Non-sensory Cells Research Articles

Histoarchitectural and surface ultrastructural analysis of the olfactory epithelium of Puntius ticto (Hamilton, 1822)

Organization of various cells lining the olfactory mucosa of Puntius ticto (Hamilton, 1822) were described by light as well as scanning electron microscopy. The paired olfactory chambers located antero-dorsal to the eyes and communicated outside through anterior and posterior nasal openings. The oval shaped olfactory rosette lied at the bottom of chamber and composed of 18-20 lamellae arranged on either side of median raphe. Sensory and non-sensory regions were distributed separately on each lamella. The sensory epithelium consisted mainly of two distinct morphological forms: ciliated and microvillous receptor cells. The non-sensory epithelium contained ciliated non-sensory cells, stratified epithelial cells with concentric microridges and mucous cells. Basal cells were situated at the deeper part of the epithelium, adjacent to the central core. The functional significance of cellular components of the olfactory epithelium was discussed with the habit and habitat of fish.

Mice with conditional deletion of Cx26 exhibit no vestibular phenotype despite secondary loss of Cx30 in the vestibular end organs

Connexins are components of gap junctions which facilitate transfer of small molecules between cells. One member of the connexin family, Connexin 26 (Cx26), is prevalent in gap junctions in sensory epithelia of the inner ear. Mutations of GJB2, the gene encoding Cx26, cause significant hearing loss in humans. The vestibular system, however, does not usually show significant functional deficits in humans with this mutation. Mouse models for loss of Cx26 function demonstrate hearing loss and cochlear pathology but the extent of vestibular dysfunction and organ pathology are less well characterized. To understand the vestibular effects of Cx26 mutations, we evaluated vestibular function and histology of the vestibular sensory epithelia in a conditional knockout (CKO) mouse with Cx26 loss of function. Transgenic C57BL/6 mice, in which cre-Sox10 drives excision of the Cx26 gene from non-sensory cells flanking the sensory epithelium of the inner ear (Gjb2-CKO), were compared to age-matched wild types. Animals were sacrificed at ages between 4 and 40 weeks and their cochlear and vestibular sensory organs harvested for histological examination. Cx26 immunoreactivity was prominent in the peripheral vestibular system and the cochlea of wild type mice, but absent in the Gjb2-CKO specimens. The hair cell population in the cochleae of the Gjb2-CKO mice was severely depleted but in the vestibular organs it was intact, despite absence of Cx26 expression. The vestibular organs appeared normal at the latest time point examined, 40 weeks. To determine whether compensation by another connexin explains survival of the normal vestibular sensory epithelium, we evaluated the presence of Cx30 in the Gjb2-CKO mouse. We found that Cx30 labeling was normal in the cochlea, but it was decreased or absent in the vestibular system. The vestibular phenotype of the mutants was not different from wild-types as determined by time on the rotarod, head stability tests and physiological responses to vestibular stimulation. Thus presence of Cx30 in the cochlea does not compensate for Cx26 loss, and the absence of both connexins from vestibular sensory epithelia is no more injurious than the absence of one of them. Further studies to uncover the physiological foundation for this difference between the cochlea and the vestibular organs may help in designing treatments for GJB2 mutations.

Cochlear supporting cell transdifferentiation and integration into hair cell layers by inhibition of ephrin-B2 signalling.

In mammals, cochlear sensory hair cells that are responsible for hearing are postmitotic and are not replaced after loss. One of the most promising strategies to regenerate hair cells is to identify and inhibit the factors preventing the conversion of adjacent non-sensory supporting cells into hair cells. Here we demonstrate that mammalian hair cells can be directly generated from supporting cells by inhibition of ephrin-B2 signalling. Using either ephrin-B2 conditional knockout mice, shRNA-mediated gene silencing or soluble inhibitors, we found that downregulation of ephrin-B2 signalling at embryonic stages results in supporting cell translocation into hair cell layers and subsequent switch in cell identity from supporting cell to hair cell fate. As transdifferentiation is here a result of displacement across boundary, this original finding presents the interest that newly generated hair cells directly integrate either hair cell layer, then would be likely more rapidly able to fit into functional circuitry.

