The lhfpl5 ohnologs lhfpl5a and lhfpl5b are required for mechanotransduction in distinct populations of sensory hair cells in zebrafish

Hair cells sense and transmit auditory, vestibular, and hydrodynamic information by converting mechanical stimuli into electrical signals. This process of mechano-electrical transduction (MET) requires a mechanically-gated channel localized in the apical stereocilia of hair cells. In mice, lipoma HMGIC fusion partner-like 5 (LHFPL5) acts as an auxiliary subunit of the MET channel whose primary role is to correctly localize PCDH15 and TMC1 to the mechanotransduction complex. Zebrafish have two lhfpl5 genes (lhfpl5a and lhfpl5b), but their individual contributions to MET channel assembly and function have not been analyzed. Here we show that the zebrafish lhfpl5 genes are expressed in discrete populations of hair cells: lhfpl5a expression is restricted to auditory and vestibular hair cells in the inner ear, while lhfpl5b expression is specific to hair cells of the lateral line organ. Consequently, lhfpl5a mutants exhibit defects in auditory and vestibular function, while disruption of lhfpl5b affects hair cells only in the lateral line neuromasts. In contrast to previous reports in mice, localization of Tmc1 does not depend upon Lhfpl5 function in either the inner ear or lateral line organ. In both lhfpl5a and lhfpl5b mutants, GFP-tagged Tmc1 and Tmc2b proteins still localize to the stereocilia of hair cells. Using a stably integrated GFP-Lhfpl5a transgene, we show that the tip link cadherins Pcdh15a and Cdh23, along with the Myo7aa motor protein, are required for correct Lhfpl5a localization at the tips of stereocilia. Our work corroborates the evolutionarily conserved co-dependence between Lhfpl5 and Pcdh15, but also reveals novel requirements for Cdh23 and Myo7aa to correctly localize Lhfpl5a. In addition, our data suggest that targeting of Tmc1 and Tmc2b proteins to stereocilia in zebrafish hair cells occurs independently of Lhfpl5 proteins.


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Abstract 17 Hair cells sense and transmit auditory, vestibular, and hydrodynamic information by converting 18 mechanical stimuli into electrical signals. This process of mechano-electrical transduction (MET) 19 requires a mechanically-gated channel localized in the apical stereocilia of hair cells. In mice, lipoma 20 HMGIC fusion partner-like 5 (LHFPL5) acts as an auxiliary subunit of the MET channel whose 21 primary role is to correctly localize PCDH15 and TMC1 to the mechanotransduction complex. 22 Zebrafish have two lhfpl5 genes (lhfpl5a and lhfpl5b), but their individual contributions to MET 23 channel assembly and function have not been analyzed. 24 Here we show that the zebrafish lhfpl5 genes are expressed in discrete populations of hair cells: lhfpl5a 25 expression is restricted to auditory and vestibular hair cells in the inner ear, while lhfpl5b expression 26 is specific to hair cells of the lateral line organ. Consequently, lhfpl5a mutants exhibit defects in 27 auditory and vestibular function, while disruption of lhfpl5b affects hair cells only in the lateral line 28 neuromasts. In contrast to previous reports in mice, localization of Tmc1 does not depend upon Lhfpl5 29 function in either the inner ear or lateral line organ. In both lhfpl5a and lhfpl5b mutants, GFP-tagged 30 Tmc1 and Tmc2b proteins still localize to the stereocilia of hair cells. Using a stably integrated GFP-31 Lhfpl5a transgene, we show that the tip link cadherins Pcdh15a and Cdh23, along with the Myo7aa 32 motor protein, are required for correct Lhfpl5a localization at the tips of stereocilia. Our work 33 corroborates the evolutionarily conserved co-dependence between Lhfpl5 and Pcdh15, but also reveals 34 novel requirements for Cdh23 and Myo7aa to correctly localize Lhfpl5a. In addition, our data suggest 35 that targeting of Tmc1 and Tmc2b proteins to stereocilia in zebrafish hair cells occurs independently 36 of Lhfpl5 proteins. 37 This is a provisional file, not the final typeset article 2 Introduction 38 The mechano-electrical transduction (MET) complex of sensory hair cells is an assembly of proteins 39 and lipids that facilitate the conversion of auditory, vestibular and hydrodynamic stimuli into electrical 40 signals. Our current understanding is that the proteins of the MET complex consist of the tip link 41 proteins cadherin 23 (CDH23) and protocadherin 15 (PCDH15) at the upper and lower ends of the tip 42 link respectively (Kazmierczak et al., 2007), the pore-forming subunits transmembrane channel-like 43 proteins TMC1 and TMC2 (Kawashima et  complex has also been reported (Ge et al., 2018). 63 A precise role for LHFPL5 has yet to be defined. One current hypothesis is that LHFPL5 acts as an 64 auxiliary subunit of the MET channel to stabilize the complex, similar to TARPs and the gamma 65 subunits of voltage-gated calcium channels. In mouse cochlear hair cells, PCDH15 and LHFPL5 66 require each other for stable localization at the tips of stereocilia (Mahendrasingam et , what are the molecular requirements for targeting Lhfpl5a to the hair  85  bundle? 2) Is the Lhfpl5-dependent targeting of Tmc1 to the hair bundle an evolutionarily-conserved  86 aspect of their interaction? 3) lhfpl5a mutants have defects in auditory and vestibular behaviors, yet 87 sensory hair cells of the lateral line are unaffected (Nicolson et al., 1998). What is the genetic basis for 88 the persistence of lateral line function in lhfpl5a mutants? 89 In this work, we report that teleost fish have two lhfpl5 genes, lhfpl5a and lhfpl5b. The zebrafish lhfpl5 90 ohnologs are expressed in distinct populations of larval sensory hair cells: lhfpl5a in the auditory and 91 vestibular system; lhfpl5b in the hair cells of the lateral line organ. Their divergent expression patterns 92 explain why lateral line hair cells are still mechanically-sensitive in lhfpl5a mutants. CRISPR-Cas 9 93 knockout of lhfpl5b alone silences the lateral line organ but has no effect on otic hair cell function. procedures (Westerfield, 2000). 110

