DOI: 10.5061/dryad.m63xsj4br
title: Data from: Contributions of mirror-image hair cell orientation to mouse otolith organ and zebrafish neuromast function: Part 1/2, Zebrafish data

description: Otolith organs in the inner ear and neuromasts in the fish lateral-line
 harbor two populations of hair cells oriented to detect stimuli in
 opposing directions. The underlying mechanism is highly conserved: the
 transcription factor EMX2 is regionally expressed in just one hair cell
 population and acts through the receptor GPR156 to reverse cell
 orientation relative to the other population. In mouse and zebrafish, loss
 of Emx2 results in sensory organs that harbor only one hair cell
 orientation and are not innervated properly. In zebrafish, Emx2 also
 confers hair cells with reduced mechanosensory properties. Here, we
 leverage mouse and zebrafish models lacking GPR156 to determine how
 detecting stimuli of opposing directions serves vestibular function, and
 whether GPR156 has other roles besides orienting hair cells. We find that
 otolith organs in Gpr156 mouse mutants have normal zonal
 organization and normal type I-II hair cell distribution and
 mechano-electrical transduction properties. In
 contrast, gpr156 zebrafish mutants lack the smaller
 mechanically-evoked signals that characterize Emx2-positive hair cells.
 Loss of GPR156 does not affect orientation-selectivity of afferents in
 mouse utricle or zebrafish neuromasts. Consistent with normal otolith
 organ anatomy and afferent selectivity, Gpr156 mutant mice do not
 show overt vestibular dysfunction. Instead, performance on two tests that
 engage otolith organs is significantly altered – swimming and
 off-vertical-axis rotation. We conclude that GPR156 relays hair cell
 orientation and transduction information downstream of EMX2, but not
 selectivity for direction-specific afferents. These results clarify how
 molecular mechanisms that confer bi-directionality to sensory organs
 contribute to function, from single hair cell physiology to animal
 behavior.
description: <strong>Zebrafish immunofluorescence and imaging (Figures
 5, Figures 9-10)</strong> Immunohistochemistry
 was performed on whole larvae at 5 dpf. Whole larvae were fixed with 4%
 paraformaldehyde in PBS at 4°C for 3.5 hr. For pre- and post-synaptic
 labeling all wash, block and antibody solutions were prepared with 0.1%
 Tween in PBS (PBST). For Emx2 labeling performed on sparse afferent
 labeling (see below) all wash, block and antibody solutions were prepared
 with PBS + 1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). After
 fixation, larvae were washed 4 × 5 min in PBST or PBDTT. For synaptic
 labeling, larvae were permeabilized with Acetone. For this
 permeabilization larvae were washed for 5 min with
 H<sub>2</sub>O. The H<sub>2</sub>O was removed and
 replaced with ice-cold acetone and placed at −20°C for 5 min, followed by
 a 5 min H<sub>2</sub>O wash. The larvae were then washed for 4
 × 5 min in PBST. For all immunostains larvae were blocked overnight at 4°C
 in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin
 gelatin in PBST or PBDTT). Larvae were then incubated in primary
 antibodies in antibody solution (1% bovine serum albumin in PBST or PBDTT)
 overnight, nutating at 4°C. The next day, the larvae were washed for 4 × 5
 min in PBST or PBDTT to remove the primary antibodies. Secondary
 antibodies in antibody solution were added and larvae were incubated for 2
 hrs at room temperature, with minimal exposure to light. Secondary
 antibodies were washed out with PBST or PBDTT for 4 × 5 min. Larvae were
 mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using
 No. 1.5 coverslips. Primary antibodies used were:
 Rabbit anti-Myo7a (Proteus 25-6790; 1:1000)
 Mouse anti-Ribeye b (IgG2a) (Sheets et al., 2011)
 Mouse anti-pan-MAGUK (IgG1) (Millipore MABN7; 1:500)
 Mouse anti-Myo7a (DSHB 138-1; 1:500) Rabbit
 anti-Emx2 (Trans Genic KO609; 1:250). The following
 secondaries were used at 1:1000 for synaptic labeling: goat anti-rabbit
 Alexa 488, goat anti-mouse IgG2a Alexa 546, goat anti-mouse IgG1 Alexa
 647, along with Alexa 488 Phalloidin (Thermofischer; #A-11008, #A-21133,
 #A-21240, #A12379). For Emx2 co-labeling the following secondaries were
 used at 1:1000: goat anti-mouse Alexa 488, and goat ant-rabbit Alexa 647,
 along with Alexa 488 Phalloidin (Thermofischer; #A12379, #A28175,
 #A27040). Fixed samples were imaged on an inverted
 Zeiss LSM 780 laser-scanning confocal microscope with an Airyscan
 attachment (Carl Zeiss AG, Oberkochen, Germany) using an 63 × 1.4 NA oil
 objective lens. Airyscan z-stacks were acquired every 0.18 µm. The
 Airyscan Z-stacks were processed with Zen Black software v2.1 using 2D
 filter setting of 6.0. Experiments were imaged with the same acquisition
 settings to maintain consistency between comparisons. Processed imaged
 were further processed using Fiji.
