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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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Primary MyoD−/− myogenic cells exhibit a premyoblastic cellular phenotype. (a) Immunostaining revealed that  both wild-type (Wildtype) and MyoD-deficient myogenic cells  (MyoD−/−) expressed the c-met receptor tyrosine kinase, a  marker for satellite cells and proliferating myoblasts. (b) Immunohistochemical analysis for desmin expression revealed a  marked reduction in the proportion of cells in MyoD−/− cultures expressed desmin relative to wild-type cultures. (c) Normal  level and localization of β-catenin in MyoD−/− cells as revealed  by immunofluorescence. (d) Immunohistochemical detection of  M-cadherin revealed low level expression of M-cadherin in  MyoD−/− cells relative to primary wild-type myoblasts.
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Figure 1: Primary MyoD−/− myogenic cells exhibit a premyoblastic cellular phenotype. (a) Immunostaining revealed that both wild-type (Wildtype) and MyoD-deficient myogenic cells (MyoD−/−) expressed the c-met receptor tyrosine kinase, a marker for satellite cells and proliferating myoblasts. (b) Immunohistochemical analysis for desmin expression revealed a marked reduction in the proportion of cells in MyoD−/− cultures expressed desmin relative to wild-type cultures. (c) Normal level and localization of β-catenin in MyoD−/− cells as revealed by immunofluorescence. (d) Immunohistochemical detection of M-cadherin revealed low level expression of M-cadherin in MyoD−/− cells relative to primary wild-type myoblasts.

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)

Primary MyoD−/− myogenic cells exhibit a premyoblastic cellular phenotype. (a) Immunostaining revealed that  both wild-type (Wildtype) and MyoD-deficient myogenic cells  (MyoD−/−) expressed the c-met receptor tyrosine kinase, a  marker for satellite cells and proliferating myoblasts. (b) Immunohistochemical analysis for desmin expression revealed a  marked reduction in the proportion of cells in MyoD−/− cultures expressed desmin relative to wild-type cultures. (c) Normal  level and localization of β-catenin in MyoD−/− cells as revealed  by immunofluorescence. (d) Immunohistochemical detection of  M-cadherin revealed low level expression of M-cadherin in  MyoD−/− cells relative to primary wild-type myoblasts.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Primary MyoD−/− myogenic cells exhibit a premyoblastic cellular phenotype. (a) Immunostaining revealed that both wild-type (Wildtype) and MyoD-deficient myogenic cells (MyoD−/−) expressed the c-met receptor tyrosine kinase, a marker for satellite cells and proliferating myoblasts. (b) Immunohistochemical analysis for desmin expression revealed a marked reduction in the proportion of cells in MyoD−/− cultures expressed desmin relative to wild-type cultures. (c) Normal level and localization of β-catenin in MyoD−/− cells as revealed by immunofluorescence. (d) Immunohistochemical detection of M-cadherin revealed low level expression of M-cadherin in MyoD−/− cells relative to primary wild-type myoblasts.
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.

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