Mutant and transgenic fish lines 111
The following zebrafish mutant alleles were used for this study: LSM 700 or LSM 800 laser scanning confocal microscopes. Z-stacks were analyzed using ImageJ 152 (Schneider et al., 2012). All related control and experimental images were adjusted equally for 153 brightness and contrast in Adobe Photoshop CC. 154

CRISPR-Cas9 knockout of lhfpl5b 155
An sgRNA targeting the lhfpl5b sequence 5'-CAACCCAATCACCTCGGAAT-3' was synthesized 156 essentially as described (Gagnon et al., 2014). For microinjection, 1 µg of sgRNA was mixed with 1 157 ug of Cas9 protein and warmed to 37ºC for 5 minutes to promote the formation of the Cas9-sgRNA 158 complex. Approximately 2 nl of this solution was injected into wild type embryos at the single-cell 159 stage. Efficacy of cutting was determined in two ways: 1) Single larvae genotyping -Amplification of 160 the target genomic region by PCR and running the products on 2.5% agarose gels to assay for disruption 161 of a homogenous amplicon. 2) Assaying for disruption of the mechanotransduction channel in lateral 162 line hair cells by FM 1-43 dye uptake. Larvae displaying a non-wild type pattern of FM 1-43 dye uptake 163 were raised to adulthood. Progeny from in-crosses of F0 adults were exposed to FM 1-43 dye to identify 164 founders. By outcrossing founder adults, we established a line of fish carrying a 5 base pair deletion in 165 the lhfpl5b coding region that causes a S77FfsX48 mutation (lhfpl5b vo35 ). This mutation disrupts the 166 protein in the first extracellular loop and deletes the final three of four transmembrane helices in 167 Lhfpl5b. 168

Acoustic startle response 169
Quantification of the larval acoustic startle response was performed using the Zebrabox monitoring 170 system (ViewPoint Life Sciences, Montreal, Canada) as previously described (Maeda et al., 2017) with 171 the following modifications. Each trial included six larvae which were subjected to two or three trials 172 of 6 acoustic stimuli. For each individual larva, the trial with best AEBR performance was used for 173 quantification. Positive responses where spontaneous movement occurred in the second prior to the 174 stimulus were excluded from analysis. Trials where spontaneous movement occurred for more than 6 175 of the 12 stimuli were also excluded from analysis. 176

