Extended Data Figure 9 : Manipulation of PDGFRβ signalling in human and mouse neocortex.

From: Radial glia require PDGFD–PDGFRβ signalling in human but not mouse neocortex

Extended Data Figure 9

ad, Chemical blockade of PDGFRβ signalling in cultured slices of GW14.5 human neocortex impairs RG cell cycle progression. Four pharmacological inhibitors of PDGFRβ signalling were screened at different concentrations to determine their effects on RG proliferation in cultured slices of GW17.5 human neocortex (2 days). Slices were treated with BrdU for the duration of the experiment and RG proliferation was quantified as the fraction of SOX2+ cells that incorporated BrdU after treatment with inhibitor or vehicle. Statistical significance was assessed with the Wilcoxon rank sum test using the wilcox.test R function with default settings. Images are derived from ≥3 slices in each condition. Control + DMSO, n = 18; control no DMSO, n = 9; CP673451 all concentrations, n = 9; Sutent all concentrations, n = 6; imatinib (0.1 μM, 10 μM), n = 9 (100 μM), n = 6; tivozanib (1 μM, 10 μM), n = 9 (0.1 μM), n = 6. Significance indicated by: n.s. P > 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. e, Slice cultures of E13.5 mouse neocortex were treated with BrdU and DMSO (control) or a pharmacological inhibitor of PDGFRβ signalling (CP673451) for 1 or 2 days (slices from at least three independent litters). RG (SOX2+) or intermediate progenitor (TBR2+) cell proliferation was assessed as the fraction of each population that incorporated BrdU or was Ki67+ (1d: n = 10 (DMSO) versus n = 9 (CP673451); 2d: n = 11 (DMSO) versus n = 9 (CP673451)). This experiment serves as a negative control to compare with the human. f, Ectopic PDGFRβ signalling promotes RG identity in E13.5 mouse neocortex. In utero electroporation of constitutively active TEL–PDGFRβ (ref. 23) was compared with control (mouse E13.5–E15.5) and assessed for the proportion and distribution of SOX2+ RG cells or Ki67+ progenitors (out of GFP+) in the cortical wall (quantified in g: at least n = 3 slices per embryo from two independent litters, n = 15 (control); n = 18 (TEL–PDGFRβ) or (PDGFRβ(D850V)); scale bar, 50 μm). Ki67+GFP+ cell quantification following PDGFRβ(D850V)22 electroporation was performed in a similar fashion and is also shown. The spatial distributions of RG (GFP+SOX2+) in the cortical wall were assessed by quantitative image analysis (spanning ventricle to pia). The grey band delineates a 95% confidence interval for a test of equal univariate densities based on 10,000 permutations. All error bars represent mean ± s.e.m. Statistical significance for the effects of treatment was calculated by ANOVA of multiple linear regression while controlling for individual (e) and litter (f) variability (significance indicated by: n.s. P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).