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Germline Chd8 haploinsufficiency alters brain development in mouse

Abstract

The chromatin remodeling gene CHD8 represents a central node in neurodevelopmental gene networks implicated in autism. We examined the impact of germline heterozygous frameshift Chd8 mutation on neurodevelopment in mice. Chd8+/del5 mice displayed normal social interactions with no repetitive behaviors but exhibited cognitive impairment correlated with increased regional brain volume, validating that phenotypes of Chd8+/del5 mice overlap pathology reported in humans with CHD8 mutations. We applied network analysis to characterize neurodevelopmental gene expression, revealing widespread transcriptional changes in Chd8+/del5 mice across pathways disrupted in neurodevelopmental disorders, including neurogenesis, synaptic processes and neuroimmune signaling. We identified a co-expression module with peak expression in early brain development featuring dysregulation of RNA processing, chromatin remodeling and cell-cycle genes enriched for promoter binding by Chd8, and we validated increased neuronal proliferation and developmental splicing perturbation in Chd8+/del5 mice. This integrative analysis offers an initial picture of the consequences of Chd8 haploinsufficiency for brain development.

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Figure 1: Chd8+/del5 mouse model.
Figure 2: Chd8+/del5 mice exhibit cognitive deficits but no ASD-relevant social or repetitive phenotypes.
Figure 3: Chd8 haploinsufficiency drives megalencephaly and cognitive impairment in mouse.
Figure 4: Differential gene expression in Chd8+/del5 neurodevelopment.
Figure 5: DE genes with correlated expression patterns across brain development reveal perturbations to early and later neurodevelopmental pathways.
Figure 6: An early neurodevelopmental expression network (M.1) regulated by Chd8 haploinsufficiency involved in chromatin modification, RNA processing and cell cycle.
Figure 7: Chd8+/del5 mutants exhibit altered cortical neurogenesis at E14.5.
Figure 8: RNA processing pathways are enriched for differentially expressed genes in Chd8+/del5 mice.

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Acknowledgements

Sequencing was performed at the UC Berkeley and UC Davis DNA cores. This work was supported by institutional funds from the UC Davis Center for Neuroscience, by the UC Davis MIND Institute Intellectual and Developmental Disabilities Research Center (U54 HD079125) and by NIGMS R35 GM119831. L.S.-F. was supported by the UC Davis Floyd and Mary Schwall Fellowship in Medical Research and by grant number T32-GM008799 from NIGMS-NIH. A.A.W. was supported by Training Grant number T32-GM007377 from NIH-NIGMS. R.C.-P. was supported by a Science Without Borders Fellowship from CNPq (Brazil). A.V., L.A.P. and D.E.D. were supported by National Institutes of Health grants R24HL123879, U01DE024427, R01HG003988, U54HG006997 and UM1HL098166. Research conducted at the E.O. Lawrence Berkeley National Laboratory was performed under Department of Energy Contract DE-AC02-05CH11231, University of California. J.E. and J.P.L. were supported by the Canadian Institute for Health Research, Brain Canada and the Ontario Brain Institute.

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Authors

Contributions

A.L.G., L.S.-F., J.E., N.A.C. and M.A.R. are listed as joint first authors, as each led components of the experiments and analysis. A.L.G., L.S.-F., J.E., M.A.R., N.A.C., J.P.L., J.N.C., J.L.S., K.S.Z. and A.S.N. designed the experiments. Generation of mouse model: A.S.N., D.E.D., A.V., L.A.P., B.J.M., I.P.-F. and V.A.; mouse behavior: N.A.C., M.C.P., M.D.S., J.N.C. and J.L.S.; mouse MRI: J.E. and J.P.L.; genomics and molecular genetics: L.S.-F., A.L.G., I.Z., A.A.W., R.C.-P., S.L., B.J.M. and A.S.N.; neuroanatomy: A.L.G., M.A.R., T.W.S., I.Z., G.K., K.S.Z. A.L.G., L.S.-F., J.E., N.A.C., M.A.R., K.S.Z., J.N.C., J.L.S. and A.S.N. drafted the manuscript. All authors contributed to manuscript revisions.

Corresponding author

Correspondence to Alex S Nord.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Raw western blots.