Microphonics 177
We performed the microphonic measurements as previously described (Pacentine and Nicolson, 2019). 178 In brief, we anesthetized 3 dpf larvae in extracellular solution (140 mM NaCl, 2 mM KCl, 2 mM CaCl2, the amplitude from base-to-peak of the first peak. Larvae were genotyped as described above. This is a provisional file, not the final typeset article were fixed in 4% paraformaldehyde at 5 dpf. In experiment 2, larvae (n = 6 each genotype) were imaged 205 immediately. lhfpl5a tm290d/+ fish were in-crossed to produce wild type and lhfpl5a tm290d siblings. Larvae 206 were sorted based on the auditory and vestibular defects associated with the lhfpl5a tm290d homozygous 207 mutants. Larvae were labelled with FM 1-43 (n = 6 each). For all experiments, specimens were 208 mounted in low melting point agarose and the L1, MI1, and O2 neuromasts were imaged on a Zeiss 209 LSM 800 confocal. Cell counts were performed using the Z-stack data. 210

Statistics 211
Statistical analyses were done using the R stats package in RStudio (R Core Team, 2019; RStudio 212 Team, 2018). P-values of less than 0.05 were considered to be statistically significant. Plots were made 213 with ggplot2 (Wickham, 2016). 214

Data availability 215
The raw data supporting the conclusions of this manuscript will be made available by the 216 corresponding author, without undue reservation, to any qualified researcher. 217 4 Results 218

Teleost fish have ohnologous lhfpl5 genes. 219
Genes that have been duplicated as a result of a whole genome duplication (WGD) event are known as 220 ohnologs (Ohno, 1970;Wolfe, 2000). Due to the teleost-specific WGD, it is not uncommon to find 221 ohnologous genes in teleost fish where other vertebrate classes possess a single gene. The Danio rerio 222 (zebrafish) genome contains two lhfpl5 genes: lhfpl5a (ENSDARG00000045023) and lhfpl5b 223 (ENSDARG00000056458). To determine if lhfpl5 duplication is general to the teleost lineage, we 224 queried either GenBank or Ensembl databases to collect Lhfpl5 protein sequences for humans, mice, 225 chick, frogs, and representative sequences from 14 phylogenetic orders of ray-finned fish 226 (Actinopterygii), including 13 orders from the Teleostei infraclass and one from the Holostei infraclass 227 (Supplemental Table 1). The latter (Spotted gar, Lepisosteus oculatus) is commonly used to infer the 228 consequences of the teleost WGD, since the Holostei and Teleostei infraclasses diverged before the 229 teleost WGD (Braasch et al., 2016). Phylogenetic analysis of the Lhfpl5 protein sequences supports 230 the idea that duplicate lhfpl5 genes originated from the teleost WGD event ( Figure 1A). In all 13 teleost 231 orders surveyed, there are two lhfpl5 genes whose protein products cluster with either the lhfpl5a or 232 lhfpl5b ohnolog groups. Based on available genomic data, there is no evidence that spotted gar fish 233 have duplicated lhfpl5 genes. Nor is there evidence that the Salmonid-specific WGD (Allendorf and 234 Thorgaard, 1984) lead to further expansion of the lhfpl5 family. These analyses suggest that the 235 ancestral lhfpl5 gene was duplicated in the teleost WGD and that the lhfpl5 ohnologs were retained in 236 all teleost species examined here. 237 238 Alignment of the zebrafish Lhfpl5a and Lhfpl5b proteins with those from human, mouse and chicken 239 reveals that both zebrafish ohnologs retain the same protein structure ( Figure 1B) To characterize the spatial and temporal patterns of lhfpl5a/b gene expression, we performed whole 249 mount mRNA in situ hybridization on zebrafish larvae at 1, 2, and 5-days post-fertilization (dpf) 250 ( Figure 2). After one day of development, there are nascent hair cells of the presumptive anterior and 251 posterior maculae in the developing ear, but no lateral line hair cells at this stage. We detect lhfpl5a 252 expression in the presumptive anterior and posterior maculae at 1 dpf ( Figure 2A). No signal for lhfpl5b 253 was observed at this time point ( Figure 2B). At 2 dpf, we observe a clear distinction in the expression 254 patterns of the lhfpl5a and lhfpl5b genes ( Figure 2C, D). lhfpl5a continues to be expressed in the ear, 255 but expression is not observed in the newly deposited neuromasts. Conversely, we detect lhfpl5b 256 expression exclusively in neuromasts at this stage, both on the head ( Figure 2D) and trunk (data not 257 shown). This divergence in lhfpl5 ohnolog expression continues at 5 dpf, with lhfpl5a found 258 exclusively in the sensory patches of the ear and lhfpl5b restricted to lateral line hair cells ( Figure 2E-259 J). Taken together, our results suggest that both lhfpl5a and lhfpl5b have been retained since the teleost 260 whole genome duplication because of their non-overlapping mRNA expression patterns. 261

