Hh pathway activation expands the population of cells with stem cell-like properties and promotes symmetric proliferative divisions of radial glial cells.
(A) E14.5 Ptc1Lox/Lox;NestinCre neocortex (n = 10) showed an 8-fold increase (***p<0.0001) in the number of primary neurosphere colonies per cell seeded compared to littermate controls (n = 11). For RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre samples, n = 3 and n = 4 respectively. * and ** p<0.005 by unpaired Student's t-test (with Welch's correction). (B) Clonal expansion of neurospheres derived from E14.5 neocortex. Cell numbers obtained at each passage were transformed into LOG and linear regression analysis was performed on resulting curves. The rate of clonal expansion of the neurospheres is represented as the slope of the line. The number of stem cells was significantly increased in Ptc1Lox/Lox;NestinCre neurospheres compared to Ptc1Lox/Lox neurospheres (slope: 0.8877±0.06774 vs 0.7060±0.03302, p<0.05). However, RbpjLox/Lox;NestinCre and Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neurospheres showed a significant reduction in stem cells compared to the wild type control (slope: −1.337±3.400e+038, p<0.0001 and slope: −0.08099±0.04816, p value compared to control <0.0001, respectively). Error bars indicate standard deviation (A, B). (C–F): Co-immunofluorescence of GLAST (red) and TuJ1 (green) on neocortical progenitors isolated from E14.5 Rbpj and Ptc1 mutant and wild types neocortices. Cells were counterstained with DAPI (blue). Arrows (in C, E, F) indicate TuJ1+ cells and arrowheads (D) indicate GLAST+ radial glial cells. RG cells from Ptc1Lox/Lox;NestinCre neocortex rarely differentiate into TuJ1+ neurons after 24 hours (D, G), whereas concomitant loss of the Rbpj and Ptc1 alleles results in an increased rate of neurogenesis in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortical cells (F, G). On the other hand, cells from RbpjLox/Lox;NestinCre neocortex mostly differentiate into TuJ1+ neurons after 24 hours. Also, (G): Quantitative analysis for GLAST and TuJ1 revealed that the percentage of RG cells undergoing symmetric neurogenic divisions was significantly increased (p<0.05) in the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to the Ptc1Lox/Lox;NestinCre neocortices. On the other hand, there was a reduction in the number of symmetric proliferative cell divisions in Ptc1Lox/Lox;RbpjLox/Lox;NestinCre compared to Ptc1Lox/Lox;NestinCre (p<0.05) samples. Bars represent standard errors. ***p<0.0001. Scale bar (C–F), 10 µm.