Jonathan I. Matsui

Differential Expression of Unconventional Myosins in Apoptotic and Regenerating Chick Hair Cells Confirms Two Regeneration Mechanisms

Hair cells of the inner ear are damaged by intense noise, aging, and aminoglycoside antibiotics. Gentamicin causes oxidative damage to hair cells, inducing apoptosis. In mammals, hair cell loss results in a permanent deficit in hearing and balance. In contrast, avians can regenerate lost hair cells to restore auditory and vestibular function. This study examined the changes of myosin VI and myosin VIIa, two unconventional myosins that are critical for normal hair cell formation and function, during hair cell death and regeneration. During the late stages of apoptosis, damaged hair cells are ejected from the sensory epithelium. There was a 4–5‐fold increase in the labeling intensity of both myosins and a redistribution of myosin VI into the stereocilia bundle, concurrent with ejection. Two separate mechanisms were observed during hair cell regeneration. Proliferating supporting cells began DNA synthesis 60 hours after gentamicin treatment and peaked at 72 hours postgentamicin treatment. Some of these mitotically produced cells began to differentiate into hair cells at 108 hours after gentamicin (36 hours after bromodeoxyuridine (BrdU) administration), as demonstrated by the colabeling of myosin VI and BrdU. Myosin VIIa was not expressed in the new hair cells until 120 hours after gentamicin. Moreover, a population of supporting cells expressed myosin VI at 78 hours after gentamicin treatment and myosin VIIa at 90 hours. These cells did not label for BrdU and differentiated far too early to be of mitotic origin, suggesting they arose by direct transdifferentiation of supporting cells into hair cells. J. Comp. Neurol. 499:691–701, 2006.

Regeneration and replacement in the vertebrate inner ear

Deafness affects more than 40 million people in the UK and the USA, and many more world-wide. The primary cause of hearing loss is damage to or death of the sensory receptor cells in the inner ear, the hair cells. Birds can readily regenerate their cochlear hair cells but the mammalian cochlea has shown no ability to regenerate after damage. Current research efforts are focusing on gene manipulation, gene therapy and stem cell transplantation for repairing or replacing damaged mammalian cochlear hair cells, which could lead to therapies for treating deafness in humans.

Sensory hair cell death and regeneration: two halves of the same equation.

Purpose of reviewSensory hair cells are susceptible to ototoxic damage from a variety of sources, including antibiotic treatment. Unfortunately, this often results in permanent hearing and/or balance problems in humans. By understanding how sensory hair cells die in response to aminoglycoside treatment, preventive strategies may be developed. This review will discuss some of the key recent findings in sensory hair cell death and regeneration. Recent findingsAminoglycosides induce hair cell death through the initiation of apoptosis. Early and late stages of hair cell apoptosis have been defined, and several of the key molecules involved in the cascade have been identified. Moreover, specific inhibitors of apoptosis rescue hair cells from death and preserve function. Hair cell death has been shown to induce regeneration through supporting cell transdifferentiation, proliferation, and new hair cell differentiation in birds and lower vertebrates. Regeneration in the mammalian cochlea does not occur spontaneously, but genetic manipulation of cell cycle genes, induction of new hair cells through gene therapy, and introduction of stem cells into damaged cochleas suggest that repair and replacement of lost hair cells in the organ of Corti may be possible. Finally, continuing investigations of the mouse, zebrafish, and human genomes may one day enable manipulation of the cochlea so that functional regeneration is readily available as a therapeutic intervention. SummaryThe discovery that hair cells can regenerate in birds and other nonmammalian vertebrates has fueled a wide range of studies to find ways to restore hearing and balance in mammals. The demonstration that apoptosis and proliferation are coupled as controlling factors in regeneration and the advent of new approaches such as gene therapy, stem cell transplantation, and genomics may lead to methods for inducing hair cell regeneration and repair in the mammalian cochlear and vestibular systems.

Specificity of the horizontal cell‐photoreceptor connections in the zebrafish (Danio rerio) retina

Horizontal cells (HCs) are involved in establishing the center‐surround receptive field organization of photoreceptor and bipolar cells. In many species, HCs respond differentially to colors and may play a role in color vision. An earlier study from our laboratory suggested that four types of HCs exist in the zebrafish retina: three cone HCs (H1, H2 and H3) and one rod HC. In this study, we describe their photoreceptor connections. Cones are arranged in a mosaic in which rows of alternating blue (B)‐ and ultraviolet (UV)‐sensitive single cones alternate with rows of red (R)‐ and green (G)‐sensitive double cones; the G cones are adjacent to UV cones and B cones adjacent to R cones. Two small‐field (H1 and H2) and two large‐field (H3 and rod HC) cells were observed. The cone HC dendritic terminals connected to cones with single boutons, doublets, or rosettes, whereas the rod HCs connected to rods with single boutons. The single boutons/doublets/rosettes of cone HCs were arranged in double rows separated by single rows for H1 cells, in pairs and singles for H2 cells, and in a rectilinear pattern for H3 cells. These connectivity patterns suggest that H1 cells contact R, G, and B cones, H2 cells G, B, and UV cones, and H3 cells B and UV cones. These predictions were confirmed by applying the DiI method to SWS1‐GFP retinas whose UV cones express green fluorescent protein. Each rod HC was adjacent to the soma or axon of a DiI‐labeled cone HC and connected to 50–200 rods. J. Comp. Neurol. 516:442–453, 2009.

