Phosphatase and tensin homolog deleted on chromosome 10 regulates sensory cell proliferation and differentiation of hair bundles in the mammalian cochlea.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that regulates cell proliferation, differentiation and growth. It regulates neural and glioma stem/progenitor cell renewal and PTEN deletion can drive expansion of epithelial progenitors in the lung, enhancing their capacity for regeneration. Because it is expressed at relatively high levels in developing mammalian auditory hair cells we have analyzed the phenotype of the auditory epithelium in PTEN knock-out mice. PTEN(+/-) heterozygous littermates have only one functional copy of the gene and show clear evidence for haploinsufficiency in the organ of Corti. Auditory sensory epithelial progenitors withdraw from the cell cycle later than in wild-type animals and this is associated with increases in the numbers of both inner and outer hair cells. The cytoskeletal differentiation of hair cells was also affected. While many hair bundles on the hair cells appeared to develop normally, others were structurally disorganized and a number were missing, apparently lost after they had been formed. The results show that PTEN plays a novel role in regulating cell proliferation and differentiation of hair bundles in auditory sensory epithelial cells and suggest that PTEN signaling pathways may provide therapeutic targets for auditory sensory regeneration.

A model for mammalian cochlear hair cell differentiation in vitro: effects of retinoic acid on cytoskeletal proteins and potassium conductances.

We have established a model for the in-vitro differentiation of mouse cochlear hair cells and have used it to explore the influence of retinoic acid on proliferation, cytoskeletal proteins and voltage-gated potassium conductances. The model is based on the conditionally immortal cell line University of Sheffield/ventral otocyst-epithelial cell line clone 36 (US/VOT-E36), derived from ventral otic epithelial cells of the mouse at embryonic day 10.5 and transfected with a reporter for myosin VIIa. Retinoic acid did not increase cell proliferation but led to up-regulation of myosin VIIa and formation of prominent actin rings that gave rise to numerous large, linear actin bundles. Cells expressing myosin VIIa had larger potassium conductances and did not express the cyclin-dependent kinase inhibitor p27(kip1). US/VOT-E36 endogenously expressed the voltage-gated potassium channel alpha-subunits Kv1.3 and Kv2.1, which we subsequently identified in embryonic and neonatal hair cells in both auditory and vestibular sensory epithelia in vivo. These subunits could underlie the embryonic and neonatal delayed-rectifiers recorded in nascent hair cells in vivo. Kv2.1 was particularly prominent on the basolateral membrane of cochlear inner hair cells. Kv1.3 was distributed throughout all hair cells but tended to be localized to the cuticular plates. US/VOT-E36 recapitulates a coherent pattern of cell differentiation under the influence of retinoic acid and will provide a convenient model for screening the effects of other extrinsic factors on the differentiation of cochlear epithelial cell types in vitro.

Information for gene networks in inner ear development: a study centered on the transcription factor gata2.

The search for molecular mechanisms to stimulate sensory regeneration in the mammalian inner ear is commonly based upon developmental studies. This has revealed many genes that regulate the differentiation of sensory cells. A major challenge is to place these genes into the context of functional networks that describe developmental processes more fully and increase the chances of identifying useful therapeutic targets. We used a novel approach to identify genes that are functionally related to the transcription factor gata2. Temporal profiles of gene expression were derived from three conditionally immortal cell lines and clustered to those of gata2 by applying the gamma model for oligonucleotide signals, a statistical method that allows quantitative analysis of oligonucleotide array data. We derived an objective list of 28 genes that clustered with gata2 in all three cell lines. A number of these genes have known functional links with gata2. Genes encoding CCAAT/enhancer binding proteins (C/EBP) and signal transducer and activation of transcription 3 (Stat3) are especially interesting as they are known to bind gata proteins directly. The results provide strong evidence that our experimental approach can reveal functional relationships between genes that regulate fundamental processes in the differentiation of sensory cells in the inner ear.

Differentiation of an auditory neuronal cell line suitable for cell transplantation.

