Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice

Accepted Manuscript Identification of GLI Mutations in Patients with Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice Jessica Ai-jia Liu, Frank Pui-Ling Lai, Hong-Sheng Gui, Mai-Har Sham, Paul Kwong-Hang Tam, Maria-Mercedes Garcia-Barcelo, Chi-Chung Hui, Elly Sau-Wai Ngan PII: DOI: Reference:

S0016-5085(15)01093-8 10.1053/j.gastro.2015.07.060 YGAST 59951

To appear in: Gastroenterology Accepted Date: 31 July 2015 Please cite this article as: Liu JA-j, Lai FP-L, Gui H-S, Sham M-H, Tam PK-H, Garcia-Barcelo MM, Hui C-C, Ngan ES-W, Identification of GLI Mutations in Patients with Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice, Gastroenterology (2015), doi: 10.1053/ j.gastro.2015.07.060. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.

ACCEPTED MANUSCRIPT Identification of GLI Mutations in Patients with Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice Jessica Ai-jia LIU1, Frank Pui-Ling LAI1, Hong-Sheng GUI2,3, Mai-Har SHAM4, Paul Kwong-Hang TAM1, Maria-Mercedes GARCIA-BARCELO1 , Chi-Chung HUI5, Elly Sau-Wai NGAN1* 1

Department of Surgery, 2Department of Psychiatry, 3Center for Genomic Sciences, Department of Biochemistry, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong. 5 Program in Developmental & Stem Cell Biology, The Hospital for Sick Children and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5G1L7.

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*Corresponding author: Dr. Elly SW Ngan Associate Professor Department of Surgery University of Hong Kong, Pokfulam Faculty of Medicine Building 21 Sassoon Road, Hong Kong, SAR, China. Tel: (852) 2819-9641 Fax: (852) 2816-9621 Email: [email protected]

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Short Title: GLI in ENS development and HSCR pathogenesis

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Author contributions: J.A.J.L. analyzed the mutant mice. F.P.L.L. and J.A.J.L. performed the live imaging studies. J.A.J.L. performed in vitro differentiation assay. H.S.G. and M.G. performed genetic analysis. M.H.S. provided Sox10N/+ mouse line. E.S.W.N. and C.C.H. supervised the project and prepared the manuscript.

Grant Support: This work was supported by research grants from the Research Grants Council of Hong Kong Special Region, China Hong Kong (HKU17116914 and T12C-714/14-R) and a seed grant for basic research from the University of Hong Kong.

Disclosures: The authors declare no conflicts of interest.

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ACCEPTED MANUSCRIPT ABSTRACT:

BACKGROUND & AIMS: Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling

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disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis.

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METHODS: We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and

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searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system (ENS) was studied in Sufuf/f, Gli3∆699, Wnt1-Cre, and Sox10NGFP mice using immunohistochemical and whole-mount staining procedures to

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quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging.

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RESULTS: We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10

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form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufuf/f mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity.

CONCLUSIONS: We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU–GLI–SOX10 regulatory loop disrupts

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ACCEPTED MANUSCRIPT migration of NCCs and development of the enteric nervous system in mice.

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KEYWORDS: mouse model; nervous system development, aganglionic megacolon, HSCR

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ACCEPTED MANUSCRIPT INTRODUCTION Neural crest cells (NCCs) represent a transient population of multipotent stem cells in vertebrate. They arise from the dorsal neural tube, migrate over a long distance to the gut and give rise to the enteric nervous system (ENS). This

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developmental process requires a precise regulation of the size of the stem cell pool and their subsequent differentiation processes. Perturbations in these processes directly affect the gut colonization by enteric NCCs, and may cause Hirschsprung’s

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disease (HSCR, aganglionic megacolon) in humans1. Indeed, deleterious mutations or aberrant expression of genes implicated in either neuronal or glial lineage

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differentiation have been shown to impair ENS development and confer higher susceptibility to HSCR2-5.

A battery of transcriptional factors organized into gene regulatory circuits controls cell fate specification of NCCs. They work cooperatively to specify enteric

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NCCs to neural progenitors, and then further differentiate into neurons and glia in the ENS. Sox10, a member of the SRY-like HMG-box family of transcription factor, is essential for the maintenance of progenitor states of NCCs6 and their glial

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differentiation7, 8. Regulation of Sox10 level determines the neuronal versus glial lineage differentiation3,

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and is of particular importance in ENS development.

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Mutations in SOX10 are associated with Waardenburg-HSCR syndrome10. In mice, a null mutation of Sox10 generated by targeted insertion of lacZ (Sox10lacZ) and a spontaneous mutation in Sox10 referred to as Dominant megacolon (Sox10Dom), both display colonic aganglionosis and hypopigmentation11, 12. Hedgehog (Hh) regulates gut organogenesis and ENS development. Depletion of Hh leads to partial intestinal aganglionosis accompanied by megacolon or ectopic ganglia formation in mice13-15. In mammals, Gli1, Gli2 and Gli3 are the transcription

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ACCEPTED MANUSCRIPT factors mediating Hh signaling output. Gli1 is a constitutive activator, while Gli2 and Gli3 act as both activator and repressor. Gli2 functions primarily as an activator (Gli2A), albeit also as a weak repressor in some circumstances, while Gli3 is predominantly a repressor (Gli3R) and only full length Gli3 possesses activator

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function. Ectopic overexpression of human GLI1 in transgenic mice resulted in HSCR-like phenotypes, in which the severity of the ENS phenotype correlates with the expression level of the GLI1 transgene16. Similarly, aberrant activation of Hh

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signaling results in premature gliogenesis of enteric NCCs and specific SNP in PTCH1 gene imparts susceptibility to HSCR17. These observations suggest that GLI

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expression level or activity is critical for ENS development and HSCR pathogenesis. Nonetheless, how Gli activator/repressor or their ratio contributes to ENS development and HSCR disease still remain largely unclear.

Suppressor of Fused (Sufu) is a key negative regulator of Gli transcription

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factors, it reduces Gli transcriptional activity by promoting the formation of Gli3 repressor (Gli3R) and the cytoplasmic sequestration of Gli activators (GliA)18,

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Intriguingly, Sox10 has been shown to promote glial differentiation of CNS

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progenitors by down-regulating Sufu expression20. This raises the possibility that

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Sufu/Gli work together with Sox10 to determine the fate of NCCs. In this study, we present evidence for a SOX10-SUFU-GLI regulatory nexus in

ENS development and its perturbation in HSCR pathogenesis. In mice, we demonstrate a mechanistic link between Sufu-Gli and Sox10 in a regulatory loop that orchestrates neuronal versus glial lineage differentiation as well as migration of NCCs. Importantly, sequencing and functional studies of HSCR samples revealed several novel mutations in GLI1, GLI2 and GLI3, which all lead to increased GLI transcriptional activity and Sox10 expression. Together, our observations provide

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ACCEPTED MANUSCRIPT evidence that links GLI mutations to HSCR patients and demonstrate that perturbed SOX10-SUFU-GLI regulatory nexus contributes to HSCR pathogenesis.

Patients

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MATERIALS AND METHODS

In total 20 sporadic (no family history) HSCR patients devoid of RET coding sequence (CDS) mutations (16 males and 4 females) were included in this study.

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These patients had been consecutively recruited at Queen Mary Hospital, where they

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had undergone surgery. Characteristics of the patients can be found in Supplementary method and Supplementary Table 2. HSCR diagnosis was based on histological examination of either biopsy or surgical resection material for absence of enteric plexuses. As controls, we included 20 Chinese individuals (16 males and 4 females) with no HSCR.

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The study was approved the institutional review board of The University of Hong Kong together with the Hospital Authority (IRB: UW 09-360). Blood samples were drawn after obtaining written informed consent. Parents gave written consent on

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behalf of their children.

Targeted sequencing

The next generation targeted sequencing was used to identify rare variants

(Minor Allele Frequency –MAF- <1% in the general population) from pooled DNA of HSCR patients. SUFU, SOX10, GLI1, GLI2 and GLI3 were selected for capture and sequencing. The study was done in Centre of Genomic Sciences at the University of Hong Kong. Analysis was performed using standard bioinformatics protocols and as described in the Supplementary Method.

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Plasmid constructions

Luciferase assays

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Expression constructs of human GLI1-3, their mutants21 and the reporter constructs (Supplementary method) were transfected into HeLa or neuroblastoma (SK-N-SH) cells. Luciferase reporter activities were monitored using the Dual-

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Luciferase® Reporter Assay System (Promega). The luminometer detected the activities of firefly (P) and Renilla luciferase (M), measuring the activities of the

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promoter constructs and the internal control, respectively. The relative luciferase activity (P:M) was calculated by normalizing the promoter firefly activity with internal Renilla luciferase activity.

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Mice

Sufuf/f, Gli3∆699, Wnt1-Cre mice were previously generated4, 22, 23. To generate Sox10NGFP mutant mice, N-terminal domain of Sox10 was replaced by an EGFP

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reporter. Swiss mice (Canadian CD-1®(ICR)) were purchased from Charles River

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Laboratories. Mice were maintained in a mix outbred background of C57 and 129/S6. Animals were maintained in the Animal Laboratory at the University of Hong Kong, and all experiments were performed in accordance with procedures approved by the committee on the Use of Live Animals, the University of Hong Kong (CULTRA 2898-12).

Enteric NCC culture

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ACCEPTED MANUSCRIPT Enteric NCCs were isolated from E11.5 Sufuf/f mouse embryonic guts as previously described (Supplementary Method)

4, 24, 25

. Cells were then transduced

with Cre-GFP (Ad-GFP-Cre) or GFP (Ad-GFP) recombinant adenovirus (Vector Biolabs). The enteric NCCs at passage 2 were harvested for Western blot, RT-qPCR,

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microarray analysis or seeded at 8 ×104 cells per 35mm well or 5×103 cells per well in 8-well chamber slide (Nunc) for the subsequent functional analyses.

