Duncan JS , Fritzsch B , Houston DW , Ketchum EM , Kersigo J , Deans MR , Elliott KL .

DC017589 U.S. Department of Health & Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD), DC013066 U.S. Department of Health & Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD), DC015333 U.S. Department of Health & Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD), AG060504 U.S. Department of Health & Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging), R03 DC015333 NIDCD NIH HHS , R21 DC017589 NIDCD NIH HHS , R01 DC013066 NIDCD NIH HHS , R01 AG060504 NIA NIH HHS

	Figure 1. Xenopus vestibular afferents display aberrant central projections following Fzd3 knockdown. (A) Schematic showing a dorsal view of a stage 46 Xenopus laevis tadpole and location of lipophilic dye placement. Lipophilic dyes were implanted into the ear (magenta) and into the anterior lateral line (aLL) and trigeminal (V) nerves (green). Brain is colored blue. (A’) Schematic showing a side view of a brain highlighting the location of the central projections from anterior lateral line (aLL), inner ear, and trigeminal afferents that would be labeled from the dye placement in (A) Dotted box represents the approximate area imaged. Confocal images of hindbrains from control animals (n = 6) (B), of animals injected with 5 ng Fzd3 morpholino (n = 12) (C), and of rescue animals injected with 5 ng Fzd3 morpholino plus 500 pg mouse Fzd3 mRNA (n = 9) (D). White arrowheads indicate aberrant projections. Yellow arrows indicate nerve entry points. (B’–D’) Higher magnification of boxed areas in (B–D). Short-dash and long-dash yellow lines represent the approximate ventral boundary of the anterior lateral line afferent projections and dorsal boundary of the trigeminal afferent projections, respectively. (B”–D”) Three-dimensional reconstructions of entire confocal stacks shown in (B–D). r4, rhombomere 4; dV, descending trigeminal tract; D, dorsal; A, anterior. Orientation for panels (B–D”) as in (B) Diagrams represent treatment (white, control; gold, Fzd3 morpholino; orange, Fzd3 morpholino plus Fzd3 mRNA) and colored wedges represent lipophilic dye placement (magenta, inner ear; green, lateral line and trigeminal nerves). Scale bars represent 100 µm.
	Figure 2. Xenopus vestibular afferents display aberrant central projections in a dose-dependent manner following Fzd3 knockdown in only the ear and associated inner ear afferents. Ears from control/wild type X. laevis embryos were transplanted to replace the ear of animals injected with Fzd3 morpholino (B,B”). Note the inner ear central projections (magenta) were between the lateral line and trigeminal projections (green), comparable to control animals (A,A”). In contrast, ears from X. laevis embryos injected with various concentration of Fzd3 morpholino transplanted to replace the ear of control animals (C–E) showed variable variations in inner ear central projections that could be rescued by an addition of mouse Fzd3 mRNA (F,F”). (A) Control X. laevis (n = 6), (B) animals injected with 5 ng Fzd3 morpholino with an ear transplanted from a control animal (n = 7), (C) control animals with an ear transplanted from an animal injected with 5 ng Fzd3 morpholino (n = 6), (D) control animals with an ear transplanted from an animal injected with 15 ng Fzd3 morpholino (n = 5), (E) control animals with an ear transplanted from an animal injected with 30 ng Fzd3 morpholino (n = 2), (F) control animals with a “rescue” ear transplanted from an animal injected with 5 ng Fzd3 morpholino plus 500 pg mouse Fzd3 mRNA (n = 6). White arrowheads indicate aberrant projections. Yellow arrows indicate nerve entry points. (A’–F’) Higher magnification of boxed areas in A-F. Short-dash and long-dash yellow lines represent the approximate ventral boundary of the anterior lateral line afferent projections and dorsal boundary of the trigeminal afferent projections, respectively. (A”–F”) Three-dimensional reconstructions of entire confocal stacks in A-F. (E” Inset) Anterior view of E”. dV, descending trigeminal tract; D, dorsal; A, anterior; L, lateral. Orientation for all panels as in A, except for E” inset. Diagrams represent treatment (white, control; gold/brown, Fzd3 morpholino; orange, Fzd3 morpholino plus Fzd3 mRNA) and colored wedges represent lipophilic dye placement (magenta, inner ear; green, lateral line and trigeminal nerves). Scale bars represent 100 µm.
	Figure 3. Mouse vestibular neurons display aberrant central projections in the absence of Fzd3. Lipophilic dyes were placed into individual sensory epithelia (cochlear apex, green; posterior crtista, magenta; saccule, cyan; utricle/anterior crista, yellow) of two Fzd3+/− control (A,C) and two Fzd3−/− mutant (B,D) mice at E18.5. All panels are coronal brain sections. (A,B) are a stack of Z-series images; (C,D) are single optical sections. DCN, dorsal cochlear nucleus; CN, cochlear nucleus; VN, vestibular nucleus; Pc, posterior crista; Ac, anterior crista; D, dorsal; M, medial. Orientation for all panels as in A. Scale bars represent 100 µm.
