Generation of human stem cell model of SETBP1-deficiency

A hESC model of SETBP1 deletion was generated by CRISPR/Cas9 assisted gene targeting in the H7 hESC line. Three gRNAs that recognize non-overlapping sequences in exon 4 were co-transfected with the targeting vector. Independent hESC clones were screened for homologous recombination firstly by PCR using primer pairs immediately outside the 5′ and 3′ homology arms, respectively (Fig. 1A). Correct homologous recombination was verified in one allele of three independent clones by PCR followed by Sanger sequencing (Fig. 1B and Additional file 2: Fig. S1A). This gene targeting introduced an early stop codon in the mutant allele and is predicted to produce a truncated protein of 475 amino acid (aa) of the full 1596aa protein sequence (www.expasy.org. Figure 1C). One of the heterozygous lines (HET1) was subjected to a second round of editing using the same gRNAs without the donor plasmid, yielding several independent clones containing a 5 bp deletion in the other allele (Additional file 2: Fig. S1B). This 5 bp deletion introduced an early stop codon in the second allele and is predicted to produce a truncated protein of 1220 aa (Additional file 2: Fig. S1C). qPCR analysis of SETBP1 mRNA levels with primers binding downstream of the region targeted by the gRNAs (Forward in exon 4–5 junction and reverse in exon 5–6 junction) shows a reduction of at least 50% in the homozygous SETBP1 mutant clones (Additional file 2: Fig. S1D).

The isogenic wild-type (WT) parental line, two homozygous SETBP1 mutant hESC lines (Homo 1 and Homo 2, referred to together as SETBP1-/-), along with two heterozygous SETBP1 mutant hESC lines (Het 1 and Het 2, referred to together as SETBP1+/-), were chosen for subsequent studies. The SETBP1 edited lines exhibited characteristic pluripotent stem cell (PSC) morphology, expressed pluripotency markers OCT4 and SOX2, and grew at a similar rate to that of H7 (Fig. 1D). Moreover, they have normal karyotype (46, XX) in 73–82% of total metaphases analysed.

Cortical neuronal differentiation is altered in SETBP1-deficient NPCs

The SETBP1 deficient and isogenic parental control hESCs were induced to differentiate toward cortical fate using a modified dual SMAD inhibition protocol as described previously (Fig. 2A) [24, 35]. Neural induction occurred efficiently in all three genotypes as indicated by the uniform transition into neural rosettes marked by N-cadherin (N-CAD) at their apical surface (Fig. 2B). To quantify the efficacy of neural induction and cortical fate commitment we performed antibody staining for markers of pan neural stem cell (SOX2, NESTIN), progenitors of telencephalon (FOXG1, OTX2) and dorsal telencephalon (PAX6) at day 18 (Fig. 2B–C). Indeed the vast majority of cells stained positive for SOX2, NESTIN, and OTX2 with comparable numbers of positive cells across the three genotypes (Fig. 2B–C), confirming normal neural induction of SETBP1-deficient hESCs. However, in the SETBP1-/- cultures we observed a ~ 70% reduction of FOXG1+ cells (P = 2.159E−06) and a moderate (~ 15%) increase in PAX6+ cells (P = 0.018) in comparison to the isogenic WT controls. No changes were found in the SETBP1+/- cultures. Consistent with immunostaining, RT-PCR analysis revealed a rapid induction of transcripts of a panel of pan-neural and forebrain-specific transcription factors and a decrease of FOXG1 in the SETBP1-/- cultures compared to the WT (Additional file 3: Fig. S2A) although the level of PAX6 appear similar until day 30 when an upregulation was observed in the SETBP1-/- cultures (Additional file 3: Fig. S2A, B).

