Fig. 5. Activation of the MAPK and PI(3)K signalling pathways is necessary for the chemoattractant response of ENCCs and enteric axons to GDNF. (A) Segments of proximal small intestine dissected from E11.0 mouse embryos were co-cultured with a clump of COS/GDNF cells (shown to the right of small intestine explants in a,c,e), in either control medium (a,b), or in medium supplemented with PI(3)K inhibitor (LY294002; c,d) or MEK1 inhibitor (PD98059; e,f). At the end of the experiment, cultures were stained for RET (a,c,e) and Tuj1 (b,d,f) and counterstained for DAPI. Reduced cell and axonal emigration were observed in the presence of inhibitors. (B) To examine whether the presence of the inhibitors resulted in increased apoptotic death, similar small intestine explants were cultured in control medium (left panel), or medium supplemented with 30 µM LY29002 (middle panel) or 60 µM PD98059 (right panel). At the end of the culture period, sections from the explants were stained for TUNEL to identify apoptotic cells. Note that under the present culture conditions, neither LY29002 nor PD98059 increases significantly the number of TUNEL+ nuclei. (C) To quantify the effect of increasing concentrations of PI(3)K and MEK1 inhibitors on ENCC migration, the number of RET-expressing cells present between the small intestine segment and COS/GDNF cells was counted. The response of explants cultured in control medium was considered as 100%. Increasing concentrations of PI(3)K inhibitor result in complete abrogation of the response. By contrast, a residual but significant response was observed even at high concentrations of MEK1 inhibitor. Nine explants were analysed for all concentrations tested for each inhibitor. Four explants were analysed in the experiments where the PI(3)K and MEK1 inhibitors were combined. The concentrations of LY294002 and PD98059 used for all explants shown in this figure were 10 µM and 30 µM, respectively.