WNT signalling control by KDM5C during development affects cognition – Nature

Reprogramming of patient-derived fibroblasts

Patient-derived fibroblasts were cultured in DMEM (Life Technologies, 11995073) supplemented with 10% FBS (Invitrogen, 12676011), 2 mM l-glutamine (Corning, 25-005-CI), 1×MEM non-essential amino acids (Invitrogen, 11140050) and 100 U ml^–1 penicillin–streptomycin (Corning, 30-002-CI).

Reprogramming was performed using non-integrating episomal plasmid vectors³⁷. In brief, 800,000 fibroblasts were washed in PBS and then resuspended in Amaxa nucleofector solution (Nucleofector kit for human dermal fibroblast; VPD-1001) with plasmids encoding OCT4, SOX2, KLF4, LMYC and LIN28A together with TP53 shRNA (Addgene, 27077, 27078 and 27080). Cells were treated with Program P22 of an Amaxa Nucleofector 2 device. Cells were plated onto a well of a 6-well plate and cultured for 6 days in fibroblast medium. On day 6, cells were dissociated in 0.05% trypsin, transferred onto 0.1% gelatin-coated 10 cm plates on irradiated mouse embryonic fibroblast (MEF) feeder cells CF-1 (GlobalStem, GSC-6201G) and cultured for 14 days in DMEM/F12 and 15 mM HEPES (StemCell Technologies, 36254) supplemented with 20% KnockOut serum replacement (Invitrogen, 10828-028), 100 mM 2-mercaptoethanol (Gibco, 21985-023), 2 mM l-glutamine (Corning, 25-005-CI), 1×MEM non-essential amino acids (Invitrogen, 11140050) and 10 ng ml^–1 bFGF (Bio Pioneer, HRP-0011-1). On day 21, smooth compact colonies were manually picked onto 24-well plates and expanded. Human iPS cell lines were investigated for expression of pluripotency markers (immunofluorescence and qPCR), lack of EBNA1 DNA integration and normal karyotype^37,38.

Generation of isogenic controls

We used CRISPR technologies to generate double-strand breaks at the mutation site to increase the frequency of homologous recombination. Simultaneously, we provided a correction template for homologous recombination that utilizes PiggyBac technology to seamlessly correct the mutation. The correction template contains arms of homology for the region, corrects the mutation and introduces a drug-selection cassette flanked by repeats recognized by PiggyBac transposase. Once iPS cell lines that have undergone homologous recombination have been selected and identified, the selection cassette was removed using a hyperactive form of the PiggyBac transposase. A detailed description of the methodology has been previously published³⁹.

Human iPS cell culture

Human iPS cells were cultured on irradiated MEF feeder cells CF-1 (GlobalStem, GSC-6201G) in DMEM/F12 and 15 mM HEPES (StemCell Technologies, 36254) supplemented with 20% KnockOut serum replacement (Invitrogen, 10828-028), 100 mM 2-mercaptoethanol (Gibco, 21985-023), 2 mM l-glutamine (Corning, 25-005-CI), 1×MEM non-essential amino acids (Invitrogen, 11140050) and 10 ng ml^–1 bFGF (Bio Pioneer, HRP-0011-1). Cells were fed and inspected daily, and differentiated cells at the edges of colonies were manually removed. Cells were passaged either manually or with collagenase (Invitrogen, 17104-019). iPS cells were routinely karyotyped and tested for mycoplasma contamination.

Neuronal differentiation

Before neuronal differentiation was started, cells were inspected daily and differentiated cells were manually removed. At least two passages before differentiation, cells were cultured at the same densities and colony size. On the day of differentiation, differentiated cells were removed and MEFs were lifted from the cultures. The time of manipulation between each line was kept identical because the timing of preparation influences the general cell state and the plating efficiency. During the cleaning and preparation stage, cells were cultured in MEF-conditioned medium supplemented with Y-27632 (Selleck Chemicals, S1049) at 10 μM. The conditioned medium containing Y-27632 was added to all cultures at and for the exact same time. The neuronal differentiation protocol was performed according to a previously published method⁴⁰ with modifications designed by V.K.-N., which gave excellent reproducibility.