Fig. 2

Neuronal differentiation is affected by loss of SETBP1 A Schematic representation of hESC cortical differentiation protocol. B Expression of NPC markers at day 18. Cultures were immunostained for N-cadherin (red) and DAPI (blue) showing the organization and size of the neural rosettes, PAX6 (green), OTX2 (red), NESTIN (NES, green), SOX2 (red) and FOXG1 (red). Dapi was used to label all nuclei. Scale bar: 100uM. C Quantitative data of marker expression presented as mean ± s.e.m for each genotype with a minimum of two independent experiments carried out per line and minimum of four per genotype (WT = 7, HET1 = 2, HET2 = 2, Homo1 = 6 and Homo 2 = 2). One-way ANOVA test with Bonferroni Post Hoc or Kruskal–Wallis non-parametric test, SOX2 P = 0.890, NES P = 0.499, OTX2 P = 0.606, FOXG1 P = 2.159E−06 (Post Hoc WT vs. SETBP1-/-, p = 0.000032; WT vs. SETBP1+/- , p = 0.331; SETBP1+/-  vs. SETBP1-/-, p = 3.77E−06), PAX6 P = 0.018 (Post Hoc WT vs. SETBP1-/-, p = 0.035; WT vs. SETBP1+/- , p = 1; SETBP1+/- vs. -/-, p = 0.052). (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). D Immunostaining of cortical layer markers TBR1 (layer VI) and CTIP2 (layers V–VI) at days 30, 40 and 50, respectively, and SATB2 (layers II–III) at day 50. Images representative of several independent experiments for each genotype E Quantitative analysis of the above. Data presented as mean ± s.e.m for each genotype with a minimum of two independent experiments carried out per line (WT = 5, Het1 = 2, 2 = 2, Homo1 = 3, and Homo2 = 2). One-way ANOVA test, Bonferroni Post Hoc; TBR1 p = 0.615 for day 30, 0.863 for day 40, and 0.585 for day 50; CTIP2 P = 0.004 for day 30 (Post Hoc WT vs. SETBP1-/-, p = 0.006; WT vs. SETBP1+/-, p = 1; SETBP1+/- vs. -/-, p = 0.007), 0.022 for day 40 (Post Hoc WT vs. SETBP1-/-, p = 0.05; WT vs. SETBP1+/-, p = 1; SETBP1+/- vs. -/-, p = 0.05), and 0.042 for day 50 (Post Hoc WT vs. SETBP1-/-, p = 0.0071; WT vs. SETBP1+/-, p = 1; SETBP1+/- vs. -/-, p = 0.182); SATB2 P = 0.491 for day 40, and 0.348 for day 50 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Scale bar: 100uM

We next examined the effect of SETBP1-deficiency on cortical neuronal production by immunostaining for a panel of commonly used cortical layer-specific markers (TBR1/layer VI, CTIP2/layers V–VI, SATB2/layers II–III) and a general neuronal marker (NeuN) at days 30, 40 and 50. We observed no significant change in TBR1+ neurons between the genotypes although the SETBP1-/- cultures exhibited a tendency of increase (Fig. 2D–E), which was observed at transcriptional level too (Additional file 3: Fig. S2B). The late born SATB2+ neurons were not detected at day 30 and were very low in number even at day 40 and 50 (~ 2%) in all cultures (Fig. 2D–E). However, evident decrease of CTIP2+ neurons was observed in SETBP1-/- cultures compared to the WT controls at all timepoints (ANOVA day 30 P = 0.004, day 40 P = 0.022, day 50 P = 0.042) (Fig. 2D–E). A decrease of CTIP2 transcripts was also observed (Additional file 3: Fig. S2B).

Reduced numbers of CTIP2+ cells were also observed in SETBP1+/- cultures from day 40, although statistical significance was only reached at day 40. Reduced numbers of NeuN+ cells were also detected in the SETBP1-/- at all three time points analysed, although statistical significance was only reached in the SETBP1+/- cultures for day 50 (ANOVA day 30 P = 0.012, day 40 P = 0.049, day 50 P = 0.009) (Additional file 4: Fig. S3A, B). Similarly, fewer MAP2+ cells can be observed in SETBP1-/- day 50 cultures while the proportion of NES+ cells was higher than the controls (Additional file 4: Fig. S3A). Together, these findings demonstrate a distorted neuronal production in SETBP1-/- cultures, while heterozygous deletion of SETBP1 had a milder effect.

Loss of SETBP1 led to a bias toward neural progenitor proliferation

The reduced neuron production in the SETBP1-/- cultures could be due to an imbalance between progenitor proliferation versus terminal differentiation. We therefore determined PAX6+ cortical progenitor numbers during neurogenesis phase between days 30–50 by antibody staining. In the WT cultures, PAX6+ cell numbers drop from ~ 75% at day 18 to 30% at day 30 and 17% by day 40 (Fig. 2C, Fig. 3A, B). However, the rate of reduction was slower in the SETBP1-/- cultures where approximately 45% cells remained PAX6+ at day 40 (Fig. 3A, B), leading to a significant increase of PAX6+ NPCs in the SETBP1-deficient cultures. This finding is mirrored by sustained higher levels of HES1 transcript in these cultures (Additional file 3: Fig. S2B).

