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First published online 8 April 2008
doi: 10.1242/jcs.024885

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β-catenin promotes self-renewal of skeletal-muscle satellite cells

¹ King's College London, Randall Division of Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, London, SE1 1UL, UK
² Department of Medical Sciences, University of Piemonte Orientale "Amedeo Avogadro", Novara, Italy

View larger version (70K): [in this window] [in a new window]   Fig. 1. Satellite cells express β-catenin. (a-f) Immunostaining of EDL myofibres immediately after isolation (a) showed that some Pax7+ satellite cells (green) expressed nuclear β-catenin (red). Similarly, once activated (T24), β-catenin could be found in the nucleus of some satellite cells (b). After the first division (T48) β-catenin was localised in the nucleus (c) and/or at the cell surface (d). When satellite cells began to differentiate or self-renew (T72), fewer were found with nuclear localised β-catenin (e,f). (g-i) To test the retroviral vectors, satellite cells were infected with either (g) pMSCV–β-catenin–IRES–eGFP or (h) pMSCV–ST-β-catenin–IRES–eGFP. Immunostaining for eGFP (green) and β-catenin (red) revealed that infected satellite cells robustly co-expressed these proteins, compared to control (pMSCV-IRES-eGFP) (i). Nuclei were counterstained with DAPI.

View larger version (60K): [in this window] [in a new window]   Fig. 2. β-catenin drives self-renewal in satellite cells. (a-i) Cultured myofibres were exposed to retrovirus and immunostained 48 hours later. Most proliferating satellite cells infected with (a) pMSCV–β-catenin–IRES–eGFP (β-catenin-RV) or (b) pMSCV–ST-β-catenin–IRES–eGFP (ST-β-catenin-RV) co-expressed eGFP (green) and Pax7 (red), which was in contrast to (c) pMSCV-IRES-eGFP-infected (Control-RV) cells. By contrast, there were fewer satellite cell progeny co-expressing wild-type or stabilised β-catenin (eGFP–green) with MyoD (red in d,e) and myogenin (red in g,h) compared with controls (f,i). Nuclei were counterstained with DAPI (blue). (j) Quantification of experiments shown in a-i. Values are the mean ± s.e.m. from three mice. *P<0.05, significantly different from control cultures.

View larger version (69K): [in this window] [in a new window]   Fig. 3. Constitutively expressed β-catenin inhibits myogenic differentiation and enhances self-renewal of satellite cells. (a-l) Plated satellite-cell-derived myoblasts were infected, switched to low-mitogen medium 24 hours later, cultured for at least another 4 days, fixed and immunostained. Only few satellite cells with retrovirally encoded wild-type or stabilised β-catenin (eGFP+, green) contained MyoD (a,b; red), myogenin (d,e; red) or MyHC protein (g,h; red), in contrast to control-infected cultures, which contained many multinucleated myotubes immunostaining for (c) MyoD, (f) myogenin and (i) MyHC. By contrast, Pax7 protein (red) was present in many satellite cells infected with (j) pMSCV–β-catenin–IRES–eGFP (catenin-RV) or (k) pMSCV–ST-β-catenin–IRES–eGFP (ST-β-catenin-RV), whereas (l) control-infected (pMSCV-IRES-eGFP) satellite cells only had the occasional unfused cell staining for Pax7. Nuclei were counterstained with DAPI (blue). (m) Quantification of experiments shown in a-l. Values are population mean ± s.e.m. from at least ten random fields. *P<0.05, significantly different from control cultures.

View larger version (93K): [in this window] [in a new window]   Fig. 4. β-catenin increases the proportion of C2 reserve cells. When C2 cells are induced to differentiate by culture in low-mitogen medium, most respond by differentiating, but some down-regulate MyoD, express Pax7 and exit the cell cycle, entering a quiescent-like state to become reserve cells (Yoshida et al., 1998; Olguin and Olwin, 2004). (a-l) Infection of proliferating C2 cells with retrovirus encoding (a) wild-type (β-catenin-RV) or (b) stabilised β-catenin (ST-β-catenin-RV) and subsequent culture in low-mitogen medium resulted in many infected eGFP+ (green) cells with Pax7 (red). Cells infected with (c) control pMSCV-IRES-eGFP, however, mostly responded by differentiating and fusing into large multi-nucleated myotubes; and Pax7 was restricted to the occasional reserve cell. As with primary satellite cells, most C2 cells that constitutively expressed β-catenin (eGFP, green) did not contain MyoD (d,e; red), myogenin (g,h; red) or MyHC protein (j,k; red). Control-infected cells formed myotubes that showed robust (f) MyoD, (i) myogenin and (l) MyHC expression. Nuclei were counterstained with DAPI (blue). (m) Quantification of experiments shown in a-l. Values are mean ± s.e.m. from at least ten random fields. *P<0.05, significantly different from control cultures.

