Role of Pcdh15 in the development of intrinsic polarity of inner ear hair cells

Raman Kaushik; Shivangi Pandey; Anubhav Prakash; Fenil Ganatra; Takaya Abe; Hiroshi Kiyonari; Raj K Ladher

doi:10.1371/journal.pgen.1011825

. 2025 Aug 13;21(8):e1011825. doi: 10.1371/journal.pgen.1011825

Role of Pcdh15 in the development of intrinsic polarity of inner ear hair cells

Raman Kaushik ¹, Shivangi Pandey ¹, Anubhav Prakash ^1,², Fenil Ganatra ¹, Takaya Abe ³, Hiroshi Kiyonari ³, Raj K Ladher ^1,^*

Editor: Nandan Nerurkar⁴

PMCID: PMC12370195 PMID: 40802839

Abstract

In vertebrates, auditory information is transduced in the cochlea by mechanosensory hair cells (HC) through an eccentrically organised structure known as the hair bundle. This consists of a true cilium, known as the kinocilium, and modified microvilli, known as stereocilia. The hair bundle has a distinct structure with stereocilia organised in graded rows, with the longest abutting the kinocilium. The hair bundles of all HC are aligned to the tissue axis and are planar polarised. Important in the development and physiology of HC are protein bridges consisting of cadherin-23 (CDH23) and protocadherin-15 (PCDH15). These link the tips of stereocilia, where they play a role in mechanotransduction, and between the kinocilia and the stereocilia, where they are involved in development. Both Cdh23 and Pcdh15 mutations result in defects in planar polarity; however, the mechanism through which this defect arises is unclear. Using a novel mutant for the Pcdh15-CD2 isoform, we show that while the initial deflection of the kinocilium occurs, its peripheral migration to register with Gαi is perturbed. Pcdh15-CD2 genetically interacts with Gpsm2, perturbing vestibular function. We find that the earliest expression of PCDH15-CD2 is at the base of the kinocilia, and the defects in morphogenesis occur before the formation of kinocilial links. By re-introducing functional PCDH15-CD2, we show that polarity can be restored. Our data suggest that, in addition to its adhesive role, PCDH15-CD2 has an early role in intrinsic hair cell polarity through a mechanism independent of kinocilial links.

Author summary

Hearing, like other sensory modalities, is essential for animals to perceive and interact with their environment. The cochlea, the auditory sensory organ, develops under tight genetic regulation. In this study, we investigate the role of Pcdh15, a gene previously implicated in maintaining hair bundle integrity in hair cells. Using mouse genetics and advanced microscopy, we uncover an extended role for Pcdh15-CD2 isoform in regulating intrinsic hair cell polarity. Our findings reveal that Pcdh15-CD2 functions not merely in forming physical links between kinocilium and stereocilia but plays a key step in establishing intrinsic hair cell polarity. Prior to forming structural connections, Pcdh15-CD2 contributes to the directionality of kinocilium positioning. Importantly, we demonstrate that restoring Pcdh15-CD2 expression in mutant mice rescues hair cell polarity in a time-sensitive manner, highlighting its dynamic and instructive role in cochlear development.

Introduction

Hearing has conferred numerous evolutionary advantages to animals, such as prey detection, predator avoidance and communication. Sound is sensed in the cochlea of the inner ear, in a specialised epithelium known as the organ of Corti (OC). OC comprises four rows of mechanosensory hair cells (HC), divided into three rows of outer (OHC) and one row of inner hair cells (IHC), all surrounded by supporting cells [1,2]. HC have hair bundles on their apical surface, which consist of graded rows of actin-based stereocilia increasing in height, with the tallest attached to a single true cilium, the kinocilium [3]. The development and orientation of the hair bundles are critical for the functioning of the inner ear as they are only mechanosensitive along one axis, the short-tall axis defined by the kinocilium [4]. Hair bundle development starts with a centrally positioned kinocilium surrounded by microvilli covering the apical surface [5–8]. The kinocilium then centrifugally repositions to the periphery of the hair cell, followed by a re-orientation along the edge towards the abneural side of the hair cells [9,10]. Microvilli near the kinocilium then elongate and form the stereocilia, and the whole hair bundle moves towards the centre of the apical surface [11]. These initial steps of hair cell development define its intrinsic polarity. This intrinsic polarity is aligned across the OC such that the intrinsic polarity of all HC is aligned to the tissue axis, a process called planar cell polarity (PCP) [12].

Mutation studies have revealed a complex interplay of genes and pathways governing these processes. PCP is controlled by a molecularly conserved pathway of genes. In the cochlea, these comprise at least six different core PCP proteins [11,12]. These proteins are expressed asymmetrically at the intercellular junctions and are crucial for the tissue-wide arrangement of hair cells. Intrinsic polarity of HC relies on the interaction of the kinocilium and a signalling complex initially defined in spindle positioning during asymmetric cell division. These proteins include the inhibitory G protein subunit alpha (Gαi), its binding partner GPSM2 (G protein signalling modulator) and Inscuteable (INSC) [13]. This interaction establishes the apical architecture of the hair cell. Mutants of any of these genes result in hair cells with varying defects in intrinsic polarity, ranging from a failure of the lateral-ward movement, hair bundle fragmentation, and decoupling of the kinocilium from the stereociliary bundle [9,10,14,15]. These mutants also show mild PCP defects, with increased deviation from the tissue axis. Similar defects have also been observed in mutants of genes involved in various aspects of cilia function, such as those of intraflagellar transport complex components or of molecules involved in kinocilia assembly [16–20]. Perturbations in the localisation of Gαi protein in the kinocilia mutants, Mkss, Bbs8 or Ift88, suggest a relationship between the kinocilia and the signalling mechanisms that are thought to underlie apical patterning of HC [10,14,18].

Protein links between its components ensure the cohesion of the hair bundle [21]. These include links between the stereocilia and between the kinocilium and stereocilia. Studies on the components of the kinocilial links, CDH23 and PCDH15, suggest a function of these molecules in the generation of HC polarity [22,23]. Pcdh15 has three major isoforms (PCDH15-CD1, PCDH15-CD2, and PCDH15-CD3) that differ from each other in their cytoplasmic domains. They are expressed through the mutually exclusive alternative splicing of exons 35, 38, and 39, respectively [24–27] (Fig 1A). Among these isoforms, only mutants of the CD2 isoform show mild planar polarity defects. In addition, these mutants also display a perturbation in the apical architecture of the hair cell, showing a decoupling of the kinocilium from the tallest stereocilia [28]. Blocking FGF signalling prevents PCDH15 from entering the kinocilium, which also results in kinocilium-stereocilia decoupling [29]. However, kinocilial links only appear after the off-centre relocation of the kinocilia [30]. This may suggest a role for PCDH15, independent of its function in kinocilial links. In this study, we generate a mouse where Pcdh15-CD2 is fused to an epitope tag. We find that PCDH15-CD2 protein is initially localised around the base of the cilium prior to the peripheral relocation. Mutation of Pcdh15-CD2 alters the localisation of Gαi, similar to kinocilial mutants. We find evidence of a genetic interaction with Gpsm2, and in mice mutant for both Gpsm2 and Pcdh15-CD2. Here not only exaggerate polarity defects and bundle fragmentation, we now find defects in vestibular function. By re-expressing Pcdh15-CD2, we find that it is possible to rescue these defects up to 2 days after peripheral relocation. Our data suggest an additional role for PCDH15-CD2 during the generation of intrinsic hair cell polarity in coupling the kinocilium to the domain of GPSM2-Gαi signalling.

Fig 1 — (A) Schematic of the *Pcdh15* exons that produce three isoforms, CD1, CD2 and CD3, by alternative splicing of exons 35, 38 and 39, respectively. (B) Schematic of the DNA construct used to generate *Pcdh15*^n38/n38 mice. (C) The organ of Corti of *Pcdh15*^n38/n38 mice at P0 (mid). Immunostaining fails to detect the PCDH15-CD2-FLAG signal (cyan), although endogenous PCDH15 (yellow) is localised at the tip of stereocilia (magenta, marked with phalloidin). (Scale bar = 5 μm). (D) Western blot fails to detect PCDH15-CD2-FLAG protein in *Pcdh15*^n38/n38 mice OC (P5/6). Positive control shows HEK293T cells lysate transfected with PCDH15-CD2-FLAG cDNA. β-actin is used as a loading control. (E-F) Fluorescence (E) and SEM (F) imaging show hair bundle polarity defects in *Pcdh15*^n38/n38 OC when compared to normal hair bundle orientation in WT & *Pcdh15*^n38/+ (P0, mid). Cilia are marked with ARL13B (green) and stereocilia with phalloidin (magenta). (Scale bar = 5 μm). (G) Kinocilium is dissociated from stereocilia in OHC and IHC of *Pcdh15*^n38/n38 OC when compared to WT. (H) Polar plots of kinocilia positions in hair cells show higher circular standard deviation, indicative of hair bundle polarity defects in *Pcdh15*^n38/n38 OC (slate blue). This is much reduced in WT (pink) & *Pcdh15*^n38/+ (green) (P0, mid). (Sample size for OHC/IHC, WT = 156/51, *Pcdh15*^n38/+ = 119/40, *Pcdh15*^n38/n38 = 184/57).

Methods

Ethics statement

Experiments on animals were performed after approval from the NCBS Institutional Animal Ethics Committee (Approval no. NCBS-IAE-2023/10 (R2)).

Animal use and care

Pcdh15-n38 mice (Accession No.: CDB0186E: https://large.riken.jp/distribution/mutant-list.html) were generated by CRISPR/Cas9-mediated knock-in in zygotes as previously described [31]. Briefly, the following guide RNAs (gRNAs) were designed by using CRISPRdirect [32] to insert the knock-in construct: 5’ gRNA target sequence – 5’-CTATATGCATAGCTTGCGTG-3’ and 3’ gRNA target sequence - 5’-CTCAGCAAGCACGTCTACGT. Founder mice were bred with C57BL/6N mice to generate heterozygous animals. Pcdh15-n38YF mice were made by crossing homozygous Pcdh15-n38 with Pgk1-Cre ([33]: JAX#020811) mice. Gpsm2 mice (Accession No. CDB1144K) were received from RIKEN BDR, and were the kind gift of Fumio Matsuzaki [34]. Atoh1-Cre/ESR1 (JAX#007684) mice were obtained from JAX [35]. All primers used for genotyping are detailed in the table (S1 Table).

