Fig. 3. Ebf1 misexpression promotes neuronal differentiation. (A-F) Stage HH15 chick embryos were either electroporated with a lacZ expression plasmid, or co-electroporated with lacZ and Ebf1 expression plasmids, incubated for the indicated periods, processed for X-gal staining and transversally sectioned. Each pattern shown reflects the situation in more than 90% of the embryos, from eight independent experiments, each involving six embryos per condition. (G-I) Stage HH15 embryos were electroporated with HA-tagged Ebf1 or R409W mutant Krox20 (K20m-HA, encoding an inactive transcription factor) and then subjected to 2-hour BrdU pulse-labelling immediately before collection at the indicated time following electroporation. Vibratome sections were then analysed by immunofluorescence with antibodies directed against HA (red) and BrdU (green). The dashed line in I indicates the separation between the mantle layer (ML) and the ventricular zone (VZ). (J) Quantification of the data obtained from experiments presented G-I. The bars represent the percentage of electroporated cells (HA-Ebf1- or Krox20m-HA-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, which are BrdU-negative (red, grey bars). The cell counts correspond to the analysis of six to nine sections from at least three independently processed embryos for each condition. The data represent mean±s.e.m. (K,L) Flat-mounted hindbrains from embryos that were either not electroporated or co-electroporated with Ebf1 and GFP expression vectors at stage HH10, then stained for neurofilaments by immunochemistry 24 hours later. (M-O) Stage HH10 embryos were electroporated with HA-tagged Ebf, sectioned 24 hours later at the level of r6 and analysed by immunofluorescence with antibodies directed against the HA epitope (red) and neurofilaments (green, N), or the HA epitope and Tuj1 (green, O). The arrowheads point to cells co-expressing the two markers. M shows the part of the sections presented in N,O. Each analysis was performed on three independent series of six embryos. Tuj1 and neurofilaments were detected in approximately 100% and 80% of the HA-Ebf1-positive cells, respectively. (P-R) Flat-mounted hindbrains from embryos co-electroporated with Ebf1 and GFP expression vectors at stage HH10, and processed 24 hours later for in situ hybridisation with N-cadherin (N-cad) and R-cadherin (R-cad) probes. In R, double in situ hybridisation was performed (N-cad, purple; R-cad, red). In K,L,P-R the patterns shown were observed in more than 90% of the embryos, from four independent experiments, each involving at least six embryos per condition. (S,T) Stage HH15 embryos were co-electroporated with GFP and chicken Id2 (S), or GFP, Id2 and Ebf1 expression vectors (T). They were then subjected to 2-hour BrdU pulse-labelling immediately before collection, 30 hours after electroporation. Vibratome sections were then analysed by immunofluorescence with antibodies directed against BrdU (green), GFP (red) and Tuj1 (blue). In S and T, 60±5% and 55±3% of transfected cells were BrdU⁺, respectively, and 98±0.6% and 88±4% were located in the VZ, respectively. The data represent mean±s.e.m. and correspond to the analysis of seven sections from three independently processed embryos for each condition. Electroporation was on the right side. Electroporated constructs are indicated at the top of each panel and immunolabelling or in situ hybridisation probes at the bottom. h, hours.