Neural crest specification by noncanonical Wnt signaling and PAR-1

Olga Ossipova 0 Sergei Y. Sokol () 0 0 Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine , New York, NY 10029 , USA SUMMARY Neural crest (NC) cells are multipotent progenitors that form at the neural plate border, undergo epithelial-mesenchymal transition and migrate to diverse locations in vertebrate embryos to give rise to many cell types. Multiple signaling factors, including Wnt proteins, operate during early embryonic development to induce the NC cell fate. Whereas the requirement for the Wnt/b-catenin pathway in NC specification has been well established, a similar role for Wnt proteins that do not stabilize bcatenin has remained unclear. Our gain- and loss-of-function experiments implicate Wnt11-like proteins in NC specification in Xenopus embryos. In support of this conclusion, modulation of b-catenin-independent signaling through Dishevelled and Ror2 causes predictable changes in premigratory NC. Morpholino-mediated depletion experiments suggest that Wnt11R, a Wnt protein that is expressed in neuroectoderm adjacent to the NC territory, is required for NC formation. Wnt11-like signals might specify NC by altering the localization and activity of the serine/threonine polarity kinase PAR-1 (also known as microtubule-associated regulatory kinase or MARK), which itself plays an essential role in NC formation. Consistent with this model, PAR-1 RNA rescues NC markers in embryos in which noncanonical Wnt signaling has been blocked. These experiments identify novel roles for Wnt11R and PAR-1 in NC specification and reveal an unexpected connection between morphogenesis and cell fate. INTRODUCTION The neural crest (NC) comprises stem-cell-like cells that form in vertebrate embryos at the neural plate border, migrate to diverse locations in the body and differentiate into multiple cell types (Anderson, 1997; Crane and Trainor, 2006; Knight and Schilling, 2006; Le Douarin and Dupin, 2003; Sauka-Spengler and BronnerFraser, 2008). NC is specified by the combined action of several embryonic signaling pathways, including the Wnt, FGF, BMP and Notch pathways, and NC fates are maintained by a network of specific transcription factors. Once formed, NC cells undergo epithelial-mesenchymal transition (EMT) and migrate to many destinations in the body to contribute to diverse cell types, including face cartilage, melanocytes and the peripheral nervous system (Acloque et al., 2009; Heeg-Truesdell and LaBonne, 2004; Kuriyama and Mayor, 2008; Thiery et al., 2009; Yang and Weinberg, 2008). The large number of human diseases that are associated with NC abnormalities, including craniosynostosis, Waardenburg and Hirschsprungs syndromes and cancers, draw considerable attention to studies of the mechanisms of NC development (Crane and Trainor, 2006; Heeg-Truesdell and LaBonne, 2004). One pathway that is essential for NC specification in all vertebrate models examined is the Wnt pathway. Canonical Wnt signaling triggers b-catenin/TCF-dependent gene transcription and regulates cell proliferation and cell fate (Cadigan and Peifer, 2009; Clevers, 2006). The involvement of this pathway in NC formation was first established by genetic studies of Wnt1/Wnt3a doubleknockout mice and in gain-of-function experiments in Xenopus (Ikeya et al., 1997; Saint-Jeannet et al., 1997), and was subsequently extended to other models (Dorsky et al., 1998; Garcia-Castro et al., 2002; Hari et al., 2002; Lewis et al., 2004; Wu et al., 2003). The transcription of many NC-specific genes, including Snail2, Snail and Twist, has been shown to depend on bcatenin/TCF (Garcia-Castro et al., 2002; Howe et al., 2003; LaBonne, 2002; Sauka-Spengler and Bronner-Fraser, 2008; Vallin et al., 2001; Wu et al., 2003), further supporting the model that NC formation involves the Wnt/b-catenin pathway. Noncanonical Wnt ligands, such as Wnt5a and Wnt11 (Angers and Moon, 2009; van Amerongen and Nusse, 2009), do not stabilize b-catenin or activate TCF-dependent transcription, but regulate morphogenetic processes that involve changes in cell shape and motility, which are sometimes referred to as planar cell polarity (PCP) (Ciani and Salinas, 2005; Komiya and Habas, 2008; Saneyoshi et al., 2002; van Amerongen et al., 2008; Winklbauer et al., 2001). The signaling from Wnt5 or Wnt11 is thought to involve Ror and Ryk receptors (Grumolato et al., 2010; Hikasa et al., 2002a; Lin et al., 2010; Lu et al., 2004; Mikels et al., 2009; Minami et al., 2010), small Rho GTPases (Habas et al., 2003; Habas et al., 2001), Rho-associated kinase (Marlow et al., 2002; Winter et al., 2001), c-Jun N-terminal kinases (Boutros et al., 1998; Lisovsky et al., 2002; Pandur et al., 2002) and intracellular calcium (Sheldahl et al., 2003; Slusarski et al., 1997; Witze et al., 2008). Although noncanonical Wnt pathways have been shown to function in NC cell migration (Carmona-Fontaine et al., 2008; De Calisto et al., 2005; Matthews et al., 2008b), their importance for NC specification has remained unclear. Craniofacial defects in Wnt5a knockout mice (Yamaguchi et al., 1999), and in wnt11 (silberblick) (Heisenberg et al., 2000; Heisenberg et al., 1996) and wnt5 (pipetail) (Piotrowski et al., 1996) zebrafish mutant embryos suggest possible roles for noncanonical Wnt signaling in NC development. The results of our study support the view that noncanonical signaling from Wnt11R is essential for NC specification in Xenopus embryos and that it might act by changing the localization and activity of the polarity kinase PAR-1. PAR proteins are conserved regulators of cell polarity that interact with several embryonic signaling pathways, including the Wnt pathway (Doe and Bowerman, 2001; Goldstein and Macara, 2007; Knoblich, 2008; Ohno, 2001). PAR-1 associates with Dishevelled (Dvl, or Dsh) and participates in Frizzled-dependent Dvl recruitment (Ossipova et al., 2005; Sun et al., 2001). We show that PAR-1 is itself required for NC specification and can rescue NC defects in embryos with inhibited Wnt5 and Wnt11 signaling. These findings identify PAR-1 as a molecular target for noncanonical Wnt signaling and reveal an unexpected causal connection between cell polarization and the NC cell fate. MATERIALS AND METHODS DNA constructs and RNA synthesis pCS2-Myc-PAR-1A, pCS2-Myc-PAR-1KD and GFP-PAR-1A in pXT7 have been described (Ossipova et al., 2005; Ossipova et al., 2007). FlagPAR-1A and Flag-PAR-1A-KD have been generated by subcloning the PAR-1 coding region into the XhoI and NotI sites of pCS2-Flag (Hikasa and Sokol, 2011). Capped synthetic RNA for microinjection was generated using the mMessage mMachine Kit (Ambion) from the following DNA templates: pCS2-Myc-PAR-1A, pCS2-nucbGal (Ossipova et al., 2005), pCS2-Ror2 and pCS2-Ror2 C (Hikasa et al., 2002b), pSP64-XWnt11, pSP64-dnWnt11, pCS2-N-Dsh, pCS2-DshDEP+ (Tada and Smith, 2000), pXT7-dnWnt5/11 (Choi and Sokol, 2009), pSP64T-XWnt5a (Moon et al., 1993), pSP64T-Xfz3 (Shi et al., 1998), pSP64T-Xwn (...truncated)