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

Disruptive variants in the chromodomain helicase CHD8, which acts as a transcriptional regulator during neurodevelopment, are strongly associated with risk for autism spectrum disorder (ASD). CHD8 haploinsufficiency is hypothesized to contribute to ASD by perturbing neurodevelopmental gene expression. However, insight into the cell-type-specific transcriptional effects of CHD8 haploinsufficiency remains limited. We utilized single-cell and single-nucleus RNA sequencing to globally profile gene expression and identify dysregulated genes in the embryonic and juvenile wild-type and Chd8+/− mouse cortex, respectively. Chd8 and other ASD risk-associated genes showed a convergent expression trajectory that was largely conserved between the mouse and human developing cortex, increasing from the progenitor zones to the cortical plate. Genes associated with risk for neurodevelopmental disorders and genes involved in neuron projection development, chromatin remodeling, signaling, and migration were dysregulated in Chd8+/− embryonic day (E) 12.5 radial glia. Genes implicated in synaptic organization and activity were dysregulated in Chd8+/− postnatal day (P) 25 deep- and upper-layer excitatory cortical neurons, suggesting a delay in synaptic maturation or impaired synaptogenesis. Our findings reveal a complex pattern of transcriptional dysregulation in the Chd8+/− developing cortex, potentially with distinct biological impacts on progenitors and maturing neurons in the excitatory neuronal lineage.

Methods
The following text describes the methods sections associated with the data in this Dryad submission. For details regarding the generation of the Chd8+/-* *line or our transcriptomics data and analyses, please refer to the manuscript, the associated GitHub page, or the GEO submissions for these datasets.

Animal breeding and tissue preparation for immunohistochemistry (IHC), imaging, and signal quantification

Embryos were collected from timed pregnancies at E12.5, E14.5, E16.0, and E17.5 (E0.5 = vaginal plug date). Embryonic brains were dissected, immersion-fixed in 4% paraformaldehyde for 18-24 hours, and cryoprotected sequentially in 15% and 30% sucrose solution for 24 hours and 48-72 hours, respectively. The brains were frozen in Tissue-Tek OCT Compound (Electron Microscopy Sciences, #62550) on a dry ice-ethanol slurry, stored at -80°C, and cryosectioned into 30μm coronal or sagittal sections (Leica Biosystems, CM3050 S). For IHC, sections were hydrated in PBS, permeabilized in 0.3% Triton X-100 in PBS, and blocked in normal donkey serum (NDS; PBS with 5% NDS and 0.3% Triton X-100) for 15 minutes each. Sections were then incubated in primary antibody solution (PBS with 5%-7.5% NDS and 0.3% Triton X-100) in a humid chamber at room temperature for 23 hours. Following three 10-minute washes with PBS, sections were incubated with one of two Cy3 fluorophore-conjugated secondary antibodies raised in donkey hosts (Jackson ImmunoResearch; anti-rabbit: #711-165-152; anti-chicken: #703-165-155; 1:300) and Hoechst 33342 (ThermoFisher Scientific; 1:300) at room temperature, then washed with PBS (3 x 10 minutes). Sections were mounted with ProLong Gold Antifade Mountant (ThermoFisher Scientific, P10144). The following primary antibodies were used: CHD8 C-terminus (Abcam, ab84527, rabbit, 1:1,000 for E12.5, E14.5, E16.0; 1:600-1:750 for E17.5), POGZ (Abcam, ab167408, rabbit, 1:750), TBR1 (EMD Millipore, AB2261, chicken, 1:500-1:750), and PAX6 (EMD Millipore, AB2237, rabbit, 1:750). Immunostained specimens were imaged on a Carl Zeiss AxioCam MRm coupled to an Axioimager Z2 epifluorescence microscope equipped with the ApoTome2 imaging system (Carl Zeiss Microimaging). Image processing was performed using Carl Zeiss Axiovision and Carl Zeiss ZEN LE software to exclude 2% of black pixels and 0.01% of white pixels for each channel (default settings for the “Best Fit” feature in ZEN LE). For images with bright non-cellular artifacts, such as secondary antibody aggregates, 0.025% of white pixels were excluded for that channel.

For quantification of CHD8 and Hoechst signal across wild-type embryonic cortical development, we performed immunohistochemistry for CHD8 on 34 WT mice of both sexes (E12.5: n=9; E14.5: n=9; E16.0: n=6; E17.5: n=10; Fig. 1D & Table S2). For each animal, we obtained two coronal section images of the frontal cortex. All images were processed using the ZEN LE software to exclude 0.1% of pixels (“Best Fit” feature). From each image, we cropped three non-overlapping rectangular strips covering the entire cortical diameter in length at medial, lateral, and intermediate positions (width: 100 mm for E12.5-E16.0, 50 mm for E17.5). All strips were scaled to the same height (1000 pixels) and divided into 10 equal bins (Fig. 1D). Quantification of the signal was performed by a custom-written, unbiased algorithm measuring the overlap of the two channels. Within each bin, the signal of the red channel (CHD8) was normalized against that of the blue channel (Hoechst 33342) and plotted using GraphPad Prism version 8 for OS X (GraphPad Software, San Diego, CA, USA; Table S2).

Western blotting

Tissue extracts were obtained by lysing E16.0 mouse cortical samples in radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor solution (cOmplete, EDTA-free Protease Inhibitor Cocktail; Roche, #11873580001) and 100mM PMSF by rotation at 4°C for 2 hours followed by sonication at amplitude 20 for 4 minutes with alternating 10 second pulses and 10 second rests, performed at 4°C. The lysates were then cleared by centrifugation at 16,000 x g, and the total protein concentration was determined with the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, #23225). Lysates were mixed with Laemmli sample buffer, and equal amounts of protein samples were resolved on a 7.5% SDS-PAGE gel (7.5% Mini-PROTEAN TGX Precast Protein Gels, 10-well; Bio-Rad, #4561023). The proteins were transferred onto a PVDF or nitrocellulose membrane and blocked in 5% nonfat dry milk (NFDM) in TBS with 0.1% Tween-20 (TBS-T). For each primary antibody, the membrane was incubated overnight at 4°C, washed with TBS-T after each primary antibody incubation, and incubated with secondary antibodies conjugated to horseradish peroxidase for one hour at room temperature.

Primary antibodies used for immunodetection included anti-CHD8 (Abcam, ab114126, rabbit), diluted 1:1,000 in TBS-T with 5% BSA, and anti-actin (Abcam, ab8226, mouse or Santa Cruz, sc-47778, mouse), diluted 1:1,000 (Abcam) or 1:2,000 (Santa Cruz) in TBS-T with 5% NFDM. Horseradish peroxidase-conjugated secondary antibodies used included donkey anti-rabbit secondary antibody (Cytiva, NA934) and sheep anti-mouse secondary antibody (Cytiva, NA931), both diluted 1:10,000 in TBS-T with 5% NFDM. Membranes were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, #34096). Western blot quantification was performed on the scanned films or images based on densitometry measured in Image Lab v6.1 (Bio-Rad). For CHD8, only the ~290KDa band corresponding to wild-type CHD8 was quantified. Actin was used as a loading control and for by-replicate normalization. Statistical significance was then determined by one-tailed Welch’s t-test, performed on the actin-normalized CHD8 signal (Fig. 1C, Table S1). For sex- and blot-matched wild type and Chd8+/– comparisons, these actin-normalized CHD8 signal values were then divided by the actin-normalized CHD8 signal in the corresponding sex- and film-matched wild type control (Table S1).