Figure 8

Fibroblast growth factor (FGF) signaling via the mitogen-activated protein kinase (MAPK) pathway is required for the maintenance of Islet1 expression in the trigeminal placodes. Cranial ectoderm explants (A-C, F, G) and midbrain to rhombomere 2 (mb-r2) (D, E) were excised at HH10 and grown overnight until approximately HH16. In situ hybridizations for Islet1 (A-C, F, G) and Fgf8 (D-E) expression are shown in blue. Explants were cultured in SU5402 (B), Wnt3A and SU5402 (C) or PD184352 (E, G). The presence of SU5402 resulted in the loss of Islet1 expression (B) compared to controls (A). In the presence of SU5402, Wnt3A did not maintain Islet1 expression (C). The presence of a selective MEK antagonist (PD184352, 2 nM) led to a loss of Fgf8 expression in mb-r2 explants (E) compared to controls (D). Inhibition of MEK activity also resulted in a complete loss of Islet1 expression (G) compared to controls (F). (D-G), control explants (left ce) and experimental explants (right ce) were isolated from the same embryo. (H) Western blots of HH10 mb-r2 explants (lanes 1, 3 and 5) and HH10 cranial ectoderm (ce; lanes 2, 4 and 6). Mb-r2 and cranial ectoderm explants lose double phosphorylated ERK1/2 (dpERK1/2) activity in the presence of 50 μM SU5402 (lanes 3 and 4). In all other lanes, isolated tissue is positive for dpErk1/2 at HH10 (lanes 1 and 2), and also for nuclear β-catenin (lanes 5 and 6). Actin is shown as a loading control. (I) Immunostain showing the expression of dpERK in the cranial ectoderm.