Limits...
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.

Show MeSH
Rescue of the differentiation deficiency of MyoD−/−  myogenic cells by forced expression of MyoD. (a) Western blot  analysis revealed the expression of MyoD protein in wild-type  cells (WT) and no expression in MyoD−/− cultures or MyoD−/−  cells transfected with a selectable vector alone (PGK-Puro).  However, MyoD-transfected pools of MyoD−/− cells (MyoD+)  expressed readily detectable MyoD protein as did C2C12 myoblasts. (b) In growth medium, MyoD+ cells exhibited a refractile  compact morphology typical of wild-type primary myoblasts and  distinct from the stellate fibroblastlike morphology of MyoD−/−  cells (growth). MyoD+ cultures exposed to differentiation medium for 3 d exhibited increased numbers of differentiated myocytes as detected with antiserum MF20 reactive with MHC, and  restored formation of elongated bipolar multinucleated myotubes (day 3/MF20). (c) Determination of fusion index indicated  a fivefold increase in the fusion potential of MyoD+ cells relative  to untransfected MyoD−/− cells and similar to wild-type (WT)  cultures. Fusion indices were calculated as described in Fig. 4.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132931&req=5

Figure 8: Rescue of the differentiation deficiency of MyoD−/− myogenic cells by forced expression of MyoD. (a) Western blot analysis revealed the expression of MyoD protein in wild-type cells (WT) and no expression in MyoD−/− cultures or MyoD−/− cells transfected with a selectable vector alone (PGK-Puro). However, MyoD-transfected pools of MyoD−/− cells (MyoD+) expressed readily detectable MyoD protein as did C2C12 myoblasts. (b) In growth medium, MyoD+ cells exhibited a refractile compact morphology typical of wild-type primary myoblasts and distinct from the stellate fibroblastlike morphology of MyoD−/− cells (growth). MyoD+ cultures exposed to differentiation medium for 3 d exhibited increased numbers of differentiated myocytes as detected with antiserum MF20 reactive with MHC, and restored formation of elongated bipolar multinucleated myotubes (day 3/MF20). (c) Determination of fusion index indicated a fivefold increase in the fusion potential of MyoD+ cells relative to untransfected MyoD−/− cells and similar to wild-type (WT) cultures. Fusion indices were calculated as described in Fig. 4.

Mentions: As suggested by our previous observations (Megeney et al., 1996), MyoD−/− cultures displayed a stellate flattened morphology with an enlarged cytoplasm and extended cytosolic processes. By contrast, wild-type cells were highly refractile under phase-contrast microscopy and displayed the rounded morphology and small compact cytoplasm characteristic of primary myoblasts (see Figs. 1 and 8).


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)

Rescue of the differentiation deficiency of MyoD−/−  myogenic cells by forced expression of MyoD. (a) Western blot  analysis revealed the expression of MyoD protein in wild-type  cells (WT) and no expression in MyoD−/− cultures or MyoD−/−  cells transfected with a selectable vector alone (PGK-Puro).  However, MyoD-transfected pools of MyoD−/− cells (MyoD+)  expressed readily detectable MyoD protein as did C2C12 myoblasts. (b) In growth medium, MyoD+ cells exhibited a refractile  compact morphology typical of wild-type primary myoblasts and  distinct from the stellate fibroblastlike morphology of MyoD−/−  cells (growth). MyoD+ cultures exposed to differentiation medium for 3 d exhibited increased numbers of differentiated myocytes as detected with antiserum MF20 reactive with MHC, and  restored formation of elongated bipolar multinucleated myotubes (day 3/MF20). (c) Determination of fusion index indicated  a fivefold increase in the fusion potential of MyoD+ cells relative  to untransfected MyoD−/− cells and similar to wild-type (WT)  cultures. Fusion indices were calculated as described in Fig. 4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132931&req=5

Figure 8: Rescue of the differentiation deficiency of MyoD−/− myogenic cells by forced expression of MyoD. (a) Western blot analysis revealed the expression of MyoD protein in wild-type cells (WT) and no expression in MyoD−/− cultures or MyoD−/− cells transfected with a selectable vector alone (PGK-Puro). However, MyoD-transfected pools of MyoD−/− cells (MyoD+) expressed readily detectable MyoD protein as did C2C12 myoblasts. (b) In growth medium, MyoD+ cells exhibited a refractile compact morphology typical of wild-type primary myoblasts and distinct from the stellate fibroblastlike morphology of MyoD−/− cells (growth). MyoD+ cultures exposed to differentiation medium for 3 d exhibited increased numbers of differentiated myocytes as detected with antiserum MF20 reactive with MHC, and restored formation of elongated bipolar multinucleated myotubes (day 3/MF20). (c) Determination of fusion index indicated a fivefold increase in the fusion potential of MyoD+ cells relative to untransfected MyoD−/− cells and similar to wild-type (WT) cultures. Fusion indices were calculated as described in Fig. 4.
Mentions: As suggested by our previous observations (Megeney et al., 1996), MyoD−/− cultures displayed a stellate flattened morphology with an enlarged cytoplasm and extended cytosolic processes. By contrast, wild-type cells were highly refractile under phase-contrast microscopy and displayed the rounded morphology and small compact cytoplasm characteristic of primary myoblasts (see Figs. 1 and 8).

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