Polarization Activity of Fgf8 in Mouse Brain Figure 3. FGF8 planar induction from IsO has initial tissue preferences in mesencephalon. Classical FGF8 soaked bead implantation in mesencephalon induces ERK1/2 activity before earliest induction of mRNA (Mkp3) could be detected (D). Interestingly asymmetric distribution is detected during the first two hours after incubation (A, A’ and B, B’). This asymmetry is lost from 3 hours onwards after bead implantation (C, C’). In cryostat sections a mesencephalic bead implantation induced highly intense ERK1/2 activation as observed at the rostral side of the bead after 1 hour of incubation (A’). After 2 hours, ERK phosphorylation is detected caudal to the bead (B’; see small asterisk). Finally ERK activity is homogeneously distributed at both rostral and caudal cells after 4 hours (C’). Red asterisks indicate an FGF8 soaked bead; blue asterisk indicate a PBS bead. Scale bars are 0,5 mm except for A’, B’, C’ that are 50 mm, in D’ is 0,25 mm. doi:10.1371/journal.pone.0039977.g003 In type 2 assays the IsO and anr were ablated. Using the same conditions as type 1 experimental assays, the remaining neural tissue (mostly mesencephalic and diencephalic regions) showed no expression of Fgf8 (n = 6/6) or Fgf8 negative feedback modulators Mkp3 (n = 7/7) or Sprouty2 (n = 3/3) before any treatment (Figure 5E). The ablated tissue still showed traces of FgfR1 transcripts in the mesencephalic territory (Figure 5I). After BAF treatment and FGF8b bead implantation into the rostral mesencephalon, phosphorylated ERK1/2 staining was observed symmetrically around the bead (n = 14/16; Figure 5F,F’). The PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e39977