Portal de Periódicos da CAPES

Hirschsprung Disease and Activation of Hedgehog Signaling via GLI1-3 Mutations

2015; Elsevier BV; Volume: 149; Issue: 7 Linguagem: Inglês

10.1053/j.gastro.2015.10.023

1528-0012

Heather M. Young, Lincon A. Stamp, Robert M.W. Hofstra,

Digestive system and related health

See “Identification of GLI mutations in patients with Hirschsprung disease that disrupt enteric nervous system development in mice,” by Liu JA-J, Lai FP-L, Gui H-S, et al, on page 1837. See “Identification of GLI mutations in patients with Hirschsprung disease that disrupt enteric nervous system development in mice,” by Liu JA-J, Lai FP-L, Gui H-S, et al, on page 1837. Hirschsprung disease (HSCR) is a congenital condition in which enteric neurons are missing from variable lengths of the distal bowel.1Heanue T.A. Pachnis V. Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies.Nat Rev Neurosci. 2007; 8: 466-479Crossref PubMed Scopus (436) Google Scholar, 2McKeown S.J. Stamp L. Hao M.M. et al.Hirschsprung disease: a developmental disorder of the enteric nervous system.WIRES Dev Biol. 2013; 2: 113-129Crossref Scopus (117) Google Scholar Because the enteric nervous system (ENS) is essential for gut motility, affected infants suffer from functional obstruction and enterocolitis, and treatment requires surgery to remove the affected, aganglionic region. HSCR is a complex, polygenic disease, and mutations or variants in ≥15 genes have been associated with HSCR.3Alves M.M. Sribudiani Y. Brouwer R.W. et al.Contribution of rare and common variants determine complex diseases-Hirschsprung disease as a model.Dev Biol. 2013; 382: 320-329Crossref PubMed Scopus (95) Google Scholar, 4Jiang Q. Arnold S. Heanue T. et al.Functional loss of semaphorin 3C and/or semaphorin 3D and their epistatic interaction with ret are critical to Hirschsprung disease liability.Am J Hum Genet. 2015; 96: 581-596Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar Coding and noncoding mutations in the RET gene account for the majority of identified mutations associated with HSCR.5Amiel J. Sproat-Emison E. Garcia-Barcelo M. et al.Hirschsprung disease, associated syndromes and genetics: a review.J Med Genet. 2008; 45: 1-14Crossref PubMed Scopus (665) Google Scholar However, mutations in genes encoding members of other signaling pathways have also been found in some HSCR patients. The hedgehog signaling pathway is an evolutionary conserved pathway that plays essential roles in the development of a variety of tissues and organs, including the spinal cord and limbs. An article published in this issue of Gastroenterology by Liu et al6Liu J.A.-J. Lai F.P.-L. Gui H.-S. et al.Identification of GLI mutations in patients with Hirschsprung disease that disrupt enteric nervous system development in mice.Gastroenterology. 2015; 149: 1837-1848Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar from the laboratory of Elly Ngan reports that defects in hedgehog signaling could also contribute to the pathogenesis of HSCR. Some HSCR patients who lacked RET coding mutations were shown to have mutations in GLI genes, which encode transcription factors that are effectors of hedgehog signaling. Furthermore, studies in mice demonstrated that hedgehog signaling is required for the normal migration of enteric neural progenitors and normal neuron:glia ratios during ENS development.6Liu J.A.-J. Lai F.P.-L. Gui H.-S. et al.Identification of GLI mutations in patients with Hirschsprung disease that disrupt enteric nervous system development in mice.Gastroenterology. 2015; 149: 1837-1848Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar It has been known for many years that sonic hedgehog, which is expressed by the endoderm at early stages of gut formation, plays an important role in the differentiation and growth of the mesodermal layers of the gut, and in the regionalization and concentric organization of the gut tube.7Roberts D.J. Smith D.M. Goff D.J. et al.Epithelial-mesenchymal signaling during the regionalization of the chick gut.Development. 1998; 125: 2791-2801Crossref PubMed Google Scholar, 8Sukegawa A. Narita T. Kameda T. et al.The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium.Development. 