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Distinct, dosage-sensitive requirements for the autism-associated factor CHD8 during cortical development - PubMed

  • ️Fri Jan 01 2021

Distinct, dosage-sensitive requirements for the autism-associated factor CHD8 during cortical development

Shaun Hurley et al. Mol Autism. 2021.

Abstract

Background: CHD8 haploinsufficiency causes autism and macrocephaly with high penetrance in the human population. Chd8 heterozygous mice exhibit relatively subtle brain overgrowth and little gene expression changes in the embryonic neocortex. The purpose of this study was to generate new, sub-haploinsufficient Chd8 mouse models to allow us to identify and study the functions of CHD8 during embryonic cortical development.

Methods: To examine the possibility that certain phenotypes may only appear at sub-heterozygous Chd8 levels in the mouse, we created an allelic series of Chd8-deficient mice to reduce CHD8 protein levels to approximately 35% (mild hypomorph), 10% (severe hypomorph) and 0% (neural-specific conditional knockout) of wildtype levels. We used RNA sequencing to compare transcriptional dysregulation, structural MRI and brain weight to investigate effects on brain size, and cell proliferation, differentiation and apoptosis markers in immunostaining assays to quantify changes in neural progenitor fate.

Results: Mild Chd8 hypomorphs displayed significant postnatal lethality, with surviving animals exhibiting more pronounced brain hyperplasia than heterozygotes. Over 2000 genes were dysregulated in mild hypomorphs, including autism-associated neurodevelopmental and cell cycle genes. We identify increased proliferation of non-ventricular zone TBR2+ intermediate progenitors as one potential cause of brain hyperplasia in these mutants. Severe Chd8 hypomorphs displayed even greater transcriptional dysregulation, including evidence for p53 pathway upregulation. In contrast to mild hypomorphs, these mice displayed reduced brain size and increased apoptosis in the embryonic neocortex. Homozygous, conditional deletion of Chd8 in early neuronal progenitors resulted in pronounced brain hypoplasia, partly caused by p53 target gene derepression and apoptosis in the embryonic neocortex. Limitations Our findings identify an important role for the autism-associated factor CHD8 in controlling the proliferation of intermediate progenitors in the mouse neocortex. We propose that CHD8 has a similar function in human brain development, but studies on human cells are required to confirm this. Because many of our mouse mutants with reduced CHD8 function die shortly after birth, it is not possible to fully determine to what extent reduced CHD8 function results in autism-associated behaviours in mice.

Conclusions: Together, these findings identify important, dosage-sensitive functions for CHD8 in p53 pathway repression, neurodevelopmental gene expression and neural progenitor fate in the embryonic neocortex. We conclude that brain development is acutely sensitive to reduced CHD8 expression and that the varying sensitivities of different progenitor populations and cellular processes to CHD8 dosage result in non-linear effects on gene transcription and brain growth. Shaun Hurley, Conor Mohan and Philipp Suetterlin have contributed equally to this work.

Keywords: Apoptosis; Autism; CHD8; Chromatin; Conditional knockout; Cortex; Gene expression; Hypomorph; Intermediate progenitor; Mouse; Neural progenitor; Proliferation; TBR2; p53.

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Conflict of interest statement

MAB consults for Merck, Sharpe & Dohme. SH is currently an employee at COMPASS Pathways plc. This work is unrelated to COMPASS Pathways plc. No other competing interests to declare.

Figures

Fig. 1
Fig. 1

Brain hyperplasia in Chd8 heterozygous and mild hypomorphic mice. a Diagrammatic representation of the Chd8 allele containing the neo cassette between exons 3 and 4. Exon 3 splicing to exon 4 yields a 150 bp product (*) by RT-PCR using primers F and R. Aberrant splicing from exon 3 into the neo cassette yields a 246 bp product (**) with primers F and neo. b Quantitative RT-PCR of Chd8 transcripts in E9.5-E10.5 neocortices of indicated genotypes. c Estimation of CHD8 protein levels in E12.5 neocortices by Western blot. *p < 0.05, **p < 0.01, ***p < 0.001.  d High-resolution 7 T structural MRI coronal images of Chd8+/− (n = 12, all males, 22 weeks old) and Chd8neo/neo brains (n = 13, 8 males, 5 females, 16 weeks old), from posterior (top) to anterior (bottom) are shown. Absolute volumetric differences, relative to wildtype controls (n = 30, 26 males, 4 females) are coloured according to the scale on the right. Some regions with enlarged volumes are labeled as follows: NCtx neocortex, EcC ectorhinal cortex, EnC entorhinal cortex, HC hippocampus, Cb cerebellum. e Absolute volumes relative to wildtypes are plotted for whole brain, neocortex and several other brain regions for the different genotypes as indicated. –FDR < 0.15, *FDR < 0.05, **FDR < 0.01. See also Additional file 2: Table S1. MRI data from Chd8+/− and littermate control mice used for comparison are from Suetterlin et al. [18]