Surface architecture of the olfactory epithelium of two Chinese cave loaches (Cypriniformes: Nemacheilidae: Oreonectes)

The surface of the olfactory epithelium of two species of cave loaches in China, Oreonectes polystigmus and O. guananensis, was studied by scanning electron microscopy (SEM). The structure of the olfactory organ of the two species is quite different from each other; as well, the distribution patterns of cell types in the olfactory epithelium of the studied species showed interesting features. A needle-like spine is visible on the lamellae of the olfactory organ of O. polystigmus, which is a unique character in this species. Ciliated non-sensory cells and microvillus sensory cells showed a different distribution in the two species, being more numerous in O. polystigmus. The non-sensory elements in O. guananensis were ciliated non-sensory cells, with microridges and chloride cells. The presence of chloride cells in the olfactory epithelium of O. guananensis may be of special interest in this species; otherwise, possible differences and physiological changes of the concerned species should be taken into account in order to explain such variability in cell types.

Uptake of fluorescent gentamicin by peripheral vestibular cells after systemic administration.

ObjectiveIn addition to cochleotoxicity, systemic aminoglycoside pharmacotherapy causes vestibulotoxicity resulting in imbalance and visual dysfunction. The underlying trafficking routes of systemically-administered aminoglycosides from the vasculature to the vestibular sensory hair cells are largely unknown. We investigated the trafficking of systemically-administered gentamicin into the peripheral vestibular system in C56Bl/6 mice using fluorescence-tagged gentamicin (gentamicin-Texas-Red, GTTR) imaged by scanning laser confocal microscopy to determine the cellular distribution and intensity of GTTR fluorescence in the three semicircular canal cristae, utricular, and saccular maculae at 5 time points over 4 hours.ResultsLow intensity GTTR fluorescence was detected at 0.5 hours as both discrete puncta and diffuse cytoplasmic fluorescence. The intensity of cytoplasmic fluorescence peaked at 3 hours, while punctate fluorescence was plateaued after 3 hours. At 0.5 and 1 hour, higher levels of diffuse GTTR fluorescence were present in transitional cells compared to hair cells and supporting cells. Sensory hair cells typically exhibited only diffuse cytoplasmic fluorescence at all time-points up to 4 hours in this study. In contrast, non-sensory cells rapidly exhibited both intense fluorescent puncta and weaker, diffuse fluorescence throughout the cytosol. The numbers and size of fluorescent puncta in dark cells and transitional cells increased over time. There is no preferential GTTR uptake by the five peripheral vestibular organs’ sensory cells. Control vestibular tissues exposed to Dulbecco’s phosphate-buffered saline or hydrolyzed Texas Red had negligible fluorescence.ConclusionsAll peripheral vestibular cells rapidly take up systemically-administered GTTR, reaching peak intensity 3 hours after injection. Sensory hair cells exhibited only diffuse fluorescence, while non-sensory cells displayed both diffuse and punctate fluorescence. Transitional cells may act as a primary pathway for trafficking of systemic GTTR from the vasculature to endolymph prior to entering hair cells.

Protein-engineered hydrogel encapsulation for 3-D culture of murine cochlea.

Elastin-like protein (ELP) hydrogel helps maintain the three-dimensional (3-D) cochlear structure in culture. Whole-organ culture of the cochlea is a useful model system facilitating manipulation and analysis of live sensory cells and surrounding nonsensory cells. The precisely organized 3-D cochlear structure demands a culture method that preserves this delicate architecture; however, current methods have not been optimized to serve such a purpose. A protein-engineered ELP hydrogel was used to encapsulate organ of Corti isolated from neonatal mice. Cultured cochleae were immunostained for markers of hair cells and supporting cells. Organ of Corti hair cell and supporting cell density and organ dimensions were compared between the ELP and nonencapsulated systems. These culture systems were then compared with noncultured cochlea. After 3 days in vitro, vital dye uptake and immunostaining for sensory and nonsensory cells show that encapsulated cochlea contain viable cells with an organized architecture. In comparison with nonencapsulated cultured cochlea, ELP-encapsulated cochleae exhibit higher densities of hair cells and supporting cells and taller and narrower organ of Corti dimensions that more closely resemble those of noncultured cochleae. However, we found compromised cell viability when the culture period extended beyond 3 days. We conclude that the ELP hydrogel can help preserve the 3-D architecture of neonatal cochlea in short-term culture, which may be applicable to in vitro study of the physiology and pathophysiology of the inner ear.

