DOI: 10.5061/dryad.0k6djhb95
title: Data from: Presynaptic Nrxn3 is essential for ribbon-synapse maturation in hair cells

description: Hair cells of the inner ear and lateral-line system rely on specialized
 ribbon synapses to transmit sensory information to the central nervous
 system. The molecules required to assemble these synapses are not fully
 understood. We show that Nrxn3, a presynaptic adhesion molecule, is
 critical for ribbon-synapse maturation in hair cells. In both mouse and
 zebrafish models, loss of Nrxn3 results in significantly fewer intact
 ribbon synapses. We show in zebrafish that initially, nrxn3 mutants have
 normal pre- and post-synapse numbers, but synapses fail to pair, resulting
 in postsynapse loss. We also demonstrate that Nrxn3 subtly influences
 synapse selectivity in zebrafish lateral-line hair cells that detect
 anterior flow. In zebrafish we show that a 60% loss of synapses in nrxn3
 mutants dramatically reduces pre- and post-synaptic responses. Despite
 fewer synapses, auditory responses in zebrafish and mice are unaffected.
 This work demonstrates that Nrxn3 is a critical and conserved molecule
 required for the maturation of ribbon synapses. Understanding how ribbon
 synapses mature is essential to generating novel therapies to treat
 synaptopathies linked to auditory or vestibular dysfunction.
description: <strong>Immunohistochemistry</strong>
 <strong>Zebrafish immunohistochemistry and
 imaging</strong> Immunohistochemistry was
 performed on whole larvae at either 3 dpf or 5 dpf. Whole larvae were
 fixed with paraformaldehyde (PFA 4%; Thermoscientific; 28906) in PBS at
 4°C for 3.5 hr. For Ca<sub>V</sub>1.3 labeling
 (Ca<sub>V</sub>1.3, Otoferlin, MAGUK or
 Ca<sub>V</sub>1.3, Paravalbumin, CTBP), all wash, block and
 antibody solutions were prepared with PBS + 0.1% Tween (PBST). For pre-
 and post-synaptic labeling (MYO7A, CTBP, MAGUK, Calretinin, SOX2, GFP),
 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 PBDDT. For Ca<sub>V</sub>1.3
 labeling, prior to block, larvae were permeabilized with acetone. For this
 permeabilization, larvae were washed for 5 min with
 H<sub>2</sub>O in glass vials. The H<sub>2</sub>O
 was removed and replaced with ice-cold acetone and larvae 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 immunolabels, 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). After block, larvae
 were 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 removed by washes with PBST or PBDTT
 for 4 × 5 min. Larvae were mounted on glass slides with Prolong Gold
 (ThermoFisher Scientific) using No. 1.5 coverslips. Fixed samples were
 imaged on an upright LSM 780 or 980 laser-scanning confocal microscope
 with an Airyscan 2 attachment using Zen (Carl Zeiss) and a 63x/1.4 NA Plan
 Apo oil immersion objective lens. Airyscan z-stacks were acquired every
 0.15 µm with a 0.043 µm X-Y pixel size for lateral-line and medial-crista
 hair cells, and every 0.15 µm with a 0.067 µm X-Y pixel size for hair
 cells in the anterior macula. The Airyscan z-stacks were autoprocessed in
 2D. Experiments were imaged with the same acquisition settings to maintain
 consistency between comparisons.  <strong>Mouse
 immunohistochemistry and imaging</strong>
 Temporal bones were isolated, and an insulin syringe was used to
 gently flush cold paraformaldehyde (PFA 4%; Electron Microscopy Sciences;
 15710) through the cleared oval and round windows after poking a small
 hole at the cochlear apex. Temporal bones were then immersion-fixed in PFA
 for 1 hour at 4°C, washed in PBS, and rotated overnight in EDTA 4% for
 decalcification. The next day, cochleae were dissected in 3 approximate
 thirds (base, mid and apex) before blocking and permeabilization for 1 hr
 at room temperature under agitation (1% bovine serum albumin; 0.5% Triton
 X-100). The following primary antibodies were used: CTBP2, GluR2 and mouse
 anti-MYO7A. Primary and secondary antibodies were incubated overnight at
 4°C in PBS. Samples were washed 3 times in PBS + 0.05% Triton X-100 after
 each antibody incubation and finally post-fixed in PFA 4% for at least 1
 hr at room temperature. Samples were then mounted flat in Mowiol mounting
 medium (Calbiochem/MilliporeSigma 4759041) using two layers of office tape
 as a spacers for the coverglass (18x18mm #1.5). Mowiol (10% w/v) was
 prepared in (25% w/v) glycerol and 0.1M Tris-Cl pH8.5. Mounted samples
 were imaged on an upright LSM 980 laser-scanning confocal microscope with
 using Zen Blue 3.4 (Carl Zeiss) and an 63x 1.4 NA oil objective lens.
 Z-stacks containing 6-9 IHCs were acquired every 0.250 µm with an 0.085 µm
 X-Y pixel size in confocal mode.  <strong>RNA
 FISH to detect <em>nrxn3a</em> and <em>nrxn3b</em>
 mRNA in lateral-line hair cells</strong> To
 detect mRNA for <em>nrxn3a and
 nrxn3b</em><strong><em> </em></strong>in
 zebrafish, we followed the Molecular Instrument-RNA FISH Zebrafish
 protocol, Revision Number 10 (https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf), with a few minor changes to the preparation of fixed whole-mount larvae. For our dehydration steps, we dehydrated using the following methanol series: 25, 50, 75, 100, 100% methanol, with 5 min for each step in the series. To permeabilize, we treated larvae with 10 µg/mL proteinase K for 20 min. RNA FISH probes were designed to target the long α form of zebrafish <em>nrxn3a</em> and <em>nrxn3b</em> (Molecular Instrument Probe lot # PRP848, PRP849).After completing the RNA FISH protocol, we mounted the larvae in ProLong Gold Antifade (ThermoFisher, P36930) under 1.5 coverglass. Samples were imaged on an upright LSM 980 laser-scanning confocal microscope with an Airyscan 2 attachment using Zen Blue 3.4 (Carl Zeiss) and a 63x/1.4 NA Plan Apo oil immersion objective lens. Airyscan z-stacks were acquired every 0.160 µm with a 0.043 µm X-Y pixel size. The Airyscan z-stacks were autoprocessed in 2D. <strong>Image processing and quantification of synapses and RNA FISH puncta</strong> To identify and quantify puncta (presynaptic, postsynaptic, Ca<sub>V</sub>1.3 cluster or RNA FISH puncta), an automated synapse quantification method using a customized Fiji-based macro, “Complete Synapse Counter v5.2” (https://github.com/KindtLab-NIDCD/CompleteSynapseCounter5.2) was used. Prior to automated puncta quantification, each channel was background subtracted using rolling-ball radius background subtraction. Then each z-stack was max-intensity projected. A mask was generated by manually outlining the region or interest (ex: hair cells) in the reference channel. This mask was then applied to the z-projection of each synaptic component or RNA FISH channel. Each masked image was then analyzed using the automated Fiji macro. In this macro, the images were thresholded using an adaptive thresholding plugin by Qingzong TSENG (https://sites.google.com/site/qingzongtseng/adaptivethreshold) to generate a binary image of the puncta. Individual synaptic or RNA FISH puncta were then segmented using the particle analysis function in Fiji. For particle analysis, the following minimum size thresholds were applied: zebrafish lateral-line images – CTBP: 0.025 μm<sup>2</sup>, MAGUK: 0.04 μm<sup>2</sup>, Ca<sub>V</sub>1.3 0.025 μm<sup>2</sup>, <em>nrxn3a</em>, <em>nrxn3b</em> and <em>dapB</em> RNA FISH particles: 0.03 μm<sup>2</sup> and 0.01 μm<sup>2</sup>, zebrafish inner ear images – CTBP: 0.025 μm<sup>2</sup>, MAGUK: 0.025 μm<sup>2</sup>, mouse IHCs – CTBP: 0.025 μm<sup>2</sup>, GluR2: 0.025 μm<sup>2</sup>. A circularity factor between 0.1-0.5 was also applied to particle analysis. A watershed was applied to the particle analysis result to break apart overlapping synaptic components. After the watershed, the particle analysis was rerun with size and circularity thresholds to generate ROIs and measurements of each synaptic or RNA FISH component. The ROIs were applied to the original z-projection to get the average intensity and area of each punctum. To recognize paired synaptic components, images were further processed using “Complete Synapse Counter v5.2”. Here, the overlap and proximity of ROIs from different channels (ex: pre- and post-synaptic puncta) were calculated. ROIs with positive overlap or ROIs within 2 pixels were counted as paired or partner components. The ROIs were applied to the original z-projection to get the average intensity and area of each paired or unpaired punctum. Some image datasets required a pre-processing step prior to entry into the “Complete Synapse Counter v5.2”. This includes zebrafish samples imaged at 3 dpf and our mouse IHCs datasets. For the pre-processing step, the volumes were segmented in VVDviewer (https://github.com/JaneliaSciComp/VVDViewer). Staining outside of the hair cell was manually segmented or removed using VVDviewer. After this segmentation, the z-stacks were then max-intensity projected and processed using the “Complete Synapse Counter v5.2” macro. <strong>Quantification of lateral-line afferent terminal areas and single afferent selectivity </strong> To quantify the area occupied by the 4-6 afferent terminals beneath lateral-line neuromasts, we examined z-stacks of Calretinin-labeled terminals.<strong> </strong>Each z-stack was opened in FIJI and max-intensity projected. Projected images were autothresholded to isolate the terminals, and the mean area within the threshold was measured<strong>.  </strong> Larvae with positive tdTomato expressions in individual afferent neurons were stained with CTBP and MAGUK to label pre- and post-synapses, as well as phalloidin to visualize hair-bundle orientations. After the immunostaining, single afferent terminals were imaged under using a Zeiss LSM 980 confocal microscope in Airyscan mode as described above. Z-stacks were then loaded in VVDviewer for 3D viewing of afferent terminals and their connections with hair cells. Hair-cells numbers and their orientations were assigned manually based on the weak membrane labeling of MAGUK and phalloidin label respectively. To assign synapses and hair cells to terminals, we manually identified all the complete synapses (paired CTPB and MAGUK label) that colocalized with each tdTomato terminal. This allowed us to determine the number of hair cells innervated and the number of complete synapses formed per terminal. To analyze the selectivity of individual terminals we used a selectivity index. This index was defined as percentage of the number of hair cells with the domination orientation innervated, divided by the total number of hair cells innervated. Here an index value of 50% indicates that the afferent terminal showed no selectivity when choosing between the two different orientations, while 100% indicates perfect selectivity towards a single orientation. <strong>Calcium imaging of lateral-line hair cells and afferents</strong> For functional imaging, 4-6 dpf larvae were anesthetized in 0.04% Tricaine-S (tricaine methanesulfonate, Western chemical, TRS1), pinned to a Sylgard-filled perfusion chamber at the head and tail, and paralyzed by injection of 125 µM a-bungarotoxin (Tocris, 2133) into the heart cavity, as previously described (Lukasz and Kindt, 2018). Larvae were then rinsed three times in E3 embryo media to remove the tricaine. Next, larvae were rinsed three times with 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) and allowed to recover. Stimulation was achieved by a fluid jet, which consisted of a pressure clamp (HSPC-1, ALA Scientific) and glass pipette, pulled and broken to an inner diameter 40-50 µm, and filled with extracellular imaging solution. A 500-ms pulse of positive or negative pressure was used to deflect the hair bundles of mechanosensitive hair cells along the anterior-posterior axis of the fish. Hair cells of the two orientations (anterior and posterior) were stimulated separately. Stimuli that deflected kinocilia 5-15 µm were included in the analysis, as these deflections represent saturating stimuli that do not induce damage. Hair-cell responses to stimuli were imaged with an A1R laser-scanning confocal scan head on an upright Nikon NI-E microscope with a resonant scanner and a 60x/1.0 NA CFI Fluor water immersion objective equipped with a z-piezo. Acquisition was controlled with Nikon Elements Advanced Research v. 5.20.02. GCaMP6s fluorescence was excited with a 488 nm solid-state laser passed through a standard 405/488/561/640 BS20/80 dichroic and collected with a 560 nm low-pass dichroic and 525/50 emission filter. Images were acquired using a GaAsP PMT and 4x averaging. Pixel size for presynaptic imaging was 0.28 µm; pixel size for MET imaging was 0.14 µm. Each neuromast (L2 or L3) was stimulated four times (starting with a posterior-to-anterior stimulus and alternating between the two directions) with an inter-stimulus interval of ~2 min. This enabled us to collect presynaptic responses (collected first) and hair-bundle responses to both stimulus directions for each neuromast. 3 z-slices (1.5 µm step size for presynaptic responses; 0.5 µm step size for hair bundle responses) were collected per timepoint for 110 timepoints at a frame rate of 33 ms for a total of ~100 ms per z-stack and a total acquisition time of ~11 sec. Stimulation began at timepoint 31; timing of the stimulus was triggered by an outgoing voltage signal from Nikon Elements. Calcium responses in the afferent process were acquired on a Swept-field confocal system built on a Nikon FN1 upright microscope (Bruker) with a 60x/1.0 NA CFI Fluor water-immersion objective. The microscope was equipped with a Rolera EM-C2 EMCCD camera (QImaging), controlled using Prairie view 5.4 (Bruker). GCaMP6s was excited using a 488 nm solid state laser. We used a dual band-pass 488/561 nm filter set (59904-ET, Chroma). Pixel size for postsynaptic imaging was 0.27 µm. Stimuli were delivered as outlined above for hair-cell responses. Each neuromast (L2, L3 or L4) was stimulated two times with an inter-stimulus interval of ~2 min. 5 z-slices (1.0 µm step) were collected per timepoint for 80 timepoints at a frame rate of 20 ms for a total of ~100 ms per Z-stack and a total acquisition time of ~8 sec. Stimulation began at timepoint 31; timing of the stimulus was triggered by an outgoing voltage signal from Prairie view. Acquired images were converted into TIFF series for processing. Researchers were blind to genotype during analysis. Z-stacks were average projected, registered, and spatially smoothed with a Gaussian filter (size = 3, sigma = 2) in custom-written MatLab software as described previously ((Zhang et al., 2018); https://github.com/KindtLab-NIDCD/ImagePro)). The first 10 timepoints (~1 sec) were removed to reduce the effect of initial photobleaching. Registered average projections were then opened in Fiji for intensity measurements. Using the Time Series Analyzer V3 plugin, circular ROIs (18x18 pixels for presynaptic responses; 8x8 pixels for hair-bundle responses, 12x12 pixels for afferent process) were placed on hair bundles or synaptic sites; average intensity measurements over time were measured for each ROI, as described previously (Lukasz and Kindt, 2018). Neuromasts were excluded in the case of motion artifacts. Hair-bundles responses were excluded if they responded to stimuli of both directions. All other data was included in analyses. Presynaptic responses were defined as &gt;10% ∆F/F0 within the 500 ms stimulus or &gt;20% within 1 sec of stimulus onset. Hair-bundle responses were defined as &gt;15% ∆F/F0 within the 500 ms stimulus and &gt;15% in the 500 ms after the stimulus. Postsynaptic responses were defined as &gt;5% ∆F/F0 and a minimum duration of 500 ms. Square wave responses indicate movement artifacts and were excluded. Calcium imaging data was further processed in Prism 10 (Graphpad). The first 20 timepoints were averaged to generate an F0 value, and all responses were calculated as ∆F/F0. Responses presented in figures represent average responses of synaptically active cells within a neuromast. The max ∆F/F0 was compared between wild-type animals and double mutants. To measure baseline GCaMP6s intensities in hair bundles, the presynaptic region or afferent terminals, the mean GCaMP6s intensity was measured during the prestimulus (2 s) time window. For the images acquired in this time window, the GCaMP6 images were autothresholded, and the mean intensity was measured at each time point. Then all the time points during the prestimulus time window were averaged for each neuromast.  <strong>Mouse auditory brainstem response (ABR) tests</strong> All tests were performed in a sound-attenuating chamber. Animals between P28 and P32 were anesthetized with a mix of ketamine and xylazine (1 mg and 0.8 mg per 10g of body weight, respectively). Body temperature was maintained at 37°C using a heating pad (FHC Inc.). Animals were then tested using the RZ6 Multi-I/O Processor System coupled to the RA4PA 4-channel Medusa Amplifier (Tucker-Davis Technologies). Subdermal needles were used as electrodes, with the active electrode inserted at the cranial vertex, the reference electrode under the left ear, and the ground electrode at the right thigh. ABRs were recorded after binaural stimulation in an open field by tone bursts at 8, 16, 32, and 40 kHz generated at 21 stimuli/second, and a waveform for each frequency/dB level was produced by averaging the responses from 512 stimuli. ABR thresholds were obtained for each frequency by reducing the sound pressure level (SPL) by 5 decibels (dB) between 90 and 20 dB. We compared waveforms by simultaneously displaying 3 or more dB levels on screen at the same time to identify the lowest level at which an ABR waveform could be recognized. Wave I amplitudes were measured by annotating the peak and trough of the first ABR waveform and calculating the difference (nV), and wave I delay was measured at the peak of the first wave (ms). In these experiments, controls consist of a pool of <em>0 (Cre-negative); Nrxn3<sup>flox/+</sup></em>, <em>0; Nrxn3<sup>flox/flox</sup></em>, and <em>Atoh1-Cre; Nrxn3<sup>flox/+</sup></em><sup> </sup>animals. Each control genotype was also compared separately (Fig S17C-D). <strong> </strong><strong>Zebrafish startle behavior</strong> A Zantiks MWP behavioral system was used to examine acoustic startle responses. Behavioral trials were performed at 5 dpf, on three independent days. For this behavioral analysis, we compared <em>nrxn3a<sup>+/-</sup>; nrxn3b<sup>+/-</sup></em><sup> </sup>double heterozygotes to <em>nrxn3a<sup>-/-</sup>; nrxn3b<sup>-/-</sup></em><sup> </sup>double mutants for an in-clutch, sibling comparison. <em>Nrxn3a<sup>+/-</sup>; nrxn3b<sup>+/-</sup></em><sup> </sup>double heterozygotes showed a slight (12%) yet significant reduction in complete synapses compared to wild-type controls. We also compared <em>nrxn3a<sup>+/-</sup>; nrxn3b<sup>+/-</sup></em><sup> </sup>double heterozygotes and <em>nrxn3a<sup>-/-</sup>; nrxn3b<sup>-/-</sup></em><sup> </sup>double mutants sibling to wild-type animals born the same day; this analysis revealed no difference in startle response between these genotypes. The Zantiks system tracked and monitored behavioral responses via a built-in infrared camera at 30 frames per second. A 12-well plate was used to house larvae during behavioral analysis. Each well was filled with E3 and 1 larva. All fish were acclimated in the plate within the Zantiks chamber in the dark for 15 min before each test. To induce startle, an integrated stepper motor was used to drive a vibration-induced startle response. A vibrational stimulus that triggered a maximal % of animals startling in controls without any tracking artifacts (due to the vibration), was used for our strongest stimuli. Each larva was presented with each vibrational stimulus 5 times with 100 s between trials. For each animal, the proportion of startle responses out of the 5 trials was plotted. During the tracking and stimulation, a Cisco router connected to the Zantiks system was used to relay <em>x</em>, <em>y</em> coordinates of each larva every frame. To qualify as a startle response, a distance above 4 pixels or ~1.9 mm was required within 2 frames after stimulus onset. Animals were excluded from our analysis if no tracking data was recorded for the animal.  