 <strong> </strong><strong>Zebrafish</strong><strong> immunostain quantification (Figure 5, Figures 9-10)</strong> Images were processed in ImageJ. Researcher was not blinded to genotype. Hair bundle orientation was scored relative to the midline of the muscle somites. HC number per neuromast were quantified based on Myo7a labeling and presence of a paired/complete synapse. For quantification of Emx2 labeling, HCs were scored as Emx2 positive if they labeled with both Emx2 and Myo7a. To quality as a ribbon or presynapse, the following minimum size filters were applied to images: Ribeye b: 0.025 μm<sup>2</sup>, MAGUK: 0.04 μm<sup>2</sup>. A complete synapse was comprised of both a Ribeye b and MAGUK puntca. An unpaired presynapse consisted of only a Ribeye b puntca, while an unpaired postsynapse consisted of only a MAGUK puncta. In each neuromast all HCs (~15 HC per neuromast) were examined for our quantifications. <strong>Zebrafish functional calcium imaging in hair bundles (Figure 5)</strong> GCaMP6s-based calcium imaging in zebrafish hair bundles has been previously described (Lukasz and Kindt, 2018). Briefly, individual 5-6 dpf larvae were first anesthetized with tricaine (0.03% Ethyl 3-aminobenzoate methanesulfonate salt, SigmaAldrich). To restrain larvae, they were then pinned to a Sylgard-filled recording chamber. To suppress the movement, alpha-bungarotoxin (125 μM, Tocris) was injected into the heart. Larvae were then rinsed and immersed in extracellular imaging solution (in mM: 140 NaCl, 2 KCl, 2 CaCl<sub>2</sub>, 1 MgCl<sub>2</sub> and 10 HEPES, pH 7.3, OSM 310 +/- 10) without tricaine. A fluid jet was used to mechanically stimulate the apical bundles of HCs of the A-P neuromasts of the primary posterior lateral-line. To stimulate the two orientations of HCs (A&gt;P and P&gt;A) a 500 ms ‘push’ was delivered. Larvae were rotated 180° to deliver a comparable ‘push’ stimulus to both the A&gt;P and P&gt;A HCs. To image calcium-dependent mechanosensitive responses in apical hair bundles, a Bruker Swept-field confocal system was used. The Bruker Swept-field confocal system was equipped with a Rolera EM-C2 CCD camera (QImaging) and a Nikon CFI Fluor 60✕ 1.0 NA water immersion objective. Images were acquired in 5 planes along the Z-axis at 0.5 mm intervals (hair bundles) at a 50 Hz frame rate (10 Hz volume rate). The 5 plane Z-stacks were projected into one plane for image processing and quantification. The method to create spatial ∆F heatmaps has been described (Lukasz and Kindt, 2018). For GCaMP6s measurements, a circular ROI with a ~1.5 mm (hair bundles) diameter was placed on the center of each individual bundle. The mean intensity (∆F/F<sub>0</sub>) within each ROI was quantified. F<sub>0 </sub>represents the GCaMP6s intensity prior to stimulation. We examined the GCaMP6s signal in each hair bundle to determine its orientation. The GCaMP6s responses for each neuromast were averaged to quantify the magnitude of the A&gt;P and P&gt;A responses. <strong>Zebrafish sparse labeling of single afferents in the lateral-line (Figure 9)</strong> To visualize the innervation pattern of single afferent neurons, a <em>neuroD1:tdTomato</em> plasmid was injected into zebrafish embryos at the 1-cell stage. This plasmid consists of a 5kb minimal promoter, <em>neurod1</em>, that drives tdTomato expression in lateral-line afferents (Ji et al., 2018). This plasmid was pressure at a concentration of 30 ng/µl. At 3 dpf larvae were anesthetized with 0.03% ethyl 3-aminobenzoate methanesulfonate (Tricaine), to screen for tdTomato expression. Larvae were screened for mosaic expression of tdTomato expression in the lateral-line afferents using a Zeiss SteREO Discovery V20 microscope (Carl Zeiss) with an X-Cite 120 external fluorescent light source (EXFO Photonic Solutions Inc). After selecting larvae with tdTomato expression, larvae were prepared for immunostaining at 5 dpf, and imaged as outlined above.
