Figure 6

AKT prevents premature differentiation of neural progenitors in vivo . (A) E13.5 forebrains were electroporated in utero with pCAG-GFP and 4-fold (by mass) excess of pcDNA control or K197M dominant negative AKT (DN-AKT) and analyzed at E14.5 (n = 4 brains each). Electroporated cells were identified using antibody staining against GFP and sections counterstained with DAPI. The pial surface is indicated by the white line. Scale bar = 100 µm. (B) To quantify changes in cortical positioning of electroporated cells, ten equal sized bins were drawn over each image covering the cortical plate. Each dot corresponds with the soma of an electroporated cell. The fraction of the total GFP + cells was then graphed. The x-axis denotes the fraction of the total number of electroporated cells in each bin. Brackets indicate 1 SEM. (C-F) Sections of electroporated brains were stained for Pax6 (C), Tbr2 (D), and Tbr1 (E). Electroporated cells are green, and the respective antigens are red. Bar = 100 µm. The dot plots show the electroporated cells co-expressing the marker as yellow and electroporated cells not expressing the marker as green. (F) Cell histograms show the fraction of electroporated cells that express each marker after electroporation (yellow/yellow + green dots). DN-AKT causes premature neuronal differentiation as defined by the alterations in Tbr1, Tbr2, and Pax6 expression. For Pax 6, DN-AKT versus control (n = 4 brains for each, **P = 0.0071), Tbr2 (n = 2 for DN-AKT, n = 4 for pcDNA control, *P = 0.0406), and Tbr1 (n = 4 for each, *P = 0.0277), unpaired t-test. Error bars represents SEM. Scale bar = 100 µm. (G) DN-AKT increases the fraction of cells that lose contact with ventricular surface. n = 4 brains, *P = 0.0368, paired t-test. Error bar represents SEM. EP’d, electroporated.