Limits...
Integrated functions of Pax3 and Pax7 in the regulation of proliferation, cell size and myogenic differentiation.

Collins CA, Gnocchi VF, White RB, Boldrin L, Perez-Ruiz A, Relaix F, Morgan JE, Zammit PS - PLoS ONE (2009)

Bottom Line: We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size.Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation.Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

View Article: PubMed Central - PubMed

Affiliation: Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, United Kingdom.

ABSTRACT
Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

Show MeSH

Related in: MedlinePlus

Inhibition of Pax3/Pax7 transcriptional targets prevents myogenic differentiation.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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2637421&req=5

pone-0004475-g004: Inhibition of Pax3/Pax7 transcriptional targets prevents myogenic differentiation.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.

Mentions: Both the presence of constitutively expressed Pax3 or Pax7, and inhibition of Pax3 and Pax7 transcriptional targets using the dominant-negative constructs, delay the induction of myogenin. We next asked whether this delay was temporary, or if myogenic differentiation was inhibited. Differentiation and fusion of myogenic cells into myotubes in vitro can be induced by allowing cultures to reach confluence. As expected, C2C12 cells or primary satellite cell-derived myoblasts infected with control RV and cultured to confluence (around 120 h under our experimental conditions) readily formed large multinucleate myotubes that expressed Myosin Heavy Chain (MyHC), a marker of sarcomere assembly during terminal differentiation (Figure 4). Reserve cells arising in post-differentiated cultures of both C2C12 (Figure 4b) and plated satellite cell-derived myoblasts were positive for Pax7. While Pax7 was robustly expressed by reserve cells, we could find no evidence of the presence of Pax3 protein (Figure 4a). Infection of C2C12 cells (Figure 4c and d) or satellite cell-derived myoblasts (Figure 4e and f) with Pax3 RV or Pax7 RV did not prevent the development of multinucleate myotubes, though in both cases there was a slight delay (∼24 h) in fusion. Immunostaining for Pax3 or Pax7 confirmed the presence of each protein in many nuclei of myotubes derived from RV-infected C2C12 and satellite cell-derived myoblasts (Figure 4c–f) which did not perturb sarcomere assembly, as shown by the presence of MyHC (Figure 4g–i and l–n). However, myotubes formed from Pax3 RV- or Pax7 RV-containing C2C12 cells were significantly thinner than those formed from control RV-infected C2C12 cells, as determined using measurements taken from the minor axis of individual myotubes (Figure 2c).


Integrated functions of Pax3 and Pax7 in the regulation of proliferation, cell size and myogenic differentiation.

Collins CA, Gnocchi VF, White RB, Boldrin L, Perez-Ruiz A, Relaix F, Morgan JE, Zammit PS - PLoS ONE (2009)

Inhibition of Pax3/Pax7 transcriptional targets prevents myogenic differentiation.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637421&req=5

pone-0004475-g004: Inhibition of Pax3/Pax7 transcriptional targets prevents myogenic differentiation.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.
Mentions: Both the presence of constitutively expressed Pax3 or Pax7, and inhibition of Pax3 and Pax7 transcriptional targets using the dominant-negative constructs, delay the induction of myogenin. We next asked whether this delay was temporary, or if myogenic differentiation was inhibited. Differentiation and fusion of myogenic cells into myotubes in vitro can be induced by allowing cultures to reach confluence. As expected, C2C12 cells or primary satellite cell-derived myoblasts infected with control RV and cultured to confluence (around 120 h under our experimental conditions) readily formed large multinucleate myotubes that expressed Myosin Heavy Chain (MyHC), a marker of sarcomere assembly during terminal differentiation (Figure 4). Reserve cells arising in post-differentiated cultures of both C2C12 (Figure 4b) and plated satellite cell-derived myoblasts were positive for Pax7. While Pax7 was robustly expressed by reserve cells, we could find no evidence of the presence of Pax3 protein (Figure 4a). Infection of C2C12 cells (Figure 4c and d) or satellite cell-derived myoblasts (Figure 4e and f) with Pax3 RV or Pax7 RV did not prevent the development of multinucleate myotubes, though in both cases there was a slight delay (∼24 h) in fusion. Immunostaining for Pax3 or Pax7 confirmed the presence of each protein in many nuclei of myotubes derived from RV-infected C2C12 and satellite cell-derived myoblasts (Figure 4c–f) which did not perturb sarcomere assembly, as shown by the presence of MyHC (Figure 4g–i and l–n). However, myotubes formed from Pax3 RV- or Pax7 RV-containing C2C12 cells were significantly thinner than those formed from control RV-infected C2C12 cells, as determined using measurements taken from the minor axis of individual myotubes (Figure 2c).

Bottom Line: We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size.Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation.Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

View Article: PubMed Central - PubMed

Affiliation: Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, United Kingdom.

ABSTRACT
Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

Show MeSH
Related in: MedlinePlus