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Reduced differentiation potential of primary MyoD-/- myogenic cells derived from adult skeletal muscle.

Sabourin LA, Girgis-Gabardo A, Seale P, Asakura A, Rudnicki MA - J. Cell Biol. (1999)

Bottom Line: However, MyoD-/- myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo.Under conditions that normally induce differentiation, MyoD-/- cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes.Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD-/- myogenic cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1.

ABSTRACT
To gain insight into the regeneration deficit of MyoD-/- muscle, we investigated the growth and differentiation of cultured MyoD-/- myogenic cells. Primary MyoD-/- myogenic cells exhibited a stellate morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD-/- myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo. Northern analysis indicated that proliferating MyoD-/- myogenic cells expressed fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain transcription factor expressed in newly activated satellite cells. Under conditions that normally induce differentiation, MyoD-/- cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD-/- myogenic cells. Mixing of lacZ-labeled MyoD-/- cells and wild-type myoblasts revealed a strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD-/- myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell.

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Northern analysis of growth-associated gene products.  (a) Primary wild-type myoblasts expressed abundant β-catenin  under growth conditions. These levels increased threefold after 2 d  of differentiation and subsequently decreased. In MyoD−/− cultures, β-catenin levels were found to continuously increase to levels that were comparable to that of wild-type cells by day 5 of differentiation. (b) Wild-type myoblasts during growth expressed  very low levels of PEA3 mRNA, and these levels increased about  twofold by day 5 of differentiation. Growing MyoD−/− cells displayed sixfold higher levels of PEA3 mRNA, which declined  steadily to wild-type levels by day 4 of differentiation. (c) No significant differences were observed in p21 mRNA levels between  wild-type and MyoD−/− cells. (d) Wild-type myoblasts in  growth conditions expressed low levels of IGF-I mRNA and  these levels were rapidly extinguished after mitogen withdrawal.  Mutant MyoD−/− myogenic cells expressed over threefold  higher levels of the small IGF-I mRNA isoforms (1 and 2) and  the 7-kb pre-IGF-I mRNA (isoform 3) was rapidly upregulated  after 3 d of differentiation. The numbered arrows adjacent to  IGF-I denote specific isoforms depicted on the corresponding  graph. (e) Expression levels as determined by densitometry were  normalized to 18S rRNA. Differentiation and graphical representation of the fold activation is as described in Fig. 5.
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Figure 6: Northern analysis of growth-associated gene products. (a) Primary wild-type myoblasts expressed abundant β-catenin under growth conditions. These levels increased threefold after 2 d of differentiation and subsequently decreased. In MyoD−/− cultures, β-catenin levels were found to continuously increase to levels that were comparable to that of wild-type cells by day 5 of differentiation. (b) Wild-type myoblasts during growth expressed very low levels of PEA3 mRNA, and these levels increased about twofold by day 5 of differentiation. Growing MyoD−/− cells displayed sixfold higher levels of PEA3 mRNA, which declined steadily to wild-type levels by day 4 of differentiation. (c) No significant differences were observed in p21 mRNA levels between wild-type and MyoD−/− cells. (d) Wild-type myoblasts in growth conditions expressed low levels of IGF-I mRNA and these levels were rapidly extinguished after mitogen withdrawal. Mutant MyoD−/− myogenic cells expressed over threefold higher levels of the small IGF-I mRNA isoforms (1 and 2) and the 7-kb pre-IGF-I mRNA (isoform 3) was rapidly upregulated after 3 d of differentiation. The numbered arrows adjacent to IGF-I denote specific isoforms depicted on the corresponding graph. (e) Expression levels as determined by densitometry were normalized to 18S rRNA. Differentiation and graphical representation of the fold activation is as described in Fig. 5.

Mentions: The receptor tyrosine kinase Musk has been suggested to be expressed in activated satellite cells (DeChiara et al., 1996), and therefore may provide an additonal marker for early myogenic cells. Northern blot analysis with a Musk-specific probe revealed the expression of three distinct isoforms in differentiating myogenic cells (Fig. 6 a). In wild-type cultures in growth medium, the large mRNA (isoform 1) was not expressed, the midsize mRNA (isoform 2) was expressed at intermediate levels, and the small mRNA (isoform 3) was expressed at higher levels. Induction of differentiation of wild-type cultures resulted in upregulation of isoforms 1 and 2, but little change in isoform 3. By contrast, MyoD−/− myogenic cells in growth medium expressed no detectable expression of Musk mRNA isoforms 1 and 2, and low levels of isoform 3. After induction of differentiation of MyoD−/− cultures, delayed upregulation of isoforms 1 and 2 was observed. Therefore, these data suggest that Musk is upregulated in a differentiation-dependent manner during muscle regeneration.


