Polarization Activity of Fgf8 in Mouse Brain Figure 1. Phosphorylation patterns of ERK1/2 (dpERK) enzymes in the anterior neural tube. Whole mount in situ hybridization (ISH) of E9.5 mouse embryo (A,B,B’) and corresponding organotypic neural tissue cultures of mouse E9.5 anterior neural tube (A’,B’’,C-E’’; ONTCs; [42]) where it shows the maintenance of gene expression profiles such us Fgf8, Tcf4 (A,A’) (used to delimit main brain subdivisions such the diencephalon (D; Tcf4 positive) and the mesencephalic anlage (M; negative staining), Pax6 D-D’’ (delimiting di-mesencephalic boundary and rhombomere 1–2 limits) and En2 E-E’’. In B-B’’) are photomicrographs of E9.5 mouse embryo with anti-dpERK Immunohistochemistry (IHC) taken from lateral (B) and caudal (B’) sides and the corresponding IHC in ONTCs. (C) double staining procedure: ISH (in blue) for Fgf8 and IHC for dpERK (dark brown) to localize inside the dpERK domain the position of the IsO, marked by the solid red line. (D-E’’) photomicrographs of same ONTCs in which first a whole mount ISH for Pax6 (D) or Tcf4/En2 (E) were made and afterwards IHC against dpERK. (D’,E’ respectively). Dashed lines mark the main transversal (in black) and longitudinal (in red) brain subdivisions. These ONTCs were cut into transversal sections to the isthmic constriction (D’’,E’’) to proof that indeed dpERK expression reaches diencephalic anlage (D’’; see asterisk; rostral is left) and has a wider expansion than En2 expression (E’’; see asterisk; rostral is left). anr is anterior neural ridge secondary organizer; ba is branchial arch; IsO is isthmic organizer; os is optic stalk; ov is otic vesicle; r is rhombomere; T is telencephalon, D, diencephalon, M, mesencephalon. Scale bars are 0,5 mm except in D’’, E’’ they are 100 mm. doi:10.1371/journal.pone.0039977.g001 hours) a significant amplification of ERK1/2 phosphorylation signal occurred (compared Figure 4G with non-treated explants bead implantation assays 3B’). PBS-beads did not produced and ERK1/2 ectopic induction (blue asterisks in Figure 4C,F). In addition, this treatment disclosed an intensification of the polarization effect (Figure 4C-I). The asymmetric distribution of ERK1/2 immunodetection around the bead was clearly localized and extended rostrally when those beads were implanted in the mesencephalon (n = 17/20; Figure 4C,D,G). Only when FGF8b beads were placed on the rhombencephalon a reversed polariza- tion distribution of phosphorylated ERK1/2 staining was detected (in these cases caudal to the bead; n = 4/5; Figure 4C,E). FGF8b beads implanted ectopically at more anterior territories in caudal diencephalic anlage showed a less evident ‘‘crescent moon’’-like ERK1/2 phosphorylation staining, with decreased expansion rostral to the bead (n = 5/5; Figure 4H). In fact, when these beads were placed at the thalamic/prethalamic boundary (n = 4/ 4; Figure 4I), the zona limitans intrathalamica (zli; [16,49,50]), they induced ERK1/2 equally distributed around the bead (symmetrical). Furthermore, when FGF8b beads were implanted in telencephalic regions, close to the mouse anterior neural ridge secondary organizer (anr; [13]) ERK1/2 activity was mainly induced in cells caudal to the beads (n = 7/9; Figure 4H). Therefore, FGF8b signal exerts differential responses on the neuroepithelium, which are encoded already at the level of the Ras-MAPK intracellular cascade activation along the anterior- posterior axis of the mouse neural tube (see below). In order to exclude any differential spatial protein release from the FGF8b beads towards rostral or caudal directions, we analyzed distribution of the ectopic FGF8b protein in the neuroepithelial cells using a specific monoclonal antibody (see materials and methods). The results demonstrated an equal distribution of the protein at both caudal and rostral sides of the FGF8 beads in the ONTC neuroepithelium (n = 4/4; Figure 4N-4O). Finally, we checked also if the endogenous FGF8 activity levels contributed to the observed ERK activity asymmetries. Thus, we implanted FGF8b soaked beads into ONTC mesencephalon made from embryos homozygous for severely hypomorphic Fgf8 alleles [23]. In these mutants, the ectopic ERK1/2 phosphorylation was always induced symmetrically after 1 hour of bead incubation (n = 10/11; Figure 4J, 4K). Thus, the asymmetric effects of FGF8b beads on ERK1/2 phosphorylation seem dependent on proper FGF8 function, likely coming from the FGF8-related secondary organizers [12,16]. Topography of Secondary Organizers Determine the Polarization of FGF8 Signaling Activity Along the Neural tube Through Negative Feed Back Modulators To prove that FGF8-related brain secondary organizers were the sources for early ERK activity polarity, we conducted neural tissue ablation assays of these morphogenetic brain areas on E9.5 mouse ONTCs (see Figure 5 drawings). In type 1 assay the IsO was ablated and the remaining tissue was incubated for 24 h before BAF treatment or implantation of FGF8b soaked beads (Figure 5A-5D’, 5H). Under these conditions Fgf8 transcripts PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e39977