Edwin W. Rubel

Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio).

Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 µM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24–48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.

Ontogeny of Structure and Function in the Vertebrate Auditory System

Why study the vertebrate auditory system? Throughout the ages, when scholars have attempted to define objectively the qualities that distinguish man from other animals, the power of human communication systems inevitably comes to the fore. Normal development of auditory perception is essential for the establishment of both expressive and receptive aspects of language. Thus, increased understanding, leading to eventual prevention or treatment of the various conditions that cause failures in the normal processing of acoustic information, has important clinical relevance. Furthermore, the auditory system is one of the primary sense modalities, involving both a highly specialized peripheral receptor organ and a complex set of central pathways.

Development of the Auditory System

1 Overview: Personal Views on the Study of Auditory System Development.- 2 Behavioral Studies of Hearing Development.- 3 Early Embryology of the Vertebrate Ear.- 4 Development of Sensory and Neural Structures in the Mammalian Cochlea.- 5 The Development of Cochlear Function.- 6 The Development of Central Auditory Processing.- 7 Structural Development of the Mammalian Central Auditory Pathways.

Mechanisms of hair cell death and protection

Purpose of reviewSensory hair cells are mechanotransducers of the inner ear that are essential for hearing and balance. Hair cell death commonly occurs following acoustic trauma or exposure to ototoxins, such as the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Loss of these inner ear sensory cells can lead to permanent sensorineural hearing loss, balance disturbance, or both. Currently, the only effective clinical intervention is prevention from exposure to known ototoxic insults. To help improve therapeutic strategies, a better understanding of the molecular mechanisms underlying hair cell degeneration is required. Current knowledge of these cell death mechanisms and potential therapeutic targets are discussed in this review. Recent findingsStudies have shown that caspase-9 and caspase-3 are key mediators of hair cell death induced by noise, aminoglycosides, and cisplatin. The Bcl-2 family consists of a group of proapoptotic and antiapoptotic molecules that act upstream of and regulate caspase activation. Recent studies have shed light on the roles of molecules acting more upstream, including mitogen-activated protein kinases and p53. SummaryThe mechanisms of sensory hair cell degeneration in response to different ototoxic stimuli share a final common pathway: caspase activation. Inhibition of caspases prevents or delays hair cell death and may preserve hearing/balance function. Inhibition of mitogen-activated protein kinases protects against noise-induced and aminoglycoside-induced but not cisplatin-induced hair cell death, which suggests divergent upstream regulatory mechanisms.

CC2D2A Is Mutated in joubert Syndrome and Interacts with the Ciliopathy-Associated Basal Body Protein CEP290

Joubert syndrome and related disorders (JSRD) are primarily autosomal-recessive conditions characterized by hypotonia, ataxia, abnormal eye movements, and intellectual disability with a distinctive mid-hindbrain malformation. Variable features include retinal dystrophy, cystic kidney disease, and liver fibrosis. JSRD are included in the rapidly expanding group of disorders called ciliopathies, because all six gene products implicated in JSRD (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67, and ARL13B) function in the primary cilium/basal body organelle. By using homozygosity mapping in consanguineous families, we identify loss-of-function mutations in CC2D2A in JSRD patients with and without retinal, kidney, and liver disease. CC2D2A is expressed in all fetal and adult tissues tested. In ciliated cells, we observe localization of recombinant CC2D2A at the basal body and colocalization with CEP290, whose cognate gene is mutated in multiple hereditary ciliopathies. In addition, the proteins can physically interact in vitro, as shown by yeast two-hybrid and GST pull-down experiments. A nonsense mutation in the zebrafish CC2D2A ortholog (sentinel) results in pronephric cysts, a hallmark of ciliary dysfunction analogous to human cystic kidney disease. Knockdown of cep290 function in sentinel fish results in a synergistic pronephric cyst phenotype, revealing a genetic interaction between CC2D2A and CEP290 and implicating CC2D2A in cilium/basal body function. These observations extend the genetic spectrum of JSRD and provide a model system for studying extragenic modifiers in JSRD and other ciliopathies.

Notch Signaling Regulates the Extent of Hair Cell Regeneration in the Zebrafish Lateral Line

Mechanosensory hair cells within the zebrafish lateral line spontaneously regenerate after aminoglycoside-induced death. Exposure of 5-d-old larvae to 400 μm neomycin for 1 h results in death of almost all lateral line hair cells. Regeneration of new hair cells is observed by 24 h after neomycin treatment with nearly complete replacement by 72 h. Using bromodeoxyuridine incorporation, we show that the majority of new hair cells are generated from a transient increase in support cell proliferation that occurs between 12 and 21 h after neomycin damage. Additional observations reveal two distinct subsets of proliferating support cells within the neuromasts that differ in position, morphology, and temporal pattern of proliferation in response to neomycin exposure. We hypothesize that proliferative hair cell progenitors are located centrally within the neuromasts, whereas peripheral support cells may have a separate function. Expression of Notch signaling pathway members notch3, deltaA, and atoh1a transcripts are all upregulated within the first 24 h after neomycin treatment, during the time of maximum proliferation of support cells and hair cell progenitor formation. Treatment with a γ-secretase inhibitor results in excess regenerated hair cells by 48 h after neomycin-induced death but has no effect without previous damage. Excess hair cells result from increased support cell proliferation. These results suggest a model where Notch signaling limits the number of hair cells produced during regeneration by regulating support cell proliferation.

