Figure 4

Ngn2 is required cell autonomously for proper axon guidance of upper layer cortical neurons to the midline. (A-J) To target upper-layer cortical neurons, the dorsal telencephalon of E15.5 wild-type embryos was electroporated with plasmids encoding the fluorescent protein Venus and either a control shRNA (B-D), shRNA targeting Ngn2 (E-G), or shRNA targeting Ngn2 plus Ngn2^Rescue mutant, which has a single non-coding point mutation that reduces the ability of the shRNA to target Ngn2 (H-J). Following electroporation, organotypic slice cultures were prepared and cultured for 5 DIV. (A) Diagrams depicting the areas that were imaged for the expression of Venus revealing the neurons and their axons containing the shRNA. (C,F,I) Regardless of the shRNA electroporated, all conditions contain medial (callosal) projecting axons. Interestingly, the neurons containing only the shRNA targeting Ngn2 had a dramatic increase in the number of lateral projecting axons (red arrowheads, G), and many of these neurons were located within the cortical plate (L). (K) Quantification of the axonal projections demonstrates a significant increase in the number of lateral projecting axons in neurons containing only the shRNA targeting Ngn2. NS, not significant; *P < 0.05 according to ANOVA test.