Neurogenin1 Defines Zebrafish Cranial Sensory Ganglia Precursors

AbstractCells delaminate from epithelial placodes to form sensory ganglia in the vertebrate head. We describe the formation of cranial neurogenic placodes in the zebrafish, Danio rerio, using bHLH transcription factors as molecular markers. A single neurogenin gene, neurogenin1 (ngn1), is required for the development of all zebrafish cranial ganglia, which contrasts with other described vertebrates. Expression of ngn1 delineates zebrafish ganglionic placodes, including trigeminal, lateral line, and epibranchial placodes. In addition, ngn1 is expressed in a subset of cells within the otic vesicle that will delaminate to form the octaval (statoacoustic) ganglion. The trigeminal placode is the first to differentiate, and forms just lateral and adjacent to the neural crest. Expression of ngn1 is transient and prefigures expression of a related bHLH transcription factor, neuroD. Interfering with ngn1 function using a specific antisense morpholino oligonucleotide blocks differentiation of all cranial ganglia but not associated glial cells. Lateral line sensory neuromasts develop independently of ngn1 function, suggesting that two derivatives of lateral line placodes, ganglia and migrating primordia, are under separate genetic control

An in vivo Biomarker to Characterize Ototoxic Compounds and Novel Protective Therapeutics

There are no approved therapeutics for the prevention of hearing loss and vestibular dysfunction from drugs like aminoglycoside antibiotics. While the mechanisms underlying aminoglycoside ototoxicity remain unresolved, there is considerable evidence that aminoglycosides enter inner ear mechanosensory hair cells through the mechanoelectrical transduction (MET) channel. Inhibition of MET-dependent uptake with small molecules or modified aminoglycosides is a promising otoprotective strategy. To better characterize mammalian ototoxicity and aid in the translation of emerging therapeutics, a biomarker is needed. In the present study we propose that neonatal mice systemically injected with the aminoglycosides G418 conjugated to Texas Red (G418-TR) can be used as a histologic biomarker to characterize in vivo aminoglycoside toxicity. We demonstrate that postnatal day 5 mice, like older mice with functional hearing, show uptake and retention of G418-TR in cochlear hair cells following systemic injection. When we compare G418-TR uptake in other tissues, we find that kidney proximal tubule cells show similar retention. Using ORC-13661, an investigational hearing protection drug, we demonstrate in vivo inhibition of aminoglycoside uptake in mammalian hair cells. This work establishes how systemically administered fluorescently labeled ototoxins in the neonatal mouse can reveal important details about ototoxic drugs and protective therapeutics

The Inner Ear Heat Shock Transcriptional Signature Identifies Compounds That Protect Against Aminoglycoside Ototoxicity

Mechanosensory hair cells of the inner ear transduce auditory and vestibular sensory input. Hair cells are susceptible to death from a variety of stressors, including treatment with therapeutic drugs that have ototoxic side effects. There is a need for co-therapies to mitigate drug-induced ototoxicity, and we showed previously that induction of heat shock proteins (HSPs) protects against hair cell death and hearing loss caused by aminoglycoside antibiotics in mouse. Here, we utilized the library of integrated cellular signatures (LINCS) to identify perturbagens that induce transcriptional profiles similar to that of heat shock. Massively parallel sequencing of RNA (RNA-Seq) of heat shocked and control mouse utricles provided a heat shock gene expression signature that was used in conjunction with LINCS to identify candidate perturbagens, several of which were known to protect the inner ear. Our data indicate that LINCS is a useful tool to screen for compounds that generate specific gene expression signatures in the inner ear. Forty-two LINCS-identified perturbagens were tested for otoprotection in zebrafish, and three of these were protective. These compounds also induced the heat shock gene expression signature in mouse utricles, and one compound protected against aminoglycoside-induced hair cell death in whole organ cultures of utricles from adult mice

Ca2+-permeable AMPARs mediate glutamatergic transmission and excitotoxic damage at the hair cell ribbon synapse