description: # Data from: Contributions of mirror-image hair cell orientation to mouse
 otolith organ and zebrafish neuromast function Summary Paper associated
 with dataset: eLife (2024)
 [https://doi.org/10.7554/eLife.97674.1](https://doi.org/10.7554/eLife.97674.1) This dataset includes raw data from the following data collected at the NIH/NIDCD: Figure 5 Figure 9 Figure 9-S1 Figure 10 This dataset includes calcium imaging experiments that measure mechanosensitive calcium responses (in response to fluid flow) in zebrafish lateral-line hair bundles (L1-L4) in gpr156 mutants and sibling animals (Figure 5) at day 6. Specifically, this dataset shows that in the posterior lateral line, control responses to anterior flow are greater than posterior flow. In addition, gpr156 mutants have larger responses, similar to control responses to anterior flow. This dataset also includes gpr156 mutants and sibling zebrafish microinjected to label single lateral line afferents with tdTomato, then immunostained to label hair cell orientation (phalloidin), Emx2 and Myo7 (Figure 9 and 9-S1) at day 5. Normal innervation patterns were observed. In addition, this dataset includes immunostaining experiments that examine whether there are more unpaired hair cell synapses in gpr156 mutants compared to siblings. There was no difference (Figure 10) at day 5. Folders included in this submission have the following names: RAW_DATA_Figure 5 RAW_DATA_Figure 9 and 9-S1 RAW_DATA_Figure 10 Outlined below is information pertaining to each of these Data folders. RAW_DATA_Figure 5 Summary of RAW_DATA_Figure 5 folder contents: This folder contains 38 .xml Prairieview data files and 1 Excel .xlxs file. There are 38 data files that contain the raw in gpr156 mutants and sibling data represented in Figure 5. This is data acquired from in gpr156(idc15) mutants and sibling larval zebrafish. These data measure mechanosensitive responses from 9 sibling and 10 gpr156(idc15) mutant neuromasts (L1,L2,L3 and L4) in the posterior lateral line. Responses were made using the following transgenic line: myo6b:GCaMP6scaax. Zebrafish were assayed at the following age: day 6. GCaMP6s signals were measured in the hair bundles (apex) of neuromast hair cells. To stimulate hair cells a fluidjet was used. A 500ms saturating fluid flow stimulus was used, towards the anterior or posterior of the fish. Fish were alternated to start with the anterior or posterior stimulus. The data 38 .xml data files are coded with the date acquired, Fish number (F) for each experimental day, the neuromast assayed (L1, L2, L3 or L4) and the direction of the stimulus (ANT= towards the anterior, POST= towards the posterior. Each neuromast was stimulated in 2 directions, anterior or posterior and is represented by the 2.xlm data files. 05-27-2021 sibling control F7L1,L2,L3; F4L1; F6L1,L2 05-27-2021 gpr156 mutant F8L1,L2,L3,L4; F9L2,L3 06-17-2021 sibling control F4L2,L3,L4 06-17-2021 gpr156 mutant F1L3,L4; F2L2,L3 On 05-27-2021 Fish 4 and Fish 9 the anterior directed stim was acquired first; Fish 6,7,8 and 9 the posterior directed stim was acquired first. On 06-17-2021 Fish 1,2,4 the anterior directed stim was acquired first. Metadata for GCaMP6s calcium imaging acquisition: Acquired on a Swept field confocal microscope using Prairie-view (Bruker) software Nikon 60x water objective 20ms per image, 0.5 microns per slice, 5 planes per Z-stack (each Z-stack or timepoint is ~0.1s) 400 images in total per acquisition (~8s), fluidjet stimulation at frame 150 (~3s) 35 microns slit EM gain 3900 2x2 binning Imaging ROI 128x128 Laser setting 100 The following images were processed to create the examples in Figure 5 gpr156 sibling control: m6bGC6scaax_061721_gpr156_F4_L4_500ms_ANT_apex-001.xml