Microtubule networks in zebrafish hair cells facilitate presynapse transport and fusion during development

Sensory cells in the retina and inner ear rely on specialized ribbon synapses for neurotransmission. Disruption of these synapses is linked to visual and auditory dysfunction, but it is unclear how these unique synapses are formed. Ribbon synapses are defined by a presynaptic density called a ribbon. Using live-imaging approaches in zebrafish, we find that early in hair-cell development, many small ribbon precursors are present throughout the cell. Later in development, fewer and larger ribbons remain, and localize at the presynaptic active zone (AZ). Using tracking analyses, we show that ribbon precursors exhibit directed motion along an organized microtubule network towards the presynaptic AZ. In addition, we show that ribbon precursors can fuse together on microtubules to form larger ribbons. Using pharmacology, we find that microtubule disruption interferes with ribbon motion, fusion, and normal synapse formation. Overall, this work demonstrates a dynamic series of events that underlies formation of a critical synapse required for sensory function.

Methods
Zebrafish animals

Zebrafish (Danio rerio) were bred and cared for at the National Institutes of Health (NIH) under animal study protocol #1362-13. Zebrafish larvae raised at 28°C in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4, buffered in HEPES, pH 7.2). All experiments were performed on larvae aged 2-3 days post fertilization (dpf). Larvae were chosen at random at an age where sex determination is not possible. The previously described mutant and transgenic lines were used in this study: Tg(myo6b:ctbp2a-TagRFP)idc11Tg referred to as myo6b:riba-TagRFP; Tg(myo6b:YFP-Hsa.TUBA)idc16Tg referred to as myo6b:YFP-tubulin (Ohta et al., 2020; Wong et al., 2019). Tg(myo6b:ctbp2a-TagRFP)idc11Tg reliably labels mature ribbons, similar to a pan-CTBP immunolabel at 5 dpf (Figure 1-S2B). This transgenic line does not alter the number of hair cells or complete synapses per hair cell (Figure 1-S2A-D). In addition, myo6b:ctbp2a-TagRFP does not alter the size of ribbons (Figure 1-S2E).

Zebrafish transgenic and CRISPR-Cas9 mutant generation

To create myo6b:EB3-GFP transgenic fish, plasmid construction was based on the tol2/gateway zebrafish kit (Kwan et al., 2007). The p5E pmyo6b entry clone (Trapani et al., 2009) was used to drive expression in hair cells. A pME-EB3-GFP clone was kindly provided by Catherine Drerup at University of Wisconsin, Madison. pDestTol2pACryGFP was a gift from Joachim Berger & Peter Currie (Addgene plasmid # 64022). These clones were used along with the following tol2 kit gateway clone, p3E-polyA (#302) to create the expression construct: myo6b:EB3-GFP. To generate the stable transgenic fish line myo6b:EB3-GFPidc23Tg, plasmid DNA and tol2 transposase mRNA were injected into zebrafish embryos as previously described (Kwan et al., 2007). The myo6b:EB3-GFPidc23Tg transgenic line was selected for single copy and low expression of EB3-GFP.

A kif1aa germline mutant (kif1aaidc24) was generated in-house using CRISPR-Cas9 technology as previously described (Varshney et al., 2016). Exon 6, containing part of the Kinesin motor domain was targeted. Guides RNAs (gRNAs) targeted to kif1aa are as follows: 5’-ACGGATGTTCTCGCACACGT(AGG)-3’, 5’-GTGCGAGAACATCCGTTGCT(AGG)-3’, 5’-TGGACTCCGGGAATAAGGCT(AGG)-3’, 5’-AGAATACCTAGCCTTATTCC(CGG)-3’. Founder fish were identified using fragment analysis of fluorescent PCR (fPCR) products. A founder fish containing a complex INDEL that destroys a BslI restriction site in exon 6 was selected (Figure 5-S1B). This INDEL disrupts the protein at amino acid 166 (Figure 5-S1A). Subsequent genotyping was accomplished using standard PCR and Bsl1 restriction enzyme digestion. Kif1aa genotyping primers used were: kif1aa_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and kif1aa_REV 5’-TGCGGTCCTAGGCTTACAAT-3’.

