Salamanders are able to fully regenerate amputated limbs as adults, but how they perform such feats of regeneration remains poorly understood. Limb regeneration does not occur if nerve fibers (axons) that normally connect with the limb are absent. The objective of this award is to elucidate the crucial-yet-poorly-understood relationship between limb regeneration and nerves. It has traditionally been thought that the role of nerves is to promote the multiplication (proliferation) of cells located at the injury site. The hypothesis of this proposal is that, while nerves tell cells to proliferate, the absence of nerves causes an increase in molecules that stop the limb from regenerating. Novel genetic approaches in the Mexican Axolotl salamander will be used to study these anti-regenerative molecules. In addition to presenting technical advantages for these studies, Axolotls are exceptionally charismatic animals that exhibit powerful regeneration responses; they are excellent for integrating teaching and research in the laboratory and classroom. Understanding the basis of nerve-limb interactions during regeneration would have a major impact on our understanding of animal regeneration, and may shape novel approaches for regenerative medicine. This project also aims to ignite interest in axolotls and regeneration, and to recruit promising future scientists into the field. This will be carried out by developing programs introducing axolotls and regeneration to local YMCA summer camps, including significant undergraduate and graduate research training, integrating research from this project into an undergraduate course on regeneration, and participating in diversity-focused training programs that center on regeneration.
Axotomy of the nerve bundles that innervate a regenerating salamander limb completely blocks regeneration. Neuregulin 1 (NRG1) has recently been implicated as a mitogenic factor emanating from nerve axons during axolotl salamander limb regeneration, but a mechanistic explanation for why NRG1 is required is lacking. This proposal will test the hypothesis that regenerating limbs with insufficient axon-derived NRG1 ligands will not regenerate due to a lack of positive mitogenic signaling as well as an increase in a peripheral nerve (Schwann cells)-derived inhibitory signals, creating a non-permissive environment. Testing the hypothesis that axons play a multifaceted role during regeneration will transform our understanding of how regeneration is regulated. Aim 1 will test the requirement of axon-derived NRG1 by studying the effects of neuron-specific morpholino knockdown and spatially controlled CRISPR/Cas9-mediated gene inactivation. To test for the sufficiency of NRG1 signaling to support regeneration in the absence of axons, a secreted form of NRG1 and a constitutively active form of its receptor will be overexpressed in axotomized and amputated limbs. Aim 2 will utilize RNA sequencing to identify signaling nodes that are specifically affected by NRG1 inhibition. Aim 3 will investigate the permissive nature of axons through the study of molecules secreted from axotomized peripheral nerve bundles using gain- and loss-of-function experiments for candidate inhibitory molecules. The findings made from the proposed experiments will increase understanding of the mechanisms promoting limb regeneration and a major technological advance in our ability to test gene function during regeneration.
