We next performed ankyrin-G staining at P4 to analyze neuronal polarity and the acquisition of a mature axonal structure18. These immunostainings demonstrated that most cells electroporated with shRNA-IGF-1R arrested at the VZ/SVZ/IZ did not express ankyrin-G (Fig. 4a bottom). Only the few cells that migrate to layers II-IV (Fig. 4a top) or layers V-VI (Fig. 4 middle) exhibited an axon as shown by immunostaining for the axonal protein ankyrin-G In contrast, in control brains close to 100% of the electroporated cells migrate to layers II-IV (see Fig. 2) and develop mature axons enriched in ankyrin-G (Fig. 4b).Figure 4shRNA-IGF-1R arrested neurons fail to acquire axonal polarity as shown by ankyrin-G staining (lack of ankyrin-G clusters). (a) Brains were electroporated with shIGF-1R/CAG-GFP at E15 and analyzed at P4 after staining with an antibody to ankyrin-G to show axons. Cells in the V-VI layers (middle) or layers II-IV (top) show co-staining of the trailing process with ankyrin-G indicating normal polarity and the correct compartmentalization/specialization of the apical neurite into anaxonal structure. Cells arrested in the VZ/SVZ/IZ show lack of ankyrin-G staining indicating failure to develop dendritic-axonal polarity. (b) Brains were electroporated with control shRNA/CAG-GFP at E15 and analyzed at P4 after staining with an antibody to ankyrin-G to show axons. Cells in layers II-IV exhibit axons stained with ankyrin G. Virtually no cells were found in the VZ/SVZ/IZ and layers V-VI under this experimental condition (see Fig. 2) Calibration bar 10 μm.