling in myoblasts. Moreover, the endocytosis of Wnt7a was surprisingly fast and the protein appeared to be present in intracellular stores for prolonged periods. Taken together, these observations suggest that a short exposure to Wnt7a has sustained effects on myogenic cells.

Wnt7a treatment facilitates the migration of primary myoblasts in vivo

We next set out to investigate whether loading of intracellular stores with Wnt7a would have long-lasting effects on cell migration. We exposed tdTomato-expressing primary myoblasts, derived from tamoxifen-treated Pax7-CreER;R26R-tdTomato mice (Yin et al., 2013a), for 3 h to Wnt7a, washed the cells, and transplanted them into the tibialis anterior muscle of immunosuppressed C57BL/6 mice (Fig. 5 A). To identify the injection site in subsequent analysis, the myoblasts were co-injected with fluorescent microspheres. 7 d later we sacrificed the mice and analyzed the behavior of the transplanted cells in the tissue by scoring the number of tdTomato-expressing myofibers that were generated by fusion events with the donor cells. Importantly, Wnt7a treatment increased the number of tdTomato-expressing myofibers by 119% (Fig. 5 B). To exclude that this effect is due to changes in the cell cycle status of the transplanted cells, we analyzed the proliferation of primary myoblasts after a 3-h exposure to various concentrations of Wnt7a. We did not observe any change in the rate of proliferation at any of the tested concentrations of Wnt7a over 5 d (Fig. S4 A). By contrast, the induction of canonical signaling with higher concentrations of Wnt3a reduced the proliferation of primary myoblasts (Fig. S4 B).

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Figure 5.

Wnt7a loading increases myoblast dispersal in muscle tissue. (A) Experimental scheme for the in vivo myoblast dispersal assay. Cells expressing tdTomato were treated with Wnt7a or vehicle for 3 h, washed, and transplanted into C57BL/6 mice. Blue fluorescent microspheres were co-injected to mark the injection site. After 7 d, the distance from the closest microsphere to myofibers expressing high (tdT+++), medium (tdT++), or low (tdT+) level of tdTomato was enumerated in muscle cross sections. (B) The total number of tdTomato-expressing myofibers generated by fusion with donor myoblasts was increased by Wnt7a treatment compared to vehicle. Error bars represent means ± SEM; n = 3. *, P < 0.05. (C) Representative images showing the abundance and fluorescent intensity of myofibers expressing tdTomato with respect to the injection site (outlined by a dashed oval). The injection site is marked by a high concentration of blue fluorescent microspheres. In the case of the cells that were treated with Wnt7a, the myofibers are more spread out and generally express reduced levels of tdTomato. Bar, 50 µm. (D–F) The minimal distance of tdT+++, tdT++, and tdT+ myofibers to the microspheres was measured in muscle cross-section according to the false-color image shown in the respective insets. Asterisks in the false-color images indicate the tdT fluorescence intensity of the fibers that were quantified. The data for the minimal distance from microspheres of the myofiber types was grouped into 200-µm bins. In the vehicle-treated condition, a large fraction of myofibers proximal to the injection site is tdT+++, whereas few distal tdT+ myofibers are present. The Wnt7a condition shows the opposite trend. Error bars represent means ± SEM; n ≥ 3. *, P < 0.05; **, P < 0.01. n.s., no significant difference.

Taken together, these data support the conclusion that the increased number of tdTomato-expressing myofibers upon transplantation of Wnt7a-treated myoblasts is not due to increased proliferation and is a consequence of enhanced dispersal. The observed increase in numbers of highly (tdT+++) tdTomato-expressing myofibers proximal to the injection site after transplantation of untreated cells (Fig. 5, C–E) relative to Wnt7a-treated cells provides strong evidence in support of this conclusion. Importantly, the myofibers generated by fusion to Wnt7a-treated cells were weaker for tdTomato (tdT+), but higher in number and more spread out with respect to the injection site (Fig. 5, C and F). Thus, Wnt7a loading results in increased migration of the cells in the host muscle and lowers the chance for multiple fusion events to the same myofiber. Taken together, our experiments suggest that short exposure of myoblasts to Wnt7a dramatically enhances engraftment by promoting the tissue dispersion of the cells.

