Canonical Wnt/β-catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β-catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β-catenin signaling, we show that this pathway is active throughout the BP (E6-E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β-catenin activity in developing HCs, we performed gain- and loss-of-function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up-regulated cell proliferation within the Sox2-positive cells of the prosensory domains. Furthermore, Wnt/β-catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside-induced HC loss. Combined, our data suggest that Wnt/β-catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β-catenin signaling could play an important role in therapeutic HC regeneration.

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.

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.

Visual Abstract

Zebrafish are increasingly used in auditory studies, in part due to the development of several transgenic lines that express hair cell-specific fluorescent proteins. However, it is largely unknown how transgene expression influences auditory phenotype. We previously observed reduced auditory sensitivity in adult Brn3c:mGFP transgenic zebrafish, which express membrane-bound green fluorescent protein (GFP) in sensory hair cells. Here, we examine the auditory sensitivity of zebrafish from multiple transgenic and background strains. We recorded auditory evoked potentials in adult animals and observed significantly higher auditory thresholds in three lines that express hair cell-specific GFP. There was no obvious correlation between hair cell density and auditory thresholds, suggesting that reduced sensitivity was not due to a reduction in hair cell density. FM1-43 uptake was reduced in Brn3c:mGFP fish but not in other lines, suggesting that a mechanotransduction defect may be responsible for the auditory phenotype in Brn3c animals, but that alternate mechanisms underlie the increased AEP thresholds in other lines. We found reduced prepulse inhibition (a measure of auditory-evoked behavior) in larval Brn3c animals, suggesting that auditory defects develop early in this line. We also found significant differences in auditory sensitivity between adults of different background strains, akin to strain differences observed in mouse models of auditory function. Our results suggest that researchers should exercise caution when selecting an appropriate zebrafish transgenic or background strain for auditory studies.