Transcription Factor 4 loss-of-function is associated with deficits in progenitor proliferation and cortical neuron content

Transcription Factor 4 (TCF4) has been associated with autism, schizophrenia, and other neuropsychiatric disorders. However, how pathological TCF4 mutations affect the human neural tissue is poorly understood. Here, we derive neural progenitor cells, neurons, and brain organoids from skin fibroblasts obtained from children with Pitt-Hopkins Syndrome carrying clinically relevant mutations in TCF4. We show that neural progenitors bearing these mutations have reduced proliferation and impaired capacity to differentiate into neurons. We identify a mechanism through which TCF4 loss-of-function leads to decreased Wnt signaling and then to diminished expression of SOX genes, culminating in reduced progenitor proliferation in vitro. Moreover, we show reduced cortical neuron content and impaired electrical activity in the patient-derived organoids, phenotypes that were rescued after correction of TCF4 expression or by pharmacological modulation of Wnt signaling. This work delineates pathological mechanisms in neural cells harboring TCF4 mutations and provides a potential target for therapeutic strategies for genetic disorders associated with this gene.

Table 1 for further information).Note that, in all patients expect PTHS #4, the mutated allele yields no protein or a truncated protein without the essential DNA-binding domain.b, Example of immunostaining of parent and PTHS iPSC colonies for pluripotency markers OCT4, NANOG, and LIN28.c, Example of digital karyotyping by SNP mapping via chip hybridization, on sample from patient PTHS #2 (Supplementary Table 1), showing large deletion on chromosome 18 (asterisk).Numbers on the left represent chromosomes.The y axis in each chromosome graph represents the logR ratio for individual hybridized chip probes (dots).d, Comparison of iPSC colony growth rate (in days to reach size required for passaging, which is 2 mm) between parents (controls), PTHS, and a control iPSC line derived from a subject not involved in the study (WT83).N = 5 subjects per group (symbols).Measurement for each subject is the median from 5 independent plates.e, Representative bright-field microscopy image showing iPSCderived neurons in culture (2 months in neuronal medium).Notice the formation of islands of progenitors and neuronal cell bodies connected by bundles of neuronal fibers in both the parent and PTHS groups.f, Fluorescence microscopy images of cultures in e stained for MAP2 (magenta) and SOX2 (green), evidencing general ability of iPSCs to differentiate into NPCs (SOX2+) and neurons (MAP2+) in both groups.g, Ratio of relative quantitation of TCF4 expression measured via RT-qPCR (RQ) between iPSC-derived PTHS and parent neuronal cultures (3 months in neuronal medium).N = 4 subjects per group (symbols), 3 independent replicates per subject, 2 technical replicates per sample.h, Ratio of relative quantitation of TCF4 expression measured via RT-qPCR (RQ) between PTHS and parent in NPCs in 2D culture.N = 5 subjects per group (symbols), 3 independent replicates per subject, 2 technical replicates per sample.Notice that for one PTHS subject (PTHS #4, circle symbol) the expression is not diminished, concordant with the expectation for this patient, whose mis-sense point mutation is not predicted to affect transcript abundance.Expression Level

. 1 :
PTHS iPSCs exhibit normal growth rate and can be differentiated into neurons.a, Structure of the TCF4 locus (exon numbers on top of each rectangle) in different patients.White rectangles symbolize missing exons due to partial or whole gene deletion.Rectangles with thick borders represent the coding sequence in each case.AD1 to AD3: transcriptional activation domains; bHLH: basic helix-loop-helix DNA-binding domain.Exons 1 and 2 are shown but they are not part of the main transcript for the TCF4 gene, called TCF4-B.Details on the types of mutation carried by each patient are given on the right (see also Supplementary

. 3 :
Annotation of subpopulations in single cell RNA-Seq experiments and associated controls.a, Dot plot showing expression of selected marker genes (see Methods for marker gene selection and validation) in the six subpopulations of cells analyzed in the single cell transcriptomic data of PTHS and parent CtOs and GbOs, as depicted in Fig. 3a.Pr-Glut: neural progenitor cells in glutamatergic lineage; IP-Glut: intermediate progenitors in glutamatergic lineage; N-Glut: glutamatergic neurons; Pr-GABA: neural progenitors in inhibitory lineage; IP-GABA: intermediate progenitors in inhibitory lineage; N-GABA: neuronal population containing GABAergic interneurons.'Others' represents a minor and heterogeneous group of cells not included in the previous