Western blots of Chd8 and Gapdh protein in adult 5-bp del and 14-bp del Chd8 mutant lines show decreased protein levels in Chd8+/del5 mice from both alleles. WT n = 3 for 280 kDa, WT n = 4 for 110 kDa, 5-bp n = 3, 14-bp n = 3. There is a significant difference in Chd8 protein abundance in Chd8+/del5 mice at 280 kDa (ANOVA *P = 0.0118, 5-bp *P = 0.0319; 14-bp *P = 0.0328) but not at 110 kDa (ANOVA P = 0.1676, 5-bp P = 0.1086, 14-bp P = 0.4509). B. Chd8 and Gapdh protein expression in WT and Chd8+/del5 (HT) mice at P0. There is a significant difference in Chd8 protein abundance in Chd8+/del5 mice at 280 kDa (n: WT = 9, Chd8+/del5 = 9; *P = 0.0089) and 110 kDa (**P = 0.001). C. Chd8 and Gapdh protein expression in Chd8+/del5 (HT) and WT mice at E14.5. There is a significant difference in Chd8 protein abundance in Chd8+/del5 mice at 280 kDa (n: WT = 6, Chd8+/del5 = 6; *P = 0.02) but not at 110 kDa (P = 0.9903). D. Raw Hnrnpa2b1 western blot from P0 forebrain. One-way ANOVA followed by unpaired post hoc t-tests used in A. Unpaired t-tests used for B-C. Error bars represent mean ± s.e.m.

Supplementary Figure 2 Chd8+/del5 mice from the replication cohort exhibit cognitive deficits, but no ASD social or repetitive phenotypes.

Chd8+/del5 mice exhibit deficits in learning and memory, including reduced freezing after fear conditioning in both context (A; t(1, 40) = 1.6607, P = 0.1046) and cued assays (B; t(1, 40) = 1.9593, P = 0.0571). Novel Object: Chd8+/del5 mice fail to show significant difference in exploration between a novel and familiarized object (C; WT F(1, 20) = 4.5583, P = 0.0453; Chd8+/del5 F(1, 19) = 0.7921, P = 0.3846). Social Approach: Chd8+/del5 mice do not exhibit differences relative to WT littermates in time spent in chamber with novel mouse (D; WT: F(1, 20) = 10.438, P = 0.0042; Chd8+/del5: F(1, 20) = 15.470, P = 0.0008), time sniffing a novel mouse (E; WT: F(1, 20) = 20.3750, P = 0.0002; Chd8+/del5: F(1, 20) = 30.3946, P = 0.00002), or chamber entries (F; F(1, 40) = 0.307, P = 0.583). Male-Female Social Interactions: Chd8+/del5 mice exhibit no differences between WT littermates in time spent engaged in nose to nose sniffs (G; t(1, 20) = 0.1520, P = 0.8807), nose to anogenital sniffs (H; t(1, 20) = 0.2492, P = 0.8057), time following (I; t(1, 20) = 1.6396, P = 0.1167), or ultrasonic vocalizations (J; t(1, 20) = 0.7010, P = 0.4914) with an estrus female. Repetitive Behavior: Chd8+/del5 mice do not exhibit any differences in time spent self-grooming (K; t(1, 40) = 0.2489, P = 0.8047). Open Field: Chd8+/del5 mice do not exhibit any differences in distance traveled (L; t(1, 40) = 0.9799, P = 0.3328). All data shown from 2nd cohort. Male mice were used in G-J; males and females were used in all other panels. Unpaired t-tests used for A-B, G-L; within genotype repeated measures ANOVA used for C-E; one-way ANOVA used for F. Error bars represent mean ± s.e.m.

Supplementary Figure 3 Fractional anisotropy (FA) differences in several coronal slices throughout the brain.

Highlighted are FA differences measured as effect size between the Chd8+/del5 brains and corresponding WT brains thresholded at 1.0. Of interest is the lateral decrease and corresponding medial increase in FA surrounding the cerebral peduncle. It should be noted that these differences are only indicative of trends as no significant differences were found in our FA measures.

Supplementary Figure 4 qRT-PCR validation of differential mRNA expression at P0.

A-J. Data from 7 WT and 5 Chd8+/del5 (HT) P0 forebrains (with the exception of G, which used 7 Chd8+/del5 P0 forebrains), with reactions performed in triplicate. For each target, the one highest and one lowest values were removed from analysis to reduce noise. P-values from Student’s t-test. Primers reported in Table S4. A-J WT n: 6, 4, 6, 6, 6, 6, 7, 6, 6, 6; Chd8+/del5 n: 7, 4, 5, 5, 4, 5, 7, 5, 5, 4.

