Interaction with ectopic cochlear crista sensory epithelium disrupts basal cochlear sensory epithelium development in Lmx1a mutant mice.

The LIM homeodomain transcription factor Lmx1a shows a dynamic expression in the developing mouse ear that stabilizes in the non-sensory epithelium. Previous work showed that Lmx1a functional null mutants have an additional sensory hair cell patch in the posterior wall of a cochlear duct and have a mix of vestibular and cochlear hair cells in the basal cochlear sensory epithelium. In E13.5 mutants, Sox2-expressing posterior canal crista is continuous with an ectopic "crista sensory epithelium" located in the outer spiral sulcus of the basal cochlear duct. The medial margin of cochlear crista is in contact with the adjacent Sox2-expressing basal cochlear sensory epithelium. By E17.5, this contact has been interrupted by the formation of an intervening non-sensory epithelium, and Atoh1 is expressed in the hair cells of both the cochlear crista and the basal cochlear sensory epithelium. Where cochlear crista was formerly associated with the basal cochlear sensory epithelium, the basal cochlear sensory epithelium lacks an outer hair cell band, and gaps are present in its associated Bmp4 expression. Further apically, where cochlear crista was never present, the cochlear sensory epithelium forms a poorly ordered but complete organ of Corti. We propose that the core prosensory posterior crista is enlarged in the mutant when the absence of Lmx1a expression allows JAG1-NOTCH signaling to propagate into the adjacent epithelium and down the posterior wall of the cochlear duct. We suggest that the cochlear crista propagates in the mutant outer spiral sulcus because it expresses Lmo4 in the absence of Lmx1a.

Glutamate transporter homolog-based model predicts that anion-π interaction is the mechanism for the voltage-dependent response of prestin.

Prestin is the motor protein of cochlear outer hair cells. Its unique capability to perform direct, rapid, and reciprocal electromechanical conversion depends on membrane potential and interaction with intracellular anions. How prestin senses the voltage change and interacts with anions are still unknown. Our three-dimensional model of prestin using molecular dynamics simulations predicts that prestin contains eight transmembrane-spanning segments and two helical re-entry loops and that tyrosyl residues are the structural specialization of the molecule for the unique function of prestin. Using site-directed mutagenesis and electrophysiological techniques, we confirmed that residues Tyr(367), Tyr(486), Tyr(501), and Tyr(508) contribute to anion binding, interacting with intracellular anions through novel anion-π interactions. Such weak interactions, sensitive to voltage and mechanical stimulation, confer prestin with a unique capability to perform electromechanical and mechanoelectric conversions with exquisite sensitivity. This novel mechanism is completely different from all known mechanisms seen in ion channels, transporters, and motor proteins.

Characterization of transcriptomes of cochlear inner and outer hair cells.

Inner hair cells (IHCs) and outer hair cells (OHCs) are the two types of sensory receptor cells that are critical for hearing in the mammalian cochlea. IHCs and OHCs have different morphology and function. The genetic mechanisms that define their morphological and functional specializations are essentially unknown. The transcriptome reflects the genes that are being actively expressed in a cell and holds the key to understanding the molecular mechanisms of the biological properties of the cell. Using DNA microarray, we examined the transcriptome of 2000 individually collected IHCs and OHCs from adult mouse cochleae. We show that 16,647 and 17,711 transcripts are expressed in IHCs and OHCs, respectively. Of those genes, ∼73% are known genes, 22% are uncharacterized sequences, and 5.0% are noncoding RNAs in both populations. A total of 16,117 transcripts are expressed in both populations. Uniquely and differentially expressed genes account for <15% of all genes in either cell type. The top 10 differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, and Map4k4 in IHCs and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, and Ankrd22 in OHCs. We analyzed commonly and differentially expressed genes with the focus on genes related to hair cell specializations in the apical, basolateral, and synaptic membranes. Eighty-three percent of the known deafness-related genes are expressed in hair cells. We also analyzed genes involved in cell-cycle regulation. Our dataset holds an extraordinary trove of information about the molecular mechanisms underlying hair cell morphology, function, pathology, and cell-cycle control.

