Hedgehog/Notch-induced premature gliogenesis represents a new disease mechanism for Hirschsprung disease in mice and humans
Elly Sau-Wai Ngan, … , Vincent Chi-Hang Lui, Paul Kwong-Hang Tam
Elly Sau-Wai Ngan, … , Vincent Chi-Hang Lui, Paul Kwong-Hang Tam
Published August 15, 2011
Citation Information: J Clin Invest. 2011;121(9):3467-3478. https://doi.org/10.1172/JCI43737.
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Research Article Gastroenterology

Hedgehog/Notch-induced premature gliogenesis represents a new disease mechanism for Hirschsprung disease in mice and humans

Abstract

Hirschsprung (HSCR) disease is a complex genetic disorder attributed to a failure of the enteric neural crest cells (ENCCs) to form ganglia in the hindgut. Hedgehog and Notch are implicated in mediating proliferation and differentiation of ENCCs. Nevertheless, how these signaling molecules may interact to mediate gut colonization by ENCCs and contribute to a primary etiology for HSCR are not known. Here, we report our pathway-based epistasis analysis of data generated by a genome-wide association study on HSCR disease, which indicates that specific genotype constellations of Patched (PTCH1) (which encodes a receptor for Hedgehog) and delta-like 3 (DLL3) (which encodes a receptor for Notch) SNPs confer higher risk to HSCR. Importantly, deletion of Ptch1 in mouse ENCCs induced robust Dll1 expression and activation of the Notch pathway, leading to premature gliogenesis and reduction of ENCC progenitors in mutant bowels. Dll1 integrated Hedgehog and Notch pathways to coordinate neuronal and glial cell differentiation during enteric nervous system development. In addition, Hedgehog-mediated gliogenesis was found to be highly conserved, such that Hedgehog was consistently able to promote gliogenesis of human neural crest–related precursors. Collectively, we defined PTCH1 and DLL3 as HSCR susceptibility genes and suggest that Hedgehog/Notch-induced premature gliogenesis may represent a new disease mechanism for HSCR.

Authors

Elly Sau-Wai Ngan, Maria-Mercè Garcia-Barceló, Benjamin Hon-Kei Yip, Hiu-Ching Poon, Sin-Ting Lau, Carmen Ka-Man Kwok, Eric Sat, Mai-Har Sham, Kenneth Kak-Yuen Wong, Brandon J. Wainwright, Stacey S. Cherny, Chi-Chung Hui, Pak Chung Sham, Vincent Chi-Hang Lui, Paul Kwong-Hang Tam

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Figure 2

Deletion of Ptch1 promotes the proliferation and gliogenesis but inhibits neurogenesis of ENCCs.

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Deletion of Ptch1 promotes the proliferation and gliogenesis but inhibit...
(A) ENCCs were isolated from the Ptch1f/f mice, which have loxP recombinase recognition sites flanking exon 3 of the Ptch1 gene. The Ptch1f/f ENCCs were then infected with Ad-GFP-Cre. 86.0% ± 1.3% of ENCCs expressed GFP 72 hours after the infection with the control virus (Ad-GFP) and Ad-GFP-Cre. Primers were designed to generate a PCR product that spanned exon 2 to 6 (ex 2-6) of Ptch1 to distinguish full-length and exon 3–deleted Ptch1 transcripts. Another pair of primers was designed at exon 20 and 22 for the comparison of Ptch1 expression. Exons are represented by rectangles, and loxP sites are represented by triangles. Scale bar: 100 μm. (B) The expression of the mutant Ptch1 and other Hh target genes was analyzed by RT-PCR in presence or absence of Shh (transduction for 4 days followed by Shh treatment for 3 days). (C) BrdU proliferation assay. Relative proliferation rates were measured by counting BrdU+/Ret+ and total Ret+ cells. (D and E) Immunocytochemistry. Neuronal and glial differentiation was monitored based on the expression of TH and S100β, respectively (GDNF treatment for 10 days). Percentages of (D) neuronal and (E) glial precursors were measured over the total number of ENCCs (DAPI). (F) Quantitative RT-PCR showed that deletion of Ptch1 (Ad-GFP-Cre) induces GFAP expression. The values reported in bar charts represent the mean ± SEM, and 3 independent assays were performed. Data were analyzed by 1-way ANOVA, followed by the Tukey post-hoc test. P values of less than 0.05 were considered to be statistically significant.