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The transcriptomic data were also used to validate the identities of the cell lines employed in the study. For patients PTHS 1 and 4, the sequences of TCF4 transcripts encompassing the respective mutated sites were retrieved from GENCODE, followed by the generation of mutated sequences to include the mutations each patient carries insertion and point mutation, respectively; see Supplementary Table 1.

The quantification results informed that the expression of each mutated TCF4 transcript is only found in the respective patient and not in the other patients nor in the parent samples Supplementary Data 5. For patients PTHS 2 and 3, we could not produce the sequences of the mutated transcripts because they carry a whole-gene deletion and a chromosome translocation, respectively Supplementary Table 1.

Next, between-sample normalization was performed using the size factors approach 63 and a local dispersion model was fit to the normalized counts. Lastly, a negative binomial generalized linear model was fit to the data, the effect sizes log2FoldChange were shrunken with the apeglm algorithm version 1.

The final list was used for gene-set enrichment assessment followed by Gene Ontology GO and pathway analyses, using the web-based WebGestalt tool version 65 , with default parameters. WebGestalt conducts permutations to obtain an over-representation Z score and enrichment p- value for each GO term. For pathway analysis, we chose the KEGG option, with default parameters.

For all analyses, a minimum of 5 genes per category was employed, with BH multiple test correction, and a significance level chosen for a false-discovery rate of 0. The signature matrix was subsequently used to impute cell fractions to each bulk neuronal RNA-Seq library mixture file , using 1, permutations.

Random images from at least 2 clones of each cell line were assessed. We used well multi-electrode array plates from Axion Biosystems to acquire electrical activity reads from organoids.

After this timeframe, the seeded organoids were kept in BrainPhys medium until the time of measurement. At least 2 independent experiments were conducted for each subject, with 3 independent replicates wells per subject in each experiment.

Organoids were assessed for electrophysiological parameters starting 7 days after switching to BrainPhys medium. Data reported in Supplementary Fig. Data reported in Fig. Spike detection was computed with an adaptive threshold of 5. The mean firing rate for a subject was calculated across active electrodes in all wells for that subject.

Similar densities of cells were achieved in all plates, and cells were randomly selected in the dishes of control and PTHS groups. Therefore, the electrophysiological and immunostaining data Fig. Filamented borosilicate glass capillaries 1.

For evoked AP recordings, current-clamp configuration was employed with the injection of small currents to maintain the membrane potential at mV. Liquid junction potentials were nulled. At least 2 experiments were conducted per subject, with three technical replicates wells per subject per experiment.

Activity levels were expressed as arbitrary units normalized against the mean activity in the respective controls. The final concentration of DMSO in all experiments was 0. In all cases, treated cells were assayed to confirm modulation of the activity of the Wnt pathway, via transfection with the TOP-Flash plasmids described above. For all experiments, we used 3 biological replicates per subject line, and similar results were obtained in at least 3 independent experiments.

Alternatively, we conducted fluorometric measurement of TCF4 immunostaining intensity Supplementary Fig.

Cells were then immediately fixed with 0. After centrifugation, cells were resuspended in 0. After 3 washes in 0. The effectiveness of this gating strategy was confirmed with negative controls not labeled with anti-TCF4 primary antibody or not labeled with both primary and secondary antibodies Supplementary Fig.

An average fluorescence intensity was then calculated for each replicate and mean TCF4 fluorescence values were computed for all subjects, as presented in the bar plot in Supplementary Fig.

Numerous alternative promoters exist in the TCF4 locus, which give rise to different transcripts 1 , 2 , 3 , 4. We chose to overexpress the TCF4-B transcript variant, because it is the most highly expressed in most tissues 4 and is transcribed from the most active promoter in NPCs, according to our promoter usage analysis Supplementary Fig.

For each promoter, we selected 3 sense and 2 antisense gRNAs based on the score generated by the computational tool designed by Hsu et al. Competent cells Stbl3 E.

The products were run on a 1. Additionally, the amplicons were deep sequenced and the percentages of clones with indels were computed.

For each gRNA, transfection was performed in triplicates. See Supplementary Data 6 for a complete list of oligonucleotides used. A melt-curve step was always included at the end of each run. G Addgene were used. Transduction of organoids CtOs was achieved by mixing 2. When using the regular organoid derivation protocol, the addition of two lentiviral vectors on the first day led to slightly impaired cellular aggregation.

This forced us to alternatively use micro-wells for these trans-epigenetic TCF4 correction experiments. This was achieved by placing the mixture of dissociated iPSCs and viruses onto a well of an Aggrewell micro-well plate Stem Cell Technologies; on the first day of the protocol. During this period, iPSCs collected at the bottom of the Aggrewell and formed very homogeneous embryoid bodies inside the micro-wells. On the following day, embryoid bodies were carefully dislodged with the aid of a tissue culture pipettor and transferred to 6-well plates.

From this point onwards, organoids were cultured under agitation on a shaker, following the same regimen applied to the regular derivation protocol.

On the second and third days of organoid derivation, medium was replaced and lentiviruses were added again. During these 3 days, embryoid bodies were formed in the presence of mTeSR1 Plus medium containing SB and dorsomorphin, as described above. From the fourth day onward, medium was replaced as per the regular protocol, without the addition of viruses.

Transduction was confirmed by the evaluation of Cas9 expression via immunostaining, using the protocol described above. We did not use statistical methods such as power analysis to determine sample size, because we were restricted by the PTHS samples available, which were chosen based on availability of detailed information about the types of TCF4 mutation carried by each patient.

However, based on the strong and consistent effect sizes observed throughout the study Supplementary Data 1 and on the level of variability across cell lines from all subjects in NPCs and organoids , further power analysis determined that increasing sample size is not expected to change statistical significance of our results.

Different types of statistical test were used throughout the study, as indicated in the corresponding figure legends. For comparing the expression of SOX4 along the differentiation trajectory pseudotime Supplementary Fig.

Sample sizes are indicated in the figure legends and in Supplementary Data 1. Supplementary Data 1 presents extended results for all statistical tests performed, including sample sizes, statistical tests employed, effect sizes, statistics metrics H, F, t , or W , along with exact p -values, listed according to the order of appearance in figure panels throughout the study.

When experimentation involved more than one independent replicate per subject cell line, or more than one technical replicate per independent replicate, the numbers of replicates are also indicated in the figure legends and Supplementary Data 1 , even though each statistical test was run based solely on the comparison between the means of different subjects. All attempts at replication were successful. For each batch, organoids were randomly selected from each well for data collection.

Blinding was used for most analyses comparing patients and control samples, including immunostaining, measurement of organoid size, cell counting, patch-clamp electrophysiological measurements, and multi-electrode array assays. Blinding was not used when analyzing results from RNA sequencing and single cell RNA sequencing experiments, due to the inherently unbiased nature of the bioinformatic approaches used for quantitating gene-expression and determining differential expression between genotypes or cell types.

Data supporting the findings in this study are included within the Supplementary Material. The source data relevant to Figs. The following public databases have been used in this study and can be accessed via the corresponding weblinks in parentheses: GRChA 10x support. Microscopy images obtained during this study were not deposited in public repositories as they contain human patient sensitive information, but requests for these data will be fulfilled by the corresponding authors upon reasonable request following appropriate procedures of the Ethics Committees of the institutions where the patient biological samples and cells were collected or are maintained.

Source data are provided with this paper. No new custom software or code has been used in this paper. Codes R programming language used for bioinformatic analyses are all strictly based on pre-existing, regularly used published codes for RNA-Seq and single cell RNA-Seq analyses, as described above. Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding authors, Fabio Papes fpapes ucsd.

When provided to others, unique reagents generated in this study will be available with a completed Materials Transfer Agreement. Kim, H. Region and cell type distribution of TCF4 in the postnatal mouse brain. Jung, M. Analysis of the expression pattern of the schizophrenia-risk and intellectual disability gene TCF4 in the developing and adult brain suggests a role in development and plasticity of cortical and hippocampal neurons.

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Camargo, A. Download references. The authors thank Drs. Nilda Villanueva for help with statistical analyses, Dr. Felipe R. We also thank Drs. Olvera, C. We are grateful to Dr. We are grateful to Drs. Jason Rihel Univ. These authors contributed equally: Antonio P. Camargo, Janaina S. Carvalho, Ryan A. Szeto, Erin LaMontagne. Fabio Papes, Antonio P.