Fig. 3

SETBP1 deficiency enhances cortical progenitor proliferation. A Cultures were immunostained for dorsal forebrain marker PAX6 (green) at days 30, 40 and 50. Dapi was used to label all nuclei. Scale bar: 100uM. B Quantitative data for PAX6 positive cells presented as mean ± s.e.m for each genotype with a minimum of two independent experiments carried out per line (WT = 5, HET2 = 2, KO1 = 3, and KO2 = 2). One-way ANOVA test with Bonferroni Post Hoc was used to compare the expression between the lines day 30 P = 0.181, day 40 P = 0.032 (Post Hoc WT vs. SETBP1-/-, p = 0.039; WT vs. SETBP1+/-, p = 0.289; SETBP1+/- vs. -/-, p = 0.385), day 50 P = 0.041 (Post Hoc WT vs. SETBP1-/-, p = 0.182; WT vs. SETBP1+/-, p = 0.050; SETBP1+/- vs. -/-, p = 0.473). C WT, SETBP1+/- (HET1) and SETBP1-/- (Homo1) day 35 cultures were immunostained for EdU (green), Ki67 (red), PH3 (red) and counterstained with DAPI (blue). Scale bar: 100uM. D Percentage of cells positive for EdU P = 4.87E−06 (Post Hoc WT vs. SETBP1-/-, p = 1.25E−05; WT vs. SETBP1+/- , p = 0.240; SETBP1+/- vs. -/-, p = 9.01E−06), Ki67 P = 0.016 (Post Hoc WT vs. SETBP1-/-, p = 0.038 WT vs. SETBP1+/- , p = 1; SETBP1+/- vs. -/-, p = 0.025), and EdU and Ki67 (cell cycle re-entry) P = 7.07E−06 (Post Hoc WT vs. SETBP1-/-, p = 1.68E−05 WT vs. SETBP1+/- , p = 0.354; SETBP1+/- vs. -/-, p = 1.38E−05). E Ratios of cell cycle exit and cell cycle length. Data presented as mean ± s.e.m from 3 independent wells with 6 random fields each. One-way ANOVA test (P = 0.055, P = 0.438). F Analysis of PAX6, TBR2 and FAM107A cycling progenitors in Ki67+ population. One-way ANOVA test with Bonferroni Post Hoc, PAX6+P = 0.00097 (Post Hoc WT vs. SETBP1-/-, p = 0.001; WT vs. SETBP1+/- , p = 0.002; SETBP1 +/- vs. SETBP1-/-, p = 1), FAM107A+P = 0.141, TBR2.+P = 0.007 (Post Hoc WT vs. SETBP1-/-, p = 0.036; WT vs. SETBP1+/- , p = 0.996; SETBP1+/- vs. -/-, p = 0.01). G Cell cycle analysis by DNA content using Flow cytometry, % of cells in each of the cell cycle phases. Data presented as mean ± s.e.m of 2 independent experiments in triplicates. Student’s T test, one tail (G1-G0 P = 0.118, S P = 0.255, G2-M P = 0.029). H Growth curve analysis from day 19 to day 45 showing the increased population growth of the SETBP1-/- (Homo1) NPCs compared to the isogenic controls. Statistical significant differences were found from day 30 onwards (Student’s T test, P = 6.71E−05 for day 30, 0.015 for day35, 0.033 for day 40, and 0.050 for day 45). Data presented as mean ± s.e.m from 3 independent wells with two technical measurements. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

