Regionalized Protein Localization Domains in the Zebrafish Hair Cell Kinocilium.

Sensory hair cells are the receptors for auditory, vestibular, and lateral line sensory organs in vertebrates. These cells are distinguished by "hair"-like projections from their apical surface collectively known as the hair bundle. Along with the staircase arrangement of the actin-filled stereocilia, the hair bundle features a single, non-motile, true cilium called the kinocilium. The kinocilium plays an important role in bundle development and the mechanics of sensory detection. To understand more about kinocilial development and structure, we performed a transcriptomic analysis of zebrafish hair cells to identify cilia-associated genes that have yet to be characterized in hair cells. In this study, we focused on three such genes-ankef1a, odf3l2a, and saxo2-because human or mouse orthologs are either associated with sensorineural hearing loss or are located near uncharacterized deafness loci. We made transgenic fish that express fluorescently tagged versions of their proteins, demonstrating their localization to the kinocilia of zebrafish hair cells. Furthermore, we found that Ankef1a, Odf3l2a, and Saxo2 exhibit distinct localization patterns along the length of the kinocilium and within the cell body. Lastly, we have reported a novel overexpression phenotype of Saxo2. Overall, these results suggest that the hair cell kinocilium in zebrafish is regionalized along its proximal-distal axis and set the groundwork to understand more about the roles of these kinocilial proteins in hair cells.

Involvement of Dmp1 in the Precise Regulation of Hair Bundle Formation in the Developing Cochlea.

Dentin matrix protein 1 (Dmp1) is a highly phosphorylated, extracellular matrix protein that is extensively expressed in bone and teeth but also found in soft tissues, including brain and muscle. However, the functions of Dmp1 in the mice cochlea are unknown. Our study showed that Dmp1 was expressed in auditory hair cells (HCs), with the role of Dmp1 in those cells identified using Dmp1 cKD mice. Immunostaining and scanning electron microscopy of the cochlea at P1 revealed that Dmp1 deficiency in mice resulted in an abnormal stereociliary bundle morphology and the mispositioning of the kinocilium. The following experiments further demonstrated that the cell-intrinsic polarity of HCs was affected without apparent effect on the tissue planer polarity, based on the observation that the asymmetric distribution of Vangl2 was unchanged whereas the Gαi3 expression domain was enlarged and Par6b expression was slightly altered. Then, the possible molecular mechanisms of Dmp1 involvement in inner ear development were explored via RNA-seq analysis. The study suggested that the Fgf23-Klotho endocrine axis may play a novel role in the inner ear and Dmp1 may regulate the kinocilium-stereocilia interaction via Fgf23-Klotho signaling. Together, our results proved the critical role of Dmp1 in the precise regulation of hair bundle morphogenesis in the early development of HCs.

Non-Canonical Wnt Signaling Regulates Cochlear Outgrowth and Planar Cell Polarity via Gsk3ß Inhibition.

During development, sensory hair cells (HCs) in the cochlea assemble a stereociliary hair bundle on their apical surface with planar polarized structure and orientation. We have recently identified a non-canonical, Wnt/G-protein/PI3K signaling pathway that promotes cochlear outgrowth and coordinates planar polarization of the HC apical cytoskeleton and alignment of HC orientation across the cochlear epithelium. Here, we determined the involvement of the kinase Gsk3ß and the small GTPase Rac1 in non-canonical Wnt signaling and its regulation of the planar cell polarity (PCP) pathway in the cochlea. We provided the first in vivo evidence for Wnt regulation of Gsk3ß activity via inhibitory Ser9 phosphorylation. Furthermore, we carried out genetic rescue experiments of cochlear defects caused by blocking Wnt secretion. We showed that cochlear outgrowth was partially rescued by genetic ablation of Gsk3ß but not by expression of stabilized ß-catenin; while PCP defects, including hair bundle polarity and junctional localization of the core PCP proteins Fzd6 and Dvl2, were partially rescued by either Gsk3ß ablation or constitutive activation of Rac1. Our results identify Gsk3ß and likely Rac1 as downstream components of non-canonical Wnt signaling and mediators of cochlear outgrowth, HC planar polarity, and localization of a subset of core PCP proteins in the cochlea.

Kcnb1 plays a role in development of the inner ear.

The function of the inner ear depends on the maintenance of high concentrations of K+ ions. The slow-inactivating delayed rectifier Kv2.1/KCNB1 channel works in the inner ear in mammals. The kcnb1 gene is expressed in the otic vesicle of developing zebrafish, suggesting its role in development of the inner ear. In the present study, we found that a Kcnb1 loss-of-function mutation affected development of the inner ear at multiple levels, including otic vesicle expansion, otolith formation, and the proliferation and differentiation of mechanosensory cells. This resulted in defects of kinocilia and stereocilia and abnormal function of the inner ear detected by behavioral assays. The quantitative transcriptional analysis of 75 genes demonstrated that the kcnb1 mutation affected the transcription of genes that are involved in K+ metabolism, cell proliferation, cilia development, and intracellular protein trafficking. These results demonstrate a role for Kv2.1/Kcnb1 channels in development of the inner ear in zebrafish.