The neuronal maintenance medium⁴⁰ comprised a 1:1 mixture of N-2 (Gibco, 17502048) and B-27 (Gibco, A35828-01). N-2 medium consists of DMEM/F-12 (Fisher Scientific, mt10092cv), 1× N-2, 5 µg ml⁻¹ insulin (Sigma-Aldrich, I9278-5M), 1 mM l-glutamine (Corning, 25-005-CL), 100 µm non-essential amino acids (Invitrogen, 11140050), 100 µM 2-mercaptoethanol (Gibco, 21985-023), 50 U ml⁻¹ penicillin and 50 mg ml⁻¹ streptomycin (Corning, 30-002-CL). B-27 medium consists of neurobasal (Life Technologies, 21103-049), 1× B-27, 200 mM l-glutamine, 50 U ml⁻¹ penicillin and 50 mg ml⁻¹ streptomycin.

The neuronal induction medium⁴⁰ comprised neural maintenance medium supplemented with 1 µM dorsomorphin (Sigma-Aldrich, P5499-5MG) and 10 µM SB431542 (Selleck Chemicals, S1067).

Neuronal differentiation in the context of rescue experiments

Two ways to perform the rescue experiment related to the data presented in the paper are described here. Cells were cultured until day 5 of differentiation in neural induction medium⁴⁰. On day 5, cells were passaged using collagenase (Invitrogen, 17104-019) at a one-quarter ratio in neuronal induction medium. On the following day, once all cells settled down, the medium was changed to neuronal induction medium supplemented with vehicle, WIF1 (R&D Systems, 1341-WF-050, final concentration of 1 µg ml^–1), IWP2 (Sigma-Aldrich, I0536-5MG) or WNT3A (R&D Systems, 5036-WN-010/CF; final concentration of 200 ng ml^–1). The medium volume was calculated for 3 days. After the first incubation, cells were washed 2 times with neuronal induction medium. Cells were then replated in neuronal induction medium overnight and were incubated again with the compounds at the same concentrations but in neuronal maintenance medium for the subsequent 3 days. In total, compounds were added only twice into the medium for the entire time of treatment. After the incubation time, cultures were washed once in PBS and twice in neuronal maintenance medium and cultured according to the protocol in standard neuronal medium (neuronal maintenance medium).

As IWP2 is an effective compound, it can be used at high cell densities. In this case, cells do not need to be passaged at day 5 or 6 but can be maintained until day 9 with a supplementation of IWP2 at a concentration of 1 µM to the neuronal induction medium from day 6 to day 9 (only added once early at day 6). After this period of time, cells were washed and replated in neuronal induction medium. On the following day, medium was changed to neuronal maintenance medium as described above and cells were cultured in these conditions for an additional day. Thereafter, cells were treated for a further 3 days with 0.25 µM of IWP2 (only added once to the culture with no daily supplementation) in neuronal maintenance medium. The same regimen was performed for WIF1. In this case WIF1 concentrations were increased to adapt to the higher cell densities and therefore higher WNT ligand production.

Immunostaining iPS cells

Human iPS cells were carefully washed with 1×PBS and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. PFA was washed out 3 × 10 min. Permeabilization was performed with 0.1% Triton X-100 in PBS for 5 min and cells were blocked with 3% serum (the same as the secondary antibody), 1% BSA in PBS 0.1% Triton X-100 at room temperature for at least 1 h. Incubation with primary antibodies diluted in blocking solutions was performed overnight at 4 °C. Cells were washed 3 × 10 min in PBS, and secondary antibodies were incubated in blocking solution for 1 h. Secondary antibodies were used at 1:1,000 dilutions (Alexa Fluor, Invitrogen). Cells were counterstained with DAPI or Hoechst (indicated in figures). The antibodies used in this study are shown in Supplementary Table 1.

