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

Abstract

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. Previously, 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.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 assembly, 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.

Keywords: auditory; axon; cochlea; frizzled; planar cell polarity; spiral ganglia.

Figure 1.

Peripheral axons from Type II SGNs project toward the cochlear base. A, Schematic diagram of the mouse cochlea illustrating the anatomy of the Type II SGN with a peripheral axon that innervates the cochlea and turns toward the cochlear base, and a central axon that exits the inner ear through the VIIIth cranial nerve. B, Surface view of the mouse organ of Corti, illustrating the morphology of the Type II SGN peripheral axon relative to the apical surfaces of hair cells and supporting cells. The peripheral axon extends beyond the single row of IHCs (ihc) and completes a 90° turn (arrow) toward the cochlear base before ascending apically (arrowheads) and extending branches to form afferent synapses (asterisk) with OHCs. Turning frequently occurs after the axon has passed between the basolateral junctions formed between neighboring supporting cells, as illustrated here for IPCs. C, Cross-section of the organ of Corti, illustrating the position of axon turning (arrow) amid the basolateral boundaries of pillar cells before apical ascension (arrowheads) and synapse formation (asterisk). D, Profile of a single row of OHCs (O2) and Deiters' cells (D2), illustrating the site of ascension (arrowheads) of the peripheral axon after turning (arrow) and before synapse formation (asterisk). IHCs, first (O1), second (O2), and third (O3) rows of OHCs, IPCs, OPCs, first (D1), second (D2), and third (D3) row of Deiters' cells.

Figure 2.

FZD3 and FZD6 are asymmetrically distributed along the basolateral sides of cochlear-supporting cells. A, HA immunofluorescence at P0 is enriched at hair cell to supporting cell junctions on the apical surface of the organ of Corti in a Fzd3 CKO mouse in which endogenous FZD3 contains an HA epitope tag (arrowheads). B, FZD6 protein is similarly enriched at hair cell to supporting cell junctions (arrowheads). C, C′, FZD3-HA and D, D′, FZD6 are also enriched along the basolateral surfaces of supporting cells at supporting cell to supporting cell junctions (arrowheads). E, Specificity of the HA antibody demonstrated by immunolabeling the basolateral surfaces of supporting cells in WT tissue and (F) specificity of the FZD6 antibody demonstrated in Fzd6 KO tissue. G, Schematic illustration of a Cre-dependent 3XHA-Fzd3 transgenic reporter and predicted distribution of the 3XHA-FZD3 protein product (magenta) in adjacent Cre-positive (blue nuclei, asterisk) and Cre-negative (green nuclei) cells. H, H′, 3XHA-FZD3 expression and localization (arrowheads) in the Pax2-Cre⁺ IPCs (asterisk) on the side facing the cochlear base. β-galactosidase immunolabel (green) marks Cre-negative cells, which do not express the FZD3 reporter. IHCs, first (O1), second (O2), and third (O3) row OHCs, IPCs, OPCs, first (D1), second (D2), and third (D3) row Deiters' cells. All images are of P0 tissue. Scale bars: A–F, 10 μm; H, 5 μm.

Figure 3.

Fzd3 and Fzd6 act redundantly to direct Type II SGN turning. A–D, All Fzd3;Fzd6 dKOs have characteristic looped tails (arrows) but variable neural tube defects ranging from craniorachischisis (B) to exencephaly (C) to normal body axes (D). C, D, Arrowheads indicate the anterior and posterior extent of the neural tube defect. A′–D′, Phalloidin staining of hair cell stereociliary bundles from cochlea dissected from Fzd3;Fzd6 dKO embryos (corresponding to B,C,D) shows that all have misoriented bundles (arrowheads) regardless of the severity of the neural tube defect. E–H, NF200 immunolabeling of Type II peripheral axons shows that the majority project toward the cochlear base in littermate controls (E), Fzd3 KOs (F), and Fzd6 KOs (G), whereas in Fzd3;Fzd6 dKOs, (H) many turn incorrectly toward the cochlear apex (arrowheads). I, The average frequency of incorrectly turned Type II peripheral axons in dKO, single KO, and littermate control animals. Data are mean ± SD with overlying circles reporting to the frequency of turning errors for individual mice. All images and quantification were collected from the basal turn of E18.5 cochlea with a single cochlea analyzed from each mouse: Control (n = 4, 159 axons), Fzd3 KO (n = 4, 178 axons), Fzd6 KO (n = 5, 240 axons), Fzd3;Fzd6 dKO (n = 5, 231 axons). ***p ≤ 0.001, Fzd3;Fzd6 dKOs versus littermate controls (Student's t test). Scale bars, 10 μm.