description: # Presynaptic Nrxn3 is essential for ribbon-synapse maturation in hair
 cells
 [https://doi.org/10.5061/dryad.0k6djhb95](https://doi.org/10.5061/dryad.0k6djhb95) ## Description of the data and file structure Title of Dataset **Presynaptic Nrxn3 is essential for ribbon-synapse maturation** **in hair cells** Summary Paper associated with dataset: Development (2024) [https://doi.org/10.1242/dev.202723](https://doi.org/10.1242/dev.202723) This dataset includes raw data from the following data collected at the NIH/NIDCD and Data collected at the Jackson Laboratory: Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure S3 Figure S4 Figure S5 Figure S6 Figure S7 Figure S8 Figure S9 Figure S10 Figure S11 Figure S13 Figure S14 Figure S15 Figure S16 Figure S17 This dataset includes immunolabeling to examine ribbon synapses in zebrafish lateral-line hair cells (L1-L4 at 5 dpf) in wild type, alpha-nrxn3a, alpha-nrxn3b, alpha-nrxn3a; alpha-nrxn3b germline mutants, and alpha-nrxn3a; alpha-nrxn3b and beta-nrxn3a; beta-nrxn3b crispants (Figure 2,3,5, Figure S5,S10). Immunolabeling of ribbon synapses in zebrafish control and alpha-nrxn3a; alpha-nrxn3b germline mutant in the inner ear (Figure S7,S8). Synapse labeling in the context of hair cell orientation or single, lateral line afferent terminals was also examined (Figure 4). This data shows that there are fewer synapse formed in alpha-nrxn3a; alpha-nrxn3b germline and crispants in zebrafish lateral line and inner ear hair cells.  RNA-FISH label of alpha-nrxn3a and alpha-nrxn3b mRNA show localization in zebrafish hair cells (Figure 1, Figure S3,S4,S9).  In zebrafish, pre- and post-synaptic calcium imaging in lateral-line hair cells and acoustic startle behavior for control and alpha-nrxn3a; alpha-nrxn3b germline zebrafish mutants are also included (Figure 7, 8, S14,S15,S16). This data shows reduced pre- and post-synaptic calcium responses, but no change in acoustic startle behavior.  Lastly zebrafish supporting cell counts, afferent terminal areas, and hair cell counts were examined in control and alpha-nrxn3a; alpha-nrxn3b germline mutants (Figure 4, S6, S11). No differences were observed in these quantifications.  This dataset also examines immunolabeling of ribbon synapses in mouse inner hair cells in controls and Nrxn3 mutants at P28 and P43 (Figure 6 and Figure S13). There are fewer ribbon synapases in Nrxn3 mutants. It also includes mouse auditory brain stem responses from controls and Nrxn3 mutants at P28-P32 (Figure 8 and Figure S17). The auditory brain stem responses were the same in controls and Nrxn3 mutants.  Folders included in this submission have the following names: RAW_DATA_Figure 1 RAW_DATA_Figure 2 RAW_DATA_Figure 3 RAW_DATA_Figure 4 RAW_DATA_Figure 5 RAW_DATA_Figure 6 RAW_DATA_Figure 7_Figure S14_Figure S15_Figure S16 RAW_DATA_Figure 8_Figure S17 RAW_DATA_Figure S3 RAW_DATA_Figure S4 RAW_DATA_Figure S5 RAW_DATA_Figure S6 RAW_DATA_Figure S7 RAW_DATA_Figure S8 RAW_DATA_Figure S9 RAW_DATA_Figure S10 RAW_DATA_Figure S11 RAW_DATA_Figure S13  Outlined below is information pertaining to each of these Data folders.  RAW_DATA_Figure 1 Summary of Figure 1 folder contents: This folder contains 2 Zeiss Airyscan processed images. There are the 2 Zeiss Airyscan processed images that represent examples shown in Figure 1. These files end in .czi. This is data acquired from wild type or transgenic myo6b:memGCaMP6s animals. Zebrafish were fixed and stained at day 5. Image of a lateral line neuromast HCR labeled with alpha-nrxn3a and alpha-nrxn3b mRNA probes: HCR_nrxn3a3b_caxax-nrxn3a-nrxn3b_20221209_S2C1_r2f4_L2-Airyscan Processing.czi Information on RNA-FISH (HCR): To detect mRNA for *nrxn3a and nrxn3b* in zebrafish, we followed the Molecular Instrument-RNA FISH Zebrafish protocol, Revision Number 10 ([https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf](https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf)), with a few minor changes to the preparation of fixed whole-mount larvae. For our dehydration steps, we dehydrated using the following methanol series: 25, 50, 75, 100, 100% methanol, with 5 min for each step in the series. To permeabilize, we treated larvae with 10 µg/mL proteinase K for 20 min. RNA FISH probes were designed to target the long α form of zebrafish *nrxn3a* and *nrxn3b* (Molecular Instrument Probe lot # PRP848, PRP849). In addition to *nrxn3a* and *nrxn3b *probes, we also used *otofb* (Figure S4I) or bacterial *dapB* (Figure S4B,D) probes as positive or negative controls respectively (Molecular Instrument Probe lot # PRP850 (*otofb*, 488 nm) RTHE541 (*dapB*, 546 nm) and RTH406 (*dapB*, 647 nm)). After completing the RNA FISH protocol, we mounted the larvae in ProLong Gold Antifade (ThermoFisher, P36930) under 1.5 coverglass. Image of a wild type lateral-line neuromast immunostained with Myo7a, CTBP and Maguk: Syn_nrxn3a3b_mag-ctbp-m7a-phallo_20220908_S1C1_r2f2_L3-Airyscan Processing.czi Information on immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5hrs at 4°C. After fixation, larvae were washed 5 x 5 min in 0.1% Tween in PBS (PBST). Prior to block, 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 -20°C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 5 x 5 min in PBST. Larvae were then blocked overnight at 4°C 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) overnight, nutating at 4°C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2hrs at room temperature. After 5 x 5 min washes min in PBST to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: rabbit anti-Myosin7a (Proteus Biosciences 25-6790; 1:1000); mouse anti-ribeyeb (IgG2a) ((Sheets *et al.*, 2011); 1:10,000)   and mouse anti-MAGUK (IgG1) (Sigma Aldrich MABN72; 1:500). With the following secondary antibodies were used at 1:1000: # A-11008, # A-21137, # A-21240 and #A-12379 (phalloidin 488 to label hair bundles) (ThermoFisher Scientific). Metadata for acquisition of the 2 .czi images: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen 3.4 Blue) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639.\ Laser powers: 0.1% for 488 and 561 0.2% for 639 Gain = 800 all laser lines Zoom: 4.5-5x Pixel size 0.043 um 633x633 pixels 0.16 microns per slice AutoProcessed in 2D in Zen 3.4 blue HCR_nrxn3a3b_caxax-nrxn3a-nrxn3b_20221209_S2C1_r2f4_L2-Airyscan Processing.czi: Channel 1= alpha-nrxn3b mRNA, Channel 2 = alpha-nrxn3a mRNA, Channel 3 = memGCaMP6s Syn_nrxn3a3b_mag-ctbp-m7a-phallo_20220908_S1C1_r2f2_L3-Airyscan Processing.czi: Channel 1= Maguk, Channel 2 = CTBP, Channel 3 = Myo7a and phalloidin These images processed in FIJI (partial max projections) and used as example images in the Figure 1. RAW_DATA_Figure 2 Summary of folder contents: This folder contains  21 Zeiss Airyscan processed images and one excel file. There are 21 Zeiss Airyscan processed images that represent the raw data in Figure 2. These files end in .czi. This is data acquired from 10 wild type and 11 nrxn3a;nrxn3b mutant neuromasts. These data are fixed samples that label hair cells (Myosin7), ribbons (CTBP), postsynapses (Maguk) and hair bundles (Phalloidin) in neuromasts (L1-L4 ) of the posterior lateral line. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the excel file .xlsx. Information on immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5hrs at 4°C. After fixation, larvae were washed 5 x 5 min in 0.1% PBS + 1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). Larvae were then blocked overnight at 4°C 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 4°C. The next day, the larvae were washed for 5 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. After 5 x 5 min washes min in PBDTT to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: rabbit anti-Myosin7a (Proteus Biosciences 25-6790; 1:1000); mouse anti-CTBP (IgG2a) (Santa Cruz sc-3878); 1:1000)  and mouse anti-MAGUK (IgG1) (Sigma Aldrich MABN72; 1:500). With the following secondary antibodies were used at 1:1000: # A-11008, # A-21137, # A-21240 along with #A-12379 (phalloidin 488 to label hair bundles)  (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen 3.4 Blue) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639.\ Laser powers: 0.1% for 488 and 561 0.2% for 639 Gain = 800 all laser lines Zoom: 4.5-5x Pixel size 0.043 um 512x512 to 704x704 pixels 0.15 microns per slice AutoProcessed in 2D in Zen 3.4 blue Channel 1= Maguk, Channel 2 = CTBP, Channel 3 = Myo7a and phalloidin The 21 Zeiss Airyscan files generated are as follows: Wild type: SO_nrxn3a3b_d5_20220908KK_S1C1_r1f1_L3-AP-01 SO_nrxn3a3b_d5_20220908KK_S1C1_r1f1_L4-AP-02 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f1_L3-AP-09 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f1_L4-AP-10 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f2_L2-AP-11 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f2_L3-AP-12 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f3_L2-AP-13 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f3_L3-AP-14 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f4_L3-AP-15 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f4_L4-AP-16 Nrxn3a;nrxn3b mutants: SO_nrxn3a3b_d5_20220908KK_S1C1_r1f2_L2-AP-03 SO_nrxn3a3b_d5_20220908KK_S1C1_r1f2_L3-AP-04 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f6_L2-AP-19 SO_nrxn3a3b_d5_20220908KK_S1C1_r2f6_L4-AP-20 SO_nrxn3a3b_d5_20220908KK_S1C2_r1f4_L2-AP-26 SO_nrxn3a3b_d5_20220908KK_S1C2_r1f4_L3-AP-27 SO_nrxn3a3b_d5_20220908KK_S1C2_r1f6_L1-AP-16 SO_nrxn3a3b_d5_20220908KK_S1C2_r1f6_L2-AP-17 SO_nrxn3a3b_d5_20220908KK_S1C2_r1f7_L1-AP-18 SO_nrxn3a3b_d5_20220908KK_S1C2_r1f8_L3-AP-26 SO_nrxn3a3b_d5_20220908KK_S1C2_r1f8_L4-AP-25 The *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro script was used to batch process the .czi images in the folder and to generate the data presented in Figure 2 G-J. The *SO_nrxn3a3b_d5_20220908_dryad.*xlsx file contains the synapse counting results derived from the *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro. This data includes a data table Syn Scores_DM-WT that lists the filename (column a), genotype (column b), hair cells per neuromast (column c), complete synapses per hair cell (column d), total presynapses per hair cell (column e), unpaired presynapses per hair cell (column f), total postsynapses per hair cell (column g), unpaired postsynapses per hair cell (column h) for each image. This data is plotted in Figure 2G-J. The following files: SO_nrxn3a3b_d5_20220908KK_S1C1_r2f2_L3-AP-12 SO_nrxn3a3b_d5_20220908KK_S1C2_r1f4_L3-AP-27 Were processed in FIJI (partial max projections) and used as example images in the Figure 2A-F. RAW_DATA_Figure 3 Summary of folder contents: This folder contains 30 Zeiss Airyscan processed images and one excel file. There are 30 Zeiss Airyscan processed images that represent the raw data in Figure 3. These files end in .czi. This is data acquired from 17 wild type and 13 nrxn3a;nrxn3b mutant neuromasts. These data are fixed samples that label hair cells (Myosin7), ribbons (CTBP) and postsynapses (Maguk) in neuromasts (L1-L4 ) of the posterior lateral line. Zebrafish were fixed and stained at day 3. The data extracted from these files is summarized in the excel file .xlsx. Information on immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5hrs at 4°C. After fixation, larvae were washed 5 x 5 min in 0.1% PBS + 1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). Larvae were then blocked overnight at 4°C 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 4°C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2hrs at room temperature. After 5 x 5 min washes min in PBDTT to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: rabbit anti-Myosin7a (Proteus Biosciences 25-6790; 1:1000); mouse anti-CTBP (IgG2a) (Santa Cruz sc-3878); 1:1000)   and mouse anti-MAGUK (IgG1) (Sigma Aldrich MABN72; 1:500). With the following secondary antibodies were used at 1:1000: # A-11008, # A-21137, # A-21240 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen 3.4 Blue) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639.\ Laser powers: 0.1% for all laser lines Gain = 800 all laser lines Zoom: 5x Pixel size 0.043 um 633x633 pixels 0.15 microns per slice AutoProcessed in 2D in Zen 3.4 blue The 30 Zeiss Airyscan files generated are as follows: Wild type: Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f2_L1-AP-40 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f2_L2-AP-39 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f2_L4-AP-38 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f3_L1-AP-37 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f3_L2-AP-36 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f3_L4-AP-35 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f8_L1-AP-24 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f8_L2-AP-23 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f8_L4-AP-22 Syn_nrxn3a3b_d3_20230527KK_S1C1_r2f1_L1-AP-21 Syn_nrxn3a3b_d3_20230527KK_S1C1_r2f1_L2-AP-20 Syn_nrxn3a3b_d3_20230527KK_S1C1_r2f1_L4-AP-19 Syn_nrxn3a3b_d3_20230527KK_S1C1_r2f6_L2-AP-06 Syn_nrxn3a3b_d3_20230527KK_S1C1_r2f6_L3-AP-05 Syn_nrxn3a3b_d3_20230527KK_S1C1_r2f6_L5-AP-04 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f4_L1-AP-08 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f6_L1-AP-04 Nrxn3a;nrxn3b mutants: Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f5_L3-AP-30 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f5_L5-AP-29 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f6_L2-AP-27 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f6_L3-AP-28 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f2_L2-AP-14 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f2_L2b-AP-12 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f2_L4-AP-13 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f3_L2-AP-11 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f3_L4-AP-10 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f3_L5-AP-09 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f7_L2-AP-03 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f7_L5-AP-02 Syn_nrxn3a3b_d3_20230527KK_S1C1_r3f8_L2-AP-01 The *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro script was used to batch process the .czi images in the folder and* *to generate the data presented in Figure 3 G-J. The *Syn_nrxn3a3b_d3_20230527_dryad.