Extended Data Fig. 8: The normal cytoarchitecture of the SVZ and olfactory bulb containing mutation-carrying cells and viral injection targeting driver mutations in the cortex.
From: Human glioblastoma arises from subventricular zone cells with low-level driver mutations
a, b, Immunostaining of S100β (a, ependymal cell marker), GFAP and nestin (b, stem-cell marker) in mutation-arising SVZ tissue from the mice with high-grade glioma in the distant cortical region (n = 6 mice with tumours). c, Targeted deep sequencing of TP53 and PTEN in tdTomato-positive neurons laser-captured from the olfactory bulb. The tdTomato-positive cells in the olfactory bulb were labelled with yellow dots in LCM images (targeting). Labelled cells were micro-dissected (LCM). d, Deep amplicon sequencing of CRISPR target sites showed that tdTomato-positive cells in the olfactory bulb contained P53/PTEN mutations. Scale bar, 50 μm. LCM and sequencing were repeated in 3 mice. e, Experimental scheme showing the procedure for viral injection of AAV5 containing sgRNAs for p53 and Pten genes, with the expression of Cas9 and Cre recombinase into the SVZ in LSL-EGFRviii; LSL-Cas9-GFP mice. f, g, Representative images of the injection site in the cortex at 1 week and 6 weeks after viral injection. Virus-expressing cells are GFP-positive. Scale bar, 500 μm. h, Quantification of the number of GFP-positive cells at the representative image. The section points were the sites where the highest number of GFP-positive cells are found (mice at 1 week: n = 3, mice at 4–6 weeks: n = 5). Not significant (NS), Student’s two-tailed t-test. Error bars represent mean ± s.e.m. i, Images from mouse at 6 weeks after viral injection to cortex. No signal was found in the distant brain as well as the SVZ. j, Images from mouse at 6 weeks after electroporation to the SVZ. For panels d and e, section points were 0.7, −1, −2.5 and −3.5 mm apart from bregma (mice at 1 week: n = 3, mice at 4–6 weeks: n = 5). Scale bars, 500 μm. k, Representative immunohistochemical images of GFAP-, OLIG2-, NeuN-, and GFP-positive cells at 1 week after viral injection) mice at 1 week: n = 3, mice at 4–6 weeks: n = 5). Scale bars, 100 μm.
