Fig. 4.

Knockdown of Aspm promotes VZ cells to adopt a non-NE fate and increases the appearance of neuron-like NE cell progeny in the neuronal layer. (A and B) Mouse E12.5 dorsal telencephalon was either coelectroporated with Aspm esiRNAs and mRFP plasmid (A and B) or electroporated with mRFP plasmid only (B), followed by 48 h in utero development. (A) Cryosections of the nontargeted (control) and targeted (Aspm RNAi) hemispheres were analyzed for RFP fluorescence (red) and γ-tubulin (green) and βIII-tubulin (blue) immunofluorescence. Note the increase in abventricular centrosomes (arrowheads) upon Aspm knockdown. The yellow lines indicate the boundary between the VZ/sub-VZ and the neuronal layers. The apical surface of the VZ is down. (Scale bar, 10 μm.) (B) Numbers of centrosomes per 10,000 μm² counted in γ-tubulin-immunostained cryosections are expressed as a ratio of targeted/nontargeted hemispheres for electroporation with mRFP plasmid only (control) and mRFP plasmid plus Aspm esiRNAs (Aspm RNAi). Data are the mean of five cryosections from three different embryos each; error bars indicate SD. (C and D) Dorsal telencephalon of E10.5 Tis21–GFP knockin mice was coelectroporated with Aspm esiRNAs and mRFP plasmid (Aspm RNAi) or electroporated with mRFP plasmid only (control), followed by 24 h of whole-embryo culture. (C) Cryosections were analyzed for the proportion of RFP-positive cells in the neuronal layer that lacked Tis21–GFP fluorescence. Data are the mean of three cryosections from two to three different embryos each (control, 70 cells; Aspm RNAi, 74 cells); error bars indicate SD. (D) Representative example of two neighboring RFP-positive cell bodies (red) in the neuronal layer, with one being positive (green circles) and one being negative (yellow circles) for Tis21–GFP fluorescence (green) but both exhibiting βIII-tubulin immunofluorescence (white). (Scale bar, 5 μm.)