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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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Enhanced proliferative potential of primary MyoD−/−  myogenic cells in growth and differentiation. (a) Analysis of  [3H]thymidine incorporation during differentiation revealed that  MyoD−/− myogenic cells continued to synthesize DNA after  mitogen withdrawal. Incorporation was normalized to protein  concentration. The error bars represent the standard error of the  mean for three different isolates. (b) Immunodetection of BrdU  incorporation in MyoD−/− cultures revealed continued DNA  synthesis after mitogen withdrawal. Differentiation was assessed  by immunostaining with antibody MF20. Taken together, these  data indicate that MyoD−/− myogenic cells inefficiently withdraw from the cell cycle under differentiation promoting conditions.
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Figure 3: Enhanced proliferative potential of primary MyoD−/− myogenic cells in growth and differentiation. (a) Analysis of [3H]thymidine incorporation during differentiation revealed that MyoD−/− myogenic cells continued to synthesize DNA after mitogen withdrawal. Incorporation was normalized to protein concentration. The error bars represent the standard error of the mean for three different isolates. (b) Immunodetection of BrdU incorporation in MyoD−/− cultures revealed continued DNA synthesis after mitogen withdrawal. Differentiation was assessed by immunostaining with antibody MF20. Taken together, these data indicate that MyoD−/− myogenic cells inefficiently withdraw from the cell cycle under differentiation promoting conditions.

Mentions: The rate of cell-cycle withdrawal after induction of differentiation was assessed by measuring [3H]thymidine incorporation at daily intervals after transfer into differentiation medium (Fig. 3 a). In growth medium, [3H]thymidine labeling experiments revealed that MyoD−/− cells exhibited an apparent twofold higher growth rate than wild-type cells (Fig. 3 a). Direct cell count experiments in growth medium revealed that numbers of primary MyoD−/− cells accumulated ∼1.5-fold faster than wild-type cells (not shown). Moreover, MyoD−/− cells displayed a 1.6-fold increase in mitotic index as determined by BrdU incorporation (Fig. 3 b). However, this increase in apparent growth can be partly accounted for by the reduced rate of spontaneous differentiation of mutant (0.10 ± 0.14%) versus wild-type cells (10.0 ± 1.4%) (see day 0, Fig. 2, a and b). In addition, interpretation of these results is also confounded by other potential variables: differences in the proportion of cells temporarily withdrawn from the cell-cycle, differences in rates of apoptosis, and differences in cell-cycle kinetics. Therefore, additional analyses are required to determine whether primary MyoD−/− myogenic cells exhibit altered cell-cycle kinetics relative to primary wild-type myoblasts.


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)

Enhanced proliferative potential of primary MyoD−/−  myogenic cells in growth and differentiation. (a) Analysis of  [3H]thymidine incorporation during differentiation revealed that  MyoD−/− myogenic cells continued to synthesize DNA after  mitogen withdrawal. Incorporation was normalized to protein  concentration. The error bars represent the standard error of the  mean for three different isolates. (b) Immunodetection of BrdU  incorporation in MyoD−/− cultures revealed continued DNA  synthesis after mitogen withdrawal. Differentiation was assessed  by immunostaining with antibody MF20. Taken together, these  data indicate that MyoD−/− myogenic cells inefficiently withdraw from the cell cycle under differentiation promoting conditions.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: Enhanced proliferative potential of primary MyoD−/− myogenic cells in growth and differentiation. (a) Analysis of [3H]thymidine incorporation during differentiation revealed that MyoD−/− myogenic cells continued to synthesize DNA after mitogen withdrawal. Incorporation was normalized to protein concentration. The error bars represent the standard error of the mean for three different isolates. (b) Immunodetection of BrdU incorporation in MyoD−/− cultures revealed continued DNA synthesis after mitogen withdrawal. Differentiation was assessed by immunostaining with antibody MF20. Taken together, these data indicate that MyoD−/− myogenic cells inefficiently withdraw from the cell cycle under differentiation promoting conditions.
Mentions: The rate of cell-cycle withdrawal after induction of differentiation was assessed by measuring [3H]thymidine incorporation at daily intervals after transfer into differentiation medium (Fig. 3 a). In growth medium, [3H]thymidine labeling experiments revealed that MyoD−/− cells exhibited an apparent twofold higher growth rate than wild-type cells (Fig. 3 a). Direct cell count experiments in growth medium revealed that numbers of primary MyoD−/− cells accumulated ∼1.5-fold faster than wild-type cells (not shown). Moreover, MyoD−/− cells displayed a 1.6-fold increase in mitotic index as determined by BrdU incorporation (Fig. 3 b). However, this increase in apparent growth can be partly accounted for by the reduced rate of spontaneous differentiation of mutant (0.10 ± 0.14%) versus wild-type cells (10.0 ± 1.4%) (see day 0, Fig. 2, a and b). In addition, interpretation of these results is also confounded by other potential variables: differences in the proportion of cells temporarily withdrawn from the cell-cycle, differences in rates of apoptosis, and differences in cell-cycle kinetics. Therefore, additional analyses are required to determine whether primary MyoD−/− myogenic cells exhibit altered cell-cycle kinetics relative to primary wild-type myoblasts.

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.

Show MeSH