Altered enteric neurodevelopment in the Ncx knockout mouse model of intestinal neuronal dysplasia.

PURPOSE: Intestinal neuronal dysplasia (IND) features abnormal enteric nervous system (ENS) development and symptom overlap with Hirschsprung's disease, yet fetal-stage mechanisms remain unclear. We established a Sox10-Venus⁺/Ncx⁻/⁻ mouse model of IND, enabling fluorescent labeling of enteric neural crest cells (ENCCs) to test the hypothesis whether neuronal/glial differentiation abnormalities arise during early fetal stages.

METHODS

ENCCs were isolated from embryonic day 13.5 (E13.5) fetal gut of Sox10-Venus⁺/Ncx⁻/⁻ (n = 6) and Sox10-Venus⁺/Ncx⁺/⁺ (n = 6) embryos, dissociated, and cultured under non-adherent conditions for 14 days to generate neurospheres. Neurosphere diameter and the proportion of SOX10+ cells were measured using epifluorescence microscopy. Differentiation on day 14 was assessed by immunofluorescence for TUJ1 (neuronal) and S100β (S100B, glial) markers.

RESULTS

Ncx⁻/⁻ neurospheres were significantly larger than controls on days 5 and 10, with no significant difference on day 14. The proportion of SOX10⁺ cells remained higher through day 10. On day 14, Ncx⁻/⁻ neurospheres exhibited a lower proportion of TUJ1⁺ cells, preserved S100β⁺ cell proportions, and disrupted spheroid organization with heterogeneous marker distribution.

CONCLUSION

These findings demonstrate that Ncx deficiency leads to abnormal ENS development beginning during the fetal period, providing a mechanistic basis for postnatal hyperganglionosis and validating this model for studying IND pathogenesis.