Nitroreductase-mediated cell ablation uncovers the pivotal role of ependymoglial cells in entire cortex regeneration in axolotls

Developing precise targeted cell ablation in the axolotl (Ambystoma mexicanum) is crucial for elucidating the roles or interactions of specific cell types in regeneration and modeling diseases. Here we establish a Nitroreductase (NTR)-based inducible cell ablation system in axolotls. Through generation of Sox2:Cherry-NTR knock-in axolotls, we achieve efficient ablation of ependymoglial cells (EGCs) in the central nervous system. Combined spinal cord and brain transplantation and injury models demonstrate regeneration failure upon EGC depletion, suggesting that EGCs are solo source of central nervous system regeneration. Additionally, EGC ablation in the spinal cord resulted in delayed tail regeneration. Moreover, we establish NeuroD6:Cherry-NTR and NeuroD6:Cherry-NTR2.0 knock-in lines to ablate postmitotic cortical neurons, to model degenerative disease and study the process of brain regeneration after large scale neuronal depletion. We found that NTR2.0 (but not NTR) leads to elimination of >95% of cortical neurons. Following the depletion of nearly all cortical neurons, all lost neuronal subtypes are chronological regenerated with layer organization mirroring developmental patterning. Finally, we create Cre-LoxP-based conditional NTR2.0 transgenic axolotls using a constitutive CAGGs promoter, enabling tissue-specific ablation of the targeted cell when combine established Cre lines. In summary, our study establishes an efficient and versatile targeted cell ablation system in axolotls, providing a valuable tool for deep dissection of tissue regeneration in axolotls.