Fig. 2. Defects in enteric nervous system development in Ret^DN/+mice. (A) The intestines from a P21 Ret^DN/+ mouse with a contracted distal colon (double arrow) and a dilated distal small bowel (triple arrowheads). The intestines of wild-type (wt), Ret^9/+ and Ret^TGM/+ mice are normal (arrow, ileocecal junction; Es, esophagus; Re, rectum). (B) Intestinal aganglionosis and hypoganglionosis in P21 Ret^DN/+ mice. Hematoxylin and Eosin (HE) staining revealed the presence of neurons (arrowheads) in the muscularis externa of the colon in wild-type (wt) but not Ret^DN/+ mice. Acetylcholinesterase (AChE) staining demonstrated neuronal (black arrows) and nerve fiber (black arrowheads) loss in Ret^DN/+ colon (the brown fibers in the Ret^DN/+ colon are extrinsic innervations characteristic of HSCR). Both AChE- and nicotinamide adenine dinucleotide phosphate (NADPH)-stained Ret^DN/+ proximal small intestines display a reduced number of neurons (arrows) and nerve fibers (arrowheads), when compared with an identical region from wild-type mice. Scale bars: 100 µm (HE); 400 µm (AChE); 200 µm (NADPH). (C) The aganglionosis is fully penetrant but variable in Ret^DN/+ mice. The schematic shows the extent of aganglionosis determined by AChE staining in various Ret^DN/+ mice (each red mark represents one Ret^DN/+ mouse, n=16). The numbers correspond to the percentage of the respective intestinal segment (small or large intestine) successfully colonized by neurons. Ret^9/+ (n=7), Ret^DNneo/+ (n=3) and Ret^TGM/+ (n=3) intestines were normal, and Ret^TGM/TGM (Ret null) had total intestinal aganglionosis. (D) Quantitative analysis of neuronal number and fiber density in the ENS of P21 wild-type and Ret^DN/+ mice demonstrated a dramatic reduction in the number of neurons and neuronal fiber density in the proximal small bowel (n=3, ^*=P<0.01, mean±s.e.m.).