2000; 127: 1971-1980Crossref PubMed Google Scholar A recent study even showed that high concentrations of sonic hedgehog at the villus tips are indirectly responsible for restricting the location of intestinal stem cells to the epithelium at the bases of the villi.9Shyer A.E. Huycke T.R. Lee C. et al.Bending gradients: how the intestinal stem cell gets its home.Cell. 2015; 161: 569-580Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar However, there was also some evidence that hedgehog signaling plays a role in development of the ENS, which arises from neural crest-derived cells that migrate from the caudal hindbrain into and along the gut.10Obermayr F. Hotta R. Enomoto H. et al.Development and developmental disorders of the enteric nervous system.Nat Rev Gastroenterol Hepatol. 2013; 10: 43-57Crossref PubMed Scopus (146) Google Scholar Patched-1 (Ptch1), the receptor for hedgehog ligands, is expressed by enteric neural progenitors11Fu M. Lui V.C. Sham M.H. et al.Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut.J Cell Biol. 2004; 166: 673-684Crossref PubMed Scopus (150) Google Scholar; mice lacking Indian hedgehog are missing neurons from some parts of the small intestine, and mice lacking sonic hedgehog have enteric neurons in ectopic locations within the gut wall,12Ramalho-Santos M. Melton D.A. McMahon A.P. Hedgehog signals regulate multiple aspects of gastrointestinal development.Development. 2000; 127: 2763-2772Crossref PubMed Google Scholar while sonic hedgehog promotes the proliferation of zebrafish enteric neural progenitors in vivo13Reichenbach B. Delalande J.M. Kolmogorova E. et al.Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish.Dev Biol. 2008; 318: 52-64Crossref PubMed Scopus (64) Google Scholar and mouse enteric neural progenitors in vitro.11Fu M. Lui V.C. Sham M.H. et al.Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut.J Cell Biol. 2004; 166: 673-684Crossref PubMed Scopus (150) Google Scholar This new study by Liu et al6Liu J.A.-J. Lai F.P.-L. Gui H.-S. et al.Identification of GLI mutations in patients with Hirschsprung disease that disrupt enteric nervous system development in mice.Gastroenterology. 2015; 149: 1837-1848Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar focused on the roles of GLI transcription factors, which are effectors in the hedgehog pathway, and SUFU, which regulates the activity of GLI transcription factors, in the pathogenesis of HSCR and the development of the ENS. The study also examined interactions between the GLI transcription factors, SUFU and SOX10. SOX10 is an essential component of the ENS development transcriptional network because it is required for RET expression, and it also maintains enteric neural progenitors and regulates the differentiation of glial cells.14Bondurand N. Sham M.H. The role of SOX10 during enteric nervous system development.Dev Biol. 2013; 382: 330-343Crossref PubMed Scopus (66) Google Scholar Liu et al first performed targeted sequencing of SUFU, SOX10, and GLI family genes in 20 HSCR patients who were devoid of RET coding sequence mutations. No mutations were found in SUFU or SOX10, but 4 rare heterozygous missense mutations were found in the coding sequences of GLI1, GLI2, and GLI3 in 5 of the HSCR patients but not in 20 control subjects. Two different GLI1 mutations occurred in 2 HSCR patients; 1 patient had a mutation in GLI2, and the same GLI3 mutation occurred in 2 HSCR patients. GLI transcription factors can be activators or repressors; GLI1 is a constitutive activator, whereas GLI2 and GLI3 can be activators and repressors. The mutations in HSCR patients were found in the repressor domain of GLI2 and the transactivation domain of GLI3. Luciferase assays revealed that all of the mutations resulted in greater GLI transcriptional activity by enhancing transactivation or reducing repressor function of GLI proteins. It is intriguing that GLI mutations were found in 20% of the HSCR patients examined, because the mutation rate for genes of the RET pathway, which is thought to be the major HSCR pathway, does not exceed this number.15Hofstra R.M. Wu Y. Stulp R.P. et al.RET and GDNF gene scanning in Hirschsprung patients using two dual denaturing gel systems.Hum Mutat. 