The Experts below are selected from a list of 3555 Experts worldwide ranked by ideXlab platform
Lisa L Cunningham - One of the best experts on this subject based on the ideXlab platform.
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lgr5 cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse Utricle
Nature Communications, 2015Co-Authors: Tian Wang, Lindsey A May, Renjie Chai, Grace S Kim, Nicole Pham, Lina Jansson, Duchuy Nguyen, Bryan R Kuo, Jian Zuo, Lisa L CunninghamAbstract:Recruitment of endogenous progenitors is critical during tissue repair. The inner ear Utricle requires mechanosensory hair cells (HCs) to detect linear acceleration. After damage, non-mammalian Utricles regenerate HCs via both proliferation and direct transdifferentiation. In adult mammals, limited transdifferentiation from unidentified progenitors occurs to regenerate extrastriolar Type II HCs. Here we show that HC damage in neonatal mouse Utricle activates the Wnt target gene Lgr5 in striolar supporting cells. Lineage tracing and time-lapse microscopy reveal that Lgr5+ cells transdifferentiate into HC-like cells in vitro. In contrast to adults, HC ablation in neonatal Utricles in vivo recruits Lgr5+ cells to regenerate striolar HCs through mitotic and transdifferentiation pathways. Both Type I and II HCs are regenerated, and regenerated HCs display stereocilia and synapses. Lastly, stabilized s-catenin in Lgr5+ cells enhances mitotic activity and HC regeneration. Thus Lgr5 marks Wnt-regulated, damage-activated HC progenitors and may help uncover factors driving mammalian HC regeneration.
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dissection of adult mouse Utricle and adenovirus mediated supporting cell infection
Journal of Visualized Experiments, 2012Co-Authors: Carlene S Brandon, Christina Voelkeljohnson, Lindsey A May, Lisa L CunninghamAbstract: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.
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Hsp70 Inhibits Aminoglycoside-Induced Hair Cell Death and is Necessary for the Protective Effect of Heat Shock
Journal of the Association for Research in Otolaryngology, 2008Co-Authors: Mona Taleb, Carlene S Brandon, Margaret I. Lomax, Wolfgang H. Dillmann, Lisa L CunninghamAbstract:Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including c-Jun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) is a highly conserved stress response that can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in a robust upregulation of Hsps in the hair cells of the adult mouse Utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin- and aminoglycoside-induced hair cell death. In our system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Therefore, we have begun to examine the role of Hsp70 in mediating the protective effect of heat shock. To determine whether Hsp70 is necessary for the protective effect of heat shock against aminoglycoside-induced hair cell death, we utilized Utricles from Hsp70.1/3 ^−/− mice. While heat shock inhibited gentamicin-induced hair cell death in wild-type Utricles, Utricles from Hsp70.1/3 ^−/− mice were not protected. In addition, we have examined the role of the major heat shock transcription factor, Hsf1, in mediating the protective effect of heat shock. Utricles from Hsf1 ^−/− mice and wild-type littermates were exposed to heat shock followed by gentamicin. The protective effect of heat shock on aminoglycoside-induced hair cell death was only observed in wild-type mice and not in Hsf1 ^−/− mice. To determine whether Hsp70 is sufficient to protect hair cells, we have utilized transgenic mice that constitutively overexpress Hsp70. Utricles from Hsp70-overexpressing mice and wild-type littermates were cultured in the presence of varying neomycin concentrations for 24 h. The Hsp70-overexpressing Utricles were significantly protected against neomycin-induced hair cell death at moderate to high doses of neomycin. This protective effect was achieved without a heat shock. Taken together, these data indicate that Hsp70 and Hsf1 are each necessary for the protective effect of heat shock against aminoglycoside-induced death. Furthermore, overexpression of Hsp70 alone significantly inhibits aminoglycoside-induced hair cell death.