Immunofluorescence

studies

were

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Immunofluorescence studies

performed

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described

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the

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Supplementary Method. In brief, the neuronal cells were detected with mouse antineuron-specific class III beta-tubulin (Tuj1), anti-neurofilament or anti-HuC/D antibodies, while the glial cells was detected with anti-Fabp. Anti-Sox10 was used to mark NCCs. The secondary antibody conjugated with Alexa Fluor 488 or 594 were

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purchased from Invitrogen. Enteric NCCs and tissue sections were photographed using a Nikon Eclipse E600 microscope with a Sony digital camera DSM1200F under

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fluorescence illumination and Zeiss LSM700/710 confocal microscope, respectively.

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Gut explant cultures and time-lapse imaging

For ex-vivo time-lapse imaging studies, E12.5 guts were placed on a filter

paper (Millipore) and cultured with DMEM medium containing 10% fetal bovine serum in a heat- and humidity-controlled chamber of 5% CO2, 37°C. GFP images were captured using a Carl Zeiss LSM510 META (Germany) laser scanning confocal microscope. Images were collected as Z stacks with a Z step size at 0.43µm and zstacks were rendered for 3D reconstruction with ZEN software (Carl Zeiss, Germany). Individual enteric NCCs in gut explants were tracked using ImageJ

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ACCEPTED MANUSCRIPT software to determine their speed of locomotion and persistence of movement. The speed of each cell was determined by dividing the total length of its trajectory by the time (a minimum of 10 hours was measured). The net speed of each cell was determined by dividing the distance between its initial and final position by the time.

by the total distance covered by the cell.

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Whole mount in situ hybridization

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Persistence was obtained by dividing the distance between its initial and final position

In situ hybridization was used to examine the expression of fatty-acid-binding E12.5 embryonic guts were

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protein-7 (Fabp7, NM_021272) in embryonic guts.

dissected out from control (Sufuf/f), Sufu (Wnt1-Cre; Sufuf/f), Sox10 (Sox10N/+) and Sufu-Sox10 compound (Wnt1-Cre; Sufuf/f; Sox10N/+) mutant embryos, then fixed with 4% PFA and cryoprotected with 30% sucrose. The whole embryonic guts were

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hybridized with DIG-labeled RNA probe encoding the coding region of Fabp7 following the standard protocol26. The signals were detected with BM-purple AP

Immunoblots

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Substrate (Roche). The embryonic guts were then sectioned and photographed.

To examine the activation of cellular signaling in enteric NCCs transduced

with adenovirus, cell lysates containing 45 µg of total protein were separated SDSpolyacrylamide gels and blotted onto nitrocellulose membranes. The membranes were then incubated with polyclonal antibodies against Sufu, Gli1, Gli2, Gli3. The same membranes were probed with anti-β-actin monoclonal antibody to ensure equal loading of cell protein per lane. All blots were incubated with secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Amersham Pharmacia

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ACCEPTED MANUSCRIPT Biotech.). Antibody-bound proteins were visualized using a chemiluminescence system (Amersham Pharmacia Biotech). The representative pictures of at least 3 independent assays were shown.

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Reverse Transcription-PCR (RT-PCR)

Total RNA was isolated using RNeasy Mini kit (Qiagen) and reverse transcribed using SuperScript™ RNA Amplification System (Invitrogen). PCR or

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quantitative RT-PCR (ABI Prism 7900, Applied Biosystems) reactions were

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performed using specific primers as listed in Supplementary table 1. Results were normalized and expressed relative to the internal control (Actin or 18S). The values reported in bar charts represent the mean ± SEM and the experiments were repeated in three independent assays.

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Statistical Analysis

The differences among multiple treatment groups were analyzed with a twosided unpaired Student t test or one-way ANOVA followed by Tukey post-test using

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GraphPad Prism 5 (GraphPad Software). A p-value less than 0.05 was interpreted to

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represent a statistically significant difference. All experiments were replicated at least three times and data are shown as means with standard error of mean (SEM).

RESULTS Identification of novel GLI mutations in HSCR patients We performed targeted sequencing study of SOX10, SUFU, and GLI family of genes in 20 HSCR patients to investigate their implications in HSCR pathogenesis.

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ACCEPTED MANUSCRIPT We identified 4 rare heterozygous missense variants in the coding sequence of GLI1, GLI2, and GLI3, but no mutation was found in SOX10 and SUFU. These 4 rare variants (Figure 1A) were observed in five of 20 HSCR patients analyzed but were not found in the control, or present at a very low frequency in the public databases

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(Supplementary Table 2). Three of four mutations were predicted to be damaging by KGGSeq27. The R557C mutation in GLI1 (rs201845227; patient HSCR#1) does not map to any known functional domain and involves a substitution of an arginine

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residue (R) with a cysteine residue (C). In patient HSCR#3, the G191R mutation (rs202141899) maps to the repressor domain of GLI2 and the H1200D mutation

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(rs145069572) is residing in the transactivation domain (TA2) of GLI3. The H1200D mutation occurred in two HSCR patients (HSCR#4 & 5).

We tested the functional consequences of these mutations using a well

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established luciferase reporter assay of Gli activity28. When GLI proteins were cotransfected with the luciferase reporter driven by Gli binding sequences (8×GBS-Luc) in a human neuroblastoma cell line (SK-N-SH), higher reporter activities were

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consistently detected with the GLI mutants when compared to the wild-type

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counterpart (Figure 1B). These assays demonstrated that all the mutations lead to enhanced transactivation activity of GLI proteins

To test the impact of the mutations residing in the repressor and transactivator

domains of GLI on their transcriptional activity, we generated a fusion protein where the repressor domain of GLI2 or the transactivation domain of GLI3 was fused with the GAL4-DNA binding domain. These fusion proteins could recognize the GAL4 binding sequence (9×GAL) of the GAL-Luc reporter and the corresponding

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ACCEPTED MANUSCRIPT transactivation or repressor function of the wild-type and mutant proteins could be measured based on the luciferase activity.

GLI2-REP-DBD fusion protein

consistently exhibited a strong repressor function, resulting in low reporter activity.

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The G191R mutation significantly reduced the repressor function of GLI2 and a significantly higher luciferase activity was detected when it was cotransfected with the reporter (Figure 1C). For GLI3, the H1200D mutant displayed a higher

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transcriptional activity (Figure 1D). In summary, all these misssense mutations resulted in high GLI transcriptional activity and likely perturbed the proper balance

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between GLI activator and repressor, representing a possible cause of HSCR disease.

Constitutive high Gli activity in Sufu mutants leads to instability of progenitors and

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promotes differentiation of enteric NCCs

It is known that deletion of Sufu promotes the formation of GliA and reduces GliR (Figure 2A), so the pathological condition (high GliA and low GliR) of the HSCR

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patient who carries the gain-of-function mutation in GLI would be mimicked by deletion of Sufu in NCCs. Enteric NCCs were isolated from E11.5 embryonic guts of

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Sufuf/f mutants as described previously29. Cre-adenovirus was then used to delete exons 3-8 of the Sufu null enteric NCCs (Figure 2B & C). This inactivation of Sufu resulted in elevated expression of Gli1, Gli2A, and reduction of Gli3R (Figure 2D), leading to an up-regulation of Hh target genes, Ptch1, Gli1 and Gli2 (Figure 2E). Both wild-type and mutant enteric NCCs response well to glial cell-derived neurotrophic factor (GDNF) and gave rise to neurons and glia of comparable capacities (data not shown). To our surprise, we found that in the untreated group (in absence of GDNF),

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ACCEPTED MANUSCRIPT significantly more neurons (Tuj1+) and glia (BFabp+) were derived from Sufu null enteric NCCs than in the control.

It suggests that deletion of Sufu induces

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spontaneous differentiation of enteric NCCs to form neurons and glia (Figure 2F).

Gli activity determines neuron to glia ratio of ENS

We then utilized Wnt1-Cre;Sufuf/f mouse mutants to further examine the

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effects of high Gli activity on neuronal and glial differentiation. Given that Sufu mutants died around E14, we analyzed the neurons and glia composition in E11-13.5

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guts. At E11.5, significant more neurons (Tuj1+) were observed in the stomach of Sufu mutants when compared to that of the control, while the difference was not found in the intestine (Figure 3A). Subsequent in situ hybridization analysis also detected robust expression of glial marker (Fabp7) in E12.5 Sufu mutants (Wnt1-

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Cre;Sufuf/f), while only weak Fabp7 expression was observed in the controls (Sufuf/f) (Figure 3B & Supplementary Figure 1). In E13.5 control guts, the glia (B-Fabp+) and neurons (HuC/D+) were well organized in a ratio of 1:1.331±0.077 and

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1:3.238±0.5117 at the proximal and distal intestine, respectively. The neurons and

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glia were highly disorganized in all Sufu mutants. More importantly, the neuron to glia ratios were significantly reduced particularly in the distal intestines (proximal intestine: 0.7037±0.0256; distal intestine: 0.7837±0.0596).

Significantly more glial

cells and reduced number of neurons were found in all Sufu mutants (Supplementary Figure 2). On the other hand, Gli3∆699/∆699 mutants, which constitutively produce only Gli3R from the Gli3 gene, had significantly fewer glia (B-Fabp+) in the intestine. The neuron to glia ratio was markedly increased compared to controls (Figure 4B). These data indicate that the Gli2A:Gli3R ratio is critical in determining the neuron versus

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ACCEPTED MANUSCRIPT glial differentiation in the ENS. In particular, high levels of Gli activity confer differentiation biased towards the glial lineage.

