In vivo limb muscle differentiation in the absence of nerves: A quantitative study

Abstract

The entire presumptive nervous system of stage 24 Amblystoma maculatum embryos was surgically excised to produce aneurogenic larvae. For controls, embryos of the same age were operated in an identical fashion except that a segment of neural tube (about 1 mm) dorsal to the limb bud anlagen was left intact. The number of peripherally situated muscle nuclei (a measure of myofiber differentiation) peaked at the four-digit stage of aneurogenic limb development. The number of peripheral nuclei in the controls also peaked at the same time but remained at that maximum. With the exception of Day 13, the number of peripheral nuclei was always greater in control than in aneurogenic muscle. The total number of muscle nuclei per limb cross section gradually decreased in aneurogenic muscle to a number significantly lower than in controls at 31 days after the operation and thereafter. The cross-sectional size of aneurogenic muscle nuclei seemed to decrease at a faster rate than control nuclei. Reflecting nuclear density, the myonuclear area per muscle area in aneurogenic limbs was smallest at 21 days after the operation. After 21 days, aneurogenic nuclear densities progressively increased whereas control densities remained at the low, Day-21 value. From Day 13 to 21 the muscle nuclear density in control and aneurogenic limbs was statistically equal. Aneurogenic myofibers in an advanced state of degeneration incorporated more [ 3H]leucine or [ 3H]uridine than fibers where cross striations were still visible with the light microscope. With the electron microscope, acid phosphatase-stained lysosomes were found adjacent to disintegrating myofibrillar material in degenerating aneurogenic myofibers. Ruthenium red staining indicated that myofiber basement laminae developed normally in aneurogenic muscle and persisted.