Heterozygous missense variant in GLI2 impairs human endocrine pancreas development

Missense variants are the most common type of coding genetic variants. Their functional assessment is fundamental for defining any implication in human diseases and may also uncover genes that are essential for human organ development. Here, we apply CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant in GLI2 identified in two siblings with early-onset and insulin-dependent diabetes of unknown cause. GLI2 is a primary mediator of the Hedgehog pathway, which regulates pancreatic β-cell development in mice. However, neither mutations in GLI2 nor Hedgehog dysregulation have been reported as cause or predisposition to diabetes. We establish and study a set of isogenic iPSC lines harbouring the missense variant for their ability to differentiate into pancreatic β-like cells. Interestingly, iPSCs carrying the missense variant show altered GLI2 transcriptional activity and impaired differentiation of pancreatic progenitors into endocrine cells. RNASeq and network analyses unveil a crosstalk between Hedgehog and WNT pathways, with the dysregulation of non-canonical WNT signaling in pancreatic progenitors carrying the GLI2 missense variant. Collectively, our findings underscore an essential role for GLI2 in human endocrine development and identify a gene variant that may lead to diabetes.


SUMMARY
Missense variants are the most common type of coding genetic variants.Their functional assessment is fundamental for defining any implication in human diseases and may also uncover genes that are essential for human organ development.Here, we applied CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant in GLI2 identified in a paediatric hyperglycaemic patient and family members.GLI2 is a primary mediator of the Hedgehog pathway, which regulates pancreatic β-cell development in mice.However, neither mutations in GLI2 nor Hedgehog dysregulation have been reported as cause or predisposition to diabetes.
A set of isogenic iPSC lines harbouring the missense variant were studied for their ability to differentiate into pancreatic β-like cells.Interestingly, iPSCs carrying the missense variant showed altered GLI2 transcriptional activity and impaired endocrine differentiation.Collectively, our findings underscore an essential role for GLI2 in human endocrine development and identify a novel variant that predisposes to diabetes.

INTRODUCTION
Diabetes is a worldwide health problem caused by the loss or dysfunction of the insulin-secreting β-cells in the pancreas. 1,2Unelucidated forms of monogenic diabetes, arising from rare mutations in one single gene, represent invaluable models for identifying new targets of β-cell development and function. 3Notably, monogenic diabetes predominantly results from mutations in transcription factors, which are involved in pancreas development and may also cause type 2 diabetes. 4To date, over 30 genes have been linked to monogenic diabetes, yet many patients are misdiagnosed.Additionally, the disease penetrance can vary substantially among individuals with the same mutation. 5Recent studies have underscored the benefits of genetic diagnosis in patients with monogenic diabetes, enabling to improve patient care by optimizing treatment strategy, to predict the disease course and to define risks for relatives. 3,5Therefore, identification of novel disease-related loci might instruct translational efforts towards new treatment of diabetes and help in defining genetic factors that increase susceptibility to type 2 diabetes.
Human pluripotent stem cell (hPSC) models are increasingly being used to study monogenic diabetes, as rodent models have often failed to recapitulate disease phenotypes observed in humans. 4,6][8][9][10] In particular, the generation of isogenic hPSC pairs, which differ only by the mutation of interest, has been key to minimize confounding results due to differences in genetic backgrounds and ensures that observed phenotypes are attributable to a specific genetic defect. 11,12[15] During pancreas organogenesis, repression of HH signalling is required for pancreas fate specification and cell differentiation, 16 while at later stages active HH signalling is essential for the maintenance of endocrine function. 17However, high HH levels in insulin-producing β-cells have been reported to impair their function by interfering with the mature β-cell differentiation state. 18Thus, a precise spatiotemporal HH regulation appears to be critical for mouse pancreas development and β-cell function, whereas its function in human embryonic pancreas is not known.0][21] Aberrant activation of the HH pathway and elevated expression of GLI2 has also been reported in pancreatic cancer. 22,23Here, we applied CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant of GLI2 gene (GLI2 P1554L ) identified in a paediatric hyperglycaemic patient and family members.A set of isogenic iPSC lines harbouring the mutation were studied for their ability to differentiate into pancreatic β-like cells in comparison to control lines.Our findings show that GLI2 plays an essential role in human endocrine cell development and identify a novel variant that predisposes to diabetes.

