Mice and genotyping
All animal procedures were approved by the Animal Care and Ethical Committee of Wuhan University. Wild-type CD-1 (ICR) and C57BL/6 mice were obtained from the Hunan SJA Laboratory Animal Company (Changsha, China). Mice were housed in a certified specific-pathogen-free (SPF) facility. The noon of the day when the vaginal plug was found was counted as embryo (E) day 0.5.
Mice with conditional deletion of Kdm2b-CxxC were obtained by first crossing Kdm2bfl/fl (generated by Applied Stem Cell) females with Emx1-Cre (Jackson Laboratories, stock number 005628), Nestin-Cre (Jackson Laboratories, stock number 003771) or Nex-Cre males [Neurod6tm1(cre)Kan, MGI:2668659]. Emx1-Cre; Kdm2bfl/+, Nestin-Cre; Kdm2bfl/+ or Nex-Cre; Kdm2bfl/+ males were crossed with Kdm2bflfl females to obtain conditional knockout mice (Kdm2bEmx1−ΔCxxC, Kdm2bNestin−ΔCxxC, Kdm2bNex−ΔCxxC). Kdm2bfl/+ and Kdm2bfl/fl were phenotypically indistinguishable from each other, and used as controls. The primer set forward 5’- cctgtagtccttggtatttcctggc-3’/reverse 5’ - cccaacttgcccttaggccg-3’ was used for mice genotyping, and band sizes for Kdm2bfl/+ mice are 364 bp (WT allele) and 404 bp (targeted allele with 5’ loxP). Forward 5’- cctgttacgtatagccgaaa-3’/reverse 5’- cttagcgccgtaaatcaatc-3’ was used for Emx1-Cre, Nestin-Cre and Nex-Cre genotyping with band size 319 bp (Cre allele).
To analyse adult neurogenesis, the Nestin-CreERT2 (Jackson Laboratories, stock number 016261) and Kdm2bfl/fl mice were crossed to generate Nestin-CreERT2; Kdm2bfl/+ animals. Nestin-CreERT2; Kdm2bfl/+ mice were further crossed with Kdm2bfl/fl mice to obtain homozygous Kdm2bNestinCreERT2−ΔCxxC animals, which were used for the experiment. Forward 5’- gaccaggttcgttcactca-3’/reverse 5’- caagttaggagcaaacagtagc-3’ was used for Nestin-CreERT2 genotyping with band size 993 bp (CreERT2 allele).
To verify the activity of Nestin-CreERT2 in hippocampal SGZ and the expression profile of Nestin-Cre in developing hippocampus, we constructed Nestin-CreERT2;Ai14 (Rosa-CAG-LSL-tdTomato-WPRE) and Nestin-Cre;Ai14 mice. The primer sets forward 5’- aagggagctgcagtggagta-3’/reverse 5’ - ccgaaaatctgtgggaagtc-3’ and forward 5’- ggcattaaagcagcgtatcc-3’/reverse 5’ - ctgttcctgtacggcatgg-3’ were used for Ai14 mice genotyping, and band sizes for Ai14+/- mice are 297 bp (WT allele) and 196 bp (Ai14 allele).
BAT-Gal mice were kind gifts from Dr. Junlei Chang (Jackson Lab, stock number 005317). To explore the Wnt signaling pathway in Kdm2bEmx1−ΔCxxC mice, the BAT-Gal and Kdm2bfl/fl mice were crossed to generate Kdm2bfl/+; BAT-Gal animals. Emx1-Cre; Kdm2bfl/+ mice were further crossed with Kdm2bfl/+; BAT-Gal mice to obtain Kdm2bEmx1−ΔCxxC; BAT-Gal animals, which were used for the experiment. The primer set forward 5′-atcctctgcatggtcaggtc-3′/reverse 5′-cgtggcctgattcattcc-3′ was used for BAT-Gal mice with band size 315 bp (LacZ allele).