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Loss of Sufu interferes with axonal fasciculation and migration of enteric NCCs

Whole mount staining of neurofilament (NF) revealed that enteric neurons in Sufu mutants are improperly organized and developed. Severe axonal fasciculation

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defects were observed in the mutant guts, where nerve bundles were barely detected and the neurite pattern was highly disorganized (Figure 5A). Consistent with this,

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neurons derived from Sufu-/- NCCs showed similar defects with thinner nerve fibers (Figure 5B).

Time-lapse imaging of the in vivo migratory behavior of enteric NCCs within the

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gut revealed that although cells at the leading edge still migrate as a chain, the directionality of these cells was more erratic. This suggest that directionality of enteric NCC migration is disrupted by the loss of Sufu ((Figure 5C & E; Supplemental

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Movie S1). Some of the leading cells did not invade the hindgut after rejoining the

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rostral chain. As a result, the persistence and net speed of migration were significantly reduced (Figure 5E). Interestingly, changes in cell shape and disrupted chain migration were found in around 24% of mutant guts (17/72 embryos), where more solitary cells were found in colonized region (Figure 5F, left panel). Obvious delay in gut colonization was observed in 14% of Sufu mutants (Wnt1-Cre;Sufuf/f;ZEG) at E12.5 (10/72 embryos) (Figure 5F). All these observations indicate that Sufu is required to retain directional migration of enteric NCCs to allow complete innervation of the gut as well as the subsequent formation of neuronal network. Loss of Sufu

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ACCEPTED MANUSCRIPT makes NCCs migrate erratically, leading to delay of migration and disorganization of ENS.

In addition, it also causes neuronal differentiation defects, resulting in

reduction of neuron and hypoganglionosis.

is present in Sufu mutants after birth.

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Since the mutants died at E14, it was not possible to examine whether megacolon Nevertheless, based on the embryonic

phenotypes including disorganization of neurites, axonal fasciculation defect,

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or HSCR-like phenotype would be expected.

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abnormal neuron to glia ratio, and delayed gut colonization, malformation of the ENS

Bi-directional regulatory loop between Sufu and Sox10 determines glial lineage differentiation of enteric NCCs

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Elevated Sox10 expression was consistently found in Sufu mutants, it raises a possibility that Gli can upregulate Sox10 expression (Figure 6A). At least two putative Gli binding site (GBS) were found in the two ENS-specific enhancers of

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Sox10, which are located at 28.5kb (MCS4) and 48.5kb (MCS7) upstream to the start

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codon30, 31 (Figure 6B). Luciferase assays were performed in a neuroblastoma line (SK-N-SH) revealed that both Gli1 and Gli2 transactivated the MCS4 and MCS7 enhancers of Sox10, and that Gli-dependent transactivation of these enhancers is greatly reduced by mutations in the putative GBS (Figure 6C & D). Gel mobility shift assays further illustrated the direct bindings of Gli on these two enhancers (Figure 6E & F and Supplementary Figure 3). These results strongly suggest that Sufu, through the control of Gli transcriptional activity, negatively regulates Sox10 expression in enteric NCCs.

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In addition, our in situ hybridization data demonstrated that reduction of Sox10 level markedly attenuate the glial differentiation defects in Sufu mutants (Wnt1-Cre;Sufuf/f;Sox10N/+) (Figure 7A-C), highlighting that elevated Sox10

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expression level is indeed driving aberrant glial differentiation in Sufu mutants. Importantly, GLI proteins carrying the HSCR associated mutations consistently exhibited higher transcriptional activity on Sox10 expression (Figure 7D). On the

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other hand, Sox10 mutant cells (Sox10N/+) showed elevated Sufu expression (Figure 7E), suggesting that a bi-directional regulatory loop is involved in the control of

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Sox10 levels (Figure 7B). Sufu-Gli regulation of Sox10 expression may represent a key nexus rendering cells responsive to external stimuli, where Sufu may regulate

DISCUSSION

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Sox10 expression by controlling the formation of GliR and the availability of GliA.

Here we report the presence of novel mutations in the GLI family of genes in

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HSCR patients. These patients are not syndromic, lacking the clinical features

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normally associated with the oral-facial-digital defects caused by GLI mutations32-35. All GLI mutations identified in these HSCR patients confer higher transcriptional activity of GLI proteins. It is conceivable that they have less damaging effects (hence on the phenotype) than those loss-of-function or truncation mutations identified previously, which is in line with the fact that some of these mutations have been inherited from unaffected parents and present at a very low frequency in the general population. HSCR is an etiologically heterogeneous condition that can be transmitted in an autosomal dominant fashion with incomplete penetrance or as a multifactorial

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ACCEPTED MANUSCRIPT trait and, as previously documented, it is therefore likely that those GLI mutations contribute to the phenotype in conjunction with mutations in other genes of the same or interacting developmental pathways. We speculate that some forms of the HSCR phenotype would result from the interplay and/or accumulation of both common and

variants may lead to a spectrum of disease severity.

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rare functional DNA variants in various genes, and different constellations of genetic

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Another key finding of our study is to define the roles of Gli in ENS development. Data from our in vitro differentiation assay demonstrated that

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constitutively high Gli activity in Sufu mutants induces an early onset of enteric NCC differentiation, severely interrupting the subsequent development of ENS. Importantly, the neuron to glia ratio of the ENS is correlated inversely to the level of Gli activity, where higher Gli activity (Gli2A:Gli3R) favors glial lineage

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differentiation of NCCs, while persistent expression of Gli3R in Gli3∆699/∆699 mutants results in retarded glial differentiation. Although there is no information about the neuron to glia ratio in HSCR patients, our unpublished data indicated that disturbed

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neuron to glia ratio alters the functionality of the ENS. Mice exhibit gut motility Similarly, mice carrying the

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problems when neuron to glia ratio is increased.

expansion mutation of PHOX2B gene identified from HSCR patient with congenital central hypoventilation syndrome (CCHS) also show aberrant neuron to glia ratio3. All these studies support the notion that HSCR associated mutations could alter neuron to glia ratio of the ENS, leading to gut motility problems.

Our study also highlights the interdependency of enteric NCC differentiation and migration during ENS development. In addition to neuron versus glia differentiation,

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ACCEPTED MANUSCRIPT loss of Sufu perturbs the migratory pattern of enteric NCCs, leading to delayed gut colonization. Dramatic change in cell shape and disrupted chain migration were also observed in 24% of the mutants. Given that the expression of cell adhesion and cytoskeleton genes are severely dysregulated in Sufu null enteric NCCs as revealed by

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our microarray data (data not shown), the migration defect is likely a result of the aberrant organization of actin cytoskeleton or actin dynamics in mutant cells, interfering the subsequent organization of NCCs in the developing gut. Therefore, the

migration of enteric NCCs.

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level of Gli activity is critical in synchronization of differentiation as well as However, the underlying mechanism that controls the

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directionality of enteric NCCs still remains to be elucidated.

Mechanistically, our data illustrate a reciprocal regulation of Sox10 and SufuGli. This regulatory loop provides mechanistic basis for signal reinforcement and

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maintenance of high level of Sox10, which dictate enteric NCCs toward the glial lineage differentiation. Given that Sufu is a potent negative regulator of the Hh

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pathway, Hh signaling from gut mesenchyme may regulate Sufu level to confer the second layer of signal refinement for enteric NCC development. In particular, Hh is

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implicated in almost every step of gut morphogenesis, so its roles in coordinating enteric NCCs and gut mesenchyme development to orchestrate the formation of a functional neuronal network for ENS are peculiarly critical.

In summary, GLI mutations are identified for the first time in HSCR patients. Aberrant Gli activity perturbs the Sox10-Sufu-Gli regulatory nexus, leading to aberrant differentiation of enteric NCCs and delayed gut colonization, contributing to HSCR pathogenesis.

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ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS The authors thank Drs Stephanie Ng and Cynthia Lau for contributing data and the technical assistance, respectively. This work was supported by research grants from the Research Grants Council of Hong Kong Special Region, China Hong Kong

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(HKU17116914 and T12C-714/14-R) and a seed grant for basic research from the University of Hong Kong to E.S.W.N. Confocal Imaging and cell sorting were performed using equipment maintained by the University of Hong Kong Li Ka Shing

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Faculty of Medicine Faculty Core Facility.

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ACCEPTED MANUSCRIPT REFERENCES: 1.

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3.