Wnt7a treatment enhances the outcome of cell therapy of dystrophic muscle

Because poor migration of myogenic cells upon intramuscular injection is a major hurdle for the development of cell-based therapies for muscular dystrophy (Skuk et al., 2007), we investigated whether ex vivo Wnt7a treatment of satellite cells before transplantation would enhance engraftment. We isolated 10,000 satellite cells from Pax7-zsGreen mice by fluorescent-activated cell sorting (Bosnakovski et al., 2008), treated them with Wnt7a for 3 h, washed them extensively, and transplanted them into the tibialis anterior muscle of immunosuppressed dystrophin-deficient mdx mice that were injured with cardiotoxin 2 d before the procedure (Fig. 6 A).

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Figure 6.

Wnt7a-loaded satellite cells have an enhanced engraftment potential. (A) Schematic of the isolation, ex vivo treatment, and transplantation of satellite cells from Pax7-zsGreen mice. (B) Representative pictures showing engraftment of zsGreen- and Pax7-expressing donor-derived cells that were either Wnt7a or vehicle treated. Bar, 20 µm. (C) Quantification of engraftment of Wnt7a- or vehicle-treated donor satellite cells (Pax7⁺/zsGreen⁺, arrowheads in B). Error bars represent means ± SEM; n = 3. **, P < 0.01. (D) Representative images showing dystrophin-expressing myofibers (asterisks) derived from transplanted Wnt7a or vehicle-treated satellite cells. Bar, 20 µm. (E) Quantification of myofibers expressing dystrophin after transplantation of Wnt7a- or vehicle-treated satellite cells. Error bars represent means ± SEM; n = 3. **, P < 0.01. (F) Engrafted satellite cells form clusters of dystrophin-expressing myofibers in host mdx muscles. The photographs show the tibialis anterior muscle that was injected with Wnt7a- or vehicle-treated cells. The distance between the two maximally spaced clusters of dystrophin-expressing myofibers (highlighted in red) is indicated with a magenta line. (G) Quantification of the maximal cluster distance in muscles injected with Wnt7a- or vehicle-treated cells. Error bars represent means ± SEM; n ≥ 4. **, P < 0.01. (H) Minimal feret measurements of dystrophin-expressing myofibers in host muscles. Myofibers that had fused with a Wnt7a-treated cell become hypertrophic. Error bars represent means ± SEM; n = 3. *, P < 0.05. (I) Quantification of the twitch force tension of mdx muscles injected with Wnt7a- or vehicle-treated cells. Error bars represent means ± SEM; n ≥ 7. **, P < 0.01. (J) Maximal specific force generated by mdx muscles injected with Wnt7a- or vehicle-treated cells. Error bars represent means ± SEM; n ≥ 7. *, P < 0.05.

As determined by immunostaining for Pax7 and zsGreen in muscle sections, Wnt7a treatment enhanced engraftment of zsGreen-expressing cells into the recipient muscles by 69% after 3 wk (Fig. 6, B and C; and Fig. S4 C). Wnt7a treatment did not alter the proportion of zsGreen⁺-engrafted cells that express Ki67 (Fig. S4 D), indicating that Wnt7a treatment does not alter the rate of in vivo proliferation. In addition, the number of endogenous satellite cells was not altered (Fig. S4 E), showing that resident satellite cells were not being stimulated by Wnt7a derived from the transplanted cells.

Under normal conditions, a portion of transplanted satellite cells will differentiate and fuse to muscle myofibers. In mdx mice, this process can be tracked by staining for restored dystrophin expression in muscle fibers. Using this assay, we observed that the number of myofibers expressing dystrophin increased on average by 486% after transplantation of Wnt7a-treated cells (Fig. 6, D and E). Demonstrating a stimulatory effect of Wnt7a on myogenic cell migration in vitro, clusters of dystrophin-expressing myofibers in muscles that were transplanted with Wnt7a-treated cells were on average located maximally 3.7 mm apart, while transplantation of untreated cells gave rise to clusters of dystrophin-expressing myofibers that were on maximally 2.2 mm apart on average (Fig. 6, F and G). We also observed that dystrophin-expressing myofibers arising after fusion with Wnt7a-treated cells were hypertrophic by 116% (Fig. 6, D and H). Importantly, transplantation of 3,000 satellite cells treated ex vivo with Wnt7a into mdx extensor digitorum longus muscles resulted in a striking 30% increase in twitch tension and in an 18% increase in maximal specific force generation when compared with the vehicle control (Fig. 6, I and J).