Immunostaining E13.5 embryos

Kdm5c WT and Kdm5c KO¹³ E13.5 male littermates were decapitated, and whole heads were washed twice in 1×PBS and fixed with 4% PFA overnight. After incubation, heads were washed 3 × 10 min in 1×PBS and incubated in 20% sucrose overnight. The next day, heads were embedded in OCT and snap-frozen by immersion in 2-methylbutane cooled on dry ice. Cryosection was performed using a Leica CM3050 S Cryostat. Serial 14 µm coronal sections were made throughout the whole cortex and slides were mounted onto charged SuperFrost Plus slides. To ensure complete collection of cortical tissue, no trimming was performed. The total number of labelled cells per fixed field per section was calculated. Results are expressed as the mean value of marker⁺ cells per field ± s.d. and were tested for significance using two-sided unpaired Student’s t-test. P < 0.05 was considered significant.

For immunostaining, slides were washed in PBS, permeabilized for 5 min in 0.04% Tween-20 in PBS and 5% serum and blocked for 2 h in the same solution. Slides were then incubated with primary antibodies diluted in 5% goat serum, 0.3% Triton X-100 in PBS overnight. The next day, primary antibodies were washed out with PBS (3 × 10 min) and incubated for 1 h at room temperature with secondary antibodies at 1:1,000 dilutions (Alexa Fluor, Invitrogen). Finally, slides were washed in PBS and coverslipped with Fluoromount-G (Southern Biotech). The antibodies used are shown in Supplementary Table 1.

Images were acquired using a Nikon Eclipse TE2000-U inverted fluorescence microscope.

Western blot analysis

Whole cell lysates were prepared using RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and 25 mM Tris pH 7.4). Lysates were run on an 8% SDS–PAGE gel and transferred to nitrocellulose membranes (Bio-Rad, 1620112). Membranes were blocked for 1 h in 0.05% Tween and PBS (PBST) with 5% non-fat dry milk, then incubated overnight with primary antibody (1:1,000 dilution of primary antibody in PBST with 5% non-fat dry milk). The next day, membranes were washed (3 washes of 5 min each with 10 ml of PBST), incubated with HRP-conjugated secondary antibodies for 1 h in PBST with 5% non-fat dry milk, then washed again (3 washes of 5 min each with 10 ml of PBST). HRP-conjugated antibodies were detected using ECL western blotting detection reagents (PerkinElmer Western Lighting Plus-ECL, NEL104001 EA) according to the manufacturer’s instructions. Full scan blots are available in Supplementary Fig. 1.

Subcellular fractionation and immunoblotting

Subcellular fractionation was performed using a previously published protocol⁴¹ with modifications. The following buffers were used: cytosolic fraction buffer: 150 mM NaCl (Invitrogen, AM9759), 50 mM Tris pH 7.5 (Invitrogen, 15567-027), 20 μg ml^–1 digitonin (TCI, D0540); membrane fraction buffer: 150 mM NaCl, 50 mM Tris pH 7.5, 1% NP40 (Sigma, I3021); nuclear fraction buffer (RIPA): 150 mM NaCl, 50 mM Tris pH 7.5, 0.5% sodium deoxycholate (Sigma, D6750), 0.1% SDS (Fisher BioReagents, BP166-500) and 250 U ml^–1 of Universal Nuclease for Cell Lysis (Pierce, 88701). Protease and phosphatase inhibitors (Pierce, A32961) were added immediately before use.