Camargo, Vinicius M. Teixeira, Thiago S. However, little is known about how alterations in TCF4 lead to impaired neural tissue development and function. The unifactorial genetic nature of PTHS offers a unique opportunity to dissect the underlying pathological molecular mechanisms and characterize the cellular abnormalities resulting from TCF4 loss-of-function. Patients with PTHS carry private TCF4 mutations 16 , 17 , 18 , 20 , 21 , which may be deletions, translocations, frameshift, nonsense, or missense changes Clinically, these individuals display profound cognitive impairment, motor delay, hypotonia, breathing abnormalities, typical autistic behaviors, constipation, and a distinctive facial gestalt 20 , Some mouse lines with mutations in Tcf4 display PTHS-like symptoms—including deficits in social interaction, associative memory, and sensorimotor gating 24 , 25 , as well as abnormal cortical development 26 , 27 , neuronal migration 28 , 29 , 30 , and oligodendrocyte differentiation 31 , However, mouse models carrying Tcf4 mutations in the clinically relevant heterozygous state exhibit mild phenotypes only, without the severe symptoms observed in patients.

In this study, we generate neural progenitor cells NPCs and neurons from induced pluripotent stem cells iPSCs from patients with PTHS to analyze the diseased cellular phenotypes under relevant genomic context. Importantly, we also derive patterned brain organoids, which have been successfully used to model cellular pathology during early neurodevelopment in several disorders 33 , 34 , 35 , Taken together, our data reveal novel cellular and molecular phenotypes in human cells with clinically relevant TCF4 mutations and show that these aberrations are reversible, providing routes for therapeutic intervention in individuals carrying genetic diseases associated with this gene.

To gain insight into the pathophysiology caused by mutations in TCF4 , we generated iPSC lines via cellular reprogramming of skin fibroblasts from five patients with PTHS and corresponding parents of matching sex Supplementary Table 1. The patients harbor mutations that either eliminate the TCF4 gene, eliminate its essential DNA-binding domain, or impact one of its transcriptional activation domains Supplementary Fig. On average, PTHS lines display a half-way reduction in TCF4 levels, in keeping with the presence of heterozygous whole-gene deletions or nonsense mutations in most lines Supplementary Fig.

Together, these data confirm that TCF4 function is impaired in our patient-derived cell lines. Arrowhead in top row shows neural rosette. Arrowhead in bottom row indicates polarized phenotype. Right: Mean CtO size at 4 weeks. Symbols in bar graphs indicate parent-patient identities: diamonds, pair 1; squares, pair 2; triangles, pair 3; circles, pair 4; crosses, pair 5.

Colors in bar graphs and violin plots represent the parents orange or PTHS blue groups. The mean expression in the parental control group was normalized to 1 in a — d and f. Blue staining is DAPI nuclear staining. See Supplementary Data 1 for statistical test results, including sample sizes, numbers of replicates, exact p -values, and effect sizes. At 4 weeks in vitro, control CtOs display the expected spheroid-shaped organoid morphology and develop clearly visible rosette-like cellular aggregates Fig.

These phenotypes are consistent across batches performed with different clones derived from the same patient Supplementary Fig. Together, these results show that PTHS brain organoids have aberrant morphology and structure, suggesting that the development of PTHS neural tissue is abnormal. Smaller organoids may result from a range of altered cellular processes, such as decreased cell division or increased apoptosis, abnormal migration, or senescence.

To identify which of these processes is defective in PTHS organoids, we analyzed the organization and contents of several key cellular subtypes. At 4 weeks in vitro, control CtOs contain a large number of rosettes composed of neural progenitors surrounding a ventricle-like lumen Fig.

See Supplementary Fig. 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 the parents orange or PTHS blue groups. See Supplementary Data 1 for sample and effect sizes and exact p -values.

Immunostaining for MAP2 revealed that, in control organoids, neurons are distributed throughout the spheroid, particularly around and between rosettes, and neuronal content increases as development proceeds Fig. Importantly, we detected similarly decreased expression of MAP2 and cortical neuron marker genes Supplementary Fig.

We chose to analyze organoids derived from parent-patient pair 4, which display large differences in size and internal structure Figs. Cross symbol indicates that mean expression log2 fold change is lower than 0.

Colors in bar graphs except in d and g and violin plots represent the parent orange or PTHS blue groups. PTHS and control organoids do not contain cells expressing mesoderm or endoderm markers Supplementary Figs. As a control, we confirmed the reproducibility of our organoid experiments by determining that the cellular compositions of replicate scRNA-Seq libraries from independently derived parent CtOs are highly concordant Supplementary Fig. We did not observe segregation of cells according to the sample of origin batch effect for these replicate libraries, and comparison between parent and PTHS organoids did not reveal segregation of the patient cells to a grossly distinct transcriptomic landscape Supplementary Fig.

Analysis of the percentages of cells assigned to each subpopulation corroborated the existence of differences in cellular composition between parent and PTHS organoids Supplementary Fig. Moreover, scRNA-Seq analyses showed that Likewise, One possibility is that the reduced cortical neuron content in PTHS organoids is due to mis-patterning.

To test this hypothesis, we conducted a comprehensive investigation of the expression of several neural lineage markers in CtOs and GbOs. Marker expression analysis in GbOs revealed that they contain a mixed population of telencephalic and non-telencephalic cells Supplementary Fig. In combination, these results show no evidence of mis-patterning in PTHS organoids. Although scRNA-Seq data from additional parent-patient pairs are needed to expand these observations, our single cell transcriptomic results concur with the histological and molecular abnormalities found in PTHS organoids from all patient lines Fig.

Several potential explanations exist for the diminished activity in PTHS organoids, including changes in neuronal diversity. To further address this issue, we measured the impact of TCF4 loss-of-function on neuronal diversity using 2D neuronal cultures. First, we confirmed that TCF4 is expressed in control neurons in this type of culture Supplementary Fig. Our iPSC-derived neuronal cultures are a mixture of different neuronal subtypes, including excitatory and inhibitory neurons, as previously reported Although we could not define the identity of neurons in these 2D cultures based on their electrophysiological properties in patch-clamp experiments see below , deconvolution of RNA sequencing data revealed that PTHS samples possess fewer glutamatergic and GABAergic neurons than parental controls Supplementary Fig.

To assess if PTHS neurons are morphologically aberrant, we performed analysis of neuronal arborization architecture in neurons in 2D culture Fig. Means are indicated by the colored lines. Representative traces are shown on the left. Cells are from pair 4. Colors in the figure represent parent orange or PTHS blue groups. Finally, a third hypothesis is that the reduced firing rate in PTHS organoids is caused by aberrant cellular-level electrophysiology.

To assess this possibility, we employed patch-clamp analysis of neurons in 2D culture derived from the most significantly impaired patient line in the MEA recordings Supplementary Fig. The three hypotheses presented here are not mutually exclusive and further studies are needed to determine how the lowered neuronal content, aberrant morphological characteristics, or cellular-level electrophysiological alterations contribute to the diminished electrical activity in PTHS.

Importantly, several genes coding for ion channels are significantly downregulated in PTHS neurons in 2D culture and in organoids Supplementary Fig. Together, these data indicate that PTHS neurons are aberrant in terms of morphology, physiology, and transcriptomic landscape, offering mechanistic insight into the PTHS neuronal intrinsic excitability defects and new opportunities for pharmacological therapeutic intervention.

To assess these possibilities, we first counted the numbers of rosettes at different organoid developmental stages Fig. These results, together with the absence of cells expressing non-neural markers Supplementary Fig. Arrowheads mark neural rosettes. 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.

The combination of aberrant morphology and diminished proliferative activity led us to hypothesize that PTHS neural progenitors are undergoing precocious replicative senescence, a process characterized by cell cycle arrest and subsequent halting of proliferation 42 , Strikingly, the expression of senescence markers is also up-regulated in the PTHS post-mortem cortex sample Fig.

