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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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Reduced expression of differentiation-specific markers  in MyoD−/− myogenic cells. Northern analysis of α-skeletal (a)  and α-cardiac actin (b) mRNA levels revealed reduced and delayed kinetics of induction upon differentiation of MyoD−/−  cultures. Similarly, acetylcholine receptor δ subunit (AchR δ) (c)  and M-cadherin mRNA levels were found to be reduced (d). A  marked reduction in the expression of Musk (e) and adhalin (f)  mRNAs was also observed. The numbered arrows for Musk correspond to the specific isoforms quantitated on the associated  graphs. The fold activation in arbitrary units for each mRNA species is shown graphically beside each Northern blot. Fold activation was measured by densitometry and was normalized to 18S  rRNA.
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Figure 5: Reduced expression of differentiation-specific markers in MyoD−/− myogenic cells. Northern analysis of α-skeletal (a) and α-cardiac actin (b) mRNA levels revealed reduced and delayed kinetics of induction upon differentiation of MyoD−/− cultures. Similarly, acetylcholine receptor δ subunit (AchR δ) (c) and M-cadherin mRNA levels were found to be reduced (d). A marked reduction in the expression of Musk (e) and adhalin (f) mRNAs was also observed. The numbered arrows for Musk correspond to the specific isoforms quantitated on the associated graphs. The fold activation in arbitrary units for each mRNA species is shown graphically beside each Northern blot. Fold activation was measured by densitometry and was normalized to 18S rRNA.

Mentions: To assess the differentiation kinetics at the level of gene expression, Northern analysis was performed with a panel of muscle-specific markers. Analysis of mRNA levels for differentiation-specific markers revealed a pattern consistent with overall delayed kinetics of differentiation in MyoD−/− myogenic cells. For example, low levels of α-skeletal and α-cardiac actin mRNAs were detected in wild-type cells in growth medium and these increased rapidly after mitogen withdrawal. By contrast, MyoD−/− cells in growth medium expressed no detectable sarcomeric actin mRNA (Fig. 5, a and b). After induction of differentiation α-skeletal actin mRNA increased about threefold in wild-type cells, whereas in MyoD−/− cells the levels increased ∼65% of wild-type levels by day 5 (Fig. 5 a). Although lower than that of α-skeletal actin, the levels of α-cardiac actin were found to increase in a similar pattern (Fig. 5 b). Cultured MyoD−/− myogenic cells displayed a twofold reduction in levels of acetylcholine receptor δ subunit (AchR δ) mRNA in growth medium. After the induction of differentiation, a 5-fold increase was observed in wild-type cultures compared to an ∼2-fold increase in MyoD−/− cells (Fig. 5 c) for a net 10-fold relative difference.


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)

Reduced expression of differentiation-specific markers  in MyoD−/− myogenic cells. Northern analysis of α-skeletal (a)  and α-cardiac actin (b) mRNA levels revealed reduced and delayed kinetics of induction upon differentiation of MyoD−/−  cultures. Similarly, acetylcholine receptor δ subunit (AchR δ) (c)  and M-cadherin mRNA levels were found to be reduced (d). A  marked reduction in the expression of Musk (e) and adhalin (f)  mRNAs was also observed. The numbered arrows for Musk correspond to the specific isoforms quantitated on the associated  graphs. The fold activation in arbitrary units for each mRNA species is shown graphically beside each Northern blot. Fold activation was measured by densitometry and was normalized to 18S  rRNA.
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Related In: Results  -  Collection

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Figure 5: Reduced expression of differentiation-specific markers in MyoD−/− myogenic cells. Northern analysis of α-skeletal (a) and α-cardiac actin (b) mRNA levels revealed reduced and delayed kinetics of induction upon differentiation of MyoD−/− cultures. Similarly, acetylcholine receptor δ subunit (AchR δ) (c) and M-cadherin mRNA levels were found to be reduced (d). A marked reduction in the expression of Musk (e) and adhalin (f) mRNAs was also observed. The numbered arrows for Musk correspond to the specific isoforms quantitated on the associated graphs. The fold activation in arbitrary units for each mRNA species is shown graphically beside each Northern blot. Fold activation was measured by densitometry and was normalized to 18S rRNA.
Mentions: To assess the differentiation kinetics at the level of gene expression, Northern analysis was performed with a panel of muscle-specific markers. Analysis of mRNA levels for differentiation-specific markers revealed a pattern consistent with overall delayed kinetics of differentiation in MyoD−/− myogenic cells. For example, low levels of α-skeletal and α-cardiac actin mRNAs were detected in wild-type cells in growth medium and these increased rapidly after mitogen withdrawal. By contrast, MyoD−/− cells in growth medium expressed no detectable sarcomeric actin mRNA (Fig. 5, a and b). After induction of differentiation α-skeletal actin mRNA increased about threefold in wild-type cells, whereas in MyoD−/− cells the levels increased ∼65% of wild-type levels by day 5 (Fig. 5 a). Although lower than that of α-skeletal actin, the levels of α-cardiac actin were found to increase in a similar pattern (Fig. 5 b). Cultured MyoD−/− myogenic cells displayed a twofold reduction in levels of acetylcholine receptor δ subunit (AchR δ) mRNA in growth medium. After the induction of differentiation, a 5-fold increase was observed in wild-type cultures compared to an ∼2-fold increase in MyoD−/− cells (Fig. 5 c) for a net 10-fold relative difference.

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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