Fig. 2. Erk1/2 activity is required for the activation of neural gene expression in the chick epiblast. Diagram (top, left) shows a schematic of a stage 3 embryo and the experimental manipulations for A and B. (A) Hamburger and Hamilton (HH) stage 3/3+ chick embryos were electroporated with an expression plasmid on one side of the prospective neural plate and incubated for 6 hours before fixation and in situ hybridisation followed by anti-EGFP-antibody staining. Overexpression of MKP3-EGFP (left) caused a reduction in the expression of Sox3 (6/7 embryos and 215/238 cells of three sectioned embryos), unlike overexpression of EGFP alone (four EGFP controls had no overt Sox3 downregulation; 53/308 cells affected in the two embryos scored). White bars mark the level of sections. Graph (right) shows the number of cells expressing both EGFP and Sox3 (blue) or only EGFP (hatched) in embryos electroporated with MKP3-EGFP or EGFP only. (B) Beads soaked in 10 mM PD184352 were implanted in the prospective neural plate of HH stage 3 (HH3) or HH stage 4 (HH4) embryos and left to develop for 6 hours. The expression of the Ets family genes Pea3 and Erm was locally inhibited around the bead at stage 3 (6/6 and 6/6 embryos, respectively), whereas Dlx5 expression was upregulated in 5/7 cases. (4/4 DMSO control embryos had normal gene expression levels.) When implanted at HH stage 3, PD184352-soaked beads caused a decrease in Sox3 expression (12/13 embryos) and, in 4/6 embryos, caused a decrease in Sox2 expression. Corresponding control DMSO-soaked beads (n=4 and 5, respectively) all showed normal expression levels. At HH stage 4, neither PD184352-(8/8 embryos) nor DMSO-soaked control (4/4 embryos) beads showed a decrease in Sox3 expression. PD184352-soaked beads also did not affect Sox2 expression at this stage (4/4 embryos). Beads soaked in 10 mM LY294002 or DMSO were placed in the prospective neural plate of HH stage 3 chick embryos, which were then left to develop for 6 hours. Expression of the early neural marker Sox3 was not affected by LY294002 treatment, whereas LY294002 did reduce phospho-PKB expression over a range of concentrations, as seen by western blotting of whole chick embryos (K. Fishwick and K.G.S., unpublished). Asterisks indicate the position of the bead in cases where it was lost during processing. Scale bars: 200 µm.