The above observation suggests an increase in neural progenitor maintenance in the SETBP1-/- cultures. To investigate whether this is due to alterations in cell cycle profile upon loss of SETBP1, we performed EdU and Ki67 double labelling at day 34 (Fig. 3C–E). EdU is a thymidine analogue hence its incorporation marks cells in the S phase, while Ki67 is a protein present during all active phases of the cell cycle (G1, S, G2 and mitosis). The SETBP1+/- and WT cultures contained a similar number of EdU+ and Ki67+ cells (14.24 ± 1.10% vs. 11.42 ± 0.11% and 19.94 ± 1.21% vs. 18.52 ± 0.93%, respectively). However, significantly more EdU+ (28.27 ± 0.22%), Ki67+ (25.45 ± 1.14%) and EdU+Ki67+ cells (SETBP1-/-, 18.28 ± 0.56% vs. WT, 12.3 ± 1.7%) were detected in the SETBP1-/- cultures (ANOVA P = 4.88E−06, P = 0.0168, P = 7.08E−06) (Fig. 3C, D). The fraction of EdU+Ki67− cells within the EdU+ population is often used as an index for cell cycle exit [36], we found that the ratio of EdU+Ki67−/EdU+ is lower in SETBP1-/- cultures than the SETBP1+/- and WT controls with a borderline p value (SETBP1-/-, 0.083 ± 0.013%, SETBP1+/- , 0.054 ± 0.036% WT, 0.133 ± 0.014%; ANOVA P = 0.055) (Fig. 3E), suggesting that SETBP1-/- NPCs were slow in exiting the cell cycle compared to their isogenic counterparts. The ratio of EdU+Ki67+/Ki67+ is inversely related to the length of cell cycle [37]. Consistent with an increase in proliferation, this ratio was slightly higher in SETBP1-/- cultures than the controls, indicating the former have shorter cell cycle (SETBP1-/-, 0.663 ± 0.043%, SETBP1+/- , 0.588 ± 0.021% WT, 0.603 ± 0.034%; ANOVA P = 0.438) (Fig. 3E). To further delineate the progenitor population targeted by SETBP1, we performed Ki67 co-staining with PAX6 (a marker for ventricular and outer radial glia), FAM107A (outer radial glia) and TBR2/EOMES (intermediate progenitor) (Fig. 3F). Within the cycling (Ki67+) progenitors, we found an enrichment of PAX6+ cells in the SETBP1-deficient lines. Moreover, there were more cycling TBR2+ intermediate progenitors in the SETBP1-/- cultures than the WT controls, providing another support on the compromised neurogenic state of the SETBP1-/- cultures.

To gain further insight into changes in cell cycle profile, we performed a flow cytometry-based cell cycle analysis (Fig. 3G). This assay identifies cells in three major phases of the cell cycle (G0/1, S and G2/M) based on their DNA content. Since the cellular defects observed were largely limited to the SETBP1-/- cultures, we focused on this genotype in the subsequent studies. Compared to the WT control, the SETBP1-/- cultures contained a higher percentage of NES + cells in S (9.24 ± 0.69% vs. 7.92 ± 1.52%) and G2/M phases (19.81 ± 0.18% vs. 18.07 ± 0.37%, P = 0.029) and fewer cells in G0/G1 (68.01 ± 0.31% vs. 70.24 ± 1.29%), although the number of cells in mitosis were similar as revealed by antibody staining for phosphorylated histone H3 (PH3) (Fig. 3C, Additional file 5: Fig. S4).

To investigate how altered cell cycle impact on the growth rate over time, we compared population growth of SETBP1-/- and WT cultures between day 19 and day 45. Consistent with EdU incorporation and cell cycle analysis, more cells were found in the SETBP1-/- cultures than the WT from day 30 onwards (P ≤ 0.05, Fig. 3H). Together, these findings demonstrate that SETBP1 deficiency leads to enhanced NPC division by regulating cell cycle.

Loss of SETBP1 compromises ventral forebrain fate induction

Despite its pan-forebrain expression, loss of Foxg1 in mice preferentially impairs lateral and medial ganglionic eminence (LGE and MGE) formation [38] while overexpression of FOXG1 in human iPSCs leads to overproduction of MGE-derived neurons [39]. The dramatic reduction of FOXG1 expression in SETBP1 mutant cells prompted the examination of LGE and MGE progenitor content in the cortical cultures by antibody staining for GSH2 and NKX2.1 at day 24. In cortically differentiated WT control cultures ~ 5% cells stained positive to NKX2.1, a transcription factor with restricted expression in the MGE (Fig. 4A, B). The SETBP1-/- cultures contained even fewer NKX2.1+ cells (P = 0.003), implicating a decrease in MGE like progenitors. In the developing human brain, PAX6 expression extends beyond the cortex into the LGE [40]. To provide further insight into the increase of PAX6+ cells in the SETBP1-/- cultures, we performed double staining of PAX6 with GSH2, a transcription factor with restricted expression in the LGE and MGE (Fig. 4A, B). We detected around 20% of GSH2+ cells in the WT control cultures, nearly all of which co-expressed PAX6. However, only 2.5% GSH2+ and 2.3% GSH2+PAX6+ cells were found in the SETBP1-/- cultures (ANOVA P = 0.006, Kruskal–Wallis Test P = 0.032) (Fig. 4A, B), demonstrating a reduced capacity of the SETBP1-/- progenitors to adopt LGE fate. This finding also suggests that the observed increase of PAX6 + cells in the SETBP1-/- cultures are most likely of cortical identity.