Heanue TA, Pachnis V. Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies. Nature reviews. Neuroscience 2007;8:466-79. Garcia-Barcelo MM, Tang CS, Ngan ES, et al. Genome-wide association study identifies NRG1 as a susceptibility locus for Hirschsprung's disease. Proc Natl Acad Sci U S A 2009;106:2694-9. Nagashimada M, Ohta H, Li C, et al. Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression. J Clin Invest 2012;122:3145-58. Ngan ES, Garcia-Barcelo MM, Yip BH, et al. Hedgehog/Notch-induced premature gliogenesis represents a new disease mechanism for Hirschsprung disease in mice and humans. J Clin Invest 2011;121:3467-78. Tang CS, Cheng G, So MT, et al. Genome-wide copy number analysis uncovers a new HSCR gene: NRG3. PLoS Genet 2012;8:e1002687. Paratore C, Eichenberger C, Suter U, et al. Sox10 haploinsufficiency affects maintenance of progenitor cells in a mouse model of Hirschsprung disease. Hum Mol Genet 2002;11:3075-85. Kelsh RN. Sorting out Sox10 functions in neural crest development. Bioessays 2006;28:788-98. Bondurand N, Natarajan D, Barlow A, et al. Maintenance of mammalian enteric nervous system progenitors by SOX10 and endothelin 3 signalling. Development 2006;133:2075-86. Okamura Y, Saga Y. Notch signaling is required for the maintenance of enteric neural crest progenitors. Development 2008;135:3555-65. Pingault V, Bondurand N, Kuhlbrodt K, et al. SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat Genet 1998;18:171-3. Herbarth B, Pingault V, Bondurand N, et al. Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease. Proceedings of the National Academy of Sciences of the United States of America 1998;95:5161-5. Southard-Smith EM, Kos L, Pavan WJ. Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model. Nat Genet 1998;18:60-4. Ramalho-Santos M, Melton DA, McMahon AP. Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 2000;127:2763-72. Mao J, Kim BM, Rajurkar M, et al. Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract. Development 2010;137:1721-9. Jin S, Martinelli DC, Zheng X, et al. Gas1 is a receptor for sonic hedgehog to repel enteric axons. Proc Natl Acad Sci U S A 2015;112:E73-80. Yang JT, Liu CZ, Villavicencio EH, et al. Expression of human GLI in mice results in failure to thrive, early death, and patchy Hirschsprung-like gastrointestinal dilatation. Mol Med 1997;3:826-35. Ngan ES, Garcia-Barcelo MM, Yip BH, et al. Hedgehog/Notch-induced premature gliogenesis represents a new disease mechanism for Hirschsprung disease in mice and humans. The Journal of clinical investigation 2011;121:3467-78.

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8.

9. 10. 11.

12.

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13.

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7.

EP

6.

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SC

5.

14.

15.

16.

17.

20

ACCEPTED MANUSCRIPT

23. 24.

25.

26. 27.

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29.

AC C

30.

RI PT

22.

SC

21.

M AN U

20.

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19.

Ngan ES, Kim KH, Hui CC. Sonic Hedgehog Signaling and VACTERL Association. Mol Syndromol 2013;4:32-45. Hui CC, Angers S. Gli proteins in development and disease. Annu Rev Cell Dev Biol 2011;27:513-37. Pozniak CD, Langseth AJ, Dijkgraaf GJ, et al. Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing Suppressor of Fused expression. Proceedings of the National Academy of Sciences of the United States of America 2010;107:21795-800. Kalff-Suske M, Wild A, Topp J, et al. Point mutations throughout the GLI3 gene cause Greig cephalopolysyndactyly syndrome. Hum Mol Genet 1999;8:1769-77. Balaskas N, Ribeiro A, Panovska J, et al. Gene regulatory logic for reading the Sonic Hedgehog signaling gradient in the vertebrate neural tube. Cell 2012;148:273-84. Bose J, Grotewold L, Ruther U. Pallister-Hall syndrome phenotype in mice mutant for Gli3. Hum Mol Genet 2002;11:1129-35. Ngan ES, Lee KY, Sit FY, et al. Prokineticin-1 modulates proliferation and differentiation of enteric neural crest cells. Biochim Biophys Acta 2007;1773:536-45. Ngan ES, Shum CK, Poon HC, et al. Prokineticin-1 (Prok-1) works coordinately with glial cell line-derived neurotrophic factor (GDNF) to mediate proliferation and differentiation of enteric neural crest cells. Biochim Biophys Acta 2008;1783:467-478 Rosen B, Beddington RS. Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions. Trends Genet 1993;9:162-7. Li MX, Gui HS, Kwan JS, et al. A comprehensive framework for prioritizing variants in exome sequencing studies of Mendelian diseases. Nucleic Acids Res 2012;40:e53. Cheung HO, Zhang X, Ribeiro A, et al. The kinesin protein Kif7 is a critical regulator of Gli transcription factors in mammalian hedgehog signaling. Sci Signal 2009;2:ra29. Chen MH, Wilson CW, Li YJ, et al. Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved. Genes Dev 2009;23:1910-28. Antonellis A, Huynh JL, Lee-Lin SQ, et al. Identification of neural crest and glial enhancers at the mouse Sox10 locus through transgenesis in zebrafish. PLoS Genet 2008;4:e1000174. Werner T, Hammer A, Wahlbuhl M, et al. Multiple conserved regulatory elements with overlapping functions determine Sox10 expression in mouse embryogenesis. Nucleic Acids Res 2007;35:6526-38. Vortkamp A, Gessler M, Grzeschik KH. GLI3 zinc-finger gene interrupted by translocations in Greig syndrome families. Nature 1991;352:539-40. Shin SH, Kogerman P, Lindstrom E, et al. GLI3 mutations in human disorders mimic Drosophila cubitus interruptus protein functions and localization. Proc Natl Acad Sci U S A 1999;96:2880-4. Johnston JJ, Sapp JC, Turner JT, et al. Molecular analysis expands the spectrum of phenotypes associated with GLI3 mutations. Hum Mutat 2010;31:1142-54. Kang S, Graham JM, Jr., Olney AH, et al. GLI3 frameshift mutations cause autosomal dominant Pallister-Hall syndrome. Nat Genet 1997;15:266-8.

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33.

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35.

21

ACCEPTED MANUSCRIPT

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Author names in bold designate shared co-first authors

22

ACCEPTED MANUSCRIPT Figure Legends: Figure 1. Missense GLI mutations in HSCR patients. (A) Summary of GLI mutations found in HSCR patients. S, short segment aganglionosis; TCA, Total colonic aganglionosis. (B) Schematic representation of GLI1, GLI2, GLI3 proteins

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with the functional domains. Arrows mark the locations of the mutations. Luciferase assays were performed to test the transactivation capacities of the wild-type and mutant forms of GLI1, GLI2 and GLI3; (C) repressor domain of GLI2; (D) activator

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domain of GLI3. 8×GBS: 8 repeats of GLI binding sequence; 9×GAL4: 9 repeats of GAL4 UAS (Upstream Activator Sequence); LUC: luciferease; RLU: relative

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luciferase activity.

Figure 2. Ablation of Sufu activates Hh pathway and promotes enteric NCC differentiation (A) Diagram illustrates Hh signaling pathway, in which Ptch1 is a

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membrane receptor negatively regulates Hh pathway, and Sufu promotes the processing of Gli3 into its Gli3R form and the cytoplasmic sequestration of Gli2. (B) Enteric NCCs were isolated from E11.5 Sufuf/f embryonic guts, which have loxP sites

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flanking the exons 3-8 of the Sufu gene. Sufuf/f enteric NCCs were then infected with

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the control (Ad-GFP) or Cre-encoding (Ad-GFP-Cre) adenovirus. Scale bars equal 200µm. (C) PCR product was generated with primers spanning exon 1 to 9 of Sufu to distinguish the full length and the exon 3-8 deleted Sufu transcripts. (D) Western Blot analysis showing Gli and Sufu expression in adenovirus infected enteric NCCs. Actin was used as a loading control. (E) The expressions of the other Hh target genes in the control and Sufu deficient enteric NCCs were analyzed by qRT-PCR. (F) Immunocytochemical analyzes with anti-Tuj1, Sox10 and B-Fabp antibodies on

23

ACCEPTED MANUSCRIPT enteric NCCs. Percentages of neuronal (Tuj1+) and glial (B-Fabp+) cells were measured over the total number of NCCs (Sox10/DAPI) and shown in the bar charts.

Figure 3. Early onset of NCC differentiation in Sufu mutant guts. (A)

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Immunohistochemical analyzes were performed on E11.5 control (Wnt1-Cre; ZEG) and conditional Sufu mutant (Wnt1-Cre; Sufuf/f; ZEG) guts with anti-Tuj1 antibody. The neurons and enteric NCCs are marked by Tuj1+ (red) and GFP (green),

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respectively. Scale bars equal 50µm. The percentages of enteric neurons in controls and mutants were measured over the total number of enteric NCCs and shown in bar

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charts (5 control and 6 mutants). (B) In situ hybridization was used to examine the expression of glial marker (Fabp7) on E12.5 control (Sufuf/f) and Sufu mutant (Wnt1Cre; Sufuf/f) guts. Abbreviations: st, stomach; mg, midgut. Scale bars equal 0.5mm. Whole mount and cross-sections of the control and mutant stomach and intestine are

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shown. Regions highlighted are magnified as shown in insets. Quantitative RT-PCR data on the expression of Fabp in E12.5 control (Sufuf/f) and mutant (Wnt1-Cre;Sufuf/f)

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guts are shown in the bar chart (n=5).

Figure 4. Manipulation of Gli activity alters neuron to glia ratio in ENS. (A)

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Immunohistochemical analyzes were performed on E13.5 control (Sufuf/f), conditional Sufu mutant (Wnt1-Cre; Sufuf/f) and Gli3∆699/ ∆699 guts with anti-HuC/D and B-Fapb antibodies. The neuronal and glial cells are HuC/D+ and B-Fabp+, respectively. Scale bars equal 50µm. (B) Diagrams illustrates the relative ratios of Gli2A to Gli3A in Sufu, Control (Ctrl) and Gli3∆699 mutants. The numbers of neurons and glia in controls and mutants were measured and the neuron to glia ratios are shown in bar charts (n=6).

