Wnt5a and PCP/Neurogenesis Table 1. The area occupied by the Islet1+, Brn3a+ or Th+ domain in coronal sections is 30–40% greater in Wnt5a2/2 mice at E18.5. Genotype Wildtype Wnt5a2/2 Student’s T-test Isl1+ domain (mm36105) n = 4 Brn3a+ domain (mm36105) n = 3 Th+ domain (mm36105) n = 3 62,469,79 s.e.m. 89,364,05 s.e.m. 0.044195531 * 347,2620,8 s.e.m. 443,2618,41 s.e.m. 0.025901993 * 805,87613,11 s.e.m. 1140,2769,34 s.e.m. 3.17277E-05 *** The more ventral Th+ population shows the greatest difference. doi:10.1371/journal.pone.0003517.t001 Shh, Foxa2 and Lmx1a domains in the Wnt5a2/2 mice. Thus, taken together, these data suggest that Wnt5a can affect both the extent and the type of cell division in VZ progenitor cells by different mechanisms. In sum, our results show that CE defects and lowered cadherin expression were the first detected phenotype at E9.5 in the Wnt5a2/2 mice. This was followed by an alteration in morphogenesis and DA differentiation, and at E12.5 by defects in cell polarization and adhesion, a shortening and a broadening of the A9–A10 nucleus and other ventral domains of the mesencephalon. We suggest that the broadening of the Shh domain may lead to an increase in Shh-signaling, increased proliferation in the FP/BP, accumulation of NR4A2+ precursors and a transient expansion of the Th+ cell population at E14.5. These defects were most pronounced in the FP, thus affecting the morphology and development of cells in the DA lineage in the Wnt5a2/2 mice. In conclusion, we show for the first time that, in the VM, Wnt5a regulates CE movements required for axial elongation, polar growth and morphogenesis as well as proliferation, neurogenesis, some aspects of differentiation, and the actual positioning of neurons in the VM. Materials and Methods Animals, Immunohistochemistry and in situ Hybridization (ISH) Wnt5atm1Amc (referred to as Wnt5a2/2 in this article) transgenic mice [38] and CD1 mice (Charles River) were housed, bred and treated in accordance with the approval of the local ethics committee (Stockholms Norra Djurfo¨ rso¨ketiska Na¨mnd). Wnt5a+/ 2 mice were kept on a C57BL/6 background. In all experiments, the Wnt5a2/2 embryos were compared to their wild-type (Wnt5a+/+ and Wnt5a+/2, heterozygotes have wild-type pheno- type) littermates, n$4 for each genotype, if not otherwise stated in the text. Mice of the relevant genotype were mated overnight and noon of day of plug was taken as E0.5. Embryos were dissected in ice-cold PBS, fixed in 4%PFA 4 hrs to overnight, cryoprotected in 20% sucrose and frozen in OCT compound on dry ice. Serial coronal 7, 14 or 16 mm sections of the brain were obtained on a cryostat. Immunohistochemistry (IHC) and in situ hybridization (ISH) were carried out as previously described [32,64]. IHC and ISH for Wnt5a were visualized with a Zeiss HBO100 microscope; images were collected with a C4742-95 Hamamatsu camera and processed with OpenLab software and/or ImageJ. Antibodies Rabbit anti-Nurr1 (NR4A2) (1:200, Santa Cruz Biotech.), rabbit anti-TH (1:500, Pel-Freeze), mouse anti-Ki67 (1:800, Abcam), anti-BrdU, Cy2-, Cy3- or Rhodamine-coupled secondary antibodies (1:250, Jackson ImmunoResearch, West Grove, PA/ USA), or biotin-conjugated goat anti-rabbit IgG (1:300) were used. Some sections were counterstained with 49, 6-Diamino-2-pheny- lindole (DAPI) (500 ng/ml, Sigma, Missouri/USA), or propidium iodide (Invitrogen). Anti-active-b-catenin (1:500, Clone 8E7 from Upstate Biotechnology/Millipore), anti Rac1 (1:1000, Upstate), anti RhoA (1:1000, sc418 Santa Cruz), anti CDC42 (1:1000, Trans Lab) and anti-b-actin (1:5000, Abcam) were used for Western blot. Radioactive in situ hybridization Paraffin sections (8 mm) of mouse embryos (E11.5/E12.5) or brains (E18.5, P56) were processed for radioactive in situ hybridization as previously described [65]. The probes used were as follows: Wnt5a [38], Shh, En1, Wnt1 [66], Th, Pitx3, Slc6a3/Dat [65], Foxa2 (bp 743–1314, Acc. Nr. NM_010446), Dbx1, Isl1, Pou4f1, Nkx2-2, Nkx6-1 [67], Ngn1, Ngn2 [68], Lmx1a (bp 412– 1211, Acc. Nr. NM_033652) Ptch1 [69] and Gli1[70]. Images were taken using darkfield optics on a stereo microscope Stemi SV6, AxioCam MRc camera and Axiovision 4.6 software (Zeiss, Jena/ Germany). BrdU treatments Pregnant females were injected intra-peritoneally with 5-bromo- 2-deoxyuridine (BrdU, 10 mg/g body weight) two hours before they were sacrificed and processed as described [71]. The number of BrdU-positive cells in a square area (2500 mm2) was counted on at least four serial sections from the midbrain of stage-matched littermates (n = 4) using the Neurolucida 6 software (MBF Bioscience, Williston, VT/USA). TH+ Cell Counts and Distribution For counts of total number or distribution of TH+ cells in the ventral midbrain, alternate 14 mm sections from coronal series of the entire A9–A10 population were counted and plotted versus position at E11.5 and E12.5 for distribution analysis. At E14.5 and E17.5, every sixth section was counted and plotted in the distribution analysis. The lateral spread of TH+ cells was measured in coronal sections through the VM, in three consecutive levels, where level 1 corresponds to the rostral midbrain-P1 boundary, level 2 corresponds to the intermediate midbrain and level 3 corresponds to the caudal midbrain (see Figure S 5). Pictures were taken with identical acquisition settings and magnification and the lateral spread was measured by drawing a horizontal line between the outermost TH+ cells. This distance was measured in pixels using ImageJ and then normalized to littermate controls. The volumes of the Th2, Brn3a2 and Isl1-expression domains at E18.5 were measured by the Cavalieri method using the Stereo Investigator 5.05.4 software (MBF Bioscience) as described [65]. Cell numbers were averaged for each genotype and subjected to a Student’s t-test for the estimation of statistical significance. PLoS ONE | www.plosone.org 11 October 2008 | Volume 3 | Issue 10 | e3517