Gene set enrichment analysis on the up-regulated genes in PTHS NPCs indicated enrichment for genes involved in cellular senescence or tissue architecture Supplementary Fig. Because the Wnt pathway has been linked to progenitor proliferation in many tissues 46 , we raised the hypothesis that abnormal Wnt activity may be causally implicated with the lower NPC proliferation rates observed in PTHS cells.

Importantly, expression of several Wnt pathway genes is markedly downregulated in the post-mortem PTHS cortex sample Fig. Left graph represents data for pair 4, and right graph shows data for pair 1. Data shown are for pair 4 see graph in Supplementary Fig. See pairwise comparisons in Supplementary Data 1. Colors in bar graphs represent parents orange , pharmacologically treated parents yellow , PTHS blue , pharmacologically treated PTHS light blue , or control post-mortem sample black groups.

Treatment of control CtOs with ICG, a diffusible small molecule that can easily penetrate the organoid, led to a polarized structure and marked reduction in organoid size Fig.

These data allow us to conclude that the rescue of proliferation defect in PTHS organoids was due to corrected Wnt signaling activation downstream of TCF4. Next, we sought to define mechanistic players downstream of TCF4 and the Wnt pathway that could control NPC proliferation and differentiation. In fact, these genes were found to be predominantly expressed in progenitors and intermediate progenitors of CtOs and GbOs Supplementary Figs. However, SOX1 was discarded as a candidate because it is not substantially expressed in organoids Supplementary Fig.

SOX gene subfamilies are shown above. Top: TPM expression. Colors in bar graphs, dot or violin plots represent parents orange , genetically manipulated parents yellow , PTHS blue , pharmacologically treated PTHS light blue , or control post-mortem sample black groups. DAPI nuclear staining in blue.

In accordance, the distribution of cells along the differentiation trajectory in PTHS organoids is skewed toward early time points, as compared with control organoids Supplementary Fig. We focused on SOX4 because it was shown to be involved in intermediate progenitor-to-neuron differentiation Based on these observations, we put forth a model according to which TCF4 loss-of-function results in Wnt downregulation and, consequently, in reduced SOX3 expression, leading to diminished proliferation and increased cellular senescence.

It is unknown if the PTHS pathophysiology can be corrected in human tissues. Our Wnt manipulation experiments indicate that some cellular phenotypes are amenable to correction, but the Wnt pathway acts downstream of TCF4 and may not correct all aberrant molecular and cellular characteristics of PTHS.

Therefore, we decided to perform genetic manipulation of TCF4 itself. Organoids are from parent—patient pair 4. Arrowheads in middle panels: polarized PTHS organoids. Arrowhead in right panel: neural rosettes. Experiments were conducted with organoids from parent-patient pair 4 circle symbols in bar graphs. Error bars represent SEM. We created a collection of expression cassettes containing 15 different gRNAs targeting the three most active alternative promoters of the TCF4 gene Supplementary Fig.

We chose this patient line because it shows the largest differences in organoid size and cellular content compared to the respective control Figs. Importantly, the PTHS phenotypic abnormalities were rescued in the genetically corrected organoids Fig. We also investigated the presence of immature neurons in PTHS organoids, using DCX doublecortin as a marker, which might indicate alterations in the formation of cortical neurons.

Importantly, PTHS organoids subjected to TCF4 OE displayed a significant improvement in two key electrophysiological parameters indicative of functional rescue—mean firing rate and number of network electrical bursts Fig. Arrowhead: neural rosette. Each row of spikes represents an electrode. Vertical red rectangles represent events of network bursts of electrical activity. Right: Quantification of mean firing rate top in transduced organoids see Supplementary Fig.

Arrowhead, neural rosette. Symbols in bar graphs indicate parent-patient identities: diamonds, pair 1; circles, pair 4. Importantly, they prove that the cellular pathology associated with TCF4 loss-of-function—including impaired progenitor proliferation, abnormal neuronal differentiation, and dysregulated cellular senescence and expression of SOX genes—can be genetically corrected during neurodevelopment. Importantly, we highlight a possible route for Wnt pharmacological intervention to correct this aberrant phenotype.

Interestingly, mutations in SOX3 have been associated with another neurodevelopmental disorder, X-linked mental retardation 52 , suggesting the existence of an overlapping molecular mechanism between such a condition and PTHS.

The lower neuron content in the post-mortem tissue is consistent with abnormalities observed in some children with PTHS via MRI, including small or absent corpus callosum Supplementary Table 1 As more samples are analyzed in vitro, statistical power will allow the identification of correlations between levels of neuronal loss and severity of clinical symptoms across patients. This suggests that mouse models are insufficient for studying the consequences of clinically relevant TCF4 mutations, which happen in heterozygosity.

Human brain organoid models provide thus a window of opportunity to observe neural abnormalities relevant to PTHS, although it should be emphasized that they are good models for neurodevelopment, and therefore other systems are necessary to illuminate PTHS pathophysiology in the fully formed neural tissue. Our manipulative experiments to genetically correct TCF4 expression in the PTHS neural tissue provide a gateway for the development of targeted therapeutics against PTHS, as well as clinically similar diseases caused by mutations in downstream TCF4-target genes 38 or even schizophrenia, which may have TCF4 as a genetic component 11 , Interestingly, because the CRISPR-mediated correction of TCF4 expression simultaneously rescued phenotypes and enhanced transcription from both the mutated and normal endogenous alleles, this experiment also unanticipatedly suggests that PTHS is caused by TCF4 haploinsufficiency and not by a dominant negative effect 16 , 17 , 18 , 22 , an important point that should be explored in future studies.

For patients harboring a point mutation, small indel, or translocation, we confirmed the details of each TCF4 mutation via directing resequencing of the TCF4 locus. These data are reported in Supplementary Table 1. To maximize comparability, we selected only male patients with PTHS for the histological and manipulative experiments in this study, and they are 4 to 14 years old patients 1 to 5 in Supplementary Table 1.

The post-mortem PTHS brain cortex sample is from a female individual who died during a surgical procedure to correct scoliosis, due to complications unrelated to the PTHS neurological symptoms patient 6; Supplementary Table 1. Written informed consent was obtained from all participating families after receiving a thorough description of the study and no compensation was provided to participants.

A total of 20 iPSC clonal lines were produced for each subject in the study, all of which were analyzed through a combination of immunostaining and SNP mapping to rule out the presence of unwanted chromosomal abnormalities and mutations example in Supplementary Fig. Most results reported in this paper are from experiments conducted with one or two P15 iPSC clones per subject, and confirmation of consistency in the observed phenotypes was obtained from 2 independent iPSC clones per subject Supplementary Fig.

Cultures were tested every two weeks for mycoplasma, and contamination was never identified at any stage. For identifying unwanted chromosomal structural alterations, genome-wide profiling for amplifications, deletions, copy number variation, and rearrangements was performed via SNP mapping-based karyotypic analysis on genomic DNA extracted from the iPSC lines, using the iScan system Illumina and the Infinium HumanCytoSNP BeadChip Illumina; , genetic markers.

Clones containing visibly large deletions and duplications were not found. An example of karyotyping conducted using this technique is presented in Supplementary Fig.

For the generation of pallial cortical brain organoids CtOs , we used our previously published protocol For every subject, most experiments were conducted with at least 3 independent batches, which were considered independent biological replicates in experiments throughout the study and in Supplementary Data 1 , with at least 3 technical replicates wells of organoids per batch.

This was followed by neuronal differentiation and organoid maturation phases, which were conducted using the same types of medium and durations used in the CtO derivation protocol. The mean number of labeled cells per sample was calculated by first averaging the number of labeled cells in each ROI to produce a mean value of labeled cells per section, and then averaging these mean values across all sections for each subject. The number of subjects and sections quantified are indicated in the figure legends and in Supplementary Data 1.

These analyses of c-Fos protein expression were performed as an additional line of investigation to support the data showing that PTHS organoids have decreased activity 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.

Hospital pathologists dissected the brain from patient 6 immediately after death and harvested cortical tissue encompassing the entire width of the cortex at the boundary between the pre-motor and prefrontal areas. Hippocampus tissue was also harvested but is not described in this study. PTHS images were compared with those obtained from control sections stained in parallel Figs.

Comparisons were performed with matching images collected from ROIs at equivalent depths measured in millimeters from the cortex surface in Supplementary Fig. No significant difference was observed by the pathologists in terms of general appearance of the brain gyri and width of the cortex tissue prior to dissection. We loaded approximately 20, cells per sample on the Chromium chip.

