Pattern of Ngn2 expression in the developing cortex. (A-H) Coronal sections from E12.5 (A-D) and E16.5 (E-H) were immunostained for Ngn2 (A,E), Tbr2 (B,F) and Draq5 (C,G). (D,H) Merged images. At E12.5, Ngn2 expression is resticted to the VZ (A) and has little colocalization with Tbr2 (B,D), a marker for intermediate progenitors. In contrast, at E16.5, a time when superficial layer neurons are generated, Ngn2 is expressed widely in the VZ and SVZ, and occasionally in the IZ (E); many cells also co-stain for Ngn2 and Tbr2 (F,H), demonstrating that many intermediate progenitors express Ngn2. Arrowheads denote cells co-expressing Ngn2 and Tbr2 located in the upper SVZ and IZ. (I) To determine when callosal axons first emerge, E15.5 cortices were electroporated with the YFP variant Venus and cultured as organotypic slices. Electroporated neurons were immunostained for GFP 36 hours after electroporation to visualize axon initiation. At this time point, several neurons have a single long neurite growing medially (red arrowheads). Interestingly, none of these neurons have formed a leading process, which is characteristic of radially migrating neurons. CP, cortical plate; IZ, intermediate zone; PP, preplate; SVZ, subventricular zone; VZ, ventricular zone.