Polarization Activity of Fgf8 in Mouse Brain Figure 6. Brefeldin A (BFA) treatment inhibits ERK1/2 activity and modulates differentially Fgf8 negative feed-back regulators. A, B and C) are 12 mM cryostat transversal sections of mouse ONTCs to the isthmic constriction. A and B) are control example before BFA administration to the culture medium showing an ISH for Fgf8 (A) and an Immunostaining for anti-FGF8 (B). Note the different domains of expression of the transcript (delineated by the solid line and arrows) and of the protein FGF8 (delineated by the red arrows). Note also that FGF8 immunodetection is detected both at basal and ventricular sides of the ONTCs (see black arrows in B). After 4 hours of BFA incubation (C-J) the mRNA of Fgf8 was maintained at the IsO (D) while the FGF8 protein profile changed dramatically being accumulated only at the ventricular side (D) as small vesicle-like, (see arrows in the figure C and the magnified insert). Moreover, ERK1/2 activity disappears in Isthmic cells and nearby cells (E). Inside this negative gap, FGF8b beads still exerts polarizing ERK1/2 effects (F). Also inside this gap, genes such us Mkp3 (G) and Sef (H) disappear in the mesencephalon while Sprouty family genes are maintained (I,J). K) represents the experiments and model by which FGF8 planar induction activity coming from mouse FGF8-related secondary organizers (IsO and anr) exerts a different tissue preferential signaling effects (based on the activation of ERK1/2). The direction of polarized ERK1/2 activity depends on the location of FGF8-related secondary organizers and the establishment of this positional information signaling is dependent mainly on FGF8 negative modulator system, particularly Sprouty2 (blue gradient). Moreover, FGF8 morphogenetic planar instruction signals coming from rostral (anr) and caudal (IsO) diminish and loose their polarization effect at the diencephalic region (zli) resulting in an equilibrium state. Scale bars is 100 mm except for D,E, G-J which is 0,5 mm. doi:10.1371/journal.pone.0039977.g006 PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e39977