Supplementary Figure 5 Chd8 RNA-seq and ChIP-seq analyses.

A. Results from RSAT de novo motif analysis on our Chd8 ChIP-seq peaks. Highlighted are three representative results for significant YY1, NRF1, and NFYB motifs (left), and corresponding predicted motif sites within Chd8 ChIP-seq peaks (middle) and the identified motif (right). B-C. Comparison of DE genes in our RNA-seq data with two recent Chd8 models, Katayama et al. (2016) (germline heterozygous Chd8), and Durak et al. (2016) (Chd8 knockdown). Dots represent genes passing FDR < 0.05 in our data and in either Katayama et al. (2016) or Durak et al. (2016) compared to our model (n = 37 and 77, respectively). The red dot indicates Chd8. DE genes in our data are significantly enriched in both datasets, but overlap between our data is stronger with Durak et al. (2016) than with Katayama et al. (2016). D. Differential expression of example synaptic genes. Top panels show expression (log2RPKM) across stages, bottom shows first four early developmental stages only. From left to right: Scn2b, Cacna1e, Cacna2d1, Cacna1b. Solid line = mean, dashed line = ±1 s.e.m. Black: WT; Red: Chd8+/del5.

Supplementary Figure 6 P0 and P7 coronal sections reveal no obvious perturbations to cortical lamination.

Single optical sections of wild type (A-D, I-M) and Chd8+/del5 (E-H, N-R) brains were imaged at 4X and paired based on corresponding anatomical depth of section. Immunofluorescence corresponding to Ctip2 (green; B, F, J, O), Tbr1 (red; C, G, K, P), and Brn2 (magenta; L, Q) was collected via sequential imaging and merged with DAPI (blue; D, H, M, R). Scale bars in E and N are 500 μm. P0: Tbr1 thickness P = 0.34, Ctip2 thickness P = 0.81, P7: Tbr1 thickness P = 0.88, Ctip2 thickness P = 0.69, Brn2 thickness P = 0.32. P-values derived from Student’s t-test. P0: WT n = 8, Chd8+/del5 n = 10; P7: WT n = 7, Chd8+/del5 n = 10.

Supplementary Figure 7 Changes in splicing in Chd8+/del5 mice.

A. DE down-regulated genes are enriched in DS genes, but not up-regulated DE genes. DS genes from our data overlap significantly with DS genes identified in a recent postmortem ASD cortex study25. B. (Left) Genome Browser representation of the alternatively spliced Srsf7 intron (chr17:80,604,601-80,603,728). (Right) Isoform-specific qRT-PCR analysis of the Srsf7 intron across development in WT and e17.5 Chd8+/del5 (HT) mice. n: e12.5 = 7, e14.5 = 9, e17.5 Chd8+/del5 = 7, e17.5 WT = 7, P0 = 11. Student’s t-test, e14.5 WT to e17.5 Chd8+/del5, P = 0.30, e17.5 Chd8+/del5 to e17.5 WT, P = 0.62. Linear regression across developmental time points, adjusted R2 = 0.37, P = 1.32E-05. Error bars represent mean ± s.e.m.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1819 kb)

Supplementary Methods Checklist (PDF 721 kb)

Supplementary Table 1

Summary of MRI volume by regions (XLSX 61 kb)

Supplementary Table 2

RNA sample and library data (XLSX 45 kb)

Supplementary Table 3

Differential expression summary table (XLSX 12110 kb)

Supplementary Table 4

Primers used for qPCR experiments (XLSX 47 kb)

Supplementary Table 5

Summary of GO enrichment (XLSX 94 kb)

Supplementary Table 6

Enrichment summary for Reactome pathways (XLSX 285 kb)

Supplementary Table 7

WGCNA results (XLSX 48 kb)

Supplementary Table 8

MISO results (XLSX 2669 kb)

Supplementary Software

Analysis scripts (ZIP 8 kb)

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Gompers, A., Su-Feher, L., Ellegood, J. et al. Germline Chd8 haploinsufficiency alters brain development in mouse. Nat Neurosci 20, 1062–1073 (2017). https://doi.org/10.1038/nn.4592

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