Frizzled3 and Frizzled6 Cooperate with Vangl2 to Direct Cochlear Innervation by Type II Spiral Ganglion Neurons

The Journal of Neuroscience, October 9, 2019 • 39(41):8013–8023 • 8013 Development/Plasticity/Repair Frizzled3 and Frizzled6 Cooperate with Vangl2 to Direct Cochlear Innervation by Type II Spiral Ganglion Neurons Satish R. Ghimire1 and Michael R. Deans1,2 1Department of Neurobiology & Anatomy, University of Utah School of Medicine, Salt Lake City, Utah 84132, and 2Department of Surgery, Division of Otolaryngology, University of Utah School of Medicine, Salt Lake City, Utah 84112 Type II spiral ganglion neurons provide afferent innervation to outer hair cells of the cochlea and are proposed to have nociceptive functions important for auditory function and homeostasis. These neurons are anatomically distinct from other classes of spiral ganglion neurons because they extend a peripheral axon beyond the inner hair cells that subsequently makes a distinct 90 degree turn toward the cochlear base. As a result, patterns of outer hair cell innervation are coordinated with the tonotopic organization of the cochlea. Previ- ously, it was shown that peripheral axon turning is directed by a nonautonomous function of the core planar cell polarity (PCP) protein VANGL2. We demonstrate using mice of either sex that Fzd3 and Fzd6 similarly regulate axon turning, are functionally redundant with each other, and that Fzd3 genetically interacts with Vangl2 to guide this process. FZD3 and FZD6 proteins are asymmetrically distributed along the basolateral wall of cochlear-supporting cells, and are required to promote or maintain the asymmetric distribution of VANGL2 and CELSR1. These data indicate that intact PCP complexes formed between cochlear-supporting cells are required for the nonautonomous regulation of axon pathfinding. Consistent with this, in the absence of PCP signaling, peripheral axons turn randomly and often project toward the cochlear apex. Additional analyses of Porcn mutants in which WNT secretion is reduced suggest that noncanonical WNT signaling establishes or maintains PCP signaling in this context. A deeper understanding of these mechanisms is necessary for repairing auditory circuits following acoustic trauma or promoting cochlear reinnervation during regeneration- based deafness therapies. Key words: auditory; axon; cochlea; frizzled; planar cell polarity; spiral ganglia Significance Statement Planar cell polarity (PCP) signaling has emerged as a complementary mechanism to classical axon guidance in regulating axon track formation, axon outgrowth, and neuronal polarization. The core PCP proteins are also required for auditory circuit assem- bly, and coordinate hair cell innervation with the tonotopic organization of the cochlea. This is a non–cell-autonomous mechanism that requires the formation of PCP protein complexes between cochlear-supporting cells located along the trajectory of growth cone navigation. These findings are significant because they demonstrate how the fidelity of auditory circuit formation is ensured during development, and provide a mechanism by which PCP proteins may regulate axon outgrowth and guidance in the CNS. Introduction outgrowth and guidance, and the establishment of planar polar- Intercellular signals relayed by planar cell polarity (PCP) proteins ity (Zou, 2004; Wang and Nathans, 2007; Goodrich and Strutt, contribute to an array of developmental events, including axon 2011; Tissir and Goffinet, 2013). In the murine cochlea, planar polarity is guided by an intercellular signaling complex com- Received July 20, 2019; revised Aug. 20, 2019; accepted Aug. 23, 2019. posed of the PCP proteins Frizzled (FZD3/6) (Wang et al., 2006), Author contributions: S.R.G. and M.R.D. designed research; S.R.G. and M.R.D. performed research; S.R.G. wrote Van Gogh-like (VANGL1/2) (Montcouquiol et al., 2003; Torban the first draft of the paper; M.R.D. edited the paper; M.R.D. wrote the paper. et al., 2008), and CELSR1 (Curtin et al., 2003). Complex forma- This work was supported by National Institute on Deafness and Other Communication Disorders Grant tion requires FZD3/6 and VANGL1/2 localization at opposite R21DC015635 and University of Utah Research Incentive Seed Grant. We thank Shinichi Aizawa, Andrew Groves, Charles Murtaugh, Rejji Kuruvilla, and Jeremy Nathans for providing mouse lines used in this study; and Orvelin sides of individual cells so that their extracellular domains appose Roman Jr and Sarah Siddoway for assistance in animal colony management and genotyping. each other at junctions between neighbors, and this complex is The authors declare no competing financial interests. maintained by homophilic CELSR1 binding across intercellular Correspondence should be addressed to Michael R. Deans at [email protected]. https://doi.org/10.1523/JNEUROSCI.1740-19.2019 space. In Drosophila, the analogous complex relays polarity infor- Copyright © 2019 the authors mation through Fzd to Van Gogh signaling (Wu and Mlodzik, 8014 • J. Neurosci., October 9, 2019 • 39(41):8013–8023 Ghimire and Deans • Frizzled Proteins Guide Cochlear Innervation 2008) or homophilic signaling by the CELSR1 ortholog Flamin- of analyses of Porcn mutants because the Porcn gene is located on the Ϫ Ϫ Ϫ Ϫ go/Starry Night (Struhl et al., 2012). Vertebrate PCP proteins X-chromosome. Fzd3 / ;Fzd6 / double mutants and littermate con- ϩ/Ϫ ϩ/Ϫ similarly relay polarity information within epithelia (Mitchell et trols were generated by intercrossing Fzd3 ;Fzd6 males and fe- al., 2009; Sienknecht et al., 2011; Stoller et al., 2018). While mech- males derived from the Fzd3 and Fzd6 KO lines. Emx2-Cre;Fzd3 CKOs and Cre-positive littermate controls were generated on a Fzd6 KO back- anisms initiating PCP may vary, an emerging consensus for ver- ground by crossing Emx2-Cre;Fzd3 ϩ/Ϫ;Fzd6ϩ/Ϫ males with Fzd3 floxed; tebrates is that noncanonical WNT signaling promotes initial Fzd6Ϫ/Ϫ females. Pax2-Cre ϩ;Vangl2 CKOs and Cre-positive littermate protein asymmetries that are relayed between neighbors by the controls were generated on a Fzd3 KO background by crossing Pax2-Cre; PCP complex (Yang, 2012; Yang and Mlodzik, 2015). Vangl2ϩ/Ϫ;Fzd3ϩ/Ϫ male mice with Vangl2floxed;Fzd3ϩ/Ϫ females. Pax2- PCP signaling is not restricted to epithelia, and PCP proteins Cre;ROR2;Vangl2 CKOs and Cre-positive littermate controls were generated by crossing Pax2-Cre ϩ;Vangl2ϩ/Ϫ;ROR2LoxP/ϩ male mice contribute to axon outgrowth (Fenstermaker et al., 2010; Morello floxed floxed ϩ 3XHA/ϩ et al., 2015), pathfinding (Lyuksyutova et al., 2003; Shafer et al., with Vangl2 ;ROR2 female mice. Pax2-Cre ;Fzd3 tissue was generated for the study of transgenic FZD3 protein localization by 2011; Hua et al., 2013; Onishi et al., 2013), and neuronal migra- ϩ ϩ crossing Pax2-Cre male mice with Fzd33XHA/ female mice. The Wnt5a tion (Qu et al., 2010; Glasco et al., 2012; Sittaramane et al., 2013; KO allele was generated by crossing Wnt5afloxed mice (Ryu et al., 2013) Davey et al., 2016). Phenotypic analyses suggest that different with CMV-Cre (Schwenk et al., 1995) to delete coding sequence exon 2 molecular mechanisms are used for these processes in different and then backcrossed with B6129SF1/J hybrid mice to obtain Cre- developmental contexts. For example, commissural neurons ex- negative Wnt5a heterozygotes. Wnt5a ϩ/ Ϫ males and females were inter- tend an axon across the ventral midline that turns anteriorly in crossed to generated Wnt5a KOs. Pax2-Cre;Porcn CKOs were generated response to a WNT gradient (Zou, 2004; Onishi and Zou, 2017). by crossing Pax2-Cre ϩ male mice with Porcn LoxP/WT female mice. Since In the absence of Vangl2 (Shafer et al., 2011), Frizzled3 (Lyuksyu- the Porcn gene is located on the X-chromosome, the analysis was re- ϩ Loxp/Y tova et al., 2003), or Celsr3 (Onishi et al., 2013), commissural stricted to males with Pax2-Cre ;Porcn embryos used as experi- ϩ; WT/Y axons cross the midline but turn incorrectly. FZD and VANGL2 mental and Pax2-Cre Porcn embryos as littermate controls. Male regulation of filopodial dynamics in the growth cone (Onishi et pups were identified by genotyping for the male-specific Sry allele. Emx2- Cre mice (Kimura et al., 2005) were provided by S. Aizawa (Riken Insti- al., 2013) indicates that this is a cell-autonomous mechanism. In tute). Fzd3 KO (Wang et al., 2002), Fzd 6 KO (Guo et al., 2004), and Fzd3 contrast, VANGL2 acts non–cell-autonomously to guide the pe- CKO (Hua et al., 2013) mice were provided by J. Nathans (John Hopkins ripheral axon of Type II spiral ganglion neurons (SGNs) in the University). Pax2-Cre mice (Ohyama and Groves, 2004) were provided cochlea (Ghimire et al., 2018). FZD3 and CELSR also act non– by A. Groves (Baylor College of Medicine). Porcn CKO mice (Barrott et cell-autonomously to promote the formation of long axon tracks al., 2011) were provided by C. Murtaugh (University of Utah). Wnt5a (Morello et al., 2015; Feng et al., 2016); however, this does not CKO line (Ryu et al., 2013) was provided by R. Kuruvilla (John Hopkins require VANGL1/2 (Qu et al., 2014) and suggests that an intact University). Generation of Vangl2floxed and Vangl2 KO lines has been intercellular PCP complex is not required for all axon guidance described previously (Yin et al., 2012; Copley et al., 2013). B6129SF1/J hybrid (strain #101043), CMV-Cre (strain #006054) (Schwenk et al., events. These analyses demonstrate that PCP proteins contribute 3XHA/ϩ to multiple guidance events but suggest they act through mecha- 1995), Fzd3 (strain #023841 (Hua et al., 2014b), and ROR2 mice (strain #018354) (Ho et al., 2012) were purchased from The Jackson nisms that differ from PCP signaling in epithelia. Laboratory. In the developing cochlea, VANGL2 ensures that the periph- Immunofluorescence. Inner ear tissues were fixed using 4% PFA in 67 eral axons of Type II SGNs complete a 90° turn toward the co- mM Sorensons’ phosphate buffer, pH 7.4, for2honice. For whole- chlear base before innervating the outer hair cells (OHCs) mount immunofluorescent labeling, cochlea were microdissected and (Ghimire et al., 2018).

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