Thalamocortical organoids enable in vitro modeling of 22q11.2 microdeletion associated with neuropsychiatric disorders

Abstract

Thalamic dysfunction has been implicated in multiple psychiatric disorders. We sought to study the mechanisms by which abnormalities emerge in the context of the 22q11.2 microdeletion, which confers significant genetic risk for psychiatric disorders. We investigated early stages of human thalamus development using human pluripotent stem cell-derived organoids and show that the 22q11.2 microdeletion underlies widespread transcriptional dysregulation associated with psychiatric disorders in thalamic neurons and glia, including elevated expression of FOXP2. Using an organoid co-culture model, we demonstrate that the 22q11.2 microdeletion mediates an overgrowth of thalamic axons in a FOXP2-dependent manner. Finally, we identify ROBO2 as a candidate molecular mediator of the effects of FOXP2 overexpression on thalamic axon overgrowth. Together, our study suggests that early steps in thalamic development are dysregulated in a model of genetic risk for schizophrenia and contribute to neural phenotypes in 22q11.2 deletion syndrome.

Keywords: 22q11 microdeletion; DiGeorge syndrome; neurodevelopmental disorders; organoid; thalamocortical; velocardiofacial syndrome.

Figure 1.. Benchmarking of human thalamic organoids derived from 22q11DS and control iPS lines.

(A) SNP array imaging results for chr22 of control and 22q11DS patient iPS lines. (B) Immunostaining against pan-neuronal marker HuC/D and thalamic neuron marker GBX2 (top), GABAergic neuron markers GAD65/67 and LHX1 (middle), and pan-thalamic marker TCF7L2 and progenitor marker PAX6 (bottom). Scale bar, low magnification: 500 μm, high magnification: 50 μm. (C) Quantification of immunostainings in (B). Mean ± SEM is shown. (D) Schematic of scRNAseq analysis strategy. (E, F) Uniform Manifold Approximation and Projection (UMAP) plots depicting cell types in the primary reference (E) and reference-mapped thalamic organoid (F) datasets. (G) Gene expression plot for major cell type markers in thalamic organoids. See also Figure S1, Table S1, and Table S2.

Figure 2.. Transcriptomic changes in 22q11DS thalamic and cortical organoids.

(A) UMAPs by genotype. (B) Cell type proportions by genotype. (C) Number of 22q11DS-associated differentially expressed genes (DEGs) by cell type. (D, E) Overlap between ASD risk genes (SFARI, risk score = 1) and DEGs in at least one cell type (D) or per cell type (E). *** FDR < 0.0005; hypergeometric test. (F) Significance of differentially expressed transcription factors implicated in ASD (SFARI, risk score = 1). (G, H) Immunostaining for pan-neuronal marker HuC/D, pan-thalamic marker TCF7L2, and DEGs FOXP2 (G) or SOX2 (H) in week 10 thalamic organoids. Left: tilescan of the entire organoid, scale bar = 250 μm. Right: high magnification images represented by insets on the left. Scale bar = 50 μm. (I, J) Percentage of TCF7L2/HuC/D+ cells expressing FOXP2 (I) or SOX2 (J) in thalamic organoids. Mean ± SEM is shown (*** p < 0.0005; Wilcoxon rank-sum test; n = 9 organoids per genotype). (K, L) Immunostaining for TCF7L2, FOXP2 (K), and SOX2 (L) in mouse thalamus. Left: tilescan of the entire thalamus (dotted line). Scale bar = 1 mm. Right: Higher magnification images represented by insets on the left. Scale bar = 50 μm. (M) Percentage of TCF7L2+ cells expressing FOXP2. Mean ± SEM is shown (* p < 0.05; linear mixed model regression; N = 3 biological replicates per genotype) (N) Percentage of NeuN+ cells that express SOX2. Mean ± SEM is shown (* p < 0.05; linear mixed model regression; N = 3 biological replicates per genotype) See also Figure S2 and Table S3.

Figure 3.. 22q11.2 microdeletion mediates thalamocortical axon overgrowth via elevated FOXP2 expression.