We report functional and structural evidence for GluA2-lacking C

In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish

Introduction: Ototoxicity is a debilitating side effect of over 150 medications with diverse mechanisms of action, many of which could be taken concurrently to treat multiple conditions. Approaches for preclinical evaluation of drug-drug interactions that might impact ototoxicity would facilitate design of safer multi-drug regimens and mitigate unsafe polypharmacy by flagging combinations that potentially cause adverse interactions for monitoring. They may also identify protective agents that antagonize ototoxic injury.Methods: To address this need, we have developed a novel workflow that we call Parallelized Evaluation of Protection and Injury for Toxicity Assessment (PEPITA), which empowers high-throughput, semi-automated quantification of ototoxicity and otoprotection in zebrafish larvae via microscopy. We used PEPITA and confocal microscopy to characterize in vivo the consequences of drug-drug interactions on ototoxic drug uptake and cellular damage of zebrafish lateral line hair cells.Results and discussion: By applying PEPITA to measure ototoxic drug interaction outcomes, we discovered antagonistic interactions between macrolide and aminoglycoside antibiotics that confer protection against aminoglycoside-induced damage to lateral line hair cells in zebrafish larvae. Co-administration of either azithromycin or erythromycin in zebrafish protected against damage from a broad panel of aminoglycosides, at least in part via inhibiting drug uptake into hair cells via a mechanism independent from hair cell mechanotransduction. Conversely, combining macrolides with aminoglycosides in bacterial inhibition assays does not show antagonism of antimicrobial efficacy. The proof-of-concept otoprotective antagonism suggests that combinatorial interventions can potentially be developed to protect against other forms of toxicity without hindering on-target drug efficacy

ORC-13661 protects sensory hair cells from aminoglycoside and cisplatin ototoxicity

Aminoglycoside (AG) antibiotics are widely used to prevent life-threatening infections, and cisplatin is used in the treatment of various cancers, but both are ototoxic and result in loss of sensory hair cells from the inner ear. ORC-13661 is a new drug that was derived from PROTO-1, a compound first identified as protective in a large-scale screen utilizing hair cells in the lateral line organs of zebrafish larvae. Here, we demonstrate, in zebrafish larvae and in mouse cochlear cultures, that ORC-13661 provides robust protection of hair cells against both ototoxins, the AGs and cisplatin. ORC-13661 also prevents both hearing loss in a dose-dependent manner in rats treated with amikacin and the loading of neomycin-Texas Red into lateral line hair cells. In addition, patch-clamp recordings in mouse cochlear cultures reveal that ORC-13661 is a high-affinity permeant blocker of the mechanoelectrical transducer (MET) channel in outer hair cells, suggesting that it may reduce the toxicity of AGs by directly competing for entry at the level of the MET channel and of cisplatin by a MET-dependent mechanism. ORC-13661 is therefore a promising and versatile protectant that reversibly blocks the hair cell MET channel and operates across multiple species and toxins

Causes and Consequences of Past and Projected Scandinavian Summer Temperatures, 500–2100 AD

Tree rings dominate millennium-long temperature reconstructions and many records originate from Scandinavia, an area for which the relative roles of external forcing and internal variation on climatic changes are, however, not yet fully understood. Here we compile 1,179 series of maximum latewood density measurements from 25 conifer sites in northern Scandinavia, establish a suite of 36 subset chronologies, and analyse their climate signal. A new reconstruction for the 1483–2006 period correlates at 0.80 with June–August temperatures back to 1860. Summer cooling during the early 17th century and peak warming in the 1930s translate into a decadal amplitude of 2.9°C, which agrees with existing Scandinavian tree-ring proxies. Climate model simulations reveal similar amounts of mid to low frequency variability, suggesting that internal ocean-atmosphere feedbacks likely influenced Scandinavian temperatures more than external forcing. Projected 21st century warming under the SRES A2 scenario would, however, exceed the reconstructed temperature envelope of the past 1,500 years

Rheotaxis in Larval Zebrafish Is Mediated by Lateral Line Mechanosensory Hair Cells

The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. This system includes cell clusters, called superficial neuromasts, located on the surface of head and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond to disturbances in the water and convey information to the brain via the lateral line ganglia. The convenient location of mechanosensory hair cells on the body surface has made the lateral line a valuable system in which to study hair cell damage and regeneration. One way to measure hair cell survival and recovery is to assay behaviors that depend on their function. We built a system in which orientation against constant water flow, positive rheotaxis, can be quantitatively assessed. We found that zebrafish larvae perform positive rheotaxis and that, similar to adult fish, larvae use both visual and lateral line input to perform this behavior. Disruption or damage of hair cells in the absence of vision leads to a marked decrease in rheotaxis that recovers upon hair cell repair or regeneration