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Research ArticleResearch Article: New Research, Development

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

Mitsumasa Okamoto, Toshihiro Uesaka, Keisuke Ito and Hideki Enomoto
eNeuro 6 May 2021, 8 (3) ENEURO.0534-20.2021; https://doi.org/10.1523/ENEURO.0534-20.2021
Mitsumasa Okamoto
Division for Neural Differentiation and Regeneration, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
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Toshihiro Uesaka
Division for Neural Differentiation and Regeneration, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
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Keisuke Ito
Division for Neural Differentiation and Regeneration, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
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Hideki Enomoto
Division for Neural Differentiation and Regeneration, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
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  • Figure 1.
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    Figure 1.

    Ret51(C618F)/51(C618F) mice display hypertrophy of enteric ganglia. A, Whole-mount Phox2b staining (green) of enteric neurons in the myenteric plexus of the small intestine from P0 Ret+/+, Ret51/51, and Ret51(C618F)/51(C618F) mice. B, Quantification of Phox2b+ enteric neuron numbers in the small intestine and the colon from P0 Ret+/+ (n = 3), Ret51/51 (n = 3), and Ret51(C618F)/51(C618F) (n = 6) mice; *p = 0.001, **p <0.0001, one-way ANOVA with Tukey’s post hoc test. C, D, Detection of EdU (magenta) incorporated into Phox2b+ ENS precursors (green) in the midgut from E12.5 (C) and E14.5 (D) Ret51/51 and Ret51(C618F)/51(C618F) fetuses. The gut was labeled by a 2-h pulse of EdU. The graphs (right panels) display the rate of ENS precursor proliferation at E12.5 and 14.5 Ret51/51 (n = 3) and Ret51(C618F)/51(C618F) (n = 3) fetuses; *p <0.05, unpaired t test. Scale bars: 50 μm (A) and 20 μm (C, D).

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    Figure 2.

    Ret51(C618F)/51(C618F) mice exhibit complete gut colonization by ENS precursors. Phox2b-labeled ENS precursors and neurons (green) in the developing gut in Ret51/51 and Ret51(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).

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    Figure 3.

    Ret51(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 Ret51/+ and Ret51(C618F)/+, Ret51/EGFP, and Ret51(C618F)/EGFP gut. Complete gut colonization by ENS cells was seen in Ret51/+, Ret51(C618F)/+, and Ret51/EGFP mice (white arrowheads), while Ret51(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 Ret51(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).

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    Figure 4.

    Detection of a few enteric neurons in the hindgut of Ret51(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).

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    Figure 5.

    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 Ret51/EGFP and Ret51(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 Ret51(C618F)/EGFP gut. B, Whole-mount images of GFP-labeled cells stained with anti-PGP9.5 in E12.5 Ret51/EGFP and Ret51(C618F)/EGFP gut. PGP9.5-labeled GFP+ cells were detected at the delayed migratory wavefront of Ret51(C618F)/EGFP gut. C, Immunohistochemical staining for GFP (green), Sox10 (blue), and activated ERK (pERK, magenta) in ENS cells of Ret51/EGFP and Ret51(C618F)/EGFP embryos at E12.5. In the migratory wavefront of Ret51(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).

  • Figure 6.
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    Figure 6.

    Reducing Gdnf gene dosage moderately rescues ENS phenotype of Ret51(C618F)/–mice. A, Representative images of P0 Ret51/EGFP/Gdnf+/– and Ret51(C618F)/EGFP/Gdnf+/– large intestine showing complete colonization with GFP-positive enteric neurons. B, Comparison of ENS wavefront location between Ret51(C618F)/EGFP and Ret51(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 Ret51/EGFP/Gdnf+/– and Ret51(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 Ret51(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).

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    Figure 7.

    The severity of interruption of the ENS migration depends on the Ednrb gene dosage in RET51(C618F)/- mice. A, Whole-mount GFP staining of P0 Ret51/EGFP/Ednrb+/− and Ret51(C618F)/EGFP/Ednrb+/− gut. White arrowheads indicate the location of wavefront of enteric neurons. B, Comparison of location of ENS wavefront between Ret51(C618F)/EGFP and Ret51(C618F)/EGFP/Ednrb+/− mice at P0. Reduction of Ednrb gene dosage increased the severity of enteric aganglionosis of Ret51(C618F)/EGFP mice. C, Whole-mount GFP staining of the enteric neurons in E12.5 Ret51(C618F)/EGFP/Ednrb+/− gut. White arrowhead indicates location of the wavefront of ENS precursors. D, Whole-mount GFP, PGP9.5, Sox10, and pERK pathway stainings of ENS cells of Ret51(C618F)/EGFP/Ednrb+/− embryos at E12.5. Activation of ERK was observed in ENS precursors at the migratory wavefront. E, Immunohistochemical detection of GFRα1 (green) in ENS progenitors (whose nuclei marked in magenta by anti-Phox2b antibody) of wild-type, Gdnf+/–, and Ednrb+/− embryos (E12.5). Ce, cecum; Co, colon; Si, small intestine; Hg, hindgut; Mg, midgut. Scale bars: 1 mm (A), 250 μm (C), and 20 μm (D), 50 μm (E).

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Increased RET Activity Coupled with a Reduction in the RET Gene Dosage Causes Intestinal Aganglionosis in Mice
Mitsumasa Okamoto, Toshihiro Uesaka, Keisuke Ito, Hideki Enomoto
eNeuro 6 May 2021, 8 (3) ENEURO.0534-20.2021; DOI: 10.1523/ENEURO.0534-20.2021

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Increased RET Activity Coupled with a Reduction in the RET Gene Dosage Causes Intestinal Aganglionosis in Mice
Mitsumasa Okamoto, Toshihiro Uesaka, Keisuke Ito, Hideki Enomoto
eNeuro 6 May 2021, 8 (3) ENEURO.0534-20.2021; DOI: 10.1523/ENEURO.0534-20.2021
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Keywords

  • cell signaling
  • enteric nervous system
  • Hirschsprung’s disease
  • neuronal differentiation
  • RET

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