*xlsx file contains the synapse counting results derived from the .czi *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro. This data includes the filename, genotype, hair cells per neuromast, complete synapses per hair cell , total presynapses per hair cell, unpaired presynapses per hair cell, total postsynapses per hair cell, unpaired postsynapses per hair cell genotype for each image. The following files: Syn_nrxn3a3b_d3_20230527KK_S1C1_r2f1_L4-AP-19 Syn_nrxn3a3b_d3_20230527KK_S1C1_r1f6_L3-AP-28 Were processed in FIJI (partial max projections) and used as example images in the Figure 3. RAW_DATA_Figure 4 Summary of folder contents: This folder contains 44 Zeiss Airyscan processed images and two excel files. There are 44 Zeiss Airyscan processed images that represent the raw data in Figure 4 D-G. These files end in .czi. This is data acquired from 21 wild type and 23 nrxn3a;nrxn3b mutant neuromasts. These data are fixed samples that label single afferent neuron via neurod1:tdTomato embryo injection, ribbons (CTBP), postsynapses (Maguk) and hair bundles (phalloidin) to detect orienation in neuromasts (L1-L5 ) of the posterior lateral line. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the excel file .xlsx. Single afferent neuron labeling: To visualize the innervation pattern of single afferent neurons, we injected a neurod1:tdTomato plasmid at 10 ng/µl, along with 10 ng/µl tol2 mRNA, into zebrafish embryos at the 1-cell stage. We screened larvae for tdTomato expression at 3 dpf. Positively identified larvae were prepared for immunostaining at 5 dpf, imaged and analyzed as outlined below. Information on immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5hrs at 4°C. After fixation, larvae were washed 5 x 5 min in 0.1% PBS + 1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). Larvae were then blocked overnight at 4°C 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 4°C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2hrs at room temperature. After 5 x 5 min washes min in PBDTT to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: mouse anti-CTBP (IgG2a) (Santa Cruz sc-3878); 1:1000)   and mouse anti-MAGUK (IgG1) (Sigma Aldrich MABN72; 1:500). With the following secondary antibodies were used at 1:1000: # A-21241,#A-21121, # A-12379/ # A-30104 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen 3.4 Blue) using a 63x 1.4 NA oil objective lens and the following lasers lines: 405, 488, 561 and 639.\ Laser powers: 0.2% for 405nm, 0.1-0.3% for 488nm, 0.7-1.5% 561nm, 0.08% for 639nm. Gain = 850 all laser lines Zoom: 5x Pixel size 0.043 um 633x633 pixels 0.15 microns per slice AutoProcessed in 2D in Zen 3.4 blue Channel configurations of Images with 3 channels: Channel 1= CTBP, Channel 2 = tdTomato, Channel 3 = Maguk and phalloidin Channel configurations of Images with 4 channels: Channel 1= CTBP, Channel 2 = tdTomato, Channel 3 = Maguk, Channel 4 = phalloidin The 44 Zeiss Airyscan files generated are as follows: Wild type: SingleAff_nrxn3a3b_d5_20221013_S1C1_r2f6_L2-AP-06 SingleAff_nrxn3a3b_d5_20221013_S1C1_r3f4_L1-AP-18 SingleAff_nrxn3a3b_d5_20221013_S1C2_r3f5_L3-AP-11 SingleAff_nrxn3a3b_d5_20221026_S1C1_r3f1_L2-AP-03 SingleAff_nrxn3a3b_d5_20221026_S1C2_r3f3_L3-AP-19 SingleAff_nrxn3a3b_d5_20221026_S2C1_r2f8_L1-AP-22 SingleAff_nrxn3a3b_d5_20240529-WT_S2C1_r1f2_L2-AP-06 SingleAff_nrxn3a3b_d5_20240529-WT_S2C1_r1f3_L2-AP-08 SingleAff_nrxn3a3b_d5_20240605-WT_S3C1_r1f3_L4-AP-14 SingleAff_nrxn3a3b_d5_20240605-WT_S3C1_r2f2_L2-AP-17 SingleAff_nrxn3a3b_d5_20240605-WT_S3C1_r2f3_L2-AP-20 SingleAff_nrxn3a3b_d5_20221013_S1C1_r1f4_L1-AP-20 SingleAff_nrxn3a3b_d5_20240529_S1C1_r1f2_L2-AP-01 SingleAff_nrxn3a3b_d5_20240529_S1C1_r1f2_L3-AP-02 SingleAff_nrxn3a3b_d5_20240605-WT_S3C1_r2f2_L4-AP-18 SingleAff_nrxn3a3b_d5_20240605-WT_S3C1_r2f2_L5-AP-19 SingleAff_nrxn3a3b_d5_20240605-WT_S3C2_r1f1_L1-AP-02 SingleAff_nrxn3a3b_d5_20240605-WT_S3C2_r1f3_L4-AP-03 SingleAff_nrxn3a3b_d5_20240605-WT_S3C2_r1f4_L1-AP-04 SingleAff_nrxn3a3b_d5_20240605-WT_S3C2_r1f4_L3-AP-05 SingleAff_nrxn3a3b_d5_20240605-WT_S3C2_r2f1_L4-AP-10 Nrxn3a;nrxn3b mutants: SingleAff_nrxn3a3b_d5_20221013_S1C2_r2f6_L4-AP-12 SingleAff_nrxn3a3b_d5_20221013_S2C1_r2f4_L2-AP-09 SingleAff_nrxn3a3b_d5_20221013_S2C1_r3f1_L4-AP-04 SingleAff_nrxn3a3b_d5_20221026_S1C2_r3f2_L3-AP-18 SingleAff_nrxn3a3b_d5_20240529_S1C1_r1f5_L3-AP-04 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C1_r1f3_L2-AP-16 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C1_r2f1_L2-AP-04 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C1_r2f2_L2-AP-05 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C1_r2f5_L5-AP-12 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C2_r2f4_L4-AP-03 SingleAff_nrxn3a3b_d5_20240605-Mut_S2C1_r1f1_L2-AP-05 SingleAff_nrxn3a3b_d5_20221013_S1C1_r2f4_L1-AP-02 SingleAff_nrxn3a3b_d5_20221013_S1C1_r2f4_L4-AP-03 SingleAff_nrxn3a3b_d5_20221013_S1C1_r2f5_L4-AP-04 SingleAff_nrxn3a3b_d5_20221013_S2C2_r3f3_L2-AP-02 SingleAff_nrxn3a3b_d5_20221026_S1C1_r2f2_L2-AP-01 SingleAff_nrxn3a3b_d5_20221026_S1C2_r1f5_L1-AP-13 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C1_r1f3_L3-AP-17 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C1_r1f6_L2-AP-02 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C1_r2f2_L3-AP-06 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C2_r1f2_L5-AP-04 SingleAff_nrxn3a3b_d5_20240605-Mut_S1C2_r2f4_L3-AP-02 SingleAff_nrxn3a3b_d5_20240605-Mut_S2C1_r1f2_L2-AP-06 Synapse counts, hair cell counts, hair cell orientation, and afferent contact sites were were scored manually from 44 .czi image files in the folder. This data is summarized in *SingleAff_nrxn3a3b_d5_scores_dryad.xlsx *in the table WT vs DM. In this table the filename, the genotype, hair cell orientation innervated by each afferent neuron (A to P) or (P to A), the number of complete synapse formed by each afferent neuron (Syn aff+), the percent of hair cells innervated by the afferent neuron out of the total hair cells in the neuromast (covering rate), and the number preferred hair cells innervated/total number of hair cells innervated by the afferent neuron (Selectivity index) is shown. The following files: SingleAff_nrxn3a3b_20240605-Mut_S1C2_r1f2_L5-AP-04.czi SingleAff_nrxn3a3b_20240605-WT_S3C1_r2f2_L4-AP-18.czi Were processed in FIJI (partial max projections) and used as example images in the Figure 4. The *SO_nrxn3a3b_d5_20220908KK_scores_AvsP_dryad.xlsx *file contains the synapse counting results derived from the same dataset in Figure 2G-J using the *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro, orientations of which are manually assigned. This data includes the filename, genotype, hair cells number per orientation in each neuromast (A to P) and (P to A) and the number complete synapses per hair cell of each orientation. RAW_DATA_Figure 5 Summary of folder contents: This folder contains 2 folders that contain in total 34 Zeiss Airyscan processed images. In addition, this folder contains 3 excel file. There are 2 subfolders cav1.3_CTBP_Myo7a and cav1.3_Maguk_Otoferlin that contain 34 Zeiss Airyscan processed images that represent the raw data in Figure 5E-L. These files end in .czi. This is data acquired from wild type and nrxn3a;nrxn3b mutant neuromasts. These data are fixed samples that label hair cells (Myosin7 or Otoferlin), ribbons (CTBP) or postsynapses (Maguk) and Cav1.3 channels in neuromasts (L1,L2 and DV1) of the posterior lateral line. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the .xlsx excel files contained in each of these two subfolders. The third excel file contains presynapse and postsynase area quantification derived from the raw data in Figure 2, shown in Figure 5A-D. Information on immunostaining for Figure 5: Whole larvae were fixed with paraformaldehyde (PFA 4%; Thermoscientific; 28906) in PBS at 4°C for 3.5hrs. For CaV1.3 labeling antibody solutions were prepared with PBS + 0.1% Tween (PBST). After fixation, larvae were washed 4 x 5 min in PBST. Prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H2O in glass vials. The H2O was removed and replaced with ice-cold acetone and larvae 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 then were blocked overnight at 4°C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). After block, larvae were incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST) overnight, nutating at 4°C. 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 2hrs at room temperature, with minimal exposure to light. Secondary antibodies were removed by washing with PBST for 4 x 5 min. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using No. 1.5 coverslips. The following primary antibodies were used: rabbit anti-Cav1.3 ((Sheets et al., 2011); 1:1000), mouse anti-CTBP (IgG2a) (Santa Cruz sc-3878); 1:1000) and mouse anti-Myosin7a (IgG1) (Developmental Study Hybridoma Bank 138-1; 1:1000). With the following secondary antibodies at 1:1000: # A-11008, # A-21127, # A-21241 (ThermoFisher Scientific). OR The following primary antibodies were used: and rabbit anti-Cav1.3 ((Sheets et al., 2011); 1:1000), mouse anti-Maguk (IgG1) (Santa Cruz sc-3878); 1:500) and mouse anti-Otoferlin (IgG2a) (Developmental Study Hybridoma Bank 138-1; 1:1000). With the following secondary antibodies at 1:1000: # A-11008, # A-21127, # A-21241 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen Black) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 543 and 633.\ Laser powers: 0.03% (Cav1.3) for 488 and 0.1% (Maguk) or 0.1% CTBP for  546 and 0.03% (myo7a or Otoferlin) for 633 Gain = 800 all laser lines Zoom: 5.0x Pixel size 0.043 um 608x608 0.17 microns per slice Processed in 3D in Zen Black with a processing factor of 7.0 Channel 1 = CTBP or Otoferlin, Channel 2 = Myo7a or Maguk, Channel 3 = Cav1.3 The 34 Zeiss Airyscan files generated are as follows: For the staining and subfolder: cav1.3_CTBP_Myo7a Wild type: S1 CS1 r1F1_L1.czi S1 CS1 r1F4_L1.czi S1 CS1 r1F5_L1.czi S1 CS1 r1F6_L1.czi S1 CS1 r1F7_L1.czi S1 CS1 r2F17_L1.czi S1 CS1 r2F19_L1.czi S1C1_r1F6_DV1.czi S1C1_r2F17_DV1.czi S1C1_r2F19_DV1.czi Nrxn3a;nrxn3b S1 CS1 r1F8_L1.czi S1 CS1 r2F11_L1.czi S1 CS1 r2F12_L1.czi S1 CS1 r2F13_L1.czi S1 CS1 r2F14_L1.czi S1 CS1 r2F16_L1.czi S1 CS1 r2F18_L1.czi S1 CS1 r2F20_L1.czi S1 CS1 r2F21_L1.czi S1C1_r2F11_DV1.czi S1C1_r3F21_DV1.czi For the staining and subfolder: cav1.3_Maguk_Otoferlin 092822_S1C1_F01_L1.czi 092822_S1C1_F08_L1.czi 092822_S1C1_F14_L1.czi 092822_S1C1_F14_L2.czi 110222_S1C2_F03_L1.czi 110222_S1C2_F10_L1.czi 092822_S1C1_F09_DV1.czi 092822_S1C1_F14_DV1.czi 110222_S1C2_F03_DV1.czi 110222_S1C2_F12_DV1.czi Nrxn3a;nrxn3b mutants: 092822_S1C1_F02_L1.czi 092822_S1C1_F02_L2.czi 092822_S1C1_F07_L1.czi 092822_S1C1_F11_L1.czi 092822_S1C1_F12_L1.czi 110222_S1C2_F07_L1.czi 110222_S1C2_F11_L1.czi 110222_S1C2_F07_DV1.czi The CompleteSynpaseCounter2Dv5.2.ijm Fiji macro*.*ijm Fiji macro script was used to batch process the .czi images in the folder and* *to generate the data presented in Figure 5 I-K. The Cav-CTBP_nrxn3a3b_d5_092822_dryad.xlsx and Cav-Mag_nrxn3a3b_d5_092822_dryad.xlsx files contain the synapse quantification results derived from the CompleteSynpaseCounter2Dv5.2.ijm Fiji macro. Cav-CTBP_nrxn3a3b_d5_092822_dryad.xlsx contains a data table that include the filename, genotype, hair cells per neuromast, Paired CTBP-Cav1.3 puncta per hair cell, area of paired Cav1.3 puncta, intensity of paired Cav.13 puncta. Cav-Mag_nrxn3a3b_d5_092822_dryad.xlsx contains a data table that includes filename, genotype, hair cells per neuromast, Paired Maguk-Cav1.3 puncta per hair cell. The following files: S1C1_r2F19_DV1.czi S1 CS1 r2F21_L1.czi 092822_S1C1_F14_L2.czi 092822_S1C1_F12_L1.czi Were processed in FIJI (partial max projections) and used as example images in the Figure 5E-H. To create the plots in Figure 5A-D, the entire dataset in Figure 2 was analyzed using the CompleteSynpaseCounter2Dv5.2.ijm Fiji macro script to obtain area information for the puncta. This data is plotted in SO_nrxn3a3b_d5_20220908_areas_dryad.xlxs and includes the filename, genotype, area of paired CTBP puncta, area of unpaired CTBP puncta, area of paired Maguk  puncta and area of unpaired Maguk puncta. RAW_DATA_Figure 6 Summary of folder contents: This folder contains 55 Zeiss Airyscan processed images and one excel file. There are 55 Zeiss confocal images that represent the raw data in Figure 6. These files end in .czi. This is data include 24 images acquired from apex, mid and base areas of cochlea in 6 *control* mice and 31 images acquired from apex, mid and base areas of cochlea in 4 *Atoh1-cre; Nrxn3flox/flox* mutant mice. These data are samples that label hair cells (Myosin7), ribbons (CTBP2), postsynapses (GluR2) of the mouse cochlear tissue, fixed and stained at P42 (6weeks). The data extracted from these files is summarized in the excel file .xlsx. Information on immunostaining: Temporal bones were isolated, and an insulin syringe was used to gently flush cold paraformaldehyde (PFA 4%; Electron Microscopy Sciences; 15710) through the cleared oval and round windows after poking a small hole at the cochlear apex. Temporal bones were then immersion-fixed in PFA for 1hr at 4°C, washed in PBS, and rotated overnight in EDTA 4% for decalcification. The next day, cochleae were dissected in 3 approximate thirds (base, mid, and apex) before blocking and permeabilization for 1hr at room temperature under agitation (1% bovine serum albumin; 0.5% Triton X-100). Primary and secondary antibodies were incubated overnight at 4°C in PBS. Samples were washed 3 times in PBS + 0.05% Triton X-100 after each antibody incubation and finally post-fixed in PFA 4% for at least 1hr at room temperature. Samples were then mounted flat in Mowiol mounting medium (Calbiochem/MilliporeSigma 4759041) using two layers of office tape as a spacer for the coverglass (18x18mm #1.5). Mowiol (10% w/v) was prepared in (25% w/v) glycerol and 0.1M Tris-Cl pH8.5. The following primary antibodies were used: mouse anti-Myosin7a (IgG1) (Developmental Study AB_2282417; 1:400); Rabbit anti-CTBP2 (Synaptic Systems 192103; 1:400) and mouse anti-GluR2 (IgG2a) (Millipore/Sigma MAB397; 1:100). With the following secondary antibodies were used at 1:1000: # A-21202, # A-31572, # A-21447 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Mounted samples were imaged on an upright LSM 980 laser-scanning confocal microscope using Zen Blue 3.4 (Carl Zeiss) and an 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639. Z-stacks containing 6-9 IHCs were acquired in confocal mode.