m6bGC6scaax_061721_gpr156_F4_L4_500ms_POST_apex-003.xml Fish 3 L3-Airyscan Processing-08_wt emx2_neg.czi Gpr156 mutant: m6bGC6scaax_061721_gpr156_F2_L3_500ms_ANT_apex-001.xml m6bGC6scaax_061721_gpr156_F2_L3_500ms_POST_apex-003.xml Each of the .xml file was processed using the GUI-based Matlab 2014Rb program IOS_Software.m and IOS_Software.fig. This program average projects each z-stack. Then registered the z-stack. The first second (10 frames) of the recording were removed for baseline stability. Then the resulting 70 frames per recording were opened in FIJI/ImageJ. The Time Series Analyzer V3 plugin was used to create size 6 circular ROIs. ROIs were placed on each hair bundle. The multi-measure feature in the ROI manager was used to obtain the mean gray value from each ROI and each image in the series. Background subtracted plots were created. The background was the first 20 rows. The calculation to subtract background was Percentage different 100*(Value-Baseline)/Baseline. From the background subtracted plots, for each neuromast the anterior and posterior hair cells were determined. For each of the 38 acquisitions the average response for anterior and posterior cells was determined and plotted in 'Source Data for gpr156 vs control Calcium imaging .xlxs' Data Tables deltaF F0 per NM traces P to A and delta F F0 per NM traces P to A. Here the neuromast responses are listed according to date and animal # and neuromast # (rows 2 and 3). Row statistics was applied to these Data Tables to obtain the mean response (and SEM) per neuromast for each direction, this is in 'Source Data for gpr156 vs control Calcium imaging .xlxs' Data Table deltaF Fo Final AVG. From this Data Table the peak of the GCaMP6 signals were measured. This is data is found in Data Table' Max deltaF F0' columns F and H. In Max deltaF F0 the date, fish and neuromast #, genotype, age, and file names analyzed are listed in Columns A, B, C, D, E and G respectively. RAW_DATA_Figure 9_and 9-S1 Summary of Figure 9 folder contents: This folder contains 5 data folders containing 40 Zeiss Airyscan processed .czi images, and 1 Excel .xlxs file. There are 40 Zeiss Airyscan processed images that represent the raw data in Figure 9 and 9-S1. These files end in .czi. This is data acquired from gpr156(idc15) mutants and sibling control neuromasts. These samples were microinjected at day 0 with a neurod:tdTomato construct to sparsely label lateral line afferent neurons. At day 5 larvae were sorted for tdTomato and fixed and immunostained to label hair cells, hair bundle orientation and Emx2 in neuromasts (L1,L2,L3,L4,L5,L6) in the posterior lateral line. Information on immunostaining: Whole larvae were fixed with 4% paraformaldehyde in PBS at 4C for 3.5 hr. For Emx2 labeling performed on sparse afferent labeling all wash, block and antibody solutions were prepared with PBS?+?1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). After fixation, larvae were washed 4 x 5 min in PBDTT. Larvae were blocked overnight at 4C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBDTT). Larvae were then incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBDTT) overnight, nutating at 4C. The next day, the larvae were washed for 4 x 5 min in PBDTT to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2 hrs at room temperature, with minimal exposure to light. Secondary antibodies were washed out with PBDTT for 4 x 5 min. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using No. 1.5 coverslips. Primary antibodies used were: Mouse anti-Myo7a (DSHB 138-1; 1:500) Rabbit anti-Emx2 (Trans Genic KO609; 1:250). The following secondaries were used at 1:1000: goat anti-mouse Alexa 488, and goat ant-rabbit Alexa 647, along with Alexa 488 Phalloidin (Thermofischer; #A12379, #A28175, #A27040).  