Figure 4.

Type II turning is regulated by nonautonomous Fzd3/Fzd6 function. A, Schematic diagram showing the pattern of Emx2-Cre-mediated recombination in the cochlear duct. B, C, Conventional ISH showing that the reduction of Fzd3 mRNA at E16.5 in the organ of Corti (bracket) but not the SGN (outlined) from Emx2-Cre;Fzd3 CKOs. D, D′, E, E′, FZD3-HA expression is also lost from the basolateral junctions between neighboring supporting cells in Emx2-Cre;Fzd3 CKOs compared with littermate controls. F, G, NF200 immunolabeling of Type II SGN peripheral axons in Emx2-Cre;Fzd3 CKO;Fzd6 KOs (Emx2-Cre;Fzd3/6 CKO) reveals multiple turning errors (arrowheads) compared with littermate controls in the basal turn of the E18.5 cochlea. H, Average frequency of incorrectly turned Type II fibers projecting toward the cochlear apex. Data are mean ± SD with overlying circles reporting to the frequency of turning errors for individual mice. A single cochlea was analyzed from each animal: Control (n = 3, 127 axons), CKOs (n = 3, 142 axons). ***p ≤ 0.001, CKOs versus littermate controls (Student's t test). Supporting cell nuclei are identified based upon DAPI staining of nuclei: IPCs, OPCs, first (D1), second (D2), and third (D3) row Deiters' cells. Scale bars, 10 μm.

Figure 5.

Loss of Fzd3 and Fzd6 disrupts the polarized distribution of the remaining core PCP proteins. A, A′, B, B′, Immunofluorescent labeling of VANGL2 or CELSR1 (magenta) on the apical surface of the organ of Corti shows the asymmetric distribution of these proteins at hair cell to supporting cell junctions (arrowheads), and between nonsensory cell boundaries (arrows) in the greater epithelial ridge of littermate controls. C, C′, D, D′, In Fzd3;Fzd6 dKOs, VANGL2 and CELSR1 distribution is disrupted around hair cells (examples marked by asterisk) and throughout the GER region (bracket). Moreover, the localization of VANGL2 and CELSR1 appear to be completely lost in the OHC region. E, E′, F, F′, The asymmetric distribution of VANGL2 and CELSR1 at basolateral membrane of supporting cells in control tissue resembles FZD distribution at this location. G, G′, H, H′, In Fzd3;Fzd6 dKOs, VANGL2 and CELSR1 levels appear reduced and the proteins encircle many supporting cells. All images are from E18.5 cochlea. Supporting cell nuclei are identified based upon DAPI staining of nuclei: IPCs, OPCs, first (D1), second (D2), and third (D3) row Deiters' cells. Scale bars, 10 μm.

Figure 6.