Immunofluorescence

Inner ear dissections were performed as previously described [36,37]. Fixation was based on the choice of antibody used (S2 Table). Fixed inner ears were washed with 1X PBS and further dissected to expose the sensory epithelium. Tectorial membrane was removed before permeabilisation with PBS + 0.3%Tween-20. Tissues were blocked with blocking buffer (10% goat serum, 1% bovine serum albumin (BSA) in permeabilisation buffer) and then incubated with primary antibodies (S2 Table) overnight at 4°C. Tissues were then washed 5–6 times with PBST before incubating with secondary antibodies and phalloidin (S3 Table) at room temperature for 1 hour. Tissues were washed 5–6 times before mounting with aqueous mounting media.

Image acquisition and processing

Confocal images were captured by an Olympus FV3000 inverted confocal microscope with a 60x objective (1.42 NA) using its software Olympus FV31S-SW. All images were captured at sub-saturation level under Nyquist sampling criteria. High-resolution images were taken on a Zeiss LSM 980 Airyscan 2 microscope with a 63x objective (1.4 NA). All the raw files were processed by ImageJ software.

Immunoprecipitation and western blotting

For immunoprecipitation (IP), 20 mouse cochleae at P5 stage were dissected and homogenised in lysis buffer (0.5% Triton X-100, 150mM NaCl, 5mM EDTA and 1X MS-SAFE protease and phosphatase inhibitor in PBS). Lysates were cleared by centrifugation at 15,000 g for 10 mins. Anti-HA or normal mouse IgG antibody was incubated with Dynabead Protein G beads (Invitrogen) for 1 hr at 4°C to form the Dynabead-bound antibody complexes. Parallelly, the lysate was incubated with Dynabeads to reduce the non-specific binding. The cleared lysates were incubated with Dynabead-bound antibody complexes of either HA-specific antibody (Sigma H3663) or with normal mouse IgG overnight at 4°C. The proteins were purified and washed 5 times with lysis buffer before eluting in 1X SDS loading buffer. Proteins were resolved by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA in 0.1% TBST buffer and incubated with primary antibodies overnight at 4°C. After 5 washes with 0.1% TBST buffer, membranes were incubated with peroxidase-conjugated secondary antibodies. Immunoreactive proteins were detected with Western Bright ECL detection reagents.

Vestibular test

Littermate mice (5 months old) of different genotypes (Pcdh15^n38/+:: Gpsm2^+/-, Pcdh15^n38/n38:: Gpsm2^+/-, Pcdh15^n38/+:: Gpsm2^-/- and Pcdh15^n38/n38:: Gpsm2^-/-) were kept in a controlled environment. Vestibular test experiments were performed in a box (22cm X 15 cm X 13 cm), and the activity of mice was recorded for 5 min. The total distance covered, mean speed and no. of rotations were measured. Analyses were performed using ANY-maze software.

Preyer’s reflex test

Adult mice (3 months old) of different genotypes (wild-type, Pcdh15^n38/n38 and Pcdh15^n38YF/n38YF) were kept in a controlled environment. Preyer’s reflex test was performed in a box (22cm X 15 cm X 13 cm), and the startle response was recorded for 10 stimuli (spaced by 2 min). The presence of the startle response was measured as a percentage of positive Preyer’s reflex.

Image analysis and statistics

Data were collected from at least three animals (from at least two different breeder pairs) for each experiment. The sample size is mentioned with each individual graph. Images were analysed using ImageJ and Imaris.

For hair bundle defects, kinocilium position (marked with ARL13B or acetylated tubulin antibodies) was measured with respect to the hair cell cortex (marked by phalloidin). A vector was drawn from the centre of the hair cell to the kinocilium base, whose length (r) is normalised with cell radius and the angle (θ) from the proximal to distal (P-D) axis. The kinocilium position in different hair cells was visualised by using polar plots in the Matplotlib library of Python. Similarly, the frequencies of kinocilium orientation were measured and plotted from 0°-360° within 10° brackets. For measurement of stereocilia bundle orientation, the angle tool in ImageJ software was utilised, where a line was drawn from the centre of the stereocilia bundle and then plotted with the tissue axis.

For spatial expression measurement of HA staining, a line (ROI) of width equal to the kinocilium (marked by acetylated tubulin) was taken, and the fluorescence intensity of acetylated tubulin and HA was measured with respect to the distance (on the line drawn). The fluorescence intensity of both signals was plotted together to give their colocalisation pattern.

For alpha – theta (α-θ) correlation analysis, θ angles were plotted against α angles. θ angles were calculated for the angle of the kinocilia base from 0° (along the P-D axis), and α angles were calculated for the centre of the Gαi expression domains from the horizontal (0°) on the P-D axis. The Python script is available as supporting information.

Box and whisker graphs were plotted using GraphPad Prism 9.5.0. The same software was used for statistical analyses. We analysed data using ordinary one-way or two-way ANOVA test (Tukey’s multiple comparison test). P values were represented in the graphs; p < 0.05 was considered statistically significant.

Scanning electron microscopy (SEM)

Samples for SEM were prepared as described previously [37]. After inner ear dissection in 1X PBS, tissues were fixed in 2.5% glutaraldehyde in sodium cacodylate buffer (0.1M) with calcium chloride (3mM) for 48 hours. After fixation, samples were micro-dissected to expose the sensory epithelium. Samples were again processed with the OTOTO method for an alternate series of fixation with 1% osmium tetroxide (O) and 0.5% thiocarbohydrazide (T). Samples were dehydrated first with serially increased alcohol percentage, followed by critical point dehydration (Leica EM CPD300). Mounting was done on double-sided carbon adhesive tapes, followed by sputter coating. Samples were imaged on Zeiss Merlin Compact VP with SE2 detectors.

Results

Generation of Pcdh15^n38/n38 and Pcdh15^n38YF/n38YF mice

Our previous work had suggested that tyrosine phosphorylation on the cytoplasmic domain of the PCDH15-CD2 isoform was important for PCDH15 function [29]. We hypothesised that finding interactions specific to phosphorylation of PCDH15 would help understand this function. To do this, we engineered a mouse where the endogenous exon 38 was replaced by a construct that consisted of a CD2 domain with an in-frame FLAG tag. In addition, the construct contained a mutant CD2 in which all 6 tyrosine residues were changed to phenylalanine and fused to an HA tag. These sequences were flanked by LoxP, Lox71 and Lox66 sites. Without Cre recombination, the wild-type PCDH15-CD2 tagged with the FLAG epitope should be expressed (Pcdh15^n38/n38) (Figs 1B and S1A).

To check for PCDH15-CD2 expression in hair cells of the organ of Corti, we performed immuno-localisation with a FLAG-tag specific antibody in Pcdh15^n38/n38 mice. While PCDH15 expression could be visualised in stereocilia tips, we were unable to detect immuno-reactivity for the FLAG-tag (Fig 1C). The lack of FLAG immunoreactivity in the tissue was confirmed by Western blotting. We failed to detect the FLAG epitope in P5/6 Pcdh15^n38/n38 organ of Corti lysates, despite detection in HEK cells (Figs 1D and S1B). RT-PCR indicated no difference in mRNA expression between WT and Pcdh15^n38/n38 mice (S1C Fig). This suggested that translation of the mutant PCDH15-CD2 isoform was impaired, indicating that the Pcdh15^n38/n38 mice are functionally null and act similar to Pcdh15-∆CD2 mutants reported earlier [28,38].

Defects in hair bundle polarity in Pcdh15^n38/n38 mice

Pcdh15-∆CD2 mutants show defects in hair bundle orientation and mechanotransduction [28,38]. To ask if Pcdh15^n38/n38 mice show a similar phenotype to Pcdh15-ΔCD2, we immunostained the P0 organ of Corti with ARL13B and phalloidin to determine hair bundle polarity. The average kinocilium deviation from the tissue axis was comparable in P0 wild-type and Pcdh15^n38/+ animals (Fig 1E, 1F and 1H). In OHC, this deviation, at P0, is 13˚ ± 11˚ in Pcdh15^n38/+; however, in Pcdh15^n38/n38 animals, this deviation is 59˚ ± 32˚, indicating a defect in planar polarity. While hair bundle polarity is affected equally in all three rows of OHC, IHC show a less severe defect in hair bundle polarity, with the average kinocilium deviation of 23˚ ± 15˚ in Pcdh15^n38/n38 as compared to 8˚ ± 6˚ in Pcdh15^n38/+ animals (Fig 1H). Despite the defect in polarity, the expression of the core PCP protein, VANGL2, is unchanged in Pcdh15^n38/n38 mice (S1D Fig). Closer inspection using SEM revealed a decoupling of the kinocilium from the stereocilia (Fig 1F). In Pcdh15^n38/+, almost all kinocilia are attached to the longest stereocilia and are found at the vertex of the stereocilia bundle. In Pcdh15^n38/n38 mice, we were unable to detect any hair cell with a kinocilium coupled to the stereocilia. Additionally, we observed misoriented stereocilia bundles of varying degrees from the tissue axis, as well as occasional circular bundles with a frequency <1% (Fig 1G). Given that the kinocilium is decoupled from the stereocilia bundle in the mutants, we quantified orientation defects by measuring the angular deviation of stereocilia relative to the tissue axis. The average stereocilia bundle deviation, 26˚ ± 12˚ in Pcdh15^n38/38 mice OHC is significantly higher when compared to 15˚ ± 8˚ in Pcdh15^n38/+ controls (S1E Fig).