Fig. 4

SETBP1 deficiency impairs acquisition of ventral forebrain identity. A Expression of dorsal forebrain marker PAX6 (green), dorso-ventral boundary forebrain marker GHS2 (red) and ventral marker NKX2.1 (red) in day 24 cortical NPCs. Dapi was used to label all nuclei. Scale bar: 100uM. B Quantitative data presented as mean ± s.e.m for each genotype with two biological replicas per line and genotype. One-way ANOVA or Kruskal–Wallis test: PAX6 P = 0.021 (Post Hoc WT vs. SETBP1-/-, p = 0.047 WT vs. SETBP1+/- , p = 0.912; SETBP1+/- vs. SETBP1-/-, p = 0.040), NKX2.1 P = 0.003 (Post Hoc WT vs. SETBP1-/-, p = 0.221 WT vs. SETBP1+/- , p = 0.138; SETBP1+/- vs. SETBP1-/-, p = 0.003), GSH2 P = 0.006 (Post Hoc WT vs. SETBP1-/-, p = 0.011 WT vs. SETBP1+/- , p = 0.803; SETBP1+/- vs. SETBP1-/-, p = 0.022), PAX6+GSH2+/PAX6+P = 0.032 (Post Hoc WT vs. SETBP1-/-, p = 0.022 WT vs. SETBP1 +/- , p = 0.567; SETBP1+/- vs. SETBP1-/-, p = 0.036) (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). C Expression of pan-neural forebrain marker FOXG1 (red), neural radial glia marker NES (green), dorsal forebrain marker PAX6 (red), and ventral markers NKX2.1 (green) in ventrally derived (MGE) NPCs at day 20. Dapi was used to label all nuclei. Scale bar: 100uM. D Quantitative data presented as mean ± s.e.m for each genotype with three biological replicas per line and genotype. One-way ANOVA test: NKX2.1 P = 0.038 (Post Hoc WT vs. SETBP1-/-, p = 0.042 WT vs. SETBP1+/- , p = 0.098; SETBP1+/- vs. SETBP1, p = 1), FOXG1 P = 4.77E−06 (Post Hoc WT vs. SETBP1-/-, p = 3.86E−06 WT vs. SETBP1+/- , p = 4.8E−05; SETBP1+/- vs. SETBP1-/-, p = 0.103) (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). E Schematic representation of hESC ventral forebrain (MGE) differentiation protocol to generate ventral NPC population

Since the cortical protocol yielded limited number of ventral cell types, we also differentiated the WT and SETBP1-deficient hESCs using a MGE induction protocol (Fig. 4E). A reduction of NKX2.1+ cells was detected in both the SETBP1-/- and SETBP1+/- cultures compared to WT controls (ANOVA P = 0.038, Fig. 4C, D). We didn’t detect a change in the number of EdU+ and Ki67+ cells in the SETBP1 mutant cultures (Additional file 6: Fig. S5). However, a marked reduction of FOXG1+ cells was also observed in MGE differentiated SETBP1-/- cultures (P = 4.77E−06, Fig. 4C, D). Interestingly, unlike in the cortical cultures where no significant reduction of FOXG1+ were observed, a reduction was also detected in the SETBP1+/- cultures. These observations provide further support that SETBP1-deficiency compromise ventral forebrain fate induction.

