Ependymoglial cells are critical for 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 sole 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, enable the investigation of brain regeneration after large-scale neuronal depletion. We found that NTR2.0 (but not NTR) leads to elimination of >95% of targeted neurons in the dorsal pallium. All lost neuronal subtypes are chronologically regenerated with laminar-like distribution mirroring developmental patterning. Finally, we create Cre-LoxP-based conditional NTR2.0 transgenic axolotls using a constitutive CAGGs promoter, enabling tissue-specific ablation of specific cell types when crossed to existing 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.