The entire process of subcellular fractionation was performed at 4 °C. Cells were washed with 1×PBS and incubated with cytosolic fraction buffer for 5 min. Cells were resuspended and spun down for 10 min at 2,000g. The supernatant was collected as the cytosol-enriched fraction. The pellet was washed once with 1×PBS and resuspended with membrane fraction buffer for 30 min. Cells were spun down for 10 min at 7,000g. The supernatant was collected as the membrane-enriched fraction. The pellet was washed once with 1×PBS and resuspended with nuclear fraction buffer for 1 h. Cells were vortexed and spun down for 10 min at 7,000g. The supernatant was collected as the nuclear-enriched fraction. Samples were denatured in Laemmli SDS sample buffer (Boston BioProducts, BP-111R) for 5 min at 97 °C, run on an SDS–PAGE and transferred to an Immobilon-P membrane (Millipore, IPVH85R). Membranes were blocked with 5% BSA (Jackson ImmunoResearch, 001-000-162) in TBST (Boston BioProducts, IBB-180). Primary and secondary antibodies (conjugated with HRP) were diluted in 5% BSA. Chemiluminescence was detected using ECL Prime western blotting detection reagent (Cytiva, RPN2236) on a ChemiDoc MP imaging system (Bio-Rad, 12003154). For samples from brother 1: 3 biological replicates were performed per day. For brother 2: day 14: n = 3 biological replicates; day 7 and day 30: n = 2 biological replicates. Full scan blots are available in Supplementary Fig. 1.

Dual luciferase assay

Cells were plated in 48-well plates and transfected with 225 ng Super TOPFLASH (Addgene, 12457) or FOPFLASH (Addgene, 12456) and 4.5 ng EF1α-Renilla⁴². Cells were transfected in triplicate using Lipofectamine 3000 (Invitrogen, L3000015). Dual luciferase reporter assays were performed using a Dual-Luciferase Reporter assay system (Promega, E1960) according to the manufacturer’s instructions. Luminescence was measured using an EnSight Multimode plate reader (PerkinElmer). For clearer presentation, values from mutant cells were set to 1, and values for corrected clones were calculated to the mutant value = 1. Error bars for luciferase represent the s.d.; n = 3 biological replicates. Significance was determined using Student’s t-test (values are presented in the figure legends).

RNA analysis

Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Life Technologies, 15596018). cDNA was synthesized using a PrimeScript RT reagent kit (Takara Bio, RR037Q) and qPCR analysis was performed on a LightCycler 480 (Roche) with cDNA equivalent to 200 ng total RNA. The SYBR Green (Roche, LightCycler480 SYBR Green I Master, 04887352001) protocol consisted of denaturation at 95 °C for 5 min followed by 45 cycles of 95 °C, 10 s; 60 °C, 10 s and single 72 °C with a single data acquisition during each extension cycle. Primers for qPCR are listed in Supplementary Table 2.

Gene expression was normalized to endogenous GAPDH expression, or the relative expression of the respective gene was determined after normalization to GAPDH and calculated using the following formula: relative expression level = 2ΔΔCT. Subsequently, for clearer presentation, values from mutant cells were set to 1, and values from corrected clones were calculated to mutant value = 1. All graphs containing the label ‘relative mRNA levels’ were calculated based on these considerations. Error bars in all figures represent the s.d.; n = 3–4 biological replicates (exact numbers are presented in the figure legends). Significance was determined using Student’s t-test (values are presented in figure legends). Graphs were generated using GraphPad Prism (v.10.0.3).

RNA-seq library preparation

RNA library preparation for data presented in Fig. 2 and Extended Data Fig. 5 was performed using a NEBNext Ultra Directional RNA Library Prep kit for Illumina according to the manufacture’s guidelines (New England BioLabs, E7420L, E7490L and E7335L). Multiplexed libraries were pooled in equimolar ratios and were purified from a 1.5% TBE-agarose gel using a PureLink Quick Gel extraction kit (Invitrogen, K2100-12). The libraries were sequenced to a length of 50 bases using an Illumina HiSeq 2500, High Output v4 at the Tufts Genomics Core (Tufts University) according to standard procedures. Library quality was measured on a Bioanalyzer at the Tufts Genomics Core (Tufts University).