We created kif1aa F0 crispants for our live imaging analyses. Here we injected the following kif1aa gRNAs: 5’-GTGCGAGAACATCCGTTGCT(AGG)-3’ and 5’-AGAATACCTAGCCTTATTCC(CGG)-3’, along with Cas9 protein, as previously described (Hoshijima et al., 2019). We then grew kif1aa injected F0 crispants for 2 days and then used them for our live imaging analyses. Because kif1aa mutants have no phenotype to distinguish them from sibling controls at the ages imaged, the low throughput of our live imaging approaches made using germline mutants prohibitive. Studies have shown that F0 crispants are a fast and effective way to knockdown gene function in any genetic background (Hoshijima et al., 2019; Sheets et al., 2021). After live imaging, we genotyped all kif1aa F0 crispants (Figure 6-S1) to ensure that the gRNAs cut the target robustly using fragment analysis of fluorescent PCR products and the following primers: kif1aa_FWD_fPCR 5’-TGTAAAACGACGGCCAGT-AAATAGAGATTCACTTTTAATC-3’ and kif1aa_REV_fPCR 5’- GTGTCTT-CCTAGGCTTACAATGCTTTTGG-3’ (Carrington et al., 2015). fPCR fragments were run on a genetic analyzer (Applied Biosystems, 3500XL) using LIZ500 (Applied Biosystems, 4322682) as a dye standard. Any kif1aa F0 crispants without robust genomic cutting were not included in analyses.

Zebrafish pharmacology

To destabilize or stabilize microtubules, larval zebrafish at 2 dpf were incubated in either nocodazole (Sigma-Aldrich, SML1665) or Paclitaxel (taxol) (Sigma-Aldrich, 5082270001). Both drugs were maintained in DMSO. For experiments these drugs were diluted in media for a final concentration of 0.1 % DMSO, 250-500 nM nocodazole and 25 µM taxol. For controls, larvae were incubated in media containing 0.1 % DMSO. For long-term incubation (16 hr), wild-type larvae were incubated in E3 media containing 250 nM nocodazole or 25 µM taxol at 54 hpf for 16 hrs (overnight). After this long-term treatment, larvae were fixed and prepared for immunohistochemistry (see below). For live, short-term incubations (for 3-4 hr incubations or ribbon tracking), transgenic larvae (myo6b:riba-tagRFP; Tg(myo6b:YFP-alpha-tubulin) at 48-54 hpf were embedded in 1 % low melt agarose prepared in E3 media containing 0.03 % tricaine (Sigma-Aldrich, A5040, ethyl 3-aminobenzoate methanesulfonate salt). 500 nM nocodazole, 25 µM taxol or DMSO were added to the agarose and to the E3 media used to hydrate the sample. For short-term treatments, hair cells were imaged after 30 min of embedding.

