Muscle differentiation and macromolecular synthesis

Cytodifferentiation of skeletal muscle has been studied in cell cultures derived from leg muscle of 12‐day chicken embryos. Myogenesis in cell culture closely simulates myogenesis in vivo , but is more highly synchronized. Massive cell fusion occurs in control cultures between the second and third days in vitro , during which time most of the myoblasts are swept into syncytia. On successive days, the syncytia mature into cross‐striated muscle fibers, and the cultures are progressively overgrown by fibroblastic cells. Myosin‐containing cells can be detected at any time by immunofluorescence, and myosin has been measured by quantitative immunological precipitation as early as 3 days in vitro , a few hours after fusion. Myosin in the cultures increases over the next few days, and this is reflected in the rate of incorporation of labeled amino acids into immunologically precipitable myosin. Creatine kinase, assayed spectrophotometrically by linked dehydrogenase reactions, shows a similar pattern: measurable early but rapidly increasing in activity after fusion. That this increase in myosin and creatine kinase is strictly a function of the multinuclear cells is demonstrated by experiments in which the mononuclear cell population has been drastically reduced by treatment with 5‐fluorodeoxyuridine shortly after fusion. Myosin synthesis has not been detectable in cells prevented from fusing by growth in 5‐bromo‐deoxyuridine, but low levels of creatine kinase have been demonstrated. Newly formed muscle fibers incorporate precursors into RNA at lower rates than do mononuclear cells. The relationship of this change in RNA synthesis to the formation of muscle proteins remains obscure.