Fig. 3. Inactivation of GFR1 depletes enteric neurons in the colon. (A) PGP9.5 immunostaining of the distal colon in E15.5 control and cKO mouse embryos before injection of 4-OHT. Comparable ENS staining was observed in control and cKO colon, indicating normal development of the ENS before induction of GFR1 inactivation. (B) Wholemount AChE histochemical analysis of E18.5 gut 3 days after inactivation of GFR1. The highly organized meshwork-like pattern of ENS network observed in control (top) was dramatically disrupted in cKO (bottom). ENS structure in the small intestine was well maintained in both embryos (insets). (C) Toluidine Blue staining revealed the absence of enteric ganglia (Ga) in the myenteric layer (between Lm and Cm) of the colon in cKO embryos. Cm, circular muscle; Co, colon; Ga, enteric ganglia; Lm, longitudinal muscle; SI, small intestine. Scale bars: 20 µm in A; 200 µm in B; 10 µm in C.