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Hsp70 Inhibits Aminoglycoside-Induced Hair Cell Death and is Necessary for the Protective Effect of Heat Shock
Journal of the Association for Research in Otolaryngology, 2008Co-Authors: Mona Taleb, Carlene S Brandon, Margaret I. Lomax, Wolfgang H. Dillmann, Fu-shing Lee, Lisa L CunninghamAbstract:Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including c-Jun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) is a highly conserved stress response that can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in a robust upregulation of Hsps in the hair cells of the adult mouse Utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin- and aminoglycoside-induced hair cell death. In our system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Therefore, we have begun to examine the role of Hsp70 in mediating the protective effect of heat shock. To determine whether Hsp70 is necessary for the protective effect of heat shock against aminoglycoside-induced hair cell death, we utilized Utricles from Hsp70.1/3 ^−/− mice. While heat shock inhibited gentamicin-induced hair cell death in wild-type Utricles, Utricles from Hsp70.1/3 ^−/− mice were not protected. In addition, we have examined the role of the major heat shock transcription factor, Hsf1, in mediating the protective effect of heat shock. Utricles from Hsf1 ^−/− mice and wild-type littermates were exposed to heat shock followed by gentamicin. The protective effect of heat shock on aminoglycoside-induced hair cell death was only observed in wild-type mice and not in Hsf1 ^−/− mice. To determine whether Hsp70 is sufficient to protect hair cells, we have utilized transgenic mice that constitutively overexpress Hsp70. Utricles from Hsp70-overexpressing mice and wild-type littermates were cultured in the presence of varying neomycin concentrations for 24 h. The Hsp70-overexpressing Utricles were significantly protected against neomycin-induced hair cell death at moderate to high doses of neomycin. This protective effect was achieved without a heat shock. Taken together, these data indicate that Hsp70 and Hsf1 are each necessary for the protective effect of heat shock against aminoglycoside-induced death. Furthermore, overexpression of Hsp70 alone significantly inhibits aminoglycoside-induced hair cell death.
Mark E. Warchol - One of the best experts on this subject based on the ideXlab platform.
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dynamic patterns of yap1 expression and cellular localization in the developing and injured Utricle
bioRxiv, 2020Co-Authors: Vikrant Borse, Matthew Barton, Harry Arndt, Tejbeer Kaur, Mark E. WarcholAbstract:The Hippo pathway is an evolutionarily conserved signaling pathway involved in regulating organ size, development, homeostasis and regeneration1-4. YAP1 is a transcriptional coactivator and the primary effector of Hippo signaling. Upstream activation of the Hippo pathway leads to nuclear translocation of YAP1, which then evokes changes in gene expression and cell cycle entry5. A prior study has demonstrated nuclear translocation of YAP1 in the supporting cells of the developing Utricle6, but the possible role of YAP1 in hair cell regeneration is unclear. The present study characterizes the cellular localization of YAP1 in the Utricles of mice and chicks, both under normal conditions and after hair cell injury. During neonatal development of the mouse Utricle, YAP1 expression was observed in the cytoplasm of supporting cells, and was also transiently expressed in the cytoplasm of hair cells. We also observed temporary nuclear translocation of YAP1 in supporting cells of the mouse Utricle at short time periods after placement in organotypic culture. However, little or no nuclear translocation of YAP1 was observed after injury to the Utricles of neonatal or mature mice. In contrast, a substantial degree of YAP1 nuclear translocation was observed in the chicken Utricle after streptomycin-induced hair cell damage in vitro and in vivo. Together, these data suggest that differences in YAP1 signaling may be partly responsible for the distinct regenerative abilities of the avian vs. mammalian inner ear.