Mice with conditional deletion of Rnf2 were obtained by first crossing Rnf2fl/fl (purchased from GemPharmatech, Strain NO. T014803) females with Emx1-Cre males (Jackson Laboratories, stock number 005628). The primer set forward 5’- agctgtggtcctgcgtttcatttc-3’/reverse 5’ - gctcttactgtgttacaaccctagccc-3’ was used for Rnf2fl/+ mice genotyping, and band sizes for Rnf2fl/+ mice are 289 bp (WT allele) and 391 bp (targeted allele with 5’ loxP).
Tamoxifen and BrdU administration
To activate Cre-mediated recombination, tamoxifen (TAM; Sigma-Aldrich) was used, which was made fresh daily and dissolved in sunflower oil solution (Sigma-Aldrich). 8-week-old Kdm2bNestinCreERT2−ΔCxxC mice were daily administered with 30 mg/kg prewarmed TAM intraperitoneally for 6 consecutive days (d1-d6). From day 2 to day 7, mice were injected with 50 mg/kg BrdU (Sigma-Aldrich) intraperitoneally for 6 consecutive days and were sacrificed 1 day later (day 8, Short-Term) or 4 weeks later (day 35, Long-Term) to identify BrdU-positive adult-born cells.
Tissue fixation and sectioning
The pregnant dam was anesthetized with 0.7% w/v pentobarbital sodium (105 mg/kg body weight) in 0.9% sodium chloride. Embryos were sequentially removed from the uterus. Brains of embryos were dissected out in cold PBS and immersed in 4% paraformaldehyde (PFA) overnight at 4°C. For P0, P2, P7 and adult mice, animals were anesthetized with 0.7% w/v pentobarbital sodium solution followed by trans-cardiac perfusion with 4% PFA in PBS (P0, 5 ml; P7, 10 ml; adult, 30 ml). Brains were dissected and post-fixed in 4% PFA overnight at 4°C. Next day, brains were dehydrated in 20% w/v sucrose overnight at 4°C. For sectioning, brains were embedded in OCT (SAKURA) and cut at 20 µm for adult brains and 14 µm for other stages with a cryostat (Leica CM1950).
Nissl staining
Adult brain sectionswere stained with 0.25% Cresyl Violet (Sigma-Aldrich) solution for 15 min at 65°C. Sections were then decolorized in ethanol for 0.5-1 min, dehydrated in gradient ethanol solutions for 5 min each and cleared twice in xylene for 5 min. Sections were mounted in the neutral balsam.
In situ hybridization (ISH)
Sections were dried in a hybridization oven at 50°C for 15 min and fixed in 4% PFA for 20 min at room temperature, followed by permeabilization in 2 µg/ml proteinase K in PBS for 10 min at room temperature. Prior to hybridization, sections were acylated in 0.25% acetic anhydride for 10 min. Then, sections were incubated with a digoxigenin-labeled probe diluted (0.2 ng/µl) in hybridization buffer (50% deionized formamide, 5× SSC, 5× Denhart’s, 250 µg/ml tRNA, and 500 µg/ml Herring sperm DNA) under coverslips in a hybridization oven overnight at 65°C. The next day, sections were washed 4 times for 80 min in 0.1× SSC at 65°C. Subsequently, they were treated with 20 µg/ml RNase A for 20 min at 37°C, then blocked for 3.5 h at room temperature in 10% normal sheep serum. Slides were incubated with 1:5,000 dilution of anti-digoxigenin-AP conjugated antibody (Roche) overnight at 4°C. BCIP/NBT (Roche) was used as a color developing agent. ISH primers used are listed in Table S1.
