Neonatal Hair Cells Research Articles

The Rho GTPase Cell Division Cycle 42 Regulates Stereocilia Development in Cochlear Hair Cells

Stereocilia are actin-based cell protrusions on the apical surface of inner ear hair cells, playing a pivotal role in hearing and balancing sensation. The development and maintenance of stereocilia is tightly regulated and deficits in this process usually lead to hearing or balancing disorders. The Rho GTPase cell division cycle 42 (CDC42) is a key regulator of the actin cytoskeleton. It has been reported to localize in the hair cell stereocilia and play important roles in stereocilia maintenance. In the present work, we utilized hair cell-specific Cdc42 knockout mice and CDC42 inhibitor ML141 to explore the role of CDC42 in stereocilia development. Our data show that stereocilia height and width as well as stereocilia resorption are affected in Cdc42-deficient cochlear hair cells when examined at postnatal day 8 (P8). Moreover, ML141 treatment leads to planar cell polarity (PCP) deficits in neonatal hair cells. We also show that overexpression of a constitutively active mutant CDC42 in cochlear hair cells leads to enhanced stereocilia developmental deficits. In conclusion, the present data suggest that CDC42 plays a pivotal role in regulating hair cell stereocilia development.

Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

The channel that governs mechanotransduction (MT) by hair cells in the inner ear has been investigated intensively for 4 decades, but its precise molecular composition remains enigmatic. Transmembrane channel-like protein 1 (TMC1) was recently identified as a component of the MT channel, and lipoma HMGIC fusion partner-like 5 (LHFPL5) is considered to be part of the MT complex and may functionally couple the tip link to the MT channel. As components of the MT complex, TMC1 and LHFPL5 are expected to localize at the lower end of the tip link in hair cells, a notion generally supported by previous studies on neonatal mice. However, the localization of these 2 proteins, particularly in the hair cells of adult mice, remains incompletely elucidated. Because determination of TMC1 and LHFPL5 localization at distinct developmental stages is essential for understanding their function and regulation, we used several approaches to examine the localization of these proteins in neonatal and adult hair cells in the mouse. We report several notable findings: 1) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies the previously published findings in neonatal hair cells; 2) LHFPL5 persists in the hair bundle of hair cells after postnatal day (P)7, which clarifies the previously reported unexpected absence of LHFPL5 after P7 and supports the view that LHFPL5 is a permanent component in the MT complex; and 3) TMC1 and LHFPL5 remain at the tip of the shorter rows of stereocilia in adult outer hair cells, but in adult inner hair cells, TMC1 is uniformly distributed in both the tallest row and the shorter rows of stereocilia, whereas LHFPL5 is uniformly distributed in the shorter rows of stereocilia. These findings raise intriguing questions regarding the turnover rate, regulation, additional functions, and functional interaction of TMC1 and LHFPL5. Our study confirms the previous findings in neonatal hair cells and reveals several previously unidentified aspects of TMC1 and LHFPL5 localization in more mature hair cells.-Li, X., Yu, X., Chen, X., Liu, Z., Wang, G., Li, C., Wong, E. Y. M., Sham, M. H., Tang, J., He, J., Xiong, W., Liu, Z., Huang, P. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

The Promoter and Multiple Enhancers of the pou4f3 Gene Regulate Expression in Inner Ear Hair Cells.

Few enhancers that target gene expression to inner ear hair cells (HCs) have been identified. Using transgenic analysis of enhanced green fluorescent protein (eGFP) reporter constructs and bioinformatics, we evaluated the control of pou4f3 gene expression, since it is expressed only in HCs within the inner ear and continues to be expressed throughout life. An 8.5-kb genomic DNA fragment 5' to the start codon, containing three regions of high cross-species homology, drove expression in all embryonic and neonatal HCs, and adult vestibular and inner HCs, but not adult outer HCs. Transgenes with 0.4, 0.8, 2.5, or 6.5kb of 5' DNA did not produce HC expression. However, addition of the region from 6.5 to 7.2kb produced expression in vestibular HCs and neonatal basal turn outer HCs, which also implicated the region from 7.2 to 8.5kb in inner and apical outer HC expression. Deletion of the region from 0.4 to 5.5kb 5' from the 8.5-kb construct did not affect HC expression, further indicating lack of HC regulatory elements. When the region from 1 to 0.4kb was replaced with the minimal promoter of the Ela1 gene, HC expression was maintained but at a drastically reduced level. Bioinformatics identified regions of highly conserved sequence outside of the 8.5kb, which contained POU4F3-, GFI1-, and LHX3-binding sites. These regions may be involved in maintaining POU4F3 expression in adult outer HCs. Our results identify separate enhancers at various locations that direct expression to different HC types at different ages and determine that 0.4kb of upstream sequence determines expression level. These data will assist in the identification of mutations in noncoding, regulatory regions of this deafness gene.