Tyrosine phosphorylation of the CD2 domain is not essential for PCDH15 function in mice

Our previous work had identified that FGFR1 signalling was involved in the coupling of the kinocilium to the stereocilia, through the phosphorylation of the cytoplasmic domain of PCDH15-CD2 [29]. To generate the Pcdh15^n38YF/+ germline mutant, we mated the Pcdh15^n38/n38 mice with the ubiquitously expressed Pgk1-Cre. The Pcdh15^n38YF/+ heterozygous animals were back-crossed to give homozygotes (Figs 2A, 2B and S2A). These mice showed specific epitope tag (HA) expression in the kinocilium and at stereocilia tips, indicating normal expression of the modified PCDH15-CD2-HA (Fig 2C, 2D). This expression colocalised with PCDH15 expression, validating the normal expression of the HA-tagged modified PCDH15-CD2 (S2B Fig). Although we could not resolve the hair bundle at embryonic stages, at postnatal stages, we observed PCDH15-CD2-HA expression at the tip of all three rows of stereocilia in HC (S2C Fig). This data was verified by immunoprecipitation from the cochlear lysate of Pcdh15^n38YF/n38YF mice, showing a specific band when compared to the IgG control (Figs 2E and S2D). ARL13B and phalloidin staining were used to measure hair bundle polarity, together with SEM for determining kinocilium-stereocilia coupling. Despite Pcdh15^n38/n38 showing defects in polarity, Pcdh15^n38YF/n38YF were similar to wild-type controls (Figs 2F–2H and S2E). This suggests that the phosphorylation of tyrosine in the CD2 domain of Pcdh15^n38YF/n38YF is not essential for normal PCDH15 function.

Fig 2 — (A) Schematic of the mice breeding scheme to produce *Pcdh15*^n38YF/n38YF mice. (B) Schematic of the *Pcdh15-n38* DNA construct recombination to *Pcdh15-n38YF* under Cre activity. (C) Modified PCDH15-CD2-HA (green) is expressed in the stereocilia (magenta) of *Pcdh15*^n38YF/n38YF hair cells (P0, mid), but not in WT. (Scale bar = 5 μm). (D) Surface plot of OHC shows PCDH15-CD2-HA (green) localisation at the tip of the hair bundle. (E) IP assay shows that PCDH15-CD2-HA is precipitated with anti-HA antibody. (F) Hair bundle polarity is normal in *Pcdh15*^n38YF/n38YF and *Pcdh15*^n38YF/+ hair cells (P0, mid). Cilia are marked with ARL13B (green) and stereocilia with phalloidin (magenta). (Scale bar = 5 μm). (G) Polar plots marking kinocilia positions in hair cells show normal hair bundle polarity in *Pcdh15*^n38YF/n38YF OC (green) (P0, mid). (Sample size for OHC/IHC, WT = 156/51, *Pcdh15*^n38YF/n38YF = 167/48). (H) Kinocilia are aligned normally with the tissue axis in WT (pink) and *Pcdh15*^n38YF/n38YF (green) hair cells, but significantly deviated in *Pcdh15*^n38/n38 (slate blue). The deviation is greater in OHC when compared to IHC (P0, mid). (Scale bar = 5 μm, Two-way Anova with Tukey’s multiple comparisons test, ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). (Sample size for OHC3/OHC2/OHC1/IHC, WT = 52/52/52/51, *Pcdh15*^n38/n38 = 62/60/62/57, *Pcdh15*^n38YF/n38YF = 56/57/54/48).

PCDH15-CD2 has also been implicated in mechanotransduction, although the MET channels in Pcdh15-∆CD2 are functional at early post-natal stages [28]. Using the styryl pyridinium dye, FM1–43 FX, which stains hair cells after entering through MET channels, we asked if MET channels were functional [39]. We find that HC in the P5 organ of Corti from both Pcdh15^n38/n38 and Pcdh15^n38YF/n38YF mice are stained by FM1–43, suggesting that MET channels can open (S2F Fig). Using Preyer’s reflex, we found that P60 Pcdh15^n38/n38 mice could not respond to sound, but Pcdh15^n38YF/n38YF mice responded similarly to the control animals (S2G Fig). This data suggests that, similar to Pcdh15-∆CD2 mutants [28,38], Pcdh15^n38/n38 mice cannot hear.

PCDH15-CD2 is expressed in the kinocilium prior to its off-centre movement

To understand the role of PCDH15-CD2 in intrinsic HC polarity, we utilised the HA tag on Pcdh15^n38YF/n38YF to map its earliest expression during hair bundle morphogenesis. At E15.5, we observe PCDH15-CD2-HA expression at the base of the organ of Corti, in the IHC kinocilium which at these stages is yet to move off-centre (Fig 3A). We also noted HA positive cortical punctae around the kinocilium (Fig 3A). At these stages, we did not observe HA expression in the OHC or any HC at the apex or mid of the organ of Corti. At E16.5, HA-staining could be observed in the kinocilia of both IHC and OHC at the apex of the organ of Corti (Fig 3B). HA staining was not detected in wild type E16.5 organ of Corti (S2H Fig). To understand the spatial HA expression at the kinocilium, we measured the co-staining of HA with acetylated tubulin, a marker that labels the full length of the kinocilium, including its transition zone [40]. PCDH15-CD2-HA is expressed as punctae along the length of the centrally situated kinocilium of the apex OHC (Fig 3C). In the kinocilium of more mature IHC of the base region, which had migrated eccentrically, expression is also seen in the tip and shaft of the kinocilium but is absent from its base (Fig 3D, 3E).

Fig 3 — (A) PCDH15-CD2-HA (cyan) is localised in the centrally located kinocilium (yellow, marked with acetylated tubulin) of IHC of E15.5 OC (base). (Scale bar = 10 μm). (B-C) PCDH15-CD2-HA (cyan) is localised along the entire length of the centrally located kinocilium (yellow, marked with acetylated tubulin) of OHC of E16.5 OC (apex). (Scale bar = 10 μm). (D–E) PCDH15-CD2-HA (cyan) is localised in the laterally positioned kinocilium (yellow, marked with acetylated tubulin) of the IHC of E16.5 OC (base), except at their basal end. (Scale bar = 10 μm). (F-G) PCDH15-CD2-HA (cyan) is localised in the centrally located kinocilium (yellow, marked with acetylated tubulin) of newly differentiating HC of E18.5 utricle (F) and cristae (G). As hair cells mature, this expression is enriched in hair bundles (magenta, marked with phalloidin) and gets refined. (Scale bar = 10 μm).

To ask if PCDH15-CD2-HA was also expressed on the hair bundle of vestibular HC, we assessed the utricle and cristae. At E18.5, newly differentiating hair cells can be distinguished from mature HC. In newly differentiating HC, PCDH15-CD2-HA expression is found in the kinocilium, prior to its off-centre movement. Similar to the auditory HC, PCDH15-CD2-HA expression was also observed in punctae around the HC kinocilium of the utricle (Fig 3F) and cristae at E18.5 (Fig 3G). In more mature HC, distinct punctae in kinocilia are visible. These were not detected in wild type controls (S2I Fig).

Pcdh15^n38/n38 mice exhibit early hair bundle polarity defects

During the development of the hair cell, the kinocilium initially moves to an eccentric location, adjusts along the periphery, and then relocalises towards the centroid of the HC apex with the expansion of the bare zone, a region free of microvilli, lateral to the kinocilium [11]. As PCDH15-CD2 expression could be detected in kinocilia before the off-centre movement, we asked at which point polarity defects arose. We determined kinocilia position using ARL13B at E16.5 and E18.5. In Pcdh15^n38/+ at E16.5, and prior to its off-centre movement, kinocilium position is distributed around the centroid of the HC with a slight bias to the abneural side of the OC. In contrast, the kinocilium does not show such a bias in Pcdh15^n38/n38 mice (Fig 4A–4C). We next assessed the kinocilia position pattern at E18.5. Pcdh15^n38/+ kinocilia had relocalised towards the centroid of the HC. In contrast, the kinocilia of Pcdh15^n38/n38 mice are distributed randomly and remain positioned near the periphery of hair cells (Fig 4D–4F). To further understand the randomised kinocilia position, we investigated the position of centrioles. In Pcdh15^n38/+ HC at E18.5, centrioles of the basal body are aligned orthogonally, with the mother centrioles attached to the kinocilia, and the daughter centriole extending basally. In contrast, the centrioles are separated from each other in Pcdh15^n38/n38 mice, and although the mother centriole is still in contact with the kinocilium, the daughter centriole position is randomised (Figs 4G–4I and S3A). Taken together, these data suggest that in Pcdh15^n38/n38 mice, although the initial kinocilium off-centre movement occurs, its subsequent relocalisation is impaired.

Fig 4 — (A) Hair bundle polarity is perturbed in *Pcdh15*^n38/n38 hair cells of E16.5 OC (mid). Cilia are marked with ARL13B (green) and stereocilia with phalloidin (magenta). (Scale bar = 10 μm). (B) Polar projections marking kinocilia positions in E16.5 stage (mid) hair cells show perturbed hair bundle polarity in *Pcdh15*^n38/n38 (slate blue). (Sample size for OHC/IHC, *Pcdh15*^n38/+ = 147/48, *Pcdh15*^n38/n38 = 264/91). (C) Kinocilia are deviated significantly from the tissue axis in *Pcdh15*^n38/n38 (slate blue) OHC as compared to *Pcdh15*^n38/+ (pink) (E16.5, mid). (Two-way Anova with Tukey’s multiple comparisons test, ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). (Sample size for OHC3/OHC2/OHC1/IHC, *Pcdh15*^n38/+ = 41/53/53/48, *Pcdh15*^n38/n38 = 79/95/90/91). (D) The perturbation in hair bundle polarity continues in *Pcdh15*^n38/n38 hair cells of E18.5 OC (mid). Cilia are marked with ARL13B (green) and stereocilia with phalloidin (magenta). (Scale bar = 10 μm). (E) Polar projections marking kinocilia positions in E18.5 stage (mid) hair cells show perturbed hair bundle polarity in *Pcdh15*^n38/n38 (slate blue). (Sample size for OHC/IHC, *Pcdh15*^n38/+ = 189/51, *Pcdh15*^n38/n38 = 195/50). (F) Kinocilia are deviated significantly from the tissue axis in *Pcdh15*^n38/n38 (slate blue) HC (E18.5, mid). (Two-way Anova with Tukey’s multiple comparisons test, ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). (Sample size for OHC3/OHC2/OHC1/IHC, *Pcdh15*^n38/+ = 60/65/64/51, *Pcdh15*^n38/n38 = 65/65/65/50). (G) Immunostaining and segmentation of the centrioles (marked with pericentrin) show their mispositioning in *Pcdh15*^n38/n38 mice hair cells (E18.5, base). (Scale bar = 10 μm). (H) Quantitation of the percentage of HC shows centrioles mispositioning in *Pcdh15*^n38/n38 mice as compared to the control. (Two-way Anova with Tukey’s multiple comparisons test, ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). (Sample size for OHC3/OHC2/OHC1/IHC, *Pcdh15*^n38/+ = 87/85/86/79, *Pcdh15*^n38/n38 = 62/60/63/59). (I) A zoomed image of a single optical section of the stained centrioles (marked with pericentrin) connected to the kinocilium (marked with acetylated tubulin) shows the absence of their orthogonal position (asterisk) to each other in IHC of *Pcdh15*^n38/n38 mice (E18.5, base).

