Exploring the neural control of axolotl limb regeneration

Abstract

Salamanders are capable of feats of tissue regeneration which are unmatched by other tetrapods. In contrast with mammals and virtually all other vertebrates, salamanders, among which the Mexican axolotl (Ambystoma mexicanum) is the most well-studied, can perfectly regenerate damaged organs and amputated structures. Chief among these astonishing capabilities is the phenomenon of axolotl limb regeneration. Axolotls are capable of fully regenerating amputated limbs throughout the entire course of their lives. This regeneration is perfect and demonstrates the complete return of both the original structure and function of the limb. However, although salamander limb regeneration has been studied in a scientific context for centuries, little is understood about the molecular basis of the process. What has been known for nearly two hundred years is that peripheral nerves are essential for axolotl limb regeneration. Denervation of the axolotl limb prevents the formation of the post-amputation proliferative mass called the blastema and thus completely inhibits regeneration, and the molecular biology of this nerve dependence serve as the foundation for the work described here. We have discovered that the nerve dependence of axolotl limb regeneration, a longstanding puzzle for researchers in the field, may in fact be the result of a combination of factors. Our published work demonstrates the importance of Neuregulin-1 (NRG1), a nerve-derived mitogen, for blastema formation and growth. We have found that NRG1 is localized to the regenerating blastema, capable of rescuing regeneration to digits in denervated limbs, and inhibition of this signaling pathway inhibits regeneration. While this work validates the longstanding neurotrophic hypothesis of axolotl limb regeneration while also describing the first protein known to demonstrate these characteristics in the regenerating axolotl limb, we have additionally found that there is a second component to nerve dependence. Our unpublished work, currently in preparation for submission, has shown that nerves are damaged during denervation and prevent the formation of a regeneration-permissive cellular environment by means of the secretion of inhibitory factors. Specifically, we have found that implanting axotomized nerve bundles into the wound site of amputated limbs slows down or blocks blastema formation and may lead to highly aberrant limb patterning. These effects were mitigated via overexpression of NRG1, suggesting a link between the two hypotheses by indicating that NRG1 is capable of reversing the inhibitory effects of damaged nerves. These novel findings grant us deeper insight into the molecular mechanisms utilized by a highly regenerative animal, one which may hold the key to unlocking similar abilities in less-capable organisms. Therefore, our work, both as described in this dissertation and continuing into the future, may ultimately inform future studies of regenerative medicine in humans.