\ Laser powers: 0.4-0.6% for 488nm, 0.08-0.2% for 561nm, 0.03-0.2% for 639nm. Gain = 700 all laser lines Zoom: 1.5-2x Pixel size 0.085 um 792x395 pixels 0.25 microns per slice The 55 Zeiss Airyscan files generated are as follows: controls: M6w_AN34_wt-homo_LIE_base_s1 M6w_AN34_wt-homo_RIE_apex_s1 M6w_AN34_wt-homo_RIE_base_s1 M6w_AN37_wt-het_LIE_base_s1 M6w_AN37_wt-het_RIE_apex_s1 M6w_AN37_wt-het_RIE_apex_s2 M6w_AN37_wt-het_RIE_base_s1 M6w_AN37_wt-het_RIE_mid_s1 M6w_AN54_Ctbp2_555_GluR2_488_Myo7_apex_LIE M6w_AN54_Ctbp2_555_GluR2_488_Myo7_apex_RIE M6w_AN54_Ctbp2_555_GluR2_488_Myo7_base_LIE M6w_AN54_Ctbp2_555_GluR2_488_Myo7_base_RIE M6w_AN54_Ctbp2_555_GluR2_488_Myo7_mid_LIE M6w_AN54_Ctbp2_555_GluR2_488_Myo7_mid_RIE M6w_AN56_Ctbp2_555_GluR2_488_Myo7_apex_LIE M6w_AN56_Ctbp2_555_GluR2_488_Myo7_apex_RIE M6w_AN56_Ctbp2_555_GluR2_488_Myo7_base_RIE M6w_AN56_Ctbp2_555_GluR2_488_Myo7_mid_LIE M6w_AN56_Ctbp2_555_GluR2_488_Myo7_mid_RIE M6w_AN58_Ctbp2_555_GluR2_488_Myo7_apex_LIE M6w_AN58_Ctbp2_555_GluR2_488_Myo7_apex_RIE M6w_AN58_Ctbp2_555_GluR2_488_Myo7_base_LIE M6w_AN58_Ctbp2_555_GluR2_488_Myo7_base_RIE M6w_AN58_Ctbp2_555_GluR2_488_Myo7_mid_LIE M6w_AN58_Ctbp2_555_GluR2_488_Myo7_mid_RIE M6w_AN59_Ctbp2_555_GluR2_488_Myo7_apex_LIE M6w_AN59_Ctbp2_555_GluR2_488_Myo7_apex_RIE M6w_AN59_Ctbp2_555_GluR2_488_Myo7_base_LIE M6w_AN59_Ctbp2_555_GluR2_488_Myo7_base_RIE M6w_AN59_Ctbp2_555_GluR2_488_Myo7_mid_LIE M6w_AN59_Ctbp2_555_GluR2_488_Myo7_mid_RIE Mutants* (Atoh1-cre; Nrxn3flox/flox*) M6w_AN35_hemi-homo_LIE_apex_s1 M6w_AN35_hemi-homo_LIE_base_s1 M6w_AN35_hemi-homo_LIE_mid_s1 M6w_AN35_hemi-homo_RIE_apex_s1 M6w_AN35_hemi-homo_RIE_base_s1 M6w_AN35_hemi-homo_RIE_mid_s1 M6w_AN38_hemi-homo_LIE_apex_s1 M6w_AN38_hemi-homo_LIE_base_s1 M6w_AN38_hemi-homo_LIE_mid_s1 M6w_AN38_hemi-homo_RIE_apex_s1 M6w_AN38_hemi-homo_RIE_base_s1 M6w_AN38_hemi-homo_RIE_mid_s1 M6w_AN55_Ctbp2_555_GluR2_488_Myo7_apex_LIE M6w_AN55_Ctbp2_555_GluR2_488_Myo7_apex_RIE M6w_AN55_Ctbp2_555_GluR2_488_Myo7_base_LIE M6w_AN55_Ctbp2_555_GluR2_488_Myo7_base_RIE M6w_AN55_Ctbp2_555_GluR2_488_Myo7_mid_LIE M6w_AN55_Ctbp2_555_GluR2_488_Myo7_mid_RIE M6w_AN57_Ctbp2_555_GluR2_488_Myo7_apex_LIE M6w_AN57_Ctbp2_555_GluR2_488_Myo7_apex_RIE M6w_AN57_Ctbp2_555_GluR2_488_Myo7_base_LIE M6w_AN57_Ctbp2_555_GluR2_488_Myo7_base_RIE M6w_AN57_Ctbp2_555_GluR2_488_Myo7_mid_LIE M6w_AN57_Ctbp2_555_GluR2_488_Myo7_mid_RIE The *Mouse_6w_masterlist_20230614_dryad.xlsx *file contains the synapse counting results derived from *CompleteSynpaseCounter2Dv5.2.ijm*  Fiji macro after the .czi images were manual segmentations of each channels in VVDViewer ([https://github.com/JaneliaSciComp/VVDViewer](https://github.com/JaneliaSciComp/VVDViewer)). Hair cell counting results were scored manually. This .xlsx file has three tabs, each contains data for apex, mid and base region of cochlea respectively. The data in each table includes the filename, summary genotype (mutant or control), the actual genotype, hair cells in imaging regions and the number of complete synapse per hair cell. The following files: M6w_AN59_Ctbp2_555_GluR2_488_Myo7_apex_LIE.czi M6w_AN59_Ctbp2_555_GluR2_488_Myo7_mid_LIE.czi M6w_AN59_Ctbp2_555_GluR2_488_Myo7_base_RIE.czi M6w_AN57_Ctbp2_555_GluR2_488_Myo7_apex_LIE.czi M6w_AN57_Ctbp2_555_GluR2_488_Myo7_mid_LIE.czi M6w_AN57_Ctbp2_555_GluR2_488_Myo7_base_LIE.czi Were processed in VVDViewer (for segmentation) and FIJI (partial max projections), and then used as example images in the Figure 6 A-B. RAW_DATA_Figure 7_Figure S14_Figure S15_Figure S16 Summary of Figure RAW_DATA_Figure 7_Figure S14_Figure S15_Figure S16 folder contents: This folder contains 3 subfolders and 2 Excel files. The subfolders: All Raw Data for HC BASE and All Raw data HC MET contain 112 Nikon .nd2 files, while the subfolder: All Raw Data for Afferent contains 74 folders, each of these folders contains a tif series of 400 images. The Nikon .nd2 and tif series files are the raw calcium imaging data from nrxn3a;nrxn3b mutants and wild-type animals represented in Figure 7, and Figures S14, S15 and S16. The data extracted from these files is summarized in the 2 excel files .xlsx. This is data acquired from in nrxn3a;nrxn3b mutants and wild-type larval zebrafish at 4-6 dpf. The .nd2 files are measurements of mechanosensitive and presynaptic responses from 14 wildtype and 14 nrxn3a;nrxn3b mutants mutant neuromasts (L2,L3) in the posterior lateral line. Responses were made using the following transgenic line: myo6b:GCaMP6scaax. The tif series are measurements of responses in the afferent terminal beneath lateral line hair cells from 14 wildtype and 23 nrxn3a;nrxn3b mutants mutant neuromasts (L1,L2,L3,L4) in the posterior lateral line. Responses were made using the following transgenic line: en.sill,hsp70l:GCaMP6s. To stimulate hair cells a fluidjet was used. A 500ms saturating fluid flow stimulus was used, one acquisition towards the anterior and one acquisition towards the posterior of the fish. The .nd2 data files or folders of tif series are coded with the date acquired, Fish number (F) for each experimental day, the neuromast assayed (L1, L2, L3, L4) and the direction of the stimulus (ANT/PUSH= towards the anterior, POST/PULL= towards the posterior). For afferent measurements, each neuromast was stimulated in 2 directions, anterior or posterior. For hair cell measurements, each neuromast was stimulated in 2 directions, anterior or posterior; this stimulation was applied to measurements taken at the hair cell apex (MET) and hair cell presynapse (BASE). Metadata for GCaMP6s afferent calcium imaging acquisition: Acquired on a Swept field confocal microscope using Prairie-view (Bruker) software Nikon 60x water objective 20ms per image, 1.0 microns per slice, 5 planes per Z-stack (each Z-stack or timepoint is ~0.1s) 400 tif images in total per acquisition (~8s), fluidjet stimulation at frame 150 (~3s) 70 micron slit EM gain 3900 2x2 binning Imaging ROI 128x128 Laser setting 125 The following afferent acquisitions were made: Wild type: SILLGCa6s_082922_d4_nrxn3a3b_F1_L1_500ms_POST-002 SILLGCa6s_082922_d4_nrxn3a3b_F1_L2_500ms_ANT-001 SILLGCa6s_082922_d4_nrxn3a3b_F5_L1_500ms_ANT-001 SILLGCa6s_082922_d4_nrxn3a3b_F5_L2_500ms_POST-001 SILLGCa6s_082922_d4_nrxn3a3b_F5_L3_500ms_ANT-001 SILLGCa6s_083022_d5_F1_L2_500ms_ANT-001 SILLGCa6s_083022_d5_F1_L3_500ms_POST-001 SILLGCa6s_083022_d5_F1_L4_500ms_ANT-001 SILLGCa6s_083022_d5_F5_L1_500ms_ANT-001 SILLGCa6s_083022_d5_F5_L2_500ms_POST-002 SILLGCa6s_083022_d5_F5_L3_500ms_ANT-001 101722_SILL_GC6s_nrxn3a3b_F1_L1_500ms_ANT-001 101722_SILL_GC6s_nrxn3a3b_F1_L2_500ms_POST-001 101722_SILL_GC6s_nrxn3a3b_F1_L3_500ms_ANT-001 SILLGCa6s_082922_d4_nrxn3a3b_F1_L1_500ms_ANT-003 SILLGCa6s_082922_d4_nrxn3a3b_F1_L2_500ms_POST-002 SILLGCa6s_082922_d4_nrxn3a3b_F5_L1_500ms_POST-002 SILLGCa6s_082922_d4_nrxn3a3b_F5_L2_500ms_ANT-002 SILLGCa6s_082922_d4_nrxn3a3b_F5_L3_500ms_POST-002 SILLGCa6s_083022_d5_F1_L2_500ms_POST-002 SILLGCa6s_083022_d5_F1_L3_500ms_ANT-002 SILLGCa6s_083022_d5_F1_L4_500ms_POST-002 SILLGCa6s_083022_d5_F5_L1_500ms_POST-002 SILLGCa6s_083022_d5_F5_L2_500ms_ANT-005 SILLGCa6s_083022_d5_F5_L3_500ms_POST-002 101722_SILL_GC6s_nrxn3a3b_F1_L1_500ms_POST-002 101722_SILL_GC6s_nrxn3a3b_F1_L2_500ms_ANT-002 101722_SILL_GC6s_nrxn3a3b_F1_L3_500ms_POST-002 Nrxn3a;nrxn3b mutants: SILLGCa6s_082922_d4_nrxn3a3b_F3_L1_500ms_ANT-001 SILLGCa6s_082922_d4_nrxn3a3b_F3_L2_500ms_POST-001 SILLGCa6s_082922_d4_nrxn3a3b_F3_L3_500ms_ANT-001 SILLGCa6s_082922_d4_nrxn3a3b_F3_L4_500ms_POST-001 SILLGCa6s_083022_d5_F3_L1_500ms_ANT-001 SILLGCa6s_083022_d5_F3_L2_500ms_POST-001 SILLGCa6s_083022_d5_F3_L3_500ms_ANT-001 SILLGCa6s_083022_d5_F3_L4_500ms_POST-001 SILLGCa6s_083022_d5_F4_L1_500ms_ANT-001 SILLGCa6s_083022_d5_F4_L2_500ms_POST-001 SILLGCa6s_083022_d5_F4_L3_500ms_ANT-001 SILLGCa6s_083022_d5_F4_L4_500ms_POST-001 101722_SILL_GC6s_nrxn3a3b_F4_L1_500ms_ANT-001 101722_SILL_GC6s_nrxn3a3b_F4_L2_500ms_POST-001 101722_SILL_GC6s_nrxn3a3b_F4_L3_500ms_ANT-001 101722_SILL_GC6s_nrxn3a3b_F4_L4_500ms_POST-001 101722_SILL_GC6s_nrxn3a3b_F5_L2_500ms_ANT-001 101722_SILL_GC6s_nrxn3a3b_F5_L3_500ms_POST-001 101722_SILL_GC6s_nrxn3a3b_F5_L4_500ms_ANT-001 101722_SILL_GC6s_nrxn3a3b_F6_L1_500ms_POST-001 101722_SILL_GC6s_nrxn3a3b_F6_L2_500ms_POST-002 101722_SILL_GC6s_nrxn3a3b_F6_L3_500ms_POST-001 101722_SILL_GC6s_nrxn3a3b_F6_L4_500ms_ANT-001 SILLGCa6s_082922_d4_nrxn3a3b_F3_L1_500ms_POST-002 SILLGCa6s_082922_d4_nrxn3a3b_F3_L2_500ms_ANT-003 SILLGCa6s_082922_d4_nrxn3a3b_F3_L3_500ms_POST-002 SILLGCa6s_082922_d4_nrxn3a3b_F3_L4_500ms_ANT-002 SILLGCa6s_083022_d5_F3_L1_500ms_POST-002 SILLGCa6s_083022_d5_F3_L2_500ms_ANT-002 SILLGCa6s_083022_d5_F3_L3_500ms_POST-002 SILLGCa6s_083022_d5_F3_L4_500ms_ANT-002 SILLGCa6s_083022_d5_F4_L1_500ms_POST-002 SILLGCa6s_083022_d5_F4_L2_500ms_ANT-002 SILLGCa6s_083022_d5_F4_L3_500ms_POST-002 SILLGCa6s_083022_d5_F4_L4_500ms_ANT-002 101722_SILL_GC6s_nrxn3a3b_F4_L1_500ms_POST-002 101722_SILL_GC6s_nrxn3a3b_F4_L2_500ms_ANT-002 101722_SILL_GC6s_nrxn3a3b_F4_L3_500ms_POST-002 101722_SILL_GC6s_nrxn3a3b_F4_L4_500ms_ANT-002 101722_SILL_GC6s_nrxn3a3b_F5_L2_500ms_POST-002 101722_SILL_GC6s_nrxn3a3b_F5_L3_500ms_ANT-003 101722_SILL_GC6s_nrxn3a3b_F5_L4_500ms_POST-002 101722_SILL_GC6s_nrxn3a3b_F6_L1_500ms_ANT-002 101722_SILL_GC6s_nrxn3a3b_F6_L2_500ms_ANT-003 101722_SILL_GC6s_nrxn3a3b_F6_L3_500ms_ANT-002 101722_SILL_GC6s_nrxn3a3b_F6_L4_500ms_POST-002 Each acquisition is a series of 400 tif files. Each tif series was processed using the GUI-based ImagePro. This program average projects each z-stack. Then registers the Z-stack. The first second (10 frames) of the recording was removed for baseline stability. Then the resulting 70 frames per recording was then opened in FIJI/ImageJ. The Time Series Analyzer V3 plugin was used to create size circular ROIs (12x12 pixels). ROIs were placed on each responding terminal. 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 (F0). The calculation to subtract background was Percentage different 100*(Value-Baseline)/Baseline. From the background subtracted plots (deltaF/F0), for each neuromast the anterior and posterior responsive sites were determined. For each genotype, the average response magnitude (Max deltaF/F0) and number of responsive sites was determined for each terminal. To measure the baseline GCaMP6s signal in the afferent terminal, the images from the first acquisition were cropped to remove extraneous signals and the intensity in the terminal was measured during the first 20 of the 70 frame. These images were autothresholded in FIJI and the intensity from these 20 frames was averaged to obtain a single baseline measurement. These analyses are summarized in Source Data for nrxn3ab vs WT afferent Calcium Imaging.xlxs file according to date, fish # and neuromast and genotype. The table Afferent Max deltaF F0 lists each neuromast imaged (column A), the genotype (Column B), the age (column C), the Max deltaF/F0 (Column D) and the images file names for each sample (columns E and F). The table Afferent deltaF F0 Final AVG summarizes the average mutant and wild-type response at each time point, along with the SEM. Table Afferent baseline int summarize the average GCaMP6s intensity prior to stimulation (column D). Table Afferent active sites summarizes the total number of active site per neuromast (column C)as well as the number responding to each direction of flow (P to A) and (A to P) (column D and E). These tables represent the data shown in Figure 7I and J, Figure S14C and Figure S16A-B. The examples shown in Figure 7G-H and Figure S15 G-L are derived from the following raw data files: Wild type: SILLGCa6s_083022_d5_F5_L1_500ms_ANT-001 SILLGCa6s_083022_d5_F5_L1_500ms_POST-002 Nrxn3a;nrxn3b mutant: SILLGCa6s_083022_d5_F3_L3_500ms_ANT-001 SILLGCa6s_083022_d5_F3_L3_500ms_POST-002 Metadata for GCaMP6s hair cell calcium imaging acquisition: Acquired on a Nikon A1R confocal microscope using Elements (Nikon) software Nikon 60x water objective 33 ms per image, 0.5 microns for MET and 1.5 microns for BASE, per slice, 3 planes per Z-stack (each Z-stack or timepoint is ~0.1s) 330 images in total per .nd2 acquisition (~11 s), fluidjet stimulation at z-stack 31. 