The coordination of kinocilium with Gαi expression is lost in Pcdh15-CD2 mutants

Intrinsic polarity is controlled through a pathway involving GPSM2 and Gαi. Gαi expression is normally restricted to a narrow lateral crescent in HC, marking the bare zone [9,10,14]. In mutants for the ciliary genes Bbs8, Ift88 or McKusick-Kaufman syndrome (Mkks) gene, the expression domain of Gαi is expanded, such that it covers the entire lateral half of the HC [10,14,18]. Similar to ciliary mutants, we find that the loss of Pcdh15-CD2 also expands Gαi and GPSM2 expression medially (Figs 5A–5C and S4A–S4C).

Fig 5 — (A) Gαi expression (cyan) is perturbed with respect to the kinocilium position (yellow, marked with acetylated tubulin) in E18.5 (base) hair cells of *Pcdh15*^n38/n38 mice. (Scale bar = 10 μm). (B) Gαi expression domain (cyan) is extended medially in HC of *Pcdh15*^n38/n38 mice. (Scale bar = 2 μm). (C) In comparison to *Pcdh15*^n38/+ mice, the Gαi expression domain is spread significantly in HC of *Pcdh15*^n38/n38 mice (E18.5, base). (Two-way Anova with Tukey’s multiple comparisons test, ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). (Sample size for OHC3/OHC2/OHC1/IHC, *Pcdh15*^n38/+ = 51/54/50/46, *Pcdh15*^n38/n38 = 55/57/56/53). (D) Correlation between the kinocilium position and Gαi expression domain is reduced in HC of *Pcdh15*^n38/n38 mice (green) when compared to *Pcdh15*^n38/+ (blue) (E18.5, base). Alpha (α) is the angle measured for the kinocilium with the tissue axis, and theta (θ) is the angle measured for the Gαi expression domain (mid) with the tissue axis. (Sample size for OHC3/OHC2/OHC1/IHC, *Pcdh15*^n38/+ = 51/54/50/46, *Pcdh15*^n38/n38 = 55/57/56/53).

After its off-centre movement, the kinocilium undergoes peripheral relocation, such that it is situated in the middle of a domain of Gαi expression. In Mkks mutants, this shift is perturbed [10]. To ask if a similar effect was visible in the Pcdh15-CD2 mutants, we measured the correlation between angle the kinocilium makes with the tissue axis (theta) and the angle that the midpoint of Gαi expression makes with the tissue axis (alpha). In E18.5 Pcdh15^n38/+ organ of Corti, a strong angular correlation between centre of the Gαi expression domain and kinocilia is observed, showing a Pearson correlation coefficient of 0.667. In Pcdh15^n38/n38 mutant mice, this correlation coefficient is reduced to 0.2 (Fig 5D), suggesting that Pcdh15^n38/n38 mutant HC fail to undergo a peripheral relocation during hair bundle development, remaining decoupled from the mid-point of the Gαi domain.

PCDH15-CD2 and GPSM2 proteins operate cooperatively in hair bundle development

We next asked if PCDH15-CD2 and Gαi-GPSM2 showed any genetic interaction. We used a null Gpsm2 mutant, in which almost the entire coding sequence is deleted [34]. On a Pcdh15^n38/+ background, neither Gpsm2^+/- heterozygotes (10˚ ± 8˚) nor Gpsm2^-/- homozygotes (7˚ ± 6˚) show significant defects in planar polarity at P1 (Fig 6A, 6C and 6E). Pcdh15^n38/n38 mice showed mild defects in hair bundle polarity (Figs 1H and 2H), which was similar to both Pcdh15^n38/n38:: Gpsm2^+/- and Pcdh15^n38/n38:: Gpsm2^-/- compound mutants (67˚ ± 43˚ and 59˚ ± 39˚, respectively) (Figs 6B, 6D, 6E and S5A).

Gpsm2 mutant HC show hair bundle fragmentation (24% of OHC & 14% of IHC) (Fig 6F) [9,10,14]. We thus asked if the number of hair cells showing bundle fragmentation increased in the compound mutants. We found increased fragmentation in the compound mutants, with OHC more affected than IHC (46% of OHC & 28% of IHC) (Figs 6F and S5B). Moreover, and in contrast to planar polarity, the proportion of HC in the Pcdh15^n38/n38:: Gpsm2^-/- organ of Corti that show bundle fragmentation is significantly higher than in Pcdh15^n38/n38:: Gpsm2^+/- and Pcdh15^n38/+:: Gpsm2^-/- cochlea (Figs 6F and S5B).

To further characterise the compound mutant phenotype, we asked if we could detect vestibular perturbations. Single Gpsm2 null and Pcdh15^n38/n38 mice do not exhibit vestibular defects, however, we observed that Pcdh15^n38/n38:: Gpsm2^-/- mice show circling behaviour and hyperactivity (Fig 6G and S1 Movie). In an open field test, we found that Pcdh15^n38/n38:: Gpsm2^-/- mice travel larger distances, with higher mean speed and show a larger number of rotations, with a preference to anti-clockwise turns (Fig 6H–6J).

To further investigate the vestibular defects underlying the circling behaviour in double mutant mice, we first examined the morphology of hair bundles in the utricle, saccule, and crista using scanning electron microscopy (SEM). Compared to controls, stereocilia bundles in Gpsm2^-/- and Pcdh15^n38/n38:: Gpsm2^-/- appeared shorter across all three vestibular organs. In contrast, hair bundle morphology in Pcdh15-CD2 mutant mice was comparable to that of control animals (Fig 7A). In the utricle and saccule of Pcdh15^n38/+:: Gpsm2^-/- and Pcdh15^n38/n38:: Gpsm2^-/-, bundle gradation was much reduced. In the cristae, the stereociliary bundle was still graded in Pcdh15^n38/+:: Gpsm2^-/- mutants. Interestingly, gradation was absent in the cristae of double mutants (Figs 7A and S6A). Although it remains unclear whether this loss of gradation in the cristae alone can account for the circling phenotype, these findings suggest that a cristae-specific morphological defect is found in double mutants.

Fig 7 — (A) SEM imaging shows smaller stereocilia bundles in the hair cells of all three vestibular structures (utricle, saccule and crista) of *Pcdh15*^n38/+*:: Gpsm2*^-/- & *Pcdh15*^n38/n38:: Gpsm2^-/- genotypes, but not in *Pcdh15*^n38/n38:: Gpsm2^+/-. The stereocilia gradation is preserved to some extent in the crista of *Pcdh15*^n38/+*:: Gpsm2*^-/- mice, but is lost in *Pcdh15*^n38/n38:: Gpsm2^-/- (P20, Scale bar = 1 μm). (B - C) Direction of hair cells in the cristae (marked with red arrows) shows a significant increase in their misorientation in *Pcdh15*^n38/n38:: Gpsm2^+/- and *Pcdh15*^n38/+*:: Gpsm2*^-/- as compared to *Pcdh15*^n38/+*:: Gpsm2*^+/-, which is further increased in the *Pcdh15*^n38/n38:: Gpsm2^-/- mice. (P7, Scale bar = 20 μm). (Ordinary one-way Anova with Tukey’s multiple comparisons test, ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). (Sample size for HC, *Pcdh15*^n38/+*:: Gpsm2*^+/- = 341, *Pcdh15*^n38/+*:: Gpsm2*^-/-= 350, *Pcdh15*^n38/n38:: Gpsm2^+/- = 356, *Pcdh15*^n38/n38:: Gpsm2^-/- = 354).

We next assessed planar polarity in the utricle and found comparable polarity in all the genotypes (S6B–S6F Fig). However, we found a significant increase in cristae hair cell misorientation in both Gpsm2^-/- and Pcdh15^n38/n38 single mutants relative to controls (Fig 7B, 7C). However, as neither single mutant exhibits circling, these polarity defects alone appear insufficient to explain the phenotype. Notably, hair cell misorientation was further exacerbated in double mutants, although it is uncertain whether this enhanced disorganisation is the sole cause of the circling behaviour. Together, these findings suggest a cumulative effect of Pcdh15-CD2 and GPSM2 loss on vestibular hair cell morphology and polarity, which may contribute to, but do not unequivocally explain, the circling behaviour. Taken together, these data suggest a genetic interaction between Pcdh15 and Gpsm2.

GPSM2 directs the restricted expression of Gαi, which shows a medial expansion in Pcdh15^n38/n38 (Fig 5A–5C). Gαi interacts with the coiled-coil domain containing proteins, Ccdc88a (Girdin) and Ccdc88c (Daple) [41]. To further understand the nature of this interaction, we investigated the expression of Daple and Girdin in Pcdh15 mutants. We find that their localisation is in the lateral HC junction closest to the kinocilium, and is unchanged in Pcdh15^n38/n38 cochlea (Fig 8A). We next asked if the expression of PCDH15 is altered in Gpsm2^-/- cochlea. We first used an N-terminal specific antibody that recognises all the major isoforms of PCDH15. We found that PCDH15 expression is unperturbed in Gpsm2^-/- HC (Fig 8B). To ask if the CD2 isoform was affected, we crossed Pcdh15^n38YF/n38YF into Gpsm2^-/- background and assessed the expression using HA-tag. We find that PCDH15-CD2-HA still localises to the tip of stereocilia in these mutants (Fig 8C). These data suggest that Pcdh15 and Gpsm2 function together during inner ear development.