Finally, we ligated Illumina adapters to prepare the libraries for sequencing, followed by another round of double size selection with the SPRIselect Reagent Kit. The estimated number of cells across all libraries was determined to be within the range 2, to 6, per library, with a mean number of reads per cell ranging from 58, to , Cell type subpopulations were delineated via a combination of automated annotation and refinement after manual inspection. Next, we visually inspected the expression of each marker gene in Supplementary Fig.

The combined approach of first performing unbiased determination of subpopulations followed by manual refinement maximizes the identification of biologically relevant groups of cells.

Supplementary Data 4 contains associations between cell barcodes and assigned subpopulations, to ensure reproducibility of our results. Next, we used the Seurat library version 3. Unsupervised trajectory pseudotime inference Fig. Pseudotime is the transcriptional distance abstract units between a cell and the start of the trajectory, measured along the shortest path.

Because each one of our six subpopulations probably contains many types of sub-lineages, each with its own specific temporal differentiation trajectory, it is possible that some cells within the neuronal N-Glut or N-GABA subpopulations are assigned to early pseudotime points in the overall analysis.

That does not mean that they represent early differentiation stage cells, and analyzing the behavior of the overall group of cells is more important than trying to identify the specific pseudotime position of each cell along the differentiation trajectory. For DE analysis on the cellular subpopulations of the organoid single-cell transcriptomic data, we created a subset of the main Seurat object to include just the libraries being compared for example, parent and PTHS CtOs.

The statistical algorithm used was DESeq2 version 3. The adjusted p -values were calculated by Seurat using Bonferroni correction based on the total number of genes in the dataset. We used Cell Loupe software to quantify the percentages of cells in each subpopulation and library Fig. We decided to use a threshold for identifying and objectively counting cells with detectable expression for each gene because of the existence of cells with very low expression in each group, the inclusion of which in the calculations would not make biological sense.

The percentages of cells in parent CtOs shown throughout the figures were calculated based on data from the parent replicate 2 library. After 3 to 5 days, rosettes emerged, and 7 days later the rosettes were manually picked and replated onto Matrigel-coated dishes. When neuronal processes started to grow one week later, the medium was changed to BrainPhys neuronal medium Stem Cell Technologies and cells remained under these conditions for up to 4 months, with media changes occurring every 3—4 days.

Electrophysiological measurements in Fig. Quantification of neuronal differentiation rates Fig. For each subject, RNA was extracted from 3 independently prepared biological replicates. For every sample, outliers were defined by high between-replicate Euclidean distances after transformation to achieve homoskedasticity, as described below , which led to the exclusion of just one neuron library replicate from patient 3 from the follow-up expression analysis see computational codes and quality control results in the repositories described in the Data Availability and Code Availability sections.

All remaining 56 libraries passed the quality control phase and were retained. The transcriptomic data were also used to validate the identities of the cell lines employed in the study. For patients PTHS 1 and 4, the sequences of TCF4 transcripts encompassing the respective mutated sites were retrieved from GENCODE, followed by the generation of mutated sequences to include the mutations each patient carries insertion and point mutation, respectively; see Supplementary Table 1.

Lastly, a negative binomial generalized linear model was fit to the data, the effect sizes log2FoldChange were shrunken with the apeglm algorithm version 1. Transformation of count data into an approximately homoskedastic matrix for clustering and visualization purposes Supplementary Figs. To obtain lists of DE genes across all subjects, we first derived a list of DE genes between each patient with PTHS and his respective parent Supplementary Data 2 , followed by cross examination of all lists and selection of DE genes common to all child-parent pairs.

The final list was used for gene-set enrichment assessment followed by Gene Ontology GO and pathway analyses, using the web-based WebGestalt tool version 65 , with default parameters.

WebGestalt conducts permutations to obtain an over-representation Z score and enrichment p- value for each GO term. For pathway analysis, we chose the KEGG option, with default parameters. For all analyses, a minimum of 5 genes per category was employed, with BH multiple test correction, and a significance level chosen for a false-discovery rate of 0.

The signature matrix was subsequently used to impute cell fractions to each bulk neuronal RNA-Seq library mixture file , using 1, permutations. Amplification and denaturation curves for all probes were analyzed to verify amplification of just one amplicon. Neurons were morphologically analyzed Fig. We only computed neurons whose shortest dendrite was at least 3 times longer than the diameter of the cell soma. Random images from at least 2 clones of each cell line were assessed.

We used well multi-electrode array plates from Axion Biosystems to acquire electrical activity reads from organoids. After this timeframe, the seeded organoids were kept in BrainPhys medium until the time of measurement.

At least 2 independent experiments were conducted for each subject, with 3 independent replicates wells per subject in each experiment. Organoids were assessed for electrophysiological parameters starting 7 days after switching to BrainPhys medium. Data reported in Supplementary Fig. Data reported in Fig. Spike detection was computed with an adaptive threshold of 5.

The mean firing rate for a subject was calculated across active electrodes in all wells for that subject. Similar densities of cells were achieved in all plates, and cells were randomly selected in the dishes of control and PTHS groups. Therefore, the electrophysiological and immunostaining data Fig. Filamented borosilicate glass capillaries 1. For evoked AP recordings, current-clamp configuration was employed with the injection of small currents to maintain the membrane potential at mV.

Liquid junction potentials were nulled. At least 2 experiments were conducted per subject, with three technical replicates wells per subject per experiment. Cells were incubated for another 2. The effectiveness of the gating strategy was confirmed with negative controls not labeled with EdU, not labeled with propidium iodide, or not labeled with both Supplementary Fig.

All assays were conducted on three independent replicates per NPC line per subject and three technical replicates.

For all experiments, we used 3 biological replicates per subject line, and similar results were obtained in at least 3 independent experiments. Alternatively, we conducted fluorometric measurement of TCF4 immunostaining intensity Supplementary Fig. Cells were then immediately fixed with 0. After centrifugation, cells were resuspended in 0. After 3 washes in 0. The effectiveness of this gating strategy was confirmed with negative controls not labeled with anti-TCF4 primary antibody or not labeled with both primary and secondary antibodies Supplementary Fig.

An average fluorescence intensity was then calculated for each replicate and mean TCF4 fluorescence values were computed for all subjects, as presented in the bar plot in Supplementary Fig. In all cases, treatment was performed on at least 3 independent replicates of each organoid line. Because no selection was applied after transfection, the observed effects of SOX3 or TCF4 knockdown on the expression of other genes should be interpreted as the mean variation across all cells in the transfected population.

For SOX4 knockdown in neurons Fig. Therefore, we adopted antisense oligonucleotides ASOs , two of which were used in combination in all experiments. Differentiating neuronal cultures were treated with ASOs on days 15, 20 and 25 after withdrawal of FGF-2 via direct application to the culture medium for unassisted uptake gymnosis. Numerous alternative promoters exist in the TCF4 locus, which give rise to different transcripts 1 , 2 , 3 , 4. We chose to overexpress the TCF4-B transcript variant, because it is the most highly expressed in most tissues 4 and is transcribed from the most active promoter in NPCs, according to our promoter usage analysis Supplementary Fig.

For organoid transduction experiments Fig. G Addgene Transduction of organoids CtOs was achieved by mixing 3. Next, the proActiv R package version 0. This approach identified promoters upstream of exons 3b, 8a, and 10a as the most active in both parent and PTHS samples Supplementary Fig. For each promoter, we selected 3 sense and 2 antisense gRNAs based on the score generated by the computational tool designed by Hsu et al.

Competent cells Stbl3 E. The products were run on a 1. Additionally, the amplicons were deep sequenced and the percentages of clones with indels were computed.

Transduction of organoids CtOs was achieved by mixing 2. When using the regular organoid derivation protocol, the addition of two lentiviral vectors on the first day led to slightly impaired cellular aggregation. This forced us to alternatively use micro-wells for these trans-epigenetic TCF4 correction experiments. This was achieved by placing the mixture of dissociated iPSCs and viruses onto a well of an Aggrewell micro-well plate Stem Cell Technologies; on the first day of the protocol.