New insights into regulation and function of planar polarity in the inner ear.

Acquisition of cell polarity generates signaling and cytoskeletal asymmetry and thus underpins polarized cell behaviors during tissue morphogenesis. In epithelial tissues, both apical-basal polarity and planar polarity, which refers to cell polarization along an axis orthogonal to the apical-basal axis, are essential for epithelial morphogenesis and function. A prime example of epithelial planar polarity can be found in the auditory sensory epithelium (or organ of Corti, OC). Sensory hair cells, the sound receptors, acquire a planar polarized apical cytoskeleton which is uniformely oriented along an axis orthogonal to the longitudinal axis of the cochlear duct. Both cell-intrinsic and tissue-level planar polarity are necessary for proper perception of sound. Here we review recent insights into the novel roles and mechanisms of planar polarity signaling gained from genetic analysis in mice, focusing mainly on the OC but also with some discussions on the vestibular sensory epithelia.

Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation.

RIPOR2 (previously known as FAM65B) localizes to stereocilia of auditory hair cells and causes deafness when its function is disturbed by mutations. Here, we demonstrate that during the morphogenesis of the hair cell bundle, absence of Ripor2 affects the orientation of this key subcellular structure. We show that Ripor2 interacts with Myh9, a protein encoded by a known deafness gene. Absence of Ripor2 is associated with low Myh9 abundance in the mouse cochlea despite increased amount of Myh9 transcripts. While Myh9 is mainly expressed in stereocilia, a phosphorylated form of Myh9 is particularly enriched in the kinocilium. In Ripor2-deficient mice, kinocilium shows an aberrant localization which associates with a reduced content of phosphorylated Myh9. Acetylated alpha tubulin, another specific kinociliary protein which contributes to microtubule stabilization, is reduced in the absence of Ripor2 as well. We propose that Ripor2 deficiency influences abundance and/or post-translational modifications of proteins expressed in both stereocilia and kinocilia. This effect may have a negative impact on the structure and function of the auditory hair cell bundle.

Deletion of Kncn Does Not Affect Kinocilium and Stereocilia Bundle Morphogenesis and Mechanotransduction in Cochlear Hair Cells.

Auditory hair cells possess stunning cilia structure that composes of a bundle of stereocilia for mechano-electrical transduction and a single kinocilium for guiding the polarity of hair bundle towards maturation. However, the molecules underlying kinocilium function have not yet been fully understood. Hence, the proteins involved in hair bundle development and function are of a large interest. From a fine microarray analysis, we found that kinocilin (Kncn) was enriched in hair cell specific expression profile. Consistently, it has been reported that KNCN was a protein mainly located in the kinocilium of hair cells in the inner ear. However, the hypothesis that KNCN is a kinocilium protein has not been validated in mice with Kncn gene perturbed. In this study, we generated Kncn knockout mouse lines by CRISPR/Cas9 technique and further examined the morphology and function of cochlear hair cells. Our results showed that there was no obvious hearing loss in the knockout mice, determined by audiometry. Histological study demonstrated that the inner ear and hair cell structure were intact. Especially, there was no deficit of mechanotransduction (MET) in cochlear outer hair cells (OHCs). In summary, our work suggests that KNCN is not essential for kinocilium-oriented hair bundle function in cochlear hair cells.

Evolutionary and Developmental Biology Provide Insights Into the Regeneration of Organ of Corti Hair Cells.

We review the evolution and development of organ of Corti hair cells with a focus on their molecular differences from vestibular hair cells. Such information is needed to therapeutically guide organ of Corti hair cell development in flat epithelia and generate the correct arrangement of different hair cell types, orientation of stereocilia, and the delayed loss of the kinocilium that are all essential for hearing, while avoiding driving hair cells toward a vestibular fate. Highlighting the differences from vestibular organs and defining what is known about the regulation of these differences will help focus future research directions toward successful restoration of an organ of Corti following long-term hair cell loss.

The Actin Nucleator Cobl Is Critical for Centriolar Positioning, Postnatal Planar Cell Polarity Refinement, and Function of the Cochlea.

Proper cochlear hair cell array development and sensory apparatus positioning are achieved by planar cell polarity signaling. Effectors executing proper tissue development and maturation programs are largely unknown. We show that the actin nucleator Cobl is an important effector in postnatal refinement and maintenance of planar cell polarity. During the critical time of hearing onset, these polarity defects coincided with reduced F-actin beneath the sensory apparatus and with premature kinocilium retraction. These defects were accompanied by organizational defects of the pericentriolar scaffold that coincided with basal body and centriolar mispositionings. Importantly, the pericentriolar defects observed in Cobl KO mice were demonstrated to be actin polymerization dependent and calcium/calmodulin signaling dependent. Because Cobl KO phenotypes manifested postnatally, planar cell polarity is not solely an important developmental process. The Cobl-dependent planar cell polarity maintenance and refinement processes we describe here seem critical for hearing, as Cobl KO mice show deficient cochlear amplification.

