An analysis of myogenesis by the use of fluorescent antimyosin

Abstract

Antibodies against myosin of adult chicken skeletal muscle were labelled with fluorescein and used as staining reagents to analyze the development of trunk myoblasts in the chick embryo. Myoblasts from the brachial myotomes were studied in three ways: (a) Specimens were fixed, sectioned, and stained with iron-hematoxylin. (b) Living myoblasts, and myoblasts prepared by glycerol extraction, were teased and examined by phase contrast microscopy. (c) Embryo trunks were treated with fluorescent antimyosin or with a control solution of fluorescent normal globulin, and were examined by fluorescence and phase contrast microscopy. Both glycerol-extracted and fixed materials were used. Cross-striated myofibrils appeared first in stage 16 to 17 embryos in the series studied by antimyosin staining and fluorescence microscopy. Striated myofibrils appeared first in stage 18 to 19 embryos, in the series stained by iron-hematoxylin, and at stage 22 to 23, in the series studied by glycerol extraction and phase contrast microscopy. In each series, myofibrils without apparent cross-striations were detected shortly before cross-striations were observed. Specific staining by antimyosin occurred only in differentiating myoblasts. Within the myoblasts antimyosin staining was confined to the A bands of the slender myofibrils. The following observations suggest that the first delicate striated structure to appear in the early 3 day myoblast was remarkably mature: (1) The sarcomere pattern both in length and in internal detail, was similar to that of adult muscle. (2) The distribution of myosin, as revealed by antimyosin staining, was the same in the embryonic as in the mature myofibril. (3) Glycerol-extracted myoblasts contracted vigorously on exposure to ATP. The changes in sarcomere band pattern were indistinguishable from those occurring during contraction of adult muscle induced by ATP. (4) ATP contraction was blocked by prior antimyosin staining in embryonic myoblasts as in mature muscle. It is suggested that the early myofibril grows laterally as a thin sheet associated with the sarcolemma, and that growth in length occurs in the growth tips of the elongating myoblast.