Immunohistochemistry of zebrafish samples

Immunohistochemistry to label acetylated-tubulin, tyrosinated-tubulin, Ribeyeb or pan-CTBP (ribbons and precursors), pan-Maguk (postsynaptic densities) and Myosin7a (cell bodies) was performed on whole zebrafish larvae similar to previous work. The following primary antibodies were used: rabbit anti-Myosin7a (Proteus 25-6790; 1:1000); mouse anti-pan-Maguk (IgG1) (Millipore MABN7; 1:500); mouse anti-Ribeyeb (IgG2a) ((Sheets et al., 2011); 1:10,000); mouse anti-CTPB (IgG2a) (Santa Cruz sc-55502; 1:1000); mouse anti-acetylated-tubulin (IgG2b) (Sigma-Aldrich T7451; 1:5,000); mouse anti-tyrosinated-tubulin (IgG2a) (Sigma-Aldrich MAB1864-I; 1:1,000); chicken anti-GFP (to stain YFP-tubulin) (Aves labs GFP-1010; 1:1,000). The following secondary antibodies were used at 1:1,000: (ThermoFisher Scientific, A-11008; A-21143, A-21131, A-21240; A-11-039, A-21242, A-21241, A-11039). Larvae were fixed with 4 % paraformaldehyde in PBS for 4 hr at 4°C. All wash, block and antibody solutions were prepared in 0.1 % Tween in PBS (PBST). After fixation, larvae were washed 5 × 5 min in PBST. Prior to block, larvae were permeabilized with acetone. For this permeabilization larvae were first washed for 5 min with H2O. The H2O was removed and replaced with ice-cold acetone and placed at −20°C for 3 min, followed by a 5 min H2O wash. The larvae were then washed for 5 × 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 × 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 3 hrs at room temperature. After 5 × 5 min washes min in PBST to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong gold (ThermoFisher Scientific P36930).

Confocal imaging and analysis of fixed zebrafish samples

After immunostaining, fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 (Zen 2.3 SP1) or an upright Zeiss LSM 980 (Zen 3.4) laser-scanning confocal microscope with Airyscan using a 63x 1.4 NA oil objective lens. Z-stacks encompassing the entire neuromast were acquired every 0.17 (LSM 980) or 0.18 (LSM 780) µm with an 0.04 µm x-y pixel size and Airyscan autoprocessed in 3D.

         Synaptic images from fixed samples were further processed using FIJI. Acetylated-a-tubulin or Myosin7 label was used to manually count hair cells. Complete synapses comprised of both a Ribeyeb/CTBP and Maguk puncta were also counted manually. To quantify the area of each ribbon and precursor, images were processed in a FIJI ‘IJMacro_AIRYSCAN_simple3dSeg_ribbons only.ijm’ as previously described (Wong et al., 2019). Here each Airyscan z-stack was max-projected. Background was subtracted from each projection using rolling-ball subtraction. A threshold was applied to each image, followed by segmentation to delineate individual Ribeyeb/CTBP puncta. The watershed function was used to separate adjacent puncta. A list of 2D objects of individual ROIs (minimum size filter of 0.002 μm2) was created to measure the 2D areas of each Ribeyeb/CTBP puncta. Areas for all Ribeyeb/CTBP puncta within each neuromast were then exported as a csv spreadsheet. For comparisons, all fixed images analyzed in FIJI were imaged and processed using the same parameters.

         To quantify the mean intensity of acetylated-a-tubulin after overnight nocodazole or taxol treatments, 20 slices centered on the hair cells were max-projected in FIJI. An ROI was draw around the hair cells, and this ROI was used to measure the mean intensity of the acetylated-a-tubulin label in each neuromast.

Confocal imaging and in vivo analysis of ribbon numbers in developing zebrafish hair cells

For counting ribbon numbers in developing and mature hair cells (Figure 1), double transgenic myo6b:riba-TagRFP and myo6b:YFP-tubulin larvae at 2 and 3 dpf were imaged. Transgenic larvae were pinned to a Sylgard-filled petri dish in E3 media containing 0.03 % tricaine and imaged on a Nikon A1R upright confocal microscope using a 60x 1 NA water objective lens. Denoised images were acquired using NIS Elements AR 5.20.02 with an 0.425 µm z-interval, at, 16x averaging, and 0.05 µm/pixel. Z-stacks of whole neuromasts including the kinocilium were acquired in a top-down configuration using 488 and 561 nm lasers. The 488 nm laser along with a transmitted PMT (T-PMT) detector was used to capture the kinocilial heights.

