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Combined electric and acoustic stimulation has proven to be an effective strategy to improve hearing in some cochlear implant users. We describe an acoustic microactuator to directly deliver stimuli to the perilymph in the scala tympani. The 800 µm by 800 µm actuator has a silicon diaphragm driven by a piezoelectric thin film (e.g., lead-zirconium-titanium oxide or PZT). This device could also be used as a component of a bimodal acoustic-electric electrode array. In the current study, we established a guinea pig model to test the actuator for its ability to deliver auditory signals to the cochlea in vivo. The actuator was placed through the round window of the cochlea. Auditory brainstem response (ABR) thresholds, peak latencies, and amplitude growth were calculated for an ear canal speaker versus the intracochlear actuator for tone burst stimuli at 4, 8, 16, and 24 kHz. An ABR was obtained after removal of the probe to assess loss of hearing related to the procedure. In some animals, the temporal bone was harvested for histologic analysis of cochlear damage. We show that the device is capable of stimulating ABRs in vivo with latencies and growth functions comparable to stimulation in the ear canal. Further experiments will be necessary to evaluate the efficiency and safety of this modality in long-term auditory stimulation and its ability to be integrated with conventional cochlear implant arrays., (© The Author(s) 2015.)

We developed a transgenic mouse to permit conditional and selective ablation of hair cells in the adult mouse utricle by inserting the human diphtheria toxin receptor (DTR) gene into the Pou4f3 gene, which encodes a hair cell-specific transcription factor. In adult wild-type mice, administration of diphtheria toxin (DT) caused no significant hair cell loss. In adult Pou4f3(+/DTR) mice, DT treatment reduced hair cell numbers to 6% of normal by 14 days post-DT. Remaining hair cells were located primarily in the lateral extrastriola. Over time, hair cell numbers increased in these regions, reaching 17% of untreated Pou4f3(+/DTR) mice by 60 days post-DT. Replacement hair cells were morphologically distinct, with multiple cytoplasmic processes, and displayed evidence for active mechanotransduction channels and synapses characteristic of type II hair cells. Three lines of evidence suggest replacement hair cells were derived via direct (nonmitotic) transdifferentiation of supporting cells: new hair cells did not incorporate BrdU, supporting cells upregulated the pro-hair cell gene Atoh1, and supporting cell numbers decreased over time. This study introduces a new method for efficient conditional hair cell ablation in adult mouse utricles and demonstrates that hair cells are spontaneously regenerated in vivo in regions where there may be ongoing hair cell turnover.

Loss of hair cells in humans leads to irreversible hearing deficits, since auditory hair cells are not replaced. In contrast, hair cells are regenerated in the auditory epithelium of mature birds after damage by non-sensory supporting cells that transdifferentiate into hair cells by mitotic and/or non-mitotic mechanisms. Factors controlling these processes are poorly understood. The basic helix-loop-helix transcription factor ATOH1 is both necessary and sufficient for developmental hair cell differentiation, but it is unclear if it plays the same role in the mitotic and non-mitotic pathways in hair cell regeneration. We examined Atoh1 expression and function during hair cell regeneration in chickens. Atoh1 transcripts were increased in many supporting cells in the damaged auditory epithelium shortly after ototoxin administration and later became restricted to differentiating hair cells. Fate-mapping in vitro using an Atoh1 enhancer reporter demonstrated that only 56% of the supporting cells that spontaneously upregulate Atoh1 enhancer activity after damage acquired the hair cell fate. Inhibition of notch signaling using a gamma secretase antagonist stimulated an increase in Atoh1 reporter activity and induced a higher proportion of supporting cells with Atoh1 activity (73%) to differentiate as hair cells. Forced overexpression of Atoh1 in supporting cells triggered 66% of them to acquire the hair cell fate and nearly tripled their likelihood of cell cycle entry. These findings demonstrate that Atoh1 is broadly upregulated in supporting cells after damage, but a substantial proportion of supporting cells with Atoh1 activation fails to acquire hair cell features, in part due to gamma secretase-dependent activities., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the residual spiral ganglion neurons. These neurons, however, undergo progressive degeneration after hearing loss, marked initially by peripheral fibre retraction and ultimately culminating in cell death. This research aims to use gene therapy techniques to both hold and reverse this degeneration by providing a sustained and localised source of neurotrophins to the deafened cochlea. Adenoviral vectors containing green fluorescent protein, with or without neurotrophin-3 and brain derived neurotrophic factor, were injected into the lower basal turn of scala media of guinea pigs ototoxically deafened one week prior to intervention. This single injection resulted in localised and sustained gene expression, principally in the supporting cells within the organ of Corti. Guinea pigs treated with adenoviral neurotrophin-gene therapy had greater neuronal survival compared to contralateral non-treated cochleae when examined at 7 and 11 weeks post injection. Moreover; there was evidence of directed peripheral fibre regrowth towards cells expressing neurotrophin genes after both treatment periods. These data suggest that neurotrophin-gene therapy can provide sustained protection of spiral ganglion neurons and peripheral fibres after hearing loss.