six categories (see panel e for analysis of this population and Methods for details).Dot sizes are the percentages of cells in each subpopulation that have detectable expression for the corresponding gene.Dot color intensity represents average gene expression.b,Violin plots for marker genes shown in a, displaying range of expression in the six analyzed subpopulations of cells and in the 'Others' category.Color code is the same as in Fig.3a.For GRIN2, GAD1, and GAD2, the medians were low and therefore the expression in each cell is represented as a dot.c, Single cell RNA-Seq quality control data.Violin plots represent the numbers of read counts, numbers of detected genes (features), and percentages of expressed mitochondrial genes (mtRNA) in the subpopulations listed in a. Color code is the same as in Fig. 3a and 'Others' group is shown in black.d, UMAP embedding of single cell transcriptomic data showing expression of TCF4, neural lineage markers SOX2 and MAP2, mesoderm markers MIXL1 and TBXT (Brachyury), and endoderm markers CFTR and SOX17 in PTHS and parent CtOs.Intensity of purple indicates relative expression level.e, UMAP embedding of scRNA-Seq profiling of CtOs and GbOs at 8 weeks in vitro, integrating data from all eight libraries (see Methods for details), to evidence the position of cells in the 'Others' category.Color code is the same as in Fig. 3a and 'Others' category is shown in black.f, Percentages of cells in 'Others' category in the 8 scRNA-Seq libraries.Comparisons are separated into 4 groups: CtOs, GbOs, PTHS CtOs treated with CHIR99021, and PTHS GbOs treated with CHIR99021 (see data with CHIR-treated organoids below).Notice that cells in the 'Others' category are rare in all libraries.

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Controls for single cell RNA-Seq investigation of parent and PTHS organoids.a, Left: Controls showing reproducibility in the generation of CtOs derived from the same genotype in independent experiments (replicates #1, #2, and #3 of parent CtO libraries).Right: Percentages of cells in each of the six subpopulations analyzed in replicate single cell RNA-Seq libraries, showing robustness of scRNA-Seq analyses, which detect equivalent percentages of each cell type in independent batches of organoids.Color code is the same as in Fig. 3a.

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AP2+ cells in parent and PTHS CtOs and GbOs.High magnification images are shown in insets.N = 6 sections per group; 3 sections per subject; 3 ROIs per section.j, Representative fluorescence microscopy images of CtOs and GbOs after immunostaining for FOXG1 and AP2 (TFAP2A).k, Heatmaps showing percentages of all cells in CtOs that express each neural lineage marker gene at detectable levels.Genes are divided in telencephalic, dielencephalic, mesencephalic, metencephalic, and myelencephalic categories.The numbers inside each square denote the exact percentages of cells in parent and PTHS libraries expressing each marker.l, Left: Heatmaps showing percentages of all cells in GbOs that express each neural lineage marker gene.The numbers inside each square denote the exact percentages of cells in parent and PTHS libraries.Right: Heatmaps showing percentages of GAD1+ cells (GABAergic neurons) in GbOs that express each marker gene.Similar results were obtained with GAD2+ cells.m, Examples of expression analysis conducted on the GAD1+ population in GbOs in l.The red cells in the UMAP plot and the dashed red rectangle in the violin plot on the left represent the GAD1+ population analyzed.The levels of overlap between GAD1 expression (red) and expression of FOXG1 or TFAP2A (blue) are shown as examples on the right.The colormap indicates extent of overlap, and the violin plots indicate GAD1+ cells that also express FOXG1 or TFAP2A.All single cell transcriptomic data in the figure were obtained with parent #4 and PTHS #4 CtOs and GbOs that have been maintained for 8 weeks in vitro.Bar graphs represent mean + SEM in i. n.s., not statistically significantly different, *p<0.05,**p<0.01,***p<0.001,two-tailed Wilcoxon-Mann-Whitney U test (d-f,h) or two-tailed Welch's t-test assuming unequal variances (i).Colors in bar graphs and violin plots in d-f,h,i represent parents (orange) or PTHS (blue) groups.The cross symbol indicates that the means of gene expression on a per cell basis was statistically significantly different between the parent and PTHS groups, but the log2 fold change between PTHS and parents was smaller than an arbitrary value of 0.5 (equivalent to a variation of approximately 40% compared to the parent group mean).Scale bar is 100 m.See Supplementary Data 1 for statistical test results, including sample sizes, numbers of replicates, exact p-values, and effect sizes.Metabotropic glutamate receptor pathway