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

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

Research Articles: Development/Plasticity/Repair Frizzled3 and Frizzled6 Cooperate with Vangl2 to Direct Cochlear Innervation by type II Spiral Ganglion Neurons https://doi.org/10.1523/JNEUROSCI.1740-19.2019 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1740-19.2019 Received: 20 July 2019 Revised: 20 August 2019 Accepted: 23 August 2019 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2019 the authors 1 Frizzled3 and Frizzled6 Cooperate with Vangl2 to Direct Cochlear Innervation by type II Spiral 2 Ganglion Neurons 3 4 Abbreviated title: Frizzled proteins guide cochlear innervation 5 6 Satish R. Ghimire1 and Michael R. Deans1,2 # 7 8 1 Department of Neurobiology & Anatomy, University of Utah School of Medicine, Salt Lake City, UT, 9 84132, USA 10 2 Department of Surgery, Division of Otolaryngology, University of Utah School of Medicine, Salt Lake 11 City, UT, 84112, USA 12 13 # Correspondence should be addressed to MRD at [email protected] 14 15 16 33 pages 17 9 figures 18 250 word abstract 19 109 word significance statement 20 648 word introduction 21 1482 word discussion 22 23 Conflict of Interest Statement: 24 The authors declare no competing financial interests. 25 26 Acknowledgements: 27 We thank Shinichi Aizawa, Andrew Groves, Charles Murtaugh, Rejji Kuruvilla and Jeremy Nathans for 28 providing mouse lines used in this study, and Orvelin Roman Jr. and Sarah Siddoway for assistance in 29 animal colony management and genotyping. This research was sponsored by the National Institute on 30 Deafness and Other Communication Disorders (NIDCD) grant R21DC015635 and a University of Utah 31 Research Incentive Seed Grant. 1 32 ABSTRACT 33 34 Type II spiral ganglion neurons provide afferent innervation to outer hair cells of the cochlea and are 35 proposed to have nociceptive functions important for auditory function and homeostasis. These 36 neurons are anatomically distinct from other classes of spiral ganglion neurons because they extend a 37 peripheral axon beyond the inner hair cells that subsequently makes a distinct 90 degree turn towards 38 the cochlear base. As a result, patterns of outer hair cell innervation are coordinated with the tonotopic 39 organization of the cochlea. Previously it was shown that peripheral axon turning is directed by a non- 40 autonomous function of the core PCP protein VANGL2. We demonstrate using mice of either sex that 41 Fzd3 and Fzd6 similarly regulate axon turning, are functionally redundant with each other, and that 42 Fzd3 genetically interacts with Vangl2 to guide this process. FZD3 and FZD6 proteins are 43 asymmetrically distributed along the basolateral wall of cochlear supporting cells, and are required to 44 promote or maintain the asymmetric distribution of VANGL2 and CELSR1. These data indicate that 45 intact PCP complexes formed between cochlear supporting cells are required for the non-autonomous 46 regulation of axon pathfinding. Consistent with this, in the absence of PCP signaling peripheral axons 47 turn randomly and often project towards the cochlear apex. Additional analyses of Porcn mutants in 48 which WNT secretion is reduced suggest that non-canonical WNT signaling establishes or maintains 49 PCP signaling in this context. A deeper understanding of these mechanisms is necessary for repairing 50 auditory circuits following acoustic trauma or promoting cochlear re-innervation during regeneration- 51 based deafness therapies. 52 53 SIGNIFICANCE STATEMENT 54 Planar Cell Polarity (PCP) signaling has emerged as a complementary mechanism to classical axon 55 guidance in regulating axon track formation, axon outgrowth and neuronal polarization. The core PCP 56 proteins are also required for auditory circuit assembly, and coordinate hair cell innervation with the 57 tonotopic organization of the cochlea. This is a non-cell autonomous mechanism that requires the 2 58 formation of PCP protein complexes between cochlear supporting cells located along the trajectory of 59 growth cone navigation. These findings are significant because they demonstrate how the fidelity of 60 auditory circuit formation is ensured during development, and provide a mechanism by which PCP 61 proteins may regulate axon outgrowth and guidance in the CNS. 62 63 INTRODUCTION 64 Intercellular signals relayed by Planar Cell Polarity (PCP) proteins contribute to an array of 65 developmental events including axon outgrowth and guidance, and the establishment of planar polarity 66 (Zou, 2004; Wang and Nathans, 2007; Goodrich and Strutt, 2011; Tissir and Goffinet, 2013). In the 67 murine cochlea, planar polarity is guided by an intercellular signaling complex composed of the PCP 68 proteins Frizzled (FZD3/6) (Wang et al., 2006), Van Gogh-like (VANGL1/2) (Montcouquiol et al., 2003; 69 Torban et al., 2008), and CELSR1 (Curtin et al., 2003). Complex formation requires FZD3/6 and 70 VANGL1/2 localization