DOI: 10.5061/dryad.3bk3j9kxv
title: Data from: Cell type-specific dysregulation of gene expression due to Chd8 haploinsufficiency during mouse cortical development

description: 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.
description: The following text describes the methods sections associated with
 the data in this Dryad submission. For details regarding the generation of
 the <em>Chd8</em><sup>+/-</sup>* *line or our
 transcriptomics data and analyses, please refer to the manuscript, the
 associated GitHub page, or the GEO submissions for these
 datasets. <em>Animal breeding and tissue
 preparation for immunohistochemistry (IHC), imaging, and signal
 quantification</em> 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).
 <em>Western blotting</em> 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
 <em>t</em>-test, performed on the actin-normalized CHD8 signal
 (Fig. 1C, Table S1). For sex- and blot-matched wild type and
 <em>Chd8</em><sup>+/–</sup> 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).
description: # Data from: Cell-type-specific dysregulation of gene expression due to
 Chd8 haploinsufficiency during mouse cortical development
 [https://doi.org/10.5061/dryad.3bk3j9kxv](https://doi.org/10.5061/dryad.3bk3j9kxv) ## Description of the data and file structure These files contain the raw Western blot and immunohistochemistry (IHC) image data used for figure generation and CHD8 quantification in the embryonic wild type and *Chd8*+/- mouse cortex, as described in Yim et al. *Cell Genomics* 2025. For the IHC data, raw ZVI image files and exported image files of the merged and individual channels are included, organized by time point: E12.5, E14.5, E16.0, and E17.5. Image file names include the litter ID (C#-#), embryo ID (P#), slide number (S#), and image ID information. For images of individual channels, file names include the immunostained protein or nuclear stain in that channel (e.g., `_CHD8`, `_Hoechst`). ## Files and their structure ### Western_blots.tar.gz **Description:** Western blot images used for CHD8 and actin quantification in the embryonic day (E) 16.0 cortex alongside quantification report PDFs output from Image Lab v6.1 (Bio-Rad). These files are associated with Figure 1, Figure S1, and Table S1 from Yim et al. 2025. File names indicate the date of signal detection for each Western blot (blot date), and lanes are labeled by sample ID, which includes the genotype and sex of the embryo. Exposure time for signal detection is provided on each image. **File structure:** Files are organized into two directories: `Full_images/`, which contains the raw full Western blot images, an image of the protein ladder for the 06032025 blot (`_ladder` suffix), and the rotated and white-corrected image file for the 06032025 blot, which was used for quantification (`_white-corrected` suffix); and `Quantification/`, which contains the quantification report PDF outputs from the Image Lab software for each Western blot (color inverted). #### Abbreviations * WT: wild type * Het: *Chd8*+/- * F: female * M: male ### Paired_WT_Het_CHD8_IHC.tar.gz **Description:** Image data for IHC performed in paired wild type (WT) and *Chd8*+/- (Het) mouse cortical sections. These images are associated with Figure 1E and Figure S7 from Yim et al. 2025. **File structure:** Image files are organized into subdirectories by time point: `E12.5/`, `E14.5/`, `E16.0/`, or `E17.5/`. Image files from litter-matched pairs of wild type (WT) and *Chd8*+/- (Het) embryos share the same litter ID (C#-#) in their file name. Genotype information is appended to each image file name. ### WT_CHD8_IHC_quantified_images.tar.gz **Description:** Image data used for CHD8 quantification from the embryonic wild type (WT) cortex and the table of quantified signal values, corresponding to Table S2 in Yim et al. 2025. These image files are also associated with Figure 1D, Figure S5, and Figure S6 in Yim et al. 2025. The scripts used for signal quantification are available on the Github page for this paper: [https://github.com/NoonanLab/Yim_et_al_Chd8](https://github.com/NoonanLab/Yim_et_al_Chd8). For all files, CHD8 signal is in the red channel (Cy3.5) and Hoechst 33342 in the blue channel (DAPI). For the following image files in the subdirectories within `Full_images/`, sections were also immunostained for either TBR1 or calretinin in the green channel (Cy2), specified in parentheticals below: * `E14.5/C15-7-P1-S1a-2018-0029.zvi` (TBR1) * `E14.5/C15-7-P1-S1a-2018-0034.zvi` (TBR1) * `E14.5/c15-7-P3-SA-2018-0020.zvi` (calretinin) * `E14.5/c15-7-P3-Sb-2018-0033.zvi` (calretinin) * `E14.5/c15-7-p9-sa-2018-0042.zvi` (TBR1) * `E17.5/c15-3-P4-SA-2018-0004-2.zvi` (TBR1) * `E17.5/c15-3-p6-sb-2018-0019.zvi` (calretinin) * `E17.5/c15-3-p6-sb-2018-0037.zvi` (calretinin) **File structure:** Image files are organized into two directories: `Full_images/`, which contains the original ZVI files and exported, uncropped images; and `Quantified_columns/`, the cropped images spanning the apicobasal cortex that were used as input into the quantification scripts described in the paper. These image files are further divided into four subdirectories based on time point (`E12.5/`, `E14.5/`, `E16.0/`, and `E17.5/`). ### WT_IHC_images.tar.gz **Description:** Raw image data for IHC performed in embryonic wild type (WT) mouse cortical sections, corresponding to panels in Figure 1, Figure 3, Figure S3, Figure S4, and Figure S5 in Yim et al. 2025. This file includes data for CHD8, PAX6, POGZ, and TBR1 immunostaining. Embryonic age is indicated in each image file name (E12.5, E14.5, E16.0, or E17.5). **File structure:** Image files are organized into directories based on the name of their associated figure: `Figure_1D/`, `Figure_3/`, `Figure_S3/`, and `Figure_S4/`. Image files in the `Figure_1D/` directory also correspond to Figure S5 in Yim et al. 2025. For the `Figure_3/` directory, image file names are appended with the IHC target protein: CHD8, PAX6, POGZ, or TBR1. For the `Figure_S3/` directory, image files are further organized into subdirectories based on their associated figure panel: `Panel_A/` and `Panel_B`, corresponding to negative controls performed using secondary antibody alone or CHD8 immunostaining by sex, respectively. File names in `Panel_B/` are appended with the sex of the imaged wild type embryo.