Polarization Activity of Fgf8 in Mouse Brain Figure 7. Initial FGF8 planar instruction effects in IsO after a short signaling deprivation. Schematic representation of isthmic FGF8 feedback modulator genes regulation given by the isthmic gradiental FGF8 morphogen (and signaling) on the mouse mesencephalon. The graphic describes the distribution of dosage (activity) versus time and space of FGF8 and of Fgf8 negative signal modulators. A) represents the normal FGF8 signal activity in the IsO (high levels of FGF8 protein; red solid wedge), during which FGF8 maintains at dose-dependent manner the different FGF negative modulators expression profile starting from Mkp3 (purple solid curve), Sef (green) and finishing Sprouty genes (where low levels of FGF8 protein). The yellow background represents ERK activity. B) describes the presumed situation during Brefeldin a (BFA) treatment (4 hours) on the FGF8 morphogenetic activity. Thus, at isthmus the FGF8 protein level (a therefore morphogenetic activity: red solid bell-shaped curve) would be cero but some residual protein away from the source would still activate ERK (yellow solid slope curve). Inside this negative gap of ERK1/2 activity the expression of Mkp3 (purple dashed curve line) and Sef (green dashed curved line) at the mesencephalon is completely absent. Nonetheless the residual FGF8 morphogen apart from the isthmus is enough to maintain Sprouty1/2 expression in the mesencephalon in the absence of ERK activity (red asterisks’; see also [32,76]). doi:10.1371/journal.pone.0039977.g007 example EGFR, remain ligand bound, phosphorylated and active in endosomes until late stages of endosomal trafficking, including the presence of a MAPK scaffold complex (reviewed in [47]). Following the recent findings on the endocytotic mechanism for Fgf8 morphogen in zebrafish IsO [41], we used Bafilomicin A1 (BAF), a highly specific inhibitor of vacuolar type H+-ATPase (V- ATPase; [66]) to amplify the ERK1/2 phosphorylation in cells induced by FGF8b signaling. Under these conditions we disclosed an asymmetrical distribution of ERK1/2 activity, but not of the FGF8 protein itself, around the FGF8 releasing bead. This differential planar tissue behavior in the ERK intracellular response depended on the position of the FGF8b bead relative to anterior neural ridge (anr) and isthmic organizer (IsO) (the FGF8-related secondary organizers; Figure 6K; [13,19]). Impor- tantly, the diencephalon behaved very differently from the mesencephalon, particularly the boundary between prethalamus and thalamus (the zona limitans intrathalamica; [49,50]). Here, the activation of ERK1/2 by the FGF8b bead was distributed symmetrical around it. In addition, this non-polarized ERK1/2 activity related to FGF8b signal was also detected in FGF8 hypomorphic mice (see Figure 4 and [45]) and in ONTCs experimental assays where both the anr and the IsO were ablated (see Figure 5 type 2 experiments). Importantly, when the IsO was ablated we could reverse the polarization effect of ERK1/2 activity, suggesting that in fact these FGF8-related morphogenetic centers are implicated in the differential ERK1/2 response. Interestingly, in these IsO ablation assays we did not detect any trace of FGF8 negative modulators gene expression on the remaining mesencephalon, raising the question whether other unknown rostral factors might also contribute to the reversed ERK activity. Viera et al., [67] using chick neural tube embryos and FGF8-beads implantation assays studied the molecular mecha- nisms by which Pax2 gene expression pattern was restricted from diencephalic/mesencephalic boundary to the isthmic territory. 24 hours after FGF8b bead implantation in the caudal diencephalon, the authors showed a heterogeneous ectopic Pax2 expression, which was consistently more intense on the caudal side of the FGF8b bead compared to the rostral side. These authors explained this phenomenon as a mechanism by which the putative signal that progressively restricted Pax2 into the isthmic region could be of positive character (necessary to maintain Pax2 expression). Indeed, this signal would probably come from the caudal part of the mesencephalon or the isthmus, needed for normal antero-posterior polarity of the epithelium and therefore would not be directly related to an FGF8 signal. Ubiquitination by c-Cbl on the intracellular domain of the FGFR1 receptor leads to differential recycling of the receptor and modifies the duration of its signal [41,68]. Based on the high affinity and sensitivity of this receptor to FGF8b signaling during neural development, we analyzed its expression profile in our secondary organizers ablation assays (Type 2 assays; Figure 5G; [51,52]). In these experiments, the rather uniform expression of FgfR1 in the mesencephalon was not affected during the time of the experiments (4hours; Figure 5H-I). Thus, our results suggest that instructions of FGF8 signal activity in mouse secondary organizers confer planar positional information from the IsO and from the anr by differentially di-phosphorylating ERK1/2 nearby neuroepithelial cells away from them without affecting the FgfR1 gene expression (Figure 6K). Actually, this polarization coming from the two transversal secondary organizers seem to converge at the central diencephalic anlage (the zli) where no ERK differential polarizing activity occurs, leaving this brain area exempted from Fgf8-related secondary organizers influence activity before E9.5 stages (Figure 6K). Then, what molecular mechanisms are behind this unbalanced activation of ERK1/2? Mutant mice have been used to understand the function of FGF8 negative feedback modulators in the mouse brain (i.e. Mkp3, Sprouty1/2, Sef). However, this powerful approach faces the difficulty of dissecting the function of each modulator because of their redundancy in FGF8 signal PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e39977