Metadata for Zeiss Airyscan acquisition: Fixed samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with an Airyscan attachment (Carl Zeiss AG, Oberkochen, Germany) using an 63 x 1.4 NA oil objective lens. Airyscan z-stacks were acquired every 0.18um. The Airyscan Z-stacks were processed with Zen Black software v2.1 using 2D filter setting of 6.0. The following lasers lines were used: 488, 546 and 647. Gain = 800 all laser lines ch1= myo7a and phalloidin ch2=tdTomato ch3=Emx2 The 40 Airyscan files generated are in the 5 data folders (11172020_Slide1_RS, 11172020_Slide2_RS, 11172020_Slide3_RS, 11172020_Slide1_LS and 03292021) and are as follows: Control: 11172020_Slide1_RS Fish 4 L3-Airyscan Processing-06_wt_emx_neg.czi Fish 7 L3-Airyscan Processing-10_wt_emx2_neg.czi Fish 7 L4-Airyscan Processing-11_wt_emx2_neg.czi 11172020_Slide2_RS Fish 4 L2-Airyscan Processing-13_wt_emx2_neg.czi Fish 4 L3-Airyscan Processing-14_wt_emx2_neg.czi 11172020_Slide3_RS Fish 3 L3-Airyscan Processing-08_wt emx2_neg.czi Fish 5 L3-Airyscan Processing-13_wt_emx2_neg.czi Fish 5 L4-Airyscan Processing-14_wt_emx2_pos.czi Fish 5 L5-Airyscan Processing-15_wt_emx2_neg.czi 11172020_Slide1_LS Fish 9 L2-Airyscan Processing-02_wt_emx2_pos.czi Fish 9 L1-Airyscan Processing-01_wt_emx2_pos.czi 3292021 Slide 1_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F4_L3_Out.czi Slide 2_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F3_L6_Out.czi Slide 2_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F4_L2_Out.czi Slide 2_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F5_L1_Out.czi Slide 2_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F5_L2_Out.czi Slide 1_LS_gpr156_my7a_phallodin_emx2_ntdtomato_F1_L5_Out.czi Slide 1_LS_gpr156_my7a_phallodin_emx2_ntdtomato_F4_L4_Out.czi Gpr156 mutants: 11172020_Slide1_RS Fish 2 L2-Airyscan Processing-01_mt.czi Fish 2 L3-Airyscan Processing-02_mt.czi Fish 2 L4-Airyscan Processing-03_mt.czi Fish 2 L5-Airyscan Processing-04_mt.czi Fish 3 L1-Airyscan Processing-05_mt.czi Fish 6 L2-Airyscan Processing-08_mt.czi Fish 6 L1-Airyscan Processing-07_mt.czi 11172020_Slide2_RS Fish 5 L3-Airyscan Processing-15_mt.czi 11172020_Slide3_RS Fish 4 L3-Airyscan Processing-10_mt_emx2_pos.czi Fish 6 L3-Airyscan Processing-16_mt.czi Fish 6 L4-Airyscan Processing-17_mt.czi 11172020_Slide1_LS Fish 10 L1-Airyscan Processing-03_mt.czi FIsh 10 L4-Airyscan Processing-04_mt.czi 3292021 Slide 1_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F2_L3_Out.czi Slide 2_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F2_L2_Out.czi Slide 2_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F2_L3_Out.czi Slide 2_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F2_L5_Out.czi Slide 2_RS_gpr156_my7a_phallodin_emx2_ntdtomato_F2_L6_Out.czi Slide 2_LS_gpr156_my7a_phallodin_emx2_ntdtomato_F2_L2_Out.czi Slide 2_LS_gpr156_my7a_phallodin_emx2_ntdtomato_F4_L1_Out.czi Slide 2_LS_gpr156_my7a_phallodin_emx2_ntdtomato_F4_L2_Out.czi Slide 2_LS_gpr156_my7a_phallodin_emx2_ntdtomato_F4_L3_Out.czi Slide 2_LS_gpr156_my7a_phallodin_emx2_ntdtomato_F4_L4_Out.czi The following images were processed (partial max projections) in FIJI/IMageJ to create the examples in Figure 9 gpr156 sibling control: 11172020_Slide3_RS Fish 3 L3-Airyscan Processing-08_wt emx2_neg.czi Gpr156 mutant: 11172020_Slide3_RS Fish 6 L3-Airyscan Processing-16_mt.czi The following images were processed (partial max projections) in FIJI/IMageJ to create the examples in Figure 9-S1 gpr156 sibling control: 11172020_Slide3_RS Fish 5 L4-Airyscan Processing-14_wt_emx2_pos.czi Gpr156 mutant: 11172020_Slide3_RS Fish 4 L3-Airyscan Processing-10_mt_emx2_pos.czi After imaging the .czi were opened in