The auditory neuroblast cell line US/VOT-N33 (N33), which is conditionally immortal, was studied as an in vitro model for the differentiation of spiral ganglion neurons (SGNs) and as a candidate for cell transplantation in rodents. It expresses numerous molecular markers characteristic of auditory neuroblasts, including the transcription factors GATA3, NeuroD, Brn3a and Islet1, as well as the neuronal cytoskeletal protein beta3-tubulin. It displays active migratory behaviour in vitro and in vivo. In the presence of the fibroblast growth factors FGF1 or FGF2 it differentiates bipolar morphologies similar to those of native SGNs. In coculture with neonatal cochlear tissue it is repelled from epithelial surfaces but not from native SGNs, alongside which it extends parallel neuronal processes. When injected into the retina in vivo, EGFP-labelled N33 cells were traced for 1-2 weeks and migrated rapidly within the subretinal space. Cells that found their way into the retinal ganglion cell layer extended multiple processes but did not express beta3-tubulin. The ability of N33 to migrate, to differentiate, to localize with native SGNs in vitro and to survive in vivo suggests that they provide an effective model for SGN differentiation and for cell transplantation into the ear.

Ventral otic cell lines as developmental models of auditory epithelial and neural precursors.

Conditionally immortal cell lines were established from the ventral otocyst of the Immortomouse at embryonic day 10.5 and selected to represent precursors of auditory sensory neural and epithelial cells. Selection was based upon dissection, tissue-specific markers, and expression of the transcription factor GATA3. Two cell lines expressed GATA3 but possessed intrinsically different genetic programs under differentiating conditions. US/VOT-E36 represented epithelial progenitors with potential to differentiate into sensory and nonsensory epithelial cells. US/VOT-N33 represented migrating neuroblasts. Under differentiating conditions in vitro the cell lines expressed very different gene expression profiles. Expression of several cell- and tissue-specific markers, including the transcription factors Pax2, GATA3, and NeuroD, differed between the cell lines in a pattern consistent with that observed between their counterparts in vivo. We suggest that these and other conditionally immortal cell lines can be used to study transient events in development against different backgrounds of cell competence.

Notch signaling and the emergence of auditory hair cells.

OBJECTIVE: Recent insights into the mechanisms that determine a hair cell's fate have emerged from studies on invertebrate sensory organs and the avian inner ear. These mechanisms have important implications for our understanding of the possible therapeutic management of sensorineural deafness. This article reviews the current state of our knowledge regarding mammalian auditory hair cell fate specification. DESIGN: Data were obtained from the MEDLINE database and data presented at the Molecular Biology of Hearing and Deafness Meeting (Bethesda, Md, October 1998). Articles reporting information about cell fate specification and Notch and its ligands were selected. MAIN OUTCOME MEASURES: Data pertaining to cell fate mechanisms, Notch and its ligands, and application to hearing were extracted. RESULTS: The Notch/ligand mechanism is responsible for the specification of the hair cell phenotype. CONCLUSIONS: Major progress has been made in understanding this fundamental process, and its application to hair cell determination is only now being realized. Possible applications could involve the "switching" of supporting cells to hair cells, thus replenishing those hair cells damaged in sensorineural hearing loss.

Calcium signalling mediated by the 9 acetylcholine receptor in a cochlear cell line from the immortomouse.

1. We have investigated the characteristics of the alpha9 acetylcholine receptor (alpha9AChR) expressed in hair cell precursors in an immortalized cell line UB/OC-2 developed from the organ of Corti of the transgenic H-2Kb-tsA58 mouse (the Immortomouse) using both calcium imaging and whole-cell recording. 2. Ratiometric measurements of fura-2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 microM ACh. The calcium increase was seen in 66 % of the cells grown at 39 degrees C in differentiated conditions. A sm aller fraction (34%) of cells grown at 33 degrees C in proliferative con ditions responded. 3. Caffeine (10mM) elevated cell calcium. In the ab sence of caffeine, the majority of imaged cells responded only once to A Ch presentations. Pretreatment with caffeine ingibited all calcium respo nses to ACh. 4. In whole-cell tight-seal recordings 10 microM ACh activa ted inward current was dependent on the extracellular calcium concentrat ion with an estimated PCa/PNa of 80 for the alpha9 receptor at physiological calcium levels. 5 . The data indicate that ACh activates a calcium-permeable channel alpha 9AChR in UB/OC-2 cells and that the channel has a significantly higher c alcium permeability than other AChRs. The results indicate that the alp ha9AChR may be able to elevate intracellular calcium levels in hair cell s both directly and via store release.

Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells.

We provide evidence from a newly established, conditionally immortal cell line (UB/UE-1) that vestibular supporting cells from the mammalian inner ear can differentiate postnatally into more than one variant of hair cell. A clonal supporting cell line was established from pure utricular sensory epithelia of H2k(b)tsA58 transgenic mice 2 d after birth. Cell proliferation was dependent on conditional expression of the immortalizing gene, the "T" antigen from the SV40 virus. Proliferating cells expressed cytokeratins, and patch-clamp recordings revealed that they all expressed small membrane currents with little time-dependence. They stopped dividing within 2 d of being transferred to differentiating conditions, and within a week they formed three defined populations expressing membrane currents characteristic of supporting cells and two kinds of neonatal hair cell. The cells expressed several characteristic features of normal hair cells, including the transcription factor Brn3.1, a functional acetylcholine receptor composed of alpha9 subunits, and the cytoskeletal proteins myosin VI, myosin VIIa, and fimbrin. Immunofluorescence labeling and electron microscopy showed that the cells formed complex cytoskeletal arrays on their upper surfaces with structural features resembling those at the apices of normal hair cells. The cell line UB/UE-1 provides a valuable in vitro preparation in which the expression of numerous structural and physiological components can be initiated or upregulated during early stages of mammalian hair cell commitment and differentiation.

Ionic currents expressed in a cell line derived from the organ of Corti of the Immortomouse.

We have investigated the maturation of adult hair cell electrophysiology in a population of precursor cells in a conditionally immortal cell line. The cell line, UB/OC-2, from the embryonic organ of Corti of the H-2Kb-tsA58 transgenic mouse, permits cells to grow proliferatively at 33 degrees C and to differentiate at 39 degrees C. Whole-cell patch-clamp recordings showed that proliferating cells had a different electrophysiology to differentiating cells. Differentiating cells had a conditionally expressed slowly activating inward current activated by hyperpolarization. The current was not blocked by extracellular application of 0.5 mM Ba2+, but was blocked reversibly by 2 mM Cs+. The current was found to be carried by both K+ and Na+ ions (PK/PNa=2.2) and activated by 10 microM forskolin. These properties identify the slowly activating current as Ih. A proportion of proliferating and differentiating cells exhibited a voltage-gated Na+ current, INa. INa was abolished in Na-free external medium and was inhibited reversibly by tetrodotoxin (TTX) with Ki=64 nM. Together these results suggest that proliferating and differentiating hair cell precursors in the immortal cochlear cell line UB/OC-2 express currents which are also found in developing hair cells.

Auditory hair cell precursors immortalized from the mammalian inner ear.

Mammalian auditory hair cells are few in number, experimentally inaccessible, and do not proliferate postnatally or in vitro. Immortal cell lines with the potential to differentiate into auditory hair cells would substantially facilitate auditory research, drug development, and the isolation of critical molecules involved in hair cell biology. We have established two conditionally immortal cell lines that express at least five characteristic hair cell markers. These markers are the transcription factor Brn3.1, the alpha 9 subunit of the acetylcholine receptor, the stereociliary protein fimbrin and the myosins VI and VIIA. These hair cell precursors permit functional studies of cochlear genes and in the longer term they will provide the means to explore therapeutic methods of stimulating auditory hair cell regeneration.

Timed markers for the differentiation of the cuticular plate and stereocilia in hair cells from the mouse inner ear.

The differentiation of the cuticular plate and stereocilia in cochleovestibular hair cells from the mouse was traced with monoclonal antibodies raised by in vitro immunization. The cuticular plate is detected first from embryonic days 14-15 (E14-E15), before cell differentiation is apparent, either with scanning electron microscopy or with actin filament labeling. A flat disc of material forms beneath the apical membrane and subsequently expands, forming a fully shaped cuticular plate at postnatal stages 3-5 (P3-P5). A second antibody labels stereocilia from stage E16 to E18. In the cochlea, the label initially appears as a punctate disc on the cell apex and then follows the development of the stereocilia until the adult shape of the bundle forms at P4-P6. Additional antibodies label stereocilia from P4 to P6 and are apparently specific for the inner ear. They do not label the cuticular plate at any stage and do not cross react with tissues of muscle, kidney, eye, tongue, gut, skin, or brain. At stage P12-P14, coinciding with the functional maturity of the ear, they label the apical regions of Deiter's cells. The temporally overlapping sequence of antibody labeling sheds new light on the development of the hair cell apex and allows us to monitor the differentiation of hair cells from their last mitotic division to the initiation of organ function, a period of over 2 weeks.