4x averaging Zoom 3x BASE 6x APEX Imaging ROI 128x128 Laser setting APEX 0.45% BASE 1.25% Gain 80 BASE 75 MET The following hair cell presynaptic acquisitions were made: Wild type: 221004_F3_WT_L2_500ms_-0-35V_pull_BASE.nd2 221005_F2_WT_L2_500ms_-0-1V_pull_BASE.nd2 221006_F1_WT_L2_500ms_-0-25V_pull_BASE.nd2 221011_F3_WT_L2_500ms_-0-25V_pull_BASE.nd2 221024_F2_L2_500ms_-0-23V_pull_BASE.nd2 221024_F3_L3_500ms_-0-23V_pull_BASE.nd2 221025_F3_L3_500ms_-0-3V_pull_BASE.nd2 221212_F2_L2_500ms_pull_BASE.nd2 221212_F3_L3_500ms_pull_BASE.nd2 221213_F3_L3_500ms_pull_BASE.nd2 221215_F11_L2_500ms_pull_BASE.nd2 221215_F2_L3_500ms_pull_BASE.nd2 221219_F3_L2_500ms_pull_BASE.nd2 221220_F2_L2_500ms_pull_BASE.nd2 221220_F5_L2_500ms_pull_BASE.nd2 221004_F3_WT_L2_500ms_0-35V_push_BASE.nd2 221005_F2_WT_L2_500ms_0-18V_push_BASE.nd2 221006_F1_WT_L2_500ms_0-25V_push_BASE.nd2 221011_F3_WT_L2_500ms_0-3V_push_BASE.nd2 221024_F2_L2_500ms_0-23V_push_BASE.nd2 221024_F3_L3_500ms_0-23V_push_BASE.nd2 221025_F3_L3_500ms_0-35V_push_BASE.nd2 221212_F2_L2_500ms_push_BASE.nd2 221212_F3_L3_500ms_push_BASE.nd2 221213_F3_L3_500ms_push_BASE.nd2 221215_F11_L2_500ms_push_BASE.nd2 221215_F2_L3_500ms_push_BASE.nd2 221219_F3_L2_500ms_push_BASE.nd2 221220_F2_L2_500ms_push_BASE.nd2 221220_F5_L2_500ms_push_BASE.nd2 Nrxn3a;nrxn3b mutants: 221214_F9_L3_500ms_pull_BASE.nd2 221214_F10_L2_500ms_pull_BASE.nd2 221214_F10_L3_500ms_pull_BASE.nd2 221215_F4_L2_500ms_pull_BASE.nd2 221215_F5_L2_500ms_pull_BASE.nd2 221215_F7_L2_500ms_pull_BASE.nd2 221215_F7_L3_500ms_pull_BASE.nd2 221219_F4_L2_500ms_pull_BASE.nd2 221219_F7_L2_500ms_pull_BASE.nd2 221220_F4_L3_500ms_pull_BASE.nd2 221220_F6_L2_500ms_pull_BASE.nd2 221220_F6_L3_500ms_pull_BASE.nd2 221220_F7_L2_500ms_pull_BASE.nd2 221220_F7_L3_500ms_pull_BASE.nd2 221214_F9_L3_500ms_push_BASE.nd2 221214_F10_L2_500ms_push_BASE.nd2 221214_F10_L3_500ms_push_BASE.nd2 221215_F4_L2_500ms_push_BASE.nd2 221215_F5_L2_500ms_push_BASE.nd2 221215_F7_L2_500ms_push_BASE.nd2 221215_F7_L3_500ms_push_BASE.nd2 221219_F4_L2_500ms_push_BASE.nd2 221219_F7_L2_500ms_push_BASE.nd2 221220_F4_L3_500ms_push_BASE.nd2 221220_F6_L2_500ms_push_BASE.nd2 221220_F6_L3_500ms_push_BASE.nd2 221220_F7_L2_500ms_push_BASE.nd2 221220_F7_L3_500ms_push_BASE.nd2 The following hair cell MET acquisitions were made: Wild type 221004_F3_WT_L2_500ms_-0-35V_pull_MET.nd2 221005_F2_WT_L2_500ms_-0-1V_pull_MET.nd2 221006_F1_WT_L2_500ms_-0-25V_pull_MET.nd2 221011_F3_WT_L2_500ms_-0-25V_pull_MET.nd2 221024_F2_L2_500ms_-0-23V_pull_MET.nd2 221025_F3_L3_500ms_-0-3V_pull_MET.nd2 221212_F2_L2_500ms_pull_MET.nd2 221212_F3_L3_500ms_pull_MET.nd2 221213_F3_L3_500ms_pull_MET.nd2 221214_F1_L2_500ms_pull_MET.nd2 221215_F11_L2_500ms_pull_MET.nd2 221215_F2_L3_500ms_pull_MET.nd2 221219_F3_L2_500ms_pull_MET.nd2 221220_F2_L2_500ms_pull_MET.nd2 221220_F5_L2_500ms_pull_MET.nd2 221004_F3_WT_L2_500ms_0-35V_push_MET.nd2 221005_F2_WT_L2_500ms_0-18V_push_MET.nd2 221006_F1_WT_L2_500ms_0-25V_push_MET.nd2 221011_F3_WT_L2_500ms_0-3V_push_MET.nd2 221024_F2_L2_500ms_0-23V_push_MET.nd2 221025_F3_L3_500ms_0-35V_push_MET.nd2 221212_F2_L2_500ms_push_MET.nd2 221212_F3_L3_500ms_push_MET.nd2 221213_F3_L3_500ms_push_MET.nd2 221214_F1_L2_500ms_push_MET.nd2 221215_F11_L2_500ms_push_MET.nd2 221215_F2_L3_500ms_push_MET.nd2 221219_F3_L2_500ms_push_MET.nd2 221220_F2_L2_500ms_push_MET.nd2 221220_F5_L2_500ms_push_MET.nd2 Nrxn3a;nrxn3b 221214_F9_L3_500ms_pull_MET.nd2 221214_F10_L2_500ms_pull_MET.nd2 221214_F10_L3_500ms_pull_MET.nd2 221215_F4_L2_500ms_pull_MET.nd2 221215_F5_L2_500ms_pull_MET.nd2 221215_F7_L3_500ms_pull_MET.nd2 221219_F4_L2_500ms_pull_MET.nd2 221219_F7_L2_500ms_pull_MET.nd2 221220_F4_L3_500ms_pull_MET.nd2 221220_F6_L2_500ms_pull_MET.nd2 221220_F6_L3_500ms_pull_MET.nd2 221220_F7_L2_500ms_pull_MET.nd2 221220_F7_L3_500ms_pull_MET.nd2 221214_F9_L3_500ms_push_MET.nd2 221214_F10_L2_500ms_push_MET.nd2 221214_F10_L3_500ms_push_MET.nd2 221215_F4_L2_500ms_push_MET.nd2 221215_F5_L2_500ms_push_MET.nd2 221215_F7_L3_500ms_push_MET.nd2 221219_F4_L2_500ms_push_MET.nd2 221219_F7_L2_500ms_push_MET.nd2 221220_F4_L3_500ms_push_MET.nd2 221220_F6_L2_500ms_push_MET.nd2 221220_F6_L3_500ms_push_MET.nd2 221220_F7_L2_500ms_push_MET.nd2 221220_F7_L3_500ms_push_MET.nd2 Each .nd2 acquisition is a series of 330 images. Each nd2 files was exported into a tif series and processed using the GUI-based ImagePro. This program average projects each z-stack. Then registered the Z-stack. The first second (10 frames) of the recording is removed for . stability. Then the resulting 100 frames per recording were opened in FIJI/ImageJ. The Time Series Analyzer V3 plugin was used to create size circular ROIs (18x18 pixels presynaptic; 8x8 pixels hair bundle). ROIs were placed on each responding hair bundle or presynaptic site. 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 (F0). The calculation to subtract background was Percentage different 100*(Value-Baseline)/Baseline. From the background subtracted plots (deltaF/F0), for each neuromast the anterior and posterior responsive sites were determined. For each genotype, the average response magnitude (Max deltaF/F0) and number of responsive sites was determined for each neuromast. To measure the baseline GCaMP6s signal in the hair bundles or presynaptic sites, the image sequence from the first acquisition were cropped to remove extraneous signals and the intensity in the hair bundles or cell soma was measured during the first 20 frames. These 20 images were autothresholded in FIJI and the intensity from these 20 frames was averaged to obtain a single baseline measurement. These analyses are summarized in Source Data for nrxn3ab vs WT Hair cell Calcium Imaging.xlxs file. The table MET Max deltaF F0 lists each neuromast imaged (column A), the genotype (column B), the Max deltaF/F0 (column D) and the images file names for each sample (columns D and E). The table MET deltaF F0 Final AVG summarizes the average mutant and wild-type response at each time point, along with the SEM. The table Presynaptic Max deltaF F0 lists each neuromast imaged (column A), the genotype (column B), the Max deltaF/F0 (Column D) and the images file names for each sample (columns D and E). The Presynaptic MET deltaF F0 Final AVG summarizes the average mutant and wild-type response at each time point, along with the SEM. Table GCaMP6s baseline INT summarize the average MET or presynaptic GCaMP6s intensity prior to stimulation (columns C and D). These tables represent the data shown in Figure 7 D and E, and Figure S14 A-B and E-F. The examples shown in Figure 7B-C and Figure S15 A-F are derived from the following raw data files: Wild type: 221219_F3_L2_500ms_pull_BASE.nd2 221219_F3_L2_500ms_push_BASE.nd2 Nrxn3a;nrxn3b mutant: 221220_F6_L3_500ms_pull_BASE.nd2 221220_F6_L3_500ms_push_BASE.nd2 RAW_DATA_Figure 8 Summary of Figure 8 folder contents: This folder contains 2 subfolders containing zebrafish startle data and mouse auditory brain stem behavior. The subfolder Zebrafish startle data contains subfolders named by date of experiment; each of these dated subfolders contains additional folders named by the plates run each day. Each plate is represented by 20 .avi videos and one .cvs data file. In total there are 400 videos (.avi files) and 20 .csv files. The subfolder Mouse ABR data contains 5 .arf files that contain auditory brain stem measurements from 4 litters of mice. For the zebrafish startle data, *nrxn3a+/-; nrxn3b+/-* double heterozygotes were compared to *nrxn3a-/-; nrxn3b-/-* double mutants at 5 dpf. The raw data is summarized by 20 excel files with extension .csv and 400 videos with extension .avi generated by the Zantiks system that represent the data in Figure 8C. The .avi videos are behavioral data contain data from 26 *nrxn3a+/-; nrxn3b+/-* double heterozygotes and 40 *nrxn3a-/-; nrxn3b-/-* double mutant animals. Video .avi files beginning with “S2-VIB2” correspond to the highest intensity stimulus, “S2-VIB3” the medium intensity stimulus, “S3-VIB4” the low intensity stimulus, and “S4-VIB5” the control with no stimulus. There is one excel file with extension .xlsx summarizing all data points derived from the zebrafish startle analysis. For the mouse auditory brain stem (ABR) data, there are 5 .arf files that were generated by the BioSigRZ software from TDT ( [https://www.tdt.com/support/downloads/](https://gcc02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.tdt.com%2Fsupport%2Fdownloads%2F&data=05%7C02%7Ckatie.kindt%40nih.gov%7C7c2bbb05ade244f9aa4f08dcd4181794%7C14b77578977342d58507251ca2dc2b06%7C0%7C0%7C638618447180780145%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C&sdata=4DpSx75zA%2Fkpj4mUBawIw6t2fc8hui8iIwtL7Tt2nMQ%3D&reserved=0 "https://gcc02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.tdt.com%2Fsupport%2Fdownloads%2F&data=05%7C02%7Ckatie.kindt%40nih.gov%7C7c2bbb05ade244f9aa4f08dcd4181794%7C14b77578977342d58507251ca2dc2b06%7C0%7C0%7C638618447180780145%7CUnknown%7CTWFp") and represent the data in Figure 8A-B and Figure S17A-E. These files contain ABR data from mice from P28 to P32 in control (*0 (Cre-negative); Nrxn3flox/+*, *0; Nrxn3flox/flox*, and *Atoh1-Cre; Nrxn3flox/+* animals) and *Atoh1-Cre; Nrxn3flox/flox* mutant animals. There is one excel file with extension .xlsx summarizing the ABR thresholds, and the ABR Wave I amplitude and delay. Information on the zebrafish startle behavior acquisition: A Zantiks MWP behavioral system was used to examine acoustic startle responses. The Zantiks system tracked and monitored behavioral responses via a built-in infrared camera at 30 frames per second. A 12-well plate was used to house larvae during behavioral analysis. Each well was filled with E3 and 1 larva. All fish were acclimated in the plate within the Zantiks chamber in the dark for 15 min before each test. To induce startle, an integrated stepper motor was used to drive a vibration-induced startle response. Each larva was presented with each 3 intensities of vibrational stimuli 5 times with 100 s between trials. For each animal, the proportion of startle responses out of the 5 trials was plotted. During the tracking and stimulation, a Cisco router connected to the Zantiks system was used to relay *x*, *y* coordinates of each larva every frame. The Zanzik systems creates video files (.avi) during each stimulus. In addition, from the x,y, coordinates a .cvs (vib-stim prefix) containing the distance traveled in pixels for each time point for each well of the plate (column I-T). This data is contained in the .cvs files for each plate. The folders by plate and date are as follows: 030922_nrxn3a3b_behavior_plate 1 030922_nrxn3a3b_behavior_plate 2 030922_nrxn3a3b_behavior_plate 3 030922_nrxn3a3b_behavior_plate 4 040622_nrxn3a3b_behavior_plate 1 040622_nrxn3a3b_behavior_plate 2 040622_nrxn3a3b_behavior_plate 3 040622_nrxn3a3b_behavior_plate 4 040622_nrxn3a3b_behavior_plate 5 040622_nrxn3a3b_behavior_plate 6 041724_nrxn3a3b_behavior_plate 2 041724_nrxn3a3b_behavior_plate 3 041724_nrxn3a3b_behavior_plate 4 041724_nrxn3a3b_behavior_plate 5 041724_nrxn3a3b_behavior_plate 6 050824_nrxn3a3b_behavior_plate 1 all WT 050824_nrxn3a3b_behavior_plate 2 050824_nrxn3a3b_behavior_plate 3 050824_nrxn3a3b_behavior_plate 4 050824_nrxn3a3b_behavior_plate 5 Each plate has 12 wells. Well position is designated by three rows A-C and 4 column 1-4; for example, the upper left well is A1 and the lower right well is C4. The data extracted from the .cvs files using Python code ‘zantiks processing-0.3s_eachtmpt.ipynb’. This code detects whether each animal travels more than 4 pixels or ~1.9 mm within 2 frames after stimulus onset. This is counted as a startle response. Then the number of times (out of 5 trials) the animal startles is determined. This data is summarized in the excel file nrxn3a3b all behavior points and genotypes. The table Summary data outlines the date, plate and well for each animal. In addition, this table lists the proportion of times (out of 5 trials) that the animal startled in response to the each vibrational stimulus (Hi, MED, LO and control (no stimulus)). This data is sorted by genotype and summarized in the table data plotted. This is the data plotted in Figure 8C. For mouse ABR measurements animals were tested using the RZ6 Multi-I/O Processor System coupled to the RA4PA 4-channel Medusa Amplifier (Tucker-Davis Technologies). ABRs were recorded after binaural stimulation in an open field by tone bursts at 8, 16, 32, and 40 kHz generated at 21 stimuli/second. ABR thresholds were obtained for each frequency by reducing the sound pressure level (SPL) by 5 decibels (dB) between 90 and 20 dB.  A waveform for each frequency/dB level was produced by averaging the responses from 512 stimuli. The following control samples were recorded: | AN139 | | :---- | | AN140 | | AN142 | | AN144 | | AN164 | | AN138 | | AN145 | | AN161 | | AN162 | | AN165 | | AN113 | | AN114 | | AN125 | | AN135 | | AN137 | | AN143 | | AN146 | | AN147 | | AN160 | | AN163 | | AN166 | The following Nrxn mouse mutants were recorded: AN115 AN122 AN124 AN134 AN136 AN141 AN148 AN167 Waveforms from these samples were compared to identify the lowest level at which an ABR waveform could be recognized to obtain the ABR threshold for each frequency. Wave I amplitudes were measured by annotating the peak and trough of the first ABR waveform and calculating the difference (nV), and wave I delay was measured at the peak of the first wave (ms). This data is represented in the the excel file Data_Atoh1creNrxn3_ABR thresholds_Dryad.xlxs. The table Fig. 8B - ABR thresholds lists the genotype, .arf containing the raw data, the age and sex of each animal. The ABR threshold for each frequency 8,12,16,32 and 40kHz are listed. The table Fig. S17A,B - Wave I lists the genotype, .arf containing the raw data, the age and sex of each animal. The amplitude and delay for Wave I of the ABR response are listed for each frequency 8,12,16,32 and 40kHz. Blue and pink colors denote control and Nrxn3 mutant samples in these tables.  RAW_DATA_Figure S3 Summary of Figure 1 folder contents: This folder contains 2 Zeiss Airyscan processed images.  There are the 2 Zeiss Airyscan processed images that represent examples shown in Figure S3. These files end in .czi. 052323_r2_F5_AM_HCR_63x-Airyscan Processing-24.czi, 052323_r2_F5_MC_HCR_63x-Airyscan Processing-23.czi This is data acquired from wild type zebrafish. Zebrafish were fixed and RNA-FISH was used to label nrxn3a and nrxn3b mRNAs at day 5. Hair cells in the anterior macula and crista were imaged.  Information on RNA-FISH (HCR): To detect mRNA for *nrxn3a and nrxn3b** ***in zebrafish, we followed the Molecular Instrument-RNA FISH Zebrafish protocol, Revision Number 10 ([https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf](https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf)), with a few minor changes to the preparation of fixed whole-mount larvae. For our dehydration steps, we dehydrated using the following methanol series: 25, 50, 75, 100, 100% methanol, with 5 min for each step in the series. To permeabilize, we treated larvae with 10 µg/mL proteinase K for 20 min. RNA FISH probes were designed to target the long α form of zebrafish *nrxn3a* and *nrxn3b* (Molecular Instrument Probe lot # PRP848, PRP849). In addition to *nrxn3a* and *nrxn3b *probes, we also used *otofb* as positive control (Molecular Instrument Probe lot # PRP850 (*otofb*, 488 nm)).  After completing the RNA FISH protocol, we mounted the larvae in ProLong Gold Antifade (ThermoFisher, P36930) under 1.5 coverglass.  Metadata for acquisition of the 2 .czi images: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen 3.4 Blue) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639.