Thermal Excitation of the Mechanotransduction Apparatus of Hair Cells.

Although a hair bundle is normally deflected by mechanical stimuli, we found that irradiation of a hair cell from the bullfrog's sacculus with ultraviolet light causes rapid motion of the hair bundle toward its tall edge. This movement is associated with opening of mechanotransduction channels and disappears when tip links are disrupted. We localized the absorptive element responsible for the motion to the region directly below the hair bundle and measured an action spectrum similar to the absorption spectra of mitochondrial constituents. Temperature measurements revealed heating around the site of absorption; direct heating of the hair bundle confirmed that the response to light is mediated through heat. Although mechanical offsets of the hair bundle revealed that heat softens gating springs, it also acts directly to open transduction channels. This study identifies an unconventional method of hair-cell stimulation and clarifies the previously unexplained sensitivity of auditory organs to thermal stimulation.

Loss of liver kinase B1 causes planar polarity defects in cochlear hair cells in mice.

The tumor suppressor gene liver kinase B1 (LKB1), also called STK11, encodes a serine/threonine kinase. LKB1 plays crucial roles in cell differentiation, proliferation, and polarity. In this study, LKB1 conditional knockout mice (LKB1Pax2 CKO mice) were generated using Pax2-Cre mice to investigate the function of LKB1 in inner ear hair cells during early embryonic period. LKB1Pax2 CKO mice died perinatally. Immunofluorescence and scanning electron microscopy revealed that stereociliary bundles in LKB1Pax2 CKO mice were clustered and misoriented, respectively. Moreover, ectopic distribution of kinocilium bundles resulting from abnormal migration of kinocilium was observed in the mutant mice. The orientation of stereociliary bundles and the migration of kinocilia are critical indicators of planar cell polarity (PCP) of hair cells. LKB1 deficiency in LKB1Pax2 CKO mice thus disrupted hair cell planar polarity during embryonic development. Our results suggest that LKB1 is required in PCP formation in cochlear hair cells in mice.

Specialized Cilia in Mammalian Sensory Systems.

Cilia and flagella are highly conserved and important microtubule-based organelles that project from the surface of eukaryotic cells and act as antennae to sense extracellular signals. Moreover, cilia have emerged as key players in numerous physiological, developmental, and sensory processes such as hearing, olfaction, and photoreception. Genetic defects in ciliary proteins responsible for cilia formation, maintenance, or function underlie a wide array of human diseases like deafness, anosmia, and retinal degeneration in sensory systems. Impairment of more than one sensory organ results in numerous syndromic ciliary disorders like the autosomal recessive genetic diseases Bardet-Biedl and Usher syndrome. Here we describe the structure and distinct functional roles of cilia in sensory organs like the inner ear, the olfactory epithelium, and the retina of the mouse. The spectrum of ciliary function in fundamental cellular processes highlights the importance of elucidating ciliopathy-related proteins in order to find novel potential therapies.

The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea.

Hair cells of the organ of Corti (OC) of the cochlea exhibit distinct planar polarity, both at the tissue and cellular level. Planar polarity at tissue level is manifested as uniform orientation of the hair cell stereociliary bundles. Hair cell intrinsic polarity is defined as structural hair bundle asymmetry; positioning of the kinocilium/basal body complex at the vertex of the V-shaped bundle. Consistent with strong apical polarity, the hair cell apex displays prominent actin and microtubule cytoskeletons. The Rho GTPase Cdc42 regulates cytoskeletal dynamics and polarization of various cell types, and, thus, serves as a candidate regulator of hair cell polarity. We have here induced Cdc42 inactivation in the late-embryonic OC. We show the role of Cdc42 in the establishment of planar polarity of hair cells and in cellular patterning. Abnormal planar polarity was displayed as disturbances in hair bundle orientation and morphology and in kinocilium/basal body positioning. These defects were accompanied by a disorganized cell-surface microtubule network. Atypical protein kinase C (aPKC), a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion. Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network. The data also suggest that defects in apical polarization are influenced by disturbed cellular patterning in the OC. In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea.

Changes in ADF/destrin expression in the development of hair cells following Atoh1-induced ectopic regeneration.

The aim of this study was to investigate the effects of actin depolymerizing factor (ADF)/destrin and position changes of kinetosomes in the development of hair cells following Atoh1-induced ectopic regeneration in the basilar membrane of mice. We observed through immunofluorescence at various time-points the expression of ADF/destrin and the specific kinetosome marker, γ-tubulin, in hair cells following ectopic regeneration induced by adenovirus transfection, overexpression of Atoh1 and in vitro culture. Changes of ADF/destrin distribution and kinetosome position during in vitro culture of new hair cells [Myo7a(+)] following Atoh1-induced ectopic regeneration are consistent with the changes in ADF/destrin expression and the polar migration of kinetosomes in hair cells of the cochlear sensory epithelium in normal development. ADF/destrin is involved in the development of the auditory epithelium and the development and structural rearrangement of ectopically regenerated hair cells in mammals. The kinetosomes of hair cells following Atoh1-induced ectopic regeneration show positional changes in vitro at different time-points.