Cellular mechanisms of mutations in Kv7.1: auditory functions in Jervell and Lange-Nielsen syndrome vs. Romano–Ward syndrome

As a result of cell-specific functions of voltage-activated K+ channels, such as Kv7.1, mutations in this channel produce profound cardiac and auditory defects. At the same time, the massive diversity of K+ channels allows for compensatory substitution of mutant channels by other functional channels of their type to minimize defective phenotypes. Kv7.1 represents a clear example of such functional dichotomy. While several point mutations in the channel result in a cardio-auditory syndrome called Jervell and Lange-Nielsen syndrome (JLNS), about 100-fold mutations result in long QT syndrome (LQTS) denoted as Romano–Ward syndrome (RWS), which has an intact auditory phenotype. To determine whether the cellular mechanisms for the diverse phenotypic outcome of Kv7.1 mutations, are dependent on the tissue-specific function of the channel and/or specialized functions of the channel, we made series of point mutations in hKv7.1 ascribed to JLNS and RWS. For JLNS mutations, all except W248F yielded non-functional channels when expressed alone. Although W248F at the end of the S4 domain yielded a functional current, it underwent marked inactivation at positive voltages, rendering the channel non-functional. We demonstrate that by definition, none of the JLNS mutants operated in a dominant negative (DN) fashion. Instead, the JLNS mutants have impaired membrane trafficking, trapped in the endoplasmic reticulum (ER) and Cis-Golgi. The RWS mutants exhibited varied functional phenotypes. However, they can be summed up as exhibiting DN effects. Phenotypic differences between JLNS and RWS may stem from tissue-specific functional requirements of cardiac vs. inner ear non-sensory cells.

Ion Channels Activated by Mechanical Forces in Bacterial and Eukaryotic Cells.

Since the first discovery of mechanosensitive ion channel (MSC) in non-sensory cells in 1984, a variety of MSCs has been identified both in prokaryotic and eukaryotic cells. One of the central issues concerning MSCs is to understand the molecular and biophysical mechanisms of how mechanical forces activate/open MSCs. It has been well established that prokaryotic (mostly bacterial) MSCs are activated exclusively by membrane tension. Thus the problem to be solved with prokaryotic MSCs is the mechanisms how the MSC proteins receive tensile forces from the lipid bilayer and utilize them for channel opening. On the other hand, the activation of many eukaryotic MSCs crucially depends on tension in the actin cytoskeleton. By using the actin cytoskeleton as a force sensing antenna, eukaryotic MSCs have obtained sophisticated functions such as remote force sensing and force-direction sensing, which bacterial MSCs do not have. Actin cytoskeletons also give eukaryotic MSCs an interesting and important function called "active touch sensing", by which cells can sense rigidity of their substrates. The contractile actin cytoskeleton stress fiber (SF) anchors its each end to a focal adhesion (FA) and pulls the substrate to generate substrate-rigidity-dependent stresses in the FA. It has been found that those stresses are sensed by some Ca2+-permeable MSCs existing in the vicinity of FAs, thus the MSCs work as a substrate rigidity sensor that can transduce the rigidity into intracellular Ca2+ levels. This short review, roughly constituting of two parts, deals with molecular and biophysical mechanisms underlying the MSC activation process mostly based on our recent studies; (1) structure-function in bacterial MSCs activation at the atomic level, and (2) roles of actin cytoskeletons in the activation of eukaryotic MSCs.

Jag1b is essential for patterning inner ear sensory cristae by regulating anterior morphogenetic tissue separation and preventing posterior cell death.