GLI2
In a consanguineous family with diabetes of unknown etiology, we identified an uncharacterized heterozygous GLI2 missense variant (hg38 chr2:120990575; c.4661C>T; p.P1554L; rs767802807) (Fig. 1a, b and Supplementary Table 1).The index child developed diabetes at the age of 5 years, the individual currently in adult age requires multiple daily insulin injections and already had complications onset.The younger sibling also carries the variant and has diabetes.Although both parents harbour the c.C4661T variant in GLI2, only the father developed diabetes.Moreover, the grandmother paternal side had diabetes, but passed away and, therefore, was not available for sequencing.
So far, mutations in GLI2 have not been associated with diabetes in publicly available databases.However, the common variant rs12617659, which is close to the locus of GLI2 (LOC105373585), has been previously associated with type 2 diabetes in a meta-analysis of GWAS. 24To assess the effect of the identified GLI2 c.4661C>T variant, we applied various independent in-silico tools (Supplementary Table 1).Specifically, the GLI2 missense variant results into a single amino acid change (p.P1554L) in the transcription activation domain (TAD) of the protein and the affected residue is highly conserved across species with a genomic evolutionary rate profiling score greater than 3 (Fig. 1a,c).Moreover, the p.P1554L missense mutation showed high CADD score and was predicted to be deleterious by all applied bioinformatics tools 25 (Supplementary Table 1).Next, we examined the spatio-temporal pattern of expression of GLI2 in human pancreas (Fig. 1d and Supplementary Fig. 1f).We found that GLI2 is present in human early fetal pancreas (Fig. 1d), specifically in a subset of progenitor cells.Consistently, GLI2 was expressed at distinct levels in human iPSCs undergoing differentiation into pancreatic cells, especially abundant at pancreatic progenitor (PP) and b-like cell stages (Supplementary Fig. 1f).Together, our results suggest that GLI2 is part of a complex regulatory network regulating human b-cell development.

The GLI2 p.P1554L patient variant exhibits decreased HH signaling activation
All three GLI proteins (GLI1, GLI2 and GLI3) share a conserved C2H2-type zinc finger DNA-binding domain. 14GLI1 functions exclusively as transcriptional activator, while GLI2 and GLI3 can act as both activators or repressors, depending on the levels of HH. 15 To start investigating the newly identified GLI2 p.P1554L (hereinafter referred to as GLI2 P>L ) patient variant, we first tested whether it interferes with the basic transactivation property of the GLI2 transcription factor using a GLI-responsive luciferase reporter assay. 26The GLI2 P>L variant displayed significantly reduced transcriptional activity compared to wild-type (WT) GLI2 activity (Fig. 1E).Next, we examined the consequences of the ectopic expression of GLI2 P>L variant in mouse Min6 insulinoma cells, a well-established in vitro β-cell model.We found that the expression of crucial β-cell genes, including Mafa and NeuroD1, was severely reduced in GLI2 P>L -transfected cells (Fig. 1f).Taken together, these results suggest that GLI2 P>L impairs HH signaling as well as the preservation of β-cell features.

