Cell-type-specific dysregulation of gene expression due to Chd8 haploinsufficiency during mouse cortical development

Abstract

Disruptive variants in the chromodomain helicase CHD8 are associated with risk for autism spectrum disorder (ASD). CHD8 haploinsufficiency is hypothesized to contribute to ASD by perturbing neurodevelopmental gene expression. However, insight into cell-type-specific transcriptional effects of CHD8 haploinsufficiency remains limited. We used single-cell and single-nucleus RNA sequencing to identify dysregulated genes in the embryonic and juvenile Chd8^+/- mouse cortex. Chd8 and other ASD risk-associated genes showed a convergent expression trajectory conserved between mouse and human developing cortex, increasing from progenitor zones to the cortical plate. Genes associated with neurodevelopmental disorders or involved in chromatin remodeling and neuron projection development were dysregulated in Chd8^+/- embryonic radial glia. Genes implicated in synaptic activity and organization were dysregulated in Chd8^+/- postnatal excitatory cortical neurons, suggesting impaired synaptogenesis. Our findings reveal complex patterns of transcriptional dysregulation due to Chd8 haploinsufficiency, potentially with distinct impacts on progenitors and maturing neurons in the excitatory neuronal lineage.

Keywords: ASD; CHD8; autism spectrum disorder; cortical development; gene regulation; mouse model; neurodevelopment; neurodevelopmental disorders; single-cell transcriptomics.

Graphical abstract

Figure 1

Generation and characterization of a Chd8^+/− mouse model (A) Schematic showing our strategy for constitutive Chd8^+/− mouse generation (STAR Methods). The Chd8 gene models (left) and comparison of the targeted sequence (right) in the wild-type (Chd8⁺) and disrupted (Chd8⁻) alleles are shown. Nucleotides adjacent to the deletion site are colored red. Triangles denote expected guide RNA (gRNA)-directed cleavage sites of the upstream (red) and downstream (yellow) gRNAs (STAR Methods). Dashed line with slashes indicates sequence omitted for clarity. (B) Boxplot of transcripts per million (TPM) quantifying Chd8 expression in wild-type (WT) and Chd8^+/− embryonic day (E) 12.5 and E17.5 cortex (n = 3 male and n = 3 female samples per genotype per time point; STAR Methods). Statistical significance and adjusted p values (p_adj) were calculated by DESeq2 (STAR Methods). (C) Western blot showing CHD8 and actin expression in male WT and Chd8^+/− E16.0 cortices (left), with quantification of CHD8 expression in the WT and Chd8^+/− E16.0 cortex (right; n = 3 male and n = 3 female samples per genotype; STAR Methods). Arrowhead indicates the ∼290-kDa CHD8 band.^,p = p value from one-tailed Welch’s t test. (D) Top and middle: Coronal sections of WT embryonic mouse cortex at the indicated stages, stained with anti-CHD8 antibody (red) and Hoechst 33342 (nuclei; blue). Scale bars: 200 μm (top), 50 μm (middle). Bottom: Quantification of the ratio of CHD8 signal to nuclear stain in bins spanning the apicobasal axis of the WT embryonic cortex (E12.5, n = 9; E14.5, n = 9; E16.0, n = 6; E17.5, n = 10; STAR Methods). (E) Spatiotemporal expression of CHD8 (red) in coronal sections of the WT and Chd8^+/− embryonic mouse cortex collected from littermates at the indicated stages. Nuclei were stained with Hoechst 33342 (blue). Scale bar: 100 μm. For all boxplots, boxes represent the interquartile range (IQR), whiskers denote the minimum and maximum data points within 1.5 × IQR, the horizontal line within each box indicates the median, and each dot represents a biological replicate. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; PP, preplate; SP, subplate; CP, cortical plate; MZ, marginal zone. See also Figures S1–S8 and S22, and Tables S1 and S2.