The sensory patches of the vertebrate inner ear, which contain hair cells and supporting cells, are essential for hearing and balance functions. How the stereotypically organized sensory patches are formed remains to be determined. In this study, we isolated a zebrafish mutant in which the jag1b gene is disrupted by an EGFP insertion. Loss of Jag1b causes cell death in the developing posterior crista and results in downregulation of fgf10a in the posterior prosensory cells. Inhibition of FGFR activity in wild-type embryos also causes loss of the posterior crista, suggesting that Fgf10a mediates Jag1b activity. By contrast, in the anterior prosensory domain, Jag1b regulates separation of a single morphogenetic field into anterior and lateral cristae by flattening cells destined to form a nonsensory epithelium between the two cristae. MAPK activation in the nonsensory epithelium precursors is required for the separation. In the jag1b mutant, MAPK activation and cell flattening are extended to anterior crista primordia, causing loss of anterior crista. More importantly, inhibition of MAPK activity, which blocks the differentiation of nonsensory epithelial cells, generated a fused large crista and extra hair cells. Thus, Jag1b uses two distinct mechanisms to form three sensory cristae in zebrafish.

Ototoxicity-induced loss of hearing and inner hair cells is attenuated by HSP70 gene transfer

The most common reason for sensorineural deafness is death of hair cells (HCs). Heat shock proteins (HSPs) are molecular chaperones that participate in folding, targeting, and degrading proteins. HSP expression is increased in response to various environmental stresses to protect cells from damage. Here, we tested whether viral-mediated overexpression of HSP70 can protect HCs and hearing from severe ototoxicity (kanamycin and furosemide) in guinea pigs. Adenovirus-HSP70 mCherry (Ad.HSP70-mCherry) was injected to experimental animals and adenovirus-mCherry to controls, 4 days before the ototoxic insult. Hearing thresholds were measured by auditory brainstem response before the insult and again before sacrificing the animals, 14 days after the insult. Epi-fluorescence immunocytochemistry showed that injection of Ad.HSP70-mCherry resulted in mCherry fluorescence in nonsensory cells of the organ of Corti. The ototoxic insult eliminated both outer HCs and inner HCs throughout most of the cochlea of control (adenovirus-mCherry-injected) ears and contralateral (uninjected) ears. Ad.HSP70-mCherry-injected ears exhibited a significant preservation of inner HCs compared to control and contralateral ears, but outer HCs were not protected. Auditory brainstem response thresholds were significantly better in Ad.HSP70-mCherry-injected ears than in control and contralateral ears. Our data show that HSP70 augmentation may represent a potential therapy attenuating ototoxic inner HC loss.

Connexins and gap junctions in the inner ear--it's not just about K⁺ recycling.

Normal development, function and repair of the sensory epithelia in the inner ear are all dependent on gap junctional intercellular communication. Mutations in the connexin genes GJB2 and GJB6 (encoding CX26 and CX30) result in syndromic and non-syndromic deafness via various mechanisms. Clinical vestibular defects, however, are harder to connect with connexin dysfunction. Cx26 and Cx30 proteins are widely expressed in the epithelial and connective tissues of the cochlea, where they may form homomeric or heteromeric gap junction channels in a cell-specific and spatiotemporally complex fashion. Despite the study of mutant channels and animal models for both recessive and dominant autosomal deafness, it is still unclear why gap junctions are essential for auditory function, and why Cx26 and Cx30 do not compensate for each other in vivo. Cx26 appears to be essential for normal development of the auditory sensory epithelium, but may be dispensable during normal hearing. Cx30 appears to be essential for normal repair following sensory cell loss. The specific modes of intercellular signalling mediated by inner ear gap junction channels remain undetermined, but they are hypothesised to play essential roles in the maintenance of ionic and metabolic homeostasis in the inner ear. Recent studies have highlighted involvement of gap junctions in the transfer of essential second messengers between the non-sensory cells, and have proposed roles for hemichannels in normal hearing. Here, we summarise the current knowledge about the molecular and functional properties of inner ear gap junctions, and about tissue pathologies associated with connexin mutations.

Aberrant Cx26 hemichannels and keratitis-ichthyosis-deafness syndrome: insights into syndromic hearing loss.