Fig 8 — (A) Daple & Girdin proteins (grey) are localised on the neural edge of the HC of both *Pcdh15*^n38/+ and *Pcdh15*^n38/n38 mice (E18.5, base). (Scale bar = 10 μm). (B) PCDH15 protein (green) is localised on the hair bundles (even in fragmented hair bundles, marked with red asterisk) of *Gpsm2* mutant HC (E18.5, mid). (Scale bar = 10 μm). (C) PCDH15-CD2-HA (cyan) is localised on both kinocilium (yellow, marked with acetylated tubulin) and stereocilia (magenta, marked with phalloidin) in *Gpsm2* mutant HC (P1, base). (Scale bar = 10 μm).

PCDH15-CD2 expression rescues the intrinsic hair bundle polarity defects

Recently, mini PCDH15-CD2 mediated rescue was reported in a Pcdh15 mutant mouse. Here, the Pcdh15 allele had been removed using a Myo15-cre [42]. Cre is active after intrinsic polarity has already developed in these mice. We asked if re-introducing the Pcdh15 function could rescue the intrinsic polarity defects observed in Pcdh15^n38/n38. We reasoned that by controlling the timing of Cre expression, we could induce recombination such that the mutant Pcdh15^n38/n38 was replaced by the functional Pcdh15^n38YF/n38YF allele (Fig 2A, 2B). We used an Atoh1-CreERT2 line, in which Cre can be activated by tamoxifen induction. We injected dams carrying E17.5 Pcdh15^n38/n38 mutants with a 5 mg/40 g dose of tamoxifen and determined the resulting phenotype at E19.5 (Fig 9A).

In Pcdh15^n38/n38 littermate controls, all HC showed kinocilia decoupling from stereocilia, and HC showed intrinsic polarity defects (Fig 9B–9D). These cochleae did not express PCDH15-CD2-HA (Fig 9B, 9C). In Atoh1-CreET2^+/-::Pcdh15^n38/n38 animals, while Atoh1-CreERT2 induction failed to induce recombination in IHC, we observed mosaic expression of PCDH15-CD2-HA in OHC (Fig 9B, 9C). In HA-positive HC, we found that kinocilia and stereocilia were coupled (Fig 9B). We quantified the kinocilium position in hair cells, differentiating whether they expressed PCDH15-CD2-HA. We found that the kinocilia of PCDH15-CD2-HA expressing OHC align with the tissue axis (26˚ ± 30˚), in contrast to the non-expressing OHC (48˚ ± 37˚) (Fig 9D).

Discussion

Our study identifies a role for PCDH15-CD2 in regulating intrinsic hair cell polarity, specifically through the coordination of basal body positioning and Gαi localisation. While PCDH15 is classically defined as a component of kinocilial and stereocilial links and is essential for mechanoelectrical transduction, our findings reveal that the CD2 isoform contributes earlier to the apical architecture of hair cells. These functions appear to be independent of tyrosine phosphorylation, as the phospho-mutant allele (n38YF) fully rescues polarity defects.

Hair cells display two distinct but interconnected types of polarity: intrinsic polarity, which defines the spatial organisation of subcellular components such as the kinocilium, G-protein domains, and actin-based stereocilia within a single cell, and planar cell polarity (PCP), which aligns this intrinsic polarity across the epithelial sheet [11]. While PCP components such as Vangl2 and Celsr1 coordinate tissue-level alignment, intrinsic polarity mechanisms must integrate cytoskeletal, centrosomal, and membrane-associated cues to generate and maintain internal asymmetry [12]. Our data demonstrate that Pcdh15-CD2 mutants exhibit defects in intrinsic polarity, particularly in the positioning of the kinocilium relative to the Gαi domain. These defects in intrinsic polarity are similar to the defects observed in the Ames Waltzer, Pcdh15 mutant mouse [23,43].

The function of Pcdh15-CD2 in intrinsic polarity has been ascribed to the absence of kinocilial links. This is supported by the intrinsic polarity defects observed in mutations in the binding partner of Pcdh15, Cdh23 [23]. However, examining the timing of kinocilium migration and the expression of PCDH15-CD2 may suggest an earlier role in this process. Kinocilial links are only observed once stereocilia begin to elongate, and are first visible in the OHC found at the base of the cochlea at E17.5 [30]. This is after the initial centrifugal relocalisation of the kinocilia [8–10]. Indeed, intrinsic polarity defects in Pcdh15-n38 mutants are already evident by E16.5, prior to the appearance of kinocilial links. Furthermore, using an HA-tagged PCDH15-CD2 allele, we detected expression of the CD2 isoform at E15.5-E16.5, localised around the base of the kinocilium. This suggests a possible earlier role for Pcdh15-CD2 independent of its role in kinocilial link formation.

In wild-type cochlear hair cells, the kinocilium relocates from a central to peripheral position in a two-step process: it first moves peripherally and then relocates radially to align with the centre of the Gαi crescent at the apical surface [9,10]. In Pcdh15-n38 mutants, the kinocilium fails to localise to the centre of the GPSM2-Gαi crescent. This process occurs between E16.5 and E17.5 [9]. It is unlikely that this positioning is initiated through kinocilium–stereocilium adhesion, as these structures remain physically distant at this stage. Furthermore, in our rescue experiment, it is difficult to envision how the kinocilium would recouple with the stereocilia through adhesion alone.

Although the initial steps in intrinsic polarity generation are independent of kinocilial links, further steps likely require functional attachments between PCDH15 in the kinocilium and CDH23 in the tallest stereocilium. These links are likely to be established once the kinocilium is at the centre of the Gai crescent. The expansion of the bare zone shifts the hair bundle medially [9,10]. In Pcdh15-n38 mutants, these links are no longer present, and the kinocilium is not anchored to the stereociliary bundle. The failure in the coordination between the kinocilium and the stereocilia is reflected in the differences between the variation of alignment of kinocilia and that of stereocilia, to the tissue axis in mutants.

In Pcdh15-n38 mutants, we observed a medial expansion of both GPSM2 and Gαi. This expansion has also been reported in basal body and ciliary mutants, including Mkks, Ift88, and Bbs8 [10,14,18]. This suggests that PCDH15-CD2 may function upstream in organising the pericentriolar architecture necessary for correct apical patterning. Support for this comes from the mispositioning of centrioles observed in Pcdh15-n38 mutants. Such positioning has also been observed in Kif3a mutants. Normally, the mother centriole (kinocilium basal body) is apically anchored, while the daughter centriole lies more basally. In Kif3a mutants, that difference in apicobasal position is lost [19]. Perturbed RAC-PAK signalling is thought to underlie the perturbed basal body position in the Kif3a mutant, with active RAC1 recruited to the pericentriolar matrix through the microtubule-associated protein, LIS1 [44]. LIS1 is also required for the dynein activity and localisation around the kinocilium centrosome [45,46]. Our data suggest that PCDH15-CD2 is also localised around the basal body, suggesting a possible link through which CD2 and the RAC-PAK pathway could influence basal body position and apical domain organisation. However, how exactly this could impact the localisation of Gai3 and GPSM2 is unclear.

Although distinct molecular pathways specify kinocilium and Gαi domains, their spatial convergence appears necessary for proper bundle morphogenesis. This loss of coordination can affect the cohesion of the stereociliary bundle. This is supported by our observation that Pcdh15-n38:: Gpsm2 double mutants exhibit exacerbated bundle fragmentation. These phenotypes parallel those observed in Daple mutants, where kinocilium-Gαi misalignment results in disorganised bundles [41]. Thus, intrinsic polarity defects in basal body and Gαi alignment appear to drive secondary consequences in bundle assembly. Moreover, the position of kinocilia can influence planar polarity. Bbs8 is known to influence the trafficking of VANGL2, and overall epithelial organisation may be affected as a result [18]. However, in our analysis of Pcdh15-n38 mutants, we did not detect an alteration in VANGL2 localisation. Recent data suggest that the kinocilium’s position alters junctional mechanics during chick cochlea extension, altering polarity [47]. Pcdh15-CD2 may change the ability of the kinocilia to influence neighbouring cellular dynamics, leading to defects in planar polarity. The restoration of PCDH15-CD2 expression via Cre-mediated recombination partially rescued intrinsic polarity, confirming that PCDH15-CD2 is likely sufficient to align the Gαi domains. This result underscores the conclusion that PCDH15-CD2 is a critical mediator of intrinsic polarity in developing hair cells.

Interestingly, although Pcdh15-n38 and Gpsm2 single mutants exhibit hearing loss, only the double mutants demonstrate vestibular-related behaviours such as circling and hyperactivity. One reason could be the shorter stereociliary bundles observed in double Pcdh15-n38::Gpsm2 mutants. However, Gpsm2 single mutants also show shorter stereocilia, but do not show a circling behaviour. This is in contrast to earlier analysis of the mPins allele of Gpsm2 [48]. Here, a vestibular phenotype was observed in the single mutant, and utricular hair cells show reduced stereocilia length. The reason for the discrepancy between the two alleles is unclear. However, the mPins allele may produce a truncated protein, which could act as a dominant negative. Double mutants show an exacerbated intrinsic polarity defect and also show reduced gradation in the stereociliary bundle of the cristae. Together with a reduction in kinociliary links, these may be sufficient to impair vestibular function.

Taken together, our findings suggest PCDH15-CD2 integrates cytoskeletal, ciliary, and membrane-associated signals to orchestrate intrinsic polarity in sensory hair cells. While planar polarity ensures tissue-wide alignment of cellular orientation, intrinsic polarity mechanisms, anchored by basal body organisation and Gαi localisation, set the stage for this higher-order coordination. Understanding how these two layers of polarity interact will be essential for decoding morphogenetic control in sensory epithelia.

Supporting information

S1 Fig. Pcdh15ⁿ 38^/n 38 mice are functionally null and act similar to Pcdh15-∆CD2 mutants.

(A) A small fragment of Pcdh15 exon 38 was amplified in WT (167 bp) and Pcdh15^n38/n38 (247 bp) animals for genotyping. (B) Full-length western blot of Fig 1D. (C) mRNA level for CD2 domain is normal in Pcdh15^n38/n38 cochlea. (D) VANGL2 protein (green) localisation (highlighted with red arrows) is unperturbed in Pcdh15^n38/n38 OC (E17.5, base). (Scale bar = 10 μm). (E) Stereocilia bundles are aligned normally with the tissue axis in Pcdh15^n38/+ (pink), but significantly deviated in Pcdh15^n38/n38 (slate blue) (P0, mid). (Two-way Anova with Tukey’s multiple comparisons test, ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). (Sample size for OHC3/OHC2/OHC1, Pcdh15^n38/+ = 69/67/72, Pcdh15^n38/n38 = 69/68/69).

