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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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Upregulation of Myf-5 and delayed expression of myogenin and MRF4 in MyoD−/− myogenic cells. Northern analysis  revealed an absence of MyoD mRNA (a) in MyoD−/− cells (−/−),  and a fourfold upregulation of Myf-5 mRNA (b) relative to wild-type myoblasts (WT). Upon differentiation, myogenin expression  was upregulated and reduced (c). Similarly, MRF4 expression  was delayed about 1 d in MyoD−/− cultures after transfer to differentiation medium (d). Day 0 denotes samples isolated from  cells in growth medium. RNA samples were prepared 1, 2, 3, 4,  and 5 d after the transfer of the cells to differentiation medium  (DM). 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. (e) Western analysis of lysates prepared from wild-type  or MyoD−/− cultures in growth medium with anti–Myf-5, anti-MyoD, and antimyogenin antibodies.
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Figure 4: Upregulation of Myf-5 and delayed expression of myogenin and MRF4 in MyoD−/− myogenic cells. Northern analysis revealed an absence of MyoD mRNA (a) in MyoD−/− cells (−/−), and a fourfold upregulation of Myf-5 mRNA (b) relative to wild-type myoblasts (WT). Upon differentiation, myogenin expression was upregulated and reduced (c). Similarly, MRF4 expression was delayed about 1 d in MyoD−/− cultures after transfer to differentiation medium (d). Day 0 denotes samples isolated from cells in growth medium. RNA samples were prepared 1, 2, 3, 4, and 5 d after the transfer of the cells to differentiation medium (DM). 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. (e) Western analysis of lysates prepared from wild-type or MyoD−/− cultures in growth medium with anti–Myf-5, anti-MyoD, and antimyogenin antibodies.

Mentions: The expression of the MRFs was investigated to elucidate the regulatory relationships and the potential for functional compensation in the absence of MyoD. Northern analysis of the expression pattern of the four MRFs confirmed that MyoD was expressed at high levels in wild-type myoblasts and was absent in the MyoD-deficient myogenic cells. Furthermore, densitometric analysis and normalization to 18S rRNA revealed that MyoD was somewhat downregulated during the differentiation of wild-type cells (Fig. 4 a). Previously, we observed a 3.5-fold increase in Myf-5 mRNA in newborn and adult muscles in vivo (Rudnicki et al., 1993). We similarly observed a fourfold increase in Myf-5 mRNA in growing MyoD−/− myogenic cells relative to wild-type myoblasts, supporting the hypothesis that MyoD negatively regulates Myf-5 expression (Fig. 4 b). In wild-type cells, Myf-5 mRNA levels decreased about twofold after differentiation, whereas in MyoD−/− cultures, Myf-5 mRNA levels were upregulated about twofold after mitogen withdrawal (Fig. 4 b). In addition, consistent with the observed 100-fold reduction in the rate of spontaneous differentiation, MyoD−/− myogenic cells expressed fivefold lower levels of myogenin mRNA in growth medium relative to wild-type myoblasts (Fig. 4 c). After the induction of differentiation, the relative levels of myogenin mRNA in MyoD−/− cells remained significantly reduced relative to wild-type cultures (Fig. 4 c). In wild-type cells, the level of MRF4 mRNA remained unchanged until day 2 of differentiation and was thereafter upregulated about sevenfold. By contrast, MRF4 mRNA was upregulated in differentiating MyoD−/− cultures to levels approximately two- to threefold lower than that of wild-type cells (Fig. 4 d).


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)

Upregulation of Myf-5 and delayed expression of myogenin and MRF4 in MyoD−/− myogenic cells. Northern analysis  revealed an absence of MyoD mRNA (a) in MyoD−/− cells (−/−),  and a fourfold upregulation of Myf-5 mRNA (b) relative to wild-type myoblasts (WT). Upon differentiation, myogenin expression  was upregulated and reduced (c). Similarly, MRF4 expression  was delayed about 1 d in MyoD−/− cultures after transfer to differentiation medium (d). Day 0 denotes samples isolated from  cells in growth medium. RNA samples were prepared 1, 2, 3, 4,  and 5 d after the transfer of the cells to differentiation medium  (DM). 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. (e) Western analysis of lysates prepared from wild-type  or MyoD−/− cultures in growth medium with anti–Myf-5, anti-MyoD, and antimyogenin antibodies.
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Figure 4: Upregulation of Myf-5 and delayed expression of myogenin and MRF4 in MyoD−/− myogenic cells. Northern analysis revealed an absence of MyoD mRNA (a) in MyoD−/− cells (−/−), and a fourfold upregulation of Myf-5 mRNA (b) relative to wild-type myoblasts (WT). Upon differentiation, myogenin expression was upregulated and reduced (c). Similarly, MRF4 expression was delayed about 1 d in MyoD−/− cultures after transfer to differentiation medium (d). Day 0 denotes samples isolated from cells in growth medium. RNA samples were prepared 1, 2, 3, 4, and 5 d after the transfer of the cells to differentiation medium (DM). 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. (e) Western analysis of lysates prepared from wild-type or MyoD−/− cultures in growth medium with anti–Myf-5, anti-MyoD, and antimyogenin antibodies.
Mentions: The expression of the MRFs was investigated to elucidate the regulatory relationships and the potential for functional compensation in the absence of MyoD. Northern analysis of the expression pattern of the four MRFs confirmed that MyoD was expressed at high levels in wild-type myoblasts and was absent in the MyoD-deficient myogenic cells. Furthermore, densitometric analysis and normalization to 18S rRNA revealed that MyoD was somewhat downregulated during the differentiation of wild-type cells (Fig. 4 a). Previously, we observed a 3.5-fold increase in Myf-5 mRNA in newborn and adult muscles in vivo (Rudnicki et al., 1993). We similarly observed a fourfold increase in Myf-5 mRNA in growing MyoD−/− myogenic cells relative to wild-type myoblasts, supporting the hypothesis that MyoD negatively regulates Myf-5 expression (Fig. 4 b). In wild-type cells, Myf-5 mRNA levels decreased about twofold after differentiation, whereas in MyoD−/− cultures, Myf-5 mRNA levels were upregulated about twofold after mitogen withdrawal (Fig. 4 b). In addition, consistent with the observed 100-fold reduction in the rate of spontaneous differentiation, MyoD−/− myogenic cells expressed fivefold lower levels of myogenin mRNA in growth medium relative to wild-type myoblasts (Fig. 4 c). After the induction of differentiation, the relative levels of myogenin mRNA in MyoD−/− cells remained significantly reduced relative to wild-type cultures (Fig. 4 c). In wild-type cells, the level of MRF4 mRNA remained unchanged until day 2 of differentiation and was thereafter upregulated about sevenfold. By contrast, MRF4 mRNA was upregulated in differentiating MyoD−/− cultures to levels approximately two- to threefold lower than that of wild-type cells (Fig. 4 d).

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