A motif of eleven amino acids is a structural adaptation that facilitates motor capability of eutherian prestin.

Cochlear outer hair cells (OHCs) alter their length in response to transmembrane voltage changes. This so-called electromotility is the result of conformational changes of membrane-bound prestin. Prestin-based OHC motility is thought to be responsible for cochlear amplification, which contributes to the exquisite frequency selectivity and sensitivity of mammalian hearing. Prestin belongs to an anion transporter family, the solute carrier protein 26A (SLC26A). Prestin is unique in this family in that it functions as a voltage-dependent motor protein manifested by two hallmarks, nonlinear capacitance and motility. Evidence suggests that prestin orthologs from zebrafish and chicken are anion exchangers or transporters with no motor function. We identified a segment of 11 amino acid residues in eutherian prestin that is extremely conserved among eutherian species but highly variable among non-mammalian orthologs and SLC26A paralogs. To determine whether this sequence represents a motif that facilitates motor function in eutherian prestin, we utilized a chimeric approach by swapping corresponding residues from the zebrafish and chicken with those of gerbil. Motility and nonlinear capacitance were measured from chimeric prestin-transfected human embryonic kidney 293 cells using a voltage-clamp technique and photodiode-based displacement measurement system. We observed a gain of motor function with both of the hallmarks in the chimeric prestin without loss of transport function. Our results show, for the first time, that the substitution of a span of 11 amino acid residues confers the electrogenic anion transporters of zebrafish and chicken prestins with motor-like function. Thus, this motif represents the structural adaptation that assists gain of motor function in eutherian prestin.

Engineered pendrin protein, an anion transporter and molecular motor.

Pendrin and prestin both belong to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Pendrin (SLC26A4) is a chloride-iodide transporter that is found at the luminal membrane of follicular cells in the thyroid gland as well as in the endolymphatic duct and sac of the inner ear, whereas prestin (SLC26A5) is expressed in the plasma membrane of cochlear outer hair cells and functions as a unique voltage-dependent motor. We recently identified a motif that is critical for the motor function of prestin. We questioned whether it was possible to create a chimeric pendrin protein with motor capability by integrating this motility motif from prestin. The chimeric pendrin was constructed by substituting residues 160-179 in human pendrin with residues 156-169 from gerbil prestin. Non-linear capacitance and somatic motility, two hallmarks representing prestin function, were measured from chimeric pendrin-transfected human embryonic kidney 293 cells using the voltage clamp technique and photodiode-based displacement measurement system. We showed that this 14-amino acid substitution from prestin was able to confer pendrin with voltage-dependent motor capability despite the amino acid sequence disparity between pendrin and prestin. The molecular mechanism that facilitates motor function appeared to be the same as prestin because the motor activity depended on the concentration of intracellular chloride and was blocked by salicylate treatment. Radioisotope-labeled formate uptake measurements showed that the chimeric pendrin protein retained the capability to transport formate, suggesting that the gain of motor function was not at the expense of its inherent transport capability. Thus, the engineered pendrin was capable of both transporting anions and generating force.

Proteomic analysis of the organ of corti using nanoscale liquid chromatography coupled with tandem mass spectrometry.

The organ of Corti (OC) in the cochlea plays an essential role in auditory signal transduction in the inner ear. For its minute size and trace amount of proteins, the identification of the molecules in pathophysiologic processes in the bone-encapsulated OC requires both delicate separation and a highly sensitive analytical tool. Previously, we reported the development of a high resolution metal-free nanoscale liquid chromatography system for highly sensitive phosphoproteomic analysis. Here this system was coupled with a LTQ-Orbitrap XL mass spectrometer to investigate the OC proteome from normal hearing FVB/N male mice. A total of 628 proteins were identified from six replicates of single LC-MS/MS analysis, with a false discovery rate of 1% using the decoy database approach by the OMSSA search engine. This is currently the largest proteome dataset for the OC. A total of 11 proteins, including cochlin, myosin VI, and myosin IX, were identified that when defective are associated with hearing impairment or loss. This study demonstrated the effectiveness of our nanoLC-MS/MS platform for sensitive identification of hearing loss-associated proteins from minute amount of tissue samples.

