Jagged-1 is induced when satellite cells are activated.

<p>EDL myofibres were cultured in suspension for 48 hrs in plating medium to elicit the activation and proliferation of satellite cells (arrows). Activated and proliferating satellite cells were identified by immunostaining for MyoD (green), and Caveolin-1 (a - red), integrin α7 (b - red) and CTR (c - red) remained expressed in the plasma membrane of activated satellite cells. The continued presence of both emerin (red, d) and lamin A/C (green, e) in the nuclear envelope of activated satellite cells expressing MyoD (green in d, red in e) was also noted. Moreover, we found that the Notch ligand Jagged-1 (red) was induced upon activation, in MyoD+ve (green) satellite cells (f). Nuclei were counterstained with DAPI (blue). Scale bar represents 20 µm.</p

Emerin and lamin A/C are expressed in the nuclear envelope of quiescent satellite cells.

<p>Freshly isolated EDL myofibres were co-immunostained for Pax7 and emerin (a). Emerin (red) was expressed in the nuclear envelope of Pax7+ve (green) satellite cells (arrow). Since a mouse monoclonal antibody was used to immunostain lamin A/C, satellite cells were identified using caveolin-1 (b). We found that the nuclear lamina of caveolin-1 (red)-expressing satellite cells (arrow) contained lamin A/C (green). All nuclei present were revealed by counterstaining with DAPI (blue). Scale bar represents 20 µm.</p

Marker protein levels reflect gene expression in quiescent and activated satellite cells.

<p>Total RNA was obtained from quiescent (T0) and activated (T48) satellite cells and transcript-specific primers used to semi-quantitatively amplify the mRNA of markers. Amplification of Gapdh transcript was used as control of the RNA content. Pax7, MyoD and myogenin transcripts levels were included as control of the activation progression. While <i>caveolin-1</i> and <i>CTR</i> transcripts appeared to be more abundant in satellite cells at T0, mRNA for <i>integrin α7</i>, <i>Emd</i> and <i>Lmna</i> were unaltered. <i>Jagged-1</i> and <i>MyoD</i> transcript were increased in activated satellite cells, reflecting the protein levels. <i>Myogenin</i> transcript, as expected, was absent in quiescent satellite cells and appeared after 48 hrs in culture. NTC is no-template control.</p

Caveolin-1 is down-regulated in differentiating satellite cell progeny.

<p>EDL myofibres were cultured in suspension for 72 hrs, by which time many begin to express myogenin as they commit to myogenic differentiation (arrows). Myogenin-containing satellite cells (green – arrowed) tended to have lower levels of caveolin-1 immunosignal (red - arrow) in their plasma membranes than uncommitted cells. Nuclei were revealed by counterstaining with DAPI (blue). Scale bar represents 20 µm.</p

Percentage of quiescent satellite cells expressing each marker

<p>Mean±SEM from at least 15 EDL myofibres from each of at least 3 mice.</p

Caveolin-1, integrin α7 and CTR are expressed by Pax7+ve quiescent satellite cells in their niche on the myofibre.

<p>Satellite cells in their niche on the surface of freshly isolated EDL myofibres were co-immunostained for Pax7 (green) and either caveolin-1 (a - red), integrin α7 (b - red) or CTR (c - red). All three proteins were expressed in the plasma membrane of Pax7+ve satellite cells (arrowed), clearly revealing their bi-polar morphology. Myofibres were also examined from the masseter of the jaw, and again Pax7+ve satellite cells expressed all three markers (caveolin-1 shown as an example in d). All nuclei were counterstained with DAPI (blue), revealing the presence of the myonuclei of the myofibres on which the satellite cells were located. Scale bar represents 20 µm.</p

Pax3/Pax7 transcriptional activity is required for myogenin expression.

<p>To test the effects of perturbing Pax3 and Pax7 function on myogenicity, cells were infected with retroviral constructs encoding Pax3, Pax7, Pax3DN or Pax7DN, together with empty vector serving as control. Cells underwent further culture and were then co-immunostained for either eGFP (green) and MyoD (red), or eGFP (green) and myogenin (red). C2C12 cells infected with control RV (a), Pax3 RV (b), Pax7 RV (c), Pax3DN RV (d) and Pax7DN RV (e) and co-immunostained for eGFP and myogenin 120 h after infection. Constitutive Pax3 or Pax7 expression resulted in more infected cells (eGFP+ve) with MyoD, while constitutive Pax3DN or Pax7DN caused less eGFP+ve cells to co-express MyoD and myogenin (quantified in p after 72 h). Satellite cells were infected with control RV (f), Pax3 RV (g), Pax7 RV (h), Pax3DN RV (i) and Pax7DN RV (j) while retained in their niche on the myofibre and cultured before co-immunostaining for eGFP (green) and myogenin (red). The presence of constitutively expressed Pax3 (g) or Pax7 (h) was compatible with myogenin expression (arrows), but there were reduced numbers of infected eGFP+ve satellite cell progeny containing myogenin (quantified in q). Many infected GFP+ve cells expressing Pax3DN (i - green - arrow) and Pax7DN (j - green - arrow) did not contain myogenin (i and j - red) (quantified in q). Similar results were obtained when plated satellite cell-derived myoblasts were infected with control RV (k), Pax3 RV (l), Pax7 RV (m), Pax3DN RV (n) and Pax7DN RV (o) and co-stained for eGFP (green) and myogenin (red), 72 h post infection (quantified in r). Counterstaining with DAPI was used to identify all nuclei present. Scale bar for each row equals 40 µm, except for k–o where it represents 100 µm. Values are population means±SEM of at least 3 independent experiments, where an asterisk denotes significant difference at <i>p</i><0.05, while two asterisks denotes significant difference at <i>p</i><0.0001, from controls using Mann-Whitney.</p

Pax3/Pax7 transcriptional targets can regulate proliferation and cell size independently of the myogenic program.

