Biology subjects

Raible, D. W.

Publications and source records attributed to Raible, D. W..

11 recordsLinked to original sources

bioRxiv2026

A comparative single-cell transcriptomic atlas for diverse populations of vertebrate hair cells

Mechanosensitive hair cells vary widely in morphology and regenerative capacity across vertebrate organs and species. To investigate their underlying transcriptomic diversity, we integrated human, mouse, chicken, and zebrafish single-cell and single-nucleus RNA sequencing datasets and assembled a cross-species atlas of hair cells spanning organs, developmental stages, and species. Analysis of 29 hair cell populations, encompassing the major cochlear, vestibular, and lateral-line hair cell types, identified approximately 5,000 genes enriched in at least one hair cell population compared to supporting cells from the same organs. Unsupervised clustering of these hair cell-enriched (HCE) genes defined species-, organ-, and hair cell state-associated cohorts as well as broadly conserved hair cell-enriched programs. Using an AUC-based scoring framework, we further defined 884 pan hair cell-enriched (pan-HCE) genes with elevated expression in most developing and/or mature hair cell populations, including genes implicated in deafness, mechanotransduction, and synaptic transmission, along with genes not previously linked to hair cell function. Independent analysis of developing hair cells using the same metrics stratified pan-HCE genes based on when they are first enriched and identified an additional 97 genes that are transiently enriched. We used the pan- and developing HCE gene sets to assess transcriptional similarity between baseline hair cell states and hair cells produced during avian hair cell regeneration and in mouse cochlear organoids, as well as hair cell-like populations produced by fibroblast reprogramming. HCE gene sets with different developmental dynamics identified young vs. more mature hair cells when projected onto independent single-cell RNA sequencing datasets from developing zebrafish, mouse, and human. We provide a web-based resource of all HCE metrics and expression profiles, enabling future exploration of vertebrate hair cell gene expression across organs, species, and experimental contexts.

neuroscience↗

bioRxiv2026

Lysosomal ion homeostasis drives delayed hair cell death after aminoglycoside uptake.

Aminoglycoside ototoxicity has been widely reported and remains an important public health issue. Unfortunately, the molecular mechanisms of ototoxicity are not well understood. Here, we report the lysosome compartment as the main driver of delayed cell death triggered by aminoglycosides. By labeling early and late endosomes we show that endocytosis is not an significant path of aminoglycoside uptake. Instead, we show that aminoglycosides are delivered to lysosomes primarily through autophagy. Hair cells can be protected from damage by activation of the dual function lysosomal Two-Pore-Channel 2 (TPC2), stimulated by NAADP agonist but not by phosphoinositide PI(3,5,)P2 agonist. These treatments neutralize lysosomal pH. Moreover, luminal pH changes are also accompanied by changes in ferrous iron availability, though classical ferroptosis inhibitors do not prevent a delayed hair cell death. These findings reveal that lysosomal-driven delayed hair cell death is ferroptosis independent, suggesting that toxicity relies on a distinct mechanism that based on the internal conditions of the lysosomal compartment.

neuroscience↗

bioRxiv2024

Transdifferentiation is uncoupled from progenitor pool expansion during hair cell regeneration in the zebrafish inner ear

Death of mechanosensory hair cells in the inner ear is a common cause of auditory and vestibular impairment in mammals, which have a limited ability to regrow these cells after damage. In contrast, non-mammalian vertebrates including zebrafish can robustly regenerate hair cells following severe organ damage. The zebrafish inner ear provides an understudied model system for understanding hair cell regeneration in organs that are highly conserved with their mammalian counterparts. Here we quantitatively examine hair cell addition during growth and regeneration of the larval zebrafish inner ear. We used a genetically encoded ablation method to induce hair cell death and observed gradual regeneration with correct spatial patterning over two weeks following ablation. Supporting cells, which surround and are a source of new hair cells, divide in response to hair cell ablation, expanding the possible progenitor pool. In parallel, nascent hair cells arise from direct transdifferentiation of progenitor pool cells uncoupled from progenitor division. These findings reveal a previously unrecognized mechanism of hair cell regeneration with implications for how hair cells may be encouraged to regenerate in the mammalian ear. SUMMARY STATEMENTHair cell regeneration in the zebrafish inner ear occurs through transdifferentiation after a transient wave of supporting cell proliferation that expands the precursor pool.

developmental biology↗

bioRxiv2023

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

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 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. 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. By applying PEPITA to characterize ototoxic drug interaction outcomes, we have 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.

pharmacology and toxicology↗

bioRxiv2023

Data-Driven 3D Shape Analysis Reveals Cell Shape-Fate Relationships in Zebrafish Lateral Line Neuromast