Immunofluorescence
Frozen brain sections were mounted onto Superfrost plus slides and then dried at room temperature. For heat-mediated antigen retrieval, slides were incubated for 15 min in 10 mM sodium citrate buffer (pH 6.0) at 95°C. For BrdU staining, sections were treated with 20 µg/ml proteinase K (Sigma) (1:1000 in PBC) for 5 min and 2 N HCl for 30 min at room temperature. Sections were then immersed in blocking buffer (3% normal sheep serum and 0.1% Triton X-100 in PBS; or 5% BSA and 0.5% Triton X-100 in PBS) for 2 h at room temperature. Sections were then incubated in primary antibodies [mouse anti-Calbindin (1:1000; Sigma, C9848), rabbit anti-ZBTB20 (1:1000; Sigma, HPA016815), mouse anti-HopX (1:200; Santa Cruz, sc-398703), rabbit anti-Wfs1 (1:1000; Proteintech, 86995), mouse anti-PROX1 (1:200; Millipore, MAB5654), rabbit anti-GFAP (1:500; DAKO, Z0334), rat anti-CTIP2 (1:500; Abcam, ab18465), rabbit anti-SATB2 (1:500; Abcam, ab92446), rat anti-BrdU (1:500; Abcam, ab6326), mouse-anti-BrdU (1:500; Roche, 11170376001), rabbit-anti-DCX (1:500; Abcam, ab18723), rabbit anti-TBR2 (1:500; Abcam, ab23345), rat anti-TBR2 (1:500; Thermo Fisher, 14-4875-82), rabbit anti-PAX6 (1:500; Millipore, ab2237), and rabbit anti-Ki67 (1:500; Abcam, ab15580] in blocking buffer overnight at 4°C. After three rinses in PBS, sections were incubated in secondary antibodies (Alexa Fluor 488-conjugated anti-mouse, A11029; Alexa Fluor 555-conjugated anti-mouse, A21422; Alexa Fluor 488-conjugated anti-rat, A11006; Alexa Fluor 555-conjugated anti-rat, A21434; Alexa Fluor 647-conjugated anti-rat, A21247; Alexa Fluor 488-conjugated anti-rabbit, A11034; Alexa Fluor 555-conjugated anti-rabbit, A21429; Alexa Fluor 647-conjugated anti-rabbit, A21245; Alexa Fluor 488-conjugated anti-chicken, A11039; Thermo Fisher Scientific; 1:1000) for 1 h at room temperature. Nuclei were labeled by incubation in PBS containing 4′,6-diamidino-2-phenylindole (DAPI) (0.1 µg/ml) (Sigma-Aldrich), and samples were mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific).
5-Ethynyl-2′-Deoxyuridine (EdU) staining
Proliferation of cells was investigated with BeyoClickTM EdU Cell Proliferation Kit (C0075S, Beyotime, China) according to the manufacturer’s protocols. In brief, frozen brain sections were dried at temperature and permeated with 0.3% Triton X-100 in PBS for 30 min. Sections were then incubated with EdU working solution for 1 h at 37°C in the dark. After incubation, regular immunofluorescence staining can be followed.
Immunohistochemical staining
Frozen brain sections were dried at room temperature, and then pretreated with 0.3% H2O2 for 15 min to deactivate endogenous peroxidase. Sections were blocked with 3% normal sheep serum with 0.1% Tween 20 at room temperature for 2 h. Sections were then incubated in primary antibodies [rabbit anti-NeuN (1:500; Abcam, ab177487), rabbit-anti-DCX (1:500; Abcam, ab18723), rabbit anti-SOX2 (1:500; Millipore, ab5603), rabbit anti-TBR2 (1:500; Abcam, ab23345), and rabbit anti-BLBP (1:500; Abcam, ab32423)] in blocking buffer overnight at 4°C, followed by addition of the avidin-biotin-peroxidase complex (1:50; VECTASTAIN Elite ABC system, Vector Laboratories). Peroxidase was reacted in 3,3′-diaminobenzidine (5 mg/ml) and 0.075% H2O2 in Tris-HCl (pH 7.2). Sections were dehydrated in gradient ethanol (75% ethanol, 95% ethanol, 100% ethanol and 100% ethanol, each for 5 min), and cleared twice in xylene for 5 min, then mounted in the neutral balsam.
