Shared effects of DISC1 disruption and elevated WNT signaling in human cerebral organoids

Fig. 1. Cerebral organoids express neural progenitor markers with ventricle, rosette, and dispersed morphologies. a Organoids were differentiated to day 19 and pulsed with 2 μM EdU during days 6–7 before being fixed, sectioned, and stained for Nestin and EdU, showing areas of ventricle (asterisk), rosette (arrowhead), and dispersed (arrow) morphology. Day 19 organoids immunostained for b–e Tbr2 and Nestin, or f–i Pax6 and Nestin, showing areas of ventricle (asterisk), rosette (arrowhead), and dispersed (arrow) morphology. Scale bars: 100 μm (a), 50 μm (b–e, j–l), 20 μm (f–i)

Fig. 2. DISC1 disruption alters cerebral organoid morphology. a–l Wild-type and DISC1-disrupted organoids were pulsed with EdU for 2 days (culture days 6–7), then fixed, sectioned, and stained for EdU at day 19. Two representative organoids are shown for wild-type (a, b, e, f, i, j) and DISC1-disrupted (c, d, g, h, k, l) lines treated with either DMSO (vehicle, a–d), WNT agonist CHIR99021 (e–h), or WNT antagonist XAV939 (i–l). Well-defined ventricles/rosettes are labeled with asterisks, small poorly defined rosettes labeled with arrowheads, and areas of dispersed morphology labeled with arrows. Scale bars = 100 μm. Example measurements of ventricles/rosettes (labeled with red arrows) with blue lines labeling length and green lines labeling width, are shown in m (same image as b) and n (same image as d). Morphology of DMSO-treated wild-type or DISC1-disrupted organoids was quantified, blinded, in sections from the middle of the organoid, by counting number of ventricles or rosettes visible per organoid (o), average ventricle/rosette area per organoid (p), average length of the long axis of ventricles/rosettes per organoid (q), and organoid area (r). Data were derived from four independent…

Fig. 3. DISC1-mutant organoids exhibit decreased BRN2 expression and reduced proliferation that is rescued by WNT antagonism. Immunostaining was performed on WT and DISC1-mutant day 19 organoids for EdU incorporation and markers as shown. Expression of cell fate markers TBR2, PAX6, P73, Reelin and polarity markers PKC-λ, and acetylated-α-tubulin were grossly unchanged (a–j). However, immunostaining of BRN2 was markedly reduced in DISC1-mutant organoids (k, l). Scale bars: a–d 100 μm, e–h 20 μm, i–j 50 μm, k–l 20 μm. m, n WT and DISC1 exon 8 wt/μ organoids were immunostained at day 19 for markers of apoptosis (TUNEL and Cleaved Caspase 3). o, p Quantification of percentage of DAPI positive nuclei positive for TUNEL or cleaved caspase-3 shows no difference with DISC1 disruption. q–u WT and DISC1-mutant organoids with WNT agonism (CHIR) or WNT antagonism (XAV) were pulsed with EdU for 2 days (culture days 6–7), then fixed, sectioned, and stained for EdU and DAPI at day 19. One representative image for each condition is shown. s Percentage of EdU-positive nuclei were quantified. Data were derived from three independent differentiations. Statistics: o, p Variance was significantly different…

Fig. 4. DISC1 disruption alters BRN2 levels in organoids and monolayer cultures. Wild-type and DISC1 ex8 wt/μ organoids were harvested at day 19 and RNA was used for Nanostring analyses. a Heat map of cell type markers for wild type (wt) and DISC1 ex8 wt/μ organoids. b Volcano plot of gene expression changes in DISC1 ex8 wt/μ vs. wild-type organoids are shown. Statistics: Student’s t-test, unadjusted p-values plotted; n = 7 for wild-type, 8 for DISC1 wt/μ, from four independent differentiations. See also Supplementary Table 1. c, d qPCR was performed for BRN2 and CALB1 on RNA derived from day 19 organoids. Data were derived from three independent differentiations. For each differentiation, over five organoids were pooled for analysis. e Wild-type and DISC1 ex8 w/μ iPSCs were differentiated to NPCs using an EB protocol7 and subsequently dissociated and plated as monolayer culture. Day 40 neuronal RNA was harvested and used for Nanostring. Data were derived from three to seven independent differentiations. Statistics: c One-way ANOVA with Sidak multiple comparisons test, d,e Student’s t-test, *p < 0.05, **p < 0.01, ****p < 0.0001. f Table summarizing gene expression changes across both…

Fig. 5. DISC1 disruption alters expression of genes implicated in neurodevelopment and migration and is phenocopied by WNT agonism. Wild-type and DISC1 ex8 wt/μ iPSCs were differentiated to NPCs and treated with vehicle (DMSO) or WNT agonist CHIR99021 (CHIR) during days 7–17, followed by withdrawal of small molecules and subsequent monolayer culture. Day 40 neuronal RNA was harvested and used for Nanostring. a Venn diagram showing genes with significantly altered expression with shared direction of fold-change compared to wild-type. Number of genes per category is shown within the diagram. Statistics: Student’s t-test with multiple comparisons correction using 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%. b–d Nanostring data for select genes are shown, including genes associated with cell fate (b), genes associated with glutamatergic neurotransmission (c), and genes associated with interneuron development (d). Statistics: Holm–Sidak; WT n = 15, WT CHIR n = 18, DISC1 ex8 wt/μ n = 7. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Supplementary Table 2