Defective endocrine progenitor specification in iPSCs carrying the GLI2 P>L patient variant
To study the GLI2 P>L patient variant in pancreatic β-cells, we established human isogenic iPSC lines with either the heterozygous or homozygous variant and differentiated them along the pancreatic cell lineage (Supplementary Fig. 1a).We used an iPSC line, which carries a doxycycline (DOX)-inducible Cas9 expression cassette inserted into the AAVS1 locus, allowing the de novo introduction of mutations into otherwise healthy iPSCs. 27This strategy not only bypasses the limitations of iPSC generation from patients that requires biopsy and reprogramming, but also overcomes the issues related to the genetic background difference of patients derived-iPSCs versus healthy controls, which can substantially influence the phenotype. 11Both heterozygous (GLI2 +/-) and homozygous (GLI2 +/+ ) gene-edited iPSCs displayed normal morphology and expressed pluripotency markers (Supplementary Fig. 1b-e).
Patient-like GLI2 +/-iPSCs also progressed to the pancreatic and endocrine progenitor stages as the GLI2 CTRL cells (Fig. 2c,d).However, the level of expression of PDX1 and NKX6.1 was significantly lower in GLI2 +/--derived progenitors (Fig. 2c) and this was accompanied by approximately 70% reduction in the fraction of PDX1/NKX6.1-doublepositive cells as compared to control cells (Fig. 2d).Additionally, the expression levels of other endocrine transcription factors, such as PAX4, NGN3 and its downstream targets NEUROD1 and NKX2.2, were reduced in GLI2 +/--derived endocrine progenitors (Fig. 2e).Consistently, the number of NKX6.1/INSULIN-double positive cells was significantly lower in GLI2 +/--derived cell clusters compared to controls (Fig. 2f and Supplementary Fig. 2a).No significant changes in apoptosis or proliferation were measured in the GLI2 +/-mutant cells compared to controls (Fig. 2f and Supplementary Fig. 2b).Altogether, our results demonstrate that the GLI2 P>L patient variant impairs the activation of genes essential for endocrine progenitor development.