Muscarinic acetylcholine receptor signaling GABA-B receptor signaling Metabotropic glutamate receptor pathway Angiotensin II signaling pathway -adrenergic receptor signaling pathway actin filament-based process trans-synaptic signaling response to culture -pair #1) neurons (2D culture -pair #2) neurons (2D culture -pair #4) Supporting data for functional investigation of neurons in 2D culture and in organoids.a, Left: CtOs seeded on multi-electrode array (MEA) plate.Right: Mean firing rate in active electrodes in MEA evaluation of CtOs.N = 4 subjects per group (symbols), 2 independent experiments per subject, 3 replicates (wells) per subject in each experiment, 10 organoids per subject in each replicate.Recordings were performed on organoids at 8 weeks in vitro.Arrowhead indicates neuronal processes on top of electrodes.b, Time course of mean firing rate in CtOs from 1 to 2 months in vitro, subjected to multi-electrode array (MEA) assay, showing comparison between parent (orange) and PTHS (blue) organoids.Each patient is represented with a different symbol (as in Supplementary Table 1).The red lines and error bars represent the mean across all subjects over time.N = 4 subjects (symbols), 6 independent replicates per subject, 10 organoids seeded per well in each replicate.c, Left: Expression of FOS in N-Glut neurons in parent and PTHS CtOs at 8 weeks in vitro.Violin plots represent distribution of gene expression in each population and each dot represents expression in a single cell.N = 1401 (parent) and 380 (PTHS) cells.Right: Percentages of FOS+ cells (expression above threshold indicated by red dashed line on the violin plot).Data were computed from scRNA-Seq data from parent-patient pair #4.d, DE genes in N-Glut and N-GABA neurons of CtOs and GbOs, comparing parent and PTHS organoids, computed from scRNA-Seq data from parent-patient pair #4.Left: MA plot showing DE genes.Each dot represents a gene, gray color indicates genes that are not DE between parent and PTHS groups, and the yellow-to-purple gradient represents DE genes, color-coded according to svalue.The x-axis represents average gene expression in the parent group.log2FC in y-axis indicates fold-change (in log2 scale).Genes with average expression below 0.1 in the parent group are not shown, since their expression is small and most are not DE.Selected DE genes described in the study are highlighted in red.Right: Dot plots representing results of Gene Set Enrichment Analysis followed by GO analysis (Biological Processes and Pathways) over DE genes that are down-regulated in PTHS N-Glut neurons in CtOs (left) or down-regulated in PTHS N-GABA neurons in GbOs (right).For each analysis, the top 10 categories in terms of adjusted p-value are shown.Dot size represents number of DE genes that fall into each classification category, dot color is the adjusted p-value, and the x-axis represents the percentage of genes in each category that are DE expressed genes in the scRNA-Seq libraries.Data were computed from scRNA-Seq data from parent-patient pair #4.e, Left: Fluorescence microscopy images of parent and PTHS CtOs at 6-8 weeks in vitro after immunostaining for indirect surrogate marker of neuronal activation c-Fos (yellow).Analysis of c-Fos protein expression is important to confirm the decreased activity in PTHS organoids and to rule out the possibility that the expression of FOS gene in organoids is a consequence of the cellular dissociation applied prior to the generation of scRNA-Seq libraries.Right: Quantification of numbers of c-Fos+ cells in an area of 300,000 m 2 and percentages of c-Fos+ cells in parent and PTHS CtOs (see Methods for a description of the ROIs used).Notice that c-Fos+ cells are concentrated at the organoid's periphery, as previously shown 1 .N = 3 parent and 5 PTHS samples (symbols), 5 sections per subject.f, Representative fluorescence microscopy image showing expression of TCF4 protein (green) in 2D cultures of neurons (MAP2 labeling shown in magenta) being differentiated from parent-derived iPSCs (1 month in neuronal medium).g, Percentages of glutamatergic (N-Glut) and GABAergic (N-GABA) neurons in 2D cultures of parent and PTHS neurons, as judged from deconvolution of bulk RNA-Seq data assisted with markers identified from the analysis of subpopulations in scRNA-Seq data (see Methods for details).These data suggest a lower percentage of N-Glut and absence of N-GABA neurons in the PTHS cultures.N = 3 subjects per groups; 3 replicate RNA-Seq libraries per subject.h, Comparison of membrane capacitance between PTHS (blue circles) and parent (orange circles) cells via patch-clamp electrophysiological analysis in 2D cultures of parent-pair #4 neurons (3 months in neuronal medium).N = 10 (parent) or 9 (PTHS) neurons.i, Interrogation of sodium (top) and potassium (bottom) current densities, comparing PTHS (blue line) and parent (orange line) neurons in 2D culture for parent-pair #4 (3 months in neuronal medium).N = 10 (parent) or 9 (PTHS) neurons.j, Heat map showing the expression levels for the 20,000 most highly expressed genes in RNA-Seq libraries of neurons in 2D culture (3 months in neuronal medium) from 2 parents and respective PTHS children (PTHS #1 and #4).The numbers of differentially expressed (DE) genes in each parent-child comparison and the number of common DE genes across all 3 parent-child pairs are shown above the plot (see Supplementary Data 2 for list of DE genes).k, Dot plot results for Gene Ontology -Biological Processes (top) and Pathway analysis (bottom), for down-regulated DE genes common to all parent-patient comparisons in j.For each analysis, the top 10 categories in terms of adjusted p-value are shown.Dot size represents number of DE genes that fall into each classification category, dot color is the adjusted p-value, and the x-axis represents the percentage of genes in each category that are DE expressed genes in the RNA-Seq libraries.Notice the presence of categories related to glutamatergic and GABAergic transmission.l, UMAP embeddings showing cells that express GAD1 (red) and rare cells that express SLC17A6 (vGLUT2) (blue) within the N-GABA subpopulation of GbOs.The blue numbers on the lower corner indicate the percentages of SLC17A6+ cells in parent and PTHS organoids.m, MA plot for genes expressed in control and PTHS neurons in 2D culture.Each parent-patient comparison is shown separately.Gray dots are genes that are not statistically significantly differentially expressed between control and PTHS.Colored dots are statistically significant DE genes, and the color indicates adjusted pvalue.Red dots represent genes coding for sodium or potassium channels with a log2 fold change superior to 2 in each direction (dashed lines).The x axes represent average normalized expression across all libraries ('baseMean').For clarity purposes, only DE channel genes with the largest fold-changes are labeled.See Supplementary Data 2 for full list of DE channelcoding genes.n, Expression levels of potassium channel KCNQ3 in neurons of parent and PTHS organoids (N-Glut neurons of CtOs and N-GABA neurons of GbOs).N = 1401 (parent) or 380 (PTHS) N-Glut cells, or N = 2661 (parent) and 988 (PTHS) N-GABA cells.Data were computed from scRNA-Seq obtained from parent-patient pair #4.Symbols in bar graphs indicate parent-patient identities: diamonds, pair #1; squares, pair #2; triangles, pair #3; circles, pair #4.Colors in the bar graphs and line and violin plots represent parents (orange) or PTHS (blue) groups.Bar graphs represent mean + SEM.ns, not statistically significantly different, *p<0.05,**p<0.01,***p<0.001;two-tailed Welch's t-test (a,e,h), two-tailed multiple t-test with Holm-Sidak multiple comparison post-hoc test (b), two-tailed Wilcoxon-Mann-Whitney U test (c,n), or one-way ANOVA followed by HSD post-hoc test (i).Scale bars are 100 m in (e) and 50 m in (f).See Supplementary Data 1 for statistical test results, including sample sizes, numbers of replicates, exact p-values, and effect sizes.Phenotypic analyses of neural progenitor cells.a, Expression of several NPC markers in iPSC-derived neural progenitor cells in 2D culture from parents (orange) and PTHS (blue) subjects, as judged from TPM expression abundance in RNA-Seq libraries.N = 4 subjects per group (symbols), 3 independent replicate libraries per subject.b, Relative TCF4 expression levels (RT-qPCR) in iPSCs and iPSC-derived NPCs and neuronal cultures, as well expression in a non-neural cell line (HEK293T).All cells are of normal genotype; iPSCs and iPSC-derived cells are from a control subject not belonging to the PTHS study cohort (WT83).N = 3 independent replicates per group, 2 technical replicates per sample.Mean expression in neural progenitor group was normalized to 1. c, Representative fluorescence microscopy image of NPCs in 2D culture after immunostaining for TCF4 (magenta) and NPC marker Nestin (green).Higher magnification image in inset.d, Representative fluorescence microscopy image showing abundant expression of TCF4 (magenta) in NPCs of rosettes in control (parent #4) CtOs at 4 weeks in vitro.e, Representative images of control and PTHS NPCs in 2D culture at early and late passages.Notice the appearance of large flat cells in PTHS NPCs at passage 20 (P20) (arrowheads).f, Ratio between the expression of transcriptomic markers of replicative senescence between PTHS and control samples of NPCs in 2D culture, at early and late passages.For each gene, the mean at each passage was determined from 3 independent biological samples.Each line connects expression for a certain gene at early and late passage conditions.Marker genes are separated according to class (down-regulated or up-regulated in senescent cells, as in Hernandez-Segura et al. 2 ).Notice the more pronounced mis-regulation in late passage conditions.Cells are from parent-child pair #4 and similar results were obtained for parent-child pairs #1 to #3 (Supplementary Data 2