lhfpl5a mediates mechanosensitivity of otic hair cells. 262
lhfpl5a tm290d (astronaut / asn) mutants were initially characterized by balance defects, an absence of 263 the acoustic startle reflex, and a lack of brainstem Ca 2+ signals after acoustic startle. However, 264 neuromast microphonic potentials were normal (Nicolson et al., 1998). In light of our in situ 265 hybridization data (Figure 2), these results suggest that lhfpl5a is required for auditory and vestibular 266 hair cell function only. To test this possibility, we performed several functional assays measuring the 267 activity of hair cells in the otic capsule. First, we tested macular hair cell activity (Lu and DeSmidt,268 2013; Yao et al., 2016) by recording extracellular microphonic potentials from the inner ears of 269 lhfpl5a tm290d mutants, along with wild type and lhfpl5b vo35 larvae at 3 dpf. We then measured baseline-270 to-peak amplitude of the first peak to quantify activity (Supplemental Figure 1A-D). Consistent with 271 the initial characterization of the behavioral defects in lhfpl5a tm290d mutants, we did not detect robust 272 microphonic potentials from the inner ear of lhfpl5a tm290d mutants ( Figure 3A). We measured an 273 average first peak microphonic of 53.1 µV from lhfpl5a tm290d mutants (n = 7), which was significantly 274 less than the 296.2 µV average value of their WT siblings (n = 5; p = 0.01, Welch's t-test). We believe 275 that at least some of the signal detected in lhfpl5a tm290d mutants may be a stimulus artefact due the 276 unusual rise time and similar observations we have made in other known transduction-null mutants. 277 For the lhfpl5b vo35 mutants (n = 6), we measured an average first peak microphonic potential of 280.9 278 µV. These values were not significantly different from their WT siblings (n = 4, average 243.8 µV; p 279 = 0.7) but were significantly greater than the lhfpl5a tm290d mutants (p = 0.036). Thus, lhfpl5a tm290d 280 mutants exhibit defects in inner ear function, while the lhfpl5b vo35 mutation has no effect on these hair 281 cells. To further confirm the lack of inner ear function in lhfpl5a tm290d mutants, we performed an 282 acoustic startle test on larvae at 6 dpf ( Figure 3B). Results confirm that lhfpl5a tm290d mutants are 283 profoundly deaf, displaying little to no response to acoustic stimuli (p < 0.001 compared to all other 284 genotypes). This is a provisional file, not the final typeset article demonstrate that the GFP-Lhfpl5a protein is functional, we assayed for rescue of the acoustic startle 293 reflex and MET channel activity in homozygous lhfpl5a tm290d mutants expressing the transgene. The 294 startle reflex of lhfpl5a tm290d mutants expressing GFP-Lhfpl5a were statistically indistinguishable from 295 non-transgenic and transgenic WT siblings (p = 0.48 and 0.26 respectively; Figure 3B). Likewise, FM 296 4-64 dye-labeling in the inner ear was restored to lhfpl5a tm290d mutants expressing GFP-Lhfpl5a ( Figure  297 3E-F'). From these results we conclude that the hair bundle-localized GFP-Lhfpl5a protein is 298 functional and can rescue the behavioral and MET channel defects in lhfpl5a tm290d mutants. cells. To test this idea, we compared hair cell counts between wild type, lhfpl5a tm290d , and lhfpl5b vo35 321 larvae at 5 dpf. As expected, wild type and lhfpl5a tm290d mutants have statistically equivalent numbers 322 of hair cells in the neuromasts surveyed (Supplemental Figure 2D, p = 0.58). In lhfpl5b vo35 mutant 323 neuromasts, we observe a statistically significant decrease in the number of hair cells ( Figure 4K, 324 n = 11 individuals per genotype, 3 NM each, p < 0.001 each NM type by Welch's t-test; representative 325 neuromasts are shown in Supplemental Figure 4). This decrease in the number of neuromast hair cells 326 is consistent with lhfpl5b mutants being deficient in mechanotransduction in this cell type. 327

