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First published online July 27, 2007
doi: 10.1242/10.1242/dev.02858

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A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification

Division of Cell and Developmental Biology, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Differentiation of ES cells is arrested in the absence of Erk1/2 signalling. (A) Activation of Sox1-EGFP expression during monolayer differentiation of 46C cells monitored daily by flow cytometry under different conditions (means of three experiments in duplicate±s.e.m). (B) Western blotting for activated signal transduction cascade components during differentiation in N2B27 media (representative blots). (C) Efficiency of differentiation (relative proportion of Sox1-EGFP-expressing cells) at day 3 in the presence of the factors shown (normalised to the no-treatment control for each of three experiments performed in duplicate +s.e.m.). (D) 46C cells were differentiated for 24 hours under the conditions shown, then lysed. Western blotting for C-terminal phosphorylated Smad1, Smad5 and Smad8 (top) shows a robust response to Bmp4 but no appreciable Smad activation in N2B27 containing 3 µM PD184352 (arrows). Arrowhead indicates a non-specific contaminating band. S214 (linker; bottom) phosphorylation of Smad1 appears to not be affected by Erk1/2 inhibition in these conditions. (E) Real-time PCR on cDNA from PDK1-/- and wild-type ES cells as well as from PDK1-/- and wild-type cells after 4 days of monolayer differentiation. Results are the expression levels relative to wild type at day 4 +s.e.m. ES, embryonic stem cell.

View larger version (70K): [in this window] [in a new window]   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.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Erk1/2 inhibition reversibly blocks ES cell differentiation at the primitive-ectoderm stage. (A) Real-time RT-PCR for Fgf5 and Nanog was performed at day 2 of differentiation. Expression of Sox1 was determined for the same conditions at day 4. Results are from duplicate determinations of three experiments+s.e.m. relative to the expression level in ES cells (Fgf5, Nanog) or day 4 of differentiation (Sox1). (B) Flow cytometry profile of 46C ES cells plated in N2B27 plus 3 µM PD184352 for 2 days before media was changed to either N2B27 (-PD184352; green line) or N2B27 containing fresh inhibitor (+PD184352; black line) for 24 hours. (C) Images of 46C cells at day 3 of differentiation under the conditions shown. Scale bar: 200 µm.

View larger version (11K): [in this window] [in a new window]   Fig. 4. A brief exposure to Erk1/2 signalling is sufficient to trigger neural specification. (A) Schematic of experiment. ES cells were differentiated for 2 days in N2B27+PD184352 before media was changed to N2B27±PD184352 for the specified time. Following this, the cells were lysed and analysed for activation of Erk1/2 (B) or media containing PD184352 was replaced and cells were cultured for 15 hours before FACS analysis (C). (B) Erk1/2 activation following inhibitor withdrawal at day 2. ES, ES cell lysate; -/+, cells differentiated for 2 days in constant absence/presence of PD184352. (C) Effect of different time periods of Erk1/2 activity on neural specification. y-axis marks the percentage increase of Sox1-EGFP-expressing cells following culture in the absence of PD184352 over culture for the same time in the presence of PD184352. Results are averages±s.e.m. from three experiments performed in triplicate.