Gpr125 modulates Dishevelled distribution and planar cell polarity signaling

Xin Li 1 2 Isabelle Roszko 0 Diane S. Sepich 0 Mingwei Ni 5 Heidi E. Hamm 2 4 Florence L. Marlow ( 1 Lilianna Solnica-Krezel 0 2 3 0 Department of Developmental Biology, Washington University School of Medicine , St Louis, MO 63110 , USA 1 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine , Bronx, NY 10461 , USA 2 Neuroscience Graduate Program, Vanderbilt University School of Medicine , Nashville, TN 37232 , USA 3 Department of Biological Sciences, Vanderbilt University , Nashville, TN 37232 , USA 4 Department of Pharmacology, Vanderbilt University School of Medicine , Nashville, TN 37232 , USA 5 Department of Surgery, New York Hospital Medical Center of Queens , Flushing, NY 11355 , USA SUMMARY During vertebrate gastrulation, Wnt/planar cell polarity (PCP) signaling orchestrates polarized cell behaviors underlying convergence and extension (C&E) movements to narrow embryonic tissues mediolaterally and lengthen them anteroposteriorly. Here, we have identified Gpr125, an adhesion G protein-coupled receptor, as a novel modulator of the Wnt/PCP signaling system. Excess Gpr125 impaired C&E movements and the underlying cell and molecular polarities. Reduced Gpr125 function exacerbated the C&E and facial branchiomotor neuron (FBMN) migration defects of embryos with reduced Wnt/PCP signaling. At the molecular level, Gpr125 recruited Dishevelled to the cell membrane, a prerequisite for Wnt/PCP activation. Moreover, Gpr125 and Dvl mutually clustered one another to form discrete membrane subdomains, and the Gpr125 intracellular domain directly interacted with Dvl in pull-down assays. Intriguingly, Dvl and Gpr125 were able to recruit a subset of PCP components into membrane subdomains, suggesting that Gpr125 may modulate the composition of Wnt/PCP membrane complexes. Our study reveals a role for Gpr125 in PCP-mediated processes and provides mechanistic insight into Wnt/PCP signaling. INTRODUCTION During embryogenesis, gastrulation establishes the three germ layers and the animal body plan. Vertebrate gastrulation relies on polarized cell behaviors to drive convergence and extension (C&E) movements that narrow embryonic tissues mediolaterally and elongate them anteroposteriorly (Keller et al., 2000; Solnica-Krezel, 2005; Yin et al., 2009; Gray et al., 2011). In dorsal regions of Xenopus and zebrafish gastrulae, cells become elongated and align along the mediolateral embryonic axis, allowing preferential intercalation between their anterior and posterior neighbors to drive C&E (Keller et al., 2000; Topczewski et al., 2001; Jessen et al., 2002; Marlow et al., 2002; Lin et al., 2005). Modulation of cell adhesion and intercellular signaling have been proposed to instruct such complex cell behaviors (Yin et al., 2009). However, the molecules implementing these actions have not been fully identified. Currently, the Wnt/PCP signaling system, which is equivalent to the PCP pathway coordinating wing hair and ommatidia orientation in Drosophila (Simons and Mlodzik, 2008; Goodrich and Strutt, 2011), is the best-studied pathway regulating C&E movements in vertebrates (Tada and Kai, 2009; Yin et al., 2009; Gray et al., 2011). Polarized cell behaviors that underlie C&E, including directed cell migration and polarized planar and radial intercalations, are exquisitely sensitive to PCP signaling levels, as excess or insufficient Wnt/PCP pathway component function impairs C&E movements (Wallingford et al., 2000; Jessen et al., 2002; Marlow et al., 2002; Carreira-Barbosa et al., 2003). In addition to regulating C&E, a subset of Wnt/PCP components also regulates the caudal tangential migration of facial branchiomotor neurons (FBMN) in zebrafish and mouse (Jessen et al., 2002; Carreira-Barbosa et al., 2003; Wada et al., 2005; Wada et al., 2006; Wada and Okamoto, 2009). PCP pathway components are known to localize asymmetrically in multiple tissues that manifest planar polarity. In the fly wing epithelia, the receptor Frizzled and cytoplasmic proteins Dishevelled (Dsh/Dvl in vertebrates) and Diego localize to the distal side of the cell, where the wing hair will eventually emerge. By contrast, the transmembrane protein Van gogh/Strabismus and cytoplasmic protein Prickle (Pk) localize proximally, whereas the seven transmembrane protocadherin Flamingo/Starry night is present at both cell edges (Axelrod, 2001; Feiguin et al., 2001; Strutt et al., 2002; Tree et al., 2002; Bastock et al., 2003). This stereotyped asymmetric localization of Pk and Dvl on opposing anterior and posterior membranes has been observed in the neural plate and dorsal mesodermal cells undergoing C&E in zebrafish (Ciruna et al., 2006; Yin et al., 2008). Such molecular asymmetries are considered to be either a consequence of cell polarization or an essential step in the process of Wnt/PCP-mediated cell polarization (Simons and Mlodzik, 2008; McNeill, 2010; Goodrich and Strutt, 2011; Gray et al., 2011). Asymmetric localization of PCP components in polarized epithelia and protein interaction studies supports a model whereby PCP components interact in asymmetric membrane complexes spanning the juxtaposed cells to generate planar polarization (McNeill, 2010; Goodrich and Strutt, 2011). Recently, Dsh was shown to cluster PCP complexes into membrane subdomains in cells of Drosophila pupal wings (Strutt et al., 2011), raising the possibility that clustering of asymmetric PCP complexes into membrane subdomains might provide a local self-enhancement mechanism that establishes and/or maintains planar polarity (Strutt et al., 2011). Interestingly, membrane clustering of PCP components occurs between Xenopus Van gogh-like 2 (Vangl2, vertebrate homolog of Van gogh/Strabismus) and Drosophila Pk expressed in Xenopus animal cap explants, and among zebrafish Frizzled7 (Fzd7), Wnt11 and Xenopus Dvl expressed in zebrafish blastula (Jenny et al., 2003; Witzel et al., 2006). In the latter case, subdomain formation correlates with increased persistence of membrane contacts partially dependent on vertebrate Flamingo homologues, Cadherin EGF LAG seven-pass G-type receptors (Celsrs) (Witzel et al., 2006). Celsrs belong to the family of adhesion G protein-coupled receptors (GPCRs), which are chimeras of adhesion molecules and transmembrane signal transducer GPCRs (Fredriksson et al., 2003). Owing to their unique structure, adhesion GPCRs are postulated to play dual roles in cell adhesion and signal transduction (Yona et al., 2008). Recent studies of GPR56, GPR124 and Gpr126 implicate adhesion GPCRs in diverse developmental processes, including brain development, blood vessel formation and myelination in zebrafish and mammals (Kuhnert et al., 2010; Piao et al., 2004; Monk et al., 2009; Monk et al., 2011). As components of the PCP pathway, Celsr adhesion GPCRs have been reported to regulate zebrafish gastrulation and FBMN migration (Formstone and Mason, 2005; Wada et al., 2006; Carreira-Barbosa et al., 2009). To bet (...truncated)