The capacity of adult mammals to regenerate sensory hair cells is not well defined. To explore early steps in this process, we examined reactivation of a transiently expressed developmental gene, Atoh1, in adult mouse utricles after neomycin-induced hair cell death in culture. Using an adenoviral reporter for Atoh1 enhancer, we found that Atoh1 transcription is activated in some hair cell progenitors (supporting cells) 3 d after neomycin treatment. By 18 d after neomycin, the number of cells with Atoh1 transcriptional activity increased significantly, but few cells acquired hair cell features (i.e., accumulated ATOH1 or myosin VIIa protein or developed stereocilia). Treatment with DAPT, an inhibitor of γ-secretase, reduced notch pathway activity, enhanced Atoh1 transcriptional activity, and dramatically increased the number of Atoh1-expressing cells with hair cell features, but only in the striolar/juxtastriolar region. Similar effects were seen with TAPI-1, an inhibitor of another enzyme required for notch activity (TACE). Division of supporting cells was rare in any control or DAPT-treated utricles. This study shows that mature mammals have a natural capacity to initiate vestibular hair cell regeneration and suggests that regional notch activity is a significant inhibitor of direct transdifferentiation of supporting cells into hair cells following damage.

A cochlear implant may be used to electrically stimulate spiral ganglion neurons (SGNs) in people with severe sensorineural hearing loss (SNHL). However, these neurons progressively degenerate after SNHL due to loss of neurotrophins normally supplied by sensory hair cells (HCs). Experimentally, exogenous neurotrophin administration prevents SGN degeneration but can also result in abnormal resprouting of their peripheral fibers. This study aimed to create a target-derived neurotrophin source to increase neuron survival and redirect fiber resprouting following SNHL. Adenoviral (Ad) vectors expressing green fluorescent protein (GFP) alone or in combination with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3) were injected into the cochlear scala tympani or scala media of guinea-pigs (GPs) deafened via aminoglycosides for 1 week. After 3 weeks, cochleae were examined for gene expression, neuron survival, and the projection of peripheral fibers in response to gene expression. Injection of vectors into the scala media resulted in more localized gene expression than scala tympani injection with gene expression consistently observed within the partially degenerated organ of Corti. There was also greater neuron survival and evidence of localized fiber responses to neurotrophin-expressing cells within the organ of Corti from scala media injections (P < 0.05), a first step in promoting organized resprouting of auditory peripheral fibers via gene therapy.

Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear.

A cascade of transcription factors is believed to regulate the coordinate differentiation of primordial inner ear cells into the subtypes of hair cells and supporting cells. While candidate genes involved in this process have been identified, the temporal and spatial patterns of expression of many of these have not been carefully described during the extended period of inner ear development and functional maturation. We systematically examined the expression of two such transcription factors, LHX3 and SOX2, from the time of hair cell terminal mitoses into adulthood. We show that LHX3 is expressed specifically in auditory and vestibular hair cells soon after terminal mitoses and persists into the adult in vestibular hair cells. While SOX2 expression is widespread in the inner ear sensory epithelia prior to hair cell differentiation, it has a unique pattern of expression in the mature auditory and vestibular organs.