24

ACCEPTED MANUSCRIPT Figure 5. Aberrantly high Gli activity in Sufu mutant results in impaired ENS development. (A) Whole mount immunostaining with neurofilament (NLF) antibody was performed on E12.5 control (Wnt1-Cre; ZEG) and mutant (Wnt1-Cre; Sufu; ZEG) guts. Scale bars equal 100µm (B) Control (Ad-GFP) and Sufu-/- (Ad-GFP-Cre)

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enteric NCCs were cultured in differentiation medium supplement with GDNF (100ng/ml) for 10 days, followed by immunocytochemistry with anti-NLF antibody. Representative pictures of higher magnification are shown on the middle panel. Scale

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bars: 50µm or 25µm. The width of axon fibers were measured and shown in the bar chart. Error bars indicated ± SEM across the top 8 thickest neurites found in the

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control and mutant groups and three independent differentiation assays were performed. (C) Still images from time-lapse movies of the hindgut of E12.5 control (Wnt1-Cre; ZEG) and Sufu mutant (Wnt1-Cre; Sufuf/f; ZEG). Time shown is in minutes. Scale bar =100µm. Colored arrows indicate the cells tracked over 8 hours at

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10-min intervals. The colored progressive tracks highlight the tracks of each of the tracked cells. (D) Polar histograms represent the trajectories of the most caudal cell at 10-min intervals in three explants of E12.5 hindgut. (E) Cell speed at which the

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migratory wave front of enteric NCCs migrated caudally along the gut, net speed and persistence are presented. (F) The whole mount images of the E12.5 control (Wnt1-

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Cre; ZEG) and mutant (Wnt1-Cre; Sufu; ZEG) guts, where enteric NCCs were GFP+. The distances from the cecal bud to the furthest NCCs (as indicated by arrows) in the control and mutant guts were measured and are shown in the bar chart (n=6).

Figure 6. Gli proteins transactivate Sox10 expression (A) Sox10 expression in E12.5 control (Wnt1-Cre; ZEG) and conditional Sufu knockout (Wnt1-Cre Sufuf/f;ZEG) guts was analyzed using a immunofluorescence for Sox10 (red). Sox10

25

ACCEPTED MANUSCRIPT positive (filled arrowheads) and negative (open arrowheads) enteric NCCs were identified in control and mutant guts. Sox10 is localized in nuclei of enteric NCCs. (B) Schematic diagram depicting the putative Gli binding motifs in enteric nervous system specific Sox10 enhancers. Luciferase assays reveals that both Gli1 and Gli2

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can transactivate the ENS-specific enhancers: (C) MCS4 and (D) MCS7 of Sox10 gene. The Gli induced luciferase activities were abolished when the putative GBSs were mutated (MCS4∆ and MCS7∆). Three independent assays were perform and

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each in triplicate. Error bars indicate SEM. Gel mobility shift assays were performed with biotin-labeled probes containing GBS identified in (E) MCS4 and (F) MCS7 of

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Sox10 gene and nuclear extract of Gli2 overexpressing cells, in presence of unlabeled probe or antibodies against Gli2 as indicated.

Figure 7. Sufu, Gli and Sox10 work coordinately to mediate gliogenesis of

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enteric NCCs. (A) In situ hybridization was used to examine the expression of glial marker (Fabp7) on E12.5 control (Sufuf/f), Sox10 (Sox10N/+), Sufu (Wnt1-Cre; Sufuf/f) and Sox10 Sufu double (Wnt1-Cre; Sufuf/f;Sox10N/+) mutant guts. Abbreviations: st,

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stomach; mg, midgut. Scale bars equal 0.5mm. Whole mount and cross-sections of the Regions highlighted are

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control and mutant stomach and intestine are shown.

magnified as shown in the bottom panel. Quantitative RT-PCR data on the expression of (B) Fabp7 and (C) Sox10 in E12.5 control (Sufuf/f), Sox10 (Sox10N/+), Sufu (Wnt1Cre; Sufuf/f) and Sox10 Sufu double (Wnt1-Cre; Sufuf/f;Sox10N/+) mutant guts are shown in the bar charts (4-5 embryonic guts). (D) Luciferase assays with the ENSspecific enhancers (MCS4 and MCS7) of Sox10 gene and GLI mutants. (E) qRT-PCR and immunoblot show elevated Sufu expression in Sox10N/+ enteric NCCs. (F)

26

ACCEPTED MANUSCRIPT Schematic diagram illustrates the regulatory loop among Sufu, Gli and Sox10

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involved in mediating gliogenesis of enteric NCCs.

27

A Gene

Chromosome

Amino acid change

Class of mutation

Gene Feature

Functional domain involved

Phenotype

HSCR#1

GLI1

12

R557C

Missense

Damaging

unknown

S-HSCR

HSCR#2

GLI1

12

P763S

Missense

Benign

---

--

HSCR#3

GLI2

3

G191R

Missense

Damaging

REP

S-HSCR

HSCR#4

GLI3

7

H1200D

Missense

Damaging

TA2

TCA

HSCR#5

GLI3

7

H1200D

Missense

Damaging

TA2

S-HSCR

GLI2 R557C TA2

ZFD

G191R TA1

REP

8×GBS LUC

ZFD

TA2

TA1

8×GBS LUC

8

p=0.0021

4

p=0.0036

5

2

1.5

RLU (fold)

M C g-

p<0.001

1.0

0.5

0.0

G LI 3 G TA LI 3 TA 2 -W T 2 -H 12 00 D

G G LI LI 2 2 R R E E P P -G 19 1R

0

GAL-DBD

9×GAL4 LUC

p<0.001

1

pF la

G li2

TA2

3

2

V G G li2 19 1R

M

C

AC C

9×GAL4 LUC

D

g-

C M V G LI G 1 LI R 1 55 7C

g-

REP GAL-DBD

V G H LI3 12 00 D

0

0

0

G LI 3

50

6

RLU (fold)

RLU (fold)

10

TE D

100

RLU (fold)

REP

p=0.0371 p=0.0072

EP

RLU (fold)

H1200D

15

p =0.0273

150

pF la

TA1

8×GBS LUC

200

C

TA2

ZFD

M AN U

250

GLI3

SC

GLI1

RI PT

Patient

pF la

B

ACCEPTED MANUSCRIPT

Figure 1

A

ACCEPTEDB MANUSCRIPT Sufuf/f

Ptch1

loxP

1

2

3

loxP 4 5 6

7 8

11 12

Ad-GFP-Cre

Ad-GFP

Sufu

9 10

Gli3R

Gli2A

200µm

Ptch1 Gli1

D

SC

C

RI PT

Gli2A

RT-PCR

WB

M AN U

Ad-GFP

Gli2

Ad-GFP Ad-GFP-Cre Sufu

995bp

140kDa

Gli3A

425bp

Ex1-9

190kDa

Gli3R

55kDa

Actin

40kDa

p=0.0023

p=0.0101

Gli2 expression (fold)

30

20

10

0

3

2

1

-G FP -G FP -C re Ad

d-

d-

G FP G FP -C re

0

A

0

Gli1 expression (fold)

2

dG A FP dG FP -C re

Ptch1 expression (fold)

4

Sufu

4

40

p=0.0013

AC C

6

A

qRT-PCR

A

E

EP

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300bp

83kDa

Ad

Actin

Ad-GFPCre

Figure 2

ACCEPTED MANUSCRIPT

F Tuj1/Sox10 Sufu-/-

Tuj1/Sox10

B-FABP/ Sufu-/DAPI

B-FABP/ DAPI

RI PT

Ctrl

50µm

TE D

25

0

Ctrl Sufu-/-

B-FABP+(%)

10

EP

20

p=0.0013

AC C

Tuj1+(%)

30

M AN U

Ctrl

SC

50µm

p<0.0001

20 15 10 5 0

Ctrl Sufu-/-

Figure 2

A

ACCEPTED MANUSCRIPT Stomach Wnt1-Cre; Z/EG

GFP/Tuj1

GFP/Tuj1

E11.5

50µm

SC

Wnt1-Cre; Sufuf/f; Z/EG

RI PT

E11.5

Intestine

40 n=5

20 0

Wnt1-Cre; ZEG

B

Whole gut E12.5

Stomach Fabp7

AC C

Sufuf/f

St

Wnt1-Cre; Sufuf/f; ZEG

EP

In Situ hybridization

Tuj1+ / GFP+ cells (%)

n=6

p>0.05

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p<0.001

60

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Tuj1+/ GFP+ cells (%)

Stomach 60

n=5

n=6

40 20 0

Wnt1-Cre; ZEG

Wnt1-Cre; Sufuf/f; ZEG

qRT-PCR Intestine Fabp7 2.5

50µm

Fabp7 level

E12.5

St

p=0.0367 n=5

2.0

int 0.5mm

Wnt1-Cre; Sufuf/f

Intestin0e

1.5

n=5

1.0 0.5

int 0.0

0.5mm

Sufuf/f

Wnt1-Cre; Sufuf/f

Figure 3

A

Proximal intestine Distal intestine ACCEPTED MANUSCRIPT

E13.5

E13.5

E13.5

E13.5

Gli3Δ699

M AN U

SC

RI PT

Wnt1-Cre; Sufuf/f

Ctrl

HuC/D B-Fabp

50µm

E13.5

TE D

Wnt1-Cre;Sufuf/f GliA

Gli R

Ctrl

GliR

Gli3Δ699/Δ699 GliA

GliR

GliA

EP

GliA:GliR

Proximal intestine

Distal intestine

AC C

10

p=0.0001

Neuron:Glia ratio

8 6

p=0.0002

4 2

p<0.0001 p<0.0001

re C

W nt 1-

9 69

trl

99

/Δ

C G li3 Δ 6

/Δ 99

;S uf u f/f

9 69

trl C G li3 Δ 6

C

re

;S uf u f/f

0

W nt 1-

B

E13.5

Figure 4

100µm

NF

Sufu-/-

D

360

480

1.5

120

p=0.0033 0.6

0.4

0.2

0.0

0.0

WT

Mutant

p=0.0133

0.4

0.8

Persistence (Arbitrary unit)

0.5

240

480

p=0.1248

1.0

MUT

Net speed (um/min)