From this point onwards, organoids were cultured under agitation on a shaker, following the same regimen applied to the regular derivation protocol. On the second and third days of organoid derivation, medium was replaced and lentiviruses were added again.

During these 3 days, embryoid bodies were formed in the presence of mTeSR1 Plus medium containing SB and dorsomorphin, as described above. From the fourth day onward, medium was replaced as per the regular protocol, without the addition of viruses. Transduction was confirmed by the evaluation of Cas9 expression via immunostaining, using the protocol described above. We did not use statistical methods such as power analysis to determine sample size, because we were restricted by the PTHS samples available, which were chosen based on availability of detailed information about the types of TCF4 mutation carried by each patient.

For comparing the expression of SOX4 along the differentiation trajectory pseudotime Supplementary Fig. Sample sizes are indicated in the figure legends and in Supplementary Data 1. Supplementary Data 1 presents extended results for all statistical tests performed, including sample sizes, statistical tests employed, effect sizes, statistics metrics H, F, t , or W , along with exact p -values, listed according to the order of appearance in figure panels throughout the study.

Adobe illustrator cs4 arrowheads free.FAQ: Google Fusion Tables

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. 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. Transcription Factor 4 TCF4 ; OMIM encodes a helix-loop-helix transcription factor highly expressed during brain development 1 , 2 , 3 , 4 and implicated in neural lineage commitment and neuronal function 5 , 6 , 7 , 8 , 9 , TCF4 gene variants have been associated with neuropsychiatric diseases such as schizophrenia, bipolar disorder, post-traumatic stress disorder, and major depressive disorder 11 , 12 , 13 , 14 , , US veterans.

However, little is known about how alterations in TCF4 lead to impaired neural tissue development and function. The unifactorial genetic nature of PTHS offers a unique opportunity to dissect the underlying pathological molecular mechanisms and characterize the cellular abnormalities resulting from TCF4 loss-of-function.

Patients with PTHS carry private TCF4 mutations 16 , 17 , 18 , 20 , 21 , which may be deletions, translocations, frameshift, nonsense, or missense changes Clinically, these individuals display profound cognitive impairment, motor delay, hypotonia, breathing abnormalities, typical autistic behaviors, constipation, and a distinctive facial gestalt 20 , Some mouse lines with mutations in Tcf4 display PTHS-like symptoms—including deficits in social interaction, associative memory, and sensorimotor gating 24 , 25 , as well as abnormal cortical development 26 , 27 , neuronal migration 28 , 29 , 30 , and oligodendrocyte differentiation 31 , However, mouse models carrying Tcf4 mutations in the clinically relevant heterozygous state exhibit mild phenotypes only, without the severe symptoms observed in patients.

Importantly, we also derive patterned brain organoids, which have been successfully used to model cellular pathology during early neurodevelopment in several disorders 33 , 34 , 35 , Taken together, our data reveal novel cellular and molecular phenotypes in human cells with clinically relevant TCF4 mutations and show that these aberrations are reversible, providing routes for therapeutic intervention in individuals carrying genetic diseases associated with this gene.

On average, PTHS lines display a half-way reduction in TCF4 levels, in keeping with the presence of heterozygous whole-gene deletions or nonsense mutations in most lines Supplementary Fig. Together, these data confirm that TCF4 function is impaired in our patient-derived cell lines. Arrowhead in top row shows neural rosette. Arrowhead in bottom row indicates polarized phenotype. Right: Mean CtO size at 4 weeks. Symbols in bar graphs indicate parent-patient identities: diamonds, pair 1; squares, pair 2; triangles, pair 3; circles, pair 4; crosses, pair 5.

At 4 weeks in vitro, control CtOs display the expected spheroid-shaped organoid morphology and develop clearly visible rosette-like cellular aggregates Fig. These phenotypes are consistent across batches performed with different clones derived from the same patient Supplementary Fig. Together, these results show that PTHS brain organoids have aberrant morphology and structure, suggesting that the development of PTHS neural tissue is abnormal. Smaller organoids may result from a range of altered cellular processes, such as decreased cell division or increased apoptosis, abnormal migration, or senescence.

As these progenitor-rich structures differentiate into several neuronal subtypes, the rosettes diminish in size Fig. In contrast, PTHS organoids display very few rosette-like structures at 4 weeks in vitro and neural progenitors are dispersed and non-clustered Fig. Arrowheads indicate rosettes. See Supplementary Fig. 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 the parents orange or PTHS blue groups. See Supplementary Data 1 for sample and effect sizes and exact p -values. Immunostaining for MAP2 revealed that, in control organoids, neurons are distributed throughout the spheroid, particularly around and between rosettes, and neuronal content increases as development proceeds Fig.

Importantly, we detected similarly decreased expression of MAP2 and cortical neuron marker genes Supplementary Fig. Together, these data strongly suggest that PTHS is characterized by severe deficits in cortical neuron content and indicate that patient-derived organoids closely match the neural phenotypes observed in vivo. We chose to analyze organoids derived from parent-patient pair 4, which display large differences in size and internal structure Figs.

Cross symbol indicates that mean expression log2 fold change is lower than 0. Colors in bar graphs except in d and g and violin plots represent the parent orange or PTHS blue groups. PTHS and control organoids do not contain cells expressing mesoderm or endoderm markers Supplementary Figs.

As a control, we confirmed the reproducibility of our organoid experiments by determining that the cellular compositions of replicate scRNA-Seq libraries from independently derived parent CtOs are highly concordant Supplementary Fig. We did not observe segregation of cells according to the sample of origin batch effect for these replicate libraries, and comparison between parent and PTHS organoids did not reveal segregation of the patient cells to a grossly distinct transcriptomic landscape Supplementary Fig.

Analysis of the percentages of cells assigned to each subpopulation corroborated the existence of differences in cellular composition between parent and PTHS organoids Supplementary Fig.

Moreover, scRNA-Seq analyses showed that Likewise, One possibility is that the reduced cortical neuron content in PTHS organoids is due to mis-patterning. To test this hypothesis, we conducted a comprehensive investigation of the expression of several neural lineage markers in CtOs and GbOs. Marker expression analysis in GbOs revealed that they contain a mixed population of telencephalic and non-telencephalic cells Supplementary Fig.

In combination, these results show no evidence of mis-patterning in PTHS organoids. Although scRNA-Seq data from additional parent-patient pairs are needed to expand these observations, our single cell transcriptomic results concur with the histological and molecular abnormalities found in PTHS organoids from all patient lines Fig.

First, we confirmed that TCF4 is expressed in control neurons in this type of culture Supplementary Fig. Our iPSC-derived neuronal cultures are a mixture of different neuronal subtypes, including excitatory and inhibitory neurons, as previously reported Although we could not define the identity of neurons in these 2D cultures based on their electrophysiological properties in patch-clamp experiments see below , deconvolution of RNA sequencing data revealed that PTHS samples possess fewer glutamatergic and GABAergic neurons than parental controls Supplementary Fig.

To assess if PTHS neurons are morphologically aberrant, we performed analysis of neuronal arborization architecture in neurons in 2D culture Fig.

Means are indicated by the colored lines. Representative traces are shown on the left. Cells are from pair 4. Colors in the figure represent parent orange or PTHS blue groups. Finally, a third hypothesis is that the reduced firing rate in PTHS organoids is caused by aberrant cellular-level electrophysiology. To assess this possibility, we employed patch-clamp analysis of neurons in 2D culture derived from the most significantly impaired patient line in the MEA recordings Supplementary Fig.

The three hypotheses presented here are not mutually exclusive and further studies are needed to determine how the lowered neuronal content, aberrant morphological characteristics, or cellular-level electrophysiological alterations contribute to the diminished electrical activity in PTHS. Differential expression DE analysis revealed a range of mis-regulated genes, and those with highest fold-changes include some involved in neurogenesis, neuronal identity, differentiation, and regulation of neuronal excitability Supplementary Fig.

To assess these possibilities, we first counted the numbers of rosettes at different organoid developmental stages Fig.

These results, together with the absence of cells expressing non-neural markers Supplementary Fig. Arrowheads mark neural rosettes. 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 and line graphs represent parents orange , PTHS blue , or control post-mortem sample black groups.

