Retinoic Acid Inhibition Alters Intestinal Composition in Zebrafish: A Non-genetic Model to Study Hirschsprung Disease?

Background: Retinoic acid (RA) is pivotal for the development of the enteric nervous system (ENS), guiding the migration and differentiation of vagal neural crest cells in the gastrointestinal tract. Despite its recognized influence on other intestinal cell types, the specific roles of RA on these cells are less understood. This study investigates the extensive impact of RA inhibition on the intestinal composition using zebrafish, while also considering its potential use as a model for Hirschsprung disease (HSCR).

Methods: Zebrafish larvae were treated with diethylaminobenzaldehyde (DEAB), an inhibitor of RA synthesis, to induce phenotypes resembling total colonic aganglionosis. Single-cell RNA sequencing was performed to compare the intestinal cell composition of DEAB-treated zebrafish with wildtype and a ret mutant HSCR model.

Results: Inhibition of RA reduced ENS cell numbers but also induced significant changes within non-neuronal cell populations. Notably, there was an increase in epithelial cells, a decrease in immune cells, and suppression of inflammatory pathways. Additionally, we observed increased myofibroblast numbers and decreased fibroblasts, which might suggest fibrosis. Comparative analysis of the ret mutant and DEAB-treated models showed profound effects on non-neuronal cells, yet most of these changes differed between the two models, underscoring the distinct roles of RA and Ret signaling on intestinal development.

Conclusion: These findings highlight RA's pivotal role in maintaining intestinal structure and immune homeostasis, while further supporting the importance of considering both neuronal and non-neuronal cell populations in HSCR research.