\ Laser powers: 5% for 488, 2% for 561 and 3% for 639 Gain = 800 all laser lines Zoom: 1.5x Pixel size 0.043 um 2087x2087 pixels 0.17 microns per slice Manual Processed in 3D in Zen 3.4 blue Channel 1= alpha-nrxn3b mRNA, Channel 2 = alpha-nrxn3a mRNA, Channel 3 = otofb mRNA Using these settings, we acquired an image of an anterior macula 052323_r2_F5_AM_HCR_63x-Airyscan Processing-24.czi And an anterior crista: 052323_r2_F5_MC_HCR_63x-Airyscan Processing-23.czi These images processed in FIJI (partial max projections) and used as example images in the Figure S3. RAW_DATA_Figure S4 Summary of folder contents: This folder contains 3 Zeiss Airyscan processed images. There are the 3 Zeiss Airyscan processed images that represent examples shown in Figure S4. These files end in .czi. This is data acquired from wild type animals. Zebrafish were fixed and stained at day 5. Image of a lateral line neuromast HCR labeled with alpha-nrxn3a and dapB mRNA: HCR-negCTRL_CHR2YFP-488_nrxn3aalpha-546_dapB-647_20240418KP_S3C1_r1f1_L1-lsm980-Airyscan Processing-05.czi Image of a lateral line neuromast HCR labeled with dapB and alpha-nrxn3b mRNA: HCR-negCTRL_CHR2YFP-488_dapB-546_nrxn3balpha-647_20240418KP_S2C1_r1f1_L1-lsm980-Airyscan Processing-02.czi Image of a lateral line neuromast HCR labeled with alpha-nrxn3a, alpha-nrxn3b and otofb mRNA: 052323_r2_F5_NM_HCR_63x-Airyscan Processing-25.czi Information on RNA-FISH (HCR) to label mRNA: To detect mRNA for *nrxn3a and nrxn3b** ***in zebrafish, we followed the Molecular Instrument-RNA FISH Zebrafish protocol, Revision Number 10 ([https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf](https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf)), with a few minor changes to the preparation of fixed whole-mount larvae. For our dehydration steps, we dehydrated using the following methanol series: 25, 50, 75, 100, 100% methanol, with 5 min for each step in the series. To permeabilize, we treated larvae with 10 µg/mL proteinase K for 20 min. RNA FISH probes were designed to target the long α form of zebrafish *nrxn3a* and *nrxn3b* (Molecular Instrument Probe lot # PRP848, PRP849). In addition to *nrxn3a* and *nrxn3b *probes, we also used *otofb* (Fig S4I) or bacterial *dapB* (Fig S4B,D) probes as positive or negative controls respectively (Molecular Instrument Probe lot # PRP850 (*otofb*, 488 nm) RTHE541 (*dapB*, 546 nm) and RTH406 (*dapB*, 647 nm)). After completing the RNA FISH protocol, we mounted the larvae in ProLong Gold Antifade (ThermoFisher, P36930) under 1.5 coverglass. Metadata for acquisition of the 3 .czi images: Wild type zebrafish were labeled at 5 dpf. Samples were imaged on an upright LSM 980 laser-scanning confocal microscope with an Airyscan 2 attachment using Zen Blue 3.4 (Carl Zeiss) and a 63x/1.4 NA Plan Apo oil immersion objective lens and the following lasers lines: 488, and 561 and 639. Laser powers: 5% for 488, 2% for 561 and 3% for 639 Gain = 800 all laser lines Zoom: 4.5x Pixel size 0.043 um 680x680pixels 0.15 microns per slice Auto Processed in 2D in Zen 3.4 blue HCR czi image channel arrangement: Channel 1= alpha-nrxn3b mRNA/dabB, Channel 2 = alpha-nrxn3a mRNA,/dabB, Channel 3 = memGCaMP6s/otofb mRNA The 3 .czi images processed in FIJI (partial max projections) and used as example images in the Figure S4. RAW_DATA_Figure S5 Summary of folder contents: This folder contains  32 Zeiss Airyscan processed images and one excel file. There are 32 Zeiss Airyscan processed images that represent the raw data in Figure S5. These files end in .czi. This is data acquired from 12 wild type, 8 nrxn3a mutant and 12 nrxn3b mutant neuromasts. These data are fixed samples that label hair cells (Myosin7), ribbons (CTBP) and postsynapses (Maguk) in neuromasts (L1-L4) of the posterior lateral line. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the excel file .xlsx. Information on immunostaining: Larvae were fixed with 4% paraformaldehyde in PBS for 3.5hrs at 4°C. After fixation, larvae were washed 5 x 5 min in 0.1% PBS + 1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). Larvae were then blocked overnight at 4°C 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 4°C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2hrs at room temperature. After 5 x 5 min washes min in PBDTT to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: rabbit anti-Myosin7a (Proteus Biosciences 25-6790; 1:1000); mouse anti-CTBP (IgG2a) (Santa Cruz sc-3878); 1:1000)  and mouse anti-MAGUK (IgG1) (Sigma Aldrich MABN72; 1:500). With the following secondary antibodies were used at 1:1000: # A-21121, # A-21134, # A-21241 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen 3.4 Blue) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639.\ Laser powers: 0.2% for 488, 0.1% for 561 and 0.2% for 639 Gain = 800 all laser lines Zoom: 5x Pixel size 0.043 um 609x609 pixels 0.15 microns per slice AutoProcessed in 2D in Zen 3.4 blue Channel 1= Myo7a, Channel 2 = CTBP, Channel 3 = Maguk The 21 Zeiss Airyscan files generated are as follows: Wild type: Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C1_r1f1_L3-AP-03 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C1_r2f5_L1-AP-41 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C1_r3f3_L1-AP-57 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C2_r3f3_L1-AP-11 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S4C1_r1f2_L1-AP-04 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S4C1_r1f2_L2-AP-05 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S2C1_r1f3_L2-AP-45 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S2C1_r3f2_L1-AP-55 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S2C2_r3f4_L1-AP-06 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S2C2_r3f4_L3-AP-04 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S3C1_r2f3_L1-AP-21 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S3C1_r2f3_L2-AP-22 Nrxn3a mutants: Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C1_r3f4_L1-AP-60 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S4C2_r1f1_L1-AP-01 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S4C2_r1f1_L2-AP-02 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S4C2_r1f1_L4-AP-03 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S4C2_r1f5_L1-AP-13 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S4C2_r1f5_L2-AP-14 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S2C1_r3f5_L1-AP-46 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S3C1_r3f6_L1-AP-13 Nrnx3b mutants: Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C2_r1f3_L1-AP-72 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C2_r2f1_L2-AP-02 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C2_r2f3_L3-AP-01 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C2_r3f1_L1-AP-05 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C2_r3f2_L1-AP-08 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S1C2_r3f2_L2-AP-09 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S4C2_r2f1_L1-AP-16 Syn_nrxn3a3b-dHet-INX_d5_20230915kk_S4C2_r2f1_L2-AP-17 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S2C1_r1f1_L1-AP-38 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S2C1_r1f1_L2-AP-39 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S2C2_r1f5_L3-AP-31 Syn_nrxn3a3b-dHet-INX_d5_20230915zc_S3C2_r1f2_L2-AP-18 The *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro script was used to batch process the .czi images in the folder and* *to generate the data presented in Figure S5. The *Syn_nrxn3a3b-dHet-INX_20230915_dryad.*xlsx file contains the synapse counting results derived from the *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro. This data includes the filename, genotype, hair cells per neuromast, complete synapses per hair cell, total presynapses and unpaired presynapses per hair cell, total postsynapses and unpaired postsynapses per hair cell. This data is plotted in Figure S5 A-D. RAW_DATA_Figure S6 Summary of folder contents: This folder contains 39 Zeiss Airyscan processed images and two excel files. There are 39 Zeiss Airyscan processed images that represent the raw data in Figure S6. These files end in .czi. This is data acquired from 15 wild type and 22 nrxn3a;nrxn3b mutant neuromasts. These data are fixed samples that label hair cells (Myosin7), support cells (Sox2) and membranes (DiI) in neuromasts (L1-L4 , D1) of the posterior lateral line. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the excel file .xlsx. Information on immunostaining: Whole larvae were fixed with paraformaldehyde (PFA 4%; Thermoscientific; 28906) in PBS at 4°C for 3.5 hr. All antibody solutions were prepared with PBS + 0.1% Tween (PBST). After fixation, larvae were washed 4 × 5 min in PBST. Prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H2O in glass vials. The H2O was removed and replaced with ice-cold acetone and larvae placed at −20°C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 4 × 5 min in PBST. Larvae then were blocked overnight at 4°C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). After block, larvae were incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST) overnight, nutating at 4°C. The next day, the larvae were washed for 4 × 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 removed by washing with PBST for 4 × 5 min. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using No. 1.5 coverslips. The following primary antibodies were used: mouse anti-Myosin7a (IgG1) (Developmental Study Hybridoma Bank 138-1; 1:1000); rabbit anti-Sox2 (Abcam ab97959; 1:1000). With the following secondary antibodies were used at 1:1000: # A-11008, # A-11030 and DiI at 0.011mg/ml (D282) (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen 3.4 Blue) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639.\ Laser powers: 0.1% for 488 and 561 0.2% for 639 Gain = 800 all laser lines Zoom: 4.5-5x Pixel size 0.043 um 512x512 to 704x704 pixels 0.15 microns per slice AutoProcessed in 2D in Zen 3.4 blue Channel 1= Sox2, Channel 2 = Myo7a, Channel 3 = DiI The Zeiss Airyscan files generated are as follows: Wild type: 05152024_488_m7a_DiO_647_sox2_s1_c2_r1_f2_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_c2_r1_f2_L3 (2)_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_c2_r2_f3_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_c2_r2_f3_L3_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_c2_r2_f3_L4_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f4_L1_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f4_L3_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f4_L4_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f8_L1_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f8_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f8_L3_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f8_L4_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f11_L1_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f11_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f11_L3_Out.czi Nrxn3a;nrxn3b mutants: 05152024_488_m7a_DiO_647_sox2_s1_c2_r1_f5_L1 (2)_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_c2_r1_f5_L3 (2)_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_c2_r1_f5_L5 (2)_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f1_L1_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f1_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f1_L3_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f1_L4_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f2_L1_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f2_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f6_L1_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f6_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f6_L3_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f6_L4_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f9_L1_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f9_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f9_L4_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f10_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f10_L3_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f10_L4_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_r2_f2_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_r2_f2_L3_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_r2_f2_L4_Out.czi The *060524_Counts_488_mta_DiO_647_sox2_SCHC_YT_dryad.*xlsx file contains the filename and the number of hair cells and supporting cells per neuromasts. These counts were made manually from the .czi images in FIJI. The following files: 05152024_488_m7a_DiO_647_sox2_s1_f1_L2_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f1_L3_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f4_L3_Out.czi 05152024_488_m7a_DiO_647_sox2_s1_f4_L4_Out.czi Were processed in FIJI (partial max projections) and used as example images in the Figure S6. In addition, the following czi files were also processed in Fiji, to create side-view images of neuromast, but were not analyzed for cell counts: Wild type: WT_052824_488_sox2_546_DiO_647_CS1_r3_F31_headNM2_Out.czi Nrxn3a; nrxn3b mutant: DM_052824_488_sox2_546_DiO_647_CS1_r1_F10_headNM_Out.czi RAW_DATA_FigureS7_FigureS8 Summary of folder contents: This folder contains 2 subfolders that contain 29 Zeiss Airyscan image files and 2 excel files. There are 2 folders Anterior macula and Medial crista that contain 29 Zeiss Airyscan processed images that represent the raw data in Figure S7 and Figure S8. These files end in .czi. This is data acquired from wild type and nrxn3a;nrxn3b mutant anterior maculae or media cristae. These data are fixed samples that label hair cells (Myosin7), ribbons (CTBP), postsynapses (Maguk) and hair bundles (Phalloidin) in  zebrafish fixed and stained at day 5. The data extracted from these files is summarized in the excel files .xlsx. Information on immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5hrs at 4°C. After fixation, larvae were washed 5 x 5 min in 0.1% PBS + 1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). Larvae were then blocked overnight at 4°C 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 4°C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2hrs at room temperature. After 5 x 5 min washes min in PBDTT to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: rabbit anti-Myosin7a (Proteus Biosciences 25-6790; 1:1000); mouse anti-CTBP (IgG2a) (Santa Cruz sc-3878); 1:1000)  and mouse anti-MAGUK (IgG1) (Sigma Aldrich MABN72; 1:500). With the following secondary antibodies were used at 1:1000: # A-11008, # A-21137, # A-21240 along with #A-12379 (phalloidin 488 to label hair bundles)  (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen 3.4 Blue) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639.