Dose-dependent effect of GLI2 P>L variant in β-cell differentiation
Co-expression of PDX1 and NKX6.1 in endocrine progenitors is a key step necessary for the transient expression of NGN3 and subsequent generation of glucoseresponsive β-like cells. 30Since GLI2 P>L patient variant affected the differentiation of iPSCs into endocrine progenitors, we asked whether this had any functional consequences on β-cell development.Heterozygous GLI2 +/--derived endocrine progenitor cells were cultured in suspension for an additional week to promote the formation of β-like cells and then analyzed for their differentiation state and functional properties.Consistent with the findings at earlier stages, GLI2 +/-β-like cells showed reduced expression of essential β-cell markers, including key transcription factors (PDX1, NKX6.1, NEUROD1, NKX2.2), genes important for insulin secretion (KIR6.2) and endocrine hormones (INSULIN) (Fig. 2g).Moreover, GLI2 +/-β-like cells showed a striking decrease of insulin release in response to glucose stimulation (Fig. 2h).Thus, these findings support impaired β-cell differentiation and β-cell function in GLI2 +/-cells, which might be responsible for a predisposition to diabetes in individuals carrying the c.C4661T variant in GLI2.
To further characterize the phenotype and assess a possible dose-dependent effect of the GLI2 P>L variant, we differentiated the homozygous GLI2 +/+ iPSC lines towards pancreatic progenitors and β-like cells (Supplementary Fig. 2c).Mutant and control lines differentiated efficiently into definitive endoderm and pancreatic foregut stages (Supplementary Fig. 2c,d).Quantification by flow cytometry showed that over 90% of GLI2 CTRL and GLI2 +/+ -derived cells were positive for PDX1 (Supplementary Fig. 2c).However, after gut tube stage, the number of GLI2 +/+ cell clusters gradually decreased and differentiation arrested at endocrine progenitor stage, failing to reach β-like cell stage.Differences between GLI2 CTRL and GLI2 +/+ became evident at pancreatic progenitor stage and exacerbated at endocrine progenitor stage, with only approximately 10% of GLI2 +/+ -derived clusters being positive for both PDX1 and NKX6.1 (Fig. S2E).Taking together, the homozygous GLI2 +/+ iPSCs show a more severe phenotype than GLI2 +/-iPSCs, failing to differentiate into β-like cells (Supplementary Fig. 2f-g).These findings suggest a dose-dependent effect of the GLI2 P>L variant on endocrine lineage differentiation.Notably, no patients homozygous for the GLI2 P>L variant were reported so far.
Finally, GLI2 +/+ -derived pancreatic progenitor cells failed to progress along endocrine differentiation also when exposed to a distinct well-established differentiation protocol (Supplementary Fig. 3) 31 .Thus, independently of the differentiation protocols applied, GLI2 +/+ cultures showed decreased expression of endocrine transcription factors and β-cell genes and overall reduction in bonafide NKX6.1/INSULIN-positive β-cells (Fig. 2 and Supplementary Fig. 3a-d).
3][34][35] We hypothesized that aberrant activation of WNT5A signaling may contribute to impaired endocrine cell development in the mutant lines.To test this hypothesis, we either stimulated or inhibited the non-canonical WNT signaling pathway in GLI2 CTRL and GLI2 +/-iPSCs, respectively (Fig. 4a).GLI2 CTRL cells were treated with WNT5A for four days after the induction of PDX1 and acquisition of pancreatic fate, and, subsequently, analyzed by RT-qPCR.After exposure to WNT5A, we observed a decrease in the expression of pancreatic (PDX1, NKX6.1) and endocrine (NGN3, NKX2.2,NEUROD1) gene markers in GLI2 CTRL -derive endocrine progenitors (Fig. 4b).RT-qPCR analysis also showed upregulation of WNT5A itself and its downstream targets, LAMC2 and TEAD4, in stimulated GLI2 CTRL cells (Fig. 4b).GLI2 CTRL cells that were further differentiated to β-like cell stage after exposure to WNT5A showed a decrease in the expression levels of NKX2.2 and NEUROD1, accompanied by a reduction of gene transcripts essential for human β-cell functionality, such as PCSK1, as well as endocrine hormones, including INSULIN, GLUCAGON, SOMATOSTATIN (Fig. 4c).
Exposure to WNT5A also affected the functional maturation of the cells, which showed reduced glucose-stimulated insulin secreting capacity as compared to GLI2 CTRLderived β-like cells, but similar to GLI2 +/-mutant cells (Fig. 4d).Together, these results demonstrate that inappropriate activation of WNT5A signaling impairs differentiation of iPSCs into β-like cells and recapitulates the phenotype of GLI2 +/-mutant cells (Supplementary Fig. 4b-d).
We next investigated if inhibition of WNT5A could instead rescue β-cell development in GLI2 +/-mutant iPSCs.Cells were treated with BOX5, a WNT5A antagonist, which was previously shown to attenuate WNT5A-mediated Ca 2+ and protein kinase C signaling 36 (Fig. 4a).GLI2 +/-clusters treated with BOX5 exhibited a significant increase in the expression of PDX1, NKX6.1 and other endocrine markers (Fig. 4b).Moreover, the expression of WNT5A and its targets was reduced after addition of BOX5 (Fig. 4b), confirming the activity of the compound.Later at day 21, BOX5-treated GLI2 +/-derived β-like cells restored the expression of β-cell genes (NKX6.1,NKX2.2,NEUROD1, PCSK1, INSULIN) to levels comparable with control cells.Consistently, we found that exposure to BOX5 ameliorates the functional defects of GLI2 +/--derived β-like cells, with the treated cells showing an increase of insulin release in response to glucose stimulation (Fig. 4d).Overall, these findings indicate that WNT5A signaling regulation is critical for endocrine development and its inhibition partially rescues the GLI2 +/-defects that may ultimately contribute to diabetes onset.

