Fig. 5. Ebf1 misexpression affects neuronal subtype specification. (A-E) Stage HH15 chick embryos were co-electroporated with Ebf1 and GFP expression vectors, incubated for 30 hours, sectioned and processed for in situ hybridisation with CRABPI (A) and Lim1 (B) probes, or for double in situ hybridisation with CRAPBI and Islet1 (C), CRABPI and Islet2 (D), or Lim1 and Islet1 (E) probes. In double-labelling experiments, the colour code is shown underneath. Note the presence of cells ectopically expressing CRABPI or Lim1 within the ventricular zone (arrows), and at the level of the motor column on the electroporated side (arrowheads). These latter cells do not express Islet1 (C,E). The cases shown are representative of more than 80% of the embryos, from six independent experiments, each involving at least six embryos per probe. (F-H) Embryos were electroporated with HA-tagged Ebf1 at stage HH13 (F), HH15 (G) and HH17 (H), and collected 30 hours later. Vibratome sections were processed for immunofluorescence analysis with antibodies directed against the HA epitope (red) and Islet1/Islet2 (green). (I) Quantification of the data obtained from the experiments presented in F-H. The bars represent the number of Islet-positive cells per section on the control side (green, black bars), the number of HA-Ebf1-positive cells per section within the estimated motor column on the electroporated side (red, grey bars) and the number of Islet-positive cells per section on the electroporated side (green, white bars). The data represent mean±s.e.m. of cell counts and correspond to the analysis of six to nine sections of at least three independently processed embryos for each condition. Only sections containing at least 10 HA-Ebf1-positive cells within the motor column have been taken into account. P<0.001 for the differences observed in the number of Islet-positive cells between control and electroporated sides. 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. MC, motor column.

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