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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.

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Pax3 and Pax7 regulate the rate of myogenic cell division.Pax3 RV and Pax7 RV were used to infect a subclone of C2C12 with low endogenous Pax7 levels, to first determine if Pax3 or Pax7 could regulate each other. Pax3 RV-infected cells were only recognized by the anti-Pax3 monoclonal antibody (a) and not by the anti-Pax7 antibody (b), whereas Pax7 RV-infected cells were recognized only by the anti-Pax7 antibody (d) and not by the anti-Pax3 (c). To determine how Pax3 or Pax7 affects myogenic cell proliferation, C2C12 and satellite cell-derived myoblasts were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN and Pax7DN, plated at clonal density, cultured and fixed 72 h later. They were then co-immunostained for either eGFP (green) and Pax3 (red), or eGFP (green) and Pax7 (red), and counterstained with DAPI to identify all nuclei. All cells present in a clone are shown (e–i). Pax3 and Pax7 RV-infected C2C12 (f and g) and satellite cell-derived myoblast clones produced more cells per colony compared to control (quantified in j and l). In contrast, constitutive expression of Pax3DN or Pax7DN generated C2C12 clones (h and i) with significantly less cells (quantified in j) with similar results obtained with satellite cell-derived myoblasts (quantified in l). Despite the different proliferation rates, constitutive expression of Pax3, Pax7 or their dominant-negative versions did not generally alter the percentage of C2C12 or plated satellite cells incorporating BrdU 72 h post-infection (quantified in k and m). Interestingly, BrdU labelling (BrdU+ve) of eGFP expressing cells (eGFP+ve) infected with Pax3DN (h′) or Pax7DN (i′) revealed that many cells had two nuclei (arrowed). Scale bar represents 40 µm (except h′ and i′). Values are population means±SEM of 15–20 clones from each of 3 independent experiments, where an asterisk denotes significant difference at p<0.05, while two asterisks denotes significant difference at p<0.0001, from controls using Mann-Whitney.
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pone-0004475-g001: Pax3 and Pax7 regulate the rate of myogenic cell division.Pax3 RV and Pax7 RV were used to infect a subclone of C2C12 with low endogenous Pax7 levels, to first determine if Pax3 or Pax7 could regulate each other. Pax3 RV-infected cells were only recognized by the anti-Pax3 monoclonal antibody (a) and not by the anti-Pax7 antibody (b), whereas Pax7 RV-infected cells were recognized only by the anti-Pax7 antibody (d) and not by the anti-Pax3 (c). To determine how Pax3 or Pax7 affects myogenic cell proliferation, C2C12 and satellite cell-derived myoblasts were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN and Pax7DN, plated at clonal density, cultured and fixed 72 h later. They were then co-immunostained for either eGFP (green) and Pax3 (red), or eGFP (green) and Pax7 (red), and counterstained with DAPI to identify all nuclei. All cells present in a clone are shown (e–i). Pax3 and Pax7 RV-infected C2C12 (f and g) and satellite cell-derived myoblast clones produced more cells per colony compared to control (quantified in j and l). In contrast, constitutive expression of Pax3DN or Pax7DN generated C2C12 clones (h and i) with significantly less cells (quantified in j) with similar results obtained with satellite cell-derived myoblasts (quantified in l). Despite the different proliferation rates, constitutive expression of Pax3, Pax7 or their dominant-negative versions did not generally alter the percentage of C2C12 or plated satellite cells incorporating BrdU 72 h post-infection (quantified in k and m). Interestingly, BrdU labelling (BrdU+ve) of eGFP expressing cells (eGFP+ve) infected with Pax3DN (h′) or Pax7DN (i′) revealed that many cells had two nuclei (arrowed). Scale bar represents 40 µm (except h′ and i′). Values are population means±SEM of 15–20 clones from each of 3 independent experiments, where an asterisk denotes significant difference at p<0.05, while two asterisks denotes significant difference at p<0.0001, from controls using Mann-Whitney.

Mentions: To both investigate cross-regulation between Pax genes in myogenic cells, and confirm the specificity of the anti-Pax3 antibody for Pax3, we infected low-density cultures of a C2C12 subclone (with low endogenous Pax7 levels) with Pax3 RV or Pax7 RV, which resulted in co-expression of Pax3, or Pax7 respectively, together with eGFP from the IRES-eGFP in the retroviral backbone (Figure 1a–d). Pax3 IgG2α monoclonal antibody bound only to the nuclei of cells infected with Pax3 RV, but not those infected with Pax7 RV, while the Pax7 IgG1 monoclonal antibody bound only to the nuclei of cells infected with Pax7 RV and not Pax3 RV (Figure 1a–d). Therefore, expression of either Pax gene did not induce expression of its paralogue, and we confirm that the Pax3 and Pax7 antibodies each have high specificity for their target protein (Figure 1a–d).


