Hirschsprung-like disease is exacerbated by reduced de novo GMP synthesis
Jonathan I. Lake, Olga A. Tusheva, Brittany L. Graham, Robert O. Heuckeroth
Jonathan I. Lake, Olga A. Tusheva, Brittany L. Graham, Robert O. Heuckeroth
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Research Article Gastroenterology

Hirschsprung-like disease is exacerbated by reduced de novo GMP synthesis

Abstract

Hirschsprung disease (HSCR) is a partially penetrant oligogenic birth defect that occurs when enteric nervous system (ENS) precursors fail to colonize the distal bowel during early pregnancy. Genetic defects underlie HSCR, but much of the variability in the occurrence and severity of the birth defect remain unexplained. We hypothesized that nongenetic factors might contribute to disease development. Here we found that mycophenolate, an inhibitor of de novo guanine nucleotide biosynthesis, and 8 other drugs identified in a zebrafish screen impaired ENS development. In mice, mycophenolate treatment selectively impaired ENS precursor proliferation, delayed precursor migration, and induced bowel aganglionosis. In 2 different mouse models of HSCR, addition of mycophenolate increased the penetrance and severity of Hirschsprung-like pathology. Mycophenolate treatment also reduced ENS precursor migration as well as lamellipodia formation, proliferation, and survival in cultured enteric neural crest–derived cells. Using X-inactivation mosaicism for the purine salvage gene Hprt, we found that reduced ENS precursor proliferation most likely causes mycophenolate-induced migration defects and aganglionosis. To the best of our knowledge, mycophenolate is the first medicine identified that causes major ENS malformations and Hirschsprung-like pathology in a mammalian model. These studies demonstrate a critical role for de novo guanine nucleotide biosynthesis in ENS development and suggest that some cases of HSCR may be preventable.

Authors

Jonathan I. Lake, Olga A. Tusheva, Brittany L. Graham, Robert O. Heuckeroth

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Figure 4

Mosaic analysis reveals that effects of GTP depletion on migration and lamellipodia are non–cell autonomous.

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Mosaic analysis reveals that effects of GTP depletion on migration and l...
(A) Mating scheme, genotypes, HPRT and EGFP expression patterns, and GTP depletion status of each population when cultured in the presence of both MPA and guanosine. These conditions create mixed cultures of GTP-depleted and EGFP-marked, guanosine-rescued ENCDCs in X-EGFP+, Hprt+/– explants, allowing migration of individual GTP-depleted ENCDCs to be examined in the context of a field of rescued ENCDCs. (B–D) BrdU labeling revealed that guanosine rescued DNA synthesis in all ENCDCs in X-EGFP+, Hprt+/+ explants (B), but only rescued DNA synthesis within the HPRT-expressing ENCDCs marked by EGFP in X-EGFP+, Hprt+/– explants (C). As expected, guanosine failed to rescue DNA synthesis in Hprt–/Y ENCDCs (D). Filled arrowheads denote BrdU+EGFP+ double-positive ENCDCs; open arrowheads denote BrdU+GFP– ENCDCs. (E) Quantification of BrdU labeling in mosaic explants. (F) ENCDC migration out of explants was impaired in Hprt–/Y explants, as expected, but Hprt+/+ and Hprt+/– explants produced similar ENCDC migration distances. (G) Quantification of migration within the depleted (EGFP–) and rescued (EGFP+) populations of female Hprt+/– cells demonstrated that GTP-depleted cells did not migrate any less efficiently than rescued cells when surrounded by rescued cells. Similarly, while lamellipodia were reduced in Hprt–/Y explants (H), they were not reduced within the GTP-depleted ENCDC population in Hprt+/– explant cultures (I). Scale bar: 50 μm (B–D). ***P < 0.001, paired t test (E and I); ANOVA (F); Wilcoxon signed-rank test (G); t test (H).