Wnt5a and PCP/Neurogenesis Figure 6. Lateral expansion of Sonic hedgehog expression and flattening of midbrain ventricle, associated with loss of N-cadherin in Wnt5a2/2 embryos. (A) Shh, Foxa2 and Lmx1a are laterally expanded by E11.5 in Wnt5a2/2 mice. The invagination at the medial hinge-point is markedly reduced/flattened in the mutants. Red bars delimit the alar plate (AP), basal plate (BP), floor plate (FP) and roof plate (RP). Scale bar 250 mm. (B) The flattened VM invagination was quantified by measuring the angle formed between the midline and the ventricular wall at several levels throughout the VM of E10.5 mice, when this morphological change first became obvious. A significant difference was found in the Wnt5a2/2 mice forming a U-shaped ventricle, with an angle of 59.4u62.8u from the midline as compared to 49.1u64.3u in WT mice. (C) Western blot of E9.5 whole brain of WT and Wnt5a2/2 mice revealed a marked reduction of N-cadherin in Wnt5a2/2 mice. doi:10.1371/journal.pone.0003517.g006 we found that NR4A2+ DA precursors were generated in excess by E12.5 (47% in the Wnt5a mutants compared to 39% in WT). These results suggest that both progenitor proliferation and the differentiation of NR4A2+ precursors were affected in the Wnt5a mutants. These alterations were transient and were not detected at E14.5, when the number of TH+ cells increased by 25%, then returned to control level at E17.5 and E18.5. Thus, our results show that Wnt5a is only transiently required for the differentiation of endogenous midbrain NR4A2+ precursors in vivo, and is sufficient for their differentiation in vitro, via Rac1 activation. Interestingly, the surprising alteration in the kinetics of DA neurogenesis in the Wnt5a2/2 mice suggests that other phenomena such as increased neurogenesis (increased number of DA postmitotic precursors) and a compensatory mechanism at E14.5, may prevent a stronger DA differentiation phenotype. Indeed, several non-canonical Wnts are expressed in the VM [37], and a functional redundancy between wnt5 and wnt11 has been described in double zebrafish pipetail (ppt)/wnt5 and silberblick (slb)/ wnt11 mutants [30], suggesting that another non-canonical Wnt may compensate for the loss of Wnt5a. A role for Wnt5a in midbrain morphogenesis The involvement of non-canonical Wnt-signaling in CE movements and neural tube morphogenesis has been widely documented [29,55,56]. Moreover, Wnt5a has recently been clearly implicated in the regulation of CE movements and PCP during cochlear development and neurulation in the mouse [4]. Interestingly, the mesencephalon undergoes unique morphogenic movements to form the cephalic flexure proper, a process that is coupled to CE movements in the ventral domain and outgrowth in the dorsal domain of the mesencephalon [57]. Moreover, Wnt5a is expressed exclusively in the VM and not dorsal midbrain throughout mouse embryonic development. Our analysis of the Wnt5a2/2 mice has revealed a large variety of subtle but clear alterations in the morphogenesis and the cytoarchitecture of the VM. Overall the VM was broader and shorter as demonstrated by: a lateral expansion of the Shh-expressing FP/BP at E11.5, accompanied by broader lateral expression of Foxa2, Lmx1a, NR4A2, Th, Pitx3 and DAT. Other markers examined were also more laterally placed such as Wnt1 and Nkx6.1. The shortened midbrain phenotype was manifested in the number of levels with TH+ cells and their distribution, and suggested by a reduced length of En1 expression in sagittal sections. In coronal sections, the mesencephalon did not acquire its distinct heartlike morphol- ogy and the ventricle in the ventral midline adopted a wide ‘‘U’’ shape instead of the typical ‘‘V’’ shape at E10.5. Thus, our findings suggest diminished CE and an involvement of Wnt/PCP signaling in the VM, as described for other structures [4,22,24,28,58]. PLoS ONE | www.plosone.org 8 October 2008 | Volume 3 | Issue 10 | e3517