Fzd3, and not ROR2, genetically interacts with Vangl2 in SGN development. A–D, NF200 immunolabeling of Type II SGN peripheral axons in Pax2-Cre;Vangl2 CKOs and littermate controls generated on a Fzd3 KO background. Arrowheads indicate Type II fibers, which have turned incorrectly toward the cochlear apex. E, F, NF200 labeled Type II peripheral axons in Pax2-Cre;ROR2 CKO and Pax2-Cre;ROR2; Vangl2 double CKOs. G, Quantification of the average frequency of turning errors for cochlea of each genotype demonstrates a genetic enhancement with the loss of Vangl2 and Fzd3. H, Similar quantification of turning errors in Pax2-Cre; ROR1; Vangl2 double CKOs did not reveal a genetic interaction between Vangl2 and ROR2. Data are mean ± SD with overlying circles reporting to the frequency of turning errors for individual mice. A single cochlea was analyzed from each animal. For Vangl2-Fzd3 interaction assays, images and quantification are from the basal turn of E18.5 cochlea: Control (n = 3, 159 axons), Fzd3 KO (n = 5, 248 axons), Fzd3 KO, Vangl2^+/− (n = 5, 234 axons), Vangl2 CKO (n = 3, 101 axons), Fzd3 KO; Vangl2 CKO (n = 3, 117 axons). For Vangl2-ROR2 interaction assays, images are from the basal turn, and quantification is summed from basal, middle, and apical turns: Control (n = 3, 375 axons), ROR2 CKO (n = 3, 380 axons), Vangl2 CKO (n = 3, 324 axons), ROR2 CKO; Vangl2 CKO (n = 3, 313 axons). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns (not significant); pairwise comparisons as indicated (Student's t test). All images are from E18.5 cochlea and scalebars are 10 μm.

Figure 7.

WNT activity is required for Type II SGN axon guidance. A, B, NF200 immunolabeling of Type II SGN peripheral axons in Wnt5a KO and littermate controls reveals no turning errors in the basal turn at E18.5. C–F, Modest impairment of inner ear development in Pax2-Cre;Porcn CKOs compared with littermate controls revealed by smaller temporal bones (C,E) and cochlea (D,F) in CKOs. G, H, Phalloidin staining of stereociliary bundles demonstrates hair cell differentiation in Pax2-Cre;Porcn CKOs in addition to an extra row of OHCs (OHC4) throughout the length of the cochlea. I, J, Type II peripheral axons are more likely to turn incorrectly (blue arrowheads) toward the cochlear apex in Porcn CKOs compared with littermate control, and stalled or branched fibers are also prevalent (orange arrowheads). K, L, Quantification of innervation defects in WNT signaling mutants their respective littermate controls. Data are mean ± SD with overlying circles reporting to the frequency of turning errors for individual mice. All images and quantification were collected from the basal turn of E18.5 cochlea, with a single cochlea analyzed from each animal: Control (n = 7, 312 axons), Wnt5a KO (n = 7, 308 axons), Porcn controls (n = 3, 139 axons), Porcn CKOs (n = 3, 115 axons). IHCs, OHCs (OHC1, OHC2, OHC3), and an extra row OHCs (OHC4). *p ≤ 0.05; ***p ≤ 0.001; mutants versus littermate controls (Student's t test). Scale bars, 10 μm.

Figure 8.

WNT mediated signaling establishes PCP axis in cochlear-supporting cells. The asymmetric distribution of VANGL2 and CELSR1 along the basolateral boundaries of cochlear-supporting cells of littermate controls (A–A″) is disrupted in Pax2-Cre;Porcn CKOs (B–B″). Arrowheads indicate examples of asymmetric protein distribution in controls. Asterisks indicate examples of supporting cells that lack asymmetric protein distributions in CKOs. Supporting cell nuclei are identified based upon DAPI staining of nuclei; IPCs, OPCs, first (D1), second (D2), and third (D3) row Deiters' cells. Scale bars, 10 μm.

Figure 9.

Planar polarity of cochlear-supporting cells directs peripheral axon turning. A, WNT signaling, perhaps acting in a gradient, promotes the asymmetric distribution of the core PCP proteins (green and magenta) within organ of Corti-supporting cells. This distribution is reinforced by intracellular feedback loops within individual cells (gray arrows) and intercellular signaling between neighboring cells (black arrows). This planar polarization mechanism may also direct the polarized subcellular distribution of membrane tethered axon guidance molecules, such as ephrins or semaphorins. B, As the growth cone of the peripheral axon enters this polarized environment, it is directed toward the cochlear base by the polarized array of proteins that it encounters before turning. Growth cone turning could occur in response to intercellular PCP signaling between supporting cells and the growth cone, or to the polarized distribution of axon guidance cues that are asymmetrically distributed through a PCP-dependent mechanism.