<p>To establish whether the effects on cell proliferation and size were a result of interactions of Pax proteins with myogenic genes, NIH 3T3 fibroblasts were also infected with either Pax3 RV (a and d), Pax7 RV (b and e), control RV (c), Pax3DN RV (f) or Pax7DN RV (g). Infected cells were plated at clonal density and analysed 72 h post-infection by co-immunostaining for either eGFP (green) and MyoD (red); eGFP (green) and Pax3 (red); eGFP (green) and Pax7 (red) or eGFP (green) and BrdU (red), before counterstaining with DAPI. NIH 3T3 fibroblasts did not express Pax3 or Pax7 (c) and constitutive expression of these proteins (a and b) did not elicit the initiation of the myogenic program, as shown by their failure to induce MyoD (a′-b′). As with myogenic cells, constitutive Pax3 (d) and Pax7 (e) expression resulted in larger clone sizes, while Pax3DN (f) and Pax7DN (g) had the opposite effect, and significantly reduced clone size (quantified in h). Infection of NIH 3T3 cells with Pax3DN RV (f′) or Pax7DN RV (g′) followed by a 2 h BrdU pulse after 72 h, indicated that S-phase length was not drastically altered (percentage of infected cells containing BrdU (BrdU+ve/eGFP+ve) quantified in i). Cells with double nuclei were present (f′ and g′). Again, as with myogenic cells, constitutive Pax3 and Pax7 also caused NIH 3T3 cells to become significantly smaller, while the presence of the dominant-negative versions resulted in an increase in mean cell size using SigmaScan Pro (quantified in j). Scale bar equals 40 µm (except d′ and e′). Values are population means±SEM of 6–20 clones from each of 3 independent experiments, where an asterisk denotes significant difference at <i>p</i><0.05, while two asterisks denotes significant difference at <i>p</i><0.0001, from controls using Mann-Whitney.</p

Pax3 and Pax7 can regulate the cell size of myogenic cells.

<p>To examine how altered Pax gene function affected cell size, C2C12 and satellite cell-derived myoblasts were infected with retroviral constructs encoding Pax3, Pax7, Pax3DN or Pax7DN, together with empty vector serving as control. Infected cells were plated at clonal density, fixed after 72 h, immunostained, counterstained with DAPI and cell size determined using SigmaScan Pro image analysis software. For both C2C12 (a) and satellite cell-derived myoblasts (b), constitutive Pax3 or Pax7 expression resulted in significantly smaller cells, while the presence of Pax3DN or Pax7DN caused the cells to get bigger. Indeed, even the myotubes formed from Pax3 RV- or Pax7 RV-infected C2C12 myoblasts were significantly smaller than controls (c). Values are population means±SEM of 17–60 cells/myotubes in total from 3 independent experiments, where an asterisk denotes significant difference (<i>p</i><0.0001) from controls using Mann Whitney.</p

Inhibition of Pax3/Pax7 transcriptional targets prevents myogenic differentiation.

<p>To test the effects of perturbing Pax3 and Pax7 function on the ability of myoblasts to fuse into multinucleated myotubes, C2C12 and plated satellite cell-derived myoblasts were infected with retroviral constructs encoding Pax3, Pax7, Pax3DN or Pax7DN, together with empty vector serving as control. Cells were then allowed to differentiate and fuse for 5 days and immunostained. Control RV-infected C2C12 co-immunostained for either eGFP (green) and Pax3 (red) or eGFP (green) and Pax7 (red) showed that Pax3 was not expressed in reserve cells (a) while Pax7 was (b), with neither protein present in myotubes. C2C12 cells still efficiently fused into myotubes in the presence of constitutively expressed Pax3 (c) and Pax7 (d), with ectopic, retroviral-driven expression of Pax3 (e) and Pax7 (f) in the nuclei of multinucleated satellite cell-derived myotubes. Infection of C2C12 cells with control RV (g), Pax3 RV (h), Pax7 RV (i), Pax3DN RV (j) and Pax7DN RV (k) and co-immunostaining for eGFP (green) and myosin heavy chain (MyHC-red) confirmed that constitutive Pax3 (h) and Pax7 (i) expression did not perturb myotube formation, in contrast to Pax3DN (j) and Pax7DN (k), which effectively prevented it, with very few myotubes containing both eGFP and MyHC (yellow). Similar results were obtained when plated satellite cell-derived myoblasts were infected with control RV (l), Pax3 RV (m), Pax7 RV (n), Pax3DN RV (o) and Pax7DN RV (p) and co-immunostained for eGFP (green) and MyHC (red). Counterstaining with DAPI was used to identify all nuclei present. All experiments were repeated at least 3 times. Scale bar represents 40 µm.</p