DAPI nuclear staining in blue except in m. To parse out between the remaining two possibilities—poor progenitor proliferation and impaired differentiation—we analyzed NPCs in 2D culture Fig. The combination of aberrant morphology and diminished proliferative activity led us to hypothesize that PTHS neural progenitors are undergoing precocious replicative senescence, a process characterized by cell cycle arrest and subsequent halting of proliferation 42 , Strikingly, the expression of senescence markers is also up-regulated in the PTHS post-mortem cortex sample Fig.

Importantly, expression of several Wnt pathway genes is markedly downregulated in the post-mortem PTHS cortex sample Fig. Left graph represents data for pair 4, and right graph shows data for pair 1.

Data shown are for pair 4 see graph in Supplementary Fig. See pairwise comparisons in Supplementary Data 1.

Colors in bar graphs represent parents orange , pharmacologically treated parents yellow , PTHS blue , pharmacologically treated PTHS light blue , or control post-mortem sample black groups. Treatment of control CtOs with ICG, a diffusible small molecule that can easily penetrate the organoid, led to a polarized structure and marked reduction in organoid size Fig.

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We decided to use a threshold for identifying and objectively counting cells with detectable expression for each gene because of the existence of cells with very low expression in each group, the inclusion of which in the calculations would not make biological sense. The percentages of cells in parent CtOs shown throughout the figures were calculated based on data from the parent replicate 2 library.

After 3 to 5 days, rosettes emerged, and 7 days later the rosettes were manually picked and replated onto Matrigel-coated dishes. When neuronal processes started to grow one week later, the medium was changed to BrainPhys neuronal medium Stem Cell Technologies and cells remained under these conditions for up to 4 months, with media changes occurring every 3—4 days.

Electrophysiological measurements in Fig. Quantification of neuronal differentiation rates Fig. For each subject, RNA was extracted from 3 independently prepared biological replicates.

For every sample, outliers were defined by high between-replicate Euclidean distances after transformation to achieve homoskedasticity, as described below , which led to the exclusion of just one neuron library replicate from patient 3 from the follow-up expression analysis see computational codes and quality control results in the repositories described in the Data Availability and Code Availability sections.

The final list was used for gene-set enrichment assessment followed by Gene Ontology GO and pathway analyses, using the web-based WebGestalt tool version 65 , with default parameters.

Random images from at least 2 clones of each cell line were assessed. We used well multi-electrode array plates from Axion Biosystems to acquire electrical activity reads from organoids. After this timeframe, the seeded organoids were kept in BrainPhys medium until the time of measurement. At least 2 independent experiments were conducted for each subject, with 3 independent replicates wells per subject in each experiment.

Therefore, the electrophysiological and immunostaining data Fig. Filamented borosilicate glass capillaries 1. For evoked AP recordings, current-clamp configuration was employed with the injection of small currents to maintain the membrane potential at mV. Liquid junction potentials were nulled. At least 2 experiments were conducted per subject, with three technical replicates wells per subject per experiment.

Cells were incubated for another 2. The effectiveness of the gating strategy was confirmed with negative controls not labeled with EdU, not labeled with propidium iodide, or not labeled with both Supplementary Fig. All assays were conducted on three independent replicates per NPC line per subject and three technical replicates. Activity levels were expressed as arbitrary units normalized against the mean activity in the respective controls. The final concentration of DMSO in all experiments was 0.

In all cases, treated cells were assayed to confirm modulation of the activity of the Wnt pathway, via transfection with the TOP-Flash plasmids described above. For all experiments, we used 3 biological replicates per subject line, and similar results were obtained in at least 3 independent experiments.

Alternatively, we conducted fluorometric measurement of TCF4 immunostaining intensity Supplementary Fig. Cells were then immediately fixed with 0. After centrifugation, cells were resuspended in 0. After 3 washes in 0. The effectiveness of this gating strategy was confirmed with negative controls not labeled with anti-TCF4 primary antibody or not labeled with both primary and secondary antibodies Supplementary Fig. An average fluorescence intensity was then calculated for each replicate and mean TCF4 fluorescence values were computed for all subjects, as presented in the bar plot in Supplementary Fig.

We chose to overexpress the TCF4-B transcript variant, because it is the most highly expressed in most tissues 4 and is transcribed from the most active promoter in NPCs, according to our promoter usage analysis Supplementary Fig. For organoid transduction experiments Fig.

G Addgene Transduction of organoids CtOs was achieved by mixing 3. Next, the proActiv R package version 0. This approach identified promoters upstream of exons 3b, 8a, and 10a as the most active in both parent and PTHS samples Supplementary Fig. For each promoter, we selected 3 sense and 2 antisense gRNAs based on the score generated by the computational tool designed by Hsu et al.

Competent cells Stbl3 E. The products were run on a 1. Additionally, the amplicons were deep sequenced and the percentages of clones with indels were computed. For each gRNA, transfection was performed in triplicates. See Supplementary Data 6 for a complete list of oligonucleotides used. A melt-curve step was always included at the end of each run.

G Addgene were used. Transduction of organoids CtOs was achieved by mixing 2. When using the regular organoid derivation protocol, the addition of two lentiviral vectors on the first day led to slightly impaired cellular aggregation. This forced us to alternatively use micro-wells for these trans-epigenetic TCF4 correction experiments. This was achieved by placing the mixture of dissociated iPSCs and viruses onto a well of an Aggrewell micro-well plate Stem Cell Technologies; on the first day of the protocol.

During this period, iPSCs collected at the bottom of the Aggrewell and formed very homogeneous embryoid bodies inside the micro-wells. On the following day, embryoid bodies were carefully dislodged with the aid of a tissue culture pipettor and transferred to 6-well plates. From this point onwards, organoids were cultured under agitation on a shaker, following the same regimen applied to the regular derivation protocol.

Transduction was confirmed by the evaluation of Cas9 expression via immunostaining, using the protocol described above.

We did not use statistical methods such as power analysis to determine sample size, because we were restricted by the PTHS samples available, which were chosen based on availability of detailed information about the types of TCF4 mutation carried by each patient.

Supplementary Data 1 presents extended results for all statistical tests performed, including sample sizes, statistical tests employed, effect sizes, statistics metrics H, F, t , or W , along with exact p -values, listed according to the order of appearance in figure panels throughout the study.

All attempts at replication were successful. For each batch, organoids were randomly selected from each well for data collection. Blinding was used for most analyses comparing patients and control samples, including immunostaining, measurement of organoid size, cell counting, patch-clamp electrophysiological measurements, and multi-electrode array assays.

Blinding was not used when analyzing results from RNA sequencing and single cell RNA sequencing experiments, due to the inherently unbiased nature of the bioinformatic approaches used for quantitating gene-expression and determining differential expression between genotypes or cell types. Microscopy images that appear in Figs. Statistical analyses were performed using Prism software GraphPad; version 9. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding authors, Fabio Papes fpapes ucsd.

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Hsu, P. Papes, F. Transcription Factor 4 loss-of-function is associated with deficits in progenitor proliferation and cortical neuron content. Camargo, A. Download references. The authors thank Drs. Nilda Villanueva for help with statistical analyses, Dr. Felipe R. We also thank Drs. Olvera, C. We are grateful to Dr. Importantly, we detected similarly decreased expression of MAP2 and cortical neuron marker genes Supplementary Fig.

Together, these data strongly suggest that PTHS is characterized by severe deficits in cortical neuron content and indicate that patient-derived organoids closely match the neural phenotypes observed in vivo. We chose to analyze organoids derived from parent-patient pair 4, which display large differences in size and internal structure Figs. Cross symbol indicates that mean expression log2 fold change is lower than 0. Colors in bar graphs except in d and g and violin plots represent the parent orange or PTHS blue groups.

PTHS and control organoids do not contain cells expressing mesoderm or endoderm markers Supplementary Figs. As a control, we confirmed the reproducibility of our organoid experiments by determining that the cellular compositions of replicate scRNA-Seq libraries from independently derived parent CtOs are highly concordant Supplementary Fig.

We did not observe segregation of cells according to the sample of origin batch effect for these replicate libraries, and comparison between parent and PTHS organoids did not reveal segregation of the patient cells to a grossly distinct transcriptomic landscape Supplementary Fig. Analysis of the percentages of cells assigned to each subpopulation corroborated the existence of differences in cellular composition between parent and PTHS organoids Supplementary Fig.