AC C

E

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360

Sufu mutant

Ctrl Sufu-/-

EP

0

2

240

MUT Direction of the most caudal cell Control

4

WT

M AN U

120

6

RI PT

100µm

0

p=0.0274

0

25µm

50µm

WT

Cell speed (um/min)

NFL

NFL

E12.5

NF

8

NFL

NFL

width of axon fibres (um)

Ctrl

SC

C

ACCEPTED MANUSCRIPT B

E12.5

Wnt1-Cre;ZEG Wnt1-Cre;Sufuf/f;ZEG

A

0.3

0.2

0.1

0.0

WT

Mutant

WT

Mutant

Figure 5

C ;Z /E G

uf u f/f ;Z /E G

;S

re

M AN U 2.0

SC

Wnt1-Cre;Sufuf/f; Wnt1-Cre;ZEG ZEG

re

C

n=10/72

Distance between cecal bud and the furtherst NCC (mm)

TE D

E12.5

W nt 1-

EP

100µm

W nt 1-

AC C

RI PT

Wnt1-Cre; Sufuf/f; Z/EG

Wnt1-Cre; Z/EG

F ACCEPTED MANUSCRIPT E12.5 GFP

n=17/72

p=0.0171

1.5

1.0

0.5

0.0

Figure 5

Stomach

A

E12.5

GFP/Sox10

Wnt1-Cre; Z/EG

GFP/Sox10

Intestine

ACCEPTED MANUSCRIPT

Sox10

Actin 50µm

RI PT

GFP/Sox10

GFP/Sox10

M AN U

SC

Wnt1-Cre; Sufuf/f; Z/EG

GFP/Sox10

5[-37.2]

6[-47.3]

-29663

-49133

20 p<0.001

p<0.001

15

li2 G

Δ S7

Δ+

C

S7

S7 C C M

S7 Δ+ Δ m G li1 S7

C M

G

li1

C M

S7 C M

S7

S4

Δ+ G

S4 C M

+G

C M

C

li2

0

Δ

0

li2

0 S4

1

S4

p=0.001

2

li2

RLU (fold)

RLU (fold)

10

5

C

S4 C

M

p<0.001

3

5

M

RLU (fold)

S4 Δ Δ+ G li1

li1

C M

+G

S4 C

M

C

S4

0

10

M

20

M

RLU (fold)

15

40

4

p<0.001

C

AC C

p<0.001

M

20

C

p<0.001

D

M

p<0.001

60

EP

C

-49431

-49338 CTGCCCCCACCCACCCCAA -49353

-29548 GCCCTGGGCGGGTGGAAA -29564

+m

-29267

MCS7

M

TE D

MCS4

7[-48.5] 8[-55.1] 9[-59.1]

+G

4[-28.5]

3[-9.5]

Putative Gli binding motif

M

Sox10

1*[-0] 2[-1.2]

ENS specific enhancers

S7

B

Figure 6

ACCEPTED MANUSCRIPT

F

MCS4 +

+ +

+ +

Anti-Gli2 Ab IgG Cold probe Nuclear extract (Glli2)

+ +

-

+

+ +

+ +

+ +

RI PT

-

EP

TE D

M AN U

SC

Anti-Gli2 Ab IgG Cold probe Nuclear extract (Glli2)

MCS7

AC C

E

Figure 6

2.5

A

B

Sufuf/f

Sox10N/+

Wnt1-Cre; Wnt1-Cre; Sufuf/f Sufuf/f;Sox10N/+

St St

int

St int

int

St int

p=0.0415

p<0.001

n=5

2.0 1.5

n=4

n=5

1.0 n=5

RI PT

0.5 0.0

50µm

C

20 15

H 12 00 D

G LI 3

G LI 3

G 21 9R

G li2

M C g-

H 12 00 D

G LI 3

G 21 9R

G li2

R 55 7C

G li1

p=0.0859

V

0

G LI 3

0

G li2

5

G li1

10

5

M

N

/+

p=0.033

10

C

n=6

40

G li2

p<0.001

g-

n=6

0.5

p<0.001

R 55 7C

20 15

pF la

1.0

60

RLU (fold)

40

n=7

N

80

p<0.001

V

RLU (fold)

60

1.5

W nt 1C C trl W re nt ;S 1uf C u f/f re So ;S x uf u f/f 10 N /+ ;S ox 10

AC C

p<0.001

Sox10-MCS7

G li1

80

n=7

Sufuf/fSox10N/+Wnt1-Cre;Wnt1-Cre; Sufuf/f Sufuf/f; Sox10N/+

G li1

Sox10-MCS4

p=0.0067 p<0.001

0.0

EP

50µm

Sox10 expression (fold)

M AN U TE D

50µm

D

p=0.0188

2.0

pF la

intestine

50µm

/+

W nt Su 1fu f/ C W f re nt ; 1Su C fu f/ re f So ;S x1 uf u f/f 0N /+ ;S ox 10

Sufuf/fSox10N/+Wnt1-Cre;Wnt1-Cre; Sufuf/f Sufuf/f; Sox10N/+

SC

stomach

0.5mm

Fabp7 expression (fold)

ACCEPTED MANUSCRIPT

Figure 7

ACCEPTED MANUSCRIPT

1.5

1.0

0.5

Sufu 0.0

0.5

55kDa

Actin

40kDa

SC

0N

Sufu

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So

So

x1

x1

0N

0 +/

/+

+

/+

+

0 +/ x1 So

NG

Sufu

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Committed NCCs

Sox10+/+Sox10N/+

1.0

0.0

Enteric NCCs

Gli

Sox10

EP

N

Early differentiating cells

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F

p<0.001

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p=0.0155

x1

Sox10

1.5

Sox10 expression (fold)

Sufu expression (fold)

2.0

So

E

G

N G Neurons vs Glia

Figure 7

ACCEPTED MANUSCRIPT Patients In total 20 sporadic (no family history) HSCR patients devoid of RET coding sequence (CDS) mutations were included in this study. These patients had been consecutively recruited at Queen Mary Hospital, where they had undergone surgery.

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Sixteen patients were male (15 with short segment aganglionosis and one with total colonic aganglionosis) and four were females (2 with short segment aganglionosis and 2 with long segment aganglionosis). Only one patient (male with short segment

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aganglionosis) had associated anomalies including delayed development, bilateral retinal detachment and hypogonadism. The rest of the patients had no associated

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anomalies (isolated HSCR). HSCR diagnosis was based on histological examination of either biopsy or surgical resection material for absence of enteric plexuses. As controls, we included 20 Chinese individuals (16 males and 4 females) with no HSCR.

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Targeted sequencing

The next generation targeted sequencing was used to identify rare variants (Minor Allele Frequency –MAF- <1% in the general population) from pooled DNA

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of HSCR patients. In brief, blood DNAs from pools of 5 cases or controls were

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enriched by two multiplex systems, Raindance Technology (RDT) and hybridizationbased capture (HBC).

SUFU, SOX10, GLI1, GLI2 and GLI3 were selected for

capture and sequencing. The 454 Genome Sequencer (GS) from Roche and Illumina’s Solexa GAIIx

were used for sequencing of the RDT or HBC enriched samples. The study was done in Centre of Genomic Sciences at the University of Hong Kong, and all the data were stored in fasta-like format (.fna for 454 and .fastq for Solexa). performed using standard bioinformatics protocols.

1

Analysis was

ACCEPTED MANUSCRIPT We used one program which integrates all predictions made by other programs: Variant pathogenecity was assessed by an in-house program developed by Prof PC Sham’s group, KGGSeq.

KGGSeq is a software platform constituted of

bioinformatics and statistical genetics functions making use of valuable biologic

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resources and knowledge providing a comprehensive and efficient framework to filter and prioritize genetic variants from whole exome and whole genome sequencing data. Importantly, KGGSeq integrates “knowledge” resources from epigenetic

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databases, biological pathways and protein-protein interaction networks to annotate the genes that harbour any post-QC variants as well as to predict the potential

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pathogenicity of their variants. For the later, KGGSeq integrates 4 prediction programs (Polyphen2, Sift, MutationTaster and Likelihood ratio) which are weighted by logistic regression.

Those rare variants predicted deleterious, unique to the patients analyzed and

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whose MAF was zero or <1% in public databases (The 1000 Genome Project, the NIH Exome Sequence Project of 6500 individuals -ESP6500-) or in-house exome sequencing projects (N=900 Chinese individuals) were selected for validation. Thus,

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Sanger sequencing of the patients’ DNA was performed to validate the next-

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generation sequencing data. Sanger sequencing of parental DNA was also performed (when available) to assess the origin of the variant.

Plasmid constructions

Full length of human GLI1 (NP_005260.1), repressor domain (REP) of GLI2 (NP_005261.2) and transactivation domain 2 (TA2) of GLI3 (NM_000168.5)

20

were

obtained by PCR with specific primer pairs (Supplementary Table 1). Human GLI1 was subcloned into a pFLAG-CMV2 expression vector (Sigma) at Hind III and XbaI

2

ACCEPTED MANUSCRIPT sites. As for GLI2 REP and GLI3 TA2, two restriction endonuclease cut sites SbfI and EcoRI with addition of a 3’ end stop codon were included in the primers for subcloning. The corresponding PCR products were then cloned into pFN26A (BIND) hRluc-neo Flexi® Vector (Promega) to obtain constructs for generation of fusion

repressor

domain

(pFN26A_GLI2_REP)

or

GLI3

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proteins, where the GAL4 DNA binding domain was placed upstream to the GLI2 transactivator

domain

(pFN26A_GLI3_TA2). Mouse Gli1 and Gli2 cDNA were subcloned into an

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expression vector pFLAG-CMV2 (pFLAG-CMV_Gli1 and pFLAG-CMV_Gli2). Luciferase reporter constructs containing the ENS specific Sox10 enhancers

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(20243:pE1b-C-F/Sox10 MCS4 and 20246:pE1b-C-F/Sox10 MCS7) were obtained from Addgene. HSCR associated mutations were then introduced into GLI expression constructs and putative Gli-binding sequences (GBS) in Sox10 enhancer reporter constructs were mutated using the QuikChange Lightning Site-Directed Mutagenesis

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Kit (Agilent Technologies, Santa Clara, CA) with specific primers listed in Supplementary Table 1 according to the manufacturer’s protocol. DNA sequence and mutations were confirmed by Sanger sequencing. The wild-type and mutated GLI

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expression constructs were used for subsequent dual luciferase assays with p8×GBS-

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luc, pGL4.35[luc2P/9XGAL4UAS/Hygro] Vector (Promega) or Sox10-enhancer reporters (MCS4/MCS7).