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S2 Fig. Tyrosine phosphorylation of the CD2 domain is not essential for PCDH15 function.

(A) Part of WT and Pcdh15^n38YF/n38YF exon 38 are amplified in different sizes (167 bp and 207 bp, respectively) with the same primer pair for genotyping. (B) PCDH15-CD2-HA protein (green) is colocalised with PCDH15 (cyan) in the hair bundles (P0, mid). (Scale bar = 10 μm). (C) PCDH15-CD2-HA protein (green) is localised at the tip of stereocilia (magenta, marked with phalloidin) at E18.5 (mid). At P5 (mid), this expression is localised in all three rows of stereocilia and is refined more in P15 stereocilia (mid). (Scale bar = 10 μm). (D) Full-length western blot of Fig 2E. (E) SEM imaging shows normal hair bundle polarity in both Pcdh15^n38/+ and Pcdh15^n38YF/n38YF HC (P0, mid). (Scale bar = 5 μm). (F) MET channels are open in HC of Pcdh15^n38/n38 and Pcdh15^n38YF/n38YF mice at P5. FM1–43 FX dye (green) is used to detect open MET channels. (Scale bar = 10 μm). (G) Positive Preyer’s reflex response measurement shows no response in Pcdh15^n38/n38 adult animals as compared to the comparable response in wild-type and Pcdh15^n38YF/n38YF mice. (H-I) PCDH15-CD2-HA protein signal (green) is not present in wild-type mice organ of Corti (H) and utricle & cristae (I) at E16.5 stage (Staining controls). (Scale bar = 10 μm).

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S3 Fig. Pcdh15^n38/n38 mice exhibit early hair bundle polarity defects.

(A) A single optical section of the stained centrioles (marked with pericentrin) connected to the kinocilium (marked with acetylated tubulin) shows the absence of their orthogonal position to each other in HC of Pcdh15^n38/n38 mice (E18.5, base).

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S4 Fig. GPSM2 expression is perturbed in Pcdh15ⁿ 38^/n 38 mice hair cells.

(A-B) GPSM2 expression domain (green) is extended medially in HC of Pcdh15^n38/n38 mice (E18.5, base). (Scale bar = 10 μm & 2 μm). (C) In comparison to Pcdh15^n38/+ mice, GPSM2 expression domain is spread significantly in HC of Pcdh15^n38/n38 mice (E18.5, base). (Two-way Anova with Tukey’s multiple comparisons test, ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). (Sample size for OHC3/OHC2/OHC1/IHC, Pcdh15^n38/+ = 59/56/57/52, Pcdh15^n38/n38 = 69/70/66/66).

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S5 Fig. Pcdh15-CD2 and Gpsm2 show genetic interaction during hair bundle development.

(A) In comparison to Pcdh15^n38/+:: Gpsm2^+/- (pink), the hair bundle polarity (polar projections) is perturbed in P1 IHC of Pcdh15^n38/n38:: Gpsm2^+/- (slate blue) and Pcdh15^n38/n38:: Gpsm2^-/- (dark magenta) OC (base). Hair bundle polarity is normal in Pcdh15^n38/+:: Gpsm2^-/- (green) OC. (B) Hair bundles are fragmented in IHC of only Pcdh15^n38/n38:: Gpsm2^-/- (dark magenta). (Ordinary one-way Anova with Tukey’s multiple comparisons test, ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). (Sample size for IHC, Pcdh15^n38/+:: Gpsm2^+/- = 36, Pcdh15^n38/+:: Gpsm2^-/-= 42, Pcdh15^n38/n38:: Gpsm2^+/- = 24, Pcdh15^n38/n38:: Gpsm2^-/- = 54).

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pgen.1011825.s005.tif^{(473.5KB, tif)}

S6 Fig. Pcdh15-CD2 and Gpsm2 show genetic interaction during hair bundle development of the vestibular system.

(A) F-actin (grey) shows smaller stereocilia bundles in the hair cells crista of Pcdh15ⁿ 38^/+:: Gpsm2^-/-, which is further reduced in Pcdh15^n38/n38:: Gpsm2^-/- mice, but not in Pcdh15^n38/n38:: Gpsm2^+/-. (P7, Scale bar = 20 μm). (B-F) Direction of hair cells in the (D) lateral, (E) LPR, and (F) medial region of the utricle shows comparable orientation in Pcdh15^n38/n38:: Gpsm2^+/-, Pcdh15^n38/+:: Gpsm2^-/- and Pcdh15^n38/n38:: Gpsm2^-/- mice as compared to littermate controls. (P7, Scale bar = 50 & 20 μm).

(TIF)

pgen.1011825.s006.tif^{(6.5MB, tif)}

S1 Movie. Pcdh15-CD2 and Gpsm2 show genetic interaction during the hair bundle development of the vestibular system.

Circling activity is observed only in Pcdh15^n38/n38:: Gpsm2^-/- mice and not in Pcdh15^n38/+:: Gpsm2^+/- or Pcdh15^n38/+:: Gpsm2^-/- mice as compared to control mice when observed under the open-field test (OFT).

(MP4)

Download video file^{(8.6MB, mp4)}

S1 Table. Genotyping and RT-PCR conditions used for different genes.

These include their specific primers, annealing temperature, and amplified product size.

(DOCX)

pgen.1011825.s008.docx^{(12.9KB, docx)}

S2 Table. List of primary antibodies used for immunofluorescence assays and western blotting.

(DOCX)

pgen.1011825.s009.docx^{(13.9KB, docx)}

S3 Table. List of secondary antibodies used for immunofluorescence assays.

(DOCX)

pgen.1011825.s010.docx^{(12.6KB, docx)}

S1 Data. The raw data of all the measurements and analyses used in this manuscript are provided as an Excel sheet under the file name Pcdh15 paper raw data.xlsx.

(XLSX)

pgen.1011825.s011.xlsx^{(174.5KB, xlsx)}

S1 Script. A Python script for measuring alpha-theta correlation is provided under the file name analyse.py.

(PY)

pgen.1011825.s012.py^{(6.6KB, py)}

Acknowledgments

We acknowledge the support of the Animal Care and Resources Centre, the Electron Microscopy Facility, and the Central Imaging Facility at NCBS. We thank Prof. Fumio Matsuzaki for providing the GPSM2 antibody and Gpsm2 mutant mice. We thank Prof. Anna Lysakowski for suggestions on the sample preparation of adult mouse vestibular structures for SEM. We thank Hrishikesh Nambisan and Arkadeep Bhattacharjee for help with the mouse behaviour analysis and Digvijay Lalwani for data analysis. We thank members of the Ear Lab at NCBS for their feedback and discussions.

Data Availability

All raw data is provide as Supporting information with the manuscript.

Funding Statement

RKL received funding from Department of Atomic Energy, Government of India, Project Identification No. RTI 4006. RK, SP, FG and RKL received a salary or stipend from this grant. RKL received funding from Royal National Institute for Deaf People G97, SERB CRG and Infosys Foundation through a TIFR Infosys-Leading Edge Grant AP: received a Simons Foundation: Simons Ashoka Early Career Fellowship. AP received a salary from this grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Genet. 2025 Aug 13;21(8):e1011825. doi: 10.1371/journal.pgen.1011825.r001

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27 Dec 2024

PLoS Genet. doi: 10.1371/journal.pgen.1011825.r002

Decision Letter 0

Nandan Nerurkar

10 Mar 2025

PGENETICS-D-24-01537

Role Of Pcdh15 In The Development Of Intrinsic Polarity Of Inner Ear Hair Cells

PLOS Genetics

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Reviewer #1: In the current manuscript, Kaushik et al. have been studying the function of PCDH15 in mechanosensory hair cells. Previous studies have shown that PCDH15 is expressed in three isoforms, which differ in their cytoplasmic domains and are generated by alternative splicing. It had also been shown that one of these isoforms, PCDH15-CD2, is a component of kinociliary links that connect the longest stereocilia of hair cells to the kinocilium. Furthermore, in PCDH15-CD2 mutant mice, planar cell polarity (PCP) of hair cells is disrupted leading to misorientation of the stereocilia bundle in the apical hair cell surface. Finally, studies from the laboratory presenting the current study have provided evidence that the CD2 cytoplasmic domain of PCHD15 is phosphorylated on tyrosine residues. The authors thus wanted to address the mechanism by which PCDH15-CD2 and its tyrosine phosphorylation affect PCP.

To conduct their studies, the authors generated mutant mice lacking the PCDH15-CD2 isoform or carrying mutations in the tyrosine residues within CD2. They demonstrated that CD2 tyrosine phosphorylation is not required for PCDH15-CD2 function in PCP, but they confirm earlier findings that PCP is disrupted in PCDH15-CD2 mutant mice. They then use immunolocalization studies for PCDH15-CD2 and other genes linked to PCP signaling such as GPSM2 and Gai to study effects of the PCDH15-CD2 mutation. They also generate PCDH15-CD2 and GPMS2 compound mutant mice to define potential genetic interactions between PCDH15-CD2 and genes linked to PCP signaling. They conclude that PCDH15-CD2 affects PCP independent from its function in the formation of kinociliary links.

Unfortunately, while the authors have done a lot of work, I do not find that the experiments support the conclusion put forward. Substantial more work would be necessary to establish what might be an interesting idea. There are also several technical limitations to the study:

1.The authors argue that PCDH15-CD2 is expressed at the base of kinocilia prior to the formation of kinociliary links. The immunohistochmical data are not convincing (Fig. 3). They use PCDH15-CD2-HA mice, which express an epitope tagged PCDH15-CD2 to reach the conclusions. They do not show non-HA controls. The PCDH15-CD2-HA mice carry several mutations in CD2, which could affect expression or localization. Also, more convincing pictures of HA staining would be required such as co-staining with markers for the base of the kinocilium and sagittal views of sectioned hair cell. There are also PCDH15 antibodies available from several laboratories to confirm the findings. Publications from the Barr-Gillespie laboratory show what kind of resolution can now be obtained to visualize subcellular distribution of proteins in hair cells. Finally, the staining data should be quantified.

