ABSTRACT. During myogenic differentiation of quail myoblasts transformed with a temperature sensitive mutant of Rous sarcoma virus (QM-RSV cells), it was observed that myogenin was continuously expressed in myotubes. In contrast, in myotubes derived from quail primary cells (parent cells of QM-RSVcells), myogenin expression was seen only in the myotubes not having striated structures comprised of myofibrils, but not in myotubes having the structures. The fact that there are not striated structures of myofibrils formed in myotubes derived from QM-RSV cells suggests that these myotubes stop at an immature state prior to the final differentiation. These results also suggest that myogenin is not only required for early steps during differentiation but also maturation steps of myotubes. To clarify the roles of myogeninafter myotubeformation and maturation, myotubes derived from QM-RSVcells were treated with N,N'-hexamethylene bisacetamide (HMBA)or incubated at 35.5°C, a permissive temperature of RSVfor suppression of myogenin expression. Ontreatment with HMBA, myogenin expression disappeared and myotubes began to incorporate 5-bromo-2'-deoxyuridine (BrdU) into the nuclei, whereas the expression remained in manymyotubes on culture at 35.5°C. These results suggest that immature myotubes can return to an up step of differentiation, prior to the commitment step with HMBA treatment, but not with culture at 35.5°C.Skeletal myogenesis involves an initial period of myoblast replication, before undergoing terminal differentiation. This process involves the cessation of DNAsynthesis, activation of muscle-specific gene expression, and the fusion of single cells into multinucleated myotubes (2, 23,27,28,32,33,34,44).The MyoDfamily, which includes MyoD (9), myogenin (10, 43), myf5 (3), and MRF4/myf6/herculin (4, 30, 36), governs myogenic differentiation, and shares the remarkable properties of myogenic differentiation when expressed in non-muscle cells (ll, 31, 35, 41, 42). Studies of gene targeting of myogenin indicate that myogenin has unique functions in the muscle differentiation, especially for the commitment to terminal differentiation and the formation of myotubes (5, 6, 17,33, 37). Further its function is apparently not complemented by other members of the MyoDfamily, suggesting that it is a key regulator amongthe muscle specific transcription factors for the terminal differentiation. Wehave been studying myoblast differentiation using quail myoblasts (QM)transformed with a temperature

Tyrosine kinase activity of v-Src from Rous sarcoma virus (RSV) inhibits the differentiation of quail myoblasts. To clarify the inhibitory mechanism, we focused on the signaling pathways from v-Src. When the activation of the Ras/MAP (mitogen-activated protein) kinase pathway was inhibited by a dominant-negative mutant of Ras or PD98059, a specific inhibitor of p42 MAP kinase kinase, differentiation was restored; muscle specific proteins were expressed and myotubes formed even under active conditions of v-Src. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (P13-kinase), showed no effects on the inhibition by v-Src. These findings suggest that v-Src activates the Ras/MAP kinase signaling pathway, but not the P13-kinase pathway, and inhibits the differentiation. However, the myotubes derived from the dominant-negative Ras did not form actin fibers, suggesting that myofibril assembly is regulated by other pathway(s) from v-Src.