modulation [40,69,70,71]. In this report, we used Brefeldin [53] to retain FGF8 molecules inside FGF8-producing cells at the IsO and anr and thus, eliminating the endogenous source of extracellular FGF8 protein along the neural tube for 4 hours. Under these deprivation conditions, the isthmic cells were the first affected cells in terms of ERK phosphorylation activity followed by the abutting territories (Figure 6 and 7). We still found traces of ERK1/2 activity outside this negative domain indicating a remanent FGF8 activity still ongoing. Importantly, Mkp3 expression was concom- itantly downregulated in the same domain where phosphorylated ERK1/2 was not immunodetected. On the contrary, expression of Sprouty1 and especially Sprouty2 was maintained. Moreover, during BFA treatment FGF8-bead implantation on caudal mesencepha- lon maintained ERK1/2 polarized activation, indicating that Mkp3 and probably Sef were not required in the specification of FGF8 differential positional planar induction activity in the mesencephalon. It has been proposed that SPROUTY 1/2 and SEF function synergistically to regulate Gbx2 expression in the anterior hindbrain (a downstream target of FGF signaling; [70]). Suzuki-Hirano and collaborators [32] elegantly demonstrated that Mkp3 was induced in chick neuroepithelial cells when a high level of ERK1/2 phosphorylation occurred. In agreement with the latter results, we were able to detect ectopic induction only of Mkp3 after 3 hours of FGF8b soaked bead implantation in mouse ONTCs (Figure S1; [46]). On the other hand, in our BFA treatment assays Mkp3 expression was the first modulator to be downregulated. Also, the same group demonstrated that Sprouty2 was involved in the downregulation of ERK1/2 activity after its initial upregulation by FGF8 signal, and that this was required for proper mid-hindbrain differentiation. In our mouse ONTCs model system, Sprouty2 may also be important for the correct early establishment of FGF8 positional information coming from the IsO by maintaining downregulated ERK1/2 phosphorylation levels. It is true that implantation of FGF8 soaked beads or ectopic gene expression by tissue electroporation may surpass the physiological levels of the protein. However, our results corrob- orated and provide a logical explanation of other previous works at which an ectopic source of the FGF8b protein either in caudal diencephalic and rostral mesencephalic territories (in terms of electroporation, grafted tissue and/or soaked beads), caused a mirror-like cerebellar tissue induction rostrally to the ectopic implanted source. Within our results we can conclude that those results were due to the first polarized ERK1/2 activity driven by the FGF8b signal [10,72,73]. Also the present work strongly suggests that the positional information given by FGF8 morpho- gen activity from IsO at E9.5 is coded already by the receptor response at the cytoplasmic level. This response is translated in distinct ERK1/2 phosphorylation states inside the neuroepithelial cells produced by the distinct levels and combinations of the FGF8 negative modulators. Actually, the decreasing gradients of FGF8 downstream regulators (mainly Sprouty1/2) at both sides of the IsO epithelium would maintain basic FGF8 intracellular activity to extend and to equilibrate the long-range distribution of active ERK1/2 along the A-P axis [32]. These results fit well with Meinhardt’s mathematical model for positional information signaling and establishment [74]. He proposed that the formation and maintenance of organizer regions would dependent of a short- ranging autocatalytic activator (the FGF8 in our model), which would catalyze in addition its long-ranging antagonist, the inhibitor (here, the SPROUTY family). Finally, the immunodetection of FGF8 protein assays in embryos and ONTCs revealed staining domains at the ventricular side and at the basal lamina (see also [54]). Interestingly, under BFA treatment conditions, we unmasked that FGF8b protein was Polarization Activity of Fgf8 in Mouse Brain highly accumulated, in a vesicular-like manner, at the ventricular side of the neuroepithelial cells, which indicates that FGF8- expressing cells may secrete the morphogen to the lumen of the ventricle. However, as the ventricular surface area is very small, the apically localized vesicles may also be released to the basolateral side. Also, other recent reports claimed that FGF8- protein is highly concentrated at the basal lamina suggesting that FGFs may act through basal processes of neuronal progenitors to maintain their progenitor status [54]. In fact, mouse Mkp3 is strongly expressed in mesenchyme compartment adjacent to the basal lamina at the isthmic region (see Figure 1 in [46]). In embryos, our monoclonal antibody immunodetection (See Figure S2 and material and methods) experiments showed positive immunolabeling of FGF8 protein at both ventricular zone and basal lamina with similar intensity. The same pattern was concomitantly observed in our ONTCs experimental model. In agreement with these observations, the high affinity FGF receptors related with the activation of FGF signaling pathways (Fgfr1 and 2) are predominantly expressed at the ventricular zone in E11.5 mouse embryos [75]. Furthermore, the resulting BFA treatment conditions in ONTCs revealed the lack of accumulating FGF8 positive staining at the basal lamina side. The release of the FGF8b protein from the ventricular side and its localization also on the basal lamina suggests a later transport of the protein from the apical to the pial side. Thus, alternative sorting and transcytosis mechanisms of FGF8b may occur inside the targets cells. Whatever the exact mechanism, our results further support association of FGF8 protein with basal lamina showing that establishment of the basal FGF8 gradient requires active exocytosis. Very recently and relevant published findings in chick claimed that FGF8b may also translocate into the nucleus, and this nuclear FGF8b could function as a transcriptional regulator to induce Sprouty2 in the isthmus independently of ERK phosphor- ylation [76]. These new data in chick IsO together with our BFA assays where Sprouty2 gene expression pattern was maintained in the absence of ERK1/2 activity provide new horizons of FGF8 function. In conclusion, FGF8 may exert distinct signal responses depending on its cellular localization. These differential planar instructions may allow the segregation of neurogenic and proliferation signaling mechanisms or alternatively facilitate diffusion of FGF8-related activity through the basal lamina during vertebrate neural tube patterning [77]. Materials and Methods Organotypic Neural Tube Culture Explants Technique (ONTCs) Timed pregnant mice were sacrificed by cervical dislocation and embryos were dissected in ice-cold phosphate-buffered saline (PBS; 0.1 M). The embryonic day (E) 0.5 was the noon of the day of the vaginal plug. The embryonic age was determined more precisely by counting the somites in which at E9.5 ranged between 21 and 29 somite pairs [78]. Anterior neural tube of E9.5 embryos was opened along the dorsal midline, placed on polycarbonate membranes (MilliCell PICMORG50), with the ventricular side facing up, and cultured in a 5% CO2, 100% humidity incubator at 37uC for up to 24 hours as previously described [42]. After experimental manipulation the explants were fixed in 4% paraformaldehyde in PBS between 2 and 4 hours. For the ablation experiments, the isthmic and anterior neural ridge regions of ONTCs were cut with the help of micro-scalpel blades (EagleLabs EG-4738) and cultivated on the same polycarbonate membranes, for 24 hours before bead implantation and/or chemical treatments (see below). PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e39977