FIJI/ImageJ and scored for hair cell counts, the presence or absence of Emx2, hair bundle orientation and contact with tdTomato positive afferent terminals These analyses are in the excel file 'Source data gpr156_neurodTdtomato Analysis.xlxs' In the single datatable in this excel file (Scoring innervation). Column A indicates the subfolder analyzed. Column B indicates the file analyzed in that subfolder. Column C indicates the genotype. Column D indicates how many mature hair cells were present per neuromast. Columns E and F indicate how many Emx2 positive and Emx2 negative hair cells were contacted by the single fiber for each neuromast. The total hair cells and the percent of hair cells innervated by the single fibers is in Columns G and H. The specificity of the fiber is in Columns I or J, based on whether the fiber was Emx2 positive or negative. The n/a indicates that the fiber did not have specificity for either Emx2 positive or negative cells. The percent of hair cells specifically innervated, combining both Emx2 positive and Emx2 negative fibers, is in Column K. RAW_DATA_Figure 10 Summary of Figure 10 folder contents: This folder contains 2 data folders containing 43 Zeiss Airyscan processed .czi images, and 2 Excel .xlxs file. There are 43 Zeiss Airyscan processed images that represent the raw data in Figure 10. These files end in .czi. This is data acquired from gpr156(idc15), emx2(idc5) and sibling control neuromasts. At day 5 larvae were immunostained to label hair cells, hair bundle orientation, presynapses and postsynapses in neuromasts (L1,L2,L3,L4) in the posterior lateral line. Whole larvae were fixed with 4% paraformaldehyde in PBS at 4C for 3.5 hr. For pre- and post-synaptic labeling all wash, block and antibody solutions were prepared with 0.1% Tween in PBS (PBST). After fixation, larvae were washed 4x 5 min in PBST. For synaptic labeling, larvae were permeabilized with Acetone. For this permeabilization larvae were washed for 5 min with H2O. The H2O was removed and replaced with ice-cold acetone and placed at 20C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 4 x 5 min in PBST. Larvae were blocked overnight at 4C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). Larvae were then incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST or PBDTT) overnight, nutating at 4C. The next day, the larvae were washed for 4 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2 hrs at room temperature, with minimal exposure to light. Secondary antibodies were washed out with PBST for 4 ? 5 min. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using No. 1.5 coverslips. Primary antibodies used were: Rabbit anti-Myo7a (Proteus 25-6790; 1:1000) Mouse anti-Ribeye b (IgG2a) (Sheets et al., 2011) Mouse anti-pan-MAGUK (IgG1) (Millipore MABN7; 1:500) The following secondaries were used at 1:1000 for synaptic labeling: goat anti-rabbit Alexa 488, goat anti-mouse IgG2a Alexa 546, goat anti-mouse IgG1 Alexa 647, along with Alexa 488 Phalloidin (Thermofischer; #A-11008, #A-21133, #A-21240, #A12379). Fixed samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with an Airyscan attachment (Carl Zeiss AG, Oberkochen, Germany) using an 63 x 1.4 NA oil objective lens. Airyscan z-stacks were acquired every 0.18 im. The Airyscan Z-stacks were processed with Zen Black software v2.1 using 2D filter setting of 6.0. ch1= myo7a and phalloidin ch2=Maguk ch3=Ribeyeb The 43 Airyscan files generated are in the 5 data folders (09282020_gpr156_control_synapses and 10142020_emx2_control_synapses) and are as follows: 09282020_gpr156_control_synapses gpr156 sibling control: Fish 2 L1 09282020-Airyscan Processing-01.czi Fish 2 L2 09282020-Airyscan Processing-02.czi Fish 2 L3 09282020-Airyscan