In a clinical setting, freshly isolated satellite cells are often not readily available. Therefore, we next tested the effect of 3 h Wnt7a loading on cultured primary mouse myoblasts that were subsequently transplanted into injured muscles of immunosuppressed mdx mice (Fig. S5 A). After transplantation of 100,000 cells, the number of dystrophin-expressing myofibers was increased by 72% in muscles that were injected with Wnt7a-treated myoblasts (Fig. S5 B). Moreover, dystrophin-expressing myofibers derived from Wnt7a-treated cells exhibited an 87% hypertrophy (Fig. S5 C). In addition, the mean maximal cluster distance increased from 1.5 mm for vehicle to 2.0 mm for Wnt7a (Fig. S5 D).

To ascertain that the improvement of engraftment efficiency is a unique property of Wnt7a, we also tested Wnt3a and Wnt5a loading of myoblasts before transplantation. Neither the number of dystrophin-expressing myofibers (Fig. S5 E) nor the mean maximal cluster distance (Fig. S5 F) was changed by these treatments. Taken together, these observations indicate that Wnt7a treatment can significantly enhance the outcome of cell therapy of skeletal muscle. Transplantation of dystrophic muscle with Wnt7a-loaded cells leads to an enhanced engraftment, enhanced tissue distribution of donor-derived myofibers, and improved force generation.

Transplantation

Transplantation of zsGreen-expressing satellite cells or myoblasts into mdx mice was performed as described previously (Bentzinger et al., 2013b). In brief, cells were resuspended in 0.9% NaCl and transplanted into the muscles of FK506-immunosupressed mice that had been injured 2 d before. Before transplantation the cells were treated for 3 h with Wnt7a, Wnt5a, or Wnt3a (R&D Systems) at a concentration of 50 ng/ml for freshly isolated satellite cells or with 100 ng/ml for cultured cells. Myoblasts expressing tdTomato were transplanted into uninjured muscles of C57BL/6 mice that were implanted with an osmotic pump (Alzet) delivering FK506 (Sigma-Aldrich) at 2.5mg/kg/day 3 d before the transplantations. In some experiments the injection site was marked with blue fluorescent microspheres (2 µm; Life Technologies) that were added at a concentration of 1:100 (vol/vol) to the cell suspension. 10 µl of the cell-microsphere suspension was injected into the tibialis anterior of each mouse using a syringe (Hamilton Company).

Muscle force measurements

Extensor digitorum longus muscles were isolated three weeks after transplantation and attached to an electrode and a force sensor (Aurora Scientific), and incubated at 25°C in buffered physiological saline (Krebs-Ringer) supplemented with glucose and oxygen. Twitch force was determined after a single electrical stimulus, whereas maximal force was generated by electrical stimulation at 100 Hz for 500 ms. Thereafter, muscle length and weight were measured and specific muscle force was calculated ([maximal force × optimal fiber length × muscle density]/muscle mass).

Live imaging

Single-cell imaging of cells transfected with Fzd7-YFP or YFP was performed on a microscope (Axio Observer.Z1; Carl Zeiss) equipped with a confocal laser scanner (LSM510 META; Carl Zeiss) and a Plan Apochromat 63×/1.40 NA oil DIC M27 objective (Carl Zeiss) under 5% CO₂ at 37°C in phenol-red free DMEM (Life Technologies) containing 10% FBS. Image acquisition was performed using Zen software (Carl Zeiss). Imaging of satellite cells on fibers was performed as described previously (Siegel et al., 2009). In brief, time-lapse microscopy of satellite cells on single collagen-embedded muscle fibers was performed using an inverted microscope (DMI 5100; Leica) and MetaMorph 7.6.1 software (Molecular Devices) with time points being acquired every 7 min in a stage-top incubator (LiveCell Imaging). ImageJ (National Institutes of Health) analyzed the time-lapse data to calculate the velocity, defined as distance (µm)/time (h), between time points, and the average velocity of each cell was calculated. The maximum velocity is defined as the highest velocity obtained by a single cell during the tracking period. For live imaging of satellite cells on single fibers, Wnt7a was used at a concentration of 1.5 µg/ml.

