Hair cell regeneration

The mammalian inner ear largely lacks the capacity to regenerate hair cells, the sensory cells required for hearing and balance. Recent studies in both lower vertebrates and mammals have uncovered genes and pathways important in hair cell development and have suggested ways that the sensory epithelia could be manipulated to achieve hair cell regeneration. These approaches include the use of inner ear stem cells, transdifferentiation of nonsensory cells, and induction of a proliferative response in the cells that can become hair cells.     

Forced activation of Wnt signaling alters morphogenesis and sensory organ identity in the chicken inner ear

Components of the Wnt signaling pathway are expressed in the developing inner ear. To explore their role in ear patterning, we used retroviral gene transfer to force the expression of an activated form of beta-catenin that should constitutively activate targets of the canonical Wnt signaling pathway. At embryonic day 9 (E9) and beyond, morphological defects were apparent in the otic capsule and the membranous labyrinth, including ectopic and fused sensory patches. Most notably, the basilar papilla, an auditory organ, contained infected sensory patches with a vestibular phenotype. Vestibular identity was based on: (1) stereociliary bundle morphology; (2) spacing of hair cells and supporting cells; (3) the presence of otoliths; (4) immunolabeling indicative of vestibular supporting cells; and (5) expression of Msx1, a marker of certain vestibular sensory organs. Retrovirus-mediated misexpression of Wnt3a also gave rise to ectopic vestibular patches in the cochlear duct. In situ hybridization revealed that genes for three Frizzled receptors, c-Fz1, c-Fz7, and c-Fz10, are expressed in and adjacent to sensory primordia, while Wnt4 is expressed in adjacent, nonsensory regions of the cochlear duct. We hypothesize that Wnt/beta-catenin signaling specifies otic epithelium as macular and helps to define and maintain sensory/nonsensory boundaries in the cochlear duct.     

Cell Cycle,Differentiation and Regeneration: Where to Begin?

Hair cells, the sensory cells of inner ear, perform essential functions in hearing and balance. However, mammalian hair cells, like most of the CNS neurons, lack the capacity to regenerate. This is in sharp contrast to lower vertebrates in which hair cell regeneration occurs spontaneously through cell division of supporting cells, which leads to hearing restoration. It is believed that the lack of regeneration in mammals is, to a large degree, due to the block of cell cycle re-entry imposed by negative cell growth genes in the inner ear. Recent studies have identified retinoblastoma gene, a well-known tumor suppressor, as the key gene involved in cell cycle exit of inner ear sensory cells. In the inner ear of pRb conditional knockout mice, hair cells undergo continuous cell division, and at the same time differentiate and become functional. Cell division continues in early postnatal cochlea and adult vestibule. Remarkably, the vestibular hair cells without pRb survive, and function at both the cellular and system levels. The time course and effects of pRb inhibition shows that there is a separation between the roles of pRb in cell cycle exit, and subsequent maturation and apoptosis. Those studies reveal distinctly different roles of pRb in the cochlear and vestibular sensory epithelia. The review discusses additional areas to be studied for regeneration of mature hair cells, and highlights the importance of transient and reversible block of pRb function as one of the routes to be explored for regeneration.     

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.     

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) ^1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions ^{7, 8}. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium ^{9, 10}. Supporting cells are also important mediators of hair cell survival and death ¹¹. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.     

Cell proliferation during the early compartmentalization of the Xenopus laevis inner ear

The auditory and vestibular endorgans of the inner ear which are essential for the senses of hearing and balance form early during development when the otocyst undergoes a period of rapid growth and compartmentalization. Here we show the spatial and temporal patterns of proliferating cells in the Xenopus laevis inner ear as this organ develops from an otic vesicle at stage 31 until stage 47, an age at which compartmentalization and the initial appearance of sensory structures are evident. Sites of new cell production were identified in specimens at stages 31, 37, 42, 45 and 47 using immunohistochemical methods to detect bromodeoxyuridine (BrdU) incorporation three hours after exposure to this thymidine analogue. Cells undergoing terminal mitosis at stages 37, 42 and 45 were detected by exposing specimens at these stages to BrdU and permitting development to proceed until stage 47. Our results show that while newly replicating cells are uniformly distributed throughout the stage 31 otic vesicle, they are spatially restricted in stages 37 through 45, with few dividing cells visible in the central patches of the emerging sensory epithelia. In contrast, no clear proliferative pattern was discerned at stage 47. BrdU-positive cells that had undergone terminal mitosis at stage 37, 42 and 45 were detected in the central regions of nascent sensory epithelia at stage 47. These findings are consistent with a developmental mechanism in which cells undergoing terminal mitosis during early X. laevis stages contribute to sensory epithelia and in which cell mixing and migration are features of inner ear compartmentalization.     

Hair cell regeneration in the avian auditory epithelium

Regeneration of sensory hair cells in the mature avian inner ear was first described just over 20 years ago. Since then, it has been shown that many other non-mammalian species either continually produce new hair cells or regenerate them in response to trauma. However, mammals exhibit limited hair cell regeneration, particularly in the auditory epithelium. In birds and other non-mammals, regenerated hair cells arise from adjacent non-sensory (supporting) cells. Hair cell regeneration was initially described as a proliferative response whereby supporting cells re-enter the mitotic cycle, forming daughter cells that differentiate into either hair cells or supporting cells and thereby restore cytoarchitecture and function in the sensory epithelium. However, further analyses of the avian auditory epithelium (and amphibian vestibular epithelium) revealed a second regenerative mechanism, direct transdifferentiation, during which supporting cells change their gene expression and convert into hair cells without dividing. In the chicken auditory epithelium, these two distinct mechanisms show unique spatial and temporal patterns, suggesting they are differentially regulated. Current efforts are aimed at identifying signals that maintain supporting cells in a quiescent state or direct them to undergo direct transdifferentiation or cell division. Here, we review current knowledge about supporting cell properties and discuss candidate signaling molecules for regulating supporting cell behavior, in quiescence and after damage. While significant advances have been made in understanding regeneration in non-mammals over the last 20 years, we have yet to determine why the mammalian auditory epithelium lacks the ability to regenerate hair cells spontaneously and whether it is even capable of significant regeneration under additional circumstances. The continued study of mechanisms controlling regeneration in the avian auditory epithelium may lead to strategies for inducing significant and functional regeneration in mammals.     

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.