2000; 15: 418-429Crossref PubMed Scopus (80) Google Scholar, 16Emison E.S. Garcia-Barcelo M. Grice E.A. et al.Differential contributions of rare and common, coding and noncoding Ret mutations to multifactorial Hirschsprung disease liability.Am J Hum Genet. 2010; 87: 60-74Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar However, it is too early to conclude that the hedgehog pathway is a major player in HSCR, similar to the RET pathway, because the number of patients and controls analyzed is small (20 cases and 20 controls). Larger studies are needed to reveal the actual importance of the hedgehog signaling pathway in HSCR. To identify the mechanisms by which the GLI family of transcription factors regulate ENS development, the authors turned to mice. Because SUFU negatively regulates the activities of GLI proteins, the effects of enhanced GLI activity can be examined by decreasing SUFU function (Figure 1). Increased Gli activity in enteric neural crest-derived cells (NCCs) in mice owing to loss of Sufu resulted in a delay in the migration of enteric NCCs along the bowel in a subpopulation of the mutants in vivo. Time lapse imaging of NCCs in cultured gut explants revealed that the reduced migration speed of the enteric NCC population seems to be due to lesser directionality in the migratory behavior of individual NCCs. Because HSCR is caused by the retarded migration of the enteric NCC population and their consequent failure to colonize the entire length of the gastrointestinal tract,2McKeown S.J. Stamp L. Hao M.M. et al.Hirschsprung disease: a developmental disorder of the enteric nervous system.WIRES Dev Biol. 2013; 2: 113-129Crossref Scopus (117) Google Scholar, 17Avetisyan M. Schill E.M. Heuckeroth R.O. Building a second brain in the bowel.J Clin Invest. 2015; 125: 899-907Crossref PubMed Scopus (97) Google Scholar the delay in the migration of enteric NCCs in Sufu mutant mice is reminiscent of HSCR. However, because mice lacking Sufu in NCCs die before the gut is completely colonized by enteric NCCs, the authors were not able to determine whether loss of Sufu results in an absence of neurons in the distal bowel and thus a HSCR-like phenotype. Nonetheless, data showing a delay in enteric NCC migration in some embryonic Sufu mutant mice are consistent with the human data in the same study showing that some HSCR patients have mutations in GLI that result in greater transcriptional activity. The study also revealed other interesting defects in the developing ENS of mice lacking Sufu in enteric NCCs; the networks of neurons and glia were highly disorganized and the neuron:glial cell ratios were reduced. Thus, Gli activity could play a role in ENS network formation and in the generation of neurons and/or glia during ENS development. Interestingly, a recent study of Sox10Dom/+ mice, another HSCR mouse model, reported that more severe aganglionosis is associated with increased glial numbers in the midcolon.18Musser M.A. Correa H. Southard-Smith E.M. Enteric neuron imbalance and proximal dysmotility in ganglionated intestine of the Hirschsprung mouse model.Cell Mol Gastroenterol Hepatol. 2015; 1: 87-101Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar To determine the mechanisms by which Sufu and the Gli family regulate ENS development, Liu et al examined possible interactions with Sox10. The expression of Sox10 was elevated in Sufu mouse mutants. Furthermore, luciferase and gel mobility assays in cell lines suggested that Gli proteins can bind to two ENS-specific enhancers to up-regulate Sox10 expression in enteric NCCs (Figure 1). These new findings from Liu et al raise some important questions. First, one of the most interesting aspects of the study was the observation of decreased neuron:glia ratios in the intestine of embryonic mice lacking Sufu in enteric NCCs. It is unclear, however, whether the mutant mice exhibit hypoganglionosis (decreased overall numbers of enteric neurons). The authors propose a role for Sufu and Gli in neuronal versus glial lineage segregation, but further investigations are required, because decreased neuron:glia ratios could also result from a delay in