Wnt5a and PCP/Neurogenesis Figure 7. Apical-basal polarity/cell orientation are affected in Wnt5a2/2 mice. (A) At E12.5, propidium iodide staining on coronal sections through the VM revealed that cell nuclei in the neuroepithelium were rounded and their orientation was more variable with some cells pointing contralaterally (red asterisks/arrows). (B) The orientation of each cell nucleus was plotted versus its distance from the ventral midline. The angle between the nucleus and the midline was measured from cell 1 (the most medial) to cell 10 (the most lateral). Cell nuclei in Wnt5a2/2 mice are oriented more ventrally compared to the more lateral orientation of cells in WT mice (two way-ANOVA for genotype and level, p = 0.0029, N = 3). (C) The frequency of cell nuclei oriented towards the contralateral ventral side (red arrows in A) was significantly increased in Wnt5a2/2 mice (paired t- test, p = 0.0198, N = 3, 10 nuclei at 3 levels/animal). doi:10.1371/journal.pone.0003517.g007 Interestingly, we also found a change in the apico-basal orientation of midbrain VZ cells in Wnt5a2/2 mice compared to WT. The axis of individual WT FP apical ventricular cells and the midline formed an average 36u angle and did not cross the midline ventrally. However, cells in the Wnt5a2/2 mice formed an average 20u angle with the midline and in some instances individual cells showed a negative angle (i.e. their axis pointed contralaterally). A possible interpretation of these results is that the increase in proliferation allows nuclei to be oriented aberrantly in Wnt5a2/2 mice during mitosis. However, misorientation was never seen in WT mice in lateral positions, regardless of cell cycle phase, but was seen in Wnt5a2/2 mice. This could also reflect a consequence of the flattened VM morphology, allowing greater deviations towards the contralateral side. A more intriguing possibility is that the alteration in PCP directly regulates or causes secondary changes in the geometry of the cells and in apicobasal polarity. PCP and apicobasal polarity require cell attachment, a process that both pathways also regulate [59,60]. In line with this, we found a decrease in the levels of N-cadherin in the Wnt5a2/2 mice at E9.5, which could contribute to decreased attachment and altered polarity. These results suggest PCP and apico-basal polarity may be coordinately regulated in certain structures by adhesion proteins. Triple slb;ppt;wnt4-morphant zebrafish embryos phenocopy the neurulation defect of trilobite (tri)/Van Gogh-like 2 (Vangl2) PCP mutants [61]. Interestingly, the defects in the Wnt5a2/2 VM resemble the tri neurulation defects with an increase in proliferation, a broadening of the Shh domain/expansion of the FP, and an ectopic accumulation of neural progenitors within the midline [61]. These findings strongly suggest that Wnt5a also regulates cell intercalation after cell division and VM morpho- genesis. Our analysis of the VM phenotype of Wnt5a2/2 mice suggests a model in which Wnt5a regulates morphogenesis by a mechanism involving cell adhesion and altered PCP signaling. This is accompanied by an alteration in apico-basal polarity, and a broader distribution of VM cell populations, but no alteration in the final number of neurons, all of which is compatible with a PCP phenotype. Proliferation: a link between differentiation and morphogenesis Our results indicate that the loss of Wnt5a directly or indirectly regulates cell division in VZ progenitors. Indeed, we found that deletion of Wnt5a increased the number of both BrdU+ and Ki67+ cells in the apical VZ, and the number of NR4A2+ postmitotic cells in the IZ, which suggested a regulation of both proliferative and neurogenic divisions. Interestingly, Wnt/PCP signaling has been previously reported to regulate oriented cell division during gastrulation, allowing axis elongation [61,62], and to regulate asymmetric cell division [63], which is the predominant mode of division used by VM progenitors during DA neurogenesis. Another mechanism that could also result in the regulation of proliferation and neurogenesis is cell adhesion. Our results indicate that Wnt5a may regulate cell adhesion in the neuroep- ithelia by regulating the levels of N-cadherin as early as E9.5. Interestingly, it has been described that a reduction in a-catenin PLoS ONE | www.plosone.org 9 October 2008 | Volume 3 | Issue 10 | e3517