Targeted ablation and regeneration of enteric nervous system neurons in zebrafish

The enteric nervous system (ENS) is the intrinsic nervous system of the gut and regulates essential gut functions, including motility, digestion, and immune response, ensuring gut homeostasis. ENS dysfunction or loss is associated with gastrointestinal disorders such as Hirschsprung disease (HSCR). Currently, surgery is the only treatment for HSCR, but it often has lifelong, severe complications. Restoring missing ENS neurons by stimulating endogenous neuronal regeneration presents a promising therapeutic approach for ENS disease. To study the cellular-molecular mechanisms of neuronal regeneration we first need to identify an animal model system with robust ENS regeneration. For this, we developed a chemical-genetic ablation model in zebrafish using the Gal4/UAS NTR 2.0 system for targeted ENS neuron ablation. Spatially and temporally controlled neuronal death was confirmed by morphological changes, complete neuronal loss, and TUNEL assays. Quantification of regenerated neurons demonstrated complete restoration of ENS neuron numbers to control levels by 9 days post treatment with recovery of gut motility. Among the regenerated neurons, nitrergic, cholinergic and vipergic subtypes showed full recovery, whereas serotonergic neurons only displayed partial recovery, indicating subtype-specific differences in the regenerative capacity and/or timing. Our study establishes a robust platform for dissecting the cellular-molecular mechanisms of ENS regeneration to develop potential treatment approaches for ENS-related diseases.