lhfpl5 is not required for Tmc localization to the hair bundle in zebrafish hair cells. 328
Previous studies have demonstrated that loss of LHFPL5 leads to a ~90% reduction in the peak 329 amplitude of the transduction current in mouse cochlear outer hair cells ( GFP and Tmc2b-GFP are still targeted to the hair bundle in lhfpl5a tm290d mutants (n = 11/11 and 9/9 341 individuals respectively; Figure 5A-D'). We also observe Tmc2b-GFP localization in the neuromast 342 hair bundles of lhfpl5b vo35 mutants (n = 7/7 individuals; Figure 5E-F'). Additionally, we do not observe 343 any rescue of hair cell function in lhfpl5b mutants expressing the Tmc2b-GFP protein (Supplemental 344 Figure 5A-C). As such, these results suggest that GFP-tagged Tmc1 and Tmc2b do not require Lhfpl5 345 for hair bundle localization in zebrafish hair cells. part of the hair bundle, with less intense punctate staining throughout the stereocilia in 3 dpf wild type 354 larvae ( Figure 6A). lhfpl5a tm290d mutants exhibit splayed hair bundles, with low levels of Pcdh15a 355 staining restricted to the tip of each stereocilium (n = 5/5 individuals; Figure 6B), confirming our 356 previous report. 357 To determine if Lhfpl5a also depends on Pcdh15a for its localization, we imaged GFP-Lhfpl5 in the 358 lateral cristae of pcdh15a psi7 homozygous mutants. In wild type larvae, GFP-Lhfpl5a is distributed 359 throughout the hair bundle at the tips of stereocilia (yellow arrow heads), with a higher intensity of 360 GFP evident in the tallest rows, possibly at the sites of kinociliary links ( Figure 6C, C', arrow). In 361 pcdh15a psi7 mutants, GFP-Lhfpl5a is absent from the hair bundle except for the tallest rows of 362 stereocilia adjacent to the kinocilium (n = 4 individuals; Figure 6D, D', arrow). We observed similar 363 results using the pcdh15a th263b allele (n = 6/6 individuals; Supplemental Figure 6A, B whether Cdh23 was required for GFP-Lhfpl5a localization. In cdh23 nl9 mutants, GFP-Lhfpl5 is 374 sparsely distributed throughout the length of the kinocilium (n = 4/4 individuals, Figure 6E, E'). We 375 also observe a redistribution of GFP-Lhfpl5a into the kinocilium using the cdh23 tj264 allele (n = 4/4 376 individuals; Supplemental Figure 6C). In both cdh23 alleles, we see occasional GFP signal in the 377 stereocilia as well, though this localization is not as robust. its role in Lhfpl5 localization has not been examined. In myo7aa ty220 (mariner) mutant hair cells, GFP-389 Lhfpl5a localization in hair bundle is severely disrupted (n = 5/5 individuals; Figure 6 F, F'). We 390 observe GFP signal near the base of hair bundles and occasionally in stereocilia (yellow arrowheads), 391 but do not see robust Lhfpl5a localization in the presumptive kinocilial links nor to the kinocilium 392 itself. Taken together, our results using the GFP-Lhfpl5a transgene suggest that Pcdh15a, Cdh23, and 393 Myo7aa all play distinct roles in Lhfpl5 localization in the bundle of zebrafish vestibular hair cells. 394

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Discussion 395 In this study, we provide support for the following points: 1) The ancestral lhfpl5 gene was likely 396 duplicated as a result of the teleost whole genome duplication, leading to the lhfpl5a and lhfpl5b 397 ohnologs described in this paper. 2) In zebrafish, there has been subfunctionalization of these genes as and functionally required in distinct, non-overlapping hair cell lineages. To our knowledge, this is the 415 first description of duplicated genes whose expression patterns have cleanly partitioned between inner 416 ear and lateral line hair cells. 417

