Regeneration of Neuronal Diversity in the Axolotl Brain

Abstract

The axolotl can regenerate multiple organs, including the brain. It remains, however, unclear whether neuronal diversity, intricate tissue architecture, and axonal connectivity can be regenerated; yet, this is critical for recovery of function and a central aim of cell replacement strategies in the mammalian central nervous system. Here, we demonstrate that, upon mechanical injury to the adult pallium, axolotls can regenerate several of the populations of neurons present before injury. Notably, regenerated neurons acquire functional electrophysiological traits and respond appropriately to afferent inputs. Interestingly, however, despite the ability to regenerate specific, molecularly-defined neuronal subtypes, we also uncovered previously unappreciated limitations by showing that newborn neurons organize within altered tissue architecture and fail to re-establish the long-distance axonal tracts and circuit physiology present before injury. The data provide a direct demonstration that diverse, neurons can be regenerated in axolotls, while challenging prior assumptions of functional brain repair in regenerative species. The molecular mechanism underlying the process of brain regeneration in regenerative organisms such as the zebrafish and the axolotl is poorly understood. Previous work in zebrafish and newt have identified factors such as gata3 and the sonic hedgehog pathway, respectively, that are critical for initiation of regeneration. To understand the molecular players involved in the process of brain regeneration in the axolotl, we have performed whole-genome RNA sequencing at 1, 2, and 4 weeks post injury. We found that transcripts associated with cell cycle regulation, immune activation, and retinoic acid signaling pathway are enriched among the differentially expressed genes during brain regeneration. Additionally, we found that genes upregulated in the blastema during limb regeneration are also highly expressed in the proliferative cells in the brain during the first four weeks of regeneration. These results identify, for the first time, key genes that may play a role in brain regeneration in the axolotl and suggest that some regeneration-specific genes may be shared among different organs. To understand the molecular mechanisms of processes such as regeneration or disease, it is critical to capture the transcriptional profile of certain cell types as opposed to profiling the whole tissue. In organisms such as the axolotl where genetic labeling of specific cell types is challenging, a new approach is necessary. Here, we developed a novel technology to quantify gene expression of specific cell types based on deep RNA sequencing of nuclei from unlabeled brain samples. This approach will, in the future, facilitate identification of cell type-specific mechanisms responsible for the regenerative process in the axolotl brain.