Reactive oxygen species in chick hair cells after gentamicin exposure in vitro

Reactive oxygen species have been invoked as a causative agent of cell death in many different developmental and pathological states. The presence of free radicals and their importance of hair cell death due to aminoglycosides is suggested by a number of studies that have demonstrated a protective effect of antioxidants. By using dichlorofluorescin (DCFH) a fluorescent compound that is a reporter of reactive oxygen species, we have shown that free radicals are rapidly produced by avian hair cells in vitro after exposure to gentamicin. In addition, free radical scavengers, catalase and glutathione, were tested with DCFH fluorescent imaging for their ability to quench the production of reactive oxygen species in hair cells after drug exposure. Both free radical scavengers were very effective in suppressing drug-induced production of free radicals. Next, we investigated the ability of these antioxidants to preserve the structural integrity of hair cells after exposure to gentamicin. We were not able to detect any attenuation of the hair cell loss using antioxidants in conjunction with gentamicin. This result must be qualified by the fact that the antioxidants used were not effective over long-term gentamicin exposure. Therefore, methodological constraints prevented adequately testing possible protective effects of the free radical scavengers in this model system.

Possible precursors of regenerated hair cells in the avian cochlea following acoustic trauma

Hair cell regeneration following acoustic trauma to the avian cochlea has been documented using DNA labeling with tritiated thymidine. The goal of this study was to identify potential precursor cell populations for regenerating hair cells. Chicks were exposed in pairs to a 1500 Hz pure tone at 120 dBSPL for 18 h. The animals received repeated injections of 3H-thymidine over a survival period of 6, 15, or 24 h, 3 days or 30 days after the completion of noise exposure. One cochlea from each animal was processed for autoradiography and the other for scanning electron microscopy. Labeled, regenerated hair cells were present by 3 days after exposure and recovery from injury was nearly complete by 30 days. Examination of animals in short survival groups suggest that two precursor populations may exist. For inferior sensory epithelial damage, cuboidal or hyaline epithelial cells appear to serve as the precursor cell population for the regeneration of both hair cells and supporting cells. With isolated superior damage, however, supporting cells may be the precursor population.

Ongoing production of sensory cells in the vestibular epithelium of the chick.

Recent studies have shown that the vestibular and auditory systems of some species of birds have the capacity to generate sensory hair cells postnatally. We used a traditional technique, 3H-thymidine autoradiography, and a newer method, bromodeoxyuridine immunocytochemistry, to determine whether ongoing proliferation of hair cells occurs in the intact chick vestibular epithelium. A ten-day course of 3H-thymidine, bromodeoxyuridine, or both was administered to twelve-day-old chicks. Both autoradiographic and immunocytochemical labeling demonstrated ongoing production of supporting cells and Type II hair cells in all chick vestibular organs. No evidence for production of Type I hair cells was seen in this investigation. New sensory cells were distributed throughout the epithelium; there was no peripheral growth zone analogous to that found in other vertebrates. Labeled Type II hair cells were frequently seen immediately above labeled supporting cells. This observation suggests that supporting cells are precursors for new hair cells. The ongoing, postnatal regeneration of vestibular epithelial cells also suggests that this epithelium may retain the potential for repair after trauma or ototoxic damage.

Patterns of cell death in mouse anteroventral cochlear nucleus neurons after unilateral cochlea removal.

Developmental changes that influence the results of removal of afferent input on the survival of neurons of the anteroventral cochlear nucleus (AVCN) of mice were examined with the hope of providing a suitable model for understanding the cellular and molecular basis for these developmental changes in susceptibility. We performed unilateral cochlear ablation on wild‐type mice at a variety of ages around the time of hearing onset to determine developmental changes in the sensitivity of AVCN neurons to afferent deprivation. In postnatal day 5 (P5) mice, cochlea removal resulted in 61% neuronal loss in the AVCN. By age P14, fewer than 1% of AVCN neurons were lost after this manipulation. This reveals a rather abrupt change in the sensitivity to disruption of afferent input, a critical period. We next investigated the temporal events associated with neuron loss after cochlea removal in susceptible animals. We demonstrate that significant cell loss occurs within 48 hours of cochlea removal in P7 animals. Furthermore, evidence of apoptosis was observed within 12 hours of cochlea removal, suggesting that the molecular events leading to cell loss after afferent deprivation begin to occur within hours of cochlea removal. Finally, we began to examine the role of the bcl‐2 gene family in regulating afferent deprivation‐induced cell death in the mouse AVCN. AVCN neurons in mature bcl‐2 knockout mice demonstrate susceptibility to removal of afferent input comparable to neonatal sensitivity of wild‐type controls. These data suggest that bcl‐2 is one effector of cell survival as these cells switch from afferent‐dependent to ‐independent survival mechanisms. J. Comp. Neurol. 426:561–571, 2000.