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development of hair cell phenotype and calyx nerve terminals in the neonatal mouse Utricle
The Journal of Comparative Neurology, 2019Co-Authors: Mark E. Warchol, Roxanna Massoodnia, Remy Pujol, Brandon C Cox, Jennifer S StoneAbstract:The vestibular organs of reptiles, birds, and mammals possess Type I and Type II sensory hair cells, which have distinct morphologies, physiology, and innervation. Little is known about how vestibular hair cells adopt a Type I or Type II identity or acquire proper innervation. One distinguishing marker is the transcription factor Sox2, which is expressed in all developing hair cells but persists only in Type II hair cells in maturity. We examined Sox2 expression and formation of afferent nerve terminals in mouse Utricles between postnatal days 0 (P0) and P17. Between P3 and P14, many hair cells lost Sox2 immunoreactivity and the density of calyceal afferent nerve terminals (specific to Type I hair cells) increased in all regions of the Utricle. At early time points, many calyces enclosed Sox2-labeled hair cells, while some Sox2-negative hair cells within the striola had not yet developed a calyx. These observations indicate that calyx maturation is not temporally correlated with loss of Sox2 expression in Type I hair cells. To determine which type(s) of hair cells are formed postnatally, we fate-mapped neonatal supporting cells by injecting Plp-CreER T2 :Rosa26 tdTomato mice with tamoxifen at P2 and P3. At P9, tdTomato-positive hair cells were immature and not classifiable by type. At P30, tdTomato-positive hair cells increased 1.8-fold compared to P9, and 91% of tdTomato-labeled hair cells were Type II. Our findings show that most neonatally-derived hair cells become Type II, and many Type I hair cells (formed before P2) downregulate Sox2 and acquire calyces between P0 and P14.
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macrophage recruitment and epithelial repair following hair cell injury in the mouse Utricle
Frontiers in Cellular Neuroscience, 2015Co-Authors: Tejbeer Kaur, Edwin W Rubel, Keiko Hirose, Mark E. WarcholAbstract:The sensory organs of the inner ear possess resident populations of macrophages, but the function of those cells is poorly understood. In many tissues, macrophages participate in the removal of cellular debris after injury and can also promote tissue repair. The present study examined injury-evoked macrophage activity in the mouse Utricle. Experiments used transgenic mice in which the gene for the human diphtheria toxin receptor (huDTR) was inserted under regulation of the Pou4f3 promoter. Hair cells in such mice can be selectively lesioned by systemic treatment with diphtheria toxin (DT). In order to visualize macrophages, Pou4f3-huDTR mice were crossed with a second transgenic line, in which one or both copies of the gene for the fractalkine receptor CX3CR1 were replaced with a gene for GFP. Such mice expressed GFP in all macrophages, and mice that were CX3CR1(GFP/GFP) lacked the necessary receptor for fractalkine signaling. Treatment with DT resulted in the death of ∼70% of utricular hair cells within 7 days, which was accompanied by increased numbers of macrophages within the utricular sensory epithelium. Many of these macrophages appeared to be actively engulfing hair cell debris, indicating that macrophages participate in the process of 'corpse removal' in the mammalian vestibular organs. However, we observed no apparent differences in injury-evoked macrophage numbers in the Utricles of CX3CR1(+/GFP) mice vs. CX3CR1(GFP/GFP) mice, suggesting that fractalkine signaling is not necessary for macrophage recruitment in these sensory organs. Finally, we found that repair of sensory epithelia at short times after DT-induced hair cell lesions was mediated by relatively thin cables of F-actin. After 56 days recovery, however, all cell-cell junctions were characterized by very thick actin cables.
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Epigenetic Influences on Sensory Regeneration: Histone Deacetylases Regulate Supporting Cell Proliferation in the Avian Utricle
Journal of the Association for Research in Otolaryngology, 2009Co-Authors: Eric L Slattery, Judith D. Speck, Mark E. WarcholAbstract:The sensory hair cells of the cochlea and vestibular organs are essential for normal hearing and balance function. The mammalian ear possesses a very limited ability to regenerate hair cells and their loss can lead to permanent sensory impairment. In contrast, hair cells in the avian ear are quickly regenerated after acoustic trauma or ototoxic injury. The very different regenerative abilities of the avian vs. mammalian ear can be attributed to differences in injury-evoked expression of genes that either promote or inhibit the production of new hair cells. Gene expression is regulated both by the binding of cis -regulatory molecules to promoter regions as well as through structural modifications of chromatin (e.g., methylation and acetylation). This study examined effects of histone deacetylases (HDACs), whose main function is to modify histone acetylation, on the regulation of regenerative proliferation in the chick Utricle. Cultures of regenerating Utricles and dissociated cells from the utricular sensory epithelia were treated with the HDAC inhibitors valproic acid, trichostatin A, sodium butyrate, and MS-275. All of these molecules prevent the enzymatic removal of acetyl groups from histones, thus maintaining nuclear chromatin in a “relaxed” (open) configuration. Treatment with all inhibitors resulted in comparable decreases in supporting cell proliferation. We also observed that treatment with the HDAC1-, 2-, and 3-specific inhibitor MS-275 was sufficient to reduce proliferation and that two class I HDACs—HDAC1 and HDAC2—were expressed in the sensory epithelium of the Utricle. These results suggest that inhibition of specific type I HDACs is sufficient to prevent cell cycle entry in supporting cells. Notably, treatment with HDAC inhibitors did not affect the differentiation of replacement hair cells. We conclude that histone deacetylation is a positive regulator of regenerative proliferation but is not critical for avian hair cell differentiation.
