All animal manipulation and experimental procedures were performed accordingly to the directives of the Spanish and European Union governments (Council Directive 86/609/EEC) and approved by the Animal Experimentation Committee of the Institute of Neuroscience UMH-CSIC. Mice from ICR strain were used as wild type. The transgenic mouse strain Fgf8neo/null (Fgf8 hypomorph mice in this paper; [45] were used as severe Fgf8 reduction level mice model [23], maintained on a C57BL/6 genetic background and generated as previously described by these last authors. En1Cre/+; Fgfr1flox/flox were generated by crossing En1Cre/+; FgfR1flox/+ males with FgfR1flox/flox females in outbred (129sv/ICR) background and genotyped as previously described by Trokovic et al., [50,51]. Implantation of FGF8b-soaked Beads Heparin acrylic beads (Sigma-Aldrich H-5263) were rinsed in PBS and soaked in FGF8b solution (1 mg/ml; R&D) for 1 h at 4uC. FGF8b-soaked beads were rinsed three times in PBS and thereafter implanted in the neural tube explants cultures as previously described [79]. Control beads were incubated in PBS and implanted in the same manner. Bafilomycin A1 and Brefeldin A Treatments Bafilomycin A1 (BAF; SigmaB1793) was used for blocking the lysosomal pathway and so, preventing FGF8 degradation after endocytosis [68]. ONTCs were incubated with BAF at a concentration of 1 mM in 0.04 mg/ml Dimethyl sulfoxide (DMSO; Sigma-Aldrich D8418; [47]), added to the culture medium (see protocol at [42]). The incubation time period was set at 2 hours, 37uC. Control explants were treated only with DMSO at same concentration and for same period of time. Brefeldin A (BFA; Sigma B5936) was used for blocking release of exocytic vesicles content (including FGF8 secretion; [55]). Culture medium solution with the diluted chemical was used to treat the ONTCs, at a concentration of 25 mg/ml (Dahl et al., 2000), in 0.04 mg/ml DMSO. The incubation time period was set at 4 hours, 37uC for optimal desired effects. Control explants were treated with DMSO only as above mentioned. In Situ Hybridization (ISH) and Immunohistochemistry (IHC) E9.5 whole-embryos were fixed in 4% PFA overnight at 4uC. Next day, samples were rinsed in PBT (PBS pH 7.4, with 0.1% Tween 20), dehydrated in an ascending ethanol series, and stored in 70% ethanol at 20uC before processing. Whole-mount ISH was performed according to Garda et al. 2001 protocol [77]. Digoxigenin-labeled RNA probes (DIG-11-UTP, Roche Diagnos- tics 11209256910) were detected by alkaline phosphatase-coupled anti-digoxigenin (Roche Diagnostics 11093274910), and incuba- tion with BM-Purple substrate (Roche Diagnostics 1442074) as chromophore. After the colorimetric detection, embryos ONTCs were washed several times in PBT. In the case of IHC procedure whole mount embryos where dissected in ice cold PBS and fixed in 4% PFA with phosphatase inhibitor tablets (Roche Diagnostics 04906837001) following companies protocol for 2–4 hours before starting procedure. In some cases and after ISH or immunostaining procedure, embryos or ONTCs were immersed in ascending sucrose to 30% concentration and then cut at 12 mm thick sections ONTCs in a cryostat at -26uC (Microm-ThermoFischer Scientific) for a cellular analysis. Whole mounts embryos, ONTCs and cryostat tissue sections were rinsed 3 times in PBS 16with 0,1% Triton (PBS-T) and then Polarization Activity of Fgf8 in Mouse Brain incubated with hydrogen peroxide (H2O2) at 3% for 30 minutes to inactivate the endogenous peroxidase activity. Then after 3 washes in PBS-T, they were blocked with goat serum at 5% and bovine serum albumin (BSA; Sigma-Aldrich A2153-50G) at 2% in PBS- T. Incubation of Rabbit anti-dpERK (1:250; Cell Signaling Technologies #9101) was done overnight at room temperature (RT). For the immunodetection in toto of the di-phosphorylated form of ERK1/2, the primary antibody was incubated for three nights at 4uC. In the case of mouse anti-FGF8b (1:250; R&D MAB323) for 2 nights at 4uC. Then, several washes in PBS-T were done before 1 hour incubation with Anti-rabbit or Anti-mouse biotinylated secondary antibodies at 1:300 (Vector Laboratories BA-1000, BA-2020). Afterwards, Avidin-Biotin Complex was added at 1:300 for 1 hour and washed in PBS-T (ABC kit; Vector Laboratories CA-94010). Colorimetric detection in em- bryos, ONTCs and tissue sections were incubated with 3,39- Diaminobenzidine (DAB; Vector Laboratories SK-4100) and 0,003% H2O2. In some cases we used combined protocols of ISH and IHC within the same tissue. Finally for immunofluores- cence detection of mouse monoclonal anti-FGF8b in cryostat sections an anti-mouse Alexa-Fluor-594; (1:500: Molecular Probes A-11032) was used for 1 hour at RT. DAPI staining (1:4000; Invitrogen #D1306) was used to visualize the nuclei of the cells. After all colorimetric detection, embryos ONTCs were washed several times in PBT. All images were photographed with Leica stereoscope (Leica MZ16FA) or an upright microscope (Leica DM6000B) for the cryostat sections, using a Leica DC500 camera or DCF350 camera for fluorescence images. All pictures were taken using Leica LAS AF software. Acknowledgments We are grateful to Dr. Constantino Sotelo for insightful and helpful suggestions during preparation of the manuscript. We would like to thank specially Drs. Laura Lahti and Joan Galcera´n for technical suggestions. We thank Dr. Gail Martin form kindly providing the mouse strain Fgf8neo/null (Fgf8 hypomorph mice in this paper). We also thank Martinezs Laboratory Staff for technical support. Supporting Information Figure S1 Maintenance of molecular isthmic organizer signal activity in mouse organotypic tissue cultures (ONTCs). Gene expression profile in mouse isthmic organizer by in situ hybridizations in mouse E9.5 ONTCs (A’-D’) in comparison to in toto mice of same age (A-D) after 6 hours of incubation. Mouse brain subdivisions at E9.5 ONTCs are described with the expression of Meis2 in blue compared to Fgf8 in red (A’) genes and one half of the explant. The transversal black dashed lines illustrate the boundaries depicted by the genes on the mouse brain tissue. B-D) FGF8 negative feedback modulators, Sprouty2 (B), Mkp3 (C), and Sef (D). Note the similarities of these genes with respect to that of Fgf8 expression but the wider territory occupancy of their signals when compared to that of Fgf8, arguing indirectly the long range of FGF8 signal activity through the neuroepithelium from organizer centers. (TIF) Figure S2 Expression pattern profile of Fgf8 mRNA versus FGF8 protein in mouse E9.5 embryo. An anti- FGF8b immunohistochemistry was made onto 12mm cryostat longitudinal sections to the isthmus (see drawing) to visualize the intracellular and extracellular FGF8b protein (see arrows for the expansion of the protein in C) and compared with the Fgf8 mRNA PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e39977