We carried out an analysis of the expression of Prox1, a homeo-domain transcription factor, during mouse inner ear development with particular emphasis on the auditory system. Prox1 is expressed in the otocyst beginning at embryonic day (E)11, in the developing vestibular sensory patches. Expression is down regulated in maturing (myosin VIIA immunoreactive) vestibular hair cells and subsequently in the underlying support cell layer by E16.5. In the auditory sensory epithelium, Prox1 is initially expressed at embryonic day 14.5 in a narrow stripe of cells at the base of the cochlea. This stripe encompasses the full thickness of the sensory epithelium, including developing hair cells and support cells. Over the next several days the stripe of expression extends to the apex, and as the sensory epithelium differentiates Prox1 becomes restricted to a subset of support cells. Double labeling for Prox1 and cell-type-specific markers revealed that the outer hair cells transiently express Prox1. After E18, Prox1 protein is no longer detectable in hair cells, but it continues to be expressed in support cells for the rest of embryogenesis and into the second postnatal week. During this time, Prox1 is not expressed in all support cell types in the organ of Corti, but is restricted to developing Deiters' and pillar cells. The expression is maintained in these cells into the second week of postnatal life, at which time Prox1 is dynamically down regulated. These studies form a baseline from which we can analyze the role of Prox1 in vertebrate sensory development., (J. Comp. Neurol. 496:172-186, 2006. (c) 2006 Wiley-Liss, Inc.)

In humans, hair cell loss often leads to hearing and balance impairments. Hair cell replacement is vigorous and spontaneous in avians and nonmammalian vertebrates. In mammals, in contrast, it occurs at a very low rate, or not at all, presumably because of a very low level of supporting cell proliferation following injury. Heregulin (HRG), a member of the epidermal growth factor (EGF) family of growth factors, is reported to be a potent mitogen for neonatal rat vestibular sensory epithelium, but its effects in adults are unknown. We report here that HRG-alpha stimulates cell proliferation in organotypic cultures of neonatal, but not adult, mouse utricular sensory epithelia. Our findings support the idea that the proliferative capabilities of the adult mammalian vestibular sensory epithelia differ significantly from that seen in neonatal animals. Immunohistochemistry reveals that HRG-binding receptors (erbBs 2-4) and erbB1 are widely expressed in vestibular and auditory sensory epithelia in neonatal and adult mouse inner ear. The distribution of erbBs in the neonatal and adult mouse ear is consistent with the EGF receptor/ligand family regulating diverse cellular processes in the inner ear, including cell proliferation and differentiation.

Growth factors and other extracellular signals regulate cell division in many tissues. Consequently, growth factors may have therapeutic uses to stimulate the production of replacement sensory hair cells in damaged human inner ears, thereby assisting in alleviating hearing loss and vestibular dysfunction. Assessment of the ability of growth factors to stimulate cell proliferation in inner ear sensory epithelia is at an early stage. This paper provides a brief account of what we know regarding growth factor regulation of cell proliferation in developing and mature inner ear sensory epithelia.

In the chick embryo, the primitive streak is the first axial structure to develop. The initiation of primitive streak formation in the posterior area pellucida is influenced by the adjacent posterior marginal zone (PMZ). We show here that chick Vg1 (cVg1), a member of the TGFbeta family of signalling molecules whose homolog in Xenopus is implicated in mesoderm induction, is expressed in the PMZ of prestreak embryos. Ectopic expression of cVg1 protein in the marginal zone chick blastoderms directs the formation of a secondary primitive streak, which subsequently develops into an ectopic embryo. We have used cell marking techniques to show that cells that contribute to the ectopic primitive streak change fate, acquiring two distinct properties of primitive streak cells, defined by gene expression and cell movements. Furthermore, naive epiblast explants exposed to cVg1 protein in vitro acquire axial mesodermal properties. Together, these results show that cVg1 can mediate ectopic axis formation in the chick by inducing new cell fates and they permit the analysis of distinct events that occur during primitive streak formation.