Processing-03.czi Fish 2 L4 09282020-Airyscan Processing-04.czi Fish 3 L2 09282020-Airyscan Processing-05.czi Fish 3 L3 09282020-Airyscan Processing-06.czi Fish 6 L2 09292020-Airyscan Processing-03.czi Fish 6 L3 09292020-Airyscan Processing-04.czi Fish 6 L4 09292020-Airyscan Processing-05.czi gpr156 mutants: Fish 0 L2 09292020-Airyscan Processing-01.czi Fish 0 L3 09292020-Airyscan Processing-02.czi Fish 0 L4 09292020-Airyscan Processing-03.czi Fish 1 L2 09282020-Airyscan Processing-01.czi Fish 1 L3 09282020-Airyscan Processing-02.czi Fish 4 L2 09282020-Airyscan Processing-08.czi Fish 4 L3 09282020-Airyscan Processing-09.czi Fish 4 L5 09282020-Airyscan Processing-10.czi Fish 5 L2 09292020-Airyscan Processing-01.czi Fish 5 L3 09292020-Airyscan Processing-01.czi Fish 5 L4 09292020-Airyscan Processing-02.czi Fish 7 L3 09292020-Airyscan Processing-06.czi Fish 7 L4 09292020-Airyscan Processing-01.czi Fish 7 L5 09292020-Airyscan Processing-02.czi Fish 8 L3 09292020-Airyscan Processing-03.czi Fish 8 L4 09292020-Airyscan Processing-01.czi 10142020_emx2_control_synapses Emx2 sibling control: Fish 1 L3-Airyscan Processing-04.czi Fish 2 L1-Airyscan Processing-05.czi Fish 4 L2-Airyscan Processing-01.czi Fish 6 L5-Airyscan Processing-03.czi Fish 9 L3-Airyscan Processing-05.czi Fish 1 L2-Airyscan Processing-01.czi Fish 4 L3-Airyscan Processing-01.czi Fish 1 L4-Airyscan Processing-02.czi Fish 18 L3-Airyscan Processing-12.czi Emx2 mutants: Fish 5 L2-Airyscan Processing-07.czi Fish 5 L5-Airyscan Processing-02.czi Fish 8 L1-Airyscan Processing-08.czi Fish 8 L3-Airyscan Processing-04.czi Fish 13 L2-Airyscan Processing-01.czi Fish 13 L3-Airyscan Processing-03.czi Fish 14 L2-Airyscan Processing-01.czi Fish 26 L2-Airyscan Processing-04.czi Fish 26 L3-Airyscan Processing-03.czi The following images were processed (partial max projections) in FIJI/IMageJ to create the examples in Figure 10 gpr156 sibling control: Fish 2 L1 09282020-Airyscan Processing-01.czi Gpr156 mutant: Fish 8 L3 09292020-Airyscan Processing-03.czi Emx2 mutant: Fish 5 L4-Airyscan Processing-02.czi  Images were processed in ImageJ. Researcher was not blinded to genotype. HC number per neuromast were quantified based on Myo7a labeling and presence of a paired/complete synapse. To quality as a ribbon or presynapse, the following minimum size filters were applied to images: Ribeye b: 0.025 um2 using the FIJI macro IJMacro_ribbons_0.025.ijm. A complete synapse was comprised of both a Ribeye b and MAGUK puntca. An unpaired presynapse consisted of only a Ribeye b puntca, while an unpaired postsynapse consisted of only a MAGUK puncta. These analyses are in the 2 excel files 'emx2 and sibling control synapse data.xlxs' and 'gpr156 and sibling control synapse data.xlxs' In the single datatable in this excel file (Synpase scoring). Column A indicates the file that is analyzed. Column B indicates the genotype. Columns C and D indicates how many mature hair cells were present per neuromast based on hair bundles (C) and cell body (D). Columns E and F indicate how many A to P and P to A orientated hair cell were present for each neuromast. The total number of complete synapses per neuromast is indicated in Columns G. The number of unpaired postsynapses (Maguk) and presynapses (ribbon) is indicated in Columns H or I. The number of complete synpases per hair cell is indicated in Column J. Sharing/Access information Please contact Katie Kindt, [katie.kindt@nih.gov](mailto:katie.kindt@nih.gov) for additional information or for access to the data. Code/Software Included in submission: IOS_fig.m FIJI macros IJMacro_ribbons_0.025.ijm This submission is a part submission of DOI: [https://doi.org/10.5061/dryad.905qfttvj](https://doi.org/10.5061/dryad.905qfttvj)