Rho:
0.06 0.07 0.08 0.09 0.10 0.11 Differential gene expression analysis in neural progenitor cells.a, Heat map showing the expression levels for the 20,000 most highly expressed genes in RNA-Seq libraries of NPCs in 2D culture from parents #1 and #4 and respective PTHS children, as an example (see Supplementary Data 2 for expression abundances in all 4 parent-child pairs and independent replicates).Three independent RNA-Seq libraries were sequenced per subject (columns in heatmap).The numbers of DE genes in the intersection among comparisons for all four parent-child pairs are shown above the plot (see Supplementary Data 2 for list of DE genes in each parent-child pair and across all 4 pairs).b, Dot plot results for Gene Ontology -Biological Processes (top) and Pathway analysis (bottom), for up-regulated DE genes represented in a resulting from the intersection among all 4 parent-child pairs.For each analysis, the top 10 categories in terms of adjusted p-value are shown.Dot size represents number of DE genes that fall into each classification category, dot color is the adjusted p-value, and the x-axis represents the percentage of genes in each category that are DE expressed genes in the RNA-Seq libraries.Notice the presence of up-regulated genes involved in cellular senescence (related to Fig. 5) and tissue architecture.c, TPM expression abundances of HOPX in parent and PTHS NPCs in 2D culture.Notice the tendency for increased expression in all lines, but lack of statistical significance across groups.N = 4 subjects per group (symbols), 3 independent libraries per subject.d, MA plot depicting DE genes between control and PTHS NPCs in 2D culture, to evidence upregulation of HOPX in the patient line.Only parent-patient pair #4 is shown as an example (see Supplementary Data 2 for list of DE genes in other pairs).Gray dots are genes that are not statistically significantly differentially expressed between control and PTHS.Colored dots are statistically significant DE genes, and the color indicates adjusted p-value.Red dot represents HOPX.The x-axis represents average normalized expression across all libraries ('baseMean').e, Left: Violin plots showing comparison between parent and PTHS CtOs and GbOs in terms of HOPX expression.N = 3544 (parent) or 2944 (PTHS) CtO cells, or 6466 (parent) or 3792 (PTHS) GbO cells.Right: Bar plots show percentages of cells in CtOs and GbOs expressing HOPX above threshold corresponding to 40% of the respective mean (shown by the red dashed line in the corresponding violin plots).Notice the diverging regulation patterns of HOPX in CtOs and GbOs.Data were obtained from scRNA-Seq results from parent #4 and PTHS #4 CtOs and GbOs that have been maintained for 8 weeks in vitro.The hatched pattern in the bar plots indicates the fraction of HOPX+ cells that are also positive for astrocytic marker S100B expression.f, Left: Fluorescence microscopy images of PTHS and control CtOs and GbOs, after immunostaining for HOPX (yellow).High magnification insets show overlap between HOPX and DAPI staining.(Right): Quantification of percentages of HOPX+ cells in PTHS and parent organoids.N = 6 sections per group (symbols); 3 sections per subject; 3 ROIs per section.g, Relative expression (RT-qPCR) of HOPX in the post-mortem PTHS cortex sample (PTHS #6 patient; Supplementary Fig. 6j for similar results with parent-pair #4 cells.h, Treatment of CtOs with Wnt pathway agonist CHIR99021 at the beginning of the progenitor proliferation phase (light blue bar) rescues organoid size measured at 4 weeks in vitro.N = 3 independent replicates (circles), 15-20 measured organoids per experiment in each group.Organoids were from parent-patient pair #4.Symbols in bar graphs indicate parent-patient identities: diamonds, pair #1; squares, pair #2; triangles, pair #3; circles, pair #4.Colors in bar graphs represent parents (orange), pharmacologically treated parents (yellow), PTHS (blue), or pharmacologically treated PTHS (light blue) groups.Bar graphs represent mean + SEM.n.s., not statistically significantly different, *p<0.05,**p<0.01,***p<0.001;two-sample Welch's t-test in a and one-way ANOVA followed by HSD post-hoc test elsewhere.In d, mean gene expression was normalized to 1 in the parents group.In f, Wnt signaling activity in parent + DMSO group was set to 1. Scale bars are 100 m.See Supplementary Data 1 for statistical test results, including sample sizes, numbers of replicates, exact p-values, and effect sizes.