Regulation of POU4F3 gene expression in hair cells by 5′ DNA in mice

The POU-domain transcription POU4F3 is expressed in the sensory cells of the inner ear. Expression begins shortly after commitment to the hair cell (HC) fate, and continues throughout life. It is required for terminal HC differentiation and survival. To explore regulation of the murine Pou4f3 gene, we linked enhanced green fluorescent protein (eGFP) to 8.5 kb of genomic sequence 5' to the start codon in transgenic mice. eGFP was uniformly present in all embryonic and neonatal HCs. Expression of eGFP was also observed in developing Merkel cells and olfactory neurons as well as adult inner and vestibular HCs, mimicking the normal expression pattern of POU4F3 protein, with the exception of adult outer HCs. Apparently ectopic expression was observed in developing inner ear neurons. On a Pou4f3 null background, the transgene produced expression in embryonic HCs which faded soon after birth both in vivo and in vitro. Pou4f3 null HCs treated with caspase 3 and 9 inhibitors survived longer than untreated HCs, but still showed reduced expression of eGFP. The results suggest the existence of separate enhancers for different HC types, as well as strong autoregulation of the Pou4f3 gene. Bioinformatic analysis of four divergent mammalian species revealed three highly conserved regions within the transgene: 400 bp immediately 5' to the Pou4f3 ATG, a short sequence at -1.3 kb, and a longer region at -8.2 to -8.5 kb. The latter contained E-box motifs that bind basic helix-loop-helix (bHLH) transcription factors, including motifs activated by ATOH1. Cotransfection of HEK293 or VOT-E36 cells with ATOH1 and the transgene as a reporter enhanced eGFP expression when compared with the transgene alone. Chromatin immunoprecipitation of the three highly conserved regions revealed binding of ATOH1 to the distal-most conserved region. The results are consistent with regulation of Pou4f3 in HCs by ATOH1 at a distal enhancer.

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.

Voltage-gated Na+ channel activation induces both action potentials in utricular hair cells and brain-derived neurotrophic factor release in the rat utricle during a restricted period of development.

The mammalian utricular sensory receptors are commonly believed to be non-spiking cells with electrical activity limited to graded membrane potential changes. Here we provide evidence that during the first post-natal week, the sensory hair cells of the rat utricle express a tetrodotoxin (TTX)-sensitive voltage-gated Na+ current that displays most of the biophysical and pharmacological characteristics of neuronal Na+ current. Single-cell RT-PCR reveals that several alpha-subunit isoforms of the Na+ channels are co-expressed within a single hair cell, with a major expression of Nav1.2 and Nav1.6 subunits. In neonatal hair cells, 30 % of the Na+ channels are available for activation at the resting potential. Depolarizing current injections in the range of the transduction currents are able to trigger TTX-sensitive action potentials. We also provide evidence of a TTX-sensitive activity-dependent brain-derived neurotrophic factor (BDNF) release by early post-natal utricle explants. Developmental analysis shows that Na+ currents decrease dramatically from post-natal day 0 (P0) to P8 and become almost undetectable at P21. Concomitantly, depolarizing stimuli fail to induce both action potential and BDNF release at P20. The present findings reveal that vestibular hair cells express neuronal-like TTX-sensitive Na+ channels able to generate Na+-driven action potentials only during the early post-natal period of development. During the same period an activity-dependent BDNF secretion by utricular explants has been demonstrated. This could be an important mechanism involved in vestibular sensory system differentiation and synaptogenesis.

E-cadherin and the Differentiation of Mammalian Vestibular Hair Cells

E-cadherin is expressed in vestibular, mechanosensory epithelia during early embryonic development. During late embryonic and neonatal stages it is expressed in supporting cells but down-regulated in differentiating sensory hair cells. We used a conditionally immortal cell line (UB/UE-1) from the neonatal mouse utricle to test the hypothesis that constitutive expression of E-cadherin inhibits the progression of hair cell differentiation. Under differentiating culture conditions, transfected E-cadherin inhibited expression of the cytoskeletal protein myosin VIIa and functional expression of both acetylcholine receptors and potassium channels, which are normally expressed by neonatal hair cells. However, it had no effect on the expression of the transcription factor Brn3c or the cytoskeletal protein fimbrin, which are also expressed by neonatal hair cells. The number of adherens junctions increased significantly under differentiating conditions but there was no detectable change in formation of tight junctions or gap junctions. However, E-cadherin expression led to density-dependent cell death under differentiating conditions. We have shown that E-cadherin is expressed in vestibular supporting cells, which form the basis of the sensory epithelium, but that constitutive expression inhibits the full differentiation of hair cells. Down-regulation of E-cadherin is thus likely to be a key element in the regeneration of hair cells.