For RNA-seq, input RNA samples were first subjected to quality check using an Agilent Fragment Analyzer. Only RNA samples that passed quality control were then used for library preparation using an Illumina mRNA Sample Preparation kit per the manufacturer’s instructions. The molar concentrations of the resulting libraries were then quantified on the Fragment Analyzer, adjusted and mixed to equal molar mixture. The pooled libraries were sequenced on an Illumina NextSeq 550 using v.2.5 High Output chemistry and single-read 75 bases format. The base-calling and demultiplexing were performed on the raw data using Illumina bcl2fastq.

CUT&RUN

CUT&RUN for KDM5C was performed according to a Cell Signaling Cut&Run Assay kit (86652) with modifications developed by V.K.-N. Libraries were performed using a NEBNext UltraII DNA Library prep kit for Illumina according to the manufacture’s guidelines (New England Biolabs, E7645, E7335 and E7500). Multiplexed libraries were pooled in equimolar ratios. Library quality was measured on a Bioanalyzer at the Molecular Genetics Core at Boston Children’s Hospital. The libraries were sequenced on an Illumina NextSeq 500 System using a NextSeq 500/550 High Output kit v2.5 (75 cycles) at the Molecular Genetics Core at Boston Children’s Hospital.

ATAC–seq

Cryopreserved cells were sent to Active Motif. The cells were then thawed in a 37 °C water bath, pelleted, washed with cold PBS and tagmented as previously described⁴³, with some modifications based on ref. ⁴⁴. In brief, cell pellets were resuspended in lysis buffer, pelleted and tagmented using the enzyme and buffer provided in the ATAC–seq kit (Active Motif). Tagmented DNA was then purified using a MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR and purified using Agencourt AMPure SPRI beads (Beckman Coulter). The resulting material was quantified using a KAPA Library Quantification kit for Illumina platforms (KAPA Biosystems) and sequenced with PE42 sequencing on a NextSeq 500 sequencer (Illumina).

Bioinformatics

RNA-seq data

RNA-seq data relating to Fig. 2 and Extended Data Fig. 5 were mapped against the human genome version hg19 with STAR (v.2.5.2b)⁴⁵. R (v.3.4.1)⁴⁶ and Bioconductor (v.3.6)⁴⁷ were used for the RNA-seq analysis. Reads were counted using the R package GenomicAlignments (v.1.14.0)⁴⁸ (mode=‘Union’, inter.feature=FALSE) and only primary read alignments were retained. rlog-transformed values of the counts and differential expression values were calculated using DESeq2 (v.1.18.0)⁴⁹. Figure 2 was created using ggplot2 (v.2.2.1)⁵⁰.

The GSEA was done according to a previously published method⁵¹. GO analysis results were prepared using the goseq (v.1.28.0) package⁵².

For the data relating to Fig. 4 and Extended Data Fig. 7, the resulting demultiplexed data were aligned to the human reference genome hg38 using HISAT (v2.1.0). Read count normalization (FPKM) and differential expression analysis were performed using Cufflinks (v.2.1.0). The resulting normalized count table was modified and used as input for visualization and GSEA using Qlucore Omics Explorer (v.3.8)^53,54.

CUT&RUN data

CUT&RUN reads (Fig. 3 and Extended Data Fig. 6) were mapped to hg38 using Bowtie (v.2.3.5.1), with the parameters –no-unal –local –very-sensitive-local –no-mixed –no-discordant –phred33 -I 10 -X 700. Bigwig files were generated using BamCoverage in Deeptools (v.3.3.1), with the parameters –binSize 20 — normalizeUsing BPM. MACS (v.2.2.6) was used to call KDM5C peaks on each replicate individually, with the –nolambda parameter. Bedtools (v.2.28.0) was used to subtract blacklist regions (using bedtools subtract) and to identify peaks called in both replicates (using bedtools intersect). The blacklist file was downloaded from GitHub (https://github.com/Boyle-Lab/Blacklist/blob/master/lists/hg38-blacklist.v2.bed.gz). Peaks mapping to chrM and chrUn were removed, and peak annotation was performed using homer-4.11, with annotatePeaks.pl, and the fraction of peaks associated with each genomic feature was plotted in Prism 9. IGV version 2.11.9 was used to visualize data.