Mutation of the GJB2 gene, which encodes the connexin 26 (Cx26) gap junction (GJ) protein, is the most common cause of hereditary, sensorineural hearing loss. Cx26 is not expressed in hair cells, but is widely expressed throughout the non-sensory epithelial cells of the cochlea. Most GJB2 mutations produce non-syndromic deafness, but a subset produces syndromic deafness in which profound hearing loss is accompanied by a diverse array of infectious and neoplastic cutaneous disorders that can be fatal. Although GJ channels, which are assembled by the docking of two, so-called hemichannels (HCs), have been the main focus of deafness-associated disease models, it is now evident that the HCs themselves can function in the absence of docking and contribute to signaling across the cell membrane as a novel class of ion channel. A notable feature of syndromic deafness mutants is that the HCs exhibit aberrant behaviors providing a plausible basis for disease that is associated with excessive or altered contributions of Cx26 HCs that, in turn, lead to compromised cell integrity. Here we discuss some of the aberrant Cx26 HC properties that have been described for mutants associated with keratitis-ichthyosis-deafness (KID) syndrome, a particularly severe Cx26-associated syndrome, which shed light on genotype-phenotype relationships and causes underlying cochlear dysfunction.

Deficiency of transcription factor Brn4 disrupts cochlear gap junction plaques in a model of DFN3 non-syndromic deafness.

Brn4, which encodes a POU transcription factor, is the gene responsible for DFN3, an X chromosome–linked, non-syndromic type of hearing loss. Brn4-deficient mice have a low endocochlear potential (EP), hearing loss, and ultrastructural alterations in spiral ligament fibrocytes, however the molecular pathology through which Brn4 deficiency causes low EP is still unclear. Mutations in the Gjb2 and Gjb6 genes encoding the gap junction proteins connexin26 (Cx26) and connexin30 (Cx30) genes, respectively, which encode gap junction proteins and are expressed in cochlear fibrocytes and non-sensory epithelial cells (i.e., cochlear supporting cells) to maintain the proper EP, are responsible for hereditary sensorineural deafness. It has been hypothesized that the gap junction in the cochlea provides an intercellular passage by which K+ is transported to maintain the EP at the high level necessary for sensory hair cell excitation. Here we analyzed the formation of gap junction plaques in cochlear supporting cells of Brn4-deficient mice at different stages by confocal microscopy and three-dimensional graphic reconstructions. Gap junctions from control mice, which are composed mainly of Cx26 and Cx30, formed linear plaques along the cell-cell junction sites with adjacent cells. These plaques formed pentagonal or hexagonal outlines of the normal inner sulcus cells and border cells. Gap junction plaques in Brn4-deficient mice did not, however, show the normal linear structure but instead formed small spots around the cell-cell junction sites. Gap junction lengths were significantly shorter, and the level of Cx26 and Cx30 was significantly reduced in Brn4-deficient mice compared with littermate controls. Thus the Brn4 mutation affected the assembly and localization of gap junction proteins at the cell borders of cochlear supporting cells, suggesting that Brn4 substantially contributes to cochlear gap junction properties to maintain the proper EP in cochleae, similar to connexin-related deafness.

Acid-sensing ion channel 2 (ASIC2) is selectively localized in the cilia of the non-sensory olfactory epithelium of adult zebrafish.

Ionic channels play key roles in the sensory cells, such as transducing specific stimuli into electrical signals. The acid-sensing ion channel (ASIC) family is voltage-insensitive, amiloride-sensitive, proton-gated cation channels involved in several sensory functions. ASIC2, in particular, has a dual function as mechano- and chemo-sensor. In this study, we explored the possible role of zebrafish ASIC2 in olfaction. RT-PCR, Western blot, chromogenic in situ hybridization and immunohistochemistry, as well as ultrastructural analysis, were performed on the olfactory rosette of adult zebrafish. ASIC2 mRNA and protein were detected in homogenates of olfactory rosettes. Specific ASIC2 hybridization was observed in the luminal pole of the non-sensory epithelium, especially in the cilia basal bodies, and immunoreactivity for ASIC2 was restricted to the cilia of the non-sensory cells where it was co-localized with the cilia marker tubulin. ASIC2 expression was always absent in the olfactory cells. These findings demonstrate for the first time the expression of ASIC2 in the olfactory epithelium of adult zebrafish and suggest that it is not involved in olfaction. Since the cilium sense and transduce mechanical and chemical stimuli, ASIC2 expression in this location might be related to detection of aquatic environment pH variations or to detection of water movement through the nasal cavity.

