Fig. 4: HMGB1 ablation in newborn astrocytes perturbs endfoot placement and endothelial morphology.

a Left, Fluorescence micrographs of immunostained cortical sections (ALDH1L1 green, CD31 red) from P14 control and Hmgb1^ΔAstro mice. Middle: Binarized images of ALDH1L1 staining with superimposed CD31 mask (red outlines). Higher magnification images illustrating detail of astrocyte endfeet coverage on CD31 ROI are displayed in yellow lines. Right: Distribution graph reveals significant differences in the frequency distribution of CD31 coverage by ALDH1L1 immunoreactivity between control and Hmgb1^ΔAstro mice (**p < 0.01, two-tailed Kolmogorov-Smirnov test). b Electron micrographs illustrating ultrastrucutral differences between control and Hmgb1^ΔAstro mice in astroglial endfeet (pseudocoloured in two shades of green for better distinction between individual endfeet) and endothelial cells (pseudocoloured in light red). Pericyte processes were pseudocoloured in light yellow. White arrows indicate macropynocytotic extensions; white arrowheads indicate macropinocytotic vacuoles; red arrowheads point endothelial protrusions extending into astroglial endfeet; red asterisks indicate regions of vessel wall lacking endfoot coverage. Graph display quantifications of the average number of endfeet /µm basement membrane (BM); average endfoot size (µm²); average proportion of macropynocytic vessels, and average number of vacuoles per endothelial cells (EC). L lumen, RBC red blood cell. Data are whisker boxes (min to max, center line indicating median). **p < 0.01 (two-tailed Mann–Whitney’s test). c Fluorescence micrographs of cortical sections immunostained for CD31 (green) and cadaverine Alexa Fluor-555 (red) in P14 Hmgb1^flx/flx (left) and Hmgb1^ΔAstro (middle) mice. Left, positive control: extravasation of cadaverine in the cerebral cortex 20 min after a stab wound with a 30 G needle. White arrows indicate cells that have taken up the dye. d Unchanged levels of inter-endothelial junction proteins VE-Cadherin, β-Catenin, Claudin-5, and Occludin as revealed by western blot from cerebral cortex extracts at P14. CTL control (Hmgb1^flx/flx), MUT mutant (Hmgb1^ΔAstro). All microscopy images displayed in this figure are representative of experiments repeated in at least 5 mice per group, with similar results. Source data are provided as a Source Data file.