46;-spectrin, encoded in C. elegans by the gene
unc-70, is abundant in neurons throughout development and adult life. Several lines of evidence suggest that 46;-spectrin might have an essential function in axonal outgrowth. First,
unc-70 mutants have aberrant axonal morphology. Second, spectrin is found in growth cones of cultured vertebrate neurons. Third, injection of spectrin peptides prevents neurite extension in neuroblastoma cells.To assess the axon outgrowth phenotype of animals that lack 46;-spectrin, we used confocal microscopy to image growth cones in
unc-70 mutants. Surprisingly, we found that growth cone morphology was normal. Further, growth cones usually reached their normal targets, and initial patterning of the nervous system was similar to wild type. Thus, 46;-spectrin is not required for axon outgrowth in C. elegans. Neurons in
unc-70 animals must degrade after patterning is complete.To characterize neuronal degradation in
unc-70 animals, we imaged animals at various time points. The nervous system was more severely degenerated in older animals. To determine the steps involved in degradation, we performed a longitudinal study. We found that neuronal processes that initially appeared normal later displayed breaks. Some of the terminal ends of the breaks appeared quiescent. Other terminal ends appeared to have re-initiated growth. However, this second growth often produced wandering and elaborately branched structures. Further, isolated axon fragments often disappeared, reminiscent of Wallerian degeneration. Thus, the defective nervous system in adult
unc-70 animals is a result of axonal breaking, regrowth and retraction.Axons of
unc-70 animals could break for two reasons. First, lack of 46;-spectrin could render neuronal membranes insufficiently elastic to cope with stretching during movement. Alternatively, lack of 46;-spectrin could result in reduced ability to add interstitial membrane during animal growth. To distinguish between these models, we are testing the neuronal phenotype of
unc-70;
unc-54 animals. These animals move less, but are longer, than
unc-70 alone. Thus, the nervous system will be less defective if the elasticity model is correct, and more defective if the membrane addition model is correct.Use-dependent, peripheral neuropathies are present in human disorders such as multiple sclerosis. Axon breaking in
unc-70 animals could provide insight into the progression of this phenomenon, and perhaps even suggest a molecular model.