The transition from proliferation to differentiation is delayed in satellite cells from mice lacking MyoD

Satellite cells from adult rat muscle coexpress proliferating cell nuclear antigen and MyoD upon entry into the cell cycle, suggesting that MyoD plays a role during the recruitment of satellite cells. Moreover, the finding th at muscle regeneration is compromised in MyoD-/- mice, has provided evidenc e for the role of MyoD during myogenesis in adult muscle. In order to gain further insight into the role of MyoD during myogenesis in the adult, we co mpared satellite cells from MyoD-/- and wildtype mice as they progress thro ugh myogenesis in single-myofiber cultures and in tissue-dissociated cell c ultures (primary cultures). Satellite cells undergoing proliferation and di fferentiation were traced immunohistochemically using antibodies against va rious regulatory proteins. In addition, an antibody against the mitogen-act ivated protein kinases ERK1 and ERK2 was used to localize the cytoplasm of the fiber-associated satellite cells regardless of their ability to express specific myogenic regulatory factor proteins. We show that during the init ial days in culture the myofibers isolated from both the MyoD-/- and the wi ldtype mice contain the same number of proliferating, ERK+ satellite cells. However, the MyoD-/- satellite cells continue to proliferate and only a ve ry small number of cells transit into the myogenin+ state, whereas the wild type cells exit the proliferative compartment and enter the myogenin+ stage . Analyzing tissue-dissociated cultures of MyoD-/- satellite cells, we iden tified numerous cells whose nuclei were positive for the Myf5 protein. In c ontrast, quantification of Myf5+ cells in the wildtype cultures was difficu lt due to the low level of Myf5 protein present. The Myf5+ cells in the Myo D-/- cultures were of ten positive for desmin, similar to the MyoD+ cells i n the wildtype cultures. Myogenin+ cells were identified in the MyoD-/- pri mary cultures, but their appearance was delayed compared to the wildtype ce lls. These "delayed" myogenin+ cells can express other differentiation mark ers such as MEF2A and cyclin D3 and fuse into myotubes. Taken together, our studies suggest that the presence of MyoD is critical for the normal progr ession of satellite cells into the myogenin+, differentiative state. It is further proposed that the Myf5+/MyoD- phenotype may represent the myogenic stem cell compartment which is capable of maintaining the myogenic precurso r pool in the adult muscle. (C) 1999 Academic Press.