Spontaneous recovery of cochlear fibrocytes after severe degeneration caused by acute energy failure.

Cochlear fibrocytes in the lateral wall region play a critical role in the regulation of inner ear ion and fluid homeostasis, although these are non-sensory cells. Along with other non-sensory cells, fibrocytes in the spiral ligament have been reported to repopulate themselves after damage. However, the studies of regeneration of cochlear fibrocytes have been difficult because a suitable fibrocyte-specific degeneration model did not exist. Therefore, we analyzed cochlear fibrocytes using a rat model of acute cochlear energy failure induced by a mitochondrial toxin. This model is unique because hearing loss is caused by apoptosis of fibrocytes in the cochlear lateral wall not by damage to sensory cells. Although this model involves severe damage to the cochlear lateral wall, delayed spontaneous regeneration occurs without any treatment. Moreover, partial hearing recovery is accompanied by morphological remodeling of the cochlear lateral wall. Two hypotheses are conceivable regarding this spontaneous recovery of cochlear fibrocytes. One is that residual cochlear fibrocytes proliferate spontaneously, followed by remodeling of the functional region of the lateral wall. Another is that some foreign cells such as bone marrow-derived cells promote morphological and functional recovery of the lateral wall. Acceleration of the lateral wall recovery promoted by these mechanisms may be a new therapeutic strategy against hearing loss.

Localization of sex hormone binding globulin in the rat vomeronasal organ

Volatile and non-volatile derivates of gonadal steroids are known to act as pheromones in many mammalian species. Pheromones have multiple effects on the brain via the olfactory system. Their primary port of entry seems to be the vomeronasal organ (VNO) but the underlying cellular and molecular mechanisms are unclear so far. Recently we localized sex hormone binding globulin (SHBG) in both the main and the accessory olfactory system of rat with immunocytochemistry and RT-PCR. The accessory olfactory system consisting of VNO and accessory olfactory bulb showed high expression of SHBG.In the present paper we studied SHBG expression in the VNO in greater detail. In semithin sections we found SHBG immunostaining in the perinuclear cytoplasm of some of the sensory neurons, in sensory cilia and in their axons. A portion of the basal cells and some of the goblet cells in the non-sensory epithelium showed intense SHBG staining. SHBG was abundant in exocrine cells of the vomeronasal glands, perhaps compartimentalized in secretory vesicles. In situ hybridization revealed specific signals in sensory and non-sensory cells of the VNO. Our findings indicate that SHBG expressed in the VNO may be liberated into nasal secretions to bind aerosolic steroids. SHBG in sensory cells may be involved in signaling actions of pheromones.

LMO4 functions as a negative regulator of sensory organ formation in the mammalian cochlea.

In mammals, formation of the auditory sensory organ (the organ of Corti) is restricted to a specialized area of the cochlea. However, the molecular mechanisms limiting sensory formation to this discrete region in the ventral cochlear duct are not well understood, nor is it known whether other regions of the cochlea have the competence to form the organ of Corti. Here we identify LMO4, a LIM-domain-only nuclear protein, as a negative regulator of sensory organ formation in the cochlea. Inactivation of Lmo4 in mice leads to an ectopic organ of Corti (eOC) located in the lateral cochlea. The eOC retains the features of the native organ, including inner and outer hair cells, supporting cells, and other nonsensory specialized cell types. However, the eOC shows an orientation opposite to the native organ, such that the eOC appears as a mirror-image duplication to the native organ of Corti. These data demonstrate a novel sensory competent region in the lateral cochlear duct that is regulated by LMO4 and may be amenable to therapeutic manipulation.

Hair cell regeneration after ATOH1 gene therapy in the cochlea of profoundly deaf adult guinea pigs.