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Lectin from Griffonia simplicifolia identifies an immature-appearing subpopulation of sensory hair cells in the avian Utricle
Journal of Neurocytology, 2001Co-Authors: Mark E. WarcholAbstract:The sensory hair cells of the inner ear are coated with a variety of glycoproteins and glycolipids which can be identified by the binding of specific lectins. The present study examined the binding patterns of three lectins–Wheat Germ Agglutinin, Peanut Agglutinin, and lectin from Griffonia simplicifolia (Isoform B_4)–in the avian Utricle. Each of the lectins exhibited a distinct pattern of hair cell labeling. Wheat Germ Agglutinin (WGA) appeared to label the ciliary bundles of all sensory hair cells. In contrast, the binding of Peanut Agglutinin (PNA) was mainly confined to the ciliary bundles of extrastriolar hair cells. Finally, lectin from Griffonia simplicifolia (GS-IB_4) labeled a subpopulation of hair cells in all regions of the chick Utricle. Those bundles were much smaller than the majority of ciliary bundles labeled by either WGA or PNA, and the density of GS-IB_4-labeled bundles in the normal mature Utricle was relatively low. Increased densities of GS-IB_4-labeled hair cells were observed in the embryonic Utricle and during the process of hair cell regeneration. The observations suggest that GS-IB_4 labels a glycoprotein that is expressed preferentially on the ciliary bundles of immature hair cells.
Christian Chabbert - One of the best experts on this subject based on the ideXlab platform.
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Development/Plasticity/Repair Control of Hair Cell Excitability by Vestibular Primary Sensory Neurons
2016Co-Authors: Christian ChabbertAbstract:In the rat Utricle, synaptic contacts between hair cells and the nerve fibers arising from the vestibular primary neurons form during the first week after birth. During that period, the sodium-based excitability that characterizes neonate Utricle sensory cells is switched off. To investigate whether the establishment of synaptic contacts was responsible for the modulation of the hair cell excitability, we used an organotypic culture of rat Utricle in which the setting of synapses was prevented. Under this condition, the voltage-gated sodium current and the underlying action potentials persisted in a large proportion of nonafferented hair cells. We then studied whether impairment of nerve terminals in the Utricle of adult rats may also affect hair cell excitability. We induced selective and transient damages of afferent terminalsusingglutamate excitotoxicity in vivo. The efficiencyof the excitotoxic injurywasattestedby selective swellingsof the terminals and underlying altered vestibular behavior. Under this condition, the sodium-based excitability transiently recovered in hair cells. These results indicate that the modulation of hair cell excitability depends on the state of the afferent terminals. In adult Utricle hair cells, this property may be essential to set the conditions required for restoration of the sensory network after damage. This is achieved via re-expression of a biological process that occurs during synaptogenesis. Key words: voltage-gated sodium channel; hair cells; Utricle; excitability; development; excitotoxicity; nerve impairment; repai
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Control of hair cell excitability by vestibular primary sensory neurons.
