Extended Data Fig. 9: In vivo location of EdU-labelled cells in the SVZ neurogenic niche.
a, Representative image of vinculin immunofluorescence staining of old coronal section from GFAP-GFP mouse (Fig. 4c) indicating how vinculin (white) was used to demarcate ventricle border. The ventricular lining is indicated by a dotted white line. Green, GFAP (astrocyte/NSC). Pink, Ki67 (proliferation). Blue, DAPI. Scale bar, 50 μm. b, Representative image of S100a6 immunofluorescent staining of old coronal section from GFAP-GFP mouse. White arrows: qNSC (S100a6 + /Ki67- and GFAP + /Ki67-); white arrowheads aNSC (S100a6 + /Ki67+ and GFAP + /Ki67+). Green, GFAP (astrocyte/NSC). Pink, S100a6 (NSC marker in the SVZ neurogenic niche). Red, Ki67 (proliferation). Blue, DAPI. Scale bar, 50 μm. c, NSC distance to the ventricle was calculated for qNSCs (S100a6 + /Ki67-) and aNSCs (S100a6 + /Ki67+) in coronal sections of young and old SVZs. Each dot represents the mean distance from the ventricle of 140–574 cells in 6-11 fields per section (3 sections per dot) for an individual mouse. n = 4 mixed-sex young mice, and n = 4 mixed-sex old mice, combined over 4 independent experiments. d, NSC distance to the ventricle was calculated for qNSCs/astrocytes (GFAP + /Ki67-) and aNSCs (GFAP + /Ki67+) in coronal sections (left) of young and old SVZs from mixed-sex GFAP-GFP mice and sagittal sections (right) of young and old SVZs from male C57BL/6 mice. Each grey dot represents the distance of a single cell from the ventricular lining. Each colored dot represents the mean distance from the ventricle of 19-231 cells in 3-7 fields per section (3 sections for coronal sections or 1 section for sagittal section) per individual mouse. Coronal sections: n = 4 young, and n = 4 old mixed-sex GFAP-GFP mice, combined over 4 independent experiments. Sagittal sections: n = 5 young male mice, and n = 5 old male C57BL/6 mice combined over 2 independent experiments. e, Single cell RNA-seq analysis of 28 C57BL/6 mice ranging in age from 3.3 months to 29 months (see Methods). Out of 21,458 cells, there are 4 cells expressing all markers of ependymal-repairing SVZ astrocytes (S100b, Gfap, CD24a, Ctnnb1) (see Methods). These cells are colored by age (4.7 months, 5.4 months, 16.83 months, and 18.87 months). Ependymal-repairing SVZ astrocytes are absent from qNSC/astrocyte, aNSC/NPC, and neuroblast clusters but are present in ependymal cell cluster. f, Distance to the ventricle was calculated for EdU+ aNSC/NPC (Ki67 + /DCX-) in sagittal sections of young and old SVZs 4 hours after EdU injection. Each grey dot represents the distance of a single aNSC/NPC from the ventricle. Each colored dot represents the mean aNSC/NPC distance from the ventricle of 2-35 cells from 1 section per individual mouse. n = 5 young male mice, and n = 5 old male mice, combined over 2 independent experiments. g, Distance to the ventricle was calculated for EdU+ neuroblasts (DCX+) in sagittal sections of young and old SVZs 4 hours after EdU injection. Each grey dot represents the distance of a single neuroblast from the ventricle. Each colored dot represents the mean neuroblast distance from the ventricle of 3-25 cells from 1 section per individual mouse. n = 5 young male mice, and n = 5 old male mice, combined over 2 independent experiments. h, Quantification of number of EdU+ cells counted in the SVZ (along the entire length of the ventricle) from young and old sagittal sections 4 hours post-injection of EdU. n = 5 young male mice, and n = 5 old male mice, combined over 2 independent experiments. I, Quantification of percent of Ki67+ cells that are EdU+ 4 hours post-injection of EdU in the SVZ (counting cells along the entire length of the ventricle). n = 5 young male mice, and n = 5 old male mice, combined over 2 independent experiments. j, Representative images of immunofluorescence staining of sagittal sections encompassing the SVZ, RMS, and OB regions from a young or old male C57BL/6 mouse 4 hrs or 7 days after intraperitoneal EdU injection. White inset denotes SVZ. Dotted white line indicates ventricle lining. n = 5 old male mice 4 hours post-injection, n = 3 old male mice 2 days post-injection, and n = 4 old male mice 7 days post-injection, combined over 2 independent experiments. Green, EdU (proliferation). Pink, Ki67 (aNSC/NPC/neuroblast); Red, DCX (neuroblast). Scale bar, 500 μm. All data are mean ± SEM. All statistical comparisons made using a two-tailed Mann-Whitney test comparing sample means. Data from independent experiments are in Source Data. Ages of animals used provided in Source Data.
