Fig. 1. Isolated adult muscle fibres and the Wnt-signalling pathway. (A) RT-PCR of Wnt genes from single fibres cultured for 48 hours. Lane 1, Wnt11; Lane 2, Wnt5a; Lane 3, Wnt3a; Lane 4, Wnt1. The position of the 500 bp size marker is indicated. (B) TOPFlash Wnt reporter activity of isolated single muscle fibres. Control activity was normalised to 1. Isolated fibres gave 2.15 times the control stimulation (^*P<0.01). LiCl (positive control) gave stimulation of 2.5 times control (^**P<0.001). (C) EGCG, a compound that promotes β-catenin degradation inhibits satellite-cell proliferation. Control fibres cultured for 72 hours resulted in 75.3±7.8 (s.e.m.) satellite cells per myofibre, compared with 31.9±3.2 on myofibres treated with EGCG (^*P<0.0001, n=25 control and n=35 EGCG-treated myofibres). (D) Pan β-catenin expression on freshly isolated myofibres (t=0 hours). Pan β-catenin was localised at the periphery of the satellite cell (green arrowhead). (E) Double staining for pan β-catenin and the nucleus. Pan β-catenin localised around the edge of the nucleus at t=0 (green arrowhead). (F) Double staining for Act-β-Cat and the nuclei at t=0 hours. Act-β-Cat was not detected in freshly isolated fibres. (G) Double staining for Act-β-Cat and MyoD after 6 hours in culture. MyoD-positive satellite cells do not show the presence of Act-β-Cat (red arrowhead). Adjacent myonucleus (MyoD-negative) shows faint Act-β-Cat expression (green arrowhead). (H) Double staining for Act-β-Cat and MyoD after 24 hours in culture. MyoD-positive satellite cells coexpress low levels of Act-β-Cat (white arrowhead). (I) Satellite cells prior to division, double stained for Act-β-Cat and MyoD after 48 hours in culture. MyoD-positive satellite cells robustly coexpress Act-β-Cat (white arrowheads). (J) Double staining for Act-β-Cat and MyoD after 48 hours in culture in satellite cells after division. MyoD-positive nuclei show weak coexpression of Act-β-Cat (red arrowhead). However, strong Act-β-Cat expression localised between two divided cells (green arrowhead). (K) Double staining for Act-β-Cat and myogenin after 72 hours in culture. Rare large cluster of satellite cells expressing Act-β-Cat but not myogenin is shown. (L) Image shown in K overlaid with DAPI. Large cluster of satellite cells express Act-β-Cat localised to the nuclei. (M) Double staining for Act-β-Cat and myogenin after 72 hours in culture. A small cluster showing mutually exclusive expression of Act-β-Cat (green arrowhead) and myogenin (red arrowhead). (N) Same image shown in M overlaid with DAPI, confirming the nuclear localisation. Myogenin was expressed in cells that failed to express Act-β-Cat (red arrowhead). Adjacent cells show two profiles of Act-β-Cat expression, either at the membrane (green arrowhead), reminiscent of J, or predominantly in the nucleus (white arrowhead) similar to I. (O) Double staining for Act-β-Cat and BrdU after 36 hours in culture. Single activated satellite cell shows coexpression of both markers (yellow arrowhead). (P) Same image shown in O overlaid with DAPI, confirming the nuclear localisation of Act-β-Cat and BrdU (white arrowhead). (Q) Satellite-cell progeny at 72 hours stained for BrdU and DAPI. Culture was pulsed with BrdU at 48 hours. Two BrdU-positive nuclei are indicated (red arrowheads). (R) Same image shown in Q overlaid with Act-β-Cat expression, which localises predominantly to the BrdU-positive nuclei (white arrowheads). (S) Percentage of satellite-cell nuclei positive for BrdU alone, BrdU and Act-β-Cat and neither marker was quantified at 48 and 72 hours of culture. At 48 hours, 91.8% of satellite-cell nuclei were Act-β-Cat⁺ BrdU⁺, 6.3% were Act-β-Cat^– BrdU⁺, and 1.5% were Act-β-Cat^– BrdU^–. At 72 hours, 74.5% of satellite-cell nuclei were Act-β-Cat⁺ BrdU⁺, 24.8% were Act-β-Cat^– BrdU⁺, and 0.7% were Act-β-Cat^– BrdU^–. n=24 myofibres for each time point.