GATA3 is downregulated during hair cell differentiation in the mouse cochlea.

GATA3 is a transcription factor expressed in the inner ear during the early stages of development. A monoclonal antibody revealed that it is expressed in spiral ganglion cells and in all cells of the developing auditory sensory epithelium in the mouse before the hair cells differentiate at embryonic days 14-16. Expression decreases selectively in the hair cells as they differentiate progressively from the base to the apex of the developing organ of Corti. GATA3 subsequently decreases in the supporting cells and cannot be detected by immunofluorescence in any cell of the adult sensory epithelium. It is not expressed in the vestibular sensory epithelia or surrounding tissues from embryonic day 14. We suggest that GATA3 could act as a repressor of critical genes involved in cell differentiation in the organ of Corti, enabling a progressive formation of the adult cellular pattern.

Mechanics of microtubule bundles in pillar cells from the inner ear.

The mechanical properties of cross-linked microtubule bundles were measured from outer pillar cells isolated from the mammalian inner ear. Measurements were made using a three-point bending test and were incorporated into a mathematical model designed to distinguish between the stiffness contributions from microtubules and their cross-linking proteins. Outer pillar cells were composed of 1000-3000 parallel bundled microtubules in a square array that was interdigitated and cross-linked with actin filaments. The average midpoint bending stiffness of intact cells was 7 x 10(-4) N/m. After removal of both the actin filaments and cross-links with detergent in the presence of DNase I, the square array was disrupted and the stiffness decreased by a factor of 4, to 1.7 x 10(-4) N/m. The bending modulus for individual microtubules was calculated to be 7 x 10(-23) Nm2, and the Young's modulus for these 15 protofilament microtubules was 2 x 10(9) Pa. The shear modulus between microtubules in intact cells was calculated to be 10(3) Pa. It was concluded that cross-linking proteins provided shear resistance between microtubules, which resulted in a fourfold increase in stiffness. The model can be used to estimate the mechanical properties of cross-linked microtubule bundles in cells from which direct measurements are not available.

Conditional immortalization of hair cells from the inner ear.

The aim of this work was to culture conditionally immortalized cells that possess the potential to differentiate into mechanosensory hair cells. Utricular epithelia at embryonic stage E16 were cultured from the vestibular system of the H2kbtsA58 transgenic mouse (Immortomouse) that carries a conditionally expressed immortalizing gene derived from the simian virus 40. Immunolabelling showed that the immortalizing transgene product, the T antigen (Tag), was expressed in utricular cells under permissive conditions and that it was inactivated under non-permissive conditions. Several morphologically distinct cell types proliferated when Tag was expressed, including those that resembled fibroblasts, nerve cells and epithelial cells. Mixed cultures of cells from the utricle, passaged up to 50 times every 3-4 days over a period of 5 months, were subsequently allowed to differentiate for 10 days by transferring them to non-permissive conditions. Monoclonal antibody markers were used to locate expression of hair cell specific antigens. One antibody that normally labels stereociliary bundles from postnatal stage P4-6 labelled cellular projections from a population of spheroid cells that were distributed across the culture surface. A second antibody that normally labels stereociliary bundles did not label the same structures. We conclude that utricular hair cell progenitors can be derived from the H2kbtsA58 transgenic mouse but that under the experimental conditions used they do not follow the normal pattern of differentiation.

Production of conditionally immortalised cell lines from a transgenic mouse.

This review describes the H2kbtsA58 transgenic mouse (Immortomouse) and its application to the production of conditionally immortalised cell lines from sensory epithelia within the mammalian inner ear. Established cell lines should overcome many of the technical difficulties associated with experimental procedures in auditory and vestibular research. These include the limited amount of tissue available and the relatively complex and laborious dissection. Conditional immortalisation should also allow essential studies on the molecular and cellular mechanisms that govern both the differentiation of sensory cells and the development of sensory epithelia.

