Figure 5. The ventral midbrain progenitor domains are expanded in Wnt5a2/2 embryos. (A) Detection of Th, Ngn1, and Ngn2 on representative serial coronal sections at the level of the midbrain of WT and Wnt5a2/2 embryos at E11.5. Red brackets delimit the ventral Ngn1 and Ngn2 domain in the WT embryo, respectively. (Scale bar in A: 500 mm.). (B) Increase in NR4A2+ cells in the VM of Wnt5a2/2 mice, which occupy a broader region laterally and dorsoventrally. (C) A 47% increase in the number of NR4A2+ cells was detected in the Wnt5a2/2 mice at E12.5 (unpaired t-test, p = 0.044, WT N = 3, Wnt5a2/2 N = 4). (D) Analysis of the proportion of NR4A2+ cells differentiating into TH+ DA neurons revealed that the differentiation of NR4A2+ precursors into TH+ cells was impaired in the Wnt5a2/2 mice at E12.5 (paired t-test, p = 0.0098, N = 3). doi:10.1371/journal.pone.0003517.g005 anteroposterior levels of the VM. This angle changed from very acute to less acute as the cells were positioned further away from the midline in the wild type. This angle reached almost 50u in wild type, but did not exceed 30u degrees in Wnt5a2/2 mice (Figure 7B). These results reflect the fact that, in Wnt5a2/2 mice, the nuclei of apical FP cells are oriented ventrally, while the nuclei of control mice are oriented more ventro-laterally (Figure 7A, B). Furthermore, when the frequency of nuclei aberrantly oriented towards the contralateral side was examined, Wnt5a2/2 mice showed a 7-fold increase compared to WT (Figure 7A, C). Note that while cells in the midline of both WT and Wnt5a2/2 mice exhibited nuclei oriented contralaterally, only Wnt5a2/2 mice showed nuclei with contralateral orientation in lateral positions of the FP (Figure 7A). Several neuronal populations in the VM are redistributed following the PCP defect A more detailed analysis of the distribution of A9–A10 DA neurons, in the very same animals that did not show any change in total DA cell number at E12.5, revealed changes in the mediolateral, rostro-caudal, and dorsoventral axis (Figure 8A). Sections at regular intervals throughout the A9–A10 nucleus were examined. The three levels analyzed in WT and Wnt5a2/2 mice Wnt5a and PCP/Neurogenesis (rostral, intermediate and caudal) are shown at E12.5 in (Figure S4). In the mediolateral axis, TH+ cells extend more laterally in the Wnt5a2/2 mice than in WT mice, especially in the rostral portion of the A9–A10 nuclei (Figure 8A–D). This phenotype persisted until E17.5, and followed the earlier morphological defects in the distribution of progenitor (Shh, Foxa2, Lmx1a, Ngn2) and precursor (NR4A2) markers, all of which were expanded already at E11.5. In the rostro-caudal axis, the distribution of TH+ cells was unchanged at E11.5 (Figure 8E). However, at E12.5 and E14.5, the A9–A10 nuclei were shortened in Wnt5a2/2 mice, seemingly at the expense of the most anterior portion of the nuclei (Figure 8F, G). Finally, by E17.5 the distribution of VM DA neurons in the anteroposterior axis tended to normalize but remained broad in the dorsoventral axis (Figure 3B). This shortening of the anterior neural tube at E12.5 was also apparent when the length of the En1 expression domain was measured in sagittal sections through the VM of Wnt5a2/2 mice, and compared to WT (390 mm compared to 540 mm, Figure S5). To distinguish between a deregulation of TH and a true misplacement of the DA neurons, we examined the spatio-temporal expression pattern of other DA neuron markers such as Pitx3 and Slc6a3/Dat. Both marker genes were expressed within the marginal zone (MZ) of the Wnt5a2/2 ventral midbrain at E12.5 (Figure 8H). A mediolateral expansion of the Th, Pitx3 and Slc6a3/DAT expression domains was detected in the Wnt5a2/2 mice, confirming the redistribution of DA neurons within the VM (Figure 8H). Since Wnt5a regulates PCP in several structures and the Shh, Foxa2 and Ngn1 domains were also broadened in the BP, we examined whether deletion of Wnt5a altered the distribution of other mature ventral neuronal populations in coronal midbrain sections at E18.5, when no difference in number of DA neurons was detected (data not shown). Interestingly, the area occupied by Th2, Islet12 or Brn3a2 expressing cells was increased in Wnt5a2/2 mutants (30–40%, Table 1). These results indicate that while the absolute numbers of midbrain neuron populations such as DA neurons are unchanged, the cells are re-distributed and positioned in a laterally and dorsoventrally enlarged domain in the Wnt5a2/2 VM (Table 1 and Figure 3). Discussion The impact of deletions of Wnt/PCP signaling components [2,29], including ligands such as Wnt5a [4], have been studied in very specific structures that have become standard Wnt/PCP functional assays. These include studying the orientation of bristles on the Drosophila wing, and in mammals the orientation of hair cells in the inner ear, convergent extension movements during embryo elongation, or neural tube closure. However, Wnt/PCP signaling components and ligands are expressed in very diverse tissues and at multiple developmental stages, where no previous PCP phenotypes have been described. In our study we investigated the function of Wnt5a in one such structure and developmental time, the VM during neurogenesis. We report that Wnt5a regulates VM morphogenesis, limits DA progenitor proliferation and enhances DA precursor differentiation. Regulation of DA precursor differentiation by Wnt5a Despite the clear effects of Wnt5a on DA differentiation in both gain and loss of function experiments in vitro [32,54], Wnt5a2/2 mice exhibited only a mild and transient DA differentiation phenotype in vivo. Moreover, in addition to a decrease in the proportion of NR4A2+ precursors differentiating into TH+ DA neurons (25% decrease in the Wnt5a mutants compared to WT), PLoS ONE | www.plosone.org 7 October 2008 | Volume 3 | Issue 10 | e3517