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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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Strict cell autonomy in differentiated cocultures of lacZ-expressing MyoD−/− myogenic cells and  wild-type myoblasts. Primary cells  were plated in ratios of 1:0 (a), 1:4 (b),  4:1 (c), and 0:1 (d) of MyoD−/− to  wild-type cells. After 5 d of differentiation, cells were fixed, and stained for  β-galactosidase and with antibody  MF20 reactive with MHC. Note the  complete absence of lacZ-labeled nuclei in myotubes containing greater  than two nuclei and the normal differentiation of wild-type myotubes.
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Figure 7: Strict cell autonomy in differentiated cocultures of lacZ-expressing MyoD−/− myogenic cells and wild-type myoblasts. Primary cells were plated in ratios of 1:0 (a), 1:4 (b), 4:1 (c), and 0:1 (d) of MyoD−/− to wild-type cells. After 5 d of differentiation, cells were fixed, and stained for β-galactosidase and with antibody MF20 reactive with MHC. Note the complete absence of lacZ-labeled nuclei in myotubes containing greater than two nuclei and the normal differentiation of wild-type myotubes.

Mentions: The reduced fusion and continued proliferation of MyoD−/− myogenic cells under conditions that normally promote cell-cycle withdrawal and terminal differentiation can be hypothesized to be a consequence of cell autonomous attributes. For example, the marked reduction in M-cadherin expression (Figs. 1 d and 6 b) and the overexpression of IGF-I (Fig. 6 d) in MyoD−/− myogenic cells could both act to inhibit differentiation. Alternatively, MyoD−/− cells may have a unique developmental identity that precludes participation in the myogenic precursor cell differentiation program. To explore these possibilities, we mixed different proportions of wild-type myoblasts with lacZ-expressing MyoD−/− myogenic cells, and induced differentiation by culturing the cells in 5% horse serum for 5 d (Fig. 7). Importantly, PGK-lacZ expression is unaffected by terminal differentiation in transfected wild-type myoblasts (not shown). Strikingly, lacZ-labeled nuclei were never detected within any myotubes containing more than two nuclei (Fig. 7, b and c). Conversely, the differentiation of wild-type myocytes was completely unaffected by the presence of high numbers of MyoD−/− myogenic cells (compare Fig. 7, b and c, with Fig. 7 d). Taken together, these data support the notion that Myf-5 expression may define a distinct cell identity in the satellite cell developmental program.


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)

Strict cell autonomy in differentiated cocultures of lacZ-expressing MyoD−/− myogenic cells and  wild-type myoblasts. Primary cells  were plated in ratios of 1:0 (a), 1:4 (b),  4:1 (c), and 0:1 (d) of MyoD−/− to  wild-type cells. After 5 d of differentiation, cells were fixed, and stained for  β-galactosidase and with antibody  MF20 reactive with MHC. Note the  complete absence of lacZ-labeled nuclei in myotubes containing greater  than two nuclei and the normal differentiation of wild-type myotubes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132931&req=5

Figure 7: Strict cell autonomy in differentiated cocultures of lacZ-expressing MyoD−/− myogenic cells and wild-type myoblasts. Primary cells were plated in ratios of 1:0 (a), 1:4 (b), 4:1 (c), and 0:1 (d) of MyoD−/− to wild-type cells. After 5 d of differentiation, cells were fixed, and stained for β-galactosidase and with antibody MF20 reactive with MHC. Note the complete absence of lacZ-labeled nuclei in myotubes containing greater than two nuclei and the normal differentiation of wild-type myotubes.
Mentions: The reduced fusion and continued proliferation of MyoD−/− myogenic cells under conditions that normally promote cell-cycle withdrawal and terminal differentiation can be hypothesized to be a consequence of cell autonomous attributes. For example, the marked reduction in M-cadherin expression (Figs. 1 d and 6 b) and the overexpression of IGF-I (Fig. 6 d) in MyoD−/− myogenic cells could both act to inhibit differentiation. Alternatively, MyoD−/− cells may have a unique developmental identity that precludes participation in the myogenic precursor cell differentiation program. To explore these possibilities, we mixed different proportions of wild-type myoblasts with lacZ-expressing MyoD−/− myogenic cells, and induced differentiation by culturing the cells in 5% horse serum for 5 d (Fig. 7). Importantly, PGK-lacZ expression is unaffected by terminal differentiation in transfected wild-type myoblasts (not shown). Strikingly, lacZ-labeled nuclei were never detected within any myotubes containing more than two nuclei (Fig. 7, b and c). Conversely, the differentiation of wild-type myocytes was completely unaffected by the presence of high numbers of MyoD−/− myogenic cells (compare Fig. 7, b and c, with Fig. 7 d). Taken together, these data support the notion that Myf-5 expression may define a distinct cell identity in the satellite cell developmental program.

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
Related in: MedlinePlus