Polarization Activity of Fgf8 in Mouse Brain Figure 2. Low threshold of FGF8 protein levels disrupts ERK1/2 phosphorylation patterns. DpERK immunodetection was absent in the isthmic domain using ONTCs severe hypomorphic mouse mutant (A, B; Fgf8 neo/null [45]). Yet a small tip of En2 (B) positive expression was visible at the most dorsal parts, probably by the maintenance of Fgf8 (C) expression. Under these mutant conditions, none of the FGF8 signal negative modulators Mkp3 (D), Sef (E) Sprouty2 (F) were observed at IsO. Asterisks indicate the position of abolished isthmic region and solid line the boundary between diencephalon/mesencephalon. Scale bar in C is 0,5 mm for all images. doi:10.1371/journal.pone.0039977.g002 (n = 12/15) and negative modulators of Fgf8 signaling such Mkp3 (n = 4/4) and Sprouty2 were not longer expressed at caudal mesencephalon (n = 5/5; Figure 5A, 5B). Meanwhile, Fgf8 expression was still maintained at the anr and at the upper mesenchymal branchial arches (ba). In these ablation assays the main signaling receptor for FGF8, FGF receptor 1 (FgfR1; Figure 5G, 5H); [33,51,52]) was also maintained uniformly at the mesencephalon. Here, after 2 hours of FGF8b bead implantation in the midbrain and BAF treatment, we detected effects opposite to the previously observed polarized response of ERK1/2 in this region. Immunodetection of dpERK appeared now stronger at the caudal parts of the FGF8b bead than at rostral one (n = 11/14; Figure 5C, 5C’; compare with Figure 4C,H). Interestingly, FGF8b beads implanted at the central diencephalon (putative zli; Figure 5D, 5D’) still maintained the symmetrical distribution of ERK1/2 activity around the bead. Thus, these results support the idea that at E9.5 the murine isthmic organizer region must be the source for the initial polarizing cue on ERK1/2 activity related to FGF8b signal in the entire mesencephalon, caudal diencephalon and most probably rhombomere 1. PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e39977