Wnt5a Regulates Ventral Midbrain Morphogenesis and the Development of A9–A10 Dopaminergic Cells In Vivo Emma R. Andersson 1 , Nilima Prakash2, Lukas Cajanek1., Eleonora Minina2., Vitezslav Bryja1,3,4, Lenka Bryjova1,3,4, Terry P. Yamaguchi5, Anita C. Hall6, Wolfgang Wurst2,7.*, Ernest Arenas1.* 1 Laboratory of Molecular Neurobiology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet, Stockholm, Sweden, 2 Helmholtz Centre Munich, German Research Centre for Environmental Health, and Technical University Munich, Institute of Developmental Genetics, Munich/Neuherberg, Germany, 3 Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic, 4 Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic, 5 Cancer and Developmental Biology Laboratory, National Cancer Institute-Frederick, Frederick, Maryland, United States of America, 6 Division of Cell and Molecular Biology, Imperial College London, London, United Kingdom, 7 Max-Planck-Institute of Psychiatry, Munich, Germany Abstract Wnt5a is a morphogen that activates the Wnt/planar cell polarity (PCP) pathway and serves multiple functions during development. PCP signaling controls the orientation of cells within an epithelial plane as well as convergent extension (CE) movements. Wnt5a was previously reported to promote differentiation of A9–10 dopaminergic (DA) precursors in vitro. However, the signaling mechanism in DA cells and the function of Wnt5a during midbrain development in vivo remains unclear. We hereby report that Wnt5a activated the GTPase Rac1 in DA cells and that Rac1 inhibitors blocked the Wnt5a- induced DA neuron differentiation of ventral midbrain (VM) precursor cultures, linking Wnt5a-induced differentiation with a known effector of Wnt/PCP signaling. In vivo, Wnt5a was expressed throughout the VM at embryonic day (E)9.5, and was restricted to the VM floor and basal plate by E11.5–E13.5. Analysis of Wnt5a2/2 mice revealed a transient increase in progenitor proliferation at E11.5, and a precociously induced NR4A2+ (Nurr1) precursor pool at E12.5. The excess NR4A2+ precursors remained undifferentiated until E14.5, when a transient 25% increase in DA neurons was detected. Wnt5a2/2 mice also displayed a defect in (mid)brain morphogenesis, including an impairment in midbrain elongation and a rounded ventricular cavity. Interestingly, these alterations affected mostly cells in the DA lineage. The ventral Sonic hedgehog- expressing domain was broadened and flattened, a typical CE phenotype, and the domains occupied by Ngn2+ DA progenitors, NR4A2+ DA precursors and TH+ DA neurons were rostrocaudally reduced and laterally expanded. In summary, we hereby describe a Wnt5a regulation of Wnt/PCP signaling in the DA lineage and provide evidence for multiple functions of Wnt5a in the VM in vivo, including the regulation of VM morphogenesis, DA progenitor cell division, and differentiation of NR4A2+ DA precursors. Citation: Andersson ER, Prakash N, Cajanek L, Minina E, Bryja V, et al. (2008) Wnt5a Regulates Ventral Midbrain Morphogenesis and the Development of A9–A10 Dopaminergic Cells In Vivo. PLoS ONE 3(10): e3517. doi:10.1371/journal.pone.0003517 Editor: Patrick Callaerts, Katholieke Universiteit Leuven, Belgium Received July 9, 2008; Accepted September 16, 2008; Published October 27, 2008 Copyright: 2008 Andersson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Swedish Foundation for Strategic Research, Swedish Royal Academy of Sciences, Knut and Alice Wallenberg Foundation, European Union (Eurostemcell), Swedish MRC and Karolinska Institutet (to EA), and by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN), Fo¨ rderkennzeichen 01GS0476, the BMBF Fo¨ rderkennzeichen 01GN0512, the Deutsche Forschungsgemeinschaft (DFG) WU 164/3-1 and WU 164/3-2, the Bayerische Forschungsverbund ForNeuroCell, the Helmholtz Virtual Institute of Neurodegeneration&Aging (VH-VI-252) and by European Union (Eumorphia) (to WW). The authors are responsible for the contents of this publication. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wurst@helmholtz-muenchen.de (WW); ernest.arenas@ki.se (EA) . These authors contributed equally to this work. Introduction Wnts comprise a family of 19 lipid-modified secreted glycopro- teins that signal via different pathways and regulate multiple aspects of development [1,2]. These pathways include the canonical Wnt/b-catenin, noncanonical Wnt/Ca2+ and noncano- nical Wnt/planar cell polarity (PCP) pathways. Wnt5a has been reported to activate both canonical and noncanonical signaling depending on receptor, cellular and tissue context [3–5]. However, Wnt5a is generally considered a noncanonical Wnt that activates PCP or Ca2+ signaling [6]. Most PCP genes were initially identified in Drosophila or Xenopus and their homologues were subsequently found in mammals. These include genes for transmembrane proteins, such as frizzled (fz/Fz) [7–9], Van Gogh/Strabismus (Vang/Vangl/stbm) [10,11], and starry night/flamingo/Celsr (stan/fmi/Celsr) [12,13]. Some cytoplasmic components of this pathway are shared with the Wnt/b-catenin pathway, such as Dishevelled (dsh/Dvl) [14,15] and Casein kinase 1 (Ck1) [16–18]. Specific Wnt/PCP cytoplasmic components include Daam, small GTPases of the Rho family: Cdc42, Rac1, RhoA, the Rho kinase and JNK [19–22]. Mutations in PCP genes produce specific and distinctive phenotypes. These include the general convergent extension (CE) defects seen in the overall shortened and broadened morphology of mutants and in Keller explants of the Xenopus dorsal marginal zone [23,24], the Drosophila wing with misdirected bristles and disorganization of the compound eye [2,25], the murine cochlea with misdirected hair cells [4,26], and the murine PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3517