CELL HETEROGENEITY UPON MYOGENIC DIFFERENTIATION - DOWN-REGULATION OFMYOD AND MYF-5 GENERATES RESERVE CELLS

When a proliferating myoblast culture is induced to differentiate by d eprivation of serum in the medium, a significant proportion of cells e scape from terminal differentiation, while the rest of the cells diffe rentiate. Using C2C12 mouse myoblast cells, this heterogeneity observe d upon differentiation was investigated with an emphasis on the myogen ic regulatory factors. The differentiating part of the cell population followed a series of well-described events, including expression of m yogenin, p21(WAF1), and contractile proteins, permanent withdrawal fro m the cell cycle and cell fusion, whereas the rest of the cells did no t initiate any of these events, Interestingly, the latter cells showed an undetectable or greatly reduced level of MyoD and Myf-5 expression , which had been originally expressed in the undifferentiated prolifer ating myoblasts. When these undifferentiated cells mere isolated and r eturned to the growth conditions, they progressed through the cell cyc le and regained MyoD expression. These cells demonstrated identical fe atures with the original culture on the deprivation of serum. They pro duced both MyoD-positive differentiating and MyoD-negative undifferent iated populations once again. Thus the undifferentiated cells in the s erum-deprived culture were designated 'reserve cells'. Upon serum depr ivation, MyoD expression rapidly decreased as a result of down-regulat ion in approximately 50% of the cells, After this heterogenization, My oD positive cells expressed myogenin, which is the earliest known even t of terminal differentiation and marks irreversible commitment to thi s, while MyoD-negative cells did not differentiate and became the rese rve cells. We also demonstrated that ectopic expression of MyoD conver ted the reserve cells to differentiating cells, indicating that down-r egulation of MyoD is a causal event in the formation of reserve cells.