Journal of Neuroscience, 2007Co-Authors: Aurore Brugeaud, Cécile Travo, Danielle Demêmes, Marc Lenoir, Jordi Llorens, Jean-luc Puel, Christian ChabbertAbstract:In the rat Utricle, synaptic contacts between hair cells and the nerve fibers arising from the vestibular primary neurons form during the first week after birth. During that period, the sodium-based excitability that characterizes neonate Utricle sensory cells is switched off. To investigate whether the establishment of synaptic contacts was responsible for the modulation of the hair cell excitability, we used an organotypic culture of rat Utricle in which the setting of synapses was prevented. Under this condition, the voltage-gated sodium current and the underlying action potentials persisted in a large proportion of nonafferented hair cells. We then studied whether impairment of nerve terminals in the Utricle of adult rats may also affect hair cell excitability. We induced selective and transient damages of afferent terminals using glutamate excitotoxicity in vivo. The efficiency of the excitotoxic injury was attested by selective swellings of the terminals and underlying altered vestibular behavior. Under this condition, the sodium-based excitability transiently recovered in hair cells. These results indicate that the modulation of hair cell excitability depends on the state of the afferent terminals. In adult Utricle hair cells, this property may be essential to set the conditions required for restoration of the sensory network after damage. This is achieved via re-expression of a biological process that occurs during synaptogenesis.
Yehoash Raphael - One of the best experts on this subject based on the ideXlab platform.
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sirna targeting hes5 augments hair cell regeneration in aminoglycoside damaged mouse Utricle
Molecular Therapy, 2013Co-Authors: Jae Yun Jung, Matthew R Avenarius, Swetlana Adamsky, Evgenia Alpert, Elena Feinstein, Yehoash RaphaelAbstract:Notch signaling is active during the development of mosaic epithelial sheets and during their turnover and regeneration. After the loss of hair cells in the mosaic sheet of the vestibular sensory epithelium, new hair cells can be spontaneously generated by transdifferentiation of supporting cells. This regenerative process involves downregulation of the Hes5 gene and is known to be limited and incomplete, especially when the lesion is severe. Here, we test whether further downregulation of Hes5 gene accomplished by the use of siRNA after a severe lesion induced by an aminoglycoside in the mouse Utricle can enhance the transdifferentiation of supporting cells and lead to the increased production of new hair cells. We demonstrate that Hes5 levels in the Utricle decreased after the application of siRNA and that the number of hair cells in these Utricles was significantly larger than following control treatment. The data suggest that siRNA technology may be useful for inducing repair and regeneration in the inner ear and that the Notch signaling pathway is a potentially useful target for specific gene expression inhibition.
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spontaneous hair cell regeneration in the mouse Utricle following gentamicin ototoxicity
Hearing Research, 2009Co-Authors: Kohei Kawamoto, Masahiko Izumikawa, Lisa A Beyer, Graham Atkin, Yehoash RaphaelAbstract:Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine Utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in Utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the Utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the Utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.
James F Martin - One of the best experts on this subject based on the ideXlab platform.
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transcriptomic and epigenetic regulation of hair cell regeneration in the mouse Utricle and its potentiation by atoh1
eLife, 2019Co-Authors: Hsini Jen, Matthew C Hill, Litao Tao, Kuanwei Sheng, Wenjian Cao, Hongyuan Zhang, Juan Llamas, Chenghang Zong, James F MartinAbstract:The mammalian cochlea loses its ability to regenerate new hair cells prior to the onset of hearing. In contrast, the adult vestibular system can produce new hair cells in response to damage, or by reprogramming of supporting cells with the hair cell transcription factor Atoh1. We used RNA-seq and ATAC-seq to probe the transcriptional and epigenetic responses of Utricle supporting cells to damage and Atoh1 transduction. We show that the regenerative response of the Utricle correlates with a more accessible chromatin structure in Utricle supporting cells compared to their cochlear counterparts. We also provide evidence that Atoh1 transduction of supporting cells is able to promote increased transcriptional accessibility of some hair cell genes. Our study offers a possible explanation for regenerative differences between sensory organs of the inner ear, but shows that additional factors to Atoh1 may be required for optimal reprogramming of hair cell fate.
