Fig. 8: Progeny of WM astrocytes and regulatory signals controlling their proliferation.

a, t-SNE plot of the isolated astrocytes showing RNA velocities. b, PAGA analysis showing differentiation from cluster 5 to cluster 4. c, Pseudotime heatmap of the top 3,000 most variable genes along the differentiation axis. d, Analysis of S100a6⁺/Sox9⁺ astrocytes in the WM at 2 and 4 months. n = 4 animals. e, Quantifications of Sox9⁺ astrocytes over time in the WM. n ≥ 3 animals (for details, see ‘Statistics and reproducibility’ in the Methods). One-way ANOVA with Tukey post-hoc test. f, Confocal images immunostained for Sox9. 5-EdU was administered in drinking water for 24 h and staining was performed 24 h, 3 days and 5 days after 5-EdU administration. Scale bar, 100 μm. g, Closer view of Sox9⁺/5-EdU⁺ astrocytes in the GM after 3 days. Scale bar, 20 μm. h, Analysis of Sox9⁺/5-EdU⁺ astrocytes over time. n = 4 animals. One-way ANOVA with Tukey post-hoc test. i, Position of Sox9⁺/5-EdU⁺ astrocytes over time from the WM (bin 1) to the superficial layer of the cortex (bin 5). n = 3 animals. One-way (middle) and two-way (right) ANOVA with Tukey post-hoc test. j, Cell–cell communication mediated by the epidermal growth factor (EGF) pathway, with the arrow thickness indicating strength. k, Contribution of ligand–receptor pair in EGF signaling. l, t-SNE plots showing Egfr, Hbegf and Tgfa expression in astrocytes (left) and all cells (middle and right). m, Confocal images of WM immunostained for Sox9,5-EdU and Cd31. Scale bar, 20 μm. n, Analysis of Sox9⁺/5-EdU⁺ astrocytes in contact (0 µm), closer (<5 µm) and far away (>5 µm) from blood vessels (n = 4 animals). The graphs show the mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

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