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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 differentiation potential of MyoD−/− myogenic cells.  (a) Differentiated myocytes were detected by immunostaining with antibody MF20 reactive with MHC. Incubation of wild-type (WT) myoblast  cultures in differentiation medium resulted in a rapid increase in MHC synthesis and formation of elongated  multinucleated myotubes. By contrast  MyoD-deficient cells (MyoD−/−) differentiated with reduced kinetics and  failed to form multinucleated elongated myotubes. Note the 100-fold reduced rate of spontaneous differentiation observed in MyoD−/− cultures  under growth conditions (day 0). Days  correspond to the time spent in differentiation medium before staining,  whereas day 0 represents cultures in  growth medium. (b) Percent MF20  positive cells was determined by enumeration of MHC expressing differentiated myocytes by immunostaining  with antibody MF20. Note the delayed  and reduced kinetics of differentiation  in the absence of MyoD. (c) Calculation of fusion indices as percent cells  containing two or more nuclei within a  differentiated myocyte confirmed that  MyoD−/− myoblasts were severely  deficient in fusion capacity with the  majority of differentiated myocytes  containing a single nuclei. The error  bars represent the standard error of  the mean from three independently derived primary cultures.
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Figure 2: Reduced differentiation potential of MyoD−/− myogenic cells. (a) Differentiated myocytes were detected by immunostaining with antibody MF20 reactive with MHC. Incubation of wild-type (WT) myoblast cultures in differentiation medium resulted in a rapid increase in MHC synthesis and formation of elongated multinucleated myotubes. By contrast MyoD-deficient cells (MyoD−/−) differentiated with reduced kinetics and failed to form multinucleated elongated myotubes. Note the 100-fold reduced rate of spontaneous differentiation observed in MyoD−/− cultures under growth conditions (day 0). Days correspond to the time spent in differentiation medium before staining, whereas day 0 represents cultures in growth medium. (b) Percent MF20 positive cells was determined by enumeration of MHC expressing differentiated myocytes by immunostaining with antibody MF20. Note the delayed and reduced kinetics of differentiation in the absence of MyoD. (c) Calculation of fusion indices as percent cells containing two or more nuclei within a differentiated myocyte confirmed that MyoD−/− myoblasts were severely deficient in fusion capacity with the majority of differentiated myocytes containing a single nuclei. The error bars represent the standard error of the mean from three independently derived primary cultures.

Mentions: To evaluate the differentiation potential of the MyoD−/− myogenic cultures the extent of myogenic differentiation was assessed at the cellular level by immunostaining cultures fixed at daily intervals after mitogen withdrawal with antibody MF20 reactive with MHC (Fig. 2 a). Importantly, this analysis was performed on three independently isolated wild-type and MyoD−/− primary myogenic cultures. After MF20 immunostaining, the proportion of MHC-positive cells and the fusion index of both wild-type and MyoD−/− cultures were assessed (Fig. 2, b and c).


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 differentiation potential of MyoD−/− myogenic cells.  (a) Differentiated myocytes were detected by immunostaining with antibody MF20 reactive with MHC. Incubation of wild-type (WT) myoblast  cultures in differentiation medium resulted in a rapid increase in MHC synthesis and formation of elongated  multinucleated myotubes. By contrast  MyoD-deficient cells (MyoD−/−) differentiated with reduced kinetics and  failed to form multinucleated elongated myotubes. Note the 100-fold reduced rate of spontaneous differentiation observed in MyoD−/− cultures  under growth conditions (day 0). Days  correspond to the time spent in differentiation medium before staining,  whereas day 0 represents cultures in  growth medium. (b) Percent MF20  positive cells was determined by enumeration of MHC expressing differentiated myocytes by immunostaining  with antibody MF20. Note the delayed  and reduced kinetics of differentiation  in the absence of MyoD. (c) Calculation of fusion indices as percent cells  containing two or more nuclei within a  differentiated myocyte confirmed that  MyoD−/− myoblasts were severely  deficient in fusion capacity with the  majority of differentiated myocytes  containing a single nuclei. The error  bars represent the standard error of  the mean from three independently derived primary cultures.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132931&req=5

Figure 2: Reduced differentiation potential of MyoD−/− myogenic cells. (a) Differentiated myocytes were detected by immunostaining with antibody MF20 reactive with MHC. Incubation of wild-type (WT) myoblast cultures in differentiation medium resulted in a rapid increase in MHC synthesis and formation of elongated multinucleated myotubes. By contrast MyoD-deficient cells (MyoD−/−) differentiated with reduced kinetics and failed to form multinucleated elongated myotubes. Note the 100-fold reduced rate of spontaneous differentiation observed in MyoD−/− cultures under growth conditions (day 0). Days correspond to the time spent in differentiation medium before staining, whereas day 0 represents cultures in growth medium. (b) Percent MF20 positive cells was determined by enumeration of MHC expressing differentiated myocytes by immunostaining with antibody MF20. Note the delayed and reduced kinetics of differentiation in the absence of MyoD. (c) Calculation of fusion indices as percent cells containing two or more nuclei within a differentiated myocyte confirmed that MyoD−/− myoblasts were severely deficient in fusion capacity with the majority of differentiated myocytes containing a single nuclei. The error bars represent the standard error of the mean from three independently derived primary cultures.
Mentions: To evaluate the differentiation potential of the MyoD−/− myogenic cultures the extent of myogenic differentiation was assessed at the cellular level by immunostaining cultures fixed at daily intervals after mitogen withdrawal with antibody MF20 reactive with MHC (Fig. 2 a). Importantly, this analysis was performed on three independently isolated wild-type and MyoD−/− primary myogenic cultures. After MF20 immunostaining, the proportion of MHC-positive cells and the fusion index of both wild-type and MyoD−/− cultures were assessed (Fig. 2, b and c).

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