Figure 4.

Defective GNP proliferation in the NF1^Nes EGL. (A) Representative confocal images of BrdU incorporation in the EGL of control and mutant P7 pups that were sacrificed 2 h after BrdU administration. Bar, 50 μm. (B) Quantitative analysis revealed a decreased number of BrdU-incorporating cells among total cells in the NF1^Nes EGL. Mean ± SEM; n = 4; (****) P < 0.0001. (C) Assessment of progenitor cell proliferation at the EGL by Ki67 immunostaining. Arrows indicate the few Ki67-positive cells in the mutant EGL. Bar, 50 μm. (D) Quantitative analysis indicated reduced Ki67 expression among the total number of cells in the NF1^Nes EGL. Mean ± SEM; n = 4; (****) P < 0.0001. (E) Analysis of the transcript levels of genes involved in GNP proliferation (Ki67 and Shh signaling pathway) in control and mutant cerebella. Mean ± SEM; n = 3; (*) P < 0.05. (F) Quantitative Western blot analysis indicating sustained reduction of Gli1 protein levels in NF1^Nes cerebella. Mean ± SEM; n = 3; (*) P < 0.05. (G) Representative Western blot examining the Shh signaling pathway in NF1^Nes cerebella. (H) Quantitative analysis indicates unaltered levels of Shh, Ptch1, and Cyclin D1 but significantly reduced levels of MycN in mutant mice at P7. Mean ± SEM; n = 3; (*) P < 0.05. Molecular weight markers (in kilodaltons) are shown in F and G.