Discussion
We report a previously uncharacterized heterozygous missense variant p.P1554L in the GLI2 gene in patients with early-onset diabetes.Isogenic iPSC lines with either a heterozygous or homozygous p.P1554L mutation in the GLI2 gene showed impaired endocrine progenitor development in a dose-dependent manner.The iPSC models have allowed us to move beyond the discovery of the mutant variant to an understanding of the causative variant and the molecular mechanisms of the disease.Indeed, the diminished expression of NGN3 observed in patient-like iPSCs leads to endocrine progenitor pool depletion, which might ultimately result in reduced b-cell mass at birth and explain the onset of the diabetes in young age.Hence, these results support the likely pathogenicity of the p.P1554L variant, possibly through impaired GLI2 transcriptional activity.
18]37 Here, we uncovered a role for GLI2 in human β-cell development, expanding our current knowledge of human pancreatic development.
We found that GLI2 is expressed in a sub-set of pancreatic progenitors, before endocrine specification, and its expression persists at later stages in human islet cells.
Early GLI2 transcriptional activity regulates a set of genes, including NGN3, which are essential for the activation of the endocrine program.RNASeq and network analyses also revealed a crosstalk between HH/GLI2 and WNT pathways in human pancreatic progenitors, which results into dysregulation of non-canonical WNT signaling in GLI2 +/- mutant cells.Notably, rescue experiments (by blocking WNT5A) as well as mimicking experiments (by adding WNT5A) showed that GLI2 functions in endocrine progenitors through mechanisms that regulate Wnt signaling.A crosstalk between HH/GLI and WNT pathways has been reported in other contexts, such as cancer, 38 neurogenesis 39 and differentiation. 40However, the precise mechanisms that regulate HH and WNT activity seem to be highly context-specific and the events by which changes in HH/GLI2 signaling in pancreatic cells determines WNT regulation remain to be fully elucidated.2][43] Recent studies reported a non-cell autonomous WNT5Amediated role for b-cell differentiation from endocrine progenitors in mouse 44 and human stem cell models. 33By contrast, WNT5A/PCP role in beta-cell maturation is still not fully understood and might be different between mouse and human. 32,45reover, WNT5a is expressed in the pancreatic epithelial component, beside the mesenchyme, and it is unclear how the expression of this ligand is coordinated in the epithelial/mesenchyme compartment to regulate endocrine cell formation.Here, we identified an additional stage-specific response to cell autonomous WNT that corresponds to the time when human pancreatic progenitors acquire endocrine identity.At this specific stage, it is likely that the crosstalk between the two pathways confers temporal control to WNT, wherein GLI2 activation might sustain canonical WNT and prevent aberrant non-canonical WNT signaling during the acquisition of human endocrine progenitor fate.The diverse roles and mechanisms regulating HH and WNT crosstalk during pancreatic lineage differentiation remain to be fully elucidated.Also, while we have presented evidence for HH regulation of noncanonical WNT, the mechanism by which GLI2 affects WNT responsiveness is not known in pancreatic progenitors and is a subject for future research.The other family members were tested by Sanger sequencing.Mutations that are pathogenic for early onset diabetes are expected to be very rare in the population.
Accordingly, AF of p.P1554L was 0.000044 in gnomAD and MAF <0.01. 25 The missense variant is annotated as of uncertain significance and there is no functional    Technologies).The medium was changed daily, and cells were passaged every ∼3 days as cell clumps or single cells using 0.5 mM EDTA (Invitrogen) or Accutase (Invitrogen), respectively.Medium was supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Sigma) when iPSCs were thawed or passaged as single cells.
All cell lines tested negative for mycoplasma contamination which was carried out routinely.