(A) Thalamocortical organoid co-culture model for visualizing axon outgrowth. (B) Immunostaining against thalamus marker TCF7L2 and telencephalon marker FOXG1 in thalamocortical organoid co-cultures. Scale bar: 500 μm. (C) Visualization of thalamic (green) or cortical (magenta) axons crossing the fusion boundary after six days post-fusion (dpf). Scale bar: 500 μm. (D, E) Quantification of thalamocortical (D) or corticothalamic (E) axon outgrowth. *** adj. p < 0.0005, ns = not significant; Wilcoxon rank-sum test with Bonferroni-Holm correction. In (D), n = 21 control organoids, 9 CAG-FOXP2 organoids, and 29 patient organoids. In (E), n = 16 control organoids, 10 CAG-FOXP2 organoids, and 20 patient organoids. (F) Immunostaining against FOXP2, TCF7L2, and HuC/D in a CRISPR-engineered 22q11DS thalamic organoid and isogenic control. Scale bar, low magnification: 250 μm, high magnification: 50 μm. (G) Percentage of TCF7L2/HuC/D+ cells expressing FOXP2. * adj. p < 0.05; Wilcoxon Rank-sum test with Bonferroni-Holm correction; n = 5 control organoids and 6 CRISPR-engineered organoids. (H) Visualization of thalamic (green) or cortical (magenta) axons crossing the fusion boundary. Scale bar: 500 μm. (I, J) Quantification of thalamocortical (I) or corticothalamic (J) axon outgrowth. ** adj. p < 0.005, *** adj. p < 0.0005, ns = not significant; Wilcoxon rank-sum test with Bonferroni-Holm correction. In (I), n = 4 isogenic control organoids, 3 CAG-FOXP2 organoids, and 11 CRISPR-engineered organoids. In (J), n = 4 isogenic control organoids, 4 CAG-FOXP2 organoids, and 5 CRISPR-engineered organoids. (K) Immunostaining against thalamocortical axon marker PKCδ in parasagittal sections of P56 wildtype or Df(h22q11)/+ mouse brain. Scale bar, low magnification: 1 mm, high magnification: 500 μm. (L) Percent area in the striatum occupied by thalamocortical axons across eight sections per biological replicate, comparing wildtype (n = 3) or Df(h22q11)/+ mice (n = 3). (* adj. p < 0.05; linear mixed regression model). See also Figure S3.

Figure 4.. ROBO2 is a putative downstream target of FOXP2 whose downregulation mediates thalamic axon overgrowth.

(A) FOXP2-targeting sgRNA validation. (* adj. p < 0.05; Wilcoxon rank-sum test with Bonferroni-Holm correction) (B) Schematic of FOXP2 knockdown in thalamic organoids followed by co-culture with cortical organoids. (C, D) Thalamocortical axon outgrowth with or without FOXP2 knockdown after six days post-fusion (dpf) in 22q11DS patient lines (C) and CRISPR-engineered lines (D). Scale bar: 500 μm. (E, F) Quantification of thalamocortical axon outgrowth after FOXP2 knockdown in patient (E) or CRISPR-engineered (F) lines. * adj. p < 0.05, ** adj. p < 0.005, *** adj. p < 0.0005, ns = not significant; Wilcoxon rank-sum test with Bonferroni-Holm correction. In (E): n = 12 control organoids with non-targeting sgRNA, 8 patient organoids with FOXP2 sgRNA #1, 11 patient organoids with FOXP2 sgRNA #2, and 11 patient organoids with non-targeting sgRNA. In (F): n = 4 control organoids with non-targeting sgRNA, 13 CRISPR-engineered organoids with FOXP2 sgRNA #2, and 13 CRISPR-engineered organoids with non-targeting sgRNA. (G) CUT&Tag was performed to identify FOXP2 binding sites in week 5 control thalamic organoids. (H) Number of detected CUT&Tag peaks by functional annotation. (I) Overlap between genes within 500 kb of CUT&Tag peaks and thalamic organoid DEGs. (J) Volcano plot visualizing DEGs implicated in axon outgrowth that overlap with genes within 500 kb of FOXP2 binding sites. (K) ROBO2-targeting sgRNA validation. (* p < 0.05; Wilcoxon rank-sum test). (L) Schematic of ROBO2 knockdown in thalamic organoids followed by co-culture with cortical organoids. (M) Visualization of thalamocortical axon outgrowth with or without ROBO2 knockdown in control organoids, compared to 22q11DS organoids. Scale bar: 500 μm. (N) Quantification of thalamocortical axon outgrowth after ROBO2 knockdown. (** adj. P < 0.005, *** adj. P < 0.0005; Wilcoxon rank-sum test with Bonferroni-Holm correction; n = 12 control organoids with non-targeting sgRNA, 5 control organoids with ROBO2 sgRNA, 11 patient organoids with non-targeting sgRNA, and 14 CRISPR-engineered organoids with non-targeting sgRNA). (O-Q) Working model for how 22q11.2 microdeletion mediates axon overgrowth phenotypes in thalamocortical organoid co-cultures. See also Figure S4 and Table S4.