Morphological types and connectivity of horizontal cells found in the adult zebrafish (Danio rerio) retina

We describe here different types of horizontal cells in the zebrafish retina and how they connect to photoreceptors. To label horizontal cells, crystals of DiI were placed onto the tips of pulled glass pipettes and inserted into the inner nuclear layer of fixed whole‐mount retinas. The DiI‐labeled horizontal cells were imaged by confocal microscopy and analyzed according to dendritic arborization, cell depth, dendritic terminal morphology, and connectivity with photoreceptors. Three types of horizontal cells were unequivocally identified: two cone‐connecting (H1/2 and H3) and one rod‐related cell. H1/2 cells have dendritic terminals that are arranged in “rosette” clusters and that connect to cone photoreceptors without any apparent specificity. H3 cells are larger and have dendritic terminal doublets arranged in a rectilinear pattern. This pattern corresponds to the mosaic of the single cones in the zebrafish photoreceptor mosaic and indicates that H3 cells connect specifically to either the blue‐sensitive (long‐single) or ultraviolet‐sensitive (short‐single) cones. Thus, H3 cells are likely to be chromaticity‐type cells that process specific color information, whereas H1/2 cells are probably luminosity‐type cells that process luminance information. Rod horizontal cells were identified by their shape and dendritic pattern, and they connect with numerous rod photoreceptors via small spherical terminals. J. Comp. Neurol. 506:328–338, 2008.

Hair cell regeneration: an exciting phenomenon...but will restoring hearing and balance be possible?

Sensory hair cells of the inner ear are susceptible to damage from a variety of sources including aging, genetic defects, and environmental stresses such as loud noises or chemotherapeutic drugs. Unfortunately, the consequence of this damage in humans is often permanent hearing/balance problems. The discovery that hair cells can regenerate in birds and other nonmammalian vertebrates has fueled a wide range of studies that are designed to find ways of restoring hearing and balance after such damage. In this review, we will discuss some of the key recent findings in sensory hair cell regeneration and what they mean for audiologists and other hearing healthcare practitioners.

myosin 7aa(-/-) mutant zebrafish show mild photoreceptor degeneration and reduced electroretinographic responses.

Mutations in myosin VIIa (MYO7A) cause Usher Syndrome 1B (USH1B), a disease characterized by the combination of sensorineural hearing loss and visual impairment termed retinitis pigmentosa (RP). Although the shaker-1 mouse model of USH1B exists, only minor defects in the retina have been observed during its lifespan. Previous studies of the zebrafish mariner mutant, which also carries a mutation in myo7aa, revealed balance and hearing defects in the mutants but the retinal phenotype has not been described. We found elevated cell death in the outer nuclear layer (ONL) of myo7aa(-/-) mutants. While myo7aa(-/-) mutants retained visual behaviors in the optokinetic reflex (OKR) assay, electroretinogram (ERG) recordings revealed a significant decrease in both a- and b-wave amplitudes in mutant animals, but not a change in ERG threshold sensitivity. Immunohistochemistry showed mislocalization of rod and blue cone opsins and reduced expression of rod-specific markers in the myo7aa(-/-) ONL, providing further evidence that the photoreceptor degeneration observed represents the initial stages of the RP. Further, constant light exposure resulted in widespread photoreceptor degeneration and the appearance of large holes in the retinal pigment epithelium (RPE). No differences were observed in the retinomotor movements of the photoreceptors or in melanosome migration within the RPE, suggesting that myo7aa(-/-) does not function in these processes in teleosts. These results indicate that the zebrafish myo7aa(-/-) mutant is a useful animal model for the RP seen in humans with USH1B.

Dimethyl Sulfoxide (DMSO) Exacerbates Cisplatin-induced Sensory Hair Cell Death in Zebrafish (Danio rerio)

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.

Ethanol Affects the Development of Sensory Hair Cells in Larval Zebrafish (Danio rerio)

Children born to mothers with substantial alcohol consumption during pregnancy can present a number of morphological, cognitive, and sensory abnormalities, including hearing deficits, collectively known as fetal alcohol syndrome (FAS). The goal of this study was to determine if the zebrafish lateral line could be used to study sensory hair cell abnormalities caused by exposure to ethanol during embryogenesis. Some lateral line sensory hair cells are present at 2 days post-fertilization (dpf) and are functional by 5 dpf. Zebrafish embryos were raised in fish water supplemented with varying concentrations of ethanol (0.75%–1.75% by volume) from 2 dpf through 5 dpf. Ethanol treatment during development resulted in many physical abnormalities characteristic of FAS in humans. Also, the number of sensory hair cells decreased as the concentration of ethanol increased in a dose-dependent manner. The dye FM 1-43FX was used to detect the presence of functional mechanotransduction channels. The percentage of FM 1-43-labeled hair cells decreased as the concentration of ethanol increased. Methanol treatment did not affect the development of hair cells. The cell cycle markers proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) demonstrated that ethanol reduced the number of sensory hair cells, as a consequence of decreased cellular proliferation. There was also a significant increase in the rate of apoptosis, as determined by TUNEL-labeling, in neuromasts following ethanol treatment during larval development. Therefore, zebrafish are a useful animal model to study the effects of hair cell developmental disorders associated with FAS.