Fig. 5. Shh signaling was required for formation of the deep cortical layer and subplate. Cresyl violet staining of the E18.5 dorsal pallium (A-C). In situ hybridization on parasagittal sections of the E18.5 dorsal pallium with Wnt7b (D-F) and Tbr1 probes (G-I). In wild type, Wnt7b and Tbr1 were intensely expressed in the deep cortical layer and SP, and weakly expressed in SVZ/VZ, but not in IZ (D,G). In Shh-CKO, the unstained gap of Wnt7b expression was not observed (E). Tbr1 expression was comparable with wild type (H). In Smo-CKO, Wnt7b expression in SP and SVZ/VZ was undetectable (F). Tbr1 expression in SP and SVZ/VZ was not detected either (I). MAP2 and chondroitin sulfate were detected by immunohistochemistry. In wild type and Shh-CKO, MAP2 (J-L) and chondroitin sulfate (CS56) (M-O) expression in SP neurons was detectable at E18.5 and E15.5, respectively (J,K; arrow in M, N). In Smo-CKO, MAP2 and Chondroitin sulfate were weakly expressed in SP neurons at E18.5 (L) and E15.5 (arrow in O), respectively. Scale bar: 100 µm.