Fig. 4. Positioning of postmitotic neurons was affected in CKO mutants. (A-C) Birth-date analysis was carried out by CldU and IdU double staining of E18.5 the dorsal pallium sections after a single pulse of CldU at E13.5 and IdU at E15.5. (D-I) Quantification of CldU- and IdU-positive cell index in each Bin. In wild-type embryos, E13.5-born neurons (CldU+, red) mostly settled down in the deep cortical layers with a peak in Bin4 (^**29.92±2.45%; n=3, in D) and E15.5-born neurons (IdU+, green) distributed mainly to the superficial layer, Bin5 (#14.12±2.51%; n=3, in G). In Shh-CKO embryos, E13.5-neurons positioned similarly to wild type though neurons in Bin4 were significantly decreased (^**11.24±1.89% in E compared with D; n=3, P<0.01). E15.5 neurons in Shh-CKO were positioned in the almost same pattern as in wild type (compare G with H). In Smo-CKO embryos, E13.5 neurons were differently scattered throughout the dorsal pallium (Bin1^****, 4.06±0.669%; Bin2^***, 18.38±3.69%; Bin5^*, 28.07±0.76%; n=3, P<0.01 in F) compared with wild type (Bin1^****, 0.09±0.09%; Bin2^***, 2.56±1.86%; Bin5^*, 14.00±3.29%; n=3, in D). E15.5 cells mostly remained in SVZ/VZ (Bin1 and 2 in I) and significantly decreased in CP (Bin3##, 3.63±1.97%; n=3, P<0.05 and Bin5#, 1.51±0.38%; n=3, P<0.01 in I) compared with wild type (Bin3##, 7.37±1.02% and Bin5#, 14.12±2.51%; n=3, in G). Scale bar: 50 µm.