Marker expression analysis in GbOs revealed that they contain a mixed population of telencephalic and non-telencephalic cells Supplementary Fig. In combination, these results show no evidence of mis-patterning in PTHS organoids. Although scRNA-Seq data from additional parent-patient pairs are needed to expand these observations, our single cell transcriptomic results concur with the histological and molecular abnormalities found in PTHS organoids from all patient lines Fig.

Differential expression DE analysis revealed a range of mis-regulated genes, and those with highest fold-changes include some involved in neurogenesis, neuronal identity, differentiation, and regulation of neuronal excitability Supplementary Fig. Importantly, several genes coding for ion channels are significantly downregulated in PTHS neurons in 2D culture and in organoids Supplementary Fig.

Together, these data indicate that PTHS neurons are aberrant in terms of morphology, physiology, and transcriptomic landscape, offering mechanistic insight into the PTHS neuronal intrinsic excitability defects and new opportunities for pharmacological therapeutic intervention.

Colors in bar and line graphs represent parents orange , PTHS blue , or control post-mortem sample black groups. DAPI nuclear staining in blue except in m. To parse out between the remaining two possibilities—poor progenitor proliferation and impaired differentiation—we analyzed NPCs in 2D culture Fig. The combination of aberrant morphology and diminished proliferative activity led us to hypothesize that PTHS neural progenitors are undergoing precocious replicative senescence, a process characterized by cell cycle arrest and subsequent halting of proliferation 42 , Strikingly, the expression of senescence markers is also up-regulated in the PTHS post-mortem cortex sample Fig.

Left graph represents data for pair 4, and right graph shows data for pair 1. Data shown are for pair 4 see graph in Supplementary Fig. See pairwise comparisons in Supplementary Data 1. Colors in bar graphs represent parents orange , pharmacologically treated parents yellow , PTHS blue , pharmacologically treated PTHS light blue , or control post-mortem sample black groups. Treatment of control CtOs with ICG, a diffusible small molecule that can easily penetrate the organoid, led to a polarized structure and marked reduction in organoid size Fig.

First, we confirmed that Wnt signaling was increased in the treated cells Supplementary Fig. These results suggest that fate restriction does not cause the phenotypic corrections after Wnt signaling activation in PTHS NPCs, although further experiments are needed to confirm this hypothesis. These data allow us to conclude that the rescue of proliferation defect in PTHS organoids was due to corrected Wnt signaling activation downstream of TCF4.

Next, we sought to define mechanistic players downstream of TCF4 and the Wnt pathway that could control NPC proliferation and differentiation. In fact, these genes were found to be predominantly expressed in progenitors and intermediate progenitors of CtOs and GbOs Supplementary Figs.

However, SOX1 was discarded as a candidate because it is not substantially expressed in organoids Supplementary Fig. SOX gene subfamilies are shown above. Top: TPM expression. Colors in bar graphs, dot or violin plots represent parents orange , genetically manipulated parents yellow , PTHS blue , pharmacologically treated PTHS light blue , or control post-mortem sample black groups.

DAPI nuclear staining in blue. In accordance, the distribution of cells along the differentiation trajectory in PTHS organoids is skewed toward early time points, as compared with control organoids Supplementary Fig.

We focused on SOX4 because it was shown to be involved in intermediate progenitor-to-neuron differentiation Based on these observations, we put forth a model according to which TCF4 loss-of-function results in Wnt downregulation and, consequently, in reduced SOX3 expression, leading to diminished proliferation and increased cellular senescence.

Organoids are from parent—patient pair 4. Arrowheads in middle panels: polarized PTHS organoids. Arrowhead in right panel: neural rosettes. Experiments were conducted with organoids from parent-patient pair 4 circle symbols in bar graphs. Error bars represent SEM. We created a collection of expression cassettes containing 15 different gRNAs targeting the three most active alternative promoters of the TCF4 gene Supplementary Fig.

We also investigated the presence of immature neurons in PTHS organoids, using DCX doublecortin as a marker, which might indicate alterations in the formation of cortical neurons. Importantly, PTHS organoids subjected to TCF4 OE displayed a significant improvement in two key electrophysiological parameters indicative of functional rescue—mean firing rate and number of network electrical bursts Fig. Arrowhead: neural rosette. Each row of spikes represents an electrode.

Vertical red rectangles represent events of network bursts of electrical activity. Right: Quantification of mean firing rate top in transduced organoids see Supplementary Fig. Arrowhead, neural rosette. Symbols in bar graphs indicate parent-patient identities: diamonds, pair 1; circles, pair 4. Importantly, they prove that the cellular pathology associated with TCF4 loss-of-function—including impaired progenitor proliferation, abnormal neuronal differentiation, and dysregulated cellular senescence and expression of SOX genes—can be genetically corrected during neurodevelopment.

Importantly, we highlight a possible route for Wnt pharmacological intervention to correct this aberrant phenotype. Interestingly, mutations in SOX3 have been associated with another neurodevelopmental disorder, X-linked mental retardation 52 , suggesting the existence of an overlapping molecular mechanism between such a condition and PTHS.

Mechanistic model to explain aberrant cellular phenotypes in PTHS neural structures. We also hypothesize that TCF4 loss-of-function mechanistically leads to SOX4 downregulation, resulting in decreased neuronal differentiation Fig. Importantly, we provide a direct report of the histological characteristics in the human PTHS brain Figs. The lower neuron content in the post-mortem tissue is consistent with abnormalities observed in some children with PTHS via MRI, including small or absent corpus callosum Supplementary Table 1 As more samples are analyzed in vitro, statistical power will allow the identification of correlations between levels of neuronal loss and severity of clinical symptoms across patients.

This suggests that mouse models are insufficient for studying the consequences of clinically relevant TCF4 mutations, which happen in heterozygosity. Human brain organoid models provide thus a window of opportunity to observe neural abnormalities relevant to PTHS, although it should be emphasized that they are good models for neurodevelopment, and therefore other systems are necessary to illuminate PTHS pathophysiology in the fully formed neural tissue.

Our manipulative experiments to genetically correct TCF4 expression in the PTHS neural tissue provide a gateway for the development of targeted therapeutics against PTHS, as well as clinically similar diseases caused by mutations in downstream TCF4-target genes 38 or even schizophrenia, which may have TCF4 as a genetic component 11 , Interestingly, because the CRISPR-mediated correction of TCF4 expression simultaneously rescued phenotypes and enhanced transcription from both the mutated and normal endogenous alleles, this experiment also unanticipatedly suggests that PTHS is caused by TCF4 haploinsufficiency and not by a dominant negative effect 16 , 17 , 18 , 22 , an important point that should be explored in future studies.

Subjects are members of volunteering families recruited through the Pitt Hopkins Research Foundation or the University of Campinas. Patients with PTHS Supplementary Table 1 were selected based on availability of detailed clinical and molecular diagnostics information, including the types of TCF4 mutation they carry. For patients harboring a point mutation, small indel, or translocation, we confirmed the details of each TCF4 mutation via directing resequencing of the TCF4 locus.

These data are reported in Supplementary Table 1. To maximize comparability, we selected only male patients with PTHS for the histological and manipulative experiments in this study, and they are 4 to 14 years old patients 1 to 5 in Supplementary Table 1.

Clones containing visibly large deletions and duplications were not found. An example of karyotyping conducted using this technique is presented in Supplementary Fig. For the generation of pallial cortical brain organoids CtOs , we used our previously published protocol For every subject, most experiments were conducted with at least 3 independent batches, which were considered independent biological replicates in experiments throughout the study and in Supplementary Data 1 , with at least 3 technical replicates wells of organoids per batch.

For phenotypic evaluations conducted on 4 or more separate batches, we used two or more independent clones of iPSCs to produce the organoids and NPCs and to confirm the effect of genotype, as depicted in Supplementary Fig. This was followed by neuronal differentiation and organoid maturation phases, which were conducted using the same types of medium and durations used in the CtO derivation protocol.

The mean number of labeled cells per sample was calculated by first averaging the number of labeled cells in each ROI to produce a mean value of labeled cells per section, and then averaging these mean values across all sections for each subject. The number of subjects and sections quantified are indicated in the figure legends and in Supplementary Data 1. These analyses of c-Fos protein expression were performed as an additional line of investigation to support the data showing that PTHS organoids have decreased activity 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.

