the duration of Fgf8 expression in the IsO, and the strength of its signal activity seem to be crucial for the specification of these brain regions [35]. Three major intracellular signaling pathways can carry out the transduction of FGF signal during embryogenesis: PI3Kinase, PLC-gamma and Ras/MAPK (reviewed by [12,36]). Phosphor- ylation of Extracellular signal Regulated Kinase 1/2 (ERK1/2) is a crucial step of the Ras-MAPK intracellular pathway. In early frog, fish, chick and mouse embryos, ERK1/2 activity depends on FGF signaling making the detection of di-phosphorylated forms of ERK1 and ERK2 (dpERK) useful readouts of FGF activity. In vertebrate embryos, ERK1/2 phosphorylation pattern profile is discrete, dynamic and it largely correlates and with Fgf8 gene expression domains [24,37–39]. The proposed mechanism by which the signaling of FGF8 spreads over a field of target cells in zebrafish is established and maintained by two essential factors: firstly, free diffusion of single FGF8 molecules away from the secretion source through the extracellular space and secondly, a sink function of the receiving cells regulated by receptor-mediated endocytosis [40,41]. Howev- er, the precise shape of the FGF8 morphogenetic activity is still unclear during the early mammalian brain regionalization. It is also important to understand how the FGF8 signaling expands from the IsO in order to be interpreted as positional information by the nearby neuroepithelial cells. Here, we address these questions using the mouse IsO as experimental model system. The study discloses position related preferences of neuroepithelial cells to FGF8 planar signal activity. This differential orientation and polarity of the FGF8 signal is directly dependent on the spatial position of mouse Fgf8-related secondary organizers and on the activity of a negative modulator Sprouty2. Our findings reaffirm the existence of positional information encoded by the FGF8 morphogenetic activity in neuroepithelial cells along the vertebrate neural tube. Results ERK1/2 Phosphorylation Reveals the Longest-range Form of FGF8 Morphogenetic Activity From the Mouse IsO We first compared the distribution of phosphorylated forms of ERK1/2 activity (Figure 1Bs; [38]) to the expression pattern of Fgf8 (Figure 1A,A’’) and the main Fgf8 downstream genes (Figure 1C-E’’) in E9.5 wild-type mouse embryos and organotypic cultures of neural tube explants (ONTCs; [42]). In both models, we also corroborated the expression patterns of the Fgf8 downstream negative modulators Sprouty2, Sef, Mkp3 showing their gradient distribution being strong near the FGF8-related second- ary organizers (Figure S1 and [25,43,44]). In E9.5 whole mount embryos, immunodetection of ERK1/2 did not show the same distribution as the FGF8 modulators (Figure 1B,B’). In fact, when using E9.5 ONTCs the immuno- staining against phosphorylated forms of ERK1/2 showed an almost non-gradient pattern, facing now the ventricular side of the IsO territory (Figure 1B’’,C; [42]). Moreover, in E9.5 ONTCs ERK1/2 phosphorylation was detected over almost the entire mesencephalon (also positive for Meis2 transcription factor; see Figure S1A) and the entire rhombomere 1 (r1; positive for En2 and limited caudally by Pax6; Figure 1D’,D’’). Rostrally, ERK1/2 activity staining reached the ventral parts of the mesencephalic- diencephalic boundary (based on the caudal gene expression limit of either Pax6 or Tcf4; n = 28/33; Figure 1D’-E’’) leaving a mesencephalic alar plate wedge domain free of expression. Caudally, the immunodetection adjoined to the expression of Pax6 at rhombomere 2. Therefore, at this developmental stage Polarization Activity of Fgf8 in Mouse Brain phosphorylated forms of ERK1/2 appeared to be the longest- range marker for FGF8 activity. To demonstrate that ERK1/2 phosphorylation was controlled by FGF8 activity on these territories at E9.5, we used mutant mice with reduced levels of FGF8 (Fgf8 hypomorphs; [23,45]). Immunostaining of dpERK at mid- and hindbrain regions was completely absent in these mutant mice (n = 7/7; Figure 2A, 2B). This absence was concomitant with downregulation of Fgf8 expression and of FGF8 downstream negative modulators genes such as Sef, Mkp3 and Sprouty2 (n = 6/6; Figure 2C-F; for comparison see Figure S1). In the Fgf8 hypomorphs Tcf4 expression pattern (used as a landmark for caudal diencephalic limit) did not change. Surprisingly, at this developmental stage we still found a small portion at the most dorsal area of the isthmic constriction where Fgf8, Sef and En2 transcripts were expressed (asterisks in Figure 2B,C,E). However, this reduced expression was not enough to maintain full IsO morphogenetic activity [23]. Therefore, ERK1/2 phosphorylation at mouse midbrain and rostral hindbrain seems to be strongly linked to the FGF8 signaling activity coming from the IsO. FGF8 Signaling Activity Exerts Different Tissue Preferences Along the Anterior Posterior Neural Tube Axis We next characterized the molecular dynamics of FGF8 signaling activity coming from the IsO, analyzing ERK1/2 activity after ectopic implantation of FGF8 sources (Figure 3 and 4; see material and methods). Ectopic induction of Mkp3 was the first transcript detected only after 3 hours of FGF8b soaked bead implantation to the mesencephalon (Figure 3D,D’ and [46]). On the other hand, ectopic ERK1/2 activity was detected already before one hour of incubation with FGF8b beads (Figure 3A). Interestingly, the ERK1/2 phosphorylation staining was distrib- uted asymmetrically around the bead in the mesencephalon (Figure 3A,A’). High intensity of staining was detected only at the rostral side of the bead (n = 9/13). With longer incubation time periods (2 hours; Figure 3B,B’), ectopic dpERK immunostaining started to be detected also caudal to the bead but was still induced higher in rostral cells. Only after 3 to 4 hours of FGF8b bead incubation, ERK1/2 activity was detected symmetrically around the bead (Figure 3C,C’). In all cases, control PBS soaked beads implanted on the same neuroepithelial positions and same time periods neither showed induction of ERK1/2 activity nor molecular induction of Fgf8 downstream genes (blue asterisk in Figure 3C,D). This early asymmetric phosphorylation of ERK1/2 raised the possibility that FGF8 morphogenetic activity may confer positional information to the neural tube encoded already at the intracellular signaling pathway level along its anterior-posterior axis. To further investigate the causal mechanisms of this unbalanced distribution of ERK1/2 activity at early steps of FGF8 signaling, we searched for amplification of the intracellular ERK1/2 activity. Recent work has proposed as an explanation for the establishment of FGF8 morphogen gradients by endocytosis and degradation of the Fgf8 protein [41]. Therefore, we decided to pharmacologically block the lysosomal pathway to prevent FGF8 degradation after endocytosis. Using Bafilomycin A1 compound (BAF; see material and methods) endocytosed FGF8 should maintain within the endosomes and still trigger ERK1/2 activity [47]; while the extracellular FGF8 protein should continue to be taken up by the cells. After 2 hours of 1 mM BAF treatment [48] E9.5 ONTCs still maintained similar molecular IsO activity and gene expression patterns to those observed in controls (Figure 4A,B). When we implanted FGF8b soaked beads during the BAF treatment (2 PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e39977