Figure 2

Analysis of embryonic cortical development using scRNA-seq in WT and Chd8^+/− mice (A) Study design, including dissection schema and number of cells collected at each time point in each genotype. (B) UMAP embedding of 135,926 cells colored by cell-type clusters (left), genotype (top right), and time point (bottom right; STAR Methods). (C) Cell-type composition at each time point in each genotype. (D) PHATE embedding of 128,112 cells in the excitatory neuronal lineage, colored by cell type (left) and pseudotime (right). Black arrow indicates the principal curve through the primary trajectory. Cells excluded from further analysis are shown in gray (STAR Methods). (E) Cell-type representation along pseudotime, color coded by genotype (STAR Methods). Below each distribution, the position of each cell along pseudotime is plotted as a vertical line, colored by cell-type label. See also Figures S8–S14 and Table S3. Sample information and sequencing summary statistics for the wild-type and Chd8+/− embryonic cortex scRNA-seq dataset, related to Figure 2 and STAR Methods, Table S4. Marker genes identified for each cluster in the embryonic scRNA-seq dataset, using the Wilcoxon rank-sum test, related to Figure 2, Table S5. Summary of cell-type assignment for each sample within the embryonic scRNA-seq dataset, related to Figure 2, Table S6. Significance testing results for comparison of cell-type numbers in the wild-type and Chd8+/− het embryonic cortex scRNA-seq dataset, related to Figure 2. IP, intermediate progenitors; UL, upper-layer; DL, deep-layer; RBC, red blood cells; OPC, oligodendrocyte precursor cells.

Figure 3

Inferring spatial and developmental gene expression gradients in the developing mouse cortex using scRNA-seq Expression gradients for (A) Chd8, (B) Pogz, (C) Tbr1, and (D) Pax6 inferred at E14.5 (left) and E17.5 (right). For each time point, the expression of each protein visualized using IHC is shown at the left (CHD8, red; POGZ, cyan; TBR1, green; PAX6, yellow; STAR Methods). The PHATE embedding of cells in the excitatory neuronal lineage colored by normalized gene expression is shown at the center. The inferred expression trajectories for each gene are shown at the right (STAR Methods). Mean expression per bin (open circles) is plotted against pseudotime. Smoothed lines are drawn with loess (span = 0.75) and a confidence interval of 0.95. See also Figures 2 and S15. Scale bars: 100 μm. Abbreviations match those in Figure 1.

Figure 4

Metagenes reveal neurodevelopmental, ASD risk-associated genes, and developmental disorder risk-associated genes with common transcriptional trajectories (A) Number of genes assigned to each metagene for each time point and genotype (top), with examples of gene expression trajectories assigned to each metagene (bottom; STAR Methods). The metagene center is plotted as a dashed line, and the example gene expression trajectory from the E17.5 WT cortex data is plotted as a solid line. r = Pearson correlation between the example gene trajectory and metagene center. (B) Gene Ontology Biological Process (GO:BP) terms significantly enriched in metagenes H and I (up to 10 most significant terms per metagene; black line, g:SCS-adjusted p value = 0.05; STAR Methods). (C) Enrichment of autism spectrum disorder risk-associated genes (ASD), the Deciphering Developmental Disorder (DDD) gene set, and CHD8 binding targets in the E17.5 WT mouse cortex for each metagene. Circle size corresponds to the number of genes within each metagene that intersect the given gene set at each time point and genotype. Circle color corresponds to the Benjamini-Hochberg-adjusted p value (STAR Methods). See also Figures S15–S17 and Table S7. Metagene assignments of 33,516 genes in the E14.5, E16.0, and E17.5 wild-type and Chd8+/− cortex scRNA-seq datasets, related to Figure 4, Table S8. GO:BP term enrichment for mouse metagenes identified from the E14.5, E16.0, and E17.5 wild-type cortex scRNA-seq datasets, related to Figure 4, Table S9. Enrichment of ASD risk-associated genes, DDD genes, and CHD8 target genes among mouse metagenes identified from E14.5, E16.0, and E17.5 wild-type and Chd8+/− cortex scRNA-seq datasets, related to Figure 4, Table S10. Lists of CHD8 target genes, neurodevelopmental disorder-associated gene sets, and FMRP target genes used for assessing enrichment in metagene and differential expression gene lists, related to Figures 4–6.