\ Laser powers: 0.4% for 488 0.2% for 561 and 0.2% for 639 Gain = 800 all laser lines Zoom: 2x ( anterior macula) 4x (medial crista) Pixel size: 0.067 um ( anterior macula) 0.043 um (medial crista) 1000x1000 ( anterior macula) 792x792 (medial crista) 0.15 microns per slice AutoProcessed in 2D in Zen 3.4 blue Channel 1= Maguk, Channel 2 = CTBP, Channel 3 = Myo7a and phalloidin In the folder Anterior macula, there are 14  Zeiss Airyscan files  as follows: Wild type: 091422_M7_CTBP_MAGUK_S1_r1_F1_AM-Airyscan Processing-16.czi 091422_M7_CTBP_MAGUK_S1_r1_F3_AM-Airyscan Processing-18.czi 091422_M7_CTBP_MAGUK_S1_r1_F6_AM-Airyscan Processing-22.czi 091422_M7_CTBP_MAGUK_S1_r2_F7_AM-Airyscan Processing-23.czi 091422_M7_CTBP_MAGUK_S1_r2_F8_AM-Airyscan Processing-25.czi 091422_M7_CTBP_MAGUK_S1_r2_F9_AM-Airyscan Processing-27.czi 091422_M7_CTBP_MAGUK_S1_r2_F10_AM-Airyscan Processing-29.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F14_AM-Airyscan Processing-30.czi Nrxn3a;nrxn3b mutants: 091422_M7_CTBP_MAGUK_S1_r1_F2_AM-Airyscan Processing-17.czi 091422_M7_CTBP_MAGUK_S1_r2_F12_AM-Airyscan Processing-33.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F17_AM-Airyscan Processing-37.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F19_AM-Airyscan Processing-41.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F20_AM-Airyscan Processing-01.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F21_AM-Airyscan Processing-46.czi In the folder Medial Crista, there are 15 Zeiss Airyscan files  as follows: Wild type: 091422_M7_CTBP_MAGUK_S1_r1_F1_MC-Airyscan Processing-01.czi 091422_M7_CTBP_MAGUK_S1_r1_F3_MC-Airyscan Processing-09.czi 091422_M7_CTBP_MAGUK_S1_r1_F6_MC-Airyscan Processing-13.czi 091422_M7_CTBP_MAGUK_S1_r2_F7_MC-Airyscan Processing-20.czi 091422_M7_CTBP_MAGUK_S1_r2_F8_MC-Airyscan Processing-24.czi 091422_M7_CTBP_MAGUK_S1_r2_F9_MC-Airyscan Processing-26.czi 091422_M7_CTBP_MAGUK_S1_r2_F10_MC-Airyscan Processing-28.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F14_MC-Airyscan Processing-29.czi Nrxn3a;nrxn3b mutants: 091422_M7_CTBP_MAGUK_S1_r1_F2_MC-Airyscan Processing-03.czi 091422_M7_CTBP_MAGUK_S1_r1_F4_MC-Airyscan Processing-07.czi 091422_M7_CTBP_MAGUK_S1_r2_F12_MC-Airyscan Processing-32.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F17_MC-Airyscan Processing-36.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F19_MC-Airyscan Processing-40.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F20_MC-Airyscan Processing-42.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F21_MC-Airyscan Processing-44.czi The Syn-EAR_nrxn3a3b_mag-ctbp-m7a-phallo_d5_091422*.*xlsx file contains the synapse counting results derived from the *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro. This excel file contains data tables, Anterior macula and Media crista and summarizes the file name (column a), genotype (column b), hair cells per organ (column c), complete synapses per hair cell (column d), total unpaired presynapses per hair cell (column e), unpaired presynapses per hair cell (column f) for each image. These numbers were used to generate the plots in Figure S8. The following files: 091422_M7_CTBP_MAGUK_S1_r1_F6_MC-Airyscan Processing-13.czi 091422_M7_CTBP_MAGUK_S1CS2_r1_F20_AM-Airyscan Processing-01.czi Were processed in FIJI (partial max projections) and used as example images in the Figure S7. RAW_DATA_Figure S9 Summary of folder contents: This folder contains 22 Zeiss Airyscan processed images and one excel file. There are the 22 Zeiss Airyscan processed images that represent examples shown in Figure S9. These files end in .czi. This is data acquired from in transgenic myo6b:memGCaMP6s background in wild type or Nrxn3a;nrxn3b mutant animals. Zebrafish were fixed and stained to label alpha-nrxn3a and alpha-nrxn3b mRNA at day 5. Information on RNA-FISH (HCR) to label mRNA: To detect mRNA for *nrxn3a and nrxn3b** ***in zebrafish, we followed the Molecular Instrument-RNA FISH Zebrafish protocol, Revision Number 10 ([https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf](https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf)), with a few minor changes to the preparation of fixed whole-mount larvae. For our dehydration steps, we dehydrated using the following methanol series: 25, 50, 75, 100, 100% methanol, with 5 min for each step in the series. To permeabilize, we treated larvae with 10 µg/mL proteinase K for 20 min. RNA FISH probes were designed to target the long α form of zebrafish *nrxn3a* and *nrxn3b* (Molecular Instrument Probe lot # PRP848, PRP849). In addition to *nrxn3a* and *nrxn3b *probes, we used m6b-GCaMP-caxx to visualize hair cell in the lateral line. After completing the RNA FISH protocol, we mounted the larvae in ProLong Gold Antifade (ThermoFisher, P36930) under 1.5 coverglass. Metadata for acquisition of the 22 .czi images: Wild type zebrafish were labeled at 5 dpf. Samples were imaged on an upright LSM 980 laser-scanning confocal microscope with an Airyscan 2 attachment using Zen Blue 3.4 (Carl Zeiss) and a 63x/1.4 NA Plan Apo oil immersion objective lens and the following lasers lines: 488, and 561 and 639. Laser powers: 0.7% for 488, 0.4% for 561 and 0.1% for 639 Gain = 800 all laser lines Zoom: 4x Pixel size 0.043 um 768x768 pixels 0.16 microns per slice Auto Processed in 2D in Zen 3.4 blue Channel 1= alpha-nrxn3b mRNA, Channel 2 = alpha-nrxn3a mRNA, Channel 3 = memGCaMP6s The 22 Zeiss Airyscan files generated are as follows: Wild type: HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S1C1_r2f4_L1-Airyscan Processing-13 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S1C1_r2f4_L2-Airyscan Processing-14 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C1_r1f1_L1-Airyscan Processing-06 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C1_r1f1_L2-Airyscan Processing-08 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C1_r2f6_L2-Airyscan Processing-30 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C1_r2f6_L3-Airyscan Processing-31 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C2_r1f5_L2-Airyscan Processing-01 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C2_r1f5_L3-Airyscan Processing-02 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C2_r2f1_L2-Airyscan Processing-05 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C2_r2f1_L3-Airyscan Processing-06 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C2_r2f6_L2-Airyscan Processing-15 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C2_r2f6_L3-Airyscan Processing-16 Nrxn3a;nrxn3b mutants: HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S1C1_r1f4_L1-Airyscan Processing-03 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S1C1_r1f4_L2-Airyscan Processing-04 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S1C1_r2f5_L1-Airyscan Processing-15 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S1C1_r2f5_L2-Airyscan Processing-16 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S1C1_r3f1_L1-Airyscan Processing-17 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S1C1_r3f1_L2-Airyscan Processing-18 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C1_r2f1_L2-Airyscan Processing-20 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C1_r2f1_L3-Airyscan Processing-21 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C1_r2f4_L1-Airyscan Processing-26 HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S2C1_r2f4_L2-Airyscan Processing-27 The *HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_dryad.*xlsx file contains the HCR puncta counting results derived from the *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro. This data includes the genotype, hair cells per neuromast, Nrxn3a mRNA puncta per hair cell and Nrxn3b mRNA puncta per hair. This data is plotted in Figure S9 D-E. The following files: HCR_nrxn3a3b_caxx-nrxn3a-nrxn3b_20221209_S1C1_r3f1_L2-Airyscan-0003 was processed in FIJI (partial max projections) and used as example images in the Figure S9 A-C. RAW DATA Figure S10 Summary of folder contents: This folder contains 47 Zeiss Airyscan processed images in two subfolders and two excel files. There are 21 Zeiss Airyscan processed images in subfolder ‘raw double alpha’ that represent the raw data in Figure S10B-K. These files end in .czi. This is data acquired from 10 wild type, and 11 α-nrxn3a;nrxn3b crispant neuromasts. Zebrafish were fixed and stained at 5 days post fertilization. Samples are labeled for hair cells (Myosin7), ribbons (CTBP), and postsynapses (Maguk) along the posterior lateral line (L1-L4). Crispr/Cas9 guides used for α-nrxn3a;nrxn3b crispants: α-nrxn3a gRNA 5’-GACCACGACAGGCTACACGC(AGG)-3’ α-nrxn3b gRNA 5’-GCACAACTTGCGAACCGTGT(TGG)-3’ genotyping primers: α-nrxn3a_FWD_fPCR 5’-TGTAAAACGACGGCCAGTGACAAGAACGGCCTACTCAAAGTCT-3’, α-nrxn3a_REV_fPCR 5’-GTGTCTTCAACCCATAAAGTTGTTGCTGA-3’, α-nrxn3b_FWD_fPCR 5’-TGTAAAACGACGGCCAGTGCGTGGACTGTGCAGAAACC-3’, α-nrxn3b_REV_fPCR 5’-GTGTCTTGCCATGCTGCAACTGCCTCCGCCATGCTGCAACTGCCTCC-3’ There are 27 Zeiss Airyscan processed images in subfolder ‘raw double beta’ that represent the raw data in Figure S10 L-O. These files end in .czi. This is data acquired from 15 wild type, and 12 β-nrxn3a;nrxn3b crispant neuromasts. Zebrafish were fixed and stained at 5 days post fertilization. Samples are labeled for hair cells (Myosin7), ribbons (CTBP), and postsynapses (Maguk) along the posterior lateral line (L1-L4). Crispr/Cas9 guides used for α-nrxn3a;nrxn3b crispants: β-nrxn3a gRNA 5’-AACACCCGGTCCACAACCCT(CGG)-3’ β-nrxn3b gRNA 5’-AGAGGACGACTGTGCTATCA(AGG)-3’ genotyping primers β-nrxn3a_FWD_fPCR 5’-TGTAAAACGACGGCCAGT-AGCATGGGGTTTTCTGCATC-3’, β -nrxn3a_REV_fPCR 5’-GTGTCTT-CCCCTATCGCAATTAACAGCAAG-3’, β-nrxn3b_FWD_fPCR 5’-TGTAAAACGACGGCCAGT-ATGCGCCCCCACTTTAAGAC-3’, β -nrxn3ba_REV_fPCR 5’-GTGTCTTCGTGGCCACCTCGTAAAGAGG-3’ Information on immunostaining: Whole larvae were fixed with paraformaldehyde (PFA 4%; Thermoscientific; 28906) in PBS at 4°C for 3.5hrs. All antibody solutions were prepared with PBS + 0.1% Tween (PBST). After fixation, larvae were washed 4 × 5 min in PBST. Prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H2O in glass vials. The H2O was removed and replaced with ice-cold acetone and larvae placed at −20°C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 4 × 5 min in PBST. Larvae then were blocked overnight at 4°C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). After block, larvae were incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST) overnight, nutating at 4°C. The next day, the larvae were washed for 4 × 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added, and larvae were incubated for 2hrs at room temperature, with minimal exposure to light. Secondary antibodies were removed by washing with PBST for 4 × 5 min. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using No. 1.5 coverslips. The following primary antibodies were used: Mouse anti MYO7A (Proteus 25-6790); rabbit anti-CTBP2 (Synaptic Systems 192103); mouse anti- MAGUK (Millipore MABN7)\ The following secondary antibodies were used at 1:1,000: \# A-21202,  # A-21241, #A-31572 (Thermofisher Scientific) Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen Black) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 543 and 633.\ \ Laser powers: 488= 0.3% 543= 1.5% 633= 0.4% Gain = 800 all laser lines Zoom: 4.5x Pixel size 0.043 um 512x512 to 704x704 pixels 0.185 microns per slice AutoProcessed in 2D in Zen 3.4 blue  Channel 1= CTBP, Channel 2 = Myo7a, Channel 3 = Maguk For the alpha-nrxn3 double crispants Zeiss Airyscan files generated are as follows: Wild type: Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r1f2_L1_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r1f2_L2_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r1f2_L3_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r1f3_L1_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r1f3_L2_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r2f1_L1_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r2f1_L2_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r2f3_L1_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r2f3_L2_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r2f3_L4_Out Nrxn3ab-Alpha_12212023-YTinj_S1C1_r2f3_L1_Out Nrxn3ab-Alpha_12212023-YTinj_S1C1_r1f2_L1_Out α-nrxn3a;nrxn3b crispants: Nrxn3ab-Alpha_12212023-YTinj_S1C1_r2f1_L1_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f1_L1_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f1_L2_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f2_L1_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f2_L2_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f2_L3_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f3_L1_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f3_L2_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f3_L3_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f4_L1_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f4_L2_Out Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f6_L1_Out For the beta-nrxn3 double crispants Zeiss Airyscan files generated are as follows: Wild type: Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F1_L1_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F3_L1_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F3_L2_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F3_L3_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F4_L1_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F4_L2_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F5_L2_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F5_L3_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F5_L4_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F6_L1_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F6_L3_R_Out Nrxn3ab_Beta_051624_KP_UNINJ_S1C2_R1_F6_L4_R_Out Nrxn3ab_Beta_043024_KP_UNINJ_S1C1_R1_F1_L1_Out Nrxn3ab_Beta_043024_KP_UNINJ_S1C1_R1_F1_L2_Out Nrxn3ab_Beta_043024_KP_UNINJ_S1C1_R1_F3_L2_Out β -nrxn3a;nrxn3b crispants: Nrxn3ab_Beta_043024_KP_S1C1_R1_F3_L2_Out Nrxn3ab_Beta_043024_KP_S1C1_R2_F5_L1_Out Nrxn3ab_Beta_043024_KP_S1C1_R3_F3_L2_Out Nrxn3ab_Beta_043024_KP_S1C1_R3_F4_L2_Out Nrxn3ab_Beta_050724_KP_S1C2_R1_F1_L3_Out Nrxn3ab_Beta_050724_KP_S1C2_R1_F3_L1_Out Nrxn3ab_Beta_050724_KP_S1C2_R1_F3_L3_Out Nrxn3ab_Beta_050724_KP_S1C2_R2_F1_L2_Out Nrxn3ab_Beta_050724_KP_S1C2_R2_F1_L3_Out Nrxn3ab_Beta_050724_KP_S1C2_R2_F4_L1_Out Nrxn3ab_Beta_050724_KP_S1C2_R2_F4_L4_Out Nrxn3ab_Beta_050724_KP_S1C2_R2_F5_L1_Out The *051724_Nrxn3_Double-alpha_YT_dryad.xlsx *and* 051924_Nrxn3_Double-beta_YT_dryad.xlsx *files contains the synapse counting results derived from the *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro. This data includes the genotype, hair cells per neuromast, complete synapses per hair cell, total presynapses per hair cell, unpaired presynapses per hair cell, total postsynapses per hair cell, unpaired postsynapses per hair cell genotype for each image. This data is plotted in Figure S10H-O. The following files: Nrxn3ab-Alpha_12212023-YTinj_S1C2_r1f1_L1_Out Nrxn3ab-Alpha_12212023-YTUNINJ_S2C1_r2f1_L1_Out Were processed in FIJI (partial max projections) and used as example images in the Figure S10B-G. RAW_DATA_Figure S11 Summary of folder contents: This folder contains 2 folders containing 34 Zeiss Airyscan processed images and two excel files. There are 34 Zeiss Airyscan processed images that represent the raw data in Figure S11. These files end in .czi. This is data acquired from wild type and nrxn3a;nrxn3b mutant neuromast terminals or posterior lateral line ganglia (pLLg). For the terminal images the samples are fixed samples that label hair cells (Myosin7), ribbons (CTBP), and terminals (Calretinin) in neuromasts (L1-L4 ) of the posterior lateral line. For pLLg images, fixed samples expressing the transgene en.sill,hsp70l:GCamp6s were imaged. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the 2 excel files .xlsx. Information on immunostaining: Larvae were fixed with 4% paraformaldehyde in PBS for 3.5hrs at 4°C. After fixation, larvae were washed 5 x 5 min in 0.1% PBS + 1% DMSO, 0.5% Triton-X100, 0.1% Tween-20 (PBDTT). Larvae were then blocked overnight at 4°C 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 4°C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2hrs at room temperature. After 5 x 5 min washes min in PBDTT to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used for afferent terminal labeling: rabbit anti-Myosin7a (Proteus Biosciences 25-6790; 1:1000); mouse anti-Calretinin (IgG1) (Swant 6B3; 1:1000)  and mouse anti-CTBP (IgG2a) (Santa Cruz sc-3878; 1:1000). With the following secondary antibodies were used at 1:1000: # A-21121, # A-11011, # A-21241 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition for pLLg images: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope in confocal mode using a 63x 1.4 NA oil objective lens and the following lasers lines: 488 nm Laser powers: 0.2% Gain = 650 Zoom: 4.5-5x Pixel size 0.088 um 1024x1024 pixels 0.25 microns per slice Metadata for Zeiss Airyscan acquisition for afferent terminal images: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 980 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen 3.4 Blue) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639.\ Laser powers: 0.04% for 488, 0.1% for 561 and 0.03% for 639 Gain = 850 all laser lines Zoom: 5x Pixel size 0.043 um 512x512 to 704x704 pixels 0.15 microns per slice Auto Processed in 2D in Zen 3.4 blue pLLg images: Channel 1= en.sill,hsp70l:GCamp6s Afferent terminal images Channel 1= CTBP, Channel 2 = Myosin7, Channel 3 = Calretinin The following .czi pLLg images were acquired: Wild type: Aff_SILL-nrxn3a3b_20221013_S1C1_r1f2_AG.czi Aff_SILL-nrxn3a3b_20221013_S1C1_r1f3_AG.czi Aff_SILL-nrxn3a3b_20221013_S1C1_r1f5_AG.czi Aff_SILL-nrxn3a3b_20221013_S1C1_r2f4_AG.czi Aff_SILL-nrxn3a3b_20221013_S1C1_r3f5_AG.czi Aff_SILL-nrxn3a3b_20221013_S1C1_r3f7_AG.czi Nrxn3a;nrxn3b Aff_SILL-nrxn3a3b_20221013_S1C1_r2f1_AG.czi Aff_SILL-nrxn3a3b_20221013_S1C1_r2f3_AG.czi Aff_SILL-nrxn3a3b_20221013_S1C1_r2f6_AG.czi Aff_SILL-nrxn3a3b_20221013_S1C1_r2f7_AG.czi Aff_SILL-nrxn3a3b_20221013_S1C1_r3f6_AG.czi This images were opened in FIJI/ImageJ and the number of cell bodies were counted manually. These counts are summarized in the excel file  Summary_pLLg counts. The single table pLLg counts lists the file name (column a), the genotype (column b) and the number of neurons per pLLg (column c). This data is shown in Figure S11A. The following .czi pLLg images were acquired: Wild type: Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C1_r3f1_L2-Airyscan Processing-16.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C1_r3f1_L3-Airyscan Processing-17.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r2f4_L2-Airyscan Processing-12.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r2f4_L3-Airyscan Processing-13.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C2_r1f1_L2-Airyscan Processing-17.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C2_r1f3_L2-Airyscan Processing-14.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C2_r1f3_L3-Airyscan Processing-13.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C2_r1f5_L1-Airyscan Processing-10.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C2_r1f5_L2-Airyscan Processing-09.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C2_r2f3_L2-Airyscan Processing-04.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C2_r2f3_L4-Airyscan Processing-03.czi Nrxn3a;nrxn3b Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C1_r2f3_L3-Airyscan Processing-12.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C1_r3f3_L2-Airyscan Processing-20.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C1_r3f3_L3-Airyscan Processing-21.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r1f2_L4-Airyscan Processing-25.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r1f5_L2-Airyscan Processing-19.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r1f5_L3-Airyscan Processing-18.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r3f1_L3-Airyscan Processing-08.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r3f1_L4-Airyscan Processing-09.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r3f2_L2-Airyscan Processing-01.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r3f2_L3-Airyscan Processing-07.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r3f4_L1-Airyscan Processing-04.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r3f4_L3-Airyscan Processing-03.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C2_r1f4_L3-Airyscan Processing-14.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C2_r1f4_L4_4_5x-Airyscan Processing-13.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C2_r2f1_L3-Airyscan Processing-10.czi Each z-stack was opened in FIJI and max-intensity projected. Projected images were autothresholded to isolate the terminals, and the mean area within the threshold was measured. These areas are summarized in the excel file  Summary_terminal areas. The single table Summary_terminal areas lists the file name (column a), the genotype (column b) and the area per terminal (column c). This data is shown in Figure S11B. The following images were processed in FIJI (partial max projections) and used as example images in the Figure S11C-D. Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C2_r1f3_L2-Airyscan Processing-14.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S1C2_r1f5_L1-Airyscan Processing-10.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r2f4_L2-Airyscan Processing-12.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C2_r1f4_L3-Airyscan Processing-14.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r3f2_L3-Airyscan Processing-07.czi Afferent_nrxn3a3b_Calretinin-myo7a-rbb_20220428_S2C1_r1f5_L3-Airyscan Processing-18.czi RAW_DATA_Figure S13 Summary of folder contents: This folder contains 48 Zeiss Airyscan processed images and one excel file. There are 48 Zeiss confocal images that represent the raw data in Figure S13. These files end in .czi. This is data include 24 images acquired from apex, mid and base areas of cochlea in  4 *control* mice and 24 images acquired from apex, mid and base areas of cochlea of 4 *Atoh1-cre; Nrxn3flox/flox* mice. These data are samples that label hair cells (Oncomodulin), ribbons (CTBP2), postsynapses (GluR2) of the mouse cochlear fixed and stained at P28. The data extracted from these files is summarized in the excel file .xlsx. Information on immunostaining: Temporal bones were isolated, and an insulin syringe was used to gently flush cold paraformaldehyde (PFA 4%; Electron Microscopy Sciences; 15710) through the cleared oval and round windows after poking a small hole at the cochlear apex. Temporal bones were then immersion-fixed in PFA for 1hr at 4°C, washed in PBS, and rotated overnight in EDTA 4% for decalcification. The next day, cochleae were dissected in 3 approximate thirds (base, mid, and apex) before blocking and permeabilization for 1hr at room temperature under agitation (1% bovine serum albumin; 0.5% Triton X-100). Primary and secondary antibodies were incubated overnight at 4°C in PBS. Samples were washed 3 times in PBS + 0.05% Triton X-100 after each antibody incubation and finally post-fixed in PFA 4% for at least 1 hr at room temperature. Samples were then mounted flat in Mowiol mounting medium (Calbiochem/MilliporeSigma 4759041) using two layers of office tape as a spacer for the coverglass (18x18mm #1.5). Mowiol (10% w/v) was prepared in (25% w/v) glycerol and 0.1M Tris-Cl pH8.5. The following primary antibodies were used: goat anti-Oncomodulin (IgG) (Developmental Study AB_2282417; 1:400); Rabbit anti-CTBP2 (Synaptic Systems 192103; 1:400) and mouse anti-GluR2 (IgG2a) (Millipore/Sigma MAB397; 1:100). With the following secondary antibodies were used at 1:1000: # A-21131, # A-21240, # A-21428 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Mounted samples were imaged on an upright LSM 980 laser-scanning confocal microscope using Zen Blue 3.4 (Carl Zeiss) and an 63x 1.4 NA oil objective lens and the following lasers lines: 488, 561 and 639. Z-stacks containing 6-9 IHCs were acquired in confocal mode.\ Laser powers: 0.6-1.5% for 488nm, 0.08% for 561nm, 0.05% for 639nm. Gain = 700 all laser lines Zoom: 1.5-2x Pixel size 0.085 um 792x395 pixels 0.25 microns per slice Channel 1 = oncodulin Channel 2 = CTBP2 Channel 3 = Glur2 The 48 Zeiss Airyscan files generated are as follows: Control: P28_AN3_hemi-het_LIE_apex_s1 P28_AN3_hemi-het_LIE_base_s1 P28_AN3_hemi-het_LIE_mid_s1 P28_AN3_hemi-het_RIE_apex_s1 P28_AN3_hemi-het_RIE_base_s1 P28_AN3_hemi-het_RIE_mid_s1 P28_AN5_hemi-het_LIE_apex_s1_hp P28_AN5_hemi-het_LIE_base_s1_hp P28_AN5_hemi-het_LIE_mid_s1_hp P28_AN5_hemi-het_RIE_apex_s1_hp P28_AN5_hemi-het_RIE_base_s1_hp P28_AN5_hemi-het_RIE_mid_s1_hp P28_AN8_hemi-het_LIE_mid_s1 P28_AN8_hemi-het_RIE_apex_s1 P28_AN8_hemi-het_RIE_apex_s2 P28_AN8_hemi-het_RIE_base_s2 P28_AN8_hemi-het_RIE_mid_s1 P28_AN8_hemi-het_RIE_mid_s2 P28_AN9_wt-het_LIE_base_s1 P28_AN9_wt-het_RIE_apex_s1 P28_AN9_wt-het_RIE_apex_s2 P28_AN9_wt-het_RIE_base_s2 P28_AN9_wt-het_RIE_mid_s1 P28_AN9_wt-het_RIE_mid_s2 Mutant* (Atoh1-cre; Nrxn3flox/flox*) P28_AN1_hemi-homo_LIE_apex_s1 P28_AN1_hemi-homo_LIE_apex_s2 P28_AN1_hemi-homo_LIE_base_s1 P28_AN1_hemi-homo_LIE_mid_s1 P28_AN1_hemi-homo_RIE_base_s1 P28_AN1_hemi-homo_RIE_mid_s1 P28_AN2_hemi-homo_LIE_apex_s1 P28_AN2_hemi-homo_LIE_base_s1 P28_AN2_hemi-homo_LIE_mid_s1 P28_AN2_hemi-homo_RIE_apex_s1 P28_AN2_hemi-homo_RIE_base_s1 P28_AN2_hemi-homo_RIE_mid_s1 P28_AN4_hemi-homo_LIE_apex_s1 P28_AN4_hemi-homo_LIE_base_s1 P28_AN4_hemi-homo_LIE_mid_s1 P28_AN4_hemi-homo_RIE_apex_s1 P28_AN4_hemi-homo_RIE_base_s1 P28_AN4_hemi-homo_RIE_mid_s1 P28_AN6_hemi-homo_LIE_apex_s1 P28_AN6_hemi-homo_LIE_base_s1_hp P28_AN6_hemi-homo_LIE_mid_s1 P28_AN6_hemi-homo_RIE_apex_s1_hp P28_AN6_hemi-homo_RIE_base_s1 P28_AN6_hemi-homo_RIE_mid_s1 The *Mouse_p28_masterlist_20230905_dryad.xlsx *file contains the synapse counting results derived from *CompleteSynpaseCounter2Dv5.2.ijm*  Fiji macro after the .czi images were manual segmentations of each channels in VVDViewer ([https://github.com/JaneliaSciComp/VVDViewer](https://github.com/JaneliaSciComp/VVDViewer)). Hair cell counting results were scored manually. This .xlsx file has three tabs, each contains data for apex, mid and base region of cochlea respectively. The data in each table includes the filename, summary genotype (mutant or control), the actual genotype, hair cells in imaging regions and the number of complete synapse per hair cell. The following files: P28_AN2_hemi-homo_RIE_apex_s1czi P28_AN2_hemi-homo_RIE_base_s1.czi P28_AN2_hemi-homo_RIE_mid_s1.czi P28_AN3_hemi-het_RIE_apex_s1.czi P28_AN3_hemi-het_RIE_mid_s1.czi P28_AN3_hemi-het_LIE_base_s1.czi Were processed in VVDViewer (for segmentation) and FIJI (partial max projections), and then used as example images in the Figure S13A-B. **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 ## Code/software All the excel files were made using 16.78.3 The .czi and .nd2 files can be opened using FIJI/ImageJ software version 2.14.0 The mouse ABR data (.arf files) can be opened using the BioSigRZ software from TDT ( [https://www.tdt.com/support/downloads/](https://gcc02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.tdt.com%2Fsupport%2Fdownloads%2F&data=05%7C02%7Ckatie.kindt%40nih.gov%7C7c2bbb05ade244f9aa4f08dcd4181794%7C14b77578977342d58507251ca2dc2b06%7C0%7C0%7C638618447180780145%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C&sdata=4DpSx75zA%2Fkpj4mUBawIw6t2fc8hui8iIwtL7Tt2nMQ%3D&reserved=0 "https://gcc02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.tdt.com%2Fsupport%2Fdownloads%2F&data=05%7C02%7Ckatie.kindt%40nih.gov%7C7c2bbb05ade244f9aa4f08dcd4181794%7C14b77578977342d58507251ca2dc2b06%7C0%7C0%7C638618447180780145%7CUnknown%7CTWFp") The zebrafish startle behavior can be opened using Quicktime 10.5