MicroRNA-183 family expression in hair cell development and requirement of microRNAs for hair cell maintenance and survival.

MicroRNAs (miRNAs) post-transcriptionally repress complementary target gene expression and can contribute to cell differentiation. The coordinate expression of miRNA-183 family members (miR-183, miR-96, and miR-182) has been demonstrated in sensory cells of the mouse inner ear and other vertebrate sensory organs. To further examine hair cell miRNA expression in the mouse inner ear, we have analyzed miR-183 family expression in wild type animals and various mutants with defects in neurosensory development. miR-183 family member expression follows neurosensory cell specification, exhibits longitudinal (basal-apical) gradients in maturating cochlear hair cells, and is maintained in sensory neurons and most hair cells into adulthood. Depletion of hair cell miRNAs resulting from Dicer1 conditional knockout (CKO) in Atoh1-Cre transgenic mice leads to more disparate basal-apical gene expression profiles and eventual hair cell loss. Results suggest that hair cell miRNAs subdue cochlear gradient gene expression and are required for hair cell maintenance and survival.

Molecular and cytological profiling of biological aging of mouse cochlear inner and outer hair cells.

Age-related hearing loss (ARHL) negatively impacts quality of life in the elderly population. The prevalent cause of ARHL is loss of mechanosensitive cochlear hair cells (HCs). The molecular and cellular mechanisms of HC degeneration remain poorly understood. Using RNA-seq transcriptomic analyses of inner and outer HCs isolated from young and aged mice, we show that HC aging is associated with changes in key molecular processes, including transcription, DNA damage, autophagy, and oxidative stress, as well as genes related to HC specialization. At the cellular level, HC aging is characterized by loss of stereocilia, shrinkage of HC soma, and reduction in outer HC mechanical properties, suggesting that functional decline in mechanotransduction and cochlear amplification precedes HC loss and contributes to ARHL. Our study reveals molecular and cytological profiles of aging HCs and identifies genes such as Sod1, Sirt6, Jund, and Cbx3 as biomarkers and potential therapeutic targets for ameliorating ARHL.

From zebrafish to mammal: functional evolution of prestin, the motor protein of cochlear outer hair cells.

Prestin is the motor protein of cochlear outer hair cells. It belongs to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Members of this family serve two fundamentally distinct functions. Although most members transport different anion substrates across a variety of epithelia, prestin (SLC26A5) is unique, functioning as a voltage-dependent motor protein. Recent evidence suggests that prestin orthologs from zebrafish and chicken are electrogenic divalent/chloride anion exchangers/transporters with no motor function. These studies appear to suggest that prestin was evolved from an anion transporter. We examined the motor and transport functions of prestin and its orthologs from four different species in the vertebrate lineage, to gain insights of how these two physiological functions became distinct. Somatic motility, voltage-dependent nonlinear capacitance (NLC), and transporter function were measured in transfected human embryonic kidney (HEK) cells using voltage-clamp and anion uptake techniques. Zebrafish and chicken prestins both exhibited weak NLC, with peaks significantly shifted in the depolarization (right) direction. This was contrasted by robust NLC with peaks left shifted in the platypus and gerbil. The platypus and gerbil prestins retained little transporter function compared with robust anion transport capacities in the zebrafish and chicken orthologs. Somatic motility was detected only in the platypus and gerbil prestins. There appears to be an inverse relationship between NLC and anion transport functions, whereas motor function appears to have emerged only in mammalian prestin. Our results suggest that motor function is an innovation of therian prestin and is concurrent with diminished transporter capabilities.

The role of bHLH genes in ear development and evolution: revisiting a 10-year-old hypothesis.