Figure 5

ASD risk-associated genes and developmental disorder risk-associated genes show convergent expression trajectories in mouse and human cortical development (A) PHATE embedding of 30,132 cells from the fetal human cortex at gestational weeks (GW) 17 and 18 (n = 4), colored by cell types as defined in Polioudakis et al. (left) and by pseudotime (right). vRG, ventral radial glia; oRG, outer radial glia; PgS, S phase cycling progenitors; PgG2M, G2M phase cycling progenitors; ExN, newborn excitatory neurons; ExM, maturing excitatory neurons; ExM-U, upper-layer-enriched maturing excitatory neurons; ExDp1 and ExDp2, deep-layer excitatory neurons (subclusters 1 and 2). (B) Cell-type representation along pseudotime, labeled as in (A). Circle size corresponds to the percentage of cells of the indicated cell type per pseudotime bin (STAR Methods). (C) Number of genes in each human cortex metagene. (D) Heatmap of Pearson correlation (r) between mouse cortex metagene centers and human cortex metagene centers (STAR Methods). (E) Gene Ontology Biological Processes (GO:BP) terms significantly enriched in human metagenes A′, C′, H′, and I′ (≤10 most significant terms per metagene; black line, g:SCS-adjusted p value = 0.05; STAR Methods). (F) Metagene enrichment for ASD genes, the DDD gene set, and CHD8 binding targets in the human mid-fetal cortex. Circle size corresponds to the number of genes within each metagene that intersect the given gene set. Circle color corresponds to Benjamini-Hochberg-adjusted p value (STAR Methods). (G) Overlap of ASD genes identified in mouse metagenes H and I at E16.0 and E17.5, and ASD genes identified in human metagenes H′ and I′. The 39 ASD genes that were assigned to these metagenes in both species are shown. See also Figure S18 and Tables S7 and Table S10. Lists of CHD8 target genes, neurodevelopmental disorder-associated gene sets, and FMRP target genes used for assessing enrichment in metagene and differential expression gene lists, related to Figures 4–6, Table S11. Metagene membership of 10,393 genes in the human fetal cortex scRNA-seq dataset from Polioudakis et al., related to Figure 5, Table S12. Summary of human metagenes in the human fetal cortex scRNA-seq dataset from Polioudakis et al., related to Figure 5, Table S13. GO:BP term enrichment for human metagenes identified in the human fetal cortex scRNA-seq dataset from Polioudakis et al., related to Figure 5, Table S14. Enrichment of ASD risk-associated genes, DDD genes, and CHD8 target genes among human metagenes identified in the human fetal cortex scRNA-seq dataset from Polioudakis et al., related to Figure 5. Abbreviations match those in Figure 4.

