PERIPHERAL NERVES PROVIDE MITOGENIC & PERMISSIVE SUPPORT DURING AXOLOTL LIMB REGENERATION

Background: The Mexican axolotl (Ambystoma mexicanum) is a salamander that is capable of complete limb regeneration throughout adulthood. However, axotomizing the limb prevents the formation of the post-injury proliferative mass called the blastema, and the mechanisms behind this nerve-dependency remain largely unknown. Here we provide evidence that peripheral nerves have both a mitogenic and permissive role during axolotl limb regeneration. Methods: Beads soaked in the nerve-derived mitogen Neuregulin-1 (NRG1) were implanted underneath the wound epithelium of denervated limbs, while NRG1 signaling was inhibited in innervated limbs with the drug Mubritinib. Axotomized peripheral nerve bundles were implanted into fully-innervated amputated limbs as well as underneath the wound site of limbs which underwent accessory blastema surgery. Blastemal growth was monitored and limbs were collected for sectioning and histology. Results: Supplementation with NRG1-soaked beads induced blastema formation and supported regeneration in denervated axolotl limbs, while inhibition of NRG1 signaling completely blocked blastema formation. Axotomized peripheral nerve grafts inhibited blastema formation and regenerative growth in both accessory and regenerating limbs, while histology of these inhibited limbs showed aberrant wound closure and inflammatory cell recruitment. Conclusion: We have shown that the nerve dependency of axolotl limb regeneration may have multiple underlying causes. Our results suggest that nerve-derived NRG1 signaling is essential for blastema proliferation, and the loss of this pro-proliferative signal may impede regeneration. Furthermore, we have shown that axotomized nerves induce inflammation and inhibit regeneration in fully-innervated limbs, thus suggesting that denervation may also impair regeneration via the secretion of inflammatory factors into the wound environment.