Sabourin L A, Girgis-Gabardo A, Seale P, Asakura A, Rudnicki M A
Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1.
J Cell Biol. 1999 Feb 22;144(4):631-43. doi: 10.1083/jcb.144.4.631.
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.
为深入了解MyoD基因敲除肌肉的再生缺陷，我们研究了培养的MyoD基因敲除肌原性细胞的生长和分化情况。原代MyoD基因敲除肌原性细胞呈现出星状形态，与野生型成肌细胞紧密的形态不同，并且表达c-met，一种在卫星细胞中表达的受体酪氨酸激酶。然而，MyoD基因敲除肌原性细胞不表达结蛋白，一种通常在体外培养的成肌细胞和体内肌原性前体细胞中表达的中间丝蛋白。Northern分析表明，增殖的MyoD基因敲除肌原性细胞中Myf-5的表达水平高四倍，PEA3（一种在新激活的卫星细胞中表达的ETS结构域转录因子）的表达水平高六倍。在正常诱导分化的条件下，MyoD基因敲除细胞继续增殖，动力学延迟，产生的主要为单核肌细胞数量减少。Northern分析显示，肌细胞生成素、MRF4和其他分化特异性标志物的诱导延迟，尽管p21正常上调。在MyoD基因敲除肌原性细胞中，M-钙黏蛋白mRNA的表达严重降低，而IGF-1的表达显著增加。用lacZ标记的MyoD基因敲除细胞与野生型成肌细胞混合，显示出分化潜能的严格自主性。转染MyoD表达盒可恢复细胞形态并挽救分化缺陷。我们对这些数据的解释是，MyoD基因敲除肌原性细胞代表静止卫星细胞和肌原性前体细胞之间的一个中间阶段。
