Fig 1: 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 differentiation methods: organoid day 19 qPCR (from c, d and other data not shown) and day 40 monolayer Nanostring data. Asterisks indicate significance by two-tailed Student’s t-test; #indicates data published in ref. 7
Fig 2: Downregulated Pax3 probably resulted in reduced expression of FPN1 in PE placental tissues.Both GSE50783 and GSE44667 data sets provided the data of genes that were aberrantly expressed in placental tissues from PE patients and physiologically normal pregnancies. Transcript factors that were implicated in the transcription of FPN1 were predicted using a Bioinformatics analysis website (http://bioinfo.life.hust.edu.cn/hTFtarget#!/). We analyzed the intersection between the aberrantly expressed genes and transcript factors targeting FPN1. As indicated by the Venn diagram, three of the transcript factors (Pax3, POU3F2 and BHLHE40) were found to be aberrantly expressed in placental tissues from PE patients (A). Bioinformatics analysis website Jasper (http://jaspar2016.genereg.net/) gave the highest score on POU3F2 followed by Pax3 (B). We performed western blot to test the protein levels of these three transcript factors in placental tissues from PE patients and physiologically normal pregnancies (C). Pax3 protein level was down-regulated in PE placental tissues, while BHLHE40 protein level was significantly up-regulated. Results from immunohistochemistry assay (D) and GSE10588 data set (E) also confirmed the down-regulation of PAX3 in PE placental tissues. Another transcript factor-predicting websit, Jasper (http://jaspar2016.genereg.net/), showed the motif, a sequence in gene promoter that is recognized by Pax3 (F). Moreover, Jasper revealed two potential binding sites of transcription factor Pax3 at the promoter of FPN1 and SLC7A11 genes. Our following study was performed to identify the regulatory effect of Pax3 on FPN1 and SLC7A11 expression. *p < 0.05, **p < 0.01, compared with the normal group.
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 between conditions, Welch’s t-test. s Variance was significantly different between conditions, one-way ANOVA with Geiser–Greenhouse correction, Sidak’s multiple comparisons test. ***p < 0.001, ****p < 0.0001. Scale bars: m, n, q, p, t, u: 50 μm
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