Behavior tests
We used 12- to 16-week-old age-matched male mice for all behavioral tests. Mice were housed (3–5 animals per cage) in standard filter-top cages with access to water and rodent chow at all times, maintained on a 12:12 h light/dark cycle (09:00–21:00 h lighting) at 22°C, with relative humidity of 50–60%. All behavioral assays were done blind to genotypes.
Open field test. The test mouse was gently placed near the wall-side of a length of 50 cm, a width of 50 cm, and a height of 50 cm open-field arena and allowed to explore freely for 20 min. Only the last 10 min of the movement of the mouse was recorded by a video camera and analyzed with Ethovision XT 13 (Noldus).
Rotarod test. The test consists of 4 trials per day for 10 days with the rotarod (3 cm in diameter) set to accelerate from 4 rpm to 40 rpm over 5 minutes. The trial started once mice were placed on the rotarod rotating at 4 rpm in partitioned compartments. The time for each mouse spent on the rotarod were recorded. At least 20 min recovery time was allowed between trials. The rotarod apparatus was cleaned with 70% ethanol and wiped with paper towels between each trial.
Morris water maze. Mice were introduced into a stainless water-filled circular tank, which is 122 cm in diameter and 51 cm in height with non-reflective interior surfaces and ample visual cues. Two principal axes were drawn on the floor of the tank, each line bisecting the maze perpendicular to one another to create an imaginary ‘+’. The end of each line demarcates four cardinal points: North, South, East and West. To enhance the signal-to-noise ratio, the tank was filled with water colored with powdered milk. A 10-cm circular plexiglass platform was submerged 1 cm below the surface of the water in middle of the southwest quadrant. Mice started the task at fixed points, varied by day of testing66. Four trials were performed per mouse per day with 20 min intervals for 5 days. Each trial lasted 1 min and ended when the mouse climbed onto and remained on the hidden platform for 10 s. The mouse was given 20 s rest on the platform during the inter-trial interval. The time taken by the mouse to reach the platform was recorded as its latency. Times for four trials were averaged and recorded as a result for each mouse. On day 6, the mouse was subjected to a single 60-s probe trial without a platform to test memory retention. The mouse started the trial from northeast, the number of platform crossings was counted, and the swimming path was recorded and analyzed using the Ethovision XT 13 (Noldus).
Fear conditioning (FC). The FC apparatus consisted of a conditioning box (18 × 18 × 30 cm), with a grid floor wired to a shock generator surrounded by an acoustic chamber and controlled by Ethovision XT 13 (Noldus). On Day 1, each mouse was placed in the conditioning box for 2 min, and then a pure tone (80 db) was sounded for 30 s followed by a 2 s foot shock (0.4 mA). Two minutes later, this procedure was repeated. After the delivery of the second shock, mice were returned to their home cages. On Day 2, each mouse was first placed in the fear conditioning chamber containing the exact same context, but there was no administration of a tone or foot shock. Freezing was analyzed for 4 min. One hour later, the mice were placed in a new context (containing a different odor, cleaning solution, floor texture, walls and shape) where they were allowed to explore for 3 min before being re-exposed to the fear conditioning tone and freezing was assessed for an additional 3 min. FC was assessed through the continuous measurement of freezing (complete immobility), which is the dominant behavioral fear response. Freezing was measured using the Noldus Ethovision video tracking system (Ethovision XT 13).
Forced swimming test. For the forced swimming test, the test mouse was placed into a 20 cm height and 17 cm diameter glass cylinder filled with water to a depth of 10 cm at 22°C. The test continues 6 min and the immobility time of the last 5 minutes was recorded for further processing.
Tail suspension test. The test mouse was suspended in the middle of a tail suspension box (55 cm height × 60 cm width × 11.5 cm depth) above the ground by its tail. The mouse tail was adhered securely to the suspension bar using adhesive tapes. After 1 min accommodation, the immobility time was recorded by a video camera and analyzed by Ethovision XT 13 (Noldus).