In mouse ear development, two bHLH genes, Atoh1 and Neurog1, are essential for hair cell and sensory neuron differentiation. Evolution converted the original simple atonal-dependent neurosensory cell formation program of diploblasts into the derived developmental program of vertebrates that generates two neurosensory cell types, the sensory neuron and the sensory hair cell. This transformation was achieved through gene multiplication in ancestral triploblasts resulting in the expansion of the atonal bHLH gene family. Novel genes of the Neurogenin and NeuroD families are upregulated prior to the expression of Atoh1. Recent data suggest that NeuroD and Neurogenin were lost or their function in neuronal specification reduced in flies, thus changing our perception of the evolution of these genes. This sequence of expression changes was accompanied by modification of the E-box binding sites of these genes to regulate different downstream genes and to form inhibitory loops among each other, thus fine-tuning expression transitions.

PLCγ-activated signalling is essential for TrkB mediated sensory neuron structural plasticity.

BACKGROUND: The vestibular system provides the primary input of our sense of balance and spatial orientation. Dysfunction of the vestibular system can severely affect a person's quality of life. Therefore, understanding the molecular basis of vestibular neuron survival, maintenance, and innervation of the target sensory epithelia is fundamental. RESULTS: Here we report that a point mutation at the phospholipase Cγ (PLCγ) docking site in the mouse neurotrophin tyrosine kinase receptor TrkB (Ntrk2) specifically impairs fiber guidance inside the vestibular sensory epithelia, but has limited effects on the survival of vestibular sensory neurons and growth of afferent processes toward the sensory epithelia. We also show that expression of the TRPC3 cation calcium channel, whose activity is known to be required for nerve-growth cone guidance induced by brain-derived neurotrophic factor (BDNF), is altered in these animals. In addition, we find that absence of the PLCγ mediated TrkB signalling interferes with the transformation of bouton type afferent terminals of vestibular dendrites into calyces (the largest synaptic contact of dendrites known in the mammalian nervous system) on type I vestibular hair cells; the latter are normally distributed in these mutants as revealed by an unaltered expression pattern of the potassium channel KCNQ4 in these cells. CONCLUSIONS: These results demonstrate a crucial involvement of the TrkB/PLCγ-mediated intracellular signalling in structural aspects of sensory neuron plasticity.

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

Evolutionary insights into the unique electromotility motor of mammalian outer hair cells.

Prestin (SLC26A5) is the molecular motor responsible for cochlear amplification by mammalian cochlea outer hair cells and has the unique combined properties of energy-independent motility, voltage sensitivity, and speed of cellular shape change. The ion transporter capability, typical of SLC26A members, was exchanged for electromotility function and is a newly derived feature of the therian cochlea. A putative minimal essential motif for the electromotility motor (meEM) was identified through the amalgamation of comparative genomic, evolution, and structural diversification approaches. Comparisons were done among nonmammalian vertebrates, eutherian mammalian species, and the opossum and platypus. The opossum and platypus SLC26A5 proteins were comparable to the eutherian consensus sequence. Suggested from the point-accepted mutation analysis, the meEM motif spans all the transmembrane segments and represented residues 66-503. Within the eutherian clade, the meEM was highly conserved with a substitution frequency of only 39/7497 (0.5%) residues, compared with 5.7% in SLC26A4 and 12.8% in SLC26A6 genes. Clade-specific substitutions were not observed and there was no sequence correlation with low or high hearing frequency specialists. We were able to identify that within the highly conserved meEM motif two regions, which are unique to all therian species, appear to be the most derived features in the SLC26A5 peptide.

Lmx1a is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis.

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1a (dr)) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a "cochlear-gravistatic" endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants.

Pocket proteins and cell cycle regulation in inner ear development.

