Characterization of Myogenin Expression in Myotubes Derived from Quail Myoblasts Transformed with a Temperature Sensitive Mutant of Rous Sarcoma Virus.

Abstract

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

Characterization of Heterokaryons between Skeletal Myoblasts and Somatic Cells Formed by Fusion with HVJ (Sendai Virus): Effects on Myogenic Differentiation.

“…2C-d). This expression pattern of myogenin was similar to that in the course of spontaneous differentiation of mononucleated QM-RSV cells at 41°C (Hirayama et al, 1997;Isobe et al, 1998). Myofibrils were also formed normally within 24 hr and developed along the long axis of myotubes until 48 hr in the homokaryons (Fig.…”

“…Nuclei, F-actin, and myogenin were triply stained as described previously with some modifications (Kim et al, 1995;Isobe et al, 1998). The cells were fixed with 3.7% formaldehyde in PBS for 30 min at 4°C and then permeabilized with 0.5% Triton X-100 for 5 min at 4°C.…”

“…The cells were treated with a mixture of mouse serum against chick myogenin (´100) and the supernatant of a hybridoma, F5D, that produces an antibody to rat myogenin, for 1 hr at 4°C. Mouse serum to chick myogenin was prepared in our laboratory (Isobe et al, 1998) and did not react with rodent myogenin. F5D developed by Dr. W. E. Wright (University of Texas Southwestern Medical Center, Dallas, TX, USA) (Wright et al, 1996) was obtained from the Developmental Studies Hybridoma Bank (DSHB) maintained by the University of Iowa (Department of Biological Sciences, Iowa City, IA).…”

“…We have been studying myogenic differentiation, mainly focusing on the step of myoblast fusion for myotube formation, using quail myoblasts (QM) transformed with a temperature-sensitive mutant of Rous sarcoma virus (RSV), QM-RSV cells (Kim et al, 1992a, b;Saiuchi et al, 1993;Hyodo and Kim, 1994;Kim et al, 1995;Inoue-Hyodo and Kim, 1996;Hirayama et al, 1997;Isobe et al, 1998;Hirayama et al, 1999Hirayama et al, , 2000a. The differentiation of QM-RSV cells can be controlled by the culture temperature (Kim et al, 1992a, b).…”

“…However, the cells begin to differentiate on shift up to 41°C, a non-permissive temperature for RSV. After shift up to 41°C, the cells express myogenin from about 8 hr (Hirayama et al, 1997;Isobe et al, 1998), acquire commitment for myotube formation in 10-12 hr (Kim et al, 1992b), and form myotubes within 24 hr (Kim et al, 1992a, b). Myofibrils are constructed with mature myotubes (Kim et al, 1995;Hirayama et al, 2000a).…”