Genome-wide transcriptome analysis identified Wnt/β-catenin signaling as a target of SETBP1 function

To gain further insight into the molecular mechanisms underlying prolonged proliferation window of SETBP1-deficient NPCs, we performed a genome-wide transcriptome analysis of neural cells derived from the SETBP1-/- and isogenic WT control lines by RNAseq. To cover all stages of cellular abnormality, samples were collected from day 15 and day 21 (early and peak neural progenitor stage, respectively) and day 34, when abnormal NPC division and neurogenesis was becoming evident. Principle Component Analysis (PCA) showed that 100% of the variance is attributed to SETBP1 genotypes, while the biological replicates within SETBP1-/- or the control samples exhibit 0% variance statistically (Fig. 5A). Comparison of our hESCs-derived cortical gene expression to the BrainSpan Atlas of the Developing Human Brain (http://www.brainspan.org/), found the highest degree of correlation being at days 21 and 34 (Fig. 5B).

Fig. 5

Genome-wide transcriptome profiling revealed SETBP1 regulation of Wnt signaling. A Principle component analysis (PCA) of the samples. B Heatmap depicting correlation with Allen Brain atlas at early, mid and late gestational trimesters and prenatal trimester. C Heatmap depicting 17,654 differentially expressed transcripts at day 34 (padj ≤ 0.1). D Example of neurogenic and proliferation related genes differentially expressed at day 34. E Example of neuronal marker genes downregulated at day 34, and upregulation of neuronal marker NES. F, G Differentially expressed genes associated with canonical non-canonical Wnt pathway at day 34. H Representative images of Western blot analysis for Wnt signaling proteins for WT and SETBP1-/- (Homo1). I Relative protein level of B-catenin, B-catenin p-S552 and p-S675, and LRP6 co-receptor and p-LRP6 at day 21, 30 and 40 to WT basal levels. Data from 3 independent differentiations analysed in duplicates or triplicates. Student’s T test was used to compare the expression between the two lines. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). All tested proteins were normalized to GAPDH

At a significant level of adjusted P ≤ 0.1, we identified 6060, 9997 and 17,654 differentially expressed transcripts at the three analysed time points, respectively (see example of day 34 in Fig. 5C). Consistent with the observed bias in NPC proliferation in the SETBP1-/- cultures, we observed an up-regulation of a number of neural proliferation marker genes such as FGF8, DISC1, HES1, CDC20B and CCND1 at day 34 (Fig. 5D). This is complemented by a down-regulation of genes involved in neurogenesis such as DLL1, ASCL1, NEUROD1, NEUROG2 and DCX (Fig. 5D). Moreover, at the same timepoint, basal progenitor (TBR2), pan neuronal (TUBB3, MAP2) and cortical layers specific marker genes (TBR1, CTIP2/BCL11B, CUX1/2, SATB2, RELN) were found down-regulated in the SETBP1-deficient cultures (Fig. 5E). In contrast, NES was twofold higher in the SETBP1-/- samples than the controls (Fig. 5E). These findings support the observed NPC SETBP1-deficient phenotype and demonstrate a further role for SETBP1 in cortical NPC proliferation and neurogenesis.

Using DAVID 6.8 gene functional classification tool [33] on the top 1000 differentially expressed protein coding genes, we identified that the top enriched gene ontology (GO) terms concerned mainly biological processes such as regulation of transcription, cell adhesion and extracellular matrix organization. KEGG (Kyoto Encyclopedia of Genes and Genomes, https://www.genome.jp/kegg) pathway analysis revealed Wnt, hippo, PI3K-Akt and ECM-receptor interaction signaling amongst the top enriched pathways up-regulated in SETBP1-/- cultures (Additional file 7: Fig. S6). All these pathways are highly relevant to the regulation of NPCs proliferation and neurogenesis [41,42,43,44,45].

