The loss of Ngn2 results in cortical axons aberrantly projecting to cortical and subcortical targets in vivo. In utero electroporation of plasmids encoding the YFP variant Venus and either control shRNA or shRNA targeting Ngn2 were used to determine the final location of aberrantly projecting axons. Electroporated embryos were allowed to be born and survive to P14. Coronal sections were prepared and immunostained for GFP to enhance and reveal electroporated neurons and their axonal projections. (A) A diagram depicting the areas imaged. (B-F') Several areas were found to contain axons from neurons electroporated with control shRNA (B-F) and shRNA targeting Ngn2 (B'-F'). These areas include the zone of electroporation (B,B'), the contralateral cortex (C,C'), the ipsilateral cortex (D,D'), the internal capsule (E,E') and the thalamus (F,F'). Arrowheads indicate limits between layers. We found that several areas containing axons from neurons electroporated with the shRNA targeting Ngn2 (D',E',F') but not the control shRNA (D,E,F). (G) We have developed a model for the regulation of cortical axon guidance by Ngn2. At early stages (E10 to E14) when deep layer neurons (layers 5 to 6) are being generated, most of the axons initially project laterally. At later stages when superficial layer neurons (layers 2 to 4) are being generated, neurons differentially respond to cues within the cortex and project medially towards the corpus callosum. We believe Ngn2 is involved in this differential response. In the absence of Ngn2, there is a reduction in callosal axons and some of these axons project laterally towards cortical and subcortical targets.