Fig. 7. Ebf uncouples cell cycle exit from neuronal differentiation. (A-C) Stage HH15 chick embryos were co-electroporated with GFP and mutant Krox20 (K20m-HA, Kr), Ebf (Ebf, E) or Ebf1 (HA-Ebf1, E1) expressing constructs as indicated, subjected to 2-hour BrdU pulse-labelling 28 hours later, and vibratome sectioned for direct detection of GFP fluorescence (shown in red) and immunofluorescence analysis of incorporated BrdU (green). The mutant Krox20 protein is inactive and was used as a control. The dashed line in A indicates the separation between the mantle layer (ML) and the ventricular zone (VZ). (D) Quantification of the data obtained from the experiments presented in A-C. The bars represent the percentage of electroporated cells (GFP-positive, red or yellow) that are located in the ventricular zone (black bars), the percentage of electroporated cells that are BrdU-positive (yellow, white bars), and the percentage of electroporated cells within the ventricular zone that are BrdU-negative (red, grey bars). (E-G) Stage HH15 chick embryos were electroporated with Flag-Ngn2 (n2) alone or together with Ebf or Ebf1 expression constructs as indicated, subjected to 2-hour BrdU pulse-labelling 28 hours later, and vibratome sectioned for immunofluorescence analysis of incorporated BrdU (green), Flag (blue) and HA (red) epitopes, marking Ngn2 and Ebf1, respectively. (H) Quantification of the data obtained from the experiments presented in E-G. The bars represent the percentage of electroporated cells that are Ngn2-positive (blue, purple or white), which are located in the ventricular zone (black bars), and the percentage of electroporated cells that are BrdU-positive (white, white bars). In D and H the data represent mean±s.e.m. and correspond to the analysis of six to nine sections from at least three independently processed embryos for each condition. (I-K) Stage HH15 chick embryos were co-electroporated with GFP and Ebf, Flag-Ngn2 alone, and Flag-Ngn2 and Ebf expressing constructs as indicated, incubated for 30 hours and then vibratome sectioned for immunofluorescence analysis of Tuj1 (green) and Flag epitope (Blue), or direct detection of GFP fluorescence (red). (L,M) Stage HH15 chick embryos were co-electroporated with lacZ and Krox20m or Ebf expression plasmids, respectively, incubated for 40 hours and then vibratome sectioned for immunofluorescence analysis of Tuj1 (blue), and ß-galactosidase (ß-gal, red) and TUNEL analysis (green). In sections electroporated to the same extent, as judged by lacZ expression, apoptosis is significantly increased (3.5±1-fold) in Ebf-versus Krox20m-electroporated embryos. More than half of the TUNEL-positive cells in M are also lacZ-positive, although only weakly. Cell counts were performed on eight sections from three independently processed embryos. Electroporation was on the right side.