Transgenic embryos expressing Cwnt8C under the control of the human beta-actin promoter exhibit duplicated axes or a severely dorsalised phenotype. Although the transgene was introduced into fertilised eggs all duplications occurred within a single amnion and, therefore, arose from the production of more than one primitive streak at the time of gastrulation. Morphological examination and the expression of diagnostic markers in transgenic embryos suggested that ectopic Cwnt8C expression produced only incomplete axis duplication: axes were always fused anteriorly, there was a reduction in tissue rostral to the anterior limit of the notochord, and no duplicated expression domain of the forebrain marker Hesx1 was observed. Anterior truncations were evident in dorsalised transgenic embryos containing a single axis. These results are discussed in the light of the effects of ectopic Xwnt8 in Xenopus embryos, where its early expression leads to complete axis duplication but expression after the mid-blastula transition causes anterior truncation. It is proposed that while ectopic Cwnt8C in the mouse embryo can duplicate the primitive streak and node this only produces incomplete axis duplication because specification of the anterior aspect of the axis, as opposed to maintenance of anterior character, is established by interaction with anterior primitive endoderm rather than primitive streak derivatives.

Malignant tumors involving the structures of the temporal bone represent formidable diagnostic and therapeutic challenges for clinicians involved in the treatment of otologic disease. This article offers a perspective on the current understanding of the biology of malignancies involving the external auditory canal, middle ear space, and temporal bone, and reviews the often confusing and contradictory literature on this topic.

To begin to examine the possibility that Wnt proteins act as cell signalling molecules during chick embryogenesis, PCR was used to identify Wnt genes expressed in Hensen's node. We have identified a novel member of the Wnt gene family, Cwnt-8C, which is expressed prior to gastrulation in the posterior marginal zone, the primitive streak and Hensen's node. Injection of Cwnt-8C mRNA into Xenopus embryos caused axis duplication and dorsalization of mesodermal tissues. During neurulation, Cwnt-8C is expressed transiently in a restricted domain of the prospective hindbrain neurectoderm that will give rise to rhombomere 4. This domain is defined prior to the formation of rhombomere boundaries and also precedes the up-regulation and restriction of expression of Hox B1 in the same region. Thus, Cwnt-8C is potentially involved in the regulation of axis formation and hindbrain patterning.

In an effort to identify trans-acting factors regulating specific genes, we cloned a novel human gene, DBP-5. The cDNA clone contains a predicted open reading frame coding for a potential 1,179 amino acid protein. The mRNA corresponding to DBP-5 is ubiquitously distributed, and the gene is phylogenetically conserved. Immunofluorescence analyses with several cell lines indicate that the protein is localized to the nucleus. Sequence analysis revealed unusual features of the predicted protein structure, including four completely conserved repeats. The phylogenetic conservation of DBP-5, the ubiquity of its expression, its nuclear localization, and its ability to bind DNA sequences, raise the possibility that DBP-5 may play a role in the organization of interphase chromatin and/or in transcriptional regulation.

Two consensus sequences, called X and Y boxes, capable of binding nuclear proteins and regulating expression in B cells have been defined within the immediate upstream region of major histocompatibility complex (MHC) class II promoters. Unlike other class II promoters, the HLA-DR alpha (DRA) promoter also contains one element identical to the "octamer" motif of immunoglobulin variable region promoters that is responsible for B cell-specific transcription. This "octamer" in the context of DRA appears capable of binding both the ubiquitous (OTF-1) and lymphoid-specific (OTF-2) "octamer" binding proteins, but at least one other distinct "octamer" complex was found. In order to characterize the function of cis-acting elements, we have developed an in vitro system in which a DRA promoter construct is transcribed more efficiently in extracts from B cells than in extracts from class II-negative HeLa cells. 5' deletion constructs which lacked the Y box, but retained the "octamer" motif and TATA box were completely inactive, and internal deletion of the Y box reduced transcription by 95%. Using supercoiled, but not linear templates, we observed differences in transcription efficiencies from templates lacking or disrupting the X consensus element that reflect effects of random replacement of X box sequences in transient expression assays. Demonstration of the complete dependence on the Y box in this system suggests that, despite its demonstrated importance in the DRA promoter, the DRA "octamer" does not utilize OTF-2 in a manner analogous to immunoglobulin promoters in B cells.

