ESC models of autism with copy-number variations reveal cell-type-specific translational vulnerability

Abstract

Human genetics has identified numerous copy-number variations (CNVs) associated with autism spectrum disorders (ASDs). However, the lack of standardized biological resources impedes understanding of the cell-type-specific common features of ASD. Here, we establish a biological resource including 63 genetically modified mouse embryonic stem cell (ESC) lines as genetic models of ASD. We perform neural differentiation using 12 representative cell lines, and their comprehensive analyses, including single-cell RNA sequencing, uncover cell-type-specific susceptible pathways. Moreover, we find that a common phenotype in glutamatergic and GABAergic neurons is reduced expression of Upf3b, a core member of the translational termination and nonsense-mediated decay (NMD). This finding emphasizes that the dysfunction of translational machinery in the developing neurons can be a possible target of early intervention for ASD. This ESC model bank becomes an invaluable resource for studies in vitro and in vivo of ASD or other neuropsychiatric disorders.

Keywords: autism spectrum disorder; cell library; chromosome engineering; copy number variation; embryonic stem cell; genome editing; neurodevelopmental disorders; non-sense mRNA decay; single-cell RNA sequence; translation.

Graphical abstract

Figure 1

ASD cell bank for functional analysis (A) Schematic diagram to generate a cellular model of ASD with chromosomal abnormalities and strategy to identify ASD-associated pathogenic phenotypes using representative ASD cell lines. (B) Representative image of chromosome targeting locus in mouse chromosome 7 (7qC) and human 15q13.3 locus. (C) Schematic of the sgRNA targeting sites in Chrna7 and Otud7a, respectively. The target sequences are underlined and labeled in blue. The protospacer adjacent motif (PAM) sequence is underlined and labeled in red.

Figure 2

Transcriptional features and heterogeneity of cells harboring ASD-associated CNVs (A) Schematic presentation of neural differentiation from mouse ESCs. (B) UMAP dimensionality reduction embedding of differentiated neurons and the derivatives from mESCs harboring ASD-associated CNVs. Whole cells were colored by annotated cell types. (C) Heatmap shows the expression pattern of cell-type-specific genes. Columns represent individual cells, and rows represent individual genes. The representative differentially expressed genes (DEGs) are listed to the right. (D) Violin plot showing the expression profile of each gene in various cell types. (E) Feature plots of characteristic marker genes for the cell types are shown. Cells are color coded according to gene expression levels. (F) Cell-type-specific expression analysis (CSEA) using each glutamatergic or GABAergic cell-cluster-specific gene (top 300 independent genes, q < 0.001). It highlights the brain area with the development period in each neuronal cluster. Glutamatergic, glutamatergic neurons; glutamatergic_upper, upper-layer (2/3)-specific glutamatergic neurons; GABAergic (Sncg+), Gad1+Sncg+ GABAergic neurons; GABAergic (Npy+), Gad1+Npy+ GABAergic neurons. (G) The number of overlapping genes between genetic risk factors (SFARI genes, risk scores 1, 2, and 3, and syndromic genes) and cell-type-specific DEGs (top 300 genes, q < 0.001) in each cell cluster. Color coding is consistent with (B) and (D).

Figure 3

Significance of the glutamatergic postsynaptic density genes and upstream regulators in ASD (A) Overlap between glutamatergic (Slc17a6+) cell-cluster-specific DEGs and postsynaptic density (PSD) genes. The x axis represents the number of total DEGs in each cell cluster, and the y axis represents glutamatergic neuronal cell clusters in each CNV. The red column indicates PSD complex genes. SFARI genes (gene scores 1–3 and syndromic) in each DEG are listed to the right. The genes with underline indicate a syndromic gene. (B) Cell-type-specific upstream regulators. The heatmap rows are the upstream regulator genes, and the columns are CNVs. The gradient of color in the heatmap indicates the enrichment levels. The heatmap color indicates statistical significance (−log10(p value)). Genes highlighted in the red are SFARI ASD risk genes.

Figure 4

Cell-type- and CNV-specific pathway analysis (A) Heatmap for the cell-type-specific enriched canonical pathways was performed using Ingenuity Canonical Pathways Analysis (IPA). Each column in the figure represents an ASD-associated CNV, and each row represents a canonical pathway. The colors in the graph indicate the −log10 (p value). (B) UpSet plot showing overlap ASD risk gene among CNVs. Red is shared across more than four CNVs, blue is shared across three CNVs, orange is shared between two CNVs, and black is unique to a specific CNV.

Figure 5

Cell-type- and CNV-specific translational dysregulation (A) Heatmap showing the gene expression pattern of each phase of the translational process among ASD-associated CNVs, including whole-cell types. Columns represent individual genes, and rows represent individual CNVs. The colors in the graph indicate the log2 fold changes (log2FCs). (B) Schematic representation of a translation pathway, initiation, elongation, and termination. ASD risk genes involved in each translation phase are possibly implicated in ASD pathogenesis by regulating the quality and quantity of protein synthesis. (C) Heatmap showing associations among translation phase, cell types, and ASD-associated CNVs. The colors in the graph indicate the log2FCs. (D) Validation of both Upf3a and Upf3b expression by RT-qPCR. One-way ANOVA with Bonferroni multiple comparisons for post hoc comparisons. ∗p < 0.05 and ∗∗p < 0.01; N.S., not significant. (E) Quantification of Upf3b mRNA puncta in Tuj1 immunoreactive neurons in ctrl, 1q21.1 dup, 15q13.3, and 16p11.2 CNV cell models. One-way ANOVA with Bonferroni multiple comparisons for post hoc comparisons. ∗∗p < 0.01. The data were obtained from more than ten images from three independent samples; n = 49, 34, 30, and 32 in each genotype, ctrl, 1q21.1dup, 15q13.3, and 16p11.2, respectively. (F) Representative images of Upf3b mRNA signals in Tuj1 immunoreactive neurons. Scale bar: 10 μm.