Figure 7

Igsf3 KO results in downregulation of neuronal and smooth muscle markers and disorganized innervation of the small intestinal villi. (A) Confocal images of the WT P12.5 small intestinal villi after wholemount staining using antibodies against IGSF3 and NCAM1. IGSF3 (green) colocalizes with NCAM1 (red), a neuronal marker, in the neurons of the small intestinal villi (Merged). (B) Representative images of the intestinal villi from P12.5 WT (upper panels) and Igsf3 KO (lower panels) pups stained with antibodies against the neuron marker Tuj1 (green) and smooth muscle marker αSMA (red) on whole mount samples. (C,D) Representative images of the intestine from P12.5 WT (C) and Igsf3 KO (D) pups stained with antibodies against the E-cadherin (Ecad, green) and NCAM1 (red) on whole mount samples. Cell nuclei are visualized by Hoechst (blue). While no difference was seen in the E-cadherin expression between the WT and KO intestine, NCAM1 expression was substantially reduced in the KO compared to the WT. (E) Western blot analysis of P2.5 cerebrum extracts shows reduced expression of NCAM1 in KO samples also in the brain. Housekeeping protein GAPDH was used as a loading control. (F–K) Quantification from P12.5 intestinal immunostained images. (F) Expression of both Tuj1 and (G) αSMA was significantly reduced. (H) The αSMA:Tuj1 ratio was significantly increased in the KO villi as compared to WTs. (I) Counts of nerves per villus was significantly decreased in the KO villi as compared to the WT intestine, (J) but no difference was observed in the muscle fiber counts. (K) The colocalization of αSMA-Tuj1 was reduced in the KO villi as compared to WT intestines. Scale bars: (A–D) 100 µm.