Site-directed mutagenesis
Site-directed mutagenesis was used to introduce the patient variant GLI2 c.C4661T into the pCS2-MT GLI2 FL plasmid.Mutagenesis was performed using the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent) following manufacturer's guidelines.Mutation was generated using the following specific primer pair: GLI2 c.C4661T-F: GCGGGGATGGAGAGCAGGGTCAAGG GLI2 c.C4661T-R: CCTTGACCCTGCTCTCCATCCCCGC

GLI-responsive Luciferase assay
For luciferase assays, the dual luciferase reporter assay kit (Promega) was used.
HEK293T cells were seeded at 1.3 x 10 6 cells in 12-well plates and transiently cotransfected in duplicates with 0.5 μg of wild-type or mutant GLI2 pCS2-MT plasmids together with 0.5 μg of GLI-responsive Firefly luciferase reporter construct (8x3'Gli-BSδ51LucII) and 0.05 μg of a constitutive Renilla luciferase reporter (pRL-SV40) construct.Protein lysates were prepared 48 hrs after transfection.Firefly and Renilla Luciferase activities were quantified using the dual reporter assay kit (Promega) according to the manufacturer's instructions on an Infinite 200 Pro-luminometer (TECAN).Luciferase assay experiments were repeated three times on independent samples.As negative controls, the mutated version of the GLI-luciferase reporter construct (8xm3'Gli-BSδ51LucII) was used, as well as a GFP-expressing construct.
Firefly/Renilla activity ratio was then calculated for each sample.

Differentiation of pluripotent iPSCs into pancreatic β-like cells
Differentiation was carried out following a 21-day protocol previously described by Russ et al. 29 Briefly, iPSCs were dissociated using Accutase and seeded at a density of 5.5 x10 6 cells per well in ultra-low attachment 6-well plates (Thermo Fisher Scientific) in E8 medium supplemented with 10 μM ROCK inhibitor, 10 ng/ml Activin A (R&D Systems) and 10 ng/ml Heregulin (Peprotech).Plates were placed on an orbital shaker at 100 rpm to induce sphere formation at 37°C in a humidified atmosphere containing 5% CO2.To induce definitive endoderm differentiation, cell clusters were incubated in Day (D)1 medium [RPMI (Invitrogen) containing 0.2% FBS, 1:5000 ITS (Invitrogen), 100 ng/ml Activin A, 50 ng/ml WNT3a (R&D Systems)] into low attachment plates.Subsequently cell clusters were differentiated into β-like cells by exposure to the appropriate media as previously published. 29All recombinant proteins were purchased from R&D System unless otherwise stated (see Supplementary Table 3).

Generation of clonal HMGUi001-A2 patient-like mutant lines
Patient-like isogenic iPSC lines were generated using the DOX inducible iCRISPR system previously established by Yumlu et al. 27 Briefly, sgRNAs targeting the site of mutation were designed using the CRISPOR website (http://crispor.tefor.net/)and normalization to reference genes.RT-qPCR experiments were repeated at least three times with independent biological samples; technical triplicates were run for all samples; minus RT and no template controls were included in all experiments.
For RNA sequencing (RNA-seq), total RNA from GLI2 CTRL and GLI2 +/-cells at iPSC, gut tube and endocrine progenitor stage were extracted from two independent biological replicates.Total RNA concentration and RNA integrity of each sample were determined with NanoDrop and Qubit 4 Fluorometer (ThermoFisher Scientific).
RNASeq library preparations and sequencing reactions were conducted by GENEWIZ.

Bioinformatics
Initial bioinformatics analysis of the RNASeq was conducted by GENEWIZ.Briefly, data was generated with an Illumina HiSeq 2x150 PE HO configuration.Sequence reads were trimmed to remove adapter sequences and nucleotides with poor quality (Trimmomatic v.0.36).Using the STAR aligner v.2.5.2b the trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL.
Gene expression between distinct groups were compared using DESeq2.Gene Ontology analysis was performed using the publicly available David website (https://david.ncifcrf.gov.home.jsp).
Gene Network Analysis was based on DEGs (log2foldchange > 1) obtained when comparing gene expression at each of days 0, 5 and 14 during pancreatic differentiation between GLI2 CTRL and GLI2 +/-cells.Enrichment of Kyoto Encyclopaedia of Genes and Genomes (KEGG) ontology terms and pathways and gene network construction was conducted in Cytoscape 49 (v3.8.0) using the ClueGO (v2.3.6) and Cluepedia (v1.3.6)plugins.KEGG enrichment of proteins in the following section was carried out with the R package clusterProfiler that enables enrichment comparisons across multiple clusters of genes/proteins.All programming code was generated with the aid of package vignettes made accessible via The Comprehensive R Archive Network (CRAN).

