Matthew C. Holley

Developmental changes in the expression of potassium currents of embryonic, neonatal and mature mouse inner hair cells

Developmental changes in electrophysiological membrane properties of mouse cochlear inner hair cells (IHCs) were studied from just after terminal differentiation up to functional maturity. As early as embryonic day 14.5 (E14.5) newly differentiated IHCs express a very small outward K+ current that is largely insensitive to 4‐aminopyridine (4‐AP). One day later the inward rectifier, IK1, is first observed. These immature cells initially exhibit only slow graded voltage responses under current clamp. From E17.5 spontaneous action potentials occur. During the first week of postnatal development, the outward K+ current steadily increases in size and a progressively larger fraction of the current is sensitive to 4‐AP. During the second postnatal week, the activation of the 4‐AP‐sensitive current, by now contributing about half of the outward K+ current, shifts in the hyperpolarizing direction. Together with an increase in size of IK1, this hyperpolarizes the cell, thus inhibiting the spontaneous spike activity, although spikes could still be evoked upon depolarizing current injection. Starting at about the onset of hearing (postnatal day 12, P12) immature IHCs make the final steps towards fully functional sensory receptors with fast graded voltage responses. This is achieved mainly by the expression of the large‐conductance Ca2+‐activated K+ current IK,f, but also of a current indistinguishable from the negatively activating IK,n previously described in mature outer hair cells (OHCs). The 4‐AP‐sensitive current continues to increase after the onset of hearing to form the major part of the mature delayed rectifier, IK,s. By P20 IHCs appear mature in terms of their complement of K+ conductances.

Expression of the transcription factors GATA3 and Pax2 during development of the mammalian inner ear

The transcription factors GATA3 and Pax2 are expressed throughout development of the mouse inner ear. We have used antibodies to study their temporal and spatial expression patterns from embryonic days E8–E16.5. The two factors show reciprocal relationships in the regional patterning of the early otocyst and cellular patterning within the sensory epithelia. GATA3 is expressed in the whole otic placode at E8. In the otocyst at E9.5–10.5, the distribution is lateral and complementary to the medial expression pattern of Pax2. Only Pax2 is expressed in the endolymphatic duct, but both factors are expressed in the cochlea. At E11.5–13.5, GATA3 is expressed strongly in the cochlea, but in the dorsal, vestibular region it is downregulated. In all sensory epithelia, downregulation coincides with sensory innervation. Pax2 is expressed in all sensory and some nonsensory epithelia, but within sensory epithelia at E16.5 it is restricted to hair cells. GATA3 is expressed throughout key periods of cell proliferation, fate determination, and differentiation and is not specifically associated with any of these processes. Expression persists most strongly in the main components of the developing auditory system. These include the auditory sensory epithelium, the afferent and efferent nerves, and the mesenchymal and ectodermal cells in regions that form key parts of the middle and outer ear. GATA3 is thus expressed in functionally distinct groups of cells that integrate to form a complete sensory system. The results suggest that both factors may be involved in tissue compartmentalisation, morphogenesis, and cell signalling. J. Comp. Neurol. 442:378–391, 2002.

On the Mechanism of a High-Frequency Force Generator in Outer Hair Cells Isolated from the Guinea Pig Cochlea

Isolated mammalian outer hair cells elongate or shorten respectively by several micrometres when electrically hyperpolarized or depolarized. The experiments in this paper were designed to locate the force-generating mechanism that drives length changes in outer hair cells, and to determine some of its basic properties. The whole-cell mode of the patch-clamp technique was used to stimulate cells electrically and to perfuse them with specific drugs. The pattern of displacement of cellular organelles, and the relative displacements of the cell base and apex during electrical stimulation with the cell mechanically anchored at various points along its length, suggest that the force-generating mechanism is distributed throughout the length of the cell. Further experiments altering the shape, volume and intracellular pressure of outer hair cells suggest that the mechanism is closely associated with the plasma membrane. These experiments also demonstrate that the characteristic tubular shape of outer hair cells is maintained by membrane-associated structures with elastic properties that enable the cell to return to its original shape after deformation. The mechanism controlling length changes may, therefore, be composed of two elements in parallel, namely a force generating element and a passive elastic element. Inhibitors of ATP synthesis, or the presence of the non-hydrolysable ATP analogue AMP. PNP, perfused into outer hair cells, failed to inhibit length changes. Drugs against actin, including phalloidin, cytochalasin B and cytochalasin D, and against tubulin, including colchicine, nocodazole and colcemid, also failed to inhibit length changes. We conclude that the force-generating mechanism is, therefore, unlike most other forms of cell motility, and possible alternative hypotheses are briefly discussed.

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 α9 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.

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 Youngs 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.

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 Youngs 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 Youngs 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.

GATA3 and NeuroD distinguish auditory and vestibular neurons during development of the mammalian inner ear

The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expressed GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD and that migrate from the antero-ventral, otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3.

Keynote review: The auditory system, hearing loss and potential targets for drug development

There is a huge potential market for the treatment of hearing loss. Drugs are already available to ameliorate predictable, damaging effects of excessive noise and ototoxic drugs. The biggest challenge now is to develop drug-based treatments for regeneration of sensory cells following noise-induced and age-related hearing loss. This requires careful consideration of the physiological mechanisms of hearing loss and identification of key cellular and molecular targets. There are many molecular cues for the discovery of suitable drug targets and a full range of experimental resources are available for initial screening through to functional analysis in vivo. There is now an unparalleled opportunity for translational research.

miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells

MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system.

RNA microarray analysis in prenatal mouse cochlea reveals novel IGF-I target genes: implication of MEF2 and FOXM1 transcription factors

Background Insulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness. Methodology/Principal Findings A combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1−/− null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1−/− and Igf1+/+ transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1+/+ mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27Kip1. Conclusions/Significance We have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I.