Fig. 4: Mechanisms of CCN1 action.

a, Top, CCN1 structure and binding sites. The position of the D125A mutation is indicated. Bottom, timeline of viral injections and experiments. CT, cysteine-knot-containing domain; IGFBP, insulin-like growth factor binding protein domain; HSPGs, heparan sulfate proteoglycans; IHC, immunohistochemistry; TSP-1, thrombospondin type 1 repeat homology domain; VWC, von Willebrand factor type C repeat. b, Representative z-projection of HA tag, SOX9 and NEUN immunofluorescence in a CCN1(D125A)-OE mouse. Scale bar, 100 µm. c, Penetrance and specificity of CCN1(D125A) overexpression. n = 4 mice. d, Representative images of Arc smFISH. Scale bars, 1 mm. Dashed yellow lines indicate Arc binocular zone width. e, Quantification of Arc activation width. n = 4 mice per group. Unpaired t-test on mouse averages. c,e, Symbols show mouse averages. Data are mean ± s.e.m. f, Representative z-projection of WFA and parvalbumin immunofluorescence. Scale bars, 20 µm. g, Integrated WFA density around parvalbumin-positive cells. tdT: n = 147 cells; CCN1: n = 200 cells; CCN1(D125A): n = 284 cells. Four mice per group. Kruskal–Wallis test with post hoc Dunn’s tests on cells; *, significant P value. Dots represent cells. Lines show median ± 95% confidence interval. h, Representative z-projection of aggrecan (ACAN) immunofluorescence. Scale bars, 50 µm. i, Number of ACAN+ cells. n = 4 mice per group. One-way ANOVA with post hoc Tukey’s tests on mouse averages; *, significant P value. Symbols show mouse averages. Data are mean ± s.e.m. j, snRNA-seq experiment. See Supplementary Fig. 2 for fluorescence-activated cell sorting (FACS) gating. Created in BioRender. Allen Lab (2025) https://BioRender.com/o2k2lbh. k, Uniform manifold approximation and projection (UMAP) of MapMyCells cell types from four samples (two replicates for tdT and CCN1). BAM, border-associated macrophage; COP, committed oligodendrocyte precursor; CT, corticothalamic; ET, extratelencephalic; MFOL, myelin-forming oligodendrocyte; MOL, mature oligodendrocyte; NFOL, newly formed oligodendrocyte. Interneurons include Lamp5-, Pvalb-, Sst- and Vip-expressing subtypes. l. Integrin gene expression. Normalized counts. m, AUGUR cell-type prioritization. AUC, area under the receiver operating characteristic curve. n, Excitatory neuron overrepresentation analysis (ORA) Gene Ontology (GO) terms. o, Mean UCell gene signature scores. Kruskal–Wallis test with pairwise Wilcoxon signed rank tests with Holm’s corrections; *, adjusted P value < 0.05.

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