Fig. 2
Fig. 2

Behavioural assessment of mild Chd8 hypomorphic mice. a–k Behavioural assessments of a cohort of adult Chd8neo/neo (neo/neo, n = 24; 14 males, 10 females, a, b, d, e, g, i or n = 15; 10 males, 5 females, f, k or n = 11; 6 males, 5 females, j) and Chd8+/+ (+/+, n = 41; 26 males, 15 females, a, b, d, e, g, i or n = 31, 21 males, 10 females, f, k or n = 12; 6 males, 6 females, j) and pup Chd8neo/neo (neo/neo, n = 10; 4 males, 6 females, c) and Chd8+/+ (+/+, n = 12; 6 males, 6 females, c) mice. Chd8+/+ animals are illustrated using filled circles, dotted lines with grey bars and Chd8neo/neo animals with open triangles, solid lines and open bars. Apart from USVs (c), all other tests were conducted on young adults 6–14 weeks of age. a Duration, in seconds, spent in each chamber of the three-chamber sociability test. All mice spent a significantly higher proportion of time in the chamber with the age- and sex- matched stranger con-specific mouse compared to the other chambers. Mean ± SEM; *** < 0.001 (between-subjects ANOVA with student’s t test as post-hoc analysis). b Duration, in seconds, of social investigation over a three-minute period. Social investigation was defined as the combined total duration of head, body and anogenital sniffing of a conspecific mouse. Mean ± SEM (between-subjects ANOVA). c The mean number of ultrasonic vocalisations per minute on indicated postnatal days. Mean ± SEM (repeated-measures ANOVA). d Graph demonstrating the performance in the olfactory habituation/dishabituation test. Mean ± SEM (repeated-measures ANOVA). e The duration, in seconds, mice spent self-grooming during the 10-min self-grooming test. Mean ± SEM (between-subjects ANOVA). f The average number of marbles buried, out of a maximum of 12, within a 30-min period period. Mean ± SEM; * < 0.05, ** < 0.01 (repeated-measures ANOVA with student’s t test as post-hoc analysis). g Representative ethovision tracks of a Chd8neo/neo (neo/neo) and Chd8+/+ (+ / +) animal plotting their movements during the 10-min open field task. h The percentage of time spent in the centre of the open field arena during the 10-min test. Mean ± SEM; **p < 0.01 (between-subjects ANOVA). i The total distance travelled in the outer part of the open field arena over a 10-min time-period. Mean ± SEM; *** < 0.001 (between-subjects ANOVA). j The total activity counts per 12 h period on running wheels in the homecage during 7 days of dark-phase recording. Mean ± SEM; *p < 0.05 (repeated-measures ANOVA). k The mean latency of mice to fall from the rotarod. Mean ± SEM (repeated-measures ANOVA)

Fig. 3
Fig. 3

Gene expression changes in Chd8-deficient neocortices. a Volcano plot indicating all DEGs detected by DESeq2 in E12.5 Chd8+/− embryonic cortices. Each point represents an individual gene, and all heterozygote DEGs (FDR < 0.05) are highlighted in red. n = 3 per condition. b Volcano plot showing all DESeq2 detected DEGs in E12.5 neo/neo cortex. All differentially expressed genes (FDR < 0.05) are highlighted in red. n = 3 per condition. c Volcano plot of all DESeq2 detected DEGs in E12.5 neo/− cortex. All differentially expressed genes (FDR < 0.05) are highlighted in red. n = 3 per condition. d Heatmap of genes differentially expressed in neo/neo and neo/− embryos, indicating transformed relative expression levels in +/+, +/−, neo/neo and neo/− embryos. e Mean normalised count of aligned RNA-seq reads for a selection of genes that were differentially expressed in the mild and severe hypomorphs. f Venn diagram showing extent of overlap between neo/neo and neo/− DEGs and ASD associated genes obtained from the SFARI Human Gene database (

https://gene.sfari.org/autdb/HG_Home.do

, accessed March 2018). g Breakdown of neo/neo and neo/− DEGs that are ASD associated, DEGs with known CHD8 binding sites in E17.5 mouse neocortex, human mid foetal cortex and NSCs; and DEGs that are both ASD associated and have known CHD8 binding sites. Downregulated genes are denoted by a negative number. h Breakdown of neo/neo and neo/− DEGs with H3K27me3 in neural progenitors, DEGs with known REST binding sites, and DEGs that have been shown to have both H3K27me3 in neural progenitors and REST binding sites. Downregulated genes are denoted by a negative number. i Gene Ontology (GO) analysis of up and down-regulated neo/neo and neo/− DEGs under the “Biological Processes” category. The five most significant hits are shown for each set. See also Additional file 4: Table S3

