neural tube with a broadened Shh-expressing floor plate (FP) and neural tube closure defects [9,27,28]. PCP is defined as the organization of cells within a single layered sheet of cells. CE however, is a morphological process regulated by PCP signaling and involving the coordinated movement of cells within a 3- dimensional structure, leading to an overall elongation and narrowing of the structure [24]. Moreover, a role for PCP genes in the development of region-specific neuronal cell types outside of an epithelial plane is becoming increasingly apparent. For instance, PCP genes Celsr3 and Frizzled3 are involved in axon growth and guidance, while Celsr1 and Dvl1 also regulate dendritic arborization (for review see [29]). In zebrafish, the Wnt5 and Wnt11 mutants, pipetail and silberblick respectively, display CE defects but their roles have been suggested to be more permissive than instructive [30,31]. However, Wnt5a has been found to be required for stereocilia orientation in the cochlea, indicating that Wnt5a can play an instructive role in PCP during development [4]. We hypothesized that Wnt5a may be able to activate PCP signaling and contribute to patterning and subsequent neural differentiation in specific brain regions. We have previously reported that Wnt5a promotes DA differentiation of NR4A2 precursors in primary mesencephalic cultures [32]. However, the function of Wnt5a in the developing VM in vivo is unknown. A9–A10 dopaminergic neurons of the substantia nigra (SN) and ventral tegmental area (VTA) respec- tively, are born between E10.5 and E13.5. We therefore set out to investigate whether deletion of Wnt5a results in a Wnt/PCP phenotype and/or deficits in NR4A2 precursor differentiation in vivo at these and later stages up to E18.5. We provide evidence that Wnt5a is required for adequate morphogenesis of the midbrain by controlling the proper polarity and proliferation of VM progen- itors, and for the differentiation of postmitotic NR4A2+ precursors into DA neurons. Results Wnt5a is expressed in a restricted temporal and spatial pattern during midbrain development We have previously reported that Wnt5a is highly expressed in the VM at the time of birth of DA neurons [32]. To examine the spatial and temporal expression of Wnt5a more closely we performed in situ hybridization on E9.5–E13.5, E18.5 and P56 CD1 mice. Interestingly, in situ hybridization showed a gradient of Wnt5a expression, with higher expression of Wnt5a at E9.5 and 10.5 in rostral levels of VM as compared to caudal levels (Figure 1A). From E11.5 to E13.5, the expression of Wnt5a became progressively restricted to the FP and basal plate (BP). Until E11.5 Wnt5a was only expressed in the ventricular zone (VZ)/ subventricular zone (SVZ), but from E12.5, and mainly in caudal levels, Wnt5a was found in the intermediate and marginal zones, extending laterally (Figure 1A). We next examined the expression of Wnt5a in relation to tyrosine hydroxylase (TH - the rate-limiting enzyme in the synthesis of dopamine and a marker of DA neurons) expression. Double immunohistochemistry for TH and NR4A2 (a marker of DA neurons and precursors) on sections probed for Wnt5a revealed extensive overlap of expression, particularly in the caudal midbrain at E12.5 (Figure 1B and data not shown for other stages). From E13.5 on, the expression of Wnt5a in the ventricular zone (VZ) was reduced, and at E18.5, it became mostly restricted to DA neurons of the ventral tegmental area (VTA) (Figure 1C). In the postnatal VM, expression of Wnt5a was not detected in substantia nigra compacta (SNc) neurons, was faintly detectable in VTA neurons, and was mainly confined to the red nucleus (Figure 1C). Wnt5a and PCP/Neurogenesis Wnt5a activates Rac1, and inhibition of Rac1 blocks Wnt5a-induced DA neurogenesis We have previously shown that Wnt5a promotes the differenti- ation of dopaminergic (DA) neurons in primary midbrain cultures [32] and that Wnt5a signals via Dishevelled and Casein Kinase 1 in a dopaminergic cell line [17,33]. Wnt5a is known to activate the PCP pathway and to signal via small GTPases in different systems [34], but it is unknown which of the small GTPases transduces the Wnt5a signal in cells of the DA lineage. We therefore first investigated whether Wnt5a could activate Rac1, RhoA or Cdc42 in a DA cell line, SN4741, a validated model for studying Wnt signaling [33,35– 37]. We found that treatment with recombinant mouse Wnt5a induced the activation of Rac1 (Figure 2A), while RhoA and cdc42 activity were unchanged. In order to verify whether Rac1 mediates the pro-differentiation effects of Wnt5a, we used an in vitro assay in which Wnt5a induces the differentiation of primary DA precursors into DA neurons [32]. A dose-response curve for NSC 23766, a Rac inhibitor, showed that 10 mM had no effect on TH+ cell number. At higher doses, from 50 mM and up, cell death was seen (data not shown). Interestingly, treatment of these cultures with 10 mM of the Rac1 inhibitor NSC 23766 blocked the increase in the number of TH+ neurons otherwise induced by Wnt5a after 3 days in vitro (Figure 2 B, C), suggesting that the pro-differentiation effects of Wnt5a are mediated by Rac1. Note that the morphology of the TH+ cells is unchanged, and that the cells appear healthy in all conditions (Figure 2C). In the next part of our study we analyzed the VM phenotype of Wnt5a2/2 mice generated previously [38]. We have previously reported that Wnt5a does not activate canonical signaling in a dopaminergic neuron cell line [33]. Data in the literature suggests that Wnt/b-catenin signaling could be decreased in Wnt5a2/2 mice, since Wnt5a has been reported as capable of activating Wnt/b-catenin signaling [3], or increased since non-canonical signaling has also been reported to inhibit Wnt/b-catenin signaling [39]. To address possible regulation of canonical signaling in the Wnt5a2/2 VM, we first examined the expression of Wnt1, a Wnt expressed in the VM that activates the Wnt/b- catenin pathway and is required for VM DA neuron development [40]. In situ hybridization did not show any increase in signal, but rather a wider spacing of the two ventral stripes of Wnt1 expression in the FP (Figure S1A). This was confirmed by QPCR analysis of E12.5 WT and Wnt5a2/2 VMs tissue (Figure S1B), a finding that argues against a significant imbalance between Wnt1 and Wnt5a. To examine canonical signaling at the effector level, we investigated the levels of active b-catenin by Western blot in the VM of E10.5 and E12.5 Wnt5a2/2 mice, where no difference was detected (Figure S1C and data not shown). In sum, Wnt5a activated the small GTPase Rac1 and promoted DA differentia- tion via Rac1, but loss of Wnt5a had no effect on Wnt1 expression or canonical Wnt-signaling via b-catenin, suggesting that Wnt5a may regulate Wnt/PCP signaling in the ventral midbrain in vivo. Differentiation of dopaminergic neurons is altered in Wnt5a2/2 mice To assess the role of Wnt5a in vivo, we examined the A9–A10 DA neuron populations in Wnt5a knockout mice at E11.5, E12.5, E14.5, E17.5 and E18.5. Surprisingly, at E11.5 and E12.5, the number of TH+ DA neurons in the Wnt5a2/2 mice was not statistically different from wild-type (WT) littermate controls (Figure 3A, D). This was confirmed by quantitative PCR (QPCR) for TH and for Pitx3 (Figure S 2A,B), a homeobox transcription factor required for DA neuron survival [41–43]. A transient 25% increase in the number of Th+ DA neurons was detected at E14.5 PLoS ONE | www.plosone.org 2 October 2008 | Volume 3 | Issue 10 | e3517