Increased RET Activity Coupled with a Reduction in the RET Gene Dosage Causes Intestinal Aganglionosis in Mice

Ret^{51(C618F)/51(C618F)} mice exhibit complete gut colonization by ENS precursors. Phox2b-labeled ENS precursors and neurons (green) in the developing gut in Ret^51/51 and Ret^{51(C618F)/51(C618F)} mice at E12.5 (upper panels) and P0 (lower panels). Migration of ENS precursors is slightly delayed at E12.5, but gut colonization by them is completed at P0. Arrowheads depict the front of the migrating ENS precursors. Hg, hindgut; Mg, midgut. Scale bars: 250 μm (upper panels) and 500 μm (lower panels).

Ret^{51(C618F)/–} mice exhibit intestinal aganglionosis. A, B, Whole-mount images of the enteric neurons stained with anti-PGP-9.5 (A) or labeled by GFP (B) in P0 Ret^51/+ and Ret^51(C618F)/+, Ret^51/EGFP, and Ret^{51(C618F)/EGFP} gut. Complete gut colonization by ENS cells was seen in Ret^51/+, Ret^51(C618F)/+, and Ret^51/EGFP mice (white arrowheads), while Ret^{51(C618F)/EGFP} mice exhibited disrupted colonization of the gut by ENCCs. The wavefront (yellow arrowheads) was defined as the most caudal continuous strands of EGFP⁺ cells. Some Ret^{51(C618F)/EGFP} mice show skip segment aganglionosis where small regions of the colon contain enteric ganglia (white dotted region). C, The proportion of three types of aganglionic phenotype (small intestinal, skip segment, and colonic aganglionosis). Ce, cecum; Co, colon; Si, small intestine. Scale bars: 1 mm (A, B).

Detection of a few enteric neurons in the hindgut of Ret^{51(C618F)/EGFP} embryos. Whole-mount preparation of embryonic gut showing the presence of a few differentiating enteric neurons (A, inset) revealed by anti-PGP9.5 antibody (B). Scale bars: 100 μm (A) and 50 μm (B).

Reduced RET51(C618) expression leads to premature differentiation of ENS precursors at the migratory wavefront. A, Whole-mount images of GFP-labeled cells in the gut from E12.5 Ret^51/EGFP and Ret^{51(C618F)/EGFP} embryos (left panels). GFP⁺ cells in the migratory wavefront were stained by anti-Sox10 (right panels), whereas Sox10-negative GFP⁺ cells (white arrowheads) were found at the delayed migratory wavefront (open arrowhead) of Ret^{51(C618F)/EGFP} gut. B, Whole-mount images of GFP-labeled cells stained with anti-PGP9.5 in E12.5 Ret^51/EGFP and Ret^{51(C618F)/EGFP} gut. PGP9.5-labeled GFP⁺ cells were detected at the delayed migratory wavefront of Ret^{51(C618F)/EGFP} gut. C, Immunohistochemical staining for GFP (green), Sox10 (blue), and activated ERK (pERK, magenta) in ENS cells of Ret^51/EGFP and Ret^{51(C618F)/EGFP} embryos at E12.5. In the migratory wavefront of Ret^{51(C618F)/EGFP} embryos, pERK was mainly observed in GFP⁺ and Sox10^– differentiating neurons (white arrowheads). Hg, hindgut; Mg, midgut. Scale bars: 250 μm (A, left panel), 50 μm (A, right panel), 25 μm (B), and 20 μm (C).

Reducing Gdnf gene dosage moderately rescues ENS phenotype of Ret^{51(C618F)/–}mice. A, Representative images of P0 Ret^51/EGFP/Gdnf^+/– and Ret^{51(C618F)/EGFP}/Gdnf^+/– large intestine showing complete colonization with GFP-positive enteric neurons. B, Comparison of ENS wavefront location between Ret^{51(C618F)/EGFP} and Ret^{51(C618F)/EGFP}/Gdnf^+/– mice at P0. Reduction of Gdnf gene dosage significantly ameliorated the severity of enteric aganglionosis (χ² test, p < 0.01). C, Representative images of E12.5 Ret^51/EGFP/Gdnf^+/– and Ret^{51(C618F)/EGFP}/Gdnf^+/– gut displaying colonization by GFP-positive ENS precursors. White arrowheads indicate the location of ENS precursor wavefront. D, Whole-mount GFP, Sox10, and pERK pathway stainings of ENS cells of Ret^{51(C618F)/EGFP}/Gdnf^+/– embryos at E12.5. Activation of ERK was not observed in ENS precursors at the migratory wavefront. Ce, cecum; Co, colon; Si, small intestine; Hg, hindgut; Mg, midgut. Scale bars: 1000 μm (A), 250 μm (C), and 20 μm (D).