Figure 2. Rac1 is activated by Wnt5a and is required for Wnt5a- induced dopaminergic neuron differentiation. (A) Stimulation of SN4741 cells, a midbrain dopaminergic neuron cell line, with 200 ng/ml Wnt5a for 2 hrs activates Rac1, but not RhoA or Cdc42. (B) As previously reported, Wnt5a upregulates the number of TH+ dopaminergic neurons in E11.5 primary ventral midbrain cultures grown for three days in vitro. This differentiation is completely inhibited in the presence of 10 mM NSC23766, a Rac1 inhibitor (ANOVA for Wnt5a vs Wnt5a&NSC23766, P,0.05, N = 3 experiments, each normalized to the control conditions) (C) TH+ cells in all conditions have normal morphology, with increased numbers in the Wnt5a-treated, but not Rac-inhibited, wells. doi:10.1371/journal.pone.0003517.g002 (Figure 3A, D) but not at earlier or later stages (Figure 3B, D and data not shown). TH+ cells displayed normal morphology and relatively normal density at E12.5 (Figure 3C). Although the number of DA neurons normal at all ages except E14.5, the morphology of the midbrain and distribution of the DA neurons was altered in Wnt5a2/2 mice from E10.5, note the broader lateral and dorsoventral distribution of TH+ cells at E12.5, E14.5 and E17.5 (Figure 4A, B). We also examined the number of cleaved caspase 3+ cells (a marker of cells in apoptosis), but found no difference at E14.5 or E17.5 (data not shown). Increased proliferation and DA precursor pool in Wnt5a2/2 mice In vitro, Wnt5a promotes DA precursor differentiation, reducing the number of proliferating (in S-phase) bromodeoxyuridine Wnt5a and PCP/Neurogenesis positive (BrdU+) cells [32]. To assess the role of Wnt5a in proliferation in vivo and the reason for the transient 25% increase in Th+ cells in the Wnt5a2/2 embryos, the density of BrdU+ cells/area in the FP and the alar plate (AP) of E11.5 Wnt5a2/2 and WT embryos were compared (black squares in Figure 4A). While the density of BrdU+ cells in the AP of WT and Wnt5a2/2 embryos was not different (after a 2 hour pulse), a 35% increase in BrdU+ cells/area was detected in the FP of Wnt5a2/2 mice (Figure 4A, B). Furthermore, BrdU+ cells accumulated in the ventral midline of the Wnt5a2/2 midbrain at E11.5 (Figure 4A). The increase of proliferating cells within the FP was first evident at E11.5. This was confirmed by a 45% increase in Ki67+ cells (a marker of cells in the cell cycle) detected in Wnt5a2/2 mice (Figure 4A, B). Similarly, the number of cells that are dividing (mitotic cells, arrowheads, Figure 4A) increased by 25% in Wnt5a2/2 mice at E11.5 (Figure 4A, C). Interestingly, the increase of cells in S-phase and M-phase was maintained along the entire antero-posterior axis of the VM. Neurogenesis in the VM is controlled by a code of homeodo- main and proneural bHLH transcriptional regulators [44]. Expression of the members of the bHLH superfamily Ngn1, Ngn2 and Mash1 can be subdivided into three distinct domains within the VZ of the mouse VM [45]. Ngn2 is essential for proper DA neurogenesis, and Mash1 is required for the generation of VM GABAergic neurons [45–47]. The expression of Ngn2 (and the low levels of Mash1) in the midbrain FP therefore define the DA progenitor domain, whereas the high expression levels of Ngn2 and Ngn1 in the adjacent basal plate (BP) demarcates the oculomotor (OM) and red nucleus (RN) progenitor domains [45,46,48]. In the Wnt5a2/2 embryos, the Ngn1-positive domain within the BP and the Ngn2-positive domain comprising the FP and BP were broadened in the mutant midbrain at E11.5 (Figure 5A), whereas the expression domains of these two proneural factors remained unchanged in the dorsal midbrain. We next examined whether the increase in cell division and broadened neurogenic domain had generated more postmitotic cells, and found a 47% increase in the number of NR4A2+ postmitotic cells compared to control at E12.5, suggesting that the neurogenic process is indeed enhanced in the Wnt5a2/2 mice (Figure 5B, C). Furthermore, the 47% increase in NR4A2+ cells is consistent with a 45% increase in proliferating cells. However, a 25% excess of TH+ at E14.5 indicates that not all of the excess NR4A2+ cells differentiate into Th+ neurons at this point. Moreover, we found that the domain occupied by NR4A2+ cells was wider in Wnt5a2/2 than in wild type mice (Figure 5B). An increase in the amount of NR4A2 mRNA was also detected at E12.5 by QPCR (Figure S2C). We have previously reported that one of the functions of Wnt5a is to promote the differentiation of NR4A2+ DA precursors into DA neurons in vitro. This has been shown in diverse in vitro preparations, including rat E14.5 VM primary cultures [32], VM neurospheres derived from E10.5 mice or E12.5 rat [49] and mouse ES cells (unpublished observation). In order to investigate whether Wnt5a also promotes this differen- tiation step in vivo, we examined the proportion of TH+ cells out of NR4A2+ cells in the VM of Wnt5a2/2 mice at E12.5. In agreement with our previous in vitro results, we found that the proportion of NR4A2+ cells differentiating into Th+ cells was reduced by 23% at this stage (Figure 5C). However since the number of TH+ cells increased at E14.5, and subsequently normalized, our results suggest that differentiation is rescued by an alternative mechanism. We thus conclude that Wnt5a is only partially and transiently required for the differentiation of NR4A2+ precursors into DA neurons in vivo. PLoS ONE | www.plosone.org 4 October 2008 | Volume 3 | Issue 10 | e3517