150 mM NaCl, 0.1% Triton X-100), washed with TST, and treated with FITC-conjugated secondary antibody at 1:100 dilution in TST. After washing in TS buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl), samples were mounted in OxyFluor at 1:100 dilution in TS, and observed under confocal microscope. Whole-mount in situ hybridization Riboprobe generation and in situ hybridization were performed according to [50] with a modification in post-antibody wash as follows: after the treatment with the antibody, embryos were washed 6 times for 1 hour each and stored at 4uC overnight. This washing process was done 3 times over the course of 3 days. After color development, embryos were fixed, dehydrated, rehydrated and cleared according to the protocol available online (http:// www.med.upenn.edu/mcrc/histology_core/wholemount.shtml). Cultures of neural crest explants Embryos at E8.5 or E9.5 were collected from pregnant female mice into PBS, and neural tube area was dissected from the back of the embryos using 27 gauge needles. Dissected tissues were cultured in 1:1 of DMEM/F10 supplemented with 10% FCS and antibiotics on glass bottom dishes (Matek) pre-coated with 10 mg/ ml fibronectin in PBS for 1 hour at room temperature. Explants were cultured for 2 days and used for further analyses. Cell migration assays Immortalized wild-type and Ate1 KO embryonic fibroblasts were cultured as described in [24]. Wound healing assays to measure the migration speeds of these cells were performed by growing the cells to a confluent monolayer in tissue culture dishes, followed by scraping off a portion of the monolayer, and the migration of the cells into the resulting wound was recorded as time-lapse images at 2 min intervals over 10–15 hour observation period and analyzed using Metamorph imaging software (Molec- ular Devices). To analyze the migration of Ate1 KO cells in co-culture with wild-type cells, wound healing experiment were performed with GFP-labeled Ate1 KO cells co-plated with wild-type cells as a mixture at the ratio of KO:WT 1:2 to 1:6. For reciprocal experiments, GFP-labeled wild-type and unlabeled Ate1 KO cells were used. Immunocytochemistry in cultured cells For immunostaining, WT and Ate1 KO cell monolayers were scraped to produce the wound. After 4 hours, cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100, and stained with mouse monoclonal anti-paxillin antibody (BD Biosciences), rhodamine-phalloidin (Sigma), mouse anti-talin clone 8d4 (Sigma), and mouse anti-FAK, clone 2A7 (Upstate Biotech- nology) in different combinations. For immunostaining for fibronectin, cells attached on the cover glasses were fixed in 4% paraformaldehyde and stained with rabbit polyclonal anti- fibronectin antibody (Sigma). To observe only the extracellular fibronectin, cells were not permeabilized or washed with Triton X- 100 before or after fixation. X-gal staining in fetuses and neural crest explants Fetuses or neural crest explants were fixed with 4% parafor- maldehyde at 4C for 1 hour, rinsed for 30 minutes with rinse buffer (0.2 M sodium phosphate pH 7.3, 2 mM magnesium chloride, 0.02% IGEPAL and 0.01% sodium deoxycholate) 3 times, and incubated overnight at 37uC in the staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and Arginylation Regulates Neural Crest Migration 1 mg/ml X-gal in rinse buffer). Samples were post-fixed with 4% paraformaldehyde and stored in 70% ethanol. Detection of apoptosis in fetuses by TUNEL assay TUNEL assay were performed in fetuses at E9.5 and E12.5 using In Situ Cell Death Detection Kit, Fluorescein (Roche). Fetuses were fixed in 4% paraformaldehyde in PBS overnight at 4uC, washed 3 times in PBS and incubated with 18.7 mg/ml proteinase K in 10 mM Tris/HCl, pH 7.5 for 30 minutes at 37uC. After washing 3 times in PBS, samples were incubated in the Label Solution supplemented with Enzyme Solution for 1 hour at 37uC, then washed in PBS 3 times and photographed under a fluorescent microscope. For negative control, samples were incubated in Label Solution without Enzyme Solution. For positive control, samples were treated with 100 U/ml DNase I and 1 mg/ml BSA in 50 mM Tris/HCl, pH 7.5 for 20 minutes at room temperature, followed by incubation with Label Solution supplemented with Enzyme Solution. Detection of BrdU incorporation in neural crest explants by immunostaining Cell proliferation in the neural crest explants from E8.5 and E9.5 fetuses was analyzed by detecting BrdU incorporation using BrdU Cell Proliferation Assay kit (Calbiochem, QIA58). After 2-day culture, explants were incubated with 1:2000 BrdU Label in culture medium for 2 hours. Explants were washed twice in DMEM without serum, and fixed with Fixative/Denaturing solution at room temperature for 30minutes. After washing in 70% ethanol, samples were air-dried and incubated with 1:100 Anti-BrdU antibody in Antibody Diluent at room temperature for 1 hour. Samples were washed twice in Wash Buffer (1:20 Plate Wash Concentrate in water), twice in PBS, and treated with 1:200 Cy3 conjugated secondary antibody (Jackson ImmunoResearch Laboratories) in PBS at room temperature for 1 hour. Samples were washed 3 times in PBS and photographed under a fluorescent microscope. Supporting Information Figure S1 (A) Construction of the Ate1 conditional knockout and Wnt1-Ate1 mice. (B) Genotyping of Ate1-floxed and Wnt1-Ate1 mice. Found at: doi:10.1371/journal.pgen.1000878.s001 (0.76 MB TIF) Figure S2 Ate1 antibody characterization. Left, Coomassie- stained gel of wild-type and knockout cell extracts. Right top, immunoblots of the cell extracts shown on the left. Ate1 antibody specifically recognizes a ,55 kDa band in the wild-type but not knockout extract. Right bottom, immunoblots of bacterially expressed Ate1 isoforms show that Ate1 antibody reacts equally with all four isoforms. Found at: doi:10.1371/journal.pgen.1000878.s002 (3.06 MB TIF) Figure S3 Ate1 deletion in Wnt1-Ate1 mice in midbrain and enteric neurons. (A) An H&E-stained sagittal section of an embryo at E16.5. Large and small box outline the areas of the animal corresponding to those shown in (B,C), respectively. (B) Midbrain area of the control (WT) and Wnt1-Ate1 (KO) newborn mouse double-stained for the neuronal marker beta-III tubulin and Ate1. In KO, Ate1 is prominently missing from the midbrain, but not from other areas of the embryo. (C) Cross sections of the gut in control (WT) and Wnt1-Ate1 (KO) newborn co-stained for Ate1 and beta-III tubulin show prominent absence of Ate1 from the enteric neurons. Decrease in the Ate1 signal level compared to the control was similar in the gut neurons and in mid-brain, as verified by measurement of the fluorescence levels. Found at: doi:10.1371/journal.pgen.1000878.s003 (3.25 MB TIF) PLoS Genetics | www.plosgenetics.org 13 March 2010 | Volume 6 | Issue 3 | e1000878