Elevated plus maze test. The elevated plus maze, made of gray polypropylene and elevated about 40 cm above the ground, consists of two open arms and two closed arms (each 9.5 cm wide and 40 cm long). To assess anxiety, the test mouse was placed in the central square facing an open arm and allowed to explore freely for 5 min. The time spent in the open arm was analyzed with the Ethovision XT 13 (Noldus).
X-Gal staining
Frozen sections were fixed in fresh cold fixative (0.2% PFA) in buffer L0 (0.1M PIPES buffer (pH 6.9), 2mM MgCl2, 5mM EGTA) for 10 min. Slides were rinsed in PBS plus 2mM MgCl2 on ice, followed by a 10 min wash in the same solution. Place slides in detergent rinse [0.1M PBS (pH 7.3), 2mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40] on ice for 10 min. Slides were then moved to a freshly made and filtered X-Gal staining solution [0.1M PBS (pH 7.3), 2mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, 5mM K3Fe(CN)6, 5mM K4Fe(CN)6·3H2O and 1 mg/ml X-Gal]. Sections were incubated at 37°C from a few minutes to overnight in the dark. Sections were rinsed with water to stop the reaction. Sections were dehydrated with gradient ethanol and xylene sequentially, and mounted with the neutral balsam.
RNA isolation and reverse transcription (RT)
RNA isolation was performed using the RNAiso Plus (TAKARA) according to manufacturer’s instructions. Tissue or cells were homogenized using a glass-Teflon in 1 ml or 500 µl RNAiso Plus reagent on ice and phase separation was achieved with 200 µl or 100 µl chloroform. After centrifugation at 12,000× g for 15 min at 4°C, RNA was precipitated by mixing aqueous phase with equal volumes of isopropyl alcohol and 0.5 µl 20 mg/ml glycogen. Precipitations were dissolved in DNase/RNase-free water (not diethylpyrocarbonate treated, Ambion). 1 µg of total RNA was converted to cDNA using M-MLV reverse transcriptase (TAKARA) under standard conditions with oligo(dT) or random hexamer primers and Recombinant RNase Inhibitor (RRI, TAKARA). Then the cDNA was subjected to quantitative RT-PCR (qRT-PCR) using the SYBR green assay with 2× SYBR green qPCR master mix (Bimake). Thermal profile was 95°C for 5 min and 40 cycles of 95°C for 15 sec and 60°C for 20 sec. Gapdh was used as endogenous control genes. Relative expression level for target genes was normalized by the Ct value of Gapdh using a 2−ΔΔCt relative quantification method. Reactions were run on a CFX Connect TM Real-Time PCR Detection System (Bio-Rad). The primers used are listed in Table S2.
Neurosphere culture
Mouse hippocampal neural progenitor cells (NPCs) were enriched from P0 mouse hippocampi, cultured on ultra-low-attachment plates (Corning, New York, United States) and maintained in indicated culture media (DMEM/F12, Life Technologies) containing N2 and B27 supplements (1×, Life Technologies), 1mM Na-pyruvate, 1 mM N-acetyl-L-cysteine (NAC), human recombinant FGF2, and EGF (20 ng/mL each; Life Technologies). After cultured in vitro for three generations, neurospheres were subjected to RNA-seq and ChIP-seq analyses.
RNA-seq library construction
Total RNA was extracted as described above. The concentration and quality of RNA was measured with Nanodrop 2000c (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies), respectively. RNA-seq libraries were constructed by NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (NEB #E7775). Briefly, mRNA was extracted by poly-A selected with magnetic beads with poly-T and transformed into cDNA by first and second strand synthesis. Newly synthesized cDNA was purified by AMPure XP beads (1:1) and eluted in 50 µl nucleotide-free water. RNA-seq libraries were sequenced by Illumina NovaSeq 6000 platform with pair-end reads of 150 bp. The sequencing depth was 60 million reads per library.