Fig. 4
Fig. 4

Increased proliferation of basal neural progenitors in Chd8neo/neo embryos. a Immunohistochemistry to detect PH3B+  (blue) and TBR2+ (green) nuclei in coronal sections through the telencephalon of E14.5 embryos. White arrowheads indicate PH3B+/TBR2+nuclei. White box is shown as zoomed inset containing a double positive cell. b Quantification of TBR2+/PH3B+cells per 100 µm of neocortex in E14.5 embryos (+/+, n = 20; neo/+, n = 9; +/−, n = 7; neo/neo, n = 6; neo/−, n = 8; Mean ± SEM, *p < 0.05, ***p < 0.001, ANOVA followed by Tukey’s multiple comparisons test). c–e Cleaved caspase 3 (CC3) immunostaining of dorsal neocortex of E14.5 embryos. Black arrows indicate CC3+ cells and black box in E highlights area shown in zoomed inset containing a CC3+ cell. Scale bar = 100 μm. f Relative quantification of CC3+ cells in neocortex of E14.5 Chd8 mutant embryos, normalised to respective wildtype littermates (+/+, n = 17; neo/ + , n = 7; neo/neo, n = 6; neo/−, n = 11; Mean ± SEM **p < 0.01). Embryos used in these experiments were from 8 different litters

Fig. 5
Fig. 5

Conditional deletion of Chd8 from the embryonic neuroepithelium results in severe hypoplasia of the telencephalon and neocortex. a Schematic representation of the wildtype (wt) mouse Chd8 gene (Chd8wt), targeting construct for homologous recombination in embryonic stem cells, the Chd8 targeted allele (Chd8neo), the Chd8 conditional allele after Flp-mediated excision of the neomycin resistance cassette (Chd8flox) and the Chd8null allele produced by Cre-mediated deletion of exon 3. Boxes represent exons, with exon 1 (Ex1) to 6 and 13 shown and exon 3 filled in black. The blue box represents a neomycin resistance cassette (neo), red triangles represent loxP sites and tan triangles frt sites. The long 9.45 kb (5′) homology arm is indicated in red and the short 4.4 kb (3′) homology arm in green in the targeting construct. b X-gal staining of a Sox1-Cre; R26R embryo at E9.5 (left); and immunostaining for CHD8 protein on Chd8flox/flox (Ctrl) and conditional knockout Sox1-Cre; Chd8flox/flox (cKO) E10.5 neural tube (right). Scale bar = 50 μm. c, d Wholemount images of E18.5 brains of a representative Ctrl and cKO embryo, anterior is to the right. Scale bar = 1 mm. e, f Wholemount images of embryonic day 12.5 heads, anterior to the right. Scale bar = 1 mm. g, h Wholemount images of E10.5 embryos, anterior to the right. c′–h′) Cresyl violet-stained frontal sections through brains as indicated in c–h above. Scale bars = 500 μm (c′, d′) and 200 μm (e′–h′). The following subcortical structures are labelled in Ctrl (c′) and cKO (d′) at E18.5: NCtx: Neocortex, mhN: medial habenular nucleus, vmt: ventral medial thalamic nucleus, amN: amygdaloid nucleus. Images are representative of at least 3 independent samples