Figure 6

Gene sets associated with NDDs are enriched among dysregulated genes in E12.5 radial glia and E17.5 upper-layer neurons (A) Number of downregulated (DOWN) and upregulated (UP) differentially expressed genes (DEGs) identified in cell types of the primary trajectory at each time point (STAR Methods). (B) Volcano plots of differential expression (DE) results for radial glia at E12.5 (left) and UL neurons at E17.5 (right) in the Chd8^+/− cortex, with genes color coded by DE call (NS, not significantly different; STAR Methods). Chd8 is outlined in black. Vertical gray lines, ±log2(1.5 fold change); avg_log2FC, log2-transformed fold change of each gene’s average expression. (C) Intersection between DEGs in each cell type at E12.5 (left) or E17.5 (right) and CHD8 target genes in the E17.5 WT mouse cortex, ASD genes, DDD genes, and FMRP target genes. Asterisk (∗) indicates Benjamini-Hochberg (BH)-adjusted p value <0.05 (STAR Methods). (D) Gene set enrichment analysis (GSEA) results of differential gene expression in radial glia at E12.5 (left) or UL neurons at E17.5 (right) in the Chd8^+/− cortex, assessing enrichment of NDD risk-associated gene sets, including ASD genes, DDD genes, and genes associated with risk for epilepsy, schizophrenia, macrocephaly, or microcephaly (STAR Methods). Horizontal ticks indicate the rank of genes from each NDD risk-associated gene set, color coded by DE calls (STAR Methods). Dotted lines indicate the zero-cross rank separating positive and negative values. (E) Intersection between DEGs in the bulk RNA-seq embryonic cortex dataset at E12.5 or E17.5 and CHD8 target genes, ASD genes, DDD genes, and FMRP target genes. Asterisk (∗) indicates BH-adjusted p value <0.05 (STAR Methods). (F) GSEA results of differential gene expression in the bulk E12.5 (left) or bulk E17.5 (right) Chd8^+/− cortex, assessing enrichment of NDD risk-associated gene sets. Horizontal ticks indicate the rank of genes from each NDD risk-associated gene set, color coded by DE calls (STAR Methods). Dotted lines indicate the zero-cross rank separating positive and negative values. See also Figures S19–S21 and Tables S10 and Table S15. Summary of differential expression results determined by Monocle 3 for the wild-type and Chd8+/− scRNA-seq data, related to Figure 6, Table S16. Differential expression results for the primary trajectory in the embryonic wild-type and Chd8+/− scRNA-seq data, determined by Monocle 3, related to Figure 6, Table S17. Genes consistently called as downregulated or upregulated in the primary trajectory or a given cell type across all time points in the embryonic scRNA-seq dataset, at determined by Monocle 3, related to Figure 6, Table S18. Differential expression results for each cell type in the embryonic wild-type and Chd8+/− scRNA-seq data, determined by Monocle 3, related to Figure 6, Table S19. Summaries of differential expression results for downsampled scRNA-seq datasets from the wild-type and Chd8+/− embryonic cortex, determined by Monocle 3, related to Figure 6, Table S20. Results of enrichment testing for CHD8 target genes and neurodevelopmental disorder-associated genes among differentially expressed genes in the scRNA-seq Chd8+/− embryonic cortex data, related to Figure 6, Table S21. Results of gene set enrichment analysis for neurodevelopmental disorder-associated genes among differentially expressed genes in the scRNA-seq Chd8+/− embryonic cortex data, related to Figure 6, Table S22. Sample information for the bulk RNA-seq dataset and differential expression results from bulk RNA-seq of the embryonic wild-type and Chd8+/− cortex, determined by DESeq2, related to Figures 1, 6, and 7, Table S23. Results of enrichment testing for CHD8 target genes and neurodevelopmental disorder-associated genes among DEGs in the bulk RNA-seq Chd8+/− embryonic cortex data, related to Figure 6, Table S24. Results of gene set enrichment analysis for neurodevelopmental disorder-associated genes among differentially expressed genes in the bulk RNA-seq Chd8+/− embryonic cortex data, related to Figure 6, Table S25. GO:BP term enrichment for differentially expressed genes in the scRNA-seq Chd8+/− embryonic cortex data, related to Figure 6, Table S26. GO:BP term enrichment for differentially expressed genes in the bulk RNA-seq E12.5 Chd8+/− cortex data, related to Figure 6. Abbreviations match those in Figures 2 and 4.