Reduced differentiation potential of primary MyoD-/- myogenic cells derived from adult skeletal muscle.

Sabourin LA, Girgis-Gabardo A, Seale P, Asakura A, Rudnicki MA - J. Cell Biol. (1999)

Northern analysis of growth-associated gene products.  (a) Primary wild-type myoblasts expressed abundant β-catenin  under growth conditions. These levels increased threefold after 2 d  of differentiation and subsequently decreased. In MyoD−/− cultures, β-catenin levels were found to continuously increase to levels that were comparable to that of wild-type cells by day 5 of differentiation. (b) Wild-type myoblasts during growth expressed  very low levels of PEA3 mRNA, and these levels increased about  twofold by day 5 of differentiation. Growing MyoD−/− cells displayed sixfold higher levels of PEA3 mRNA, which declined  steadily to wild-type levels by day 4 of differentiation. (c) No significant differences were observed in p21 mRNA levels between  wild-type and MyoD−/− cells. (d) Wild-type myoblasts in  growth conditions expressed low levels of IGF-I mRNA and  these levels were rapidly extinguished after mitogen withdrawal.  Mutant MyoD−/− myogenic cells expressed over threefold  higher levels of the small IGF-I mRNA isoforms (1 and 2) and  the 7-kb pre-IGF-I mRNA (isoform 3) was rapidly upregulated  after 3 d of differentiation. The numbered arrows adjacent to  IGF-I denote specific isoforms depicted on the corresponding  graph. (e) Expression levels as determined by densitometry were  normalized to 18S rRNA. Differentiation and graphical representation of the fold activation is as described in Fig. 5.
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Figure 6: Northern analysis of growth-associated gene products. (a) Primary wild-type myoblasts expressed abundant β-catenin under growth conditions. These levels increased threefold after 2 d of differentiation and subsequently decreased. In MyoD−/− cultures, β-catenin levels were found to continuously increase to levels that were comparable to that of wild-type cells by day 5 of differentiation. (b) Wild-type myoblasts during growth expressed very low levels of PEA3 mRNA, and these levels increased about twofold by day 5 of differentiation. Growing MyoD−/− cells displayed sixfold higher levels of PEA3 mRNA, which declined steadily to wild-type levels by day 4 of differentiation. (c) No significant differences were observed in p21 mRNA levels between wild-type and MyoD−/− cells. (d) Wild-type myoblasts in growth conditions expressed low levels of IGF-I mRNA and these levels were rapidly extinguished after mitogen withdrawal. Mutant MyoD−/− myogenic cells expressed over threefold higher levels of the small IGF-I mRNA isoforms (1 and 2) and the 7-kb pre-IGF-I mRNA (isoform 3) was rapidly upregulated after 3 d of differentiation. The numbered arrows adjacent to IGF-I denote specific isoforms depicted on the corresponding graph. (e) Expression levels as determined by densitometry were normalized to 18S rRNA. Differentiation and graphical representation of the fold activation is as described in Fig. 5.
Mentions: The receptor tyrosine kinase Musk has been suggested to be expressed in activated satellite cells (DeChiara et al., 1996), and therefore may provide an additonal marker for early myogenic cells. Northern blot analysis with a Musk-specific probe revealed the expression of three distinct isoforms in differentiating myogenic cells (Fig. 6 a). In wild-type cultures in growth medium, the large mRNA (isoform 1) was not expressed, the midsize mRNA (isoform 2) was expressed at intermediate levels, and the small mRNA (isoform 3) was expressed at higher levels. Induction of differentiation of wild-type cultures resulted in upregulation of isoforms 1 and 2, but little change in isoform 3. By contrast, MyoD−/− myogenic cells in growth medium expressed no detectable expression of Musk mRNA isoforms 1 and 2, and low levels of isoform 3. After induction of differentiation of MyoD−/− cultures, delayed upregulation of isoforms 1 and 2 was observed. Therefore, these data suggest that Musk is upregulated in a differentiation-dependent manner during muscle regeneration.

Bottom Line: However, MyoD-/- myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo.Under conditions that normally induce differentiation, MyoD-/- cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes.Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD-/- myogenic cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1.

ABSTRACT
To gain insight into the regeneration deficit of MyoD-/- muscle, we investigated the growth and differentiation of cultured MyoD-/- myogenic cells. Primary MyoD-/- myogenic cells exhibited a stellate morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD-/- myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo. Northern analysis indicated that proliferating MyoD-/- myogenic cells expressed fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain transcription factor expressed in newly activated satellite cells. Under conditions that normally induce differentiation, MyoD-/- cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD-/- myogenic cells. Mixing of lacZ-labeled MyoD-/- cells and wild-type myoblasts revealed a strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD-/- myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell.

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