Loss of neurosensory cells of the ear, caused by genetic and non-genetic factors, is becoming an increasing problem as people age, resulting in deafness and vestibular disorders. Unveiling useful mechanisms of cell cycle regulation may offer the possibility to generate new cells out of remaining ones, thus providing the cellular basis to induce new hair cell differentiation in the mammalian ear. Here, we provide an overview of cell cycle regulating genes in general and of those studied in the ear in particular. We categorize those genes into regulators that act upstream of the pocket proteins and into those that act downstream of the pocket proteins. The three members of the pocket protein family essentially determine, through interaction with the eight members of the E2F family, whether or not the cell cycle will progress to the S-phase and thus cell division. The abundant presence of one or more members of these families in adult hair cells supports the notion that inhibition of cell cycle progression through these proteins is a lifelong process. Indeed, manipulating some of those proteins, unfortunately, leads to abortive entry into the cell cycle. Combined with recent success to induce hair cell differentiation through molecular therapy, these approaches may provide a viable strategy to restore lost hair cells in the inner ear.

Molecular evolution of the vertebrate mechanosensory cell and ear.

The molecular basis of mechanosensation, mechanosensory cell development and mechanosensory organ development is reviewed with an emphasis on its evolution. In contrast to eye evolution and development, which apparently modified a genetic program through intercalation of genes between the master control genes on the top (Pax6, Eya1, Six1) of the hierarchy and the structural genes (rhodopsin) at the bottom, the as yet molecularly unknown mechanosensory channel precludes such a firm conclusion for mechanosensors. However, recent years have seen the identification of several structural genes which are involved in mechanosensory tethering and several transcription factors controlling mechanosensory cell and organ development; these warrant the interpretation of available data in very much the same fashion as for eye evolution: molecular homology combined with potential morphological parallelism. This assertion of molecular homology is strongly supported by recent findings of a highly conserved set of microRNAs that appear to be associated with mechanosensory cell development across phyla. The conservation of transcription factors and their regulators fits very well to the known or presumed mechanosensory specializations which can be mostly grouped as variations of a common cellular theme. Given the widespread distribution of the molecular ability to form mechanosensory cells, it comes as no surprise that structurally different mechanosensory organs evolved in different phyla, presenting a variation of a common theme specified by a conserved set of transcription factors in their cellular development. Within vertebrates and arthropods, some mechanosensory organs evolved into auditory organs, greatly increasing sensitivity to sound through modifications of accessory structures to direct sound to the specific sensory epithelia. However, while great attention has been paid to the evolution of these accessory structures in vertebrate fossils, comparatively less attention has been spent on the evolution of the inner ear and the central auditory system. Recent advances in our molecular understanding of ear and brain development provide novel avenues to this neglected aspect of auditory neurosensory evolution.

Transcriptomes of cochlear inner and outer hair cells from adult mice.

Inner hair cells (IHCs) and outer hair cells (OHCs) are the two anatomically and functionally distinct types of mechanosensitive receptor cells in the mammalian cochlea. The molecular mechanisms defining their morphological and functional specializations are largely unclear. As a first step to uncover the underlying mechanisms, we examined the transcriptomes of IHCs and OHCs isolated from adult CBA/J mouse cochleae. One thousand IHCs and OHCs were separately collected using the suction pipette technique. RNA sequencing of IHCs and OHCs was performed and their transcriptomes were analyzed. The results were validated by comparing some IHC and OHC preferentially expressed genes between present study and published microarray-based data as well as by real-time qPCR. Antibody-based immunocytochemistry was used to validate preferential expression of SLC7A14 and DNM3 in IHCs and OHCs. These data are expected to serve as a highly valuable resource for unraveling the molecular mechanisms underlying different biological properties of IHCs and OHCs as well as to provide a road map for future characterization of genes expressed in IHCs and OHCs.

Characterization of Hair Cell-Like Cells Converted From Supporting Cells After Notch Inhibition in Cultures of the Organ of Corti From Neonatal Gerbils.

The senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Hair cells transduce mechanical stimuli into electrical activity. Loss of hair cells as a result of aging or exposure to noise and ototoxic drugs is the major cause of noncongenital hearing and balance deficits. In the ear of non-mammals, lost hair cells can spontaneously be replaced by production of new hair cells from conversion of supporting cells. Although supporting cells in adult mammals have lost that capability, neonatal supporting cells are able to convert to hair cells after inhibition of Notch signaling. We questioned whether Notch inhibition is sufficient to convert supporting cells to functional hair cells using electrophysiology and electron microscopy. We showed that pharmacological inhibition of the canonical Notch pathway in the cultured organ of Corti prepared from neonatal gerbils induced stereocilia formation in supporting cells (defined as hair cell-like cells or HCLCs) and supernumerary stereocilia in hair cells. The newly emerged stereocilia bundles of HCLCs were functional, i.e., able to respond to mechanical stimulation with mechanotransduction (MET) current. Transmission electron microscopy (TEM) showed that HCLCs converted from pillar cells maintained the pillar cell shape and that subsurface cisternae, normally observed underneath the cytoskeleton in outer hair cells (OHCs), was not present in Deiters' cells-derived HCLCs. Voltage-clamp recordings showed that whole-cell currents from Deiters' cells-derived HCLCs retained the same kinetics and magnitude seen in normal Deiters' cells and that nonlinear capacitance (NLC), an electrical hallmark of OHC electromotility, was not detected from any HCLCs measured. Taken together, these results suggest that while Notch inhibition is sufficient for promoting stereocilia bundle formation, it is insufficient to convert neonatal supporting cells to mature hair cells. The fact that Notch inhibition led to stereocilia formation in supporting cells and supernumerary stereocilia in existing hair cells appears to suggest that Notch signaling may regulate stereocilia formation and stability during development.

Cell-Specific Transcriptome Analysis Shows That Adult Pillar and Deiters' Cells Express Genes Encoding Machinery for Specializations of Cochlear Hair Cells.

The mammalian auditory sensory epithelium, the organ of Corti, is composed of hair cells and supporting cells. Hair cells contain specializations in the apical, basolateral and synaptic membranes. These specializations mediate mechanotransduction, electrical and mechanical activities and synaptic transmission. Supporting cells maintain homeostasis of the ionic and chemical environment of the cochlea and contribute to the stiffness of the cochlear partition. While spontaneous proliferation and transdifferentiation of supporting cells are the source of the regenerative response to replace lost hair cells in lower vertebrates, supporting cells in adult mammals no longer retain that capability. An important first step to revealing the basic biological properties of supporting cells is to characterize their cell-type specific transcriptomes. Using RNA-seq, we examined the transcriptomes of 1,000 pillar and 1,000 Deiters' cells, as well as the two types of hair cells, individually collected from adult CBA/J mouse cochleae using a suction pipette technique. Our goal was to determine whether pillar and Deiters' cells, the commonly targeted cells for hair cell replacement, express the genes known for encoding machinery for hair cell specializations in the apical, basolateral, and synaptic membranes. We showed that both pillar and Deiters' cells express these genes, with pillar cells being more similar to hair cells than Deiters' cells. The fact that adult pillar and Deiters' cells express the genes cognate to hair cell specializations provides a strong molecular basis for targeting these cells for mammalian hair cell replacement after hair cells are lost due to damage.

RNA-seq transcriptomic analysis of adult zebrafish inner ear hair cells.

Although hair cells are the sensory receptors of the auditory and vestibular systems in the ears of all vertebrates, hair cell properties are different between non-mammalian vertebrates and mammals. To understand the basic biological properties of hair cells from non-mammalian vertebrates, we examined the transcriptome of adult zebrafish auditory and vestibular hair cells. GFP-labeled hair cells were isolated from inner-ear sensory epithelia of a pou4f3 promoter-driven GAP-GFP line of transgenic zebrafish. One thousand hair cells and 1,000 non-sensory surrounding cells (nsSCs) were separately collected for each biological replicate, using the suction pipette technique. RNA sequencing of three biological replicates for the two cell types was performed and analyzed. Comparisons between hair cells and nsSCs allow identification of enriched genes in hair cells, which may underlie hair cell specialization. Our dataset provides an extensive resource for understanding the molecular mechanisms underlying morphology, function, and pathology of adult zebrafish hair cells. It also establishes a framework for future characterization of genes expressed in hair cells and the study of hair cell evolution.