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)

Pax3 and Pax7 regulate the rate of myogenic cell division.Pax3 RV and Pax7 RV were used to infect a subclone of C2C12 with low endogenous Pax7 levels, to first determine if Pax3 or Pax7 could regulate each other. Pax3 RV-infected cells were only recognized by the anti-Pax3 monoclonal antibody (a) and not by the anti-Pax7 antibody (b), whereas Pax7 RV-infected cells were recognized only by the anti-Pax7 antibody (d) and not by the anti-Pax3 (c). To determine how Pax3 or Pax7 affects myogenic cell proliferation, C2C12 and satellite cell-derived myoblasts were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN and Pax7DN, plated at clonal density, cultured and fixed 72 h later. They were then co-immunostained for either eGFP (green) and Pax3 (red), or eGFP (green) and Pax7 (red), and counterstained with DAPI to identify all nuclei. All cells present in a clone are shown (e–i). Pax3 and Pax7 RV-infected C2C12 (f and g) and satellite cell-derived myoblast clones produced more cells per colony compared to control (quantified in j and l). In contrast, constitutive expression of Pax3DN or Pax7DN generated C2C12 clones (h and i) with significantly less cells (quantified in j) with similar results obtained with satellite cell-derived myoblasts (quantified in l). Despite the different proliferation rates, constitutive expression of Pax3, Pax7 or their dominant-negative versions did not generally alter the percentage of C2C12 or plated satellite cells incorporating BrdU 72 h post-infection (quantified in k and m). Interestingly, BrdU labelling (BrdU+ve) of eGFP expressing cells (eGFP+ve) infected with Pax3DN (h′) or Pax7DN (i′) revealed that many cells had two nuclei (arrowed). Scale bar represents 40 µm (except h′ and i′). Values are population means±SEM of 15–20 clones from each of 3 independent experiments, where an asterisk denotes significant difference at p<0.05, while two asterisks denotes significant difference at p<0.0001, from controls using Mann-Whitney.
© Copyright Policy
Related In: Results  -  Collection

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pone-0004475-g001: Pax3 and Pax7 regulate the rate of myogenic cell division.Pax3 RV and Pax7 RV were used to infect a subclone of C2C12 with low endogenous Pax7 levels, to first determine if Pax3 or Pax7 could regulate each other. Pax3 RV-infected cells were only recognized by the anti-Pax3 monoclonal antibody (a) and not by the anti-Pax7 antibody (b), whereas Pax7 RV-infected cells were recognized only by the anti-Pax7 antibody (d) and not by the anti-Pax3 (c). To determine how Pax3 or Pax7 affects myogenic cell proliferation, C2C12 and satellite cell-derived myoblasts were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN and Pax7DN, plated at clonal density, cultured and fixed 72 h later. They were then co-immunostained for either eGFP (green) and Pax3 (red), or eGFP (green) and Pax7 (red), and counterstained with DAPI to identify all nuclei. All cells present in a clone are shown (e–i). Pax3 and Pax7 RV-infected C2C12 (f and g) and satellite cell-derived myoblast clones produced more cells per colony compared to control (quantified in j and l). In contrast, constitutive expression of Pax3DN or Pax7DN generated C2C12 clones (h and i) with significantly less cells (quantified in j) with similar results obtained with satellite cell-derived myoblasts (quantified in l). Despite the different proliferation rates, constitutive expression of Pax3, Pax7 or their dominant-negative versions did not generally alter the percentage of C2C12 or plated satellite cells incorporating BrdU 72 h post-infection (quantified in k and m). Interestingly, BrdU labelling (BrdU+ve) of eGFP expressing cells (eGFP+ve) infected with Pax3DN (h′) or Pax7DN (i′) revealed that many cells had two nuclei (arrowed). Scale bar represents 40 µm (except h′ and i′). Values are population means±SEM of 15–20 clones from each of 3 independent experiments, where an asterisk denotes significant difference at p<0.05, while two asterisks denotes significant difference at p<0.0001, from controls using Mann-Whitney.
Mentions: To both investigate cross-regulation between Pax genes in myogenic cells, and confirm the specificity of the anti-Pax3 antibody for Pax3, we infected low-density cultures of a C2C12 subclone (with low endogenous Pax7 levels) with Pax3 RV or Pax7 RV, which resulted in co-expression of Pax3, or Pax7 respectively, together with eGFP from the IRES-eGFP in the retroviral backbone (Figure 1a–d). Pax3 IgG2α monoclonal antibody bound only to the nuclei of cells infected with Pax3 RV, but not those infected with Pax7 RV, while the Pax7 IgG1 monoclonal antibody bound only to the nuclei of cells infected with Pax7 RV and not Pax3 RV (Figure 1a–d). Therefore, expression of either Pax gene did not induce expression of its paralogue, and we confirm that the Pax3 and Pax7 antibodies each have high specificity for their target protein (Figure 1a–d).

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