Fig. 2. Proliferation of progenitor/stem cells in the dorsal pallium is reduced and apoptosis is increased in the absence of Shh signaling. BrdU-incorporation analysis at E13.5 (A-C) and E15.5 (A'-C'). Cells were counted in 100 µm-wide sampling boxes (white boxes). Examples of anti-BrdU labeling (red) of parasagittal sections of the dorsal pallium of wild-type (A,A'), Shh-CKO mutant (B,B') and Smo-CKO mutant (C,C') embryos. (D) Both Shh-CKO and Smo-CKO embryos showed significantly decreased BrdU incorporation index at E13.5 (13.2±1.26% and 14.4±0.29%, respectively; n=3) compared with wild type (16.8±0.47%, n=3). At E15.5, Smo-CKO showed significantly decreased index (7.78±0.10%; n=3), while Shh-CKO showed comparable index (12.04±1.15%, n=3) to wild-type embryos (11.75±1.60%, n=3). Parasagittal sections of E13.5 dorsal pallium were immunostained with anti-cleaved caspase 3 antibody (E-G) and cells were counted on the entire sections (H). There were a few stained cells (red) in wild-type brain (E,H, 0.35±0.118%; n=3). Apoptotic cells were increased in the dorsal pallium of Shh-CKO (F,H, 1.33±0.075%; n=3) and Smo-CKO embryos (G,H, 1.07±0.210%; n=3). ^*P<0.01. Scale bar: 50 µm.