neuronal differentiation, death of neurons or their precursors, or increased proliferation of glia or their precursors without any changes in the generation of neurons. Therefore, studies to determine whether the total number of glial cells, the total number of neurons, and the total number of enteric NCCs (neurons + glia + progenitors/precursors) are altered in Sufu mutant mice are required. Patients with ulcerative colitis have decreased enteric neuron:glia ratios,19Bernardini N. Segnani C. Ippolito C. et al.Immunohistochemical analysis of myenteric ganglia and interstitial cells of Cajal in ulcerative colitis.J Cell Mol Med. 2012; 16: 318-327Crossref PubMed Scopus (72) Google Scholar but to our knowledge there are no reports of altered neuron:glia ratios in the ganglionic region of HSCR patients or in subjects with other pediatric motility disorders. It would, therefore, be interesting to examine enteric neuron:glia ratios in these patients. Second, the Sox10Dom/+ HSCR mouse model has recently been shown to exhibit alterations in neuronal subtype specification as well as changes in the number of glia in the colon.18Musser M.A. Correa H. Southard-Smith E.M. Enteric neuron imbalance and proximal dysmotility in ganglionated intestine of the Hirschsprung mouse model.Cell Mol Gastroenterol Hepatol. 2015; 1: 87-101Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar The Sufu mutant mouse used by Liu et al, in which Sufu was inactivated in all NCC, does not survive beyond E13.5, so it was not possible to examine the development of different functional classes of enteric neurons. It would be fascinating to determine whether Gli transcription factors play a role in neuronal subtype specification as little is known about the mechanisms underlying the generation of the many functional types of enteric neurons.17Avetisyan M. Schill E.M. Heuckeroth R.O. Building a second brain in the bowel.J Clin Invest. 2015; 125: 899-907Crossref PubMed Scopus (97) Google Scholar Third, this study focused on the interactions of GLI transcription factors with SUFU and SOX10, but it will also be important to identify whether there are other interactions with GLI transcription factors that are important for ENS development. Finally, Liu et al found that increased Gli activity was associated with increased Sox10 expression and defects in the migration of enteric NCCs in mice. Previous studies have shown that reduced Sox10 expression also perturbs enteric NCC migration, probably indirectly via effects on the maintenance of enteric neural progenitors.14Bondurand N. Sham M.H. The role of SOX10 during enteric nervous system development.Dev Biol. 2013; 382: 330-343Crossref PubMed Scopus (66) Google Scholar It will, therefore, be important to determine the molecular mechanisms by which increased Sox10 expression and Gli activity perturb enteric NCC migration. In conclusion, the study from Liu et al presents novel data identifying a regulatory loop involving SUFU, GLI family members and SOX10 that is required for ENS development. As HSCR is not normally diagnosed prenatally, the findings are unlikely to have any direct therapeutic potential in preventing HSCR. Because of the early death of mice lacking Sufu from all NCCs, the authors were unable to show whether loss of Sufu in mice results in a HSCR-like phenotype. So it remains to be determined whether the Sufu mutant mouse is a HSCR model or an interesting model of a non-HSCR enteric neuropathy with altered neuron:glia ratios. Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in MiceGastroenterologyVol. 149Issue 7PreviewHirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. Full-Text PDF Covering the CoverGastroenterologyVol. 149Issue 7PreviewNonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in the United States and progression to its more advanced form, nonalcoholic steatohepatitis, leads to progressive fibrosis, cirrhosis, and its complications, including hepatocellular carcinoma and the need for liver transplantation. Previous studies demonstrating a link between PNPLA-3 genotype and hepatic steatosis and features of nonalcoholic steatohepatitis have suggested a genetic predisposition to NAFLD. Full-Text PDF