Bulk RNA-Seq data analysis
P0 hippocampus and neurosphere RNA-seq data were checked for quality control by FastQC (version 0.11.9). Paired-end reads were trimmed to remove adaptors and low-quality reads and bases using cutadapt (version 3.2). Clean reads were aligned to the mouse UCSC mm10 genome using STAR (version 2.7.10b) with default parameters. The number of covering reads were counted using featureCounts (version v2.0.1). The resulting read counts were processed with R package DESeq2 (version 1.38.1) to identify differential expression genes (log2 fold change > 0.4 and p value < 0.05) between datasets. Cufflinks package (version 2.2.1) assembles individual transcripts from reads that have been aligned to reference genome. The gene expression level was normalized by fragments per kilobase of bin per million mapped reads (FPKM). Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) in this study were performed using clusterProfiler (version 4.2.2).
Chromatin immunoprecipitation (ChIP) assay
For each experiment, single-cell suspensions from P0 hippocampi were collected as described above. The hippocampal tissue was digested into single cells by Papain (20U dissolved in each mL of DMEM/F12 medium, preheated at 37°). Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature, and quenched with 0.125 M of glycine for 5 minutes. Cross-linked samples were then rinsed twice in PBS, then cells were collected by centrifugation. Next, cells were pretreated with lysis buffer (50 mM of Tris-HCl [pH 8.0], 0.1% SDS, and 5 mM of EDTA) and incubated for 5 minutes with gentle rotation at 4°C. After centrifugation, bottom cells were washed for two times with ice-cold PBS and harvested in ChIP digestion buffer (50 mM of Tris-HCl [pH 8.0], 1 mM of CaCl2, and 0.2% Triton X-100). DNA was digested to 150–300 bp by micrococcal nuclease (NEB; M0247S). Sonicate cells in EP tubes with power output 100 W, 5 min, 0.5 s on, 0.5 s off on ice. The resulting lysate was centrifugation and divided into four parts for 10% input, H2AK119Ub, H3K27me3, and H3K36me2 immunoprecipitation. After diluting each sample (in addition to input) to 1 mL with dilution buffer (20 mM of Tris-HCl [pH 8.0], 150 mM of NaCl, 2 mM of EDTA, 1% Triton X-100, and 0.1% SDS), immunoprecipitation was further performed with sheared chromatin and 3 µg rabbit anti-H2AK119Ub antibody (CST, 8240S); or rabbit anti-H3K27me3 antibody (CST, 9733S); or rabbit anti-H3K36me2 antibody (CST, 2901S), then incubated with protein A/G beads overnight at 4°C on a rotating wheel. The next day, beads were wash with Wash Buffer I (20mM Tris-HCl, pH 8.0; 1% Triton X-100; 2mM EDTA; 150mM NaCl; 0.1% SDS), Wash Buffer II (20mM Tris-HCl, pH 8.0; 1% Triton X-100; 2mM EDTA; 500mM NaCl; 0.1% SDS), Wash Buffer III (10mM Tris-HCl, pH 8.0; 1mM EDTA; 0.25M LiCl; 1% NP-40; 1% deoxycholate) and TE buffer. DNA was eluted by ChIP elution buffer (0.1 M of NaHCO3, 1% SDS, 20 µg/mL of proteinase K). The elution was incubated at 65°C overnight, and DNA was extracted with a DNA purification kit (DP214-03; TIANGEN).
