Ror2 signaling regulated by differential Wnt proteins determines pathological fate of muscle mesenchymal progenitors

Figures

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  Fig. 1. Ror2 is involved in regulating MP proliferation, differentiation, or cellular senescence. a Sorted satellite cells (SCs), mesenchymal progenitors (MPs), and unsorted cells (UCs) were obtained from (intact) skeletal muscles using FACS. Expression of Pax7, Ror1, Pdgfrα, and Ror2 mRNAs in SCs, MPs, and UCs was measured using quantitative RT-PCR analysis (n = 3). b Representative images of MPs at 3 days after transfection with the respective siRNAs, followed by staining with anti-Ki-67 antibody (magenta) and DAPI (cyan). Scale bar: 100 μm. Right graph shows proportion of Ki-67-positive MPs (n = 3). c MPs, transfected with the respective siRNAs, followed by induction of adipogenic differentiation, were visualized using Oil Red O staining. Scale bar: 100 μm. Right graph shows relative absorption of Oil Red O at 6 days after induction of adipogenic differentiation (n = 3). d Representative images of MPs at 3 days after transfection with the respective siRNAs, followed by staining with SPiDER-βGal (magenta) and DAPI (cyan). Scale bar: 50 μm. Right graph shows proportion of SPiDER-βGal-positive cells (n = 3). Data in bar graphs are expressed as mean ± SD (*p < 0.05, **p < 0.01, ***p <…

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  Fig. 2. Ror2, expressed selectively in MPs, plays a crucial role in intramuscular adipose tissue (IMAT) accumulation. a Experimental design to analyze whether Ror2 inhibits cellular senescence of MPs in the skeletal muscles obtained from either control or Ror2 cKO mice after intraperitoneal injection of tamoxifen. b Expression of Ror2 in MPs isolated (sorted) from the skeletal muscles of either control or Ror2 cKO mice was monitored using quantitative RT-PCR analysis (n = 3). c Fluorescence intensity of SPiDER-βGal in MPs sorted from the skeletal muscles of either control or Ror2 cKO mice was monitored by flow cytometric analysis. Right graph shows relative mean fluorescence intensity of SPiDER-βGal in MPs isolated from the skeletal muscles of either control or Ror2 cKO mice (control: n = 5, Ror2 cKO: n = 8). d Experimental design to analyze the role of Ror2 in IMAT accumulation. Control and Ror2 cKO mice were injected with either cardiotoxin (Ctx) or glycerol in the TA muscles after intermittent intraperitoneal injection of tamoxifen as indicated. Two weeks later, CSA of myofiber and accumulation of IMAT in the TA muscles were analyzed. Sections of the TA muscles, from either control or…

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  Fig. 3. Wnt5b-Ror2 signaling promotes MP proliferation in glycerol-injected skeletal muscles. Expression of Wnt5a and Wnt5b in the TA muscles from untreated (day 0), Ctx (C)- or glycerol (G)-injected mice at the indicated time points (a) was analyzed using quantitative RT-PCR (b, n = 3). c Cos7 cells were transfected with the indicated plasmids. Subsequently, whole-cell lysates or anti-HA immunoprecipitates were prepared and subjected to western blotting (refer to Materials and Methods). d MPs, transfected with the indicated siRNAs, followed by stimulation with Wnt5a (200 ng/ml), Wnt5b (200 ng/ml), or their vehicle (0.1% BSA, designated as (-)) were stained with EdU or an anti-Ki-67 antibody. The proportion of EdU-positive (left) or Ki-67-positive (right) cells was quantified (n = 3). e Protein expression of Ror2, αTubulin, phosphorylated p38 (p-p38), and p38 proteins in MPs, transfected with the indicated siRNAs, and stimulated with either Wnt5b (200 ng/ml) or vehicle for 1 h. Right graphs show the relative band intensity of Ror2 and p-p38 normalized using αTubulin and p38, respectively (n = 5). f Proliferation of MPs stimulated with Wnt5b (200 ng/ml), in the presence or absence of a…

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  Fig. 4. Wnt11 is involved in regulating MP proliferation, differentiation, or cellular senescence. a Expression of Wnt11 was projected on t-SNE plots obtained from Tabula Muris. Colors span a gradient from blue (high expression) to gray (low expression). b Violin plot of Wnt11 expressed in various cell ontology classes within limb muscles, as observed in the Tabula Muris dataset. c Cos7 cells were transfected with the indicated plasmids. Subsequently, whole-cell lysates or anti-Flag immunoprecipitates were prepared and subjected to western blotting. d Expression of Wnt11 in isolated MPs treated with the indicated siRNAs was assessed using quantitative RT-PCR analysis (n = 3). e Proliferation of MPs, transfected with the indicated siRNAs, was quantified using the WST-8 assay at day 0 and day 4 post-transfection (n = 3). f MPs, transfected with the indicated siRNAs, were visualized using Oil Red O staining at 6 days after induction of adipogenic differentiation (n = 3). g Fluorescence intensity of SPiDER-βGal in MPs treated with the indicated siRNAs was monitored by flow cytometric analysis (n = 3). h MPs, which had been transfected with the specified siRNAs, were co-cultured indirectly…

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  Fig. 5. Wnt11-Ror2 signaling suppresses cellular senescence of MPs by maintaining nuclear membrane integrity. a Representative images of MPs, taken 2 days after transfection with the indicated siRNAs, were obtained following staining with Phalloidin (green) and DAPI (magenta). Scale bar: 25 μm. Lower graphs show proportion of cells with stress fiber (n = 3). b Representative images of MPs, taken at 3 days after transfection with the indicated siRNAs, were obtained following staining with an anti-lamin B1 antibody (red) and DAPI (blue). Scale bar: 10 μm. Lower graphs show proportion of cells with blebbed nuclear membrane (n = 3). c MPs, transfected with the indicated siRNAs, followed by treatment with cytochalasin D, were stained with an anti-lamin B1 antibody (magenta) and DAPI (cyan). Scale bar: 10 μm. Right graph shows proportion of cells with blebbed nuclear membrane (n = 3). d MPs, transfected with the indicated siRNAs, followed by treatment with cytochalasin D, were visualized using SPiDER-βGal. Fluorescence intensity of SPiDER-βGal in MPs was monitored by flow cytometric analysis. The graph shows relative mean fluorescence intensity of SPiDER-βGal (n = 3). e Representative images…

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  Fig. 6. Co-stimulation with Wnt5b and Wnt11 induces IMAT accumulation in Ctx-injected skeletal muscles. a Proliferation of MPs, treated with respective siRNAs in the absence or presence of Wnt5b (200 ng/ml), was evaluated using WST-8 assay at the indicated time points (n = 3). b Cos7 cells were transfected with the indicated plasmids. Subsequently, whole-cell lysates (WCL) or anti-HA immunoprecipitates were prepared and subjected to western blotting. The graphs on the right show the relative band intensity of Wnt5b and Wnt11 in anti-HA immunoprecipitates, which were normalized by the corresponding levels of Wnt5b and Wnt11 in WCL (n = 5). c Cos7 cells were transfected with the respective plasmids at the indicated amounts. The amount of the plasmids transfected was adjusted to 9 μg (left) or 11 μg (right) in total with empty (mock) plasmid. Subsequently, WCL or anti-HA immunoprecipitates were prepared and subjected to western blotting. The representative data from one of two independent experiments are shown. The asterisks indicate nonspecific bands (Immunoglobulin heavy chain of antibodies), and triangles show the Wnt11-Flag bands. d, e Proportion of Ki-67-positive MPs in TA muscles…