For quantification of ribbon numbers at different developmental stages (Figure 1), a custom-written Fiji macro “Live ribbon counter” was used to batch-process the z-stacks. The red channel (Riba-TagRFP) of each z-stack was thresholded (threshold value = 97). Watershed was applied to the thresholded stack to separate ribbons near each other. The resulting mask from the thresholding and watershedding was applied to the original red channel. The number of ribbons was then counted using ‘3D Objects Counter’ plugin (Threshold = 1, min size = 0, max size = 183500). The counted objects were merged with the green channel (YFP-tubulin). Each z-stack was visually inspected to determine the localization of the ribbons. Ribbons below the nucleus were classified as ‘basal’ and the rest as ‘apical’. The number of apical and basal ribbons were counted in each hair cell.

To classify the developmental stage of each hair cell (Figure 1), the height of the kinocilium was used. The number of z-slices between the kinocilium tip and base was determine and multiplied by the z-slice interval (0.425 µm) to get the kinocilium height. Hair cells with heights < 1.5 µm were classified as ‘Early’, hair cells with heights 1.5-10 µm were classified as ‘Intermediate’ and with heights 10-18 µm were classified as ‘Late’. Hair cells with heights > 18 µm were considered ‘Mature’.

Confocal imaging and in vivo tracking EB3-GFP dynamics in zebrafish

Transgenic myo6b:EB3-GFP larvae at 2-3 dpf were mounted in 1 % LMP agarose containing 0.03 % tricaine in a glass-bottom dish. Larvae were imaged on an inverted Zeiss LSM 780 (Zen 2.3 SP1) confocal microscope using a 63x 1.4 NA oil objective lens. For timelapses, confocal z-stacks of partial cell volumes (3.5 µm, 7 z slices at 0.5 µm z interval) with an 0.07 µm x-y pixel size were taken every 7 s for 15-30 min.

The EB3-GFP timelapses were registered in FIJI using the plugin “Correct 3D drift” (Parslow et al., 2014), max-projected, and then tracked in 2D in Imaris. For spot detection, we used an estimated xy diameter of 0.534 µm with background subtraction. The detected spots were filtered by ‘Quality’ using the automatic threshold. The timelapses were visually checked to make sure the spot detection was accurate. For the tracking step, the ‘Autoregressive motion’ algorithm was used, with a maximum linking distance of 1 µm and a maximum gap size of 3 frames. To ensure accurate track detection, short tracks were removed by filtering for the number of spots in a track (> 5) and track displacement length (> automatic threshold).

To calculate the track angles relative to the cell base, we used cells that lie horizontally so we only need to consider the angles in the xy place. In Imaris, the tracks in each hair cell were selected and exported separately. Using the start and end position co-ordinates of the exported tracks, we calculated track angles in MATLAB using custom written code called, “EB3 track angle”. The angle of each hair cell was measured in Imaris. The final track angle distribution plotted was obtained by measuring the difference between each track angle and the angle of the hair cell. 

         To create movies of EB3-GFP tracks in Movie S1, the FIJI plugin “Correct 3D drift” was applied. Z-stacks were then max-projected tracks were detected using the FIJI plugin TrackMate (Parslow et al., 2014; Tinevez et al., 2017). For Movie S1, the LoG detector in TrackMate was used with an estimated object diameter of 0.6 µm, a quality threshold of 8, using a median filter and sub-pixel localization. The Linear Assignment Problem (LAP) tracker was selected using frame to frame linking max distance of 1 µm, a track segment gap closing max distance of 1 µm and max frame gap of 2 µm. Tracks were colored by Track index.  For viewing tracks over time, tracks were displayed as “Show tracks backwards in time” with a fade range of 5 time-points. To create color-coded temporal map of EB3-GFP tracks over a short time window (21 s, Figure 2C-D), the FIJI Hyperstack plugin Temporal-Color code was used with the 16 colors LUT.