ChIP-seq library construction
ChIP-seq libraries were constructed by VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme ND607). Briefly, 50 µL purified ChIP DNA (5 ng) was end-repaired for dA tailing, followed by adaptor ligation. Each adaptor was marked with a barcode of 6 bp which can be recognized after mixing different samples together. Adaptor-ligated ChIP DNA was purified by VAHTS DNA Clean Beads (Vazyme N411) and then amplified by PCR of 10 cycles with primers matching with adaptors’ universal part. Amplified ChIP DNA was purified again using VAHTS DNA Clean Beads in 35-µL EB elution buffer. For multiplexing, libraries with different barcode were mixed together with equal molar quantities by considering appropriate sequencing depth (about 30 million reads per library). Libraries were sequenced by Illumina Nova-seq 6000 platform with pair-end reads of 150 bp.
ChIP-seq data analysis
DNA libraries were sequenced on Illumina NovaSeq 6000 platform. All P0 hippocampus and neurosphere ChIP-seq data were checked and removed with adaptor sequences same as the RNA-seq data processing. Clean reads were aligned to the mouse UCSC mm10 genome using Bowtie2 (version 2.4.5). Duplicates were removed using the samtools rmdup module. Regions of peaks were called using the SICER software package, with the input genomic DNA as a background control (parameters: -w 200 -rt 1 -f 150 -egf 0.77 -fdr 0.01 -g 600 -e 1000 --significant_reads). The bigwig signal files were visualized using the computeMatrix, plotHeatmap, plotProfile modules in Deeptools (version 3.5.1). Homer was used to identify adjacent genes from the peaks obtained from SICER.
ATAC-seq library construction
ATAC-seq libraries were constructed by TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme TD501). Briefly, P0 hippocampi were dissected and gently homogenized in cold nuclear isolation buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630). Nuclei were collected by centrifugal precipitation. 50,000 nuclei were put into the tagmentation reaction for each sample (performed with 30 min incubation time at 37°C). Immediately following the tagmentation, DNA fragments were purified using VAHTS DNA Clean Beads (2X). Purified DNA fragments were added with Illumina i5 + i7 adapters with unique index to individual samples followed by PCR reaction (PCR program: 72°C for 3 min, 98°C for 30 s, 98°C for 15 s, 60°C for 30 s, 72°C for 30 s, repeat 3–5 for 13 cycles, 72°C for 5 min, and hold at 4°C). Generated libraries were purified using VAHTS DNA Clean Beads (1.2X). For multiplexing, libraries with different barcode were mixed together with equal molar quantities by considering appropriate sequencing depth (about 50 million reads per library). Libraries were sequenced by Illumina Nova-seq 6000 platform with pair-end reads of 150 bp.
ATAC-seq data analysis
P0 hippocampus ATAC-seq raw data were trimmed by Cutadapt with parameters -u 3 -u -75 -U 3 -U -75 -m 30 and then aligned to mouse mm10 genome using Bowtie2 (-X 2000 --very-sensitive). Subsequently, we downloaded blacklisted regions including a large number of repeat elements in the genome from ENCODE project and then removed these significant background noise. ATAC-seq datasets contained a large percentage of reads that were derived from mitochondrial DNA. We removed mitochondrial reads after alignment using Samtools. Then, we filtered reads to remove exact copies of DNA fragments that arise during PCR using Picard’s MarkDuplicates (version 2.26.4). All reads aligning to the + strand were offset by + 4 bp, and all reads aligning to the – strand were offset − 5 bp, since Tn5 transposase has been shown to bind as a dimer and insert two adaptors separated by 9 bp. We adjusted the shift read alignment using alignmentSieve. Next, peaks calling was finished by Macs2 (version 2.2.7.1) with parameters -f BAMPE --nomodel --keep-dup all --shift − 100 --extsize 200 -g mm --cutoff-analysis -B. We created bigwig files for visualizing using bamCoverage (parameters: --normalizeUsing RPGC –effectiveGenomeSize 2407883318) in deepTools. Homer took narrow peak files as input and checked for the enrichment of both known sequence motifs and de novo motifs.