Luciferase assays

HeLa (5.0 x 104) or neuroblastoma (SK-N-SH, 2.5 x 104) cells were seeded onto 24-well plates (Nunc) using DMEM supplemented with 10 % FBS and 1 % penicillin/streptomycin 24 hours prior to transfection. 50 or 100ng of luciferase constructs (p8×GBS-luc, pGL4.35[luc2P/9XGAL4UAS/Hygro], wild-type or mutant

3

ACCEPTED MANUSCRIPT pE1b-C-F/Sox10 MCS4 or pE1b-C-F/Sox10 MCS7) and 100-500ng GLI expression constructs were transfected into the cells using FuGene® HD Transfection Reagent (Promega) according to the manufacturer’s protocol. Luciferase activities were measured with the Dual-Luciferase® Reporter Assay System (Promega) using

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MicroLumatPlus LB 96V instrument (Berthold Technology, Germany). The luminometer detected the activities of firefly (P) and Renilla luciferase (M), measuring the activities of the promoter constructs and the internal control,

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respectively. The relative luciferase activity (P:M) was calculated by normalizing the

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promoter firefly activity with internal Renilla luciferase activity.

Enteric NCC culture

Enteric NCCs were isolated from E11.5 Sufuf/f mouse embryonic guts (from

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stomach to hindgut) and guts were dissected in L15 medium (Invitrogen). Guts were washed with Ca2+ and Mg2+ free PBS and digested with collagenase/dispase (0.2mg/ml each; 37°C for 10 min). Digested guts were triturated into single cells and

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filtered through cell strainers (100 µm and then 40µm, Falcon, BD Biosciences). Cells

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were resuspended in neural crest medium, DMEM containing 15% Chick embryo extract (SIL), FGF (20 ng/ml, Sigma, St Louis, MO), EGF (20ng/ml, Sigma), Retinoic acid (35ng/ml, Sigma), N2 (1%), B27 (2%, Invitrogen), β-mercaptoethanol (50mM, Sigma), and plated onto poly-D-lysine and fibronectin (20µg/ml each, Invitrogen)coated wells and enriched by multiple replating. The enteric NCCs at passage 2 were harvested for Western blot, RT-qPCR, microarray analysis or seeded at 8 ×104 cells per 35mm well or 5×103 cells per well in 8-well chamber slide (Nunc) for the subsequent functional analyses.

4

ACCEPTED MANUSCRIPT

Viral Transduction Cre-GFP (Ad-GFP-Cre) or GFP (Ad-GFP) recombinant adenovirus (Vector Biolabs) was added to cells in a 1:50 dilution (~1.5× 108 PFU/ml). The infection

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efficiency was estimated by counting the GFP positive cells under the fluorescence microscope. Seventy-two hours after infection, approximately 90-95% of the cells expressed GFP. Deletion of Sufu and activation of Hh signaling were confirmed by Cell proliferation and differentiation assays were

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RT-PCR and Western blot.

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performed as described below.

Immunofluorescence studies

Immunocytochemistry - To determine the differentiation capacity of enteric NCCs after deletion of Sufu, we analyzed differentiation of adenovirus infected Sufuf/f

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enteric NCCs. In brief, enteric NCCs from control and Sufu mutants were plated at clonal density and subjected to virus infection for 72 hours. After transduction, enteric NCCs were cultured without GDNF for an additional 3-10 days and then

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subjected to immunostaining as described above. The neuronal cells were detected

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with mouse anti-neuron-specific class III beta-tubulin (Tuj1) antibody (1:500, Chemicon), while the glial differentiation was detected by staining with antibodies specific for Fabp (a gift from Carmen Birchmeier , Max-Delbrueck-Centrum, Berlin, Germany, 1:1,000,000). Immunosignal was then detected using the secondary antibody conjugated with Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 680 (Invitrogen). The percentages of neuronal (Tuj1+) and glial (B-Fabp+) cells were counted in both control (Ad-GFP) and mutant (Ad-GFP-Cre) groups. For each group, a minimum of 6 random fields under 200× magnification with at least 150 cells in one

5

ACCEPTED MANUSCRIPT well of 8 well chamber slides was photographed for cell counting. The values reported in bar charts represent the mean ± SEM of three wells (i.e. 18 random fields) and the experiments were repeated three times.

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Immunohistochemistry - For section immunohistochemistry, embryos were fixed in 4% paraformaldehyde (PFA) in PBS at 4ºC, dehydrated and cryoprotected in 30% sucrose in PBS at 4ºC and embedded in OCT compound (Tissue-Tek). The sections

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were blocked in PBS containing 10% normal goat or horse serum (DAKO) for 1 hour at room temperature, then incubated overnight at 4ºC in a mixture of the anti-Tuj1

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(1:500, Chemicon), anti-neurofilament (1:200, Chemicon), anti-Sox10 (1:100, Santa Cruz), anti-HuC/D (1:200, Invitrogen), or anti-Fabp antibodies. After washing, the immunosignals were then detected using the secondary antibody conjugated with Alexa Fluor 488 or 594

(Invitrogen).

Enteric NCCs and tissue sections were

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photographed using a Nikon Eclipse E600 microscope with a Sony digital camera DSM1200F under fluorescence illumination and Zeiss LSM700/710 confocal

EP

microscope, respectively.

Immunoblots

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To examine the activation of cellular signaling in enteric NCCs transduced with adenovirus, cells from two wells of 6 well were collected and then lysed with cell lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin and 1 mM phenylmethanesulfonyl fluoride (PMSF). Cell lysates containing 45 µg of total protein were separated on 8-12% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes.

The

membranes were then incubated with polyclonal antibodies against Sufu (1:1000,

6

ACCEPTED MANUSCRIPT Abcam), Gli1 (1:1500, R&D), Gli2 (1:1000, Abcam), Gli3 (1:500, Santa Cruz). The same membranes were probed with a 1:5000 dilution of anti-β-actin monoclonal antibody (Millipore) to ensure equal loading of cell protein per lane. All blots were incubated with 1:5000 dilutions of secondary horseradish peroxidase-conjugated anti-

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mouse or anti-rabbit antibody (Amersham Pharmacia Biotech.). Antibody-bound proteins were visualized using a chemiluminescence system (Amersham Pharmacia

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Biotech). The representative pictures of at least 3 independent assays were shown.

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Reverse Transcription-PCR (RT-PCR)

Total RNA was isolated from enteric NCCs by RNeasy Mini kit (Qiagen) and reverse transcribed in 20µl reaction system using SuperScript™ RNA Amplification System (Invitrogen), in accordance with the manufacturer’s instructions.

PCR

reactions were performed using specific primers as listed in Supplementary table 1.

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Results were normalized and expressed relative to the internal control (Actin or 18S).

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Quantitative RT-PCR

RNA for qRT-PCR was extracted from mouse embryonic guts or enteric

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NCCs using RNeasy Mini kit (Qiagen) and reverse transcribed in 20µl as described above. qPCR was performed in the Cybergreen reaction mix (Applied Biosystems), which consisted of 1× mastermix, forward and reverse primers.

The reaction mix

(18µl) was aliquoted into tubes and 2 µl cDNA was added. Triplicated 20 µl samples and negative (water) controls were placed in a PCR plate and wells were sealed with optical caps.

The PCRs were carried out using an ABI Prism 7900 (Applied

Biosystems). All primers sequences were listed in Supplementary table 1. Data were analyzed and processed using Sequence Detector version 1.6.3 (Applied Biosystems) 7

ACCEPTED MANUSCRIPT in accordance with the manufacturer’s instructions.

Primers (concentration and

annealing temperature) were optimized and the linearity of the results validated in serial dilution of a cDNA pool. Results were analyzed using the 2-∆∆CT method and expressed relative to the control. 18S was used as the internal control. The values

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reported in bar charts represent the mean ± SEM and the experiments were repeated in three independent assays.