2.The authors stain hair cells around E16.5-E18.5 to support the conclusion that labeling at the base of kinocilia is observed prior to the presence of kinociliar links. However, kinociliary links are already present at this age (even in the publication from the Richardson lab they cite, which is not the most convincing example, kinociliar links are present at this age, especially at the base and top of stereocilia). If the authors want to make this point, they would have to show that unlike what is published, they cannot find kinociliary links at an age when they observe PCP defects.

3.The authors show that polar Gai localization is affected in PCDH15-CD2 mice (Fig. 5A-C). This is interesting but may not be unexpected since PCP is affected in the mutants. Thus, the localization of PCP components might be perturbed. The problem is that we do not know what is cause and what is consequence of what. No experiment directly addresses a mechanistic link between PCDH15-CD2 and Gai.

4.The authors suggest that pericentrin localization may be affected in PCDH15-CD2 mutant mice (Fig. 4G,H). The images are not convincing and would need quantification. How do we know what is the mother/daughter centriole.

5.Intercrosses between PCDH15-CD2 and GPMS2 mutant mice suggest that the two genes genetically interact. The effect is particularly striking for the vestibule, where PCDH15-CD2/GPMS2 mutant mice have a far more pronounced hyperactivity defect than single mutants. However, vestibular hair bundles are not investigated. It should also be noted that there is a profound difference in the kinocilia of cochlear and vestibular hair cells. In the cochlea kinocilia in hair cells are transiently present during development, but in the vestibule they are maintained into adulthood where they are thought to be important for stereocilia stimulation by mechanical force. Thus, the vestibular impairment could indicate a functional defect in hair cells that have to do with coupling of the hair bundle to the overlying matrix to enable efficient force sensing. Or the stereocilia might have degenerated. Or any of several other possibilities. This would likely need a separate detail study to define mechanisms.

Reviewer #2: In this manuscript by Kaushik et al the contribution of protocadherin 15 (Pcdh15) towards stereociliary bundle development is evaluated using a unique conditional allele that produces a splicing-isoform specific knockout that can be converted to an epitope tagged and phosphorylation-deficient mutant. Using this line the authors expand upon prior characterizations of Pcdh15 and propose a model in which Pcd15-CD2 contributes to hair cell planar polarity. The KO has a striking impact on stereociliary bundle morphology in which the kinocilium is frequently separated from the stereocilia (kinicilium-stereocilia decoupling). As a result, the position of the kinocilium on the apical cell surface lacks the distinctive polarized localization to adjacent to the tallest stereocilia. In contrast, other aspects of hair cell intrinsic planar polarity and planar cell polarity are not disrupted in this mutant. For example, the localization of the intrinsic polarity factor Galpha i remains polarized with the protein enriched on the lateral edge of the apical hair cell surface. In addition, the rows of stereociliary bundles appear appropriately polarized and organized in rows of increasing height that are correctly positioned adjacent to Galpha i. Lastly, the polarized distribution of Core PCP proteins like Vangl2 is not disrupted in the Pdc15-Cd2 mutants. Altogether these results raise the question of how cell intrinsic planar polarity should be defined, and what phenotype criteria are needed to categorize it as disrupted. The intellectual merit of the manuscript would be improved if the authors discussed these questions and provided a clear argument as to why Pcd15-CD2 is contributing to intrinsic planar polarity as per the title rather than maintaining stereociliary bundle integrity by linking the kinocilium to the stereocilia.

Additional concerns

1)What is Pcdh15-CD2 and how is it different from Pdch15-CD1 and Pcdh15-CD-3? The introduction would benefit from the addition of an introduction to Pcdh15 alternative splicing and isoform function.

2)Figure 1: Since the kinocilium is decoupled from the stereocilia in the n38 mutants and the kinocilium and stereocilia seem to be differentially effected, the authors should make independent polarity measures for stereocilia polarization.

3)Line 244: Since the Preyer’s reflex is not applied to n38YF mice so it is not clear whether or not they can hear based on this assay. Without that result it is not accurate for this section of the manuscript to be titled “Tyrosine phosphorylation of the CD2 domain is not essential for Pcdh15 Function in mice” since the contribution phosphorylation towards hearing has not been tested.

4)Preyer’s reflex is not a robust or rigorous assay for auditory function and the authors should consider ABRs

5)Fig.7A: unlike Galpha I, the area of cell surface containing GPSM2 does not change in Pcdh15 KOs despite the colocalization of these proteins reported by others. This result is unexpected and should be discussed.

Minor concerns

1)It would be useful to report the frequency of circular bundles since they are not common in other figure panels and so that the may be compared to the frequency of other mutants like IFT88

2)Line 44: Hearing perception does not occur in the cochlea but rather in auditory cortex. This should be corrected.

3)Line 81: Since a hair cell only has one kinocilium this phenotype should be ‘kinocilium-stereocilia decoupling’

4)It would help your readers if the text stated that Pgk1-Cre was used to make a germline mutant particularly since Cre is more often used for cell or tissue-specific gene deletion

5)Figure 1 schematic: it is not obvious what parts of the protein are encoded by exon 38 or how alternative splicing could exchanges CD1 or CD3 for CD2

6)Fig8: The genotype nomenclature “ -/- “ that is used for the wild type Atoh1 allele is confusing since “ -/- “ typically indicates a null allele or knockout. Why not say Cre-negative or nothing at all? Similarly, the abbreviations ‘-ve’ and ‘+ve’ for positive and negative can be confusing. Why not just use HA-positive and HA-negative?

Reviewer #3: In this manuscript, Kaushik et al. aimed to understand the role of Pcdh15 in planar cell polarity (PCP) establishment of hair cells in the developing cochlea. They reported a Pcdh15-n38 knock-in mouse which was designed to tag the endogenous Pcdh15-CD2 isoform, but found that the inserted FLAG tag was undetectable, and the mice were phenotypically similar to Pcdh15-CD2 knockouts in terms of PCP and centriolar defects. Upon Cre-mediated recombination, their allele did express an HA-tagged, phospho-dead CD2 domain, which partly rescued PCP defects. These results and the immunodetection of CD2 using the HA tag suggested an early role of Pcdh15 in PCP establishment independent of its interaction with the kinocilium. Furthermore, the authors generated compound mutants with Pcdh15-n38 and Gpsm2, showing genetically synergistic effects in PCP. These results are original and important to further understanding the mechanism of PCP establishment in hair cells. The paper is overall technically sound, but some conclusions are not fully supported by the data, and some interpretations do not come through (detailed below). The writing can be improved to be more understandable for general audience, as some technical terms specific to the hair cell biology were not defined when they were first used. Some essential information such as the sample size, definition of axes in the figure, and abbreviations in the figure are missing.

1. Missing definition: CD2, bare zone, ve (Fig. 8), arrows in the figures.

2. Western blot in Fig. 1C should have a loading control. Also, the authors should give some explanations/speculations about why the n38 allele did not express before Cre recombination. Was is truly equivalent to CD2 knockout at the molecular level? Is it possible to characterize the transcript at the RNA level?

3. In Fig. 4GH, it is unclear what "right angles" mean. Quantification of the angles would make more sense.

4. Line 337, why is the interaction between Pcdh15 and Gpsm2 likely to be downstream? A synergistic effect could happen with a common upstream regulator. Line 414 said their localizations were independent, which is contradictory to Fig. 5, where the n38 mutation changed Gai localization.

5. In Fig. 8D, it seems that the HA-negative cells also had a significant rescue of n38 in kinocilia deviation, which needs explanation.

**********

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy , and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No: I did not find the raw data for plotting the graphs.

**********

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PLoS Genet. 2025 Aug 13;21(8):e1011825. doi: 10.1371/journal.pgen.1011825.r003

Author response to Decision Letter 1

28 May 2025

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PLoS Genet. doi: 10.1371/journal.pgen.1011825.r004

Decision Letter 1

Nandan Nerurkar

1 Jul 2025

PGENETICS-D-24-01537R1

Role of Pchd15 in the Development of Intrinsic Polarity of Inner Ear Hair Cells

PLOS Genetics

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Reviewer #1: The authors have substantially revised the manuscript. While the data are interesting, I do not find that the data support the conclusion that “Pcdh15-CD2 couples kinocilia and stereocilia development through a mechanism independent of kinocilial links” (abstract). They also conclude that Pcdh15-CD2 is linked to Gai/Gpsm2 signaling, which in my opinion is not clearly shown.

Confirmative findings:

Using new mouse models, the authors confirm earlier findings that the Pcdh15-CD2 isoform is expressed in the kinocilium of hair cells and required for coupling of the kinocilium to stereocilia. They also confirm that hair cells of Pcdh15-CD2 mutant mice have a defect in the intrinsic polarity as revealed in the morphology of stereocilia bundles.

New findings:

1.Phosphorylation of Pcdh15-CD2 is not crucial for the development of a polar hair bundle

2.Localization of Gai is perturbed in Pcdh15-CD2 mutant mice consistent with a defect in intrinsic polarity

3. Pcdh15-CD2 mutations seem to genetically interact with Gpsm2 mutations in both the cochlea and vestibule (but see comment below).

Major critique points:

1.I do not see convincing evidence that the polarity defects cannot be explained by defects in the coupling of the stereocilia to the kinocilium via kinociliar links. The authors argue that Pcdh15-CD2 is expressed prior to the formation of kinociliary links and that polarity defects materialize prior to the presence of kinociliary links. To me the supporting data are not of sufficient quality and resolution to reach that conclusion.

2.The fact that Gai is mis-localized in Pcdh15-CD2 mutants could be a consequence of polarity defects without a direct mechanistic link.

3.The genetic interaction data are interesting but also do not provide direct evidence of a mechanistic link. Weakening two molecular pathways that run in parallel (e.g.: kinocilia-stereocilia cohesion via Pcdh15-CD2, and molecular pathways regulating the polarity of the cytoskeleton) could explain stronger phenotypes. In addition, there is an issue with the quantification of the genetic interaction data. Fig. 6E lacks the proper control, comparison of double mutants to Pcdh15-n38/n38 mutants. Pcdh15-n38/n38 mutants are shown in Fig. 1H and it seems that kinocilia mislocalization is similarly affected in Pcdh15-n38/n38 mutant mice (Fig. 1H) compared to Pcdh15-n38/n38;Gspm2-/- mice (Fig. 6E). Perhaps the effect is slightly enhanced but since there is no direct statistical comparison, it is hard to be sure. Maybe a stronger phenotype shows up for fragmentation of hair bundles in double mutants (Fig. 6F), but this has not been quantified for Pcdh15-n38/n38 mutants. Likewise, the proper control (Pcdh15-n38/n38 mutant mice) is lacking for the analysis of the vestibular phenotype. Of note, it has previously been shown that Pcdh15-CD2 mutant mice have a vestibular defect.