Defining genomic features
Mm10 CpG island (CGI) regions were downloaded from the UCSC genome browser database. Promoters were defined as all mouse UCSC mm10 gene TSSs, extended by 2 kb upstream and downstream. CGIs in promoters were defined as ± 2 kb around CGI centers overlap with promoter regions. Overlapped regions between CGIs and promoters were identified using bedtools (version 2.29.2) intersect with parameters -e -f 0.5 -F 0.5. H2AK119ub1 +/- promoter genomic loci were defined as such promoter location with or without H2AK119ub1.
In utero electroporation (IUE)
In utero microinjection and electroporation were performed as followed. Pregnant CD-1 mice with E14.5 embryos were anesthetized by injection of pentobarbital sodium (70 mg/kg), and the uteri were exposed through a 2 cm midline abdominal incision. Embryos were carefully pulled out using ring forceps through the incision and placed on sterile gauze wet with 0.9% sodium chloride. Plasmid DNA (prepared using Endo Free plasmid purification kit, Tiangen) mixed with 0.05% Fast Green (Sigma) was injected through the uterine wall into the telencephalic vesicle using pulled borosilicate needles (WPI). For gain-of-function experiments, pCIG (1 µg/µl) was mixed with pCAGGS-Wnt3a, pCAGGS-Wnt5a, pCAGGS-Wnt5b, pCAGGS-Wnt7b, pCAGGS-Wnt8b, (Wnt-mix) (0.5 µg/µl each), or with pCAGGS-SFRP2 (2 µg/µl). Control mice were injected with pCIG (1 µg/µl). Five electric pulses (33 V, 50 ms duration at 1 s intervals) were generated using CUY21VIVO-SQ (BEX) and delivered across the head of embryos using 5 mm forceps-like electrodes (BEX). The uteri were then carefully put back into the abdominal cavity, and both peritoneum and abdominal skin were sewed with surgical sutures. The whole procedure was completed within 30 min. Mice were warmed on a heating pad until they regained consciousness and were treated with analgesia (ibuprofen in drinking water) until sacrifice at E18.5.
Plasmid construction
Full-length mouse Wnt3a and Wnt8b were amplified from cDNAs of E14.5 mouse hippocampi and then cloned into pCAGGS. Full-length Wnt5a, Wnt5b, Wnt7b were amplified from cDNAs of E16.5 mouse cortex, and then cloned into pCAGGS. Full-length mouse Sfrp2 was amplified from cDNAs of P0 mouse cortex and then cloned into pCAGGS. The primers used are listed in Table S3.
Quantification and statistical analysis
Sections used for quantification were position-matched for control and experimental brains. Three corresponding brain cross sections of each control and cKO samples were selected for cell count statistics and the total number of these three cross sections was counted. Images were binned against proximal-distal transverse axis to quantify the intensity of ISH or LacZ signals of hippocampi. A plot of normalized average signal intensity with standard error of the mean across those regions was generated using ImageJ.
Statistical tests were performed using GraphPad Prism (version 8.0.2). Data analyzed by unpaired two-tailed t-test were pre-tested for equal variance by F-tests. Unpaired Student’s t-tests (two-tailed) were chosen when the data distributed with equal variance. For normally distributed data with unequal variance, an unpaired t-test with Welch’s correction was used. One-way ANOVA followed by Tukey post hoc test was used for multiple group comparison. Significant difference is indicated by a p value less than 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications. Experiments were not randomized. Investigators were blinded as to the animal genotype during tissue section staining, image acquisition, and image analysis.
Data and code availability
The GEO accession number for the RNA-seq, ChIP-seq and ATAC-seq data reported in this paper is GSE222465. RNA-seq data of P0 hippocampus, P0 neurosphere and P0 cortex have been deposited at GEO: GSE222464. ChIP-seq and ATAC-seq data of P0 hippocampus and P0 neurosphere in this study have been deposited at GEO: GSE222463 and GSE222462. All data are publicly available as of the date of publication. Custom codes were described in detail at methods part. Any additional information required to analyze the data in this paper is available from authors upon reasonable request.