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Electrophoretic Mobility Shift Assay (EMSA)

HeLa cells were seeded at a density of 3 × 105 cells per well in a 6-well plate

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and were transfected with 2.5µg of the Gli1 or Gli2 expression plasmids. Nuclear extracts containing the Gli proteins were obtained from three wells of transfected cells using nuclear and cytoplasmic extraction kit (ThermoFisher). 1µM of single-stranded oligonucleotides

(MCS4:

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5’CTTTGGCAGTGGTTCCAGCCCTGGGCGGGTGGAAAGAGTGCTGGCA-3’; MCS7:5’TCTCTGGCTCTTCTCTGCCCCCCACCCACCCCAAGTCCCTCCTCCC3’) derived from the Sox10 ENS specific enhancers were biotin-labeled using Biotin

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3' End DNA Labeling Kit (ThermoFisher). Biotin-labeled oligonucleotides were then

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heat denatured and annealed. EMSA was performed by mixing the nuclear extracts with biotin-labeled double-stranded oligonucleotides according to the manufacture protocol (LightShift® Chemiluminescent EMSA Kit, ThermoFisher). in brief, 5µg of protein nuclear extract (around 2-3µl) were incubated with 1×binding buffer, 1mM EDTA, 5 mM MgCl2, 6% glycerol, 60 mM KCl, 50 ng/µl Poly (dI.dC), 0.05% NP-40 and 125 ng/µl bovine serum albumin for 10 min for blocking. Labeled probes (20 fpm) was then added into the mixture and incubated for 30 min at room temperature. For antibody competition assays, 2µg of anti-Gli2 (AbCam ab26056), 2µg anti-Gli1

8

ACCEPTED MANUSCRIPT (Santa Cruz Biotechnology), or 2µg of rabbit IgG control(R&D Systems) was included into the mixture before adding the probes. The entire 20 µl binding reaction was resolved on a 4-20% gradient non-denaturing TBE pre-cast gel (Bio-Rad, USA). Biotin-labeled oligonucleotides were then transferred to a positively charged nylon

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membrane (ThermoFisher) in 0.5X Tris borate-EDTA buffer. After cross-linking, the blot was probed with streptavidin-HRP conjugate and biotin-labeled oligonucleotides

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EP

TE D

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SC

were detected using Chemiluminscent detection kit.

9

ACCEPTED MANUSCRIPT

Supplementary Table 1. Sequence information on PCR primers

Ptch-1 Gli2 Sox10 Fabp7 18S hGLI1 hGLI2_REP hGLI3_TA2 hGLI1-R557C hGLI2-R191C hGLI3-H1200D mGli1-R557C

Cycle

Purpose

60

35

qRT-PCR

995/ 426 300

56

35

RT-PCR

60

25

RT-PCR

80

60

40

qRT-PCR

100

60

40

qRT-PCR

186

60

40

qRT-PCR

85

60

40

qRT-PCR

80

60

40

qRT-PCR

183

64

40

qRT-PCR

3339

54

40

Subcloning

867

58

40

Subcloning

867

58

40

Subcloning

-

60

-

Mutagenesis

-

60

-

Mutagenesis

-

60

-

Mutagenesis

-

60

-

Mutagenesis

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Annealing Temperature (°°C)

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Gli1

Product Size (bp) 165

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β-actin

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Sufu ex 1-9

5’- ATTCAGCCCAACAGTGGAAC -3’ 5’- CCGTCTGTCTAATGCCTTT -3’ 5’- CCGCTATCGTCAAGTACTG -3’ 5’- CTTAGGCAGAGAGGGATG -3’ 5’-GAGAGGGAAATCGTGCGTGAC-3’ 5’-AGCTCAGTAACAGTCCGCCTA-3’ 5’- ACGCCTTGAAAACCTCAAGA-3’ 5’- GCAACCTTCTTGCTCACACA-3’ 5’- GGGTCCTCGCTTACAAACTC-3’ 5’- ATGATGCCATCTGCGTCTAC-3’ 5’-ACCATGCCTACCCAACTCAG -3’ 5’-CTGCTCCTGTGTCAGTCCAA-3’ 5’-GACCAGTACCCTCACCTC-3’ 5’-CGCTTGTCACTTTCGTTCAG-3’ 5’-CCAGCTGGGAGAAGAGTTTG-3’ 5’-GAGCTTGTCTCCATCCAACC-3’ 5’-CGGCTACCACATCCAAGGA-3’ 5’-GCTGGAATTACCGCGGCT-3’ 5’- GACAAGCTTATGTTCAACTCGATG-3’ 5’-TCCTCTAGATTAGGCACTAGAGTT-3’ 5’-TTTGCGATCGCCATGGAGACGTCTG-3’ 5’-AAAGAATTCTTAGAGGGCACCCGCTGA-3’ 5’-TTTGCGATCGCCAGTCTCGTGCTTCAG-3’ 5’-TTTGAATTCTTACAGGTACCCCTGTCCCA-3’ 5’-GCCTATACTGTCAGCTGTCGCTCCTCCCTGGCC-3’ 5’-GGCCAGGGAGGAGCGACAGCTGACAGTATAGGC-3’ 5’-CCCGCGCCCTACAGGGACCTGCTG-3’ 5’-CAGCAGGTCCCTGTAGGGCGCGGG-3’ 5’-GGCATGGTCGTCGACCCGCAGAACC-3’ 5’-GGTTCTGCGGGTCGACGACCATGCC-3’ 5’-TACACAGTCAGCTGTAGGTCCTCC-3’

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Sufu

Primer sequence (5’ to 3’)

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Gene

ACCEPTED MANUSCRIPT

5’-GGAGGACCTACAGCTGACTGTGTA-3’ 60

-

Mutagenesis

-

60

-

Mutagenesis

-

60

-

Mutagenesis

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-

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MCS7_GBS∆

EP

MCS4_GBS∆

5’-CCCACCCCTTATAGAGACCTTCTA-3’ 5’-TAGAAGGTCTCTATAAGGGGTGGG-3’ 5’-AGTGGTTCCAGCAAGTTTATTTGCACCCGAGTGCTGGCAC-3’ 5’-GTGCCAGCACTCGGGTGCAAATAAACTTGCTGGAACCACT-3’ 5’-TGGCTCTTCTCTATTTTTTGTTTGTTTTAAGTCCCTCCTC-3’ 5’-GAGGAGGGACTTAAAACAAACAAAAAATAGAGAAGAGCCA-3’

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mGli2-R191C

ACCEPTED MANUSCRIPT

Chr

Gene

HSCR#1

S, M

12

GLI1

HSCR#2

S, M

12

GLI1

HSCR#3

S, M

3

GLI2

HSCR#4

TCA, M

7

HSCR#5

S, M

7

Protein change NP_005260.1:R557C NP_001153517.1:R429C NP_001161081.1:R516C NP_005260.1: P804S NP_001153517.1: P676S NP_001161081.1:P763S

Functional domain2

Predicted impact

MAF3 (dbSNP137)

MAF4 (ESP6500)

In house exomes5

ZFD

SC

Phenotype1

Damaging

rs201845227 MAF=0.0009

Not found

Not found

Not found

Not found

Not found

Not found

Not found

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Patient*

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Supplementary Table 2: GLI mutations in HSCR patients

Benign

NP_005261.2:G191R

REP

Damaging

GLI3

NP_000159.3:H1200D

TA2

Damaging

GLI3

NP_000159.3:H1200D

TA2

Damaging

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-

rs202141899 MAF=0.0005 rs145069572 MAF=0.00538 rs145069572 MAF=0.00538

European American (MAF=0.00012) European American (MAF=0.00012)

MAF=0.021 MAF=0.021

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*: None of the patients had associated anomalies. 1: S, short segment aganglionosis; TCA, Total colonic aganglionosis; M, male; F, female. 2: ZFD, zinc finger domain; REP, repressor domain; TA, trans-activation domain. 3: minor allele frequency reported in dbSNP137 (includes latest data from the 1000 Genome Project). 4: minor allele frequency reported in the NIH Exome Sequence Project (ESP) on 6500 individuals. 5: minor allele frequency in other in-house exome sequencing projects (N=571).

ACCEPTED MANUSCRIPT

Wnt1-Cre; Sufuf/f;ZEG

Wnt1-Cre; Sufuf/f;Sox10N/+

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GFP/B-Fabp

Wnt1-Cre;ZEG

Sox10N/+

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EP

50µm

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B-Fabp

GFP/B-Fabp

B-Fabp

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SC

50µm

Supplementary Figure 1. Reduction of Sox10 level markedly attenuate the glial differentiation defects in Sufu mutants. Immunohistochemistry was used to examine the expression of glial marker (bFabp) on E12.5 control (Wnt1-Cre; ZEG), Sox10 (Sox10N/ +), Sufu (Wnt1-Cre; Sufuf/f; ZEG) and Sox10 Sufu double (Wnt1-Cre; Sufuf/f;Sox10N/+) mutant guts. Cross-sections of the control and mutant intestine are shown. Regions highlighted are magnified as shown in the bottom panel. Scale bars equal 50µm.

A

B

ACCEPTED MANUSCRIPT

Ctrl

Wnt1-Cre; Sufuf/f

Ctrl

Wnt1-Cre; Sufuf/f

50µm

p<0.0001

SC

20

Number of glia per section

n=6

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15

EP

5

0

trl

;S

uf u f/f

AC C

W nt 1-

C

re

;S

C

trl

0

n=6

10

C

5

15

re

10

n=6

C

n=6

p<0.0001

W nt 1-

20

E13.5

uf u f/f

E13.5

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number of neurons per section

50µm

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Fabp

HuC/D

Supplementary Figure 2. Reduced number of neurons and increase in number of glia in Sufu mutants. Immunohistochemical analyzes were performed on distal intestine of E13.5 control and Sufu mutant with (A) anti-HuC/D and (B) anti-Fabp antibodies. The numbers of neurons (HuC/D+) and glia (Fabp+) per section (total 24-33 sections from 6 embryos each group) are shown in the bar-chart. Scale bars equal 50µm.

A

B

ACCEPTED MANUSCRIPT

MCS4 Cold probe

-

+

Cold probe

+ +

Nuclear extract (Glli1)

-

+

+ +

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SC

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Nuclear extract (Glli1)

MCS7

AC C

EP

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Supplementary Figure 3. Gel mobility shift assays were performed with biotin-labeled probes containing GBS identified in (A) MCS4 and (B) MCS7 of Sox10 gene and nuclear extract of Gli1 overexpressing cells, in presence of unlabeled probe as indicated. Arrows mark the specific shifted band.