Optimized chemogenetic 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 functions, including motility. ENS loss is associated with gastrointestinal disorders such as Hirschsprung disease. Restoring missing neurons by stimulating neuronal regeneration presents a promising therapeutic approach for ENS disorders. To study ENS regeneration, we developed a targeted chemogenetic neuron ablation model in zebrafish using the Gal4/UAS-NTR 2.0 system. Spatially and temporally controlled neuronal death was confirmed by morphological changes, neuron loss quantification, and TUNEL assay. We observed an acute immune response that normalizes at 1 day post-treatment (dpt). We found complete restoration of ENS neuron numbers to control levels by 9 dpt, with recovery of gut motility. We identify neuronal subtype-specific differences in regenerative capacity and/or timing of cell replacement. Our study establishes a robust platform for dissecting the cellular-molecular mechanisms of ENS regeneration to develop potential treatment approaches for ENS-related disorders.