Subfunctionalization of zebrafish lhfpl5 ohnologs. 418
We constructed a phylogenetic tree using publicly available Lhfpl5 protein sequence data (Figure 1) 419 Our results suggest that the ancestral lhfpl5 gene was duplicated during the teleost WGD event and that 420 both genes have been retained throughout the teleost lineage (Figure 1). Our conclusion that lhfpl5a 421 and lhfpl5b are ohnologs is substantiated by a recent study of ohnologs in teleosts (Singh and Isambert,422 2019). In all four teleost species surveyed, Singh and Isambert show that lhfpl5a and lhfpl5b are true 423 ohnologs that arose from the teleost-specific WGD under the strictest criteria used in their study. 424 Some kind of selection pressure is required for the retention of both gene ohnologs (Glasauer and 425 Neuhauss, 2014). Our in situ hybridization results show that lhfpl5a and lhfpl5b are expressed in 426 distinct populations of hair cells and support the idea that the zebrafish lhfpl5 ohnologs were retained 427 because of their divergent expression patterns (Figure 2). Analysis of hair cell function in the different 428 lhfpl5 mutants agrees with the gene expression results, showing that lhfpl5a is required for transduction 429 in the ear while lhfpl5b plays the same role in the lateral line organs. However, rescue of the MET 430 channel defects in lhfpl5b vo35 mutants by GFP-tagged Lhfpl5a suggests that the functions of these 431 ohnologs are at least partially interchangeable. This result is not surprising given the high degree of 432 similarity between the Lhfpl5a and 5b proteins. As such, the subfunctionalization of the lhfpl5 ohnologs 433 appears to be caused by the divergence in their expression patterns rather than functional differences 434 in their protein products. It is possible that a similar mechanism is responsible for the retention of both 435 genes in other teleost species as well, though the expression patterns of the lhfpl5 ohnologs have not 436 been examined in other fish. 437 The non-overlapping expression of the lhfpl5 ohnologs provides us with a unique genetic tool to study 438 lateral line function. Most mechanotransduction mutants in zebrafish disrupt both inner ear and lateral 439 line hair cell function (Nicolson, 2017). As such, we are unable to assess the role of the lateral line 440 independently of auditory and vestibular defects, which are lethal for larval fish. The lhfpl5b mutants 441 are adult viable (data not shown) and represent a possible genetic model for understanding the lateral 442 line in both larval and adult fish. Future studies on adults will determine whether the lateral line remains 443 non-functional and whether lhfpl5b contributes to inner ear function in the mature auditory and 444 vestibular systems. 445

The molecular requirements for bundle localization of MET complex proteins differs 446
between mouse and zebrafish hair cells.  to examine Lhfpl5 localization. In wild type hair cells, GFP-Lhfpl5a is present at the tips of the shorter 497 ranked stereocilia, but the most robust signal is detected in the tallest stereocilia adjacent to the 498 kinocilium. In pcdh15a mutants, only this "kinocilial link"-like signal remains in the hair bundle. This 499 result is similar to the LHFPL5 localization reported at the tips of the tallest stereocilia of P3 cochlear 500 hair cells from wild type mice, a region where LHFPL5 would presumably not be associated with either 501 PCDH15 or the TMCs (Mahendrasingam et al., 2017). Since the kinocilium does not degenerate in 502 zebrafish hair cells, our results suggest that Lhfpl5a is normally targeted to these kinocilial links in 503 wild type cells and that its retention at this site does not require Pcdh15a. Thus, there are PCDH15-504 dependent and PCDH15-independent mechanisms of LHFPL5 localization in both mouse and 505 zebrafish hair cells. These results suggest that LHFPL5 performs as-of-yet uncharacterized PCDH15-506 independent functions as well. 507 Cdh23 is thought to form part of the linkages between the kinocilium and adjacent stereocilia, and 508 therefore may play a role in retaining Lhfpl5a at these sites. Consistent with this hypothesis, cdh23 509 mutants do not exhibit GFP-Lhfpl5a accumulation in stereocilia. Instead, GFP-Lhfpl5a is diffusely 510 distributed throughout the kinocilium. These data suggest that normal Lhfpl5a localization requires 511 both Pcdh15a and Cdh23 function, albeit in distinct ways. This result differs from previous reports 512 which found that Lhfpl5 still localized to the tips of stereocilia in CDH23-deficient cochlear hair cells 513 of mice. The same study also could not detect a biochemical interaction between Lhfpl5 and CDH23 514 via co-IP in heterologous cells (Xiong et al., 2012). Given these contrasting results for mouse and 515 zebrafish hair cells, it seems that the requirement for Cdh23 in the localization of LHFPL5 is not a 516 universal one. 517 Given the available biochemical evidence, it is possible that the association between Lhfpl5a and 518 Cdh23 is indirect through an as-of-yet unidentified protein or protein complex. Based on the defects in 519 GFP-Lhfpl5a localization in myo7aa mutants, Myo7aa is a candidate member of this uncharacterized 520 protein complex. Taken together, these results highlight the fact that, although all sensory hair cells 521 share many core genetic and biochemical features, there are important details that differ between the 522 various types of hair cells and between vertebrate species. Analyzing these differences will allow for 523 a more comprehensive understanding of vertebrate hair cell function and the underlying principles of 524 mechanotransduction. 525

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Conflict of Interest 526 The authors declare that the research was conducted in the absence of any commercial or financial 527 relationships that could be construed as a potential conflict of interest. 528   Table 1. Lhfpl5 proteins used to construct the phylogenetic tree in Figure 1A.