Fig. 6
Fig. 6

Repression of p53 target genes by CHD8 is necessary for normal brain growth. a Volcano plot of RNA-seq data illustrating in red genes that are differentially expressed (FDR < 0.05) in E10.5 cKO telencephalon, with p53 pathway genes labelled. b Pathway enrichment analysis of differentially expressed genes, with the top KEGG pathway terms shown [43]. See also Additional file 5: Table S4. c qRT-PCR validation of a selection of p53 pathway genes identified by RNA-seq (n = 4 for each condition. Mean ± SEM; *p < 0.05, ***p < 0.001, student’s t test). d Wholemount brains from E18.5 Ctrl, cKO and cKO-p53het mice are shown, anterior to the top. Data are representative of 6 embryos per genotype. Scale bar = 1 mm. e–g Cleaved caspase 3 (CC3) immunohistochemistry (brown) on frontal sections through the telencephalon of E12.5 embryos. Scale bar = 200 μm. h Quantification of CC3+ cells/μm2 of the pallium in embryos of each genotype (Ctrl, n = 4; cKO, n = 3; cKO-p53het, n = 3; Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA followed by Tukey’s multiple comparisons test). i Quantification of CC3+ cells/μm2 of the subpallium (medial ganglionic eminence) in embryos of each genotype (Ctrl, n = 4; cKO, n = 3; cKO-p53het, n = 3; Mean ± SEM; *p < 0.05, **p < 0.01, ANOVA followed by Tukey’s multiple comparisons test). j–l Immunohistochemistry to detect BrdU + (red) and phospho-histone 3B (PH3B)+ nuclei in frontal sections through the telencephalon of E12.5 embryos. Scale bar = 100 μm. j′–l′ Magnified images of the boxed neocortical regions in j–l, with the ventricular zone (vz) at the bottom and pial surface at the top. Scale bar = 25 μm. m Quantification of PH3B+ cells/μm of neocortical ventricular surface (Ctrl, n = 4; cKO, n = 3; cKO-p53het, n = 3; Mean ± SEM; *p < 0.05, **p < 0.01, ANOVA followed by Tukey’s multiple comparisons test). n, p Immunostaining of frontal E12.5 sections for DCX (green) to label differentiating neurons, with nuclei counterstained with Hoechst 33,342. Scale bar = 200 μm. n′, p′ Magnified images of n, p, with the ventricular zone (vz) at the bottom and pial surface (pp) at the top. Scale bar = 25 μm

Fig. 7
Fig. 7

The non-monotonic relationship between CHD8 protein levels, gene expression and brain size. The effects of gradual reductions in CHD8 protein levels to ~ 50% (heterozygous), ~ 35% (mild hypomorph), ~ 10% (severe hypomorph) and 0% (conditional knockout) on the transcription of neurodevelopmental, cell cycle and p53-regulated genes and brain size are depicted. CHD8 appears to function primarily as a positive regulator of neurodevelopmental genes via recruitment to H3K4me3-modified (yellow ball) histones (gray spool), presumably via enabling the recruitment of key transcription factors (TF). A sharp reduction in the expression of many of these genes (arrow) is only observed in E12.5 neocortex when CHD8 levels are reduced to below a threshold less than haploinsufficient levels. CHD8 appears to repress E2F-regulated cell cycle genes in this context, with significant induction only becoming evident at sub-haploinsufficient levels, although low expression increases (grey arrow) likely drives subtle increases in proliferation in the heterozygous state. Cell cycle genes are dysregulated in the opposite direction in the cKO, suggestive of non-monotonic effects (blue arrow). CHD8 can interact with p53 and histone 1 (H1), leading to stable heterochromatin formation and repression of p53 target genes. A few p53-regulated genes become activated in hypomorphic mice (grey arrow), but the majority remains fully repressed with de-repression only becoming evident upon complete CHD8 loss. Note the different CHD8 thresholds for different groups of genes (broken lines) and the non-monotonic effects on gene expression and over-all brain size

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References

    1. O'Roak BJ, Vives L, Fu W, Egertson JD, Stanaway IB, Phelps IG, et al. Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders. Science. 2012;338(6114):1619–1622. doi: 10.1126/science.1227764. - DOI - PMC - PubMed
    1. Neale BM, Kou Y, Liu L, Ma'ayan A, Samocha KE, Sabo A, et al. Patterns and rates of exonic de novo mutations in autism spectrum disorders. Nature. 2012;485(7397):242–245. doi: 10.1038/nature11011. - DOI - PMC - PubMed
    1. Talkowski ME, Rosenfeld JA, Blumenthal I, Pillalamarri V, Chiang C, Heilbut A, et al. Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries. Cell. 2012;149(3):525–537. doi: 10.1016/j.cell.2012.03.028. - DOI - PMC - PubMed
    1. O'Roak BJ, Vives L, Girirajan S, Karakoc E, Krumm N, Coe BP, et al. Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature. 2012;485(7397):246–250. doi: 10.1038/nature10989. - DOI - PMC - PubMed
    1. Iossifov I, O'Roak BJ, Sanders SJ, Ronemus M, Krumm N, Levy D, et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature. 2014;515(7526):216–221. doi: 10.1038/nature13908. - DOI - PMC - PubMed

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