Cell shape is a powerful readout of cell state, fate, and function. With the advent of sophisticated microscopes, image segmentation algorithms, and numerical shape representations, it is becoming more feasible to study cell shape in developing tissues. However, few studies have analyzed cell shape in three dimensions in living, intact organisms. Here, we took advantage of the favorable imaging qualities of zebrafish lateral line neuromasts to generate a dataset of high resolution images with labeled cells and nuclei. Using a custom Python-based workflow, we performed semi-automated, 3D cell and nucleus segmentation. We then used spherical harmonics and principal components analysis to distill neuromast cell and nuclear shape variation into several interpretable, biologically meaningful parameters. We found that neuromast cell and nuclear shapes vary with cell location and identity. The distinction between hair cells and support cells was discrete and accounted for much of the variation in neuromast cell and nucleus shape, which allowed us to train classifiers to predict hair cell identity from cell and nucleus shape features. Using markers for support cell subpopulations, we found that support cell subtypes also had different shapes from each other; however, shape features did not distinguish as sharply between support cell subtypes, suggesting that support cells vary continuously in shape. To investigate the effects of genetic perturbation that results in loss of a cell type on neuromast cell shape, we examined atoh1a mutants that lack hair cells. We found that neuromasts from atoh1a mutants lacked the cell shape phenotype associated with hair cells, but did not exhibit a mutant-specific cell shape. Our results demonstrate the utility of using 3D cell shape features to characterize, compare, and classify cells in a living, developing organism.

developmental biology↗

bioRxiv2022

Single-Cell Transcriptomic Profiling of the Zebrafish Inner Ear Reveals Molecularly Distinct Hair Cell and Supporting Cell Subtypes

A major cause of human deafness and vestibular dysfunction is permanent loss of the mechanosensory hair cells of the inner ear. In non-mammalian vertebrates such as zebrafish, regeneration of missing hair cells can occur throughout life. While a comparative approach has the potential to reveal the basis of such differential regenerative ability, the degree to which the inner ears of fish and mammals share common hair cells and supporting cell types remains unresolved. Here we perform single-cell RNA sequencing of the zebrafish inner ear at embryonic through adult stages to catalog the diversity of hair cell and non-sensory supporting cells. We identify a putative progenitor population for hair cells and supporting cells, as well as distinct hair cells and supporting cell types in the maculae versus cristae. The hair cell and supporting cell types differ from those described for the lateral line system, a distributed mechanosensory organ in zebrafish in which most studies of hair cell regeneration have been conducted. In the maculae, we identify two subtypes of hair cells that share gene expression with mammalian striolar or extrastriolar hair cells. In situ hybridization reveals that these hair cell subtypes occupy distinct spatial domains within the two major macular organs, the utricle and saccule, consistent with the reported distinct electrophysiological properties of hair cells within these domains. These findings suggest that primitive specialization of spatially distinct striolar and extrastriolar hair cells likely arose in the last common ancestor of fish and mammals. The similarities of inner ear cell type composition between fish and mammals also support using zebrafish as a relevant model for understanding inner ear-specific hair cell function and regeneration.

neuroscience↗

bioRxiv2022

Dermal appendage-dependent patterning of zebrafish atoh1a+ Merkel cells

Touch system function requires precise interactions between specialized skin cells and somatosensory axons, as exemplified by the vertebrate mechanosensory Merkel cell-neurite complex. Development and patterning of Merkel cells and associated neurites during skin organogenesis remains poorly understood, partly due to the in utero development of mammalian embryos. Here, we discover Merkel cells in the zebrafish epidermis and identify Atonal homolog 1a (Atoh1a) as a marker of zebrafish Merkel cells. We show that zebrafish Merkel cells derive from basal keratinocytes, express neurosecretory and mechanosensory machinery, extend actin-rich microvilli, and complex with somatosensory axons, all hallmarks of mammalian Merkel cells. Merkel cells populate all major adult skin compartments, with region-specific densities and distribution patterns. In vivo photoconversion reveals that Merkel cells undergo steady loss and replenishment during skin homeostasis. Merkel cells develop concomitant with dermal appendages along the trunk, and preventing dermal appendage formation reduces Merkel cell density by affecting both cell differentiation and maintenance. By contrast, altering dermal appendage morphology changes the distribution, but not density, of Merkel cells. Overall, our studies provide insights into touch system maturation during skin organogenesis and establish zebrafish as an experimentally accessible in vivo model for the study of Merkel cell biology.

developmental biology↗

bioRxiv2022

Activity regulates a cell type-specific mitochondrial phenotype in zebrafish lateral line hair cells

Hair cells of the inner ear are particularly sensitive to changes in mitochondria, the subcellular organelles necessary for energy production in all eukaryotic cells. There are over thirty mitochondrial deafness genes, and mitochondria are implicated in hair cell death following noise exposure, aminoglycoside antibiotic exposure, as well as in age-related hearing loss. However, little is known about the basic aspects of hair cell mitochondrial biology. Using hair cells from the zebrafish lateral line as a model and serial block-face scanning electron microscopy, we have quantifiably characterized