GTF2I dosage regulates neuronal differentiation and social behavior in 7q11.23 neurodevelopmental disorders

Abstract

Copy number variations at 7q11.23 cause neurodevelopmental disorders with shared and opposite manifestations. Deletion causes Williams-Beuren syndrome featuring hypersociability, while duplication causes 7q11.23 microduplication syndrome (7Dup), frequently exhibiting autism spectrum disorder (ASD). Converging evidence indicates GTF2I as key mediator of the cognitive-behavioral phenotypes, yet its role in cortical development and behavioral hallmarks remains largely unknown. We integrated proteomic and transcriptomic profiling of patient-derived cortical organoids, including longitudinally at single-cell resolution, to dissect 7q11.23 dosage-dependent and GTF2I-specific disease mechanisms. We observed dosage-dependent impaired dynamics of neural progenitor proliferation, transcriptional imbalances, and highly specific alterations in neuronal output, leading to precocious excitatory neuron production in 7Dup, which was rescued by restoring physiological GTF2I levels. Transgenic mice with Gtf2i duplication recapitulated progenitor proliferation and neuronal differentiation defects alongside ASD-like behaviors. Consistently, inhibition of lysine demethylase 1 (LSD1), a GTF2I effector, was sufficient to rescue ASD-like phenotypes in transgenic mice, establishing GTF2I-LSD1 axis as a molecular pathway amenable to therapeutic intervention in ASD.

Fig. 1.. 7q11.23 CNVs cause imbalances in cortical cell population composition in patient-derived COs.

(A) Experimental design: 13 reprogrammed iPSC lines from 13 different donors differentiated into COs and profiled for bulk transcriptomics, single-cell RNA sequencing (RNA-seq) and whole proteome at the specified time points. (B) Uniform manifold approximation and projection (UMAP) from (n = 97,108) single-cell transcriptomes. Leiden clustering algorithm identified eight distinct cell clusters (RG, RG/IPC, IPC1, IPC2, EN1, EN2, IN, and oRG/AST), composed by the following populations: RG, IPC, EN, IN, and oRG/AST. (C) Proportion of cells from each genotype in each cell cluster in COs at 50 and 100 days (three pooled individual organoids per genotype). (D) Density plots illustrating differences in (max-min) cell abundance in UMAP discriminated by condition; red indicates high abundance in WBS, blue indicates high abundance in 7Dup, and gray indicates high abundance in control. (E) Representative widefield fluorescence images from days 50 to 100 COs across genotypes (WBS/CTL/7Dup), immunostained with markers for different cortical populations: PAX6 (apical radial glia), microtubule-associated protein 2 (MAP2) (postmitotic neurons), TBR2 (intermediate progenitors), TBR1 (layer VI neurons), Ki67 (cycling cells), BCL11B (layer V toVI neurons). Scale bars, 50 um. (F to J) Quantification of the percentage cells positive for different population markers relative to total nuclei [4′,6-diamidino-2-phenylindole (DAPI)] at day 50 of differentiation. (K and L) Quantification of the percentage of layer V and VI neuron markers relative to total nuclei (DAPI) at day 100 of differentiation. Each data point is an individual section from five organoids per line, three independent human iPSC lines per genotype. All data are shown as means ± SEM (27 sections from three COs per cell line, three cell lines per genotype), one-way analysis of variance (ANOVA), post hoc Tukey test; *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001.

Fig. 2.. Transcriptomic and proteomic profiling of 7q11.23 CNV COs reveal imbalances in proliferative vis-a-vis neurogenic programs.

(A) DEA for 7Dup versus WBS COs. Plots report tested genes as −log₁₀ FDR and log₂FC. Purple indicates significantly modulated genes (FDR < 0.1 and absolute log₂FC > 1), black indicates genes passing FDR threshold, and gray indicates not significant. Top 30 genes are highlighted. (B) Word cloud reporting DEGs gene symbol; word size represents fold change magnitude, green represents up-regulated in 7Dup, and blue represents up-regulated in WBS. (C) STRING-based network reconstructed for modulated genes (FDR < 0.1, log₂FC > 1) in at least one time point by stage-wise DEA. Each visualization highlights the most relevant findings for the reported time point. Node size and color: magnitude and direction of log₂FC at the specified stage, green denotes up-regulation in 7Dup, and blue denotes up-regulation in WBS. Functionally relevant regions are highlighted. (D) Differentially expressed proteins (DEP) comparing 7Dup and WBS COs, (one CO per line and three lines per genotype in duplicate). DEP cutoff: FDR < 0.01 and FC > 1. (E) Top 8 enriched biological processes for up-regulated (P < 0.05, FC > 2) and down-regulated (P < 0.05, FC < −2) proteins from 7Dup versus WBS at day 50. (F) Overlap between transcriptomics and proteomics in 7Dup versus WBS at day 50. Features are selected as modulated with a P < 0.05 and absolute log₂FC > log₂(1.25). (G) Relationship of the fold change in 7Dup versus WBS in the proteome (y axis) and transcriptome (x axis) for genes tested by both approaches. Yellow indicates features modulated in both modalities; they are all modulated in the same direction except LYPLA2. Purple represents DEPs absent among DEGs, and orange represents DEGs absent among DEPs. (H) Manually curated functional annotation for genes modulated in the same direction at the transcript and protein level.

Fig. 3.. Gene dosage imbalances at 7q11.23 accelerate neuronal differentiation in 7Dup COs.

