Biochemical Research Laboratory, The Dow Chemical Co., Midland, Michigan, USA*
Muscle owes its morphological and biochemical identity to proteins such as actin, myosin, tropomyosin, etc.(1), and it is reasonable to suspect that these molecules are somehow represented in the genome. Until rather recently, it was generally believed that all new proteins are manufactured by freshly made messenger RNA's of exceedingly short half-lives. However, recently it has become evident that there are at least a few systems in which genetic messages are sent many generations before they are used (2). Is this the case with myogenesis, or is the necessary information for muscle differentiation sent shortly before it is called into play?Abstract
Actinomycin D, when appropriately timed, inhibits the regeneration of skeletal (striated) muscle in the mouse.
Actinomycin D seemed to offer an experimental to the question. The antibiotic complexes with DNA and thereby prevents the synthesis of RNA. At the same time, already-formed templates continue in the manufacture of proteins and DNA replication occurs (3).
The myogenic system selected for investigation was regenerating mouse skeletal muscle. This choice was dictated primarily by the fact that there is a great deal of synchrony attending the reaction: during the second to third day after muscle injury a class of highly proliferative mononucleated ells appears in the wound (4, 5), and on the fourth day newly developing muscle fibers are in abundance. If these mononucleated cells are arrested by colchicine new muscle fibers fail to appear on schedule (5). Moreover, if the mononucleated cells in question incorporate tritiated thymidine, the nuclei of differentiating muscle fibers show radioactivity after autoradiography (6, 7). Thus, it would seem that in the 3-4 day interval after wounding many presumptive muscle cells are achieving the differentiated state.
Single transections were inflicted in the belly of the tibialis anterior muscles of female Swiss-Webster mice, weighing 25-30 g, under light methoxyflurane anesthesia. Animals received a single intraperitoneal dose of 0.99 micrograms of actinomycin D (kindly donated by Merck and Co.) at 24, 48, 72 or 100 h after transection. Tissues were fixed for histological investigation five days after wounding. Day five was chosen because at this time there normally are scores of regenerating fibers in each section through the wound (4-6).
Large numbers of typical five-day regenerating myotubes were encountered in all sections of the 24, 48 and 100 h series (FIG. 1). In of five specimens given actinomycin at 72 h, regeneration was severely depressed (Fig. 2) and was represented by only an occasional, single, immature muscle fiber. Although few in numbers, these fibers were morphologically normal.
The findings indicated that myogenesis depends on a new round of RNA synthesis (presumably messenger) in the interval immediately preceding the histological advent of muscle as a tissue. This, in turn, suggests that muscle is specifically represented in the genome and that the information for making it is communicated shortly before it is used. Along this same line, it is noted muscle regeneration can be prevented by X-irradiation, but sensitivity to the effective does is lost somewhere on the third day; that is, after the genetic messages seem to have been sent (8).
Fig. 2. Five-day wound coagulum from an animal injected with actinomycin D 72 h after muscle transection. This representative field is devoid of newly differentiating muscle fibers. In normal regeneration and in cases injected with actinomycin D at 24, 48 and 100 h such sections exhibited scores of differentiating muscle fibers. (x 100).
**These are admitedly terrible pictures, but this is very close to how they appeared when published. Nature, in spite of being the Number One scientific journal in all the world, even today, its photographic reproductions are notoriously terrible. Still you can make out the dark rows of nuclei within the myotubes; for a better appreciation of what normal muscle regeneration looks like under the microscope, see the pictures at this location.