	Figure 4. Mouse spiral ganglion neurons display aberrant central projections in the absence of Fzd3. Lipophilic dyes were placed into the base (red) and apex (green) of Fzd3+/− control cochlea (A,C,E) and Fzd3 null mutants (B,D,F) at E18.5. A two dimensional rendering of a stack of images taken through the cochlear nuclei complex (A,B) shows more overlap of base and apex projection in the mutant compared to control. Yellow arrows indicate nerve entry points. (C,D) Single optical sections showing normal divergence to the AVCN and DCN as well as segregation between afferents from the base and apex in controls and a more profound projection to the PVCN of the apex of Fzd3 mutant mice. (E,F) Higher magnification of a single optical section showing fibers running parallel to each other in a single direction in the AVCN in a control. In contrast, there was aberrant trajectory of afferents in the AVCN in Fzd3 mutants and single fibers can be observed projecting across multiple other afferents (D,F). Magenta arrows show the course of a single fiber as it projects. White arrows show fibers projecting in the wrong direction (ventrally) (F). AVCN, anteroventral cochlear nucleus; DCN, dorsal cochlear nucleus; PVCN, posteroventral cochlear nucleus; VAS, ventral acoustic stria; D, dorsal; A, anterior. Orientation for all panels as in (A) Scale bars represent 100 µm. Two control and Four mutant animals were examined.
	Figure 5. Analysis of spiral ganglion neuron central projections following loss of Fzd3. (A) Reconstruction of ten approximately equally spaced afferents in Fig. 4E. (B) Reconstruction of ten approximately equally spaced afferents in 4F. (C,D) Selected individual axon tracings from a Fzd3+/− control mouse (C) and a Fzd3−/− mutant mouse (D), both at E18.5, overlaid onto boxes representing the mean dorsoventral limit of all control axons (dark gray), plus or minus one standard deviation (1 SD, medium gray), and plus or minus two standard deviations (2 SD, light gray) to display the analysis. Grayscale circles indicate mean/SD level crossings. (E) Linear Sholl analysis of 20 axons each from Fzd3+/− and Fzd3−/− mice. Bars represent means and standard error of the means for the number of times axons crossed the mean, one standard deviation (1 SD), and two standard deviation (2 SD) boundaries in (C,D). (F) Mean number of times an axon crossed over another axon from an analysis of 20 axons each from Fzd3+/− and Fzd3−/− mice. Error bars represent standard error of the means. **p < 0.01.
	Figure 6. Fzd3 is expressed as a gradient across the cochlea in auditory neurons. (A) Overview of a wildtype E13.5 ear following RNAscope® fluorescent in situ hybridization showing expression of Fzd3 (green), TrkC (expressed in auditory neurons, magenta), and Ubc (Ubiquitin C, control, blue). Individual puncta represent a single mRNA. U, utricle; S, saccule; Ac, anterior crista; Hc, horizontal crista; Pc, posterior crista; VG, vestibular ganglion; GG, geniculate ganglion. (B) Fzd3 expression at E13.5 in the spiral ganglion showing higher levels of expression in the apex compared with the base. The image shows the collapsed stack of the entire spiral ganglion. The spiral ganglion was outlined following the boundary of TrkC expression (magenta dotted outline). (C) Fzd3 expression in basal afferents (boxed area in B). (D) Fzd3 expression in apical afferents (boxed area in B). C and D were imaged using identical confocal settings. (E) Quantification of individual Fzd3 mRNA puncta in the base and apex following RNAscope®. Puncta were counted with ImageJ software in each of three equal sized squares (50 µm × 50 µm) per region (see yellow/cyan boxes in C and D) in a central Z series image for the base and for the apex of each animal following thresholding. Three animals were quantified. (F) Quantification of fluorescent intensity of Fzd3 mRNA in the base and apex following RNAscope®. Average intensity was determined for each of three equal sized squares (50 µm × 50 µm) per region (see yellow/cyan boxes in C and D) in the collapsed Z series for the base and for the apex of each animal. Three animals were quantified. Error bars represent standard error of the means. **p < 0.01.
	Figure 7. Hypothesis for guidance of inner ear afferents by Fzd3. We predict that the Fzd3 gradient expressed along the cochlear neurons is, in part, responsible for correct targeting to the proper dorsoventral target within the hindbrain. (A) In the absence of Fzd3, there is no gradient across cochlear neurons and thus dorsoventral targeting is disrupted. (B) Whether this gradient extends to the vestibular neurons remains to be explored.

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