Fig. 2. Although endogenous Pax7 is not present in myotubes, ectopically expressed Pax7 can still activate its transcriptional targets in myonuclei. In order to investigate Pax7 transcriptional activity as satellite cell-derived myoblasts differentiate, myofibres were plated on matrigel. This preparation allows satellite cells to emigrate from the myofibre and proliferate extensively before fusing into multi-nucleated myotubes. Such plated myofibres from the Pax3/Pax7 transcriptional activity indicator mouse line P34, gave rise to large myosin heavy chain (MyHC⁺) multinucleated myotubes, the nuclei of which were ß-gal^- (a,b). Some single cells however, often located close to myotubes, contained ß-gal protein, indicating that Pax7 remained transcriptionally active in these cells (a and b, arrows). To test the retroviral vectors (RV), P34-derived myofibres were plated and the satellite-cell-derived myoblasts infected with either control pMSCV-IRES-eGFP (c) or pMSCV-Pax7-IRES-eGFP (d) and allowed to fuse into large multinucleated myotubes. Immunostaining for ß-gal and eGFP revealed that pMSCV-IRES-eGFP-infected cultures only gave rise to eGFP⁺/ß-gal^- myotubes (c), indistinguishable from uninfected myotubes (a). However, those infected with pMSCV-Pax7-IRES-eGFP had eGFP⁺ myotubes with ß-gal⁺ myonuclei (d), showing that the introduced Pax7 protein was able to activate is transcriptional targets in myonuclei. Counterstaining with DAPI was used to identify all nuclei present. Bar, 30 µm.