Wnt5a and PCP/Neurogenesis Figure 4. Increased proliferation and accumulation of progenitor cells in Wnt5a2/2 mice. (A). Representative coronal sections at the level of the midbrain of 2 hr BrdU-pulsed WT and Wnt5a2/2 embryos at E11.5 immunostained for BrdU or Ki67, markers of cells in mitosis. The black squares in (A) depict the area within the alar plate (AP) and floor plate (FP) used for the quantification of BrdU+ cells in (B) (Squares in (A) are not drawn to scale). Note the accumulation of BrdU+ cells in the Wnt5a2/2 VM in the adjacent magnified box. Increased Ki67 staining and mitotic figures (arrowheads) in the ventricular zone. (B) The density of BrdU+ proliferating cells was significantly increased within the FP, but no significant change was found within the AP of Wnt5a2/2 embryos compared to their wt littermates (C) The number of Ki67+ cells was significantly increased at E12.5 (paired t-test, p = 0.003, N = 3). (D) Mitotic nuclei (arrowheads in (A)) were counted in the midline domain to asses the number of cells in M-phase, and this was also found to be significantly increased in the Wnt5a2/2 mutant (paired t-test, p = 0.037, N = 3). doi:10.1371/journal.pone.0003517.g004 Mediolateral/PCP and apicobasal morphogenetic defects in the VM of Wnt5a2/2 mice Several of the defects described in previous sections are reminiscent of PCP developmental defects, including the accumu- lation of BrdU+ cells in the midline, the lateral expansion of the Ki67+, Ngn2+, NR4A2+ and TH+ domains in the VM as well as a flattening of the midbrain ventricle (Figures 3, 4, 5). Previous reports of animals lacking PCP components have shown that the invagination of the VZ in the ventral midline is flattened and that the Shh domain, that defines the FP and BP in the midbrain, is broadened [50–52]. In the Wnt5a2/2 mice, a lateral expansion of the Shh and Foxa2 expression domains was first detected at E11.5 (Figure 6A, Figure S2D,E). This was associated with a lateral expansion of the Lmx1a+ DA progenitor domain (Figure 6A). Expression of the Shh-target genes (Ptch1 and Gli1) was not expanded in the midbrain AP (Figure S3). The expression of class I (Dbx1) and class II (Nkx2-2, Nkx6-1) genes was not changed in the midbrain of the Wnt5a2/2 embryos compared to WT although the aberrant morphology of the Wnt5a2/2 midbrain noted previously led to a wider separation of the Nkx6-1-positive domains in the BP of the mutant midbrain (Figure S3 and data not shown). To characterize this defect in more detail, the analysis of the angle formed between the invagination of the ventricular epithelia and the midline in the VM revealed that this angle appeared greater in Wnt5a2/2 mice (59.4u62.8u), than in WT mice (49.1u64.3u) (Figure 6B). This resulted in the midbrain ventricle adopting a ‘‘U’’-shape in the Wnt5a2/2 mice instead of the typical ‘‘V’’- shape in controls (Figure 6A, B). A similar phenotype, including a broadened Shh domain, has been observed in the neural tube of the PCP mutants Scribble and Vangl2 [50–52]. It has previously been shown that cadherins regulate intercel- lular adhesion in neural progenitors and that a disruption of the complex formed with a- and b-catenin leads to increased Shh signaling and proliferation [53]. We therefore examined the levels of N-cadherin in Wnt5a2/2 mice by Western blot and found a reduction at E9.5, prior to the expansion of the FP (Figure 6C). Moreover, since apico-basal polarity depends on PCP and adhesion, we examined apico-basal polarity of cells in the ventricular zone. Interestingly, the change in the general morphology of the VM neuroepithelia was accompanied by an alteration in the orientation of the cells, as shown by the non- uniform orientation of propidium iodide stained nuclei in the Wnt5a2/2 mice, compared to WT at E12.5 (Figure 7A). To assess this quantitatively, the angle formed by the longest axis of each nuclei with the ventral midline was measured for 10 cells (starting from the midline and counting laterally) at different PLoS ONE | www.plosone.org 6 October 2008 | Volume 3 | Issue 10 | e3517