First published online 11 April 2006
doi: 10.1242/jcs.02908
Journal of Cell Science 119, 1824-1832 (2006)
Published by The Company of Biologists 2006
Pax7 and myogenic progression in skeletal muscle satellite cells
Peter S. Zammit1,2,*,
Frederic Relaix3,
Yosuke Nagata1,
,
Ana Pérez Ruiz1,
Charlotte A. Collins1,
,
Terence A. Partridge1,¶ and
Jonathan R. Beauchamp1
1 Muscle Cell Biology Group, Medical Research Council Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK
2 Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London, SE1 1UL, UK
3 CNRS URA 2578, Département de Biologie Du Développement, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris CEDEX 15, France
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Fig. 1. Pax7 remains able to drive transcription in some satellite cell progeny. Co-immunostaining of freshly isolated (T0) EDL myofibres derived from P34 mice, that report Pax3/Pax7 transcriptional activity, demonstrate that ß-gal protein (a, arrows) is present in quiescent satellite cells, as shown by the presence of Pax7 protein (b, arrows). After 40 hours (T40) in culture, ß-gal levels are variable in proliferating MyoD+ satellite cells, even within the same cluster (c and d, arrows). Clear divergence in the fate of satellite-cell progeny is evident by  3 days (T67) in culture, with most cells downregulating Pax7 and committing to differentiation whereas others maintain Pax7 and lose MyoD. At this time, there are few satellite cell progeny with ß-gal activity after incubation in X-gal (e) and immunostaining shows these cells contain Pax7 (f-h, arrows). ß-gal levels are low/absent in cells committed to differentiation (i-l) as shown by the presence of MyoD (j, arrows) and myogenin (l, arrows). Counterstaining with DAPI was used to identify all nuclei present. Bar, 30 µm.
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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.
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Fig. 3. Maintained Pax7 expression delays the onset of myogenin expression. Myofibres were cultured in suspension, exposed to retrovirus and then immunostained between 72 and 96 hours later. Infected satellite cell progeny were readily identified by the presence of eGFP. Most cells infected with pMSCV-IRES-eGFP coexpressed eGFP myogenin (a,c,e). Cells infected with pMSCV-Pax7-IRES-eGFP also coexpressed eGFP (Pax7) and myogenin (arrows in b,d,f), showing that the presence of Pax7 did not prevent differentiation. Importantly though, fewer cells coexpressed eGFP and myogenin after pMSCV-Pax7-IRES-eGFP infection compared with control cultures (g). Values are population means ± s.e.m. from 542 (Pax7 RV) and 619 (control RV) satellite cells from two mice and *P<0.05 denotes significant difference from levels in the control using Student's t-test. Counterstaining with DAPI was used to identify all nuclei. Bar, 30 µm.
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Fig. 4. Maintained Pax7 expression does not prevent the myogenic differentiation of satellite cells. To determine the effects of Pax7 expression on satellite-cell-derived myoblasts, plated myofibres were infected with retrovirus. After a further 72 hours of culture, immunostaining showed that the presence of Pax7, as shown by eGFP expression, did not affect proliferation of satellite-cell-derived progeny as shown by BrdU incorporation (a and b arrows). Similarly, cultures infected with pMSCV-IRES-eGFP (c,d) or pMSCV-Pax7-IRES-eGFP (e,f) were indistinguishable, showing that Pax7 did not alter MyoD expression (examples indicated with arrows). After 1 week of culture, many multinucleated satellite-cell-derived myotubes could be observed. Cultures infected with control pMSCV-IRES-eGFP showed that Pax7 protein was not present in eGFP+ myotubes, but was only maintained in single cells (g). By contrast, Pax7 protein was present in the nuclei of eGFP+ myotubes in pMSCV-Pax7-IRES-eGFP-infected cultures (h). As in myotubes from non-infected and control pMSCV-IRES-eGFP infected cultures, eGFP+ myotubes from cultures infected with pMSCV-Pax7-IRES-eGFP contained MyoD and myogenin (i-l). Bar, 60 µm (a,b); 30 µm (c-l).
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Fig. 5. Ectopic Pax7 increases MyoD expression in Pax7-null myogenic cells. YMCA cells, a subclone of C2C12, did not contain detectable levels of Pax7 (a). Cycling YMCA cells were infected with pMSCV-IRES-eGFP or pMSCV-Pax7-IRES-eGFP, left for 48 hours, fixed and immunostained. Infection with pMSCV-Pax7-IRES-eGFP resulted in Pax7 protein in nuclei of YMCA cells (b). Infected cultures were also co-immunostained for eGFP and BrdU (c,d) or eGFP and MyoD (e,f). Random fields were then selected and the total number of cells containing eGFP and either BrdU (g) or MyoD determined (h). The presence of Pax7 from pMSCV-Pax7-IRES-eGFP did not significantly affect the number of cells able to incorporate BrdU compared with pMSCV-IRES-eGFP-infected or control cells (g). However, the presence of Pax7 significantly increased the number of YMCA cells expressing MyoD when compared with cells infected with control vector or non-infected cells (h). Values are from at least eight random fields and are expressed as population mean ± s.e.m., where *P<0.05 denotes significant difference from non-infected cells using Student's t-test. Bar, 30 µm.
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Fig. 6. Ectopic Pax7 perturbs differentiation in Pax7-null myogenic cells. YMCA cells infected with pMSCV-IRES-eGFP while proliferating and then stimulated to differentiate, readily fused into large multi-nucleated myotubes (a). When stimulated to differentiate however, pMSCV-Pax7-IRES-eGFP infected cells either remained single or fused into aberrantly shaped myotubes (b). As expected of a differentiated myogenic culture, most cells were refractory to BrdU incorporation when infected with control pMSCV-IRES-eGFP (c) whereas many cells infected with pMSCV-Pax7-IRES-eGFP continued to divide (d). Bar, 30 µm.
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© The Company of Biologists Ltd 2006