ATAC–seq data

ATAC–seq data ATAC–seq analysis for data relating to Fig. 4f and Extended Data Fig. 8b was performed by Active Motif. Reads were aligned using the BWA algorithm (mem mode; default settings). Duplicate reads were removed, and only reads mapping as matched pairs and only uniquely mapped reads (mapping quality &gt=1) were used for further analysis. Alignments were extended in silico at their 3′ ends to a length of 200 bp and assigned to 32-nucleotide bins along the genome. The resulting histograms (genomic ‘signal maps’) were stored in bigWig files. Peaks were identified using the MACS (v.2.1.0) algorithm at a cut-off of P = 1 × 10^–7, without control file, and with the –nomodel option. Peaks that were on the ENCODE blacklist of known false ChIP–seq peaks were removed. Signal maps and peak locations were used as input data to Active Motif’s proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations and gene annotations.

The remaining analysis was performed by the Harvard Chan Bioinformatics Core. First, quality assessment of the ATAC–seq data was performed using FASTQC (v.0.11.8) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), and the data were processed using the ATAC-seq pipeline bcbio-nextgen (v.1.2.8) that includes the following steps. Reads were filtered and trimmed using Atropos (v.1.1.29)⁵⁵. High-quality reads were mapped to the human genome (build GRCh38/hg38) using Bowtie2 (v.2.4.1)⁵⁶. Mitochondrial DNA reads were filtered from the dataset, and properly paired reads with high mapping quality (MAPQ score > 10, non-duplicates, qualified reads) were retained using Sambamba (v0.7.1)⁵⁷ for further analysis. The ‘alignmentSieve’ function of Deeptools (v.3.5.0)⁵⁸ and ‘sort’ and ‘index’ functions of Samtools (v.1.9)⁵⁹ were used to isolate fragments in nucleosome-free regions. Reads were shifted by 9 bp (+4 in positive and −5 in negative strand) to account for the dimeric binding of the Tn5 transposase that results in insertion of two adaptors separated by 9 bp. To call the peaks with unique reads, we used MACS2 (v.2.2.7.1)⁶⁰. ATAC–seq data quality was assessed using ataqv (v.1.2.1)⁶¹. CPM-normalized bigwig files (bin size = 20) were visualized using IGV (v.2.8.4)⁶². Sets of peaks were compared using BEDTools (v.2.27.1)⁶³. Statistical analysis was performed in R (v.3.6.1).

Differential accessibility was assessed using Diffbind (v.3.0.15) (http://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf) with DESeq2 (v.1.30.1)⁴⁹ and including batch in the model. Peaks were considered differentially enriched at FDR < 0.05. The genomic distribution of the peaks was annotated using ChIPseeker (v.1.26.2)⁶⁴. Functional enrichment analysis was performed using ClusterProfiler (v.3.18.1)⁶⁵.

Animal studies

All experiments involving animals were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Boston Children’s Hospital. All mice were housed in individually ventilated cages with a 12-h light–dark cycle and with ad libitum access to food and water. Mice were housed in temperatures of 18–24 °C with 40–60% humidity. Kdm5c KO mice were generated as previously described¹³.

In utero intracerebroventricular injections

All in utero experiments were performed under protocols approved by the IACUC at Boston Children’s Hospital. Timed-pregnant CD-1 dams were obtained from Charles River Laboratories. At E13.5, dams were anaesthetized by isoflurane inhalation and laparotomy was performed. Recombinant WNT3A protein (R&D Systems, 5036-WN-010/CF, 50 ng (for behavioural studies) and 33 ng (for lower dosage spine density analyses)) and the WNT signalling inhibitor IWP2 (9.34 ng; Sigma Aldrich, 10536) were dissolved in PBS. A volume of 1 µl of PBS, WNT3A or IWP2 were injected very slowly into the lateral ventricle of E13.5 embryos using fine glass capillary pipettes (Drummond Scientific, 21-176-2C) as previously described⁶⁶. Meloxicam analgesia was subcutaneously injected following surgery according to the IACUC protocol. All mice that developed a hydrocephalus or other injuries as a consequence of the surgeries were euthanized. To ensure that only healthy mice were part of our dataset, we also inspected mice brains after behavioural studies were completed.

