Abstract

Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of an enteric nervous system (ENS) in the distal gut. While the ENS is critical for normal gut function, its broader role in maintaining intestinal homeostasis remains underexplored. Using single-cell RNA sequencing, we investigated the impact of ENS loss on gut composition in wildtype and ret mutant (HSCR model) zebrafish. Significant alterations were identified, including increases in immune cells and shifts in epithelial and extracellular matrix (ECM)-producing cell populations. Immune dysregulation was highlighted by impaired TNF- signaling via NF-{kappa}B, while epithelial cell changes pointed to disrupted energy homeostasis with downregulated fatty acid metabolism and cell cycle pathways. Furthermore, the ECM producing cells showed enriched fibrotic markers. Alterations of the intestinal composition were validated in human HSCR tissues, underscoring the clinical relevance of these findings. These changes can underlie the development of secondary complications and be potentially used to improve patient outcomes.