The degeneration of hair cells in the mammalian cochlea results in permanent sensorineural hearing loss. This study aimed to promote the regeneration of sensory hair cells in the mature cochlea and their reconnection with auditory neurons through the introduction of ATOH1, a transcription factor known to be necessary for hair cell development, and the introduction of neurotrophic factors. Adenoviral vectors containing ATOH1 alone, or with neurotrophin-3 and brain derived neurotrophic factor were injected into the lower basal scala media of guinea pig cochleae four days post ototoxic deafening. Guinea pigs treated with ATOH1 gene therapy, alone, had a significantly greater number of cells expressing hair cell markers compared to the contralateral non-treated cochlea when examined 3 weeks post-treatment. This increase, however, did not result in a commensurate improvement in hearing thresholds, nor was there an increase in synaptic ribbons, as measured by CtBP2 puncta after ATOH1 treatment alone, or when combined with neurotrophins. However, hair cell formation and synaptogenesis after co-treatment with ATOH1 and neurotrophic factors remain inconclusive as viral transduction was reduced due to the halving of viral titres when the samples were combined. Collectively, these data suggest that, whilst ATOH1 alone can drive non-sensory cells towards an immature sensory hair cell phenotype in the mature cochlea, this does not result in functional improvements after aminoglycoside-induced deafness.

Transcription factors with conserved binding sites near ATOH1 on the POU4F3 gene enhance the induction of cochlear hair cells.

Overexpression of the transcription factor (TF) ATOH1 is known to induce the transformation of nonsensory cells in the organ of Corti into hair cells (HCs). Evaluating DNA 5Œ to the coding sequence of the pou4f3 gene, a target of ATOH1 in HCs, we identified in three regions containing clustered binding sites for ATOH1 and several other TFs that are expressed in developing inner ear sensory epithelia at the time of HC specification. These regions and sites are highly conserved across evolutionarily distant mammalian species. To test the hypothesis that the identified TFs act in combination to regulate the pou4f3 gene, we transfected by electroporation neonatal cochlear sensory epithelium from mice expressing green fluorescent protein (GFP) under the control of an 8.5-kb 5' pou4f3 genomic fragment. Plasmids encoding 21 TFs c-transfected with human ATOH1 (hATOH1). Cotransfection with hETV4, hNMYC, or hETS2 produced significantly more pou4f3/GFP and myosin 7A-positive nonsensory cells than hATOH1 alone. Co-transfection of hATOH1 with hHES1, hHES5, or hNEUROD1 reduced the effects of hATOH1. Chromatin immunoprecipitation (ChIP)of DNA from an inner ear cell line transfected with hNMYC,hETV4, or hETS2 revealed binding to a conserved region immediately proximal to the coding sequence. ChIP similarly revealed binding of hGATA3, hNMYC, and hTFE2 to a region several kilobases distal to the coding sequence, which we have previously shown to bind ATOH1. The results suggest that ATOH1 acts in concert with a subset of other TFs to directly regulate the pou4f3 gene and more broadly to regulate the HC phenotype.

Generation of induced neurons by direct reprogramming in the mammalian cochlea

Primary auditory neurons (ANs) in the mammalian cochlea play a critical role in hearing as they transmit auditory information in the form of electrical signals from mechanosensory cochlear hair cells in the inner ear to the brainstem. Their progressive degeneration is associated with disease conditions, excessive noise exposure and aging. Replacement of ANs, which lack the ability to regenerate spontaneously, would have a significant impact on research and advancement in cochlear implants in addition to the amelioration of hearing impairment. The aim of this study was to induce a neuronal phenotype in endogenous non-neural cells in the cochlea, which is the essential organ of hearing. Overexpression of a neurogenic basic helix–loop–helix transcription factor, Ascl1, in the cochlear non-sensory epithelial cells induced neurons at high efficiency at embryonic, postnatal and juvenile stages. Moreover, induced neurons showed typical properties of neuron morphology, gene expression and electrophysiology. Our data indicate that Ascl1 alone or Ascl1 and NeuroD1 is sufficient to reprogram cochlear non-sensory epithelial cells into functional neurons. Generation of neurons from non-neural cells in the cochlea is an important step for the regeneration of ANs in the mature mammalian cochlea.