Fig 1: Molecular analyses in mice and humans place NEUROD2 as a nexus in NDD gene regulatory network.a Scheme of the RNA-seq experiment, performed at P30. b Gene set enrichment analysis using the DAVID knowledgebase. c Fold change expression (FC; log2 scale) of 13 DEX genes belonging to the voltage-dependent ion channel family, ranked according to Padj (lowest on top). Genes associated with neuropsychiatric recurrent syndromes are depicted in blue. d Heatmap for synaptic and disease gene sets among the orthologs of DEX genes (red, higher expression; blue, lower expression). e, f Venn diagrams identifying overlaps between DEX genes and synaptic/ NDD gene sets. Number of genes for each gene set is indicated. Right in (f), differentially-expressed genes associated with ASD and their score in SFARI. g Graphical representation of % [DEX genes] (orange) and % [same number of randomly selected genes] (gray) belonging to gene sets of interest. (h) qRT-PCR for 11 neuropsychiatric-related DEX genes in cortical samples from WT, HET, and KO. These data validate the RNA-seq results and show that HET mice have an intermediate phenotype between WT and KO mice, demonstrating that Neurod2 is haploinsufficient. (i–k) NEUROD2-based coexpression network analysis in humans. i Gene module 37 with spatio-temporal co-expression was obtained from WGCNA of spatiotemporal transcriptional dynamic analyzes in the human cortex [49]. j Network representation of NEUROD2 within module 37 from Li et al. [37] showing gene connectivity based on Pearson correlation (weight of edges scaled for r between 0.7 and 1). The size of each node is proportional to degree centrality calculated with a cutoff of r = 0.7. Green edges indicate direct neighbors of NEUROD2 with threshold r = 0.7. Genes described in SFARI are colored according from SFARI score (1–3). k Circular network representation of NEUROD2 and SFARI genes interactions with weight of edges scaled for r between 0.5 and 1. The size of each node represents the same values of degree centrality as calculated in (j). For (j, k), we added a circle with nine cells. Each cell corresponds to a neuropsychiatric trait/disorder and color/position legend is at the bottom. For details on how neuropsychiatric trait/disorder cells were determined, see “Materials and methods” and File S1, sheet “GWAS_NEUROD2_GeneModule_Fig6”. N = 3 experiments per genotype for both RNA-seq and qRT-PCR. Data are means ± SEM. Statistical significance was evaluated by binomial test (g) or unpaired two-tailed Student t test (h) (*P < 0.05; **P < 0.01; ****P < 0.0001). See also Figs. S11 and S12.
Fig 2: Altered excitatory synapse density and turnover in Neurod2 KO mice.a Among the 227 human orthologs of the 263 DEX genes at P30, 39 were synaptome genes, including 4 pre-synaptic and 35 post-synaptic. This suggests synaptic alterations in Neurod2 KO mice. Bottom: fold change of DEX gene expression (log2 scale), genes are ranked according to Padj. In blue, pre-synaptic genes; in orange, post-synaptic genes. (b–f) Age-dependent spine density defects in L5 cPNs. b–c Spine density in basal dendrites. b Representative photomicrographs of GFP-expressing basal dendritic stretches with spines underscored by yellow dots. c Spine density at P30 and P120 in the basal compartment. We counted 21 dendritic segments from 4 WT and 40 segments from 6 KO mice at P30, 33 segments from 4 WT and 27 segments from 4 KO at P120 (individual cells plotted). d, e Apical tuft spine density. Representative images (d) and apical spine density (e). f Spine density variation relative to WT in basal and apical compartments. We analyzed 22 dendritic segments from 5 WT and 26 segments from 7 KO mice at P30, 34 segments from 4 WT and 27 segments from 4 KO at P120 (individual cells plotted). g–k Increased spine turnover in an apical tuft at P30. g Scheme of the experimental paradigm. h Representative 2-photon images of same dendrites at 3 days intervals. Red and blue arrowheads depict gained and lost spines, respectively. i Spine formation, j spine elimination and k net spine addition over the 3-day interval period. We counted spine changes in 42 dendritic segments from 8 WT mice and 39 segments from 6 KO mice between P30 and P33 (animals plotted). Data are represented as means ± SEM. Statistical analyses were performed using two-tailed t-tests or Mann–Whitney test depending on the normality of samples. *P < 0.05, **P < 0.01. See also Fig. S7.
