Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice
Toshihiro Uesaka, … , Shigenobu Yonemura, Hideki Enomoto
Toshihiro Uesaka, … , Shigenobu Yonemura, Hideki Enomoto
Published April 15, 2008
Citation Information: J Clin Invest. 2008;118(5):1890-1898. https://doi.org/10.1172/JCI34425.
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Research Article Gastroenterology

Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice

Abstract

Mutations in the RET gene are the primary cause of Hirschsprung disease (HSCR), or congenital intestinal aganglionosis. However, how RET malfunction leads to HSCR is not known. It has recently been shown that glial cell line–derived neurotrophic factor (GDNF) family receptor α1 (GFRα1), which binds to GDNF and activates RET, is essential for the survival of enteric neurons. In this study, we investigated Ret regulation of enteric neuron survival and its potential involvement in HSCR. Conditional ablation of Ret in postmigratory enteric neurons caused widespread neuronal death in the colon, which led to colonic aganglionosis. To further examine this finding, we generated a mouse model for HSCR by reducing Ret expression levels. These mice recapitulated the genetic and phenotypic features of HSCR and developed colonic aganglionosis due to impaired migration and successive death of enteric neural crest–derived cells. Death of enteric neurons was also induced in the colon, where reduction of Ret expression was induced after the period of enteric neural crest cell migration, indicating that diminished Ret expression directly affected the survival of colonic neurons. Thus, enteric neuron survival is sensitive to RET dosage, and cell death is potentially involved in the etiology of HSCR.

Authors

Toshihiro Uesaka, Mayumi Nagashimada, Shigenobu Yonemura, Hideki Enomoto

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Figure 1

Inactivation of Ret depletes enteric neurons in the colon.

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Inactivation of Ret depletes enteric neurons in the colon. (A) A gene ca...
(A) A gene cassette comprising floxed (black triangles, loxP sites) human Ret9 cDNA (red box) with intron polyA (IpA, black ovals), CFP reporter (blue boxes), and neomycin resistance (Neo) expression cassette (orange box) flanked by FRT sites (open triangles) was introduced into exon 1 (gray boxes) of the mouse Ret locus (black lines) to generate a floxed Ret allele. Activation of Cre recombinase resulted in the removal of floxed Ret9, simultaneously generating CFP-knockin (Ret-null) allele. (B) RET antibody staining of cKO enteric plexus showing no RET immunoreactivity (red) in CFP+ cells (green). Note the expression of RET (red) in neighboring unrecombined (CFP–) cells. (C) PGP9.5 immunostaining of the distal colon in E15.5 control and cKO mouse fetuses before injection of 4-OHT. (D) 4-OHT–induced Cre recombination in enteric neurons. Administration of 4-OHT (0.5 mg per mouse) at E15.5 induced CFP expression in a large number of neurons in the colon. (E) Whole-mount CFP (upper panels) and PGP9.5 (lower panels) staining of the small intestine and colon from E18.5 control and cKO fetuses subjected to 4-OHT treatment at E15.5. Insets show ENS morphology in the small intestine (SI). (F) Whole-mount acetylcholinesterase (AChE) histochemical analysis of E18.5 colon 3 days after inactivation of Ret. Scale bars: 10 μm in B; 100 μm in C; 20 μm in D; 200 μm in E and F.