Wnt signaling is known to play an important role in cortical development. Altered Wnt pathway was identified at all three differentiation stages, with the biggest changes observed at day 21 (FC 2.73, Padj = 0.0029) and day 34 (FC 2.53, Padj = 0.0014) (Additional file 7: Fig. S6B, C). We therefore examined further the gene set for transcripts involved in Wnt signaling (hsa04310) at day 34 (Fig. 5F, G and Additional file 8: Fig. S7A–C). Strikingly, the majority of the Wnt ligands, both canonical and non-canonical, were highly up-regulated in SETBP1-deficient cells, with fold change varying from 2.5 to 73 (Fig. 5F, G and Additional file 8: Fig. S7A, C). Also up-regulated were the canonical Wnt/β-catenin signaling responsive genes C-MYC (4.8×), CYCLIND1 (CCND1, 2x) and AXIN2 (4.7×) (Fig. 5F and Additional file 8: Fig. S7A and C). In contrast, genes involved in β-catenin degradation complex (GSK3 β, CSNK, AXIN1/2, APC) were mostly down-regulated (Additional file 6: Fig. S5A). We next used MAGMA (a tool for gene and gene-set analysis) to determine the association between risk variants identified from previously published ASD, and Intelligence GWAS, and enriched gene-sets identified from our RNAseq experiments. We chose to analyze these traits as they have clinical manifestations overlapping with SETBP1-HD. Amongst the analyzed gene-sets, “positive regulation of Wnt signaling” showed a nominal enrichment for genes associated with ASD in our day 34 dataset indicating a potential link between our in vitro model and the autistic traits observed in some of the SETBP1-HD patients (Additional file 9: Fig. S8A, B).

To ascertain that Wnt/β‐catenin signaling is indeed elevated in SETBP1-deficient NPCs at the protein level, we determined the level of β-catenin and Wnt co-receptor LRP6 in day 21, day 30 and day 40 neural cultures by Western blot (Fig. 5H). Activation of the canonical Wnt signaling results in N-terminal phosphorylation of β-catenin by GSK3β, leading to degradation of β-catenin [46, 47]. We found that the level of total β-catenin was significantly higher in SETBP1-/- cultures than the controls at day 30 (P = 0.031), although no differences were found at day 21 and 40 (Fig. 5I). It has been reported previously that C-terminal phosphorylation of β-catenin in serine 552 and serine 675 (p-S552 and p-S675) by AKT and PKA can enhance β-catenin/TCF reporter activation [48, 49]. We detected an average of 2.5-fold increase of p-S552 (p = 0.00024) and 1.5 fold increase of p-S675 (p = 0.019) in SETBP1-/- cultures than the controls at day 30 (Fig. 5I).

Another key phosphorylation event in the activation of the Wnt signaling cascade is the phosphorylation of the LRP5 and LRP6 co-receptors [50, 51], LRP6 is known to play a more dominant role during embryogenesis. We observed a near two-fold increase of phosphorylated LRP6 (p-LRP6) in day 30 SETBP1-/- NPCs than the isogenic control cells (p = 0.046, Fig. 5I). Together, these studies validated the increase of Wnt/β-catenin activation in SETBP1-deficient cells and provide the first demonstration of a regulatory role for SETBP1 in canonical WNT signaling in cortical NPCs.

Pharmacological inhibition of Wnt/β-catenin pathway rescues proliferation defect of SETBP1-/- cortical NPCs and ameliorates MGE fate induction deficit

To establish a causal relationship between the increased Wnt/β-catenin signaling and over proliferation of SETBP1-deficient NPCs, we interrogated Wnt signaling using XAV939 (XAV), a small molecule tankyrase inhibitor that stabilizes Axin and stimulatesβ-catenin degradation [52]. SETBP1-/- and WT NPC cultures were exposed to XAV for 10 days from day 11, a time window prior to the phenotypic manifestation (Fig. 6A and Additional file 10: Fig. S9A). Wnt signaling inhibition by XAV was verified by evident reduction in total β-catenin, p-S552/p-S675 as well as p-LRP6 comparing treated SETBP1-/- with respective to the no XAV sister cultures in both WT and SETBP1-/- cultures (Fig. 6B). Importantly, after XAV treatment, total β-catenin, p-S552 and p-S675 in SETBP1-/- cells were no longer different to the isogenic control cells without XAV treatment. As a control for inhibitor specificity, the levels of the GAPDH were not affected by XAV treatment.