Mechanical properties of the lateral cortex of mammalian auditory outer hair cells.

Mammalian auditory outer hair cells generate high-frequency mechanical forces that enhance sound-induced displacements of the basilar membrane within the inner ear. It has been proposed that the resulting cell deformation is directed along the longitudinal axis of the cell by the cortical cytoskeleton. We have tested this proposal by making direct mechanical measurements on outer hair cells. The resultant stiffness modulus along the axis of whole dissociated cells was 3 x 10(-3) N/m, consistent with previously published values. The resultant axial and circumferential stiffness moduli for the cortical lattice were 5 x 10(-4) N/m and 3 x 10(-3) N/m, respectively. Thus the cortical lattice is a highly orthotropic structure. Its axial stiffness is small compared with that of the intact cell, but its circumferential stiffness is within the same order of magnitude. These measurements support the theory that the cortical cytoskeleton directs electrically driven length changes along the longitudinal axis of the cell. The Young's modulus of the circumferential filamentous components of the lattice were calculated to be 1 x 10(7) N/m2. The axial cross-links, believed to be a form of spectrin, were calculated to have a Young's modulus of 3 x 10(6) N/m2. Based on the measured values for the lattice and intact cell cortex, an estimate for the resultant stiffness modulus of the plasma membrane was estimated to be on the order of 10(-3) N/m. Thus, the plasma membrane appears to be relatively stiff and may be the dominant contributor to the axial stiffness of the intact cell.

Immunologically defined component of the circumferential ring around the cuticular plate in mammalian hair cells.

A monoclonal antibody (CAR) was raised by in vitro immunisation to a component of the circumferential actin ring that is associated with the apical junctions encircling the cuticular plates of mammalian hair cells. On western blots it bound a protein band at about 42 kD, equivalent to the normal location of actin, but it did not label the paracrystalline bundle of actin filaments in the stereocilia, the complex actin filament gel that forms the cuticular plate or the filamentous actin in the cell cortex. When applied to whole mounts of the auditory sensory epithelium in the guinea pig it provided a clear, unambiguous map of the distribution of inner and outer hair cells. In this respect, it can provide an accurate guide to patterns of hair cell differentiation and repair. CAR cross-reacted with the membrane-associated cytoskeleton in selected cells from a wide range of other tissues.

Monoclonal antibody markers for early development of the stereociliary bundles of mammalian hair cells.

Two monoclonal antibodies, SC1 and SC2, were raised in vitro against antigens from the stereocilia of guinea-pig hair cells. They both labelled stereociliary antigens that were not detected in any other cell within the cochlear duct or the vestibular epithelial. SC1 cross-reacted with the tectorial membrane in the cochlea and labelled both cochlear and vestibular hair cells from both the mouse and the rat. In the mouse the SC1 antigen was labelled from embryonic days 16-18, coincident with the development of the stereociliary bundles. SC1 cross-reacted with neuromuscular junctions from striated muscle and with basal keratinocytes in skin. SC2 did not cross-react cleanly with hair cells from the mouse or the rat but it cross-reacted with proximal tubules of the guinea-pig kidney. Both antibodies can be used as cellular markers within the guinea-pig cochlea and SC1 should be particularly useful for studies of hair cell differentiation in the mouse.

Identification of a 53-kDa antigen in the fibrous sheath of avian spermatozoa.

The intracellular fibrous sheath that surrounds the proximal part of the sperm flagellar axoneme in non-passerine birds is structurally different from that of mammals. We raised a monoclonal antibody against the fibrous sheath of cockerel spermatozoa by in vitro immunisation. Indirect immunofluorescence and immunogold labelling showed that the antibody bound specifically to the fibrous sheath. It also labelled the fibrous sheath in quail spermatozoa. In both species the antibody bound an antigen that had a molecular weight of about 53 kDa. In tissue sections from adult cockerel testis the antigen was located in spermatids and spermatozoa with little cross-reactivity with the basal region of the seminiferous epithelium or interstitial tissue. The antibody may prove to be a useful tool in studies of avian spermiogenesis.