Confocal imaging and in vivo analysis of ribbon numbers after short-term pharmacological treatments in zebrafish

For counting ribbons after 3-4 hr drug treatment, transgenic zebrafish expressing myo6b:riba-TagRFP and myo6b:YFP-tubulin at 2 dpf were examined. Transgenic larvae were mounted in 1 % low melt agarose in E3 media containing 0.03 % tricaine and one of the following: 500 nM nocodazole, 25 µM taxol or 0.1 % DMSO (Control). An inverted Zeiss LSM 780 (Zen 2.3 SP1) confocal microscope with Airyscan, along with a 63x NA 1.4 oil objective lens. Z-stacks encompassing the entire neuromast were acquired every or 0.18 µm with an 0.04 µm x-y pixel size and Airyscan autoprocessed in 3D.

To quantification of ribbon numbers before and after 3-4 hr nocodazole and taxol treatment or in kif1aa F0 crispants (Figure 6, Figure 6-S1), the custom-written Fiji macro “Live ribbon counter” described above was used to batch-processed the z-stacks. The red channel (Riba-TagRFP) of each z-stack was thresholded (threshold value = 28) and segmented (watershed). The resulting mask was applied to the original red channel. The number of ribbons was then counted using ‘3D Objects Counter’ (threshold = 1, min size = 0, max size = 183500). The counted objects were merged with the green channel (YFP-tubulin). Each z-stack was visually inspected to make sure the objects counted were within hair cells. The number of ribbons per neuromast was determined and the difference in numbers pre- and post-drug treatment was plotted.

Confocal imaging and in vivo tracking of ribbons

To visualize ribbon precursor movement, timelapses of double transgenic myo6b:riba-TagRFP and myo6b:YFP-tubulin larvae at 2 dpf were imaged. For pharmacological treatments, transgenic larvae were mounted in 1 % low melt agarose in E3 media containing 0.03 % tricaine and one of the following: 500 nM nocodazole, 25 µM taxol or 0.1 % DMSO (Control) in a glass-bottom dish. Double transgenic kif1aa crispants and uninjected controls were mounted in 1 % low melt agarose in E3 media containing 0.03 % tricaine in a glass-bottom dish. Larvae were imaged on an inverted Zeiss LSM 780 or an upright LSM 980 confocal microscope with Airyscan using a 63x 1.4 NA oil objective lens. Airyscan z-stacks of partial cell volumes (~3 µm, 15-20 z-slices using 0.18 µm z-interval and an 0.04 µm x-y pixel size) were taken on the LSM 780 every 50-100 s for 30-70 min. Faster LSM 980 Airscan z-stacks of partial cell volumes (~2-3.5 µm, 12-20 z-slices using 0.17 µm z interval and an 0.04 µm x-y pixel size) were taken every 3-20 s for 5-40 minutes. Airyscan timelapses were autoprocessed in 3D. In addition, we acquired a subset of LSM 780 Airscan z-stacks every 5-8 min for 30-100 min to capture fusion events more for clearly for Movies S9-11.

The longer timelapses acquired on the Zeiss LSM 780 were registered using the FIJI plugin “Correct 3D drift”. Drift corrected timelapses were then tracked in 3D in Imaris using spot detection with estimated xy diameters of 0.427 µm (with background subtraction). The spots were filtered based on ‘Quality’, with thresholds between 3-8, chosen after visual inspection of the detected spots. For tracking, the ‘Autoregressive motion’ algorithm was used with a maximum linking distance of 1.13 µm and a maximum gap size of 1 frame. Tracks with number of spots < 5 were not included. Using the Track displacement length filter in Imaris, the number of tracks with Track displacement length > 1 µm were counted and divided by the total number of tracks to get the fractions plotted in Figure 7. For the mean squared displacement (MSD) analysis, the xyzt co-ordinates were exported in ‘csv’ format for all tracks in a timelapse. The MSD analysis was done using the prewritten MATLAB class MSDanalyzer (Tarantino et al., 2014). MSDanalyzer calculates the mean squared displacement for each track, curve-fits the MSD vs time, and provides the value of the exponent ( . The first 25 % of the MSD vs time graph was used for curve-fitting. Tracks with the number of spots < 10 were removed to ensure accuracy of the MSD analysis. Fusion events between ribbons and precursors were scored manually in these timelapses.