Fig 3: Radial over-migration of cPNs in Neurod2 KO and HET mice.a Spatio-temporal expression of Neurod2 from a longitudinal scRNA-seq study [16]. X axis is time of apical progenitor birth, Y axis represents time of neuron differentiation (1 h for VZ progenitors, 24 h for migrating neurons, 48 h for post-migratory neurons). The strongest expression of Neurod2 mRNA is in migrating neurons. b Excess migration in Neurod2 KO and HET mice. The probability density function is represented. See Fig. S4b for additional cell position analyses, which corroborate the over-migration phenotype. We analyzed 21 slices from 4 WT, 20 slices from 5 HET and 26 slices from 3 KO mice. c Post-mitotic overexpression of NEUROD2 of WT mice reduced the distance migrated by cPNs. We analyzed 21 slices from 3 pND1-GFP mice and 65 slices from 10 pND1-NEUROD2-GFP mice. d Over-migration was maintained post-migration, at P7. We analyzed 9 slices from 2 WT and 13 slices from 3 KO mice. e Over-migration of L2/3 cPNs born at E15.5 was also evident. We analyzed 26 slices from 5 WT and 15 slices from 3 KO mice. f, g At P30, somatic volume (f) and dendritic complexity (g) from 3D-reconstructed neurons were not significantly altered. For somatic volumes we analyzed 24/26 L4/L5 cells from 3 WT mice and 16/24 L4/L5 cells from 3 KO mice (individual cells plotted). For the dendritic length we measured 17/19 basal/apical dendrites from 4 WT and 14/28 basal/apical dendrites from 4 KO mice (circles represent individual dendrites). h Over-migration was already visible at 2 days post-electroporation in KO mice. We analyzed 6 slices from 2 WT and 9 slices from 3 KO mice. i Radar plot investigating morphological parameters. HET and KO cells showed decreased circularity, increased aspect ratio, increased perimeter, and increased area. 704 WT, 659 HET, and 410 KO cells were investigated from 6, 6, and 5 mice, respectively. Data are represented as means ± SEM. Statistical significance was evaluated by Anderson-Darling test [probability density graph in (b)], permutation test for a spatially adjusted two-way Anova followed by post-hoc analysis with Bonferroni correction [bin graphs in (c), (d), (e), (h)], Student t-test or Mann–Whitney test depending on normality of samples [morphometric analyses in (f), (g)], and by a two-sided permutation t-test [radar plot in (i), see also Fig. S4-S5]. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). See also Figs. S4, S5, and S6.
Fig 4: Glial cell types and development.(A) tSNE plot showing the regional differences of astrocytes in CC and pons. The expression levels of typical markers AQP4 and GFAP are shown in the bottom. (B) Costaining of NEUROD2 and GFAP in the CC and pons at GW20. NEUROD2 positive cells represent for neurons and GFAP-positive cells represent for astrocytes. The pons contains much larger abundance of astrocytes. NEUROD2, neuronal differentiation 2. (C) Expression levels of oligo subtype markers are shown by boxplot. OLIG1 is a pan marker for all oligo subtypes, PDGFRA lacks expression in Oligo_4, CDK1 is specifically expressed by Oligo_1 cells, APOD lacks expression in Oligo_2, and MOG and PDGFA are specifically expressed by Oligo_4 cells. (D) Co-immunostaining of APOD and PDGFRA in the CC at GW17. The white arrowheads show the double positive cells. DAPI, 4',6-diamidino-2-phenylindole; PDGFRA, platelet-derived growth factor receptor A. (E) Pseudotime map of the subtypes of oligodendrocytes and their progenitors. The regional identity for each cell is also shown on the bottom.
Fig 5: Administration of repetitive transcranial magnetic stimulation suppresses the expression of miR-567, and increases the expression of NEUROD2 and PSD95 in mice with Alzheimer’s disease (replicate number = 5). The expression of miR-567 was detected by RT-qPCR, and the expression of NEUROD2 and PSDD95 was detected by Western blotting. (A) Analysis of the results of RT-qPCR detection of miR-567. (B) Analysis of the results and representative images of the detection of NEUROD2 and PSDD95 by Western blotting. Sham group, mice injected with normal saline. Model group, mice injected with scopolamine. L rTMS group, mice injected with scopolamine and treated with rTMS of 1 Hz. H rTMS group, mice injected with scopolamine and treated with rTMS of 10 Hz. *P<0.05 vs Sham group, #P<0.05 vs Model group.
Supplier Page from Abcam for Anti-NeuroD2 antibody