Polarization Activity of Fgf8 in Mouse Brain Figure 4. Bafilomycin A1 (BAF) treatment demonstrates the polarization of ERK activity by FGF8 signal activity along the neural tube. A-C) BAF amplifies ectopic ERK1/2 activity-related FGF8 induction revealing a clear polarized distribution of ERK1/2 activity around FGF8 soaked beads depending of the implanted bead; rostral to IsO (C,D,G), or caudal to IsO (C,E). Nonetheles, isthmic organizer morphogenetic activity seems unaffected for Fgf8 (A) and negative regulator Sprouty 2 (B) expressions. Note that PBS bead implantation in control side (blue asterisk in C and F) did not show any ectopic induction. Two hours after bead implantation a clear amplified and almost non-homogeneous ERK1/2 activity was detected rostrally in the mesencephalon (rostral to the IsO), which was detected caudally when bead was placed in hindbrain (caudal to the IsO) territories (E). In telencephalic vesicles, caudal to the anr (H) the polarity of ERK activation was reversed. This polarized dpERK detection around the bead is lost at the zli (zona limitans intrathalamica) region (I). Similar symmetric ERK-related FGF8 signal found in zli was seen when placing a FGF8 bead in the midbrain of Fgf8 hypomorphic mice (J,K). Importantly FGF8b protein distribution (M) was observed apparently in equal intensity and range at rostral (N) and caudal (O) sides of the bead (for comparison with PBS bead in panel L). Scale bars are 0,5 mm in A, B, C, H, I, 200 mm in D, E, J, 100 mm in F, G, K, L, M, and 50 mm in N, O. doi:10.1371/journal.pone.0039977.g004 staining was very similar to the results using ONTCs of Fgf8 hypomorphic embryos or when the FGF8b beads were placed in the zli of wild-type ONTCs. Finally, as an attempt to exclude any sensitive receptor mechanism underlying this initial polarizing activity exerted by FGF8b signaling we conducted FGF8b bead implantation assays on ONTCs of mutant embryos where FgfR1 was conditionally inactivated in the midbrain-rhombomere 1 region (En1Cre/+; FgfR1 flox/flox; [51,52]). These mutant ONTCs (n = 6/6; Figure 5J) disclosed same polarized ERK1/2 activity as when FGF8b beads were implanted on rostral and caudal sides of the IsO in wt ONTCs (see Figure 4). Importantly, in these experiments we did not use Bafilomycin A1 compound to arrest late endosomal pathway. Here, ERK1/2 activity immunodetec- tion was more expanded on the neuroepithelium than in normal ONTCs suggesting a differential endosomal sorting and a high rate recycling characteristics of the FGFR1-FGF8b endocyted complex. Therefore, it seems that along the anterior neural tube, the gradient activity of FGF8b may result from different planar instructions regulated by the FGF8 feedback negative modula- tors. We therefore analyzed the contribution of the FGF8 feedback modulators (Mkp3, Sef, Sprouty1/2) during these initial planar instructions of FGF8 signaling. We decided to deprive pharma- cologically the E9.5 mouse neural tube from any endogenous secreted molecule to the extracellular space (including FGF8 protein). Brefeldin A (BFA) was chosen for the ability to inhibit protein secretion in mammalian and other eukaryotic cells by interfering with the function of the Golgi apparatus, resulting in dysregulation of membrane traffic [53]. Before that, we detected the endogenous FGF8 protein distribution in vivo by Immunohis- tochemistry in whole-mount E9.5 mice with specific antibody against FGF8. The results revealed FGF8 staining either at the neuroepithelial intracellular or at extracellular levels in cryostat sections transverse to the IsO constriction (Figure S2A,B and [54]). FGF8 positive immunostaining was detected outside the limits of its mRNA expression, both caudal and rostral to the IsO. Moreover, the FGF8 protein was detected at both ventricular PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e39977