Behavioural studies

All behavioural testing was performed by the Animal Behavior and Physiology Core Facility at Boston Children’s Hospital. All investigators were blinded. Male and female mice were between 4 and 7 months old when testing was performed on WT mice that were injected with recombinant WNT3A or PBS. Rescue experiments were performed in Kdm5c KO male mice injected with IWP2 or PBS and in WT control male mice injected with PBS. Breeding for rescue experiments was performed as previously described²⁰.

Marble-burying test

Twenty glass marbles (Dark) (approximately 15 mm in diameter) were placed equidistant in a 4 × 5 arrangement. The light intensity in the cage was adjusted to 30 lux. The number of buried marbles after 30 min was measured. n = 12 PBS-treated mice and n = 11 WNT3A-treated mice were used for WNT3A induction experiments in WT mice. n = 7 KO + PBS mice, n = 9 KO + IWP2 mice and n = 13 WT + PBS mice were used for rescue experiments in Kdm5c KO mice and WT controls as indicated.

Open-field test

For anxiety assessment, a mouse was placed in an arena of 40 × 40 cm in which the centre area measured an arena of 20 × 20 cm. The mouse explored the arena for 15 min (data were collected in 5-min bins). Video analysis and data acquisition were obtained using Noldus EthoVision XT video tracking software (v.15.0, Noldus Information Technologies). The time spent in the centre area (10 × 10 cm) and periphery was calculated as a measure of anxiety. n = 13 PBS-treated mice and n = 14 WNT3A-treated mice.

Elevated plus-maze test

Anxiety behaviour was tested using the elevated plus-maze test. The maze (Med Associates) consisted of two open arms (35.5 × 6 cm) and two closed arms (35.5 × 6 cm) radiating from a central area (6 × 6 cm). A 0.5-cm-high lip surrounded the edges of the open arms, and 20-cm-high walls enclosed the closed arms. The arms were underlit with infrared light and mice were tracked and scored using Noldus Etho-Vision XT video tracking software (v.15.0, Noldus Information Technologies). n = 14 PBS-treated mice and n = 16 WNT3A-treated mice were used for WNT3A induction experiments in WT mice. n = 9 KO + PBS mice, n = 9 KO + IWP2 mice and n = 8 WT + PBS mice were used for rescue experiments in Kdm5c KO mice and WT controls as indicated

Morris water maze test

A white, opaque, circular tub (60 cm depth × 83 cm diameter) was filled to 29 cm deep with water that was approximately 25 °C. Four visible, distinct shapes were placed in each of the four quadrants of the inner walls of the tub to form distinct quadrants. Trials were videotaped and scored using EthoVision XT video tracking software (v.12.0, Noldus Information Technologies). Acquisition training (day 1) consisted of 8 trials per mouse with a white platform (10 cm diameter) 0.5 cm above the surface of the water and marked with a red reflector. Each training trial began by lowering the mouse into the water close to the pool edge. The start location for each trial was alternated in a semi-random order for each mouse. Each mouse started in each of the quadrants twice. Hidden training (day 2–3) consisted of 20 trials per mouse (12 on day 2 and 8 on day 3) with the platform placed in a new quadrant, 1 cm below the water. Each mouse started in each of the quadrants five times. For reversal training, the platform was placed in a new quadrant, 1 cm below the water. Each mouse completed three trials, each starting from the position opposite of the new platform location. Mice were allowed a maximum of 90 s to reach the platform. During visible, hidden and reversal trials, a mouse that failed to reach the platform in 60 s was guided to the platform by the experimenter. Mice were left on the platform for 5 s before being removed. After each trial, the mouse was placed in a cage lined with absorbent paper towels and allowed to rest. n = 15 PBS-treated and n = 17 WNT3A-treated mice were used for WNT3A induction experiments in WT mice. n = 7 KO + PBS mice, n = 9 KO + IWP2 mice and n = 12 WT + PBS mice were used for rescue experiments in Kdm5c KO mice and WT controls as indicated.