Additional points:

1.Introduction: “hearing is sensed “ should be correcte. We do not sense hearing, we sense sound.

2.FM-143 uptake to study mechanotransduction: controls a lacking to show that uptake is via mechanotransduction channels and not by endocytosis. A control experiments should be performed: FM-143 uptake in the presence of channel blockers.

3.The authors conclude that the hearing defects (Preyers reflex) cannot be explained by a defect in mechanotransduction. They cannot conclude this. The FM-143 experiments were carried early postnatally, the hearing tests at P60. By that age, mechanotransduction could be affected.

4.The authors should measure ABRs, maybe at P21 and P60. Preyers reflex is insufficient to reveal if there are significant defects in auditory thresholds (maybe the phosphorylation mutants have a hearing defect not revealed by the crude assay).

5.In Fig.3A, dots for HA staining are all over the tissue, sometimes with multiple dots per cells. Staining seems to be present in hair cells and supporting cells. Is this correct? Also, why not co-stain in Fig. 3A with kinociliary markers to solidify the conclusion that the staining is in kinocilia. And show controls, in parallel with mice not expression an HA-tagged protein to exclude background. I appreciate that staining controls are shown in the response letter, but they should be part of the main figure.

Reviewer #2: While the authors have made selective improvements to the manuscript, they have left my two primary concerns unaddressed:

1. What constitutes intrinsic polarity, and what phenotype criteria must be met to categorize it as disrupted?

2. Why should Pcd15-CD2 be interpreted as a contributor to intrinsic planar polarity—as the title implies—rather than as a mediator of stereociliary bundle cohesion via kinocilium–stereocilia linkage?

I raised these points because I remain unconvinced that Pcd15-CD2 plays a direct role in establishing intrinsic hair cell planar polarity. That said, the authors should be given room to explain their rationale and clarify why they believe Pcd15-CD2 is not regulating kinocilium–stereocilium adhesion, with only indirect effects on cell orientation. They have not done so.

To their credit, they now include additional measures of hair cell orientation based on stereociliary alignment and compare them with kinocilium-based measures. They demonstrate that these two methods reveal different phenotype severities—an important observation that underscores how divergent interpretations can result from the selection of different phenotyping criteria. This discrepancy warrants explicit discussion, particularly regarding how "disruption" is defined and by what criteria. It also reinforces my own interpretation that the primary function of Pcd15-CD2 is kinocilium:stereocilia linkage and that any impacts on intrinsic hair cell polarity are indirect.

Regarding my second point, it's notable—if not puzzling—that the adhesive role of Pcd15-CD2 receives little to no attention in the discussion. Given the protein’s known function in kinocilium–stereocilium cohesion, the authors should clearly articulate why they are interpreting it to have a direct role in intrinsic polarity regulation instead, and why readers like myself should reconsider their existing expectations. A few well-placed sentences in the Discussion section would assuage my contrary interpretation.

Reviewer #3: The authors did a good job addressing my questions.

One minor point: "polarity" in the Keywords section was misspelt.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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PLoS Genet. 2025 Aug 13;21(8):e1011825. doi: 10.1371/journal.pgen.1011825.r005

Author response to Decision Letter 2

28 Jul 2025

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pgen.1011825.s016.docx^{(3.8MB, docx)}

PLoS Genet. doi: 10.1371/journal.pgen.1011825.r006

Decision Letter 2

Nandan Nerurkar

1 Aug 2025

Dear Dr Ladher,

We are pleased to inform you that your manuscript entitled "Role of Pcdh15 in the Development of Intrinsic Polarity of Inner Ear Hair Cells" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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PLoS Genet. doi: 10.1371/journal.pgen.1011825.r007

Acceptance letter

Nandan Nerurkar

PGENETICS-D-24-01537R2

Role of Pcdh15 in the Development of Intrinsic Polarity of Inner Ear Hair Cells

Dear Dr Ladher,

We are pleased to inform you that your manuscript entitled "Role of Pcdh15 in the Development of Intrinsic Polarity of Inner Ear Hair Cells" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Pcdh15ⁿ 38^/n 38 mice are functionally null and act similar to Pcdh15-∆CD2 mutants.

(TIF)

pgen.1011825.s001.tif^{(2.1MB, tif)}

S2 Fig. Tyrosine phosphorylation of the CD2 domain is not essential for PCDH15 function.

(TIF)

pgen.1011825.s002.tif^{(4.1MB, tif)}

S3 Fig. Pcdh15^n38/n38 mice exhibit early hair bundle polarity defects.

(TIF)

pgen.1011825.s003.tif^{(2.5MB, tif)}

S4 Fig. GPSM2 expression is perturbed in Pcdh15ⁿ 38^/n 38 mice hair cells.

(TIF)

pgen.1011825.s004.tif^{(2.3MB, tif)}

S5 Fig. Pcdh15-CD2 and Gpsm2 show genetic interaction during hair bundle development.

(TIF)

pgen.1011825.s005.tif^{(473.5KB, tif)}

S6 Fig. Pcdh15-CD2 and Gpsm2 show genetic interaction during hair bundle development of the vestibular system.

(TIF)

pgen.1011825.s006.tif^{(6.5MB, tif)}

S1 Movie. Pcdh15-CD2 and Gpsm2 show genetic interaction during the hair bundle development of the vestibular system.

(MP4)

Download video file^{(8.6MB, mp4)}

S1 Table. Genotyping and RT-PCR conditions used for different genes.

These include their specific primers, annealing temperature, and amplified product size.

(DOCX)

pgen.1011825.s008.docx^{(12.9KB, docx)}

S2 Table. List of primary antibodies used for immunofluorescence assays and western blotting.

(DOCX)

pgen.1011825.s009.docx^{(13.9KB, docx)}

S3 Table. List of secondary antibodies used for immunofluorescence assays.

(DOCX)

pgen.1011825.s010.docx^{(12.6KB, docx)}

S1 Data. The raw data of all the measurements and analyses used in this manuscript are provided as an Excel sheet under the file name Pcdh15 paper raw data.xlsx.

(XLSX)

pgen.1011825.s011.xlsx^{(174.5KB, xlsx)}

S1 Script. A Python script for measuring alpha-theta correlation is provided under the file name analyse.py.

(PY)

pgen.1011825.s012.py^{(6.6KB, py)}

Attachment

Submitted filename: D-24-01537.docx

pgen.1011825.s013.docx^{(14.9KB, docx)}

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Submitted filename: Response to Reviewers.docx

pgen.1011825.s015.docx^{(6.5MB, docx)}

Attachment

Submitted filename: Rebuttal 2.docx

pgen.1011825.s016.docx^{(3.8MB, docx)}

Data Availability Statement

All raw data is provide as Supporting information with the manuscript.

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PERMALINK

Role of Pcdh15 in the development of intrinsic polarity of inner ear hair cells

Raman Kaushik

Shivangi Pandey

Anubhav Prakash

Fenil Ganatra

Takaya Abe

Hiroshi Kiyonari

Raj K Ladher

Roles

Abstract

Author summary

Introduction

Fig 1. Pcdh15n38/n38 mice are functionally null and act similarly to Pcdh15-CD2 mutants.

Methods

Ethics statement

Animal use and care

Immunofluorescence

Image acquisition and processing

Immunoprecipitation and western blotting

Vestibular test

Preyer’s reflex test

Image analysis and statistics

Scanning electron microscopy (SEM)

Results

Generation of Pcdh15n38/n38 and Pcdh15n38YF/n38YF mice

Defects in hair bundle polarity in Pcdh15n38/n38 mice

Tyrosine phosphorylation of the CD2 domain is not essential for PCDH15 function in mice

Fig 2. Tyrosine phosphorylation of the CD2 domain is not essential for PCDH15 function.

PCDH15-CD2 is expressed in the kinocilium prior to its off-centre movement

Fig 3. PCDH15-CD2-HA is expressed in the kinocilium before its off-centre movement.

Pcdh15n38/n38 mice exhibit early hair bundle polarity defects

Fig 4. Pcdh15n38/n38 mice exhibit early hair bundle polarity defects.

The coordination of kinocilium with Gαi expression is lost in Pcdh15-CD2 mutants

Fig 5. Kinocilium position is not coordinated with G α i expression in Pcdh15n38/n38 mice hair cells.

PCDH15-CD2 and GPSM2 proteins operate cooperatively in hair bundle development

Fig 6. Pcdh15-CD2 and Gpsm2 show genetic interaction during hair bundle development.

Fig 7. Pcdh15-CD2 and Gpsm2 show genetic interaction during hair bundle development of the vestibular system.

Fig 8. PCDH15-CD2 and GPSM2 proteins operate in parallel pathways during hair bundle development.

PCDH15-CD2 expression rescues the intrinsic hair bundle polarity defects

Fig 9. Re-initiation of PCDH15-CD2 expression rescues the intrinsic hair bundle polarity defects.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Author response to Decision Letter 0

Transfer Alert

Decision Letter 0

Nandan Nerurkar

Roles

Author response to Decision Letter 1

Decision Letter 1

Nandan Nerurkar

Roles

Author response to Decision Letter 2

Decision Letter 2

Nandan Nerurkar

Roles

Acceptance letter

Nandan Nerurkar

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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Fig 1. Pcdh15^n38/n38 mice are functionally null and act similarly to Pcdh15-CD2 mutants.

Generation of Pcdh15^n38/n38 and Pcdh15^n38YF/n38YF mice

Defects in hair bundle polarity in Pcdh15^n38/n38 mice

Pcdh15^n38/n38 mice exhibit early hair bundle polarity defects

Fig 4. Pcdh15^n38/n38 mice exhibit early hair bundle polarity defects.

Fig 5. Kinocilium position is not coordinated with G α i expression in Pcdh15^n38/n38 mice hair cells.