Fig. 3a.Cells in 'Others' category are shown in black.Pr-Glut: neural progenitor cells in glutamatergic lineage; IP-Glut: intermediate progenitors in glutamatergic lineage; N-Glut: glutamatergic neurons; Pr-GABA: neural progenitors in inhibitory lineage; IP-GABA: intermediate progenitors in inhibitory lineage; N-GABA: neuronal population containing GABAergic interneurons.Right: quantification of percentages of neural progenitors and neurons in the corresponding types of organoids on the left.c, Relative expression of GAD1 and GAD2 in CtO organoids at 8 weeks in vitro after treatment with CHIR99021 at the beginning of the progenitor proliferation phase.N = 3 biological replicates per condition for parent/child pair #4, 3 technical replicates.d, Treatment of PTHS NPCs in 2D culture with CHIR99021 increases expression of TCF4 and TCF4 downstream target gene GADD45G.N = 4 subjects per group (symbols), 3 independent replicates per subject, 2 technical replicates.Mean expression level was normalized to 1 in 'parents+DMSO' group for each gene.e, Top: UMAP showing expression of TCF4 in PTHS GbOs treated with CHIR99021 (right) in comparison with untreated PTHS GbOs (left).Data was computed from scRNA-Seq results obtained with parent-patient pair #4 organoids at 8 weeks in vitro.Intensity of purple indicates relative expression level.Bottom: Violin plot showing expression level of TCF4 in single cells (dots) of untreated and CHIR-treated PTHS GbOs.N = 6,466 (PTHS) or 3,792 (PTHS+CHIR) cells.f, Left: Representative images of parental control and CHIR99021-treated NPCs in 2D culture seeded onto well plates, after immunostaining for TCF4 and -actin (as a normalization control).Cells in representative images are from parent-patient pair #1.Right: Quantification of normalized TCF4 expression levels, as judged by fluorometric analysis of staining on the left (see Methods for details).N = 3 subjects per group (symbols), 3 replicates (wells) per subject.g, Left: Representative plots showing the levels of TCF4 labeling fluorescence intensity via flow cytometry in PTHS NPCs in 2D culture subjected to treatment with CHIR99021 (see Methods for details), which are indicative of TCF4 protein expression levels.Each dot represents a cytometer event.The violin plots summarize the distribution of TCF4 fluorescence intensity for each group (medians are shown as thick horizontal lines and upper and lower quartiles are pair #4.g, Left, MA plot showing DE genes in IP-Glut and IP-GABA intermediate progenitors of CtOs and GbOs, comparing parent and PTHS organoids, computed from scRNA-Seq data from parent-patient pair #4.Each dot represents a gene, gray color indicates genes that are not DE between parent and PTHS groups, and the yellow-to-purple gradient represents DE genes, color-coded according to s-value.The x-axis represents average gene expression in the parent group.log2FC in the y-axis indicates fold-change (in log2 scale).Genes with average expression below 0.1 in the parent group are not shown, since their expression is small, and most are not DE.Selected DE genes described in the study are highlighted in red.h, Dot plots representing results of Gene Set Enrichment Analysis followed by GO analysis (Biological Processes and Pathways) over DE genes that are down-regulated in PTHS IP-Glut intermediate progenitors in CtOs (left) or down-regulated in PTHS IP-GABA intermediate progenitors in GbOs (right).For each analysis, the top 10 categories in terms of adjusted p-value are shown.Dot size represents number of DE genes that fall into each classification category, dot color is the adjusted p-value, and the x-axis represents the percentage of genes in each category that are DE expressed genes in the scRNA-Seq libraries.Data were computed from scRNA-Seq data from parent-patient pair #4.i, Expression of SOX4 along the differentiation trajectory path depicted in Fig. 3b, in parent and PTHS CtOs.The shaded areas around each line indicate 95% confidence intervals at each timepoint along the pseudotime scale (see Methods for details).
