Fig. 7. Effects of transitin knockdown by shRNA on neuroepithelium cell fate. (A) Transfection with pSilencer-Trans289 shRNA expression vectors successfully knockdowns transitin expression. At 24 hours after electroporation the transfected area, revealed by the expression of co-transfected EGFP, shows weak expression of transitin (purple; arrowheads). (B) Trans289-transfection results in a downregulation of Numb expression (purple). The reduction of anti-Numb immunoreactivity is evident in the basal processes of neuroepithelium (NE) cells. (C) BrdU uptake of neural tube cells co-transfected with EGFP and pSilencer-Trans289. Little overlap of GFP-fluorescence and anti-BrdU immunostaining is observed. (D) The proportion of BrdU-GFP double-positive neural tube cells co-transfected with EGFP and empty pSilencer (-; green), or pSilencer carrying mutated transitin sequence (Trans289m, mut; blue), or pSilencer carrying the wild-type transitin sequence (Trans289, wt; red), 24 hours after transfection. (E) Proportion of TUNEL-GFP double-positive neural tube cells co-transfected with EGFP and pSilencer (-), Trans289m (mut), or Trans289 (wt) 24 and 48 hours after transfection. (F) Neuronal differentiation of transfected neural tube cells co-transfected with EGFP and Trans289 (wt) 48 hours after transfection. Whereas most empty pSilencer-transfected cells remain Hu-negative in the ventricular zone (left panel), Trans289-transfected cells migrate basally, express Hu and intermingle with untransfected neurons (right panel). (G) The proportion of Hu-EGFP double-positive neural tube cells co-transfected with EGFP and pSilencer (-), Trans289m (mut), or Trans289 (wt) 24 and 48 hours after transfection. Five embryos (approximately 200-300 cells/embryo) were examined to obtain each bar in D,E,G. Error bars indicate standard deviations.