Electron microscopic observations of muscle dedifferentiation in regenerating Amblystoma limbs

Abstract

Regenerating limbs of Amblystoma punctatum larvae were fixed in osmic acid, sectioned, and studied with the electron microscope. The present report describes the changes which occur in the syncytial muscle fibers of the stump as they transform into undifferentiated, mononucleate blastema cells during the 2–6-day period after amputation. Only the nucleated cell units derived from the dissociating muscle fiber complete the process of dedifferentiation. Anucleate fragments apparently are lysed. Nuclei become rounded in shape, the chromatin more diffusely granular, and nucleoli more prominent. The nucleocytoplasmic ratio is increased by loss of cytoplasm by fragmentation during dedifferentiation. Both the intracellular and extracellular products of cell differentiation are lost during dedifferentiation. Myofibrils fragment along the A and I bands and then disintegrate. The Z substance behaves as a distinct extrafilamentous component during disorganization of the myofibril, and its behavior is compatible with the concept that it is an adhesive material serving normally to bind sarcomeres together. Individual myofibrils, undetected heretofore, persist in the cytoplasm of some of the mononucleate cells for a short period after their separation from the muscle fiber. Thus there remains no doubt that mononucleate cells in the vicinity of intact stump muscle do arise from multinucleate muscle fibers. By the time the transformation to blastema cell is completed, all the myofibrils have disappeared from the cytoplasm. The new plasma membranes separating muscle fragments appear to be formed by fusion of small vesicles which are probably derived largely from the endoplasmic reticulum. The endoplasmic reticulum, which is highly developed in differentiated muscle, breaks up into small vesicles in dedifferentiating muscle. The Golgi apparatus also becomes unorganized in appearance, whereas the mitochondria show relatively little change. One of the most striking changes in the cytoplasm is its acquisition of numerous free ribonucleoprotein granules, a feature characteristic of undifferentiated cells in general. These features together with the nuclear changes described above indicate that the dedifferentiating muscle cell is probably beginning active cytoplasmic protein synthesis. They dispel the idea that the muscle might be degenerating. The end result of the process of muscle dedifferentiation is an undifferentiated-appearing cell which cannot be distinguished from the blastema cells derived from cartilage and other tissues of the stump. By virtue of its acquisition of such embryonic features as free cytoplasmic ribonucleoprotein granules and prominent nucleoli, as well as loss of differentiated structure, the dedifferentiated blastema cell seems to be remarkably well equipped for the ensuing rapid proliferation of a large cellular mass capable of redifferentiation into a complete limb.