at opposite sides of individual cells so that their extracellular domains appose 71 each other at junctions between neighbors, and this complex is maintained by homophilic CELSR1 72 binding across intercellular space. In Drosophila, the analogous complex relays polarity information 73 through Fzd to Van Gogh signaling (Wu and Mlodzik, 2008) or homophilic signaling by the CELSR1 74 ortholog Flamingo/Starry Night (Struhl et al., 2012). Vertebrate PCP proteins similarly relay polarity 75 information within epithelia (Mitchell et al., 2009; Sienknecht et al., 2011; Stoller et al., 2018). While 76 mechanisms initiating PCP may vary, an emerging consensus for vertebrates is that non-canonical 77 WNT signaling promotes initial protein asymmetries that are relayed between neighbors by the PCP 78 complex (Yang, 2012; Yang and Mlodzik, 2015). 79 80 PCP signaling is not restricted to epithelia, and PCP proteins contribute to axon outgrowth 81 (Fenstermaker et al., 2010; Morello et al., 2015), pathfinding (Lyuksyutova et al., 2003; Shafer et al., 82 2011; Hua et al., 2013; Onishi et al., 2013), and neuronal migration (Qu et al., 2010; Glasco et al., 83 2012; Sittaramane et al., 2013; Davey et al., 2016). Phenotypic analyses suggest that different 3 84 molecular mechanisms are employed for these processes in different developmental contexts. For 85 example, commissural neurons extend an axon across the ventral midline that turns anteriorly in 86 response to a WNT gradient (Zou, 2004; Onishi and Zou, 2017). In the absence of Vangl2 (Shafer et 87 al., 2011), Frizzled3 (Lyuksyutova et al., 2003) or Celsr3 (Onishi et al., 2013) commissural axons cross 88 the midline but turn incorrectly. FZD and VANGL2 regulation of filopodial dynamics in the growth cone 89 (Onishi et al., 2013) indicate that this is a cell autonomous mechanism. In contrast, VANGL2 acts non- 90 cell autonomously to guide the peripheral axon of type II Spiral Ganglion Neurons (SGN) in the cochlea 91 (Ghimire et al., 2018). FZD3 and CELSR also act non-cell autonomously to promote the formation of 92 long axon tracks (Morello et al., 2015; Feng et al., 2016) however, this does not require VANGL1/2 (Qu 93 et al., 2014) and suggests that an intact intercellular PCP complex is not required for all axon guidance 94 events. These analyses demonstrate that PCP proteins contribute to multiple guidance events, but 95 suggest they act through mechanisms that differ from PCP signaling in epithelia. 96 97 In the developing cochlea, VANGL2 ensures that the peripheral axons of type II SGNs complete a 90° 98 turn towards the cochlear base before innervating the outer hair cells (OHC) (Ghimire et al., 2018). 99 SGNs are bipolar relay neurons that convey auditory information and consist of two major sub-types. 100 Type I SGNs innervate inner hair cells (IHC), comprise 90-95% of the SGN population, and transmit the 101 salient features of sound. Type II SGNs comprise the remainder and are thought to be neuroprotective 102 rather than sensory because they resemble nociceptive c-fibers (Flores et al., 2015; Liu et al., 2015; 103 Maison et al., 2016). Peripheral axon turning is dependent upon VANGL2 and CELSR1 and many 104 fibers turn incorrectly towards the cochlear apex in these mutants. Here we demonstrate and that Fzd3 105 and Vangl2 act in a common pathway to guide axon turning and that Fzd3 and Fzd6 are functionally 106 redundant. Reducing WNT signaling also disrupts turning, likely as a result of mislocalized core PCP 107 protein distribution. Based upon these results, we propose that a PCP signaling axis established in the 108 cochlear epithelia directs the peripheral axons of type II SGNs to turn towards the cochlear base. 109 4 110 MATERIALS AND METHODS 111 Mouse strains and husbandry: All mice were housed at the University of Utah under IACUC approved 112 guidelines, and individual lines were maintained by backcross to B6129SF1/J hybrid females and 113 genotyped using allele specific PCR reactions (available upon request). For timed breeding and tissue 114 staging, noon on the day in which vaginal plug was seen was considered embryonic day 0.5 (E0.5) and 115 the day litters were born was considered postnatal day 0 (P0). Mice from both sexes were used for 116 experimentation with the exception of analyses of Porcn mutants because the Porcn gene is located on 117 the X-chromosome. Fzd3 -/-; Fzd6 -/- double mutants and littermate controls were generated by 118 intercrossing Fzd3 +/-; Fzd6 +/- males and females derived from the Fzd3 and Fzd6 knockout lines. 119 Emx2-Cre; Fzd3 CKOs and Cre-positive littermate controls were generated on a Fzd6 KO background 120 by crossing Emx2-Cre; Fzd3 +/-; Fzd6 +/- males with Fzd3 floxed; Fzd6 -/- females. Pax2-Cre+; Vangl2 121 CKOs and Cre-positive littermate controls were generated on a Fzd3 KO background by crossing Pax2- 122 Cre; Vangl2 +/-; Fzd3 +/- male mice with Vangl2 floxed; Fzd3 +/- females.

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