Fig. 6

Phenotypic rescue of SETBP1 deficiency by pharmacological interrogation of Wnt signaling. A Experimental scheme. Differentiation cultures under basal condition (control) or exposed to 2uM XAV939 from day 11 to day 21. B Western blot analysis at day 35 for the effects of XAV treatment on WNT signaling proteins. Data was obtained from 2 independent differentiations analysed in duplicates or triplicates and shown as relative levels to the WT. Student’s T test was used to compare the expression between the two lines, B-catenin basal P = 0.033, XAV P = 0.542, S552 basal P = 0.023, XAV P = 0.906, S674 basal P = 0.024, XAV P = 0.554, LRP6 basal P = 0.153, XAV P = 0.644, P-LRP6 basal P = 0.004, XAV P = 0.012. C Effect of XAV treatment on cell cycle profile at day 35. Data presented as mean ± s.e.m of 2 independent experiments in triplicates. Student’s T test, one tail, was used to compare the expression between the two lines and the two conditions (WT basal vs. XAV G0-G1 P = 0.029, S P ≥ 0.05, G2-M P ≥ 0.05, SETBP1-/- basal vs. XAV G0-G1 P = 0.021, S P ≥ 0.05, G2-M P = 0.0045. D Immunofluorescence images of cultures in basal (DMSO) or XAV treated conditions at day 20 and 30. Cell nuclei were labelled by DAPI. Scale bar: 100uM. Bar- graphs showing quantification of FOXG1 (green) positive NPS at day 20 and CTIP2 (green), TBR1 (red) and NeuN (green) positive neurons at day 30. Student’s T test was used to compare the expression between the two lines, FOXG1+ cells: basal P = 0.002, XAV P = 0.001; CTIP2+ cells: basal P = 0.029, XAV P = 0.214; TBR1+ cells: basal P = 0.672 XAV P = 0.258, NeuN+ cells: basal P = 0.038, XAV P = 0.333 (*p ≤ 0.05, **p ≤ 0.01). E Schematic illustration depicting the role of SETBP1 in regulating NPC pool expansion and forebrain fate induction

The effect on XAV treatment in NPCs proliferation was examined via cell cycle analysis of DNA content (Fig. 6C). An increase of cells in G1 phase and a decrease of cells in G2-M was observed for both SETBP1-/- and control NES+ NPCs. In XAV treated SETBP1-/- cultures, the number of cells in G2-M phase was restored to a level similar to those in the WT cultures with or without XAV (15.18 ± 0.51 and 15.02 ± 1.07, P = 0.902). We next examined the effect of XAV on the cultures at day 20, 30, 40 and 50 (Fig. 6D and Additional file 10: Fig. S9). Compared to non-treated SETBP1-/- cultures, immunostaining revealed that, at day 20 there is a > 50% increase in FOXG1+ NPCs in the SETBP1-/- cultures (12.62 ± 8.22 vs. 37.15 ± 4.09, P = 0.027). (Fig. 6D). Moreover, as expected, XAV treatment in the WT cultures accelerated neuronal differentiation as demonstrated by a reduced number of PAX6+ and NES+ cells and concurrent increase of NeuN+ and MAP2+ cells in comparison to no XAV sister control cultures (Additional file 10: Fig. S9B, C). XAV treatment also resulted in a significant increase in CTIP2+ cells in both WT and SETBP1-/- cultures, so the CTIP2+ cell numbers in the treated SETBP1-/- cultures reached to a similar levels to those in the basal WT cultures (eg. day 30 WT untreated 16.97 ± 1.50 vs. SETBP1-/- XAV 17.82 ± 6.20, P = 0.844) (Fig. 6D and Additional file 10: Fig. S9B, C and E). XAV treatment didn’t have a significant effect on the number of TBR1+ and SATB2+ cell populations on all time points although a decrease of TBR1+ cells was detected at day 50 (Fig. 6D and Additional file 10: Fig. S9). Compared to non-treated SETBP1-/- cultures, cells in XAV treated cultures exhibited pronounced neuronal arborisation similarly to those in the WT control cultures without XAV (Additional file 10: Fig. S9B).

WNT inhibition via XAV at day 0–10 was part of the MGE differentiation paradigm (Fig. 4E). Since the production of NKX2.1+ progenitors was lower in SETBP1-deficient cultures, we investigated a potential effect of extended XAV treatment (d0-20) on MGE differentiation of SETBP1 mutant cells. We found that longer XAV exposure resulted in an increase of NKX2.1+ cells in both the WT and SETBP1 mutant cultures compare to the standard MGE protocol. Under XAV d0-20 condition, while NKX2.1+ cell numbers still appear lower in the SETBP1 mutant cultures compared to the WT controls, no statistical differences were reached (Additional file 11: Fig. S10). Together, our data demonstrates that inhibition of Wnt/β-catenin signaling can restore the neurogenesis and fate induction defects of SETBP1-/- NPCs and thus provide a functional verification that SETBP1 is playing a role in WNT signaling.