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Fig. 5. The canonical Wnt pathway promotes NPC differentiation into neurons in the developing mouse neocortex. Mouse neuroepithelial cells were subjected to in utero electroporation at E13.5 with various constructs. Embryos were fixed 2 days after electroporation and subjected to immunostaining. (A) Immunostaining with TuJ1 (red) and antibodies to GFP (green) in cells of the same region of the dorsolateral telencephalon electroporated with a vector for GFP alone (control) or for GFP and S33Y ß-catenin. Broken lines indicate the boundary of TuJ1⁺ and TuJ1^- areas, which coincides with the basal edge of the ventricular zone (VZ) judged by the shape of the cells. Scale bar: 100 µm. Most of the cells expressing S33Y ß-catenin migrated out of the VZ and only a small fraction of these cells (10.4%) remained in the VZ, whereas many control cells remained in the VZ (33.3%). The VZ of the cortex electroporated with S33Y ß-catenin was thinner than that of control. Similar results were obtained from three independent experiments. (B) Quantitative analysis of HuC/D expression in individual electroporated cells. Embryos were electroporated with a vector expressing either histone H2B-GFP alone or together with a vector for S33Y ß-catenin. The relative expression level (intensity/area) of the neuronal marker HuC/D within individual histone H2B-GFP⁺ cells was quantified by immunostaining and LSC. The percentages of cells within each range of expression are shown in the histograms. The broken lines represent the approximate expression level apparent at the border between the VZ and the intermediate zone, and the percentages of cells with expression levels less than or greater than this value are indicated (see Materials and methods). Littermates were analyzed for each experiment. (C) Immunostaining with TuJ1 (red) and antibodies to GFP (green) in cells of the same region of the dorsolateral telencephalon electroporated with vectors for histone H2B-GFP alone (control) or for histone H2B-GFP and Axin. A large number of the cells expressing Axin remained in the VZ (42.8%), compared with the number of control cells remaining (28.3%). Scale bar: 50 µm. (D,E) Quantitative analysis of HuC/D expression in individual cells electroporated with a vector for either Axin (D) or for Dkk1 (E). All data are representative of results obtained from three independent experiments.

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