(A) Force-directed graph drawing of only the EN2/IN populations (recalculating neighbors and distances subselecting only EN2/IN populations), divided in subclusters and manually annotated. This dimensionality reduction highlights the most differentiated cell population (the pointed extremity of the graph, manually annotated). (B) Differences in cell distribution in force-directed graph for day 50 organoids. WBS and CTL occupy the same space, while 7Dup has more mature cells. (C) Latent time calculated only on the EN2 and IN populations at day 50. The starting cell is identified considering the total population. (D) Representation of the EN2 and IN populations developmental path in the three genotypes in force-directed graph, overimposed with RNA velocity. (E) Differential abundance of cells in different areas of the force-directed graph. The nodes are neighborhoods of cells aggregated based on RNA expression similarity, while graph edges depict the number of cells connected between adjacent neighborhoods. White nodes are neighborhoods that are not detected as differentially abundant (FDR > 1%), red dots, on the other end, are significant (FDR < 1%).

Fig. 4.. GTF2I duplication drives precocious neuronal differentiation in 7Dup COs.

(A) Force-directed graph stratified for genotype (CTL, 7Dup, and 7DupshGTF2I) from left to right, first line at day 50. The second line shows the differences in cell distribution among genotypes, highlighting areas in the EN2 population that are unique of 7Dup and WBS, overimposed on the right. (B) Eigenvalue of the transition matrix stratified by genotype (CTL, 7Dup, and 7DupshGTF2I). In the plot, each dot represents a potential differentiation trajectory, with y axis quantifying the likelihood of solving the transition matrix (probability to be a proper differentiation trajectory); the blue line identifies the imposed probability threshold (0.95) applied to all the conditions. (C) Force-directed graph over impose with RNA velocity, depicting the developmental path of different genotypes (CTL, 7Dup, and 7DupshGTF2I). (D) Force-directed graph with color code as the probability of a cell to go to a certain endpoint, defined by the graph in (C); ***P < 0.001. Given the clustering used, we have multiple clusters that identify the same populations. The significance is defined as the probability of going from the beginning, identified as the population with minimum of total pseudo-time, to the end points in 7Dup. ns, not significant. (E) Force-directed graph with a visual representation of the differentiation trajectory and populations that are involved in the process, only 7Dup passes in regions showing upper layer markers.

Fig. 5.. Gtf2i dosage drives accelerated neuronal differentiation in the mouse developing cortex.

(A) Left: Representative images of mitosis marker Phh3 expression in Gtf2i^+/−, WT, and Gtf2i^{+/Dup E17.5} embryos. Right: Quantification of Phh3 expression at E17.5 in the three genotypes. Gtf2i^+/−, n = 9 sections from six mice; WT, n = 9 sections from five mice; Gtf2i^+/Dup, n = 9 sections from five mice. (B) Left: Representative images of cell cycle marker Ki-67 expression. Right: Scatter plot with bar quantifying the Ki-67 expression at E17.5. Gtf2i^+/−, n = 7 sections from four mice; WT, n = 13 sections from eight mice; Gtf2i^+/Dup, n = 8 sections from four mice. (C) Left: Representative images of intermediate progenitor marker Tbr2. Right: Scatter plot with bar quantifying Tbr2 expression at E17.5. Gtf2i^+/−, n = 11 sections from six mice; WT, n = 10 sections from five mice; Gtf2i^+/Dup, n = 12 sections from five mice. (D) Left: Representative images of lower layer cortical neuronal marker Bcl11b. Right: Scatter plot with bar quantifying Bcl11b expression at E17.5. Gtf2i^+/−, n = 7 sections from four mice; WT, n = 16 sections from eight mice; Gtf2i^+/Dup, n = 8 sections from four mice. (E) Left: Representative images of upper layer cortical neuronal markers Cux1. Right: Scatter plot with bar quantifying Cux1 expression at E17.5. Gtf2i^+/−, n = 7 sections from four mice; WT, n = 16 sections from eight mice; Gtf2i^+/Dup, n = 8 sections from four mice. All data are shown as means ± SEM. Statistical analyses were performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. Significance level was set to P < 0.05. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 6.. Inhibition of LSD1 rescues ASD-like phenotypes in Gtf2i^+/Dup.

(A and B) Schematic representation of the three-chamber sociability apparatus for measuring social preference and social novelty, respectively, in male mice. (C to F) Bar plots with dots depicting the time spent with a conspecific versus object and with a novel versus familiar mouse at baseline, in WT (n = 22) and Gtf2i^+/Dup (n = 20) mice. (G to J) Bar plots with dots depicting the time spent with a conspecific versus object and with a novel versus familiar mouse in Gtf2i^+/Dup mice following four oral gavage administrations (two times per week over 2 weeks) of vehicle (n = 20) or LSD1 inhibitor (10 mg/kg; n = 22). (K to N) Bar plots with dots depicting social preference and social novelty results in Gtf2i^+/Dup mice tested after the fourth administration of vehicle (n = 14) or LSD1 inhibitor (n = 17) and after a 2-week washout period. Data are shown as means ± SEM. Statistical analyses were performed using paired Student’s t test, followed by Holm-Bonferroni correction for multiple testing, except for (C), (I), and (M), where the data did not have a normal distribution and Wilcoxon signed rank test was used instead. Significance level P < 0.05. *P < 0.05, **P < 0.01, and ***P < 0.001.