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

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.

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.

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.

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.