Fig. 7. Neurons of layers II and III are not born from caNotch⁺ cells before Cre transfection. P5 cortices after electroporation of pCAG-EYFP (A-D) or pCAG-ExNotchZ (E-H) at E13.5, BrdU labeling at E14.5 and electroporation of pXCANCre at E15.5, are shown. Red and green signals show immunostaining with the anti-BrdU and anti-GFP antibodies, respectively. Arrowheads and arrows indicate neurons that were heavily and lightly labeled with BrdU, respectively. C and G are merged views of the sections shown in A and B, and E and F, respectively. (D,H) Ratios of neurons that were heavily (h) and lightly (l) labeled with BrdU in the EYFP⁺ population. Each ratio was calculated from five brains. After EYFP transfection, layers II and III contained 17.4±1.6% heavily- and 49.8±2.9% lightly-labeled neurons, whereas layer IV contained 68.8±6.6% heavily- and 21.2±5.8% lightly-labeled neurons. EYFP/caNotch transfection showed 0.7±1.1% heavily and 14.0±1.3% lightly-labeled neurons in layers II and III. After EYFP/caNotch transfection, many EYFP⁺ cells were not labeled with BrdU in the upper layers (85.8±1.8%), presumably because the amount of BrdU in nuclei was reduced to below the detection limit after multiple cell divisions. Scale bars: 200 µm for A,E; 20 µm for B,C,F,G.