Detailed two-way ANOVA analysis related to Fig. 5d: two-way ANOVA for hidden platform (H1–H5): F_(1.150) = 6,4707, P = 0.01198; and reversal (R1–R3): F_(1,96) = 6.5465, P = 0.01207.

Detailed two-way ANOVA analysis related to Fig. 5j: hidden platform (H1–H5) (two-way ANOVA: KO + PBS vs KO + IWP2, F_(1.70) = 7.6812, P = 0.007142; KO + IWP2 vs WT + PBS, F_(1.95) = 4.4292, P = 0.03797; KO + PBS vs WT + PBS, F_(1.85) = 29.2376, P = 0.0000005779). Visible platform (V1–V2) (two-way ANOVA: KO + PBS vs KO + IWP2, F_(1.28) = 0.1908, P = 0.6656; KO + IWP2 vs WT + PBS, F_(1.38) = 20.1275, P = 0.00006509; KO + PBS vs WT + PBS F_(1.34) = 28.7147, P = 0.000005877). Reversal platform (R1–R3) almost reached significance in the KO + PBS vs KO + IWP2 comparison (two-way ANOVA: KO + PBS vs KO + IWP2, F_(1.42) = 2.9032, P = 0.09579; KO + IWP2 vs WT + PBS, F_(1.57) = 2.2712, P = 0.1373; KO + PBS vs WT + PBS, F_(1.51) = 10.6433, P = 0.001974).

Spine density analysis

Spine density analysis of pyramidal cells in the CA1, the PFC and the BLA was performed in mice after in utero intracerebroventricular injections of PBS or WNT3A in E13.5 WT embryos. Brains from adult (aged 4–7 months) mice were dissected and incubated in Goldi-Cox solution according to the protocol supplied by Neurodigitech. After 2 weeks, brains were sent to Neurodigitech and investigated. In brief, each brain sample was composed of 6–8 slides that covered the range of the regions of interest (ROIs), colour-coded with the in-house alphanumerical coding system, randomly assigned to each slide folder and then distributed to the analysts who were blinded to the original slide identities. The slides included serial coronal sections that covered the anterior-to-posterior axis of the brain. The sampling of ROIs included basal and apical dendrites of pyramidal cells in the BLA, the PFC (layer III/IV) and the CA1 of the hippocampus. The dendritic segments of ROIs were then chosen and analysed using a stereology-based software called Neurolucida (MBF Bioscience), installed on a Dell PC workstation that included a Nikon Eclipse Ni microscope with a Hamamatsu CCD camera (C11440, ORCA-Flash4.0) (full resolution of 2,048 × 2048 pixels), motorized x, y and z focus for high-resolution image acquisition and digital quantitation. The following criteria were applied for selecting candidate neurons for analysis: (1) visualization of fully filled soma with no overlap of neighbouring soma and fully filled dendrites; (2) tapering of the most distal dendrites; and (3) visualization of the complete 3D profile of dendritic tress using the 3D display of the imaging software. After tracing and spine counting, the raw data were extrapolated and quantified using the NeuroExplorer program (MBF Bioscience) followed by statistical analysis (one-way ANOVA followed by Tukey’s multiple comparison test: P < 0.05 was considered significant. n = 7 PBS-treated mice for basal spine density analysis, n = 6 PBS-treated mice for apical spine density analysis, n = 4 mice for lower WNT inhibitor concentration (33 ng) and n = 3 mice for higher WNT inhibitor concentration (50 ng).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.