figure.The cross symbol indicates that the means of gene expression on a per cell basis was statistically significantly different between the parent and PTHS groups, but the log2 fold change between PTHS and parents was smaller than an arbitrary value of 0.5 (equivalent to a variation of approximately 40% compared to the parent group mean).Scale bars are 100 m.See Supplementary Data 1 for statistical test results, including sample sizes, numbers of replicates, exact p-values, and effect sizes.
All other patients have mutations expected to decrease transcript content (non-sense mutation, frameshift mutation, whole gene deletion, or translocation).i, Numbers of cells immunostained for TCF4 in a 100 × 100 m square, in Expression levels of the same mesoderm and endoderm markers shown in d, in the 'Others' category of CtOs and GbOs of parent (left in each group) and PTHS (right in each group) genotypes.All single cell transcriptomic data in the figure were obtained with parent #4 and The replicate libraries of parent CtOs are shown in the order 'replicate #3', 'replicate #1', and 'replicate #2', to highlight the comparison between parent CtO replicate #2 and PTHS CtO libraries used elsewhere in the figures.g, Top left: Percentages of cells in 'Others' category and in a group of cells exhibiting high mitochondrial RNA content (which was not included in the analyses), in parent and PTHS CtOs and GbOs.Parent CtO bar indicates data from replicate #2, as an example.Top right: Expression levels of neural lineage markers SOX2 and MAP2 in the 'Others' category of CtOs and GbOs in parent (orange) and PTHS (blue).Bottom: including sample sizes, numbers of replicates, exact p-values, and effect sizes.
). g, Initial seeding density is 1 × 10 5 cells.Right: Quantification of percentages of EdU+ NPCs in 2D culture, in the same groups as on the left.N = 3 independent replicates per group (circles).Cells were from parent-patient pair #4.j, shRNA-mediated TCF4 knockdown in NPCs in 2D culture leads to decreased expression of TCF4 and TCF4 downstream target gene GADD45G, as well subjects, 6 organoids per subject (from 3 independent experiments), 4 random ROIs per organoid.i, Left: shRNA-mediated TCF4 knockdown reduces proliferation of NPCs in 2D culture.N = 3 independent replicates (circles) per group, 3 technical replicates per sample.**p<0.01,***p<0.001;two-sample Welch's t-test assuming unequal variances (a,j) or one-way ANOVA followed by Tukey-Kramer's HSD post-hoc test (b,i).In j, mean gene expression was normalized to 1 in each parent + control shRNA group.Scale bars are 100 m.See Supplementary Data 1 for statistical test results, including sample sizes, numbers of replicates, exact p-values, and effect sizes.

Table 1 )
, in comparison with a control cortex.N = 3 independent replicates per group, 4 technical replicates.h, Dot plot results for Gene Ontology -Biological Processes (top) and Pathway analysis (bottom), for down-regulated DE genes shown in a resulting from the intersection among all 4 parent-child pairs.For each analysis, the top 10 categories in terms of adjusted p-value are shown.Dot size represents number of DE genes that fall into each classification category, dot color is the adjusted p-value, and the x-axis represents the percentage of genes in each category that are DE expressed genes in the RNA-Seq libraries.Notice the presence of down- regulated genes in the Wnt signaling pathway (related to Fig.6).Symbols in bar graphs indicate parent-patient identities: diamonds, pair #1; squares, pair #2; triangles, pair #3; circles, pair #4; gray dots, post-mortem samples.Colors in bar graphs and violin plots represent parents (orange), PTHS (blue), or control post-mortem sample (black) groups.Bar graphs represent mean + SEM.ns, not statistically significantly different, **p<0.01,***p<0.001;two-sample Welch's t-test assuming unequal variances (c,f,g) or two-tailed Wilcoxon-Mann-Whitney U test (e).Scale bar is 100 m.See Supplementary Data 1 for statistical test results, including sample sizes, numbers of replicates, exact p-values, and effect sizes.