Fig. 1 :
Fig. 1: Identification of heterozygous GLI2 mutation in a family with diabetes of unknown aetiology.a Schematic representation of human GLI2 protein and the position of the single amino acid change p.P1554L in the Transcription Activation Domain (TAD).b Family tree of patients with puberty-onset diabetes.The heterozygous GLI2 p.P1554L (c.C4661T) variant was found in four individuals (black symbols) of a consanguineous family with incomplete penetrance.The missense evidence for this variation in ClinVar [VCV001506585.4].c The amino acid residue P1554 is highly conserved among species according to the University of California Santa Cruz (UCSC) Genome Browser.d Representative immunofluorescent (IF) images for GLI2 and PDX1 on human pancreas at Carnegie Stage (CS) 20 [8 weeks post conception (wpc)].Arrows indicate GLI2/PDX1-positive cells; arrowheads indicate GLI2-positive cell cluster next to PDX1-positive epithelium.Nuclei were labeled with Hoechst.Scale bar, 20 μm. e Luciferase-based reporter assay with GLIresponsive construct in HEK 293T cells.Wild-type (8x3'Gli-BSδ51LucII) or mutated (8xm3'Gli-BSδ51LucII) luciferase reporter plasmid was co-transfected with a Renilla luciferase control plasmid and indicated DNA expression vectors (GLI2 CTRL or GLI2 P>L ).Results were normalized for transfection efficiency using Renilla luciferase and are represented as Firefly/Renilla activity ratio.The average (mean ± SD) relative light units (RLU) of one representative reaction is plotted.*p<0.05,Student's t test.n=3.ND: not determined.f RT-qPCR of β-cell markers in Min6 cells transfected with GLI2 CTRL or GLI2 P>L .Values are normalized to 36B4 and relative to cells transfected with control GFP vector.n = 3. *p<0.05,Student's t test.

Fig. 2 :
Fig. 2: Characterization of GLI2 +/--derived iPSCs undergoing pancreatic endocrine differentiation.a Schematic representation of the differentiation protocol of iPSCs into pancreatic β-like cells. 29Cells were differentiated in suspension as 3D clusters.b Representative flow cytometry plots of PDX1 + cells (shown as %) in WT GLI2 CTRL and GLI2 +/--derived cells at day (D) 5 of differentiation.c RT-qPCR analysis of selected gene transcripts in GLI2 CTRL and GLI2 +/-differentiated cells at D5 and D9.

Fig. 3 :
Fig. 3: Whole-transcriptome analysis of GLI2 +/-iPSCs undergoing differentiation into endocrine progenitor cells.a Schematic of the experimental design.RNA-seq data were obtained during differentiation of GLI2 CTRL and GLI2 +/-iPSCs at D0, D5 and endocrine progenitor (EP) D14 stages.b Volcano plots visualizing the global transcriptional change across the groups compared (D5 GLI2 CTRL vs GLI2 +/-).Each data point in the plot represents a gene.Genes with an adjusted p-value less than

Fig. 4 :
Fig. 4: Modulation of WNT5A signaling rescues b-cell differentiation in GLI2 +/- iPSCs.a Schematic representation of the differentiation protocol of iPSCs into b-like cells.Cell clusters were treated in suspension with WNT5A recombinant protein or BOX5 for 4 days at PP stage of differentiation.b RT-qPCR analysis of selected gene transcripts in GLI2 CTRL -and GLI2 +/--derived EP cells at D14 after the indicated treatments.Data are represented as fold change relative to undifferentiated cells (d0).Values shown are mean ± SEM. n=3.*p <0.05, **p < 0.01, ***p < 0.001, Student's t