View larger version (14K): [in this window] [in a new window]   Fig. 5. β-catenin perturbs cell cycle progression in myogenic cells. The graphs show the quantification of the following experiments, and represent the mean ± s.e.m. of infected cells (satellite cells) or from at least ten random fields (plated satellite cells or C2 cells), all from three independent experiments. *P<0.05, significantly different from control cultures. Myofibre-associated satellite cells, plated satellite-cell-derived myoblasts or C2 cells were infected with pMSCV–β-catenin–IRES–eGFP, pMSCV–ST-β-catenin–IRES–eGFP or pMSCV-IRES-eGFP. In myofibre-associated satellite-cell cultures, BrdU was added after 72 hours of culture for 3 hours, and cells were then fixed and immunostained. BrdU incorporation was significantly reduced by constitutively expressed β-catenin expression. For plated satellite cells and C2 cells, infection was followed the next day by culture in mitogen-poor medium for at least another 4 days before the BrdU pulse. The presence of β-catenin further reduced the number of cells able to incorporate BrdU.

View larger version (48K): [in this window] [in a new window]   Fig. 6. When degradation of endogenous β-catenin is inhibited satellite-cell self-renewal is promoted. (a,b,d,e) Myofibres were cultured in plating medium containing the GSK3β inhibitor SB216763 for 3 days, then fixed and immunostained. Inhibition of β-catenin phosphorylation and, therefore, degradation, significantly increased the number of (a) Pax7+MyoD– cells compared with (b) control cells cultured without drug. Stabilisation of β-catenin significantly reduced the number of myogenin-positive (Myog+) cells, whereas increasing (d) Pax7+–myogenin-negative (Pax7+Myog–) cells compared with (e) control cells. Nuclei were counterstained with DAPI. (c,f) Quantification of experiments shown in a,b (c), and in d,e (f). Data are the mean ± s.e.m. from three independent experiments. *P<0.05, significantly different from control cultures.

View larger version (80K): [in this window] [in a new window]   Fig. 7. Gene silencing β-catenin with siRNA enhances myogenic differentiation. (a) Western blotting analysis shows that transfection of β-catenin siRNA significantly reduced β-catenin levels in proliferating (GM) C2 cells compared to transfection with control siRNA. β-Catenin levels remained lower for at least 3 days following the switch to mitogen-poor medium (DM). (b,c) C2 cells transfected with (b) β-catenin siRNA showed enhanced myogenic differentiation compared to cells transfected with (c) control siRNA. (d,e) Immunostaining showed that satellite-cell-derived myoblasts also exhibited reduced β-catenin levels when transfected with (d) β-catenin siRNA compared with (e) control siRNA. (f,g) Silencing β-catenin in primary myoblasts later promoted differentiation and fusion into large myotubes after culture in (f) mitogen-poor medium compared with (g) control cells (quantified in h). (i) Reduced β-catenin levels also significantly decreased the number of (i) Pax7+ cells compared with (j) control cells (quantified in k). Nuclei were counterstained with DAPI (blue). (h,k) Quantification of experiments shown in f,g (h), and in i,j (k). Random fields were selected from three independent cultures and values expressed as the mean ± s.e.m.; *P<0.05, significantly different from control cultures.

View larger version (69K): [in this window] [in a new window]   Fig. 8. Repressing β-catenin transcriptional activity enhances myogenic differentiation. (a,b) Myofibre-associated satellite cells infected with pMSCV–β-catenin–ERD–IRES–eGFP (β-catenin–ERD–RV) had significantly more eGFP+ (green) cells expressing myogenin (red) compared with (b) pMSCV-IRES-eGFP-infected (control-RV). Co-immunostaining for eGFP and Pax7, or eGFP and MyoD proteins revealed that fewer satellite-cell progeny that expressed β-catenin–ERD contained either Pax7 or MyoD (quantified in c). (d,e) Transcriptional repression of β-catenin target genes by β-catenin–ERD enhanced myogenic differentiation, with myotubes with a higher fusion index than control-infected myotubes (quantified in f). Nuclei were counterstained with DAPI (blue). (c,f) Quantification of experiments shown in a,b (c), and in d,e (f). Values are population mean ± s.e.m. *P<0.05, significantly different from control cultures.

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