Asymmetric segregation of mitochondria and mortalin correlates with the multi-lineage potential of inner ear sensory cell progenitors in vitro

The sensory epithelia of the inner ear include hair cells and supporting cells that share a common precursor. One possible mechanism involved in the genesis of these cell types is through asymmetric cell division. In this work we have studied asymmetric division of inner ear sensory cell progenitors in vitro in an attempt to understand how the different cell phenotypes are generated. In the search for molecules that will segregate asymmetrically we have found that mitochondria in general, and a mitochondrial protein named mortalin in particular, are asymmetrically segregated during certain cell divisions. In one conditionally immortal cell line (UB/OC-1), which represents a population of committed hair cell precursors, mortalin is uniformly distributed in the cytoplasm and shared equally between sibling cells during division. In another cell line (UB/UE-1), which represents a bipotent, vestibular supporting cell that can produce both neonatal hair cells as well as supporting cells, mortalin segregates asymmetrically. In UB/UE-1, ∼12% of the cells display an asymmetric distribution of mortalin and mitochondria. The proportion of asymmetric cells increases immediately after the release of the immortalizing gene and before the onset of differentiation. The asymmetric segregation of mortalin in the bipotent cell line and its uniform distribution in a committed, lineage-restricted cell line raises the possibility that it may play a role in cell fate determination.

Calcium channels in mouse hair cells: function, properties and pharmacology.

Adult inner hair cells (IHCs) possess voltage-activated Ca2+ currents that couple receptor potentials to transmitter release at the afferent synapses. Before the onset of hearing both IHCs and outer hair cells (OHCs) exhibit Ca2+ currents. More than 90% of neonatal hair cell (HC) currents flow through alpha1D Ca2+ channel subunits because they are absent in both IHCs and OHCs from alpha1D-/- mice and residual currents are insensitive to L-type agonists. Since lack of the alpha1D-subunit leads to HC degeneration and profound deafness, class D L-type Ca2+ currents seem to be crucial for the development and functioning of the inner ear. Neonatal HC Ca2+ currents were studied using the whole-cell patch clamp technique. They showed rapid activation, rapid deactivation and very little inactivation. They started activating as negative as -65mV. In contrast to alpha1C-mediated (classical L-type) Ca2+ currents, they showed a rather low sensitivity to various L-type antagonists. 10 microM nifedipine e.g. blocked HC Ca2+ currents by about 40% whereas class C L-type Ca2+ currents are completely blocked by 100nM nifedipine. The L-type channel agonist Bay K 8644 increased the HC Ca2+ current by 100-200% and shifted the IV curve to more negative potentials which is similar to its effects in alpha1C-mediated Ca2+ currents.

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.

Stimulus processing by type II hair cells in the mouse utricle.

In type II and neonatal hair cells in the mouse utricle, the receptor potentials evoked by low-frequency sinusoidal deflections of the hair bundle are attenuated by adaptation of the mechanoelectrical transduction current and the voltage-dependent activation of a large potassium (K)-selective outwardly rectifying conductance, gDR. These processes may contribute to high-pass filtering of the responses of some utricular afferents to sinusoidal linear accelerations below 2 Hz. Depolarizing receptor potentials are more attenuated by gDR than are hyperpolarizing receptor potentials. It may therefore reduce nonlinear distortion introduced by mechanoelectrical transduction, which generates larger depolarizing currents than hyperpolarizing currents. The discharge properties of utricular afferents vary according to whether they innervate the striolar or extrastriolar zones of the sensory epithelium. Regional variation in hair-cell properties is likely to contribute. Preliminary results suggest that the outwardly rectifying K conductances of type II cells are slower and larger in the striola than in the extrastriola, consistent with regional variation in the relative numbers of delayed rectifier and A-current K channels.

Developmental changes in the physiology of hair cells

Mature hair cells express complements of ion channels which vary with hair cell type. Immature hair cells in the inner ears of neonatal mice and pre-hatch chicks share mechanosensitive and certain voltage-gated conductances: delayed rectifier and inwardly rectifying potassium conductances, voltage-gated calcium and sodium conductances. Over the course of several days the immature cells acquire other conductances that confer upon them the distinctive voltage-dependent properties of mature hair cells. In the mouse utricle, postnatal acquisition of additional delayed and inward rectifiers transforms the neonatal hair cells into two classes with the electrophysiological profiles of mature type I and type hair II cells. Electromotility, a highly differentiated, voltage-dependent property of mature outer hair cells from the mammalian cochlea, is also acquired after mechanosensitivity.