Mouse C3H10T1/2 cells were co-transfected with a plasmid containing the KiSV DNA and a plasmid carrying sequences coding for resistance to mycophenolic acid. Only 30% of the transfected colonies had a transformed phenotype, i.e. highly refractile rounded cells. The remaining colonies had varied morphologies with flat or slightly elongated cells. Analysis of p21 ras protein indicated that higher levels of the protein were expressed in cells with the more transformed phenotype. Tumors formed by a poorly tumorigenic clone were found to have undergone in vivo amplification of the transfected KiSV sequence. Transformed variants of this clone were also isolated in vitro. Treatment with 5-azacytidine resulted in an increase of about 10 fold in the formation of transformed variants. All transformed cells isolated, either spontaneous or 5-azacytidine induced, were tumorigenic in nude mice. The neoplastic conversion of these cells was accompanied by amplification of the transfected K-ras sequences. The data reported here indicate that a chemical agent that modifies DNA structure can enhance the frequency of amplification events in cellular DNA and, by affecting ras copy number, promote cell transformation.

Coordinate regulation of HLA class II gene expression during development and coinduction of class II genes by soluble factors suggests that common trans-acting factor(s) control expression of these genes. In B-lymphoblastoid cell lines derived from two independent class II-deficient bare lymphocyte syndrome patients, we observed a drastic decrease in transcription rates of the class II genes. When these cell lines are fused, class II genes are reexpressed, indicating that immunodeficiencies in bare lymphocyte syndrome patients are the result of two distinct mutations. Further studies show that genes governing the expression of class II antigens fall into at least three complementation groups; two of these were previously unidentified in mutant cell lines generated in vitro. In addition, we report the identification of two discrete complexes, NFX1.1 and NFX1.2, that bind to the DRA X consensus element. Though the mutation in at least one mutant line generated in vitro (RJ2.2.5) affects products functioning via interaction with the X box, clear alterations in either NFX1.1 or NFX1.2 are not found in any of the mutant cell lines.

Types II and III bare lymphocyte syndrome (BLS) are severe or lethal congenital immunodeficiencies characterized by defective cell surface expression of HLA class II antigens. We have analyzed by Southern and Northern blotting B-lymphoblastoid cell lines derived by Epstein-Barr virus (EBV) transformation from peripheral blood lymphocytes of two unrelated BLS patients and their families. While DNA analyses of both families showed no indication of rearrangement or alteration of HLA region genes, class II mRNAs were virtually absent in the patients' cell lines (BLS-1 and BLS-2). This is consistent with previous observations of different BLS patients and their families. An exception to the absence of class II mRNAs in BLS was the detection of low quantities of HLA-DQ alpha transcripts in the cell lines BLS-1. This finding provides further evidence that factors regulating HLA-DQ expression may differ from those governing expression of the other class II genes.

An interleukin-3 (IL-3) dependent mast cell line (MC) was infected with a recombinant retrovirus expressing the proto-oncogene c-myc and the drug selectable marker neo. Cells containing the transcriptionally activated c-myc gene displayed an increased growth rate in liquid culture and a higher cloning efficiency in soft agar when compared to control virus infected cells. All infected cells remained absolutely dependent on IL-3 for growth and were not tumorigenic in nude mice. Similar results were obtained with two additional IL-3 dependent cell lines, the mast cell 32D and the pre-B-cell Ea3. Thus, while constitutive expression of c-myc potentiates the response of mast cells to IL-3, it is not sufficient to eliminate their requirement for growth factors.

The human B-cell line RJ2.2.5, derived by mutagenesis from a Burkitt lymphoma cell line and selected for loss of HLA class II antigen expression, was infected with recombinant retroviruses containing either the Harvey murine sarcoma virus oncogene v-Ha-ras or the human neuroblastoma homolog NRAS. Both activated ras genes partially complemented the regulatory defect in RJ2.2.5 and specifically increased the expression of the DR and DQ subsets of HLA class II genes. Blot-hybridization analysis and RNase mapping indicated that HLA-DQ alpha-chain mRNA in the infected cell lines was increased to a level at least 50% that of the parent B-cell line, Raji. The levels of HLA-DR and -DQ beta-chain RNA also were increased but to a lesser extent. In contrast, we detected no effect of ras on the quantities of other class II, class I, or invariant-chain mRNAs. Fluorescence-activated cell sorter analysis with antibodies recognizing HLA-DR, -DQ, and class I antigens supported these observations. Enhancement of HLA class II gene expression by ras genes may have important implications for regulation of the immune system in response to transformation.