We hypothesize that this peripheral pattern of staining is a combination of the use of short exposure times and apotome-mediated imaging, which produces pixel-normalized images that capture the staining in a very thin optical section, detecting the highest concentrations of c-Fos protein at the nucleus periphery. For immunofluorescence labeling of NPCs, these cells were seeded at a density of 50, cells per well of a LabTek II 8-well chambered slide. Hospital pathologists dissected the brain from patient 6 immediately after death and harvested cortical tissue encompassing the entire width of the cortex at the boundary between the pre-motor and prefrontal areas.

Hippocampus tissue was also harvested but is not described in this study. PTHS images were compared with those obtained from control sections stained in parallel Figs. Comparisons were performed with matching images collected from ROIs at equivalent depths measured in millimeters from the cortex surface in Supplementary Fig. No significant difference was observed by the pathologists in terms of general appearance of the brain gyri and width of the cortex tissue prior to dissection.

We loaded approximately 20, cells per sample on the Chromium chip. Finally, we ligated Illumina adapters to prepare the libraries for sequencing, followed by another round of double size selection with the SPRIselect Reagent Kit. The estimated number of cells across all libraries was determined to be within the range 2, to 6, per library, with a mean number of reads per cell ranging from 58, to , Cell type subpopulations were delineated via a combination of automated annotation and refinement after manual inspection.

Next, we visually inspected the expression of each marker gene in Supplementary Fig. The combined approach of first performing unbiased determination of subpopulations followed by manual refinement maximizes the identification of biologically relevant groups of cells.

It is evident that these subpopulations could be further subdivided into other groups of cells, but we decided to focus on groups containing progenitors, intermediate progenitors, and neurons in the excitatory and inhibitory lineages shown by the single-cell data Fig. Supplementary Data 4 contains associations between cell barcodes and assigned subpopulations, to ensure reproducibility of our results.

Next, we used the Seurat library version 3. Unsupervised trajectory pseudotime inference Fig. Pseudotime is the transcriptional distance abstract units between a cell and the start of the trajectory, measured along the shortest path. Because each one of our six subpopulations probably contains many types of sub-lineages, each with its own specific temporal differentiation trajectory, it is possible that some cells within the neuronal N-Glut or N-GABA subpopulations are assigned to early pseudotime points in the overall analysis.

The statistical algorithm used was DESeq2 version 3. The adjusted p -values were calculated by Seurat using Bonferroni correction based on the total number of genes in the dataset.

We used Cell Loupe software to quantify the percentages of cells in each subpopulation and library Fig. We decided to use a threshold for identifying and objectively counting cells with detectable expression for each gene because of the existence of cells with very low expression in each group, the inclusion of which in the calculations would not make biological sense.

When neuronal processes started to grow one week later, the medium was changed to BrainPhys neuronal medium Stem Cell Technologies and cells remained under these conditions for up to 4 months, with media changes occurring every 3—4 days. Electrophysiological measurements in Fig. Quantification of neuronal differentiation rates Fig. For each subject, RNA was extracted from 3 independently prepared biological replicates.

For patients PTHS 2 and 3, we could not produce the sequences of the mutated transcripts because they carry a whole-gene deletion and a chromosome translocation, respectively Supplementary Table 1. Next, between-sample normalization was performed using the size factors approach 63 and a local dispersion model was fit to the normalized counts. Lastly, a negative binomial generalized linear model was fit to the data, the effect sizes log2FoldChange were shrunken with the apeglm algorithm version 1.

Transformation of count data into an approximately homoskedastic matrix for clustering and visualization purposes Supplementary Figs. To obtain lists of DE genes across all subjects, we first derived a list of DE genes between each patient with PTHS and his respective parent Supplementary Data 2 , followed by cross examination of all lists and selection of DE genes common to all child-parent pairs. The final list was used for gene-set enrichment assessment followed by Gene Ontology GO and pathway analyses, using the web-based WebGestalt tool version 65 , with default parameters.

Amplification and denaturation curves for all probes were analyzed to verify amplification of just one amplicon. Neurons were morphologically analyzed Fig. We only computed neurons whose shortest dendrite was at least 3 times longer than the diameter of the cell soma. Random images from at least 2 clones of each cell line were assessed. We used well multi-electrode array plates from Axion Biosystems to acquire electrical activity reads from organoids. After this timeframe, the seeded organoids were kept in BrainPhys medium until the time of measurement.

At least 2 independent experiments were conducted for each subject, with 3 independent replicates wells per subject in each experiment.

Organoids were assessed for electrophysiological parameters starting 7 days after switching to BrainPhys medium. Data reported in Supplementary Fig.

Data reported in Fig. Spike detection was computed with an adaptive threshold of 5. The mean firing rate for a subject was calculated across active electrodes in all wells for that subject. Similar densities of cells were achieved in all plates, and cells were randomly selected in the dishes of control and PTHS groups. Therefore, the electrophysiological and immunostaining data Fig. Filamented borosilicate glass capillaries 1. For evoked AP recordings, current-clamp configuration was employed with the injection of small currents to maintain the membrane potential at mV.

Alternatively, we conducted fluorometric measurement of TCF4 immunostaining intensity Supplementary Fig. Cells were then immediately fixed with 0. After centrifugation, cells were resuspended in 0.

After 3 washes in 0. The effectiveness of this gating strategy was confirmed with negative controls not labeled with anti-TCF4 primary antibody or not labeled with both primary and secondary antibodies Supplementary Fig.

An average fluorescence intensity was then calculated for each replicate and mean TCF4 fluorescence values were computed for all subjects, as presented in the bar plot in Supplementary Fig.

In all cases, treatment was performed on at least 3 independent replicates of each organoid line. Because no selection was applied after transfection, the observed effects of SOX3 or TCF4 knockdown on the expression of other genes should be interpreted as the mean variation across all cells in the transfected population. For SOX4 knockdown in neurons Fig. Therefore, we adopted antisense oligonucleotides ASOs , two of which were used in combination in all experiments.

Differentiating neuronal cultures were treated with ASOs on days 15, 20 and 25 after withdrawal of FGF-2 via direct application to the culture medium for unassisted uptake gymnosis. Numerous alternative promoters exist in the TCF4 locus, which give rise to different transcripts 1 , 2 , 3 , 4.

This was achieved by placing the mixture of dissociated iPSCs and viruses onto a well of an Aggrewell micro-well plate Stem Cell Technologies; on the first day of the protocol.

For comparing the expression of SOX4 along the differentiation trajectory pseudotime Supplementary Fig. Sample sizes are indicated in the figure legends and in Supplementary Data 1.

Microscopy images that appear in Figs. Statistical analyses were performed using Prism software GraphPad; version 9. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Data supporting the findings in this study are included within the Supplementary Material. The source data relevant to Figs. The following public databases have been used in this study and can be accessed via the corresponding weblinks in parentheses: GRChA 10x support.

Microscopy images obtained during this study were not deposited in public repositories as they contain human patient sensitive information, but requests for these data will be fulfilled by the corresponding authors upon reasonable request following appropriate procedures of the Ethics Committees of the institutions where the patient biological samples and cells were collected or are maintained.

Kim, H. Region and cell type distribution of TCF4 in the postnatal mouse brain. Jung, M. Analysis of the expression pattern of the schizophrenia-risk and intellectual disability gene TCF4 in the developing and adult brain suggests a role in development and plasticity of cortical and hippocampal neurons.

Autism 9 , 1—15 Sepp, M. Chen, E. Molecular convergence of neurodevelopmental disorders. Schmidt-Edelkraut, U. Zac1 regulates cell cycle arrest in neuronal progenitors via Tcf4.

Hill, M. Knockdown of the schizophrenia susceptibility gene TCF4 alters gene expression and proliferation of progenitor cells from the developing human neocortex.

Psychiatry Neurosci. PubMed Article Google Scholar. Forrest, M. The psychiatric risk gene transcription factor 4 TCF4 regulates neurodevelopmental pathways associated with schizophrenia, autism, and intellectual disability. Page, S. Psychiatry 23 , — Fischer, B.

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