Isolation of satellite cells and culture of primary myoblast

For myoblast culture and transplantation, satellite cells were FACS purified from Pax7-zsGreen mice through gating for zsGreen and Hoechst using a cell sorter (MoFlo XDP, Beckman Coulter; Bentzinger et al., 2013b). To derive primary myoblasts, the cells were then plated on collagen-coated dishes (BD) in Ham’s F10 medium supplemented with 20% FBS and 5 ng/ml of basic FGF (EMD Millipore). C2C12 cells and human myoblasts were cultured in DMEM supplemented with 10% FBS (von Maltzahn et al., 2012). For in vitro morphology quantifications, Wnt7a was used at a concentration of 50 ng/ml.

Scratch assays

After plasmid transfection or application of Wnts (R&D Systems), the cells were treated with 50 µg/ml mitomycin-C for 2 h. Subsequently, the monolayer of cells was scraped in a straight line. The plates were then extensively washed with culture medium and incubated for 24 h before analysis. Analysis was performed using DAPI staining after matching the reference points, and enumeration of DAPI-stained nuclei in the scar was performed. Unless indicated otherwise, Wnt7a and Wnt3a were used at a concentration of 100 ng/ml in scratch assays. Monodansylcadaverine (30432; Sigma-Aldrich) was used in scratch assays at a concentration of 50 µm.

Western blotting and immunoprecipitation

For coimmunoprecipitation (CoIP) experiments and the Rac1 activation assay, satellite cell–derived primary myoblasts were infected with retroviruses generated from empty pHAN or from pHAN-Wnt7a-HA (Kuroda et al., 2013). Dvl2 knockdown was performed with an ON-TARGETplus siRNA SMARTpool (L-040921-01, Thermo Fisher Scientific; Wu et al., 2008) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. As a control for the knockdown, a nonsilencing siRNA SMARTpool was used (D-001810-10; Thermo Fisher Scientific). Cell extracts were obtained through RIPA buffer lysis in the presence of a protease inhibitor cocktail (Nacalai Tesque). For CoIP of Rac1 with Dvl2, rabbit anti-Dvl2 antibody was coupled to protein A–Dynabeads (Life Technologies). Denaturing SDS-PAGE was performed using standard techniques. Rac1 activation assay was performed according to the manufacturer’s instructions (Termo Fisher Scientific).

Immunostaining and antibodies

Muscles frozen in liquid nitrogen were cut into 12-µm cross sections. Cross sections and cells were either fixed with 100% ethanol or 2% PFA for 5 min, permeabilized with 0.1% Triton X-100/0.1 M glycine/PBS for 10 min, blocked with 5% horse serum in PBS for several hours, and incubated with specific primary antibody in blocking buffer overnight at 4°C. Samples were subsequently washed with PBS and stained with appropriate fluorescently labeled secondary antibodies for 1 h at room temperature. After washing with PBS, samples were mounted with Permafluor (Thermo Fisher Scientific). Antibodies were as follows: chicken anti-GFP (ab13970; Abcam), mouse anti-Pax7 (Developmental Studies Hybridoma Bank [DSHB]), mouse anti-dystrophin (7A10; DSHB), mouse anti-Rac1 (05-389; EMD Millipore), mouse anti-tubulin (T9026; Sigma-Aldrich), rabbit anti-Dvl2 (3224; Cell Signaling Technology), rabbit anti-zsGreen (632474; Takara Bio Inc.), and rabbit anti-laminin (L9393; Sigma-Aldrich), rabbit anti-HA (07-221; EMD Millipore). Fluorochromes coupled to secondary antibodies were Alexa Fluor 488, 546, and 647. Images of immunostainings were taken at room temperature on a microscope (Axio Observer.Z1; Carl Zeiss) equipped with a confocal laser scanner (LSM510 META; Carl Zeiss) and an EC Plan-Neofluar 20×/0.50 NA M27, EC Plan-Neofluar 40×/1.30 NA oil DIC M27, and Plan Apochromat 63×/1.40 NA oil DIC M27 objective (all from Carl Zeiss).