Figure 7

snRNA-seq of the juvenile wild-type and Chd8^+/− cortex reveals dysregulation of synaptic genes across excitatory neuron subtypes (A) Schematic of experimental design, showing the dissection schema and number of nuclei collected for each genotype (STAR Methods). The representative coronal section of the juvenile mouse brain is from the Allen Mouse Brain Atlas, Nissl-stained (postnatal day 28, position 295; Allen Institute for Brain Science [2004], developingmouse.brain-map.org). (B) UMAP embedding of 66,840 singlet nuclei colored by cell-type assignment, with clusters excluded from downstream analyses colored in gray (STAR Methods). (C) Number of downregulated (DOWN) and upregulated (UP) DEGs per cluster (STAR Methods). Only clusters with ≥1 DEG are represented. (D) Dot plot showing enrichment of representative functional terms among DOWN and UP DEGs per cluster, restricted to terms enriched across multiple clusters (STAR Methods). Dot size corresponds to the ratio of DOWN or UP genes intersecting with the denoted functional term to all DOWN or UP genes from that cluster (GeneRatio). p_adj = g:SCS-adjusted p value (STAR Methods). (E) Volcano plots of DE results for the L4 (left) and L6-CT (right) clusters. The outline color code indicates genes encoding glutamate receptors (GluRs) or regulators of GluR localization and/or function (STAR Methods). Chd8 is outlined in purple. Vertical gray lines, ±log2(1.5 fold change). See also Figures S22–S32 and Table S27. Sample information and sequencing statistics for the P25 cortex snRNA-seq dataset for wild-type RNA-seq and Chd8+/− mice, related to Figure 7 and STAR Methods, Table S28. Marker genes identified for each cluster in the full juvenile mouse cortex snRNA-seq dataset, using the Wilcoxon rank-sum test, related to Figure 7, Table S29. Marker genes identified for each cluster in the singlet-only juvenile mouse cortex snRNA-seq dataset, using the Wilcoxon rank-sum test, related to Figure 7, Table S30. Summary of cell-type assignment for each sample within the juvenile mouse cortex snRNA-seq dataset after doublet filtering, related to Figure 7, Table S31. Significance testing results for comparison of cell-type numbers in the wild-type and Chd8+/− juvenile cortex snRNA-seq dataset post filtering, related to Figure 7, Table S32. Summary of differential expression results determined by Monocle 3 for the wild-type and Chd8+/− juvenile mouse cortex snRNA-seq data, related to Figure 7, Table S33. Differential expression results by cell type in the wild-type and Chd8+/− juvenile mouse cortex snRNA-seq data, determined by Monocle 3, related to Figure 7, Table S34. Summaries of differential expression results for downsampled snRNA-seq datasets from the wild-type and Chd8+/− juvenile cortex, determined by Monocle 3, related to Figure 7, Table S35. Results of enrichment testing for CHD8 target genes and neurodevelopmental disorder-associated genes among differentially expressed genes in the snRNA-seq Chd8+/− juvenile mouse cortex dataset, related to Figure 7, Table S36. Results of gene set enrichment analysis for neurodevelopmental disorder-associated genes among differentially expressed genes in the snRNA-seq Chd8+/− juvenile cortex dataset, related to Figure 7, Table S37. Results of functional and pathway term enrichment among differentially expressed genes in the snRNA-seq Chd8+/− juvenile cortex dataset, determined by g:Profiler, related to Figure 7, Table S38. Summary of significant functional and pathway term enrichment among differentially expressed genes in the snRNA-seq Chd8+/− juvenile cortex dataset, determined by g:Profiler, related to Figure 7. avg_log2FC, log2-transformed fold change of each gene’s average expression; L, layer of excitatory cortical neuron; IT, intratelencephalic projecting neurons; PT, pyramidal tract projecting neurons; NP, near-projecting neurons; CT, corticothalamic projecting neurons; iN, inhibitory neuron; AG, astroglia; OL, oligodendrocyte; OPC, oligodendrocyte precursor cell; MG, microglia; Vasc, vasculature; mCtx, medial cortex; Clau, claustrum; Str, striatum; Ambig, ambiguous cluster; UL, upper-layer cortical neuron; DL, deep-layer cortical neuron.