Calculation of slope of ventricular invagination and analysis of nuclei orientation E10.5 and E12.5 mice were stained with Hoechst or propidium iodide respectively. The slope of the wall of the VM at E10.5 was measured by drawing a cross bisecting the FP, where the origin coincided with the ventricular invagination. A tangent to the ventricular wall was drawn and the angle between the tangent and the midline was measured in 4 embryos of each genotype. To measure the nucleus orientation, confocal pictures of serial sections of E12.5 midbrains were taken with an optical slice of 1 mm. Ten nuclei along the ventricle were analyzed per image from the midline outwards, 3 images per mouse (rostro-caudally distribut- ed), and 3 mice per genotype were analyzed. The nucleus was outlined in Image J and the angle of the longest axis was measured compared to the vertical parallel to the midline. This gave an angle that was plotted against its position from the midline (nucleus number position). Certain nuclei were abnormally oriented towards the contralateral side, these gave a negative angle. The number of nuclei with abnormal orientation (negative angle), was compared to the total number of cells and this ratio was used for analysis of contralateral orientation. Primary ventral mesencephalic cultures and immunocytochemistry Ventral midbrains of E11.5 CD1 mice were dissected out in ice- cold PBS supplemented with 0.2%glucose, mechanically dissoci- ated in serum-free N2 through flame-narrowed Pasteur pipettes and plated at a final density of 125,000 cells/well in poly-D-lysine- coated 48-well plates as described previously [32]. Cultures were treated with recombinant mouse Wnt5a (200 ng/ml, RnD), 0.05% CHAPS (Wnt5a control, Sigma), and/or 10 mM Rac1 inhibitor (NSC 23766, Calbiochem). Treatment of cultures was initiated 1 hour after plating and cultures were incubated for 3 days in N2 at 37uC in 5%CO2. Cells were then fixed for 15 minutes with 4% paraformaldehyde, washed in PBS, and used for immunocytochemical analysis. Real-time Quantitative PCR cDNA was generated as described previously [32]. In brief, RNA from E8.5-P0 VMs of CD1 (for developmental stages) or wildtype and Wnt5a2/2 mice was extracted using RNeasy Mini Kit (Qiagen). 1 mg of RNA was reverse-transcribed using SuperScript II Reverse Transcriptase (Invitrogen) and random primers (Invitrogen). Quantitative PCR and the primers used in this study have been described previously [32] with the exception of NR4A2 primers; Forward: 59CAGCTCCGATTTCTTA ACTCCAG39 and Reverse: 59GGTGAGGTCCATGC- TAAACTTGA39. Activated GTPase assay Immunoblotting and sample preparation were performed as described [17]. SN4741cells were cultured as described previously [17]. Cells were serum starved overnight and stimulated with recombinant Wnt5a (200 ng/ml, R&D) for 2 hours. Cells were washed with ice-cold PBS and lysed in GTPase lysis buffer (10 mM Tris-HCl, pH = 7.5; 110 mM NaCl; 1 mM EDTA; 10 mM MgCl2; /well, 1% Triton – 6100; 0.1% SDS, 1 mM DTT, protease inhibitor cocktail (Roche)) for 5 minutes at 4uC. Cell lysates were cleared by centrifugation at 14 000 rpm/4uC/ 5 min and incubated for 15 min/4uC on a rotator with 25 ml of GST-PAK-CRIB beads (25% slurry) for Rac1-gtp pulldown, GST-Rhotekin for RhoA-gtp pulldown and GST-WASP for cdc42-gtp pulldown. Beads were subsequently washed 3 times with Wnt5a and PCP/Neurogenesis 0.5 ml GTPase lysis buffer (without 0.1% SDS), mixed with 26 Laemmli buffer and analyzed on SDS-PAGE for the amount of activated RhoA/Rac1/cdc42. 5% of total cell lysate was used as input control. Statistics Data were analysed using Graphpad Prism version 4.01 for Windows, GraphPad Software, San Diego California USA, www. graphpad.com. For analyses comparing WT and Wnt5a2/2 mice, t-tests were used. A paired t-test was used if 1WT/Wnt5a littermate pair was used from individual litters. If several pairs were taken from the same litter then an unpaired t-test was used. At least 3 litters were used for each analysis. Each graph value is the mean6SEM. For analyses of distribution of Rac inhibition of dopaminergic neuron differentiation and for dopaminergic neuron distribution in vivo, One-way and Two-way ANOVA were used, with Bonferroni’s post-hoc test. Supporting Information Figure S1 Canonical Wnt signaling is unaffected by loss of Wnt5a. (A) In situ hybridization at E12.5 for Wnt1 shows wider spacing between the two ventral stripes of Wnt1 expression in the Wnt5a2/2 mice (red brackets), but no enlargement of the Wnt1 domain (Scale bar is 500 mm). (B) QPCR for Wnt1 shows no difference in levels of Wnt1 in the VM of Wnt5a2/2 mice at E12.5. (C) Western blot for active (dephosphorylated) b-catenin shows little difference at E12.5 in the Wnt5a2/2 VM. Found at: doi:10.1371/journal.pone.0003517.s001 (4.57 MB TIF) Figure S2 QPCR of E12.5 WT and Wnt5a2/2 VM. No difference in Th (A) and Pitx3 (B) mRNA levels. Increase in the amount of Nr4a2 mRNA (C). Small or no increase in Shh mRNA (D) and a greater difference in Foxa2 levels (E). Found at: doi:10.1371/journal.pone.0003517.s002 (0.71 MB TIF) Figure S3 No change in dorsoventral patterning of the Wnt5a2/2 midbrain. Expression of Shh-target genes Ptch1 and Gli1 was not altered in Wnt5a2/2 mice. The expression of class I (Dbx1) and class II (Nkx6-1) genes was not changed in the midbrain of the Wnt5a2/2 embryos compared to WT. Found at: doi:10.1371/journal.pone.0003517.s003 (5.40 MB TIF) Figure S4 Anteroposterior levels used to analyze the lateral distribution of TH+ cells. Levels 1, 2 and 3 corresponding to rostral, intermediate and caudal levels are depicted on sagittal sections of WT and Wnt5a2/2 mice, probed for Th. Found at: doi:10.1371/journal.pone.0003517.s004 (3.81 MB TIF) Figure S5 The En1-expressing domain is shortened in Wnt5a2/ 2 VM (A) Bright field and dark field images of the midbrain cephalic flexure hybridized for Engrailed1 (En1) show a shorter anteroposterior extension of En1 in the Wnt5a2/2 midbrain. Red line shows the length measured from isthmus to anterior-most En1 expression. (B) Quantification of En1 expression length shows a shorter domain in Wnt5a2/2 embryos (paired t-test p = 0.0203, WT N = 3, Wnt5a2/2 N = 2). Found at: doi:10.1371/journal.pone.0003517.s005 (7.93 MB TIF) Acknowledgments The authors thank Andrew P. McMahon for the Wnt5a2/2 mice, Dr. J.H. Son for providing the SN4741 cells, to members of the Arenas lab for valuable discussions, and to S. Laass for excellent technical assistance. We thank Susanne and Cecilia in the MBB animal facility for excellent animal care. EA wishes to thank members of the K58 and B89 teams at KS for exceptional assistance. PLoS ONE | www.plosone.org 12 October 2008 | Volume 3 | Issue 10 | e3517