Real-time PCR

Total RNA was isolated (NucleoSpin RNA II; Macherey-Nagel). Reverse transcription was performed using a mixture of oligodT and random hexamer primers (iScript cDNA Synthesis kit; Bio-Rad Laboratories). SYBR Green real-time PCR analysis (iQ SYBR Supermix; Bio-Rad Laboratories) was performed using a real-time thermocycler (Mx300P; Agilent Technologies). The following primers were used: Dvl2 sense: 5′-ACGACGATGCTGTACGAGTG-3′; Dvl2 anti-sense: 5′-CGAGGGAGGGTGAAGTAGG-3′; Fzd7 sense: 5′-GCTTCCTAGGTGAGCGTGAC-3′; and Fzd7 anti-sense: 5′-AACCCGACAGGAAGATGATG-3′.

Plasmids and transfection

The UBC-Fzd7-Flag (Fzd7-Flag; backbone: pLenti-III-UBC), CMV-Fzd7-EYFP (Fzd7-YFP; backbone: pEYFP-N1), CMV-Fzd3-EYFP (Fzd3-YFP; backbone: pEYFP-N1), CMV-EYFP (YFP; backbone: pEYFP-N1), CMV-EGFP-Rac1-wt (GFP-Rac1, 12980; Addgene; backbone: pcDNA3-EGFP), CMV-EGFP-Rac1-T17N (Rac1-DN, 12982; Addgene; backbone: pcDNA3-EGFP), CMV-EGFP-RhoA-T19N (RhoA-DN, 12967; Addgene; backbone: pcDNA3-EGFP), and CMV-EGFP-Cdc42-T17N (Cdc42-DN, 12976; Addgene; backbone: pcDNA3-EGFP) constructs have been described previously (Subauste et al., 2000; von Maltzahn et al., 2011; Bentzinger et al., 2013a). The Fzd7-tdtomato plasmid was generated by replacing the C-terminal YFP in Fzd7-YFP with tdTomato. For TOP-flash and FOP-flash the TCF reporter plasmid kit (17-285; EMD Millipore) was used with the dual-luciferase reporter assay system (E1960; Promega). For normalization, pGL4.74[hRluc/TK] (E6921; Promega) was cotransfected. For TOP-flash and FOP-flash assays the cells were transfected with the GenJet Lipofection reagent (SL100499; SignaGen). Otherwise, Lipofectamine 2000 (11668019; Life Technologies) was used for all transfections.

Statistical analysis

Densitometry of gray values from Western blots was performed with ImageJ software. Compiled data are expressed as the mean ± SEM. Experiments were done with a minimum of three biological replicates. For statistical comparison of two conditions, the Student’s t test was used. The level of significance is indicated as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

We thank J. Nathans for providing us with the Fzd7 knockout mice and D. Wilson for technical assistance.

C.F. Bentzinger is supported by a grant from the Swiss National Science Foundation. Y.X. Wang is supported by fellowships from QEII-GSST and the Canadian Institutes of Health Research. N.A. Dumont is supported by Canadian Institutes of Health Research. This work was supported by grants to M.A. Rudnicki from the Canadian Institutes for Health Research, the Muscular Dystrophy Association, the National Institutes of Health, the Canadian Stem Cell Network, the Ontario Ministry of Research and Innovation, and the Canada Research Chair Program; and to D.D.W. Cornelison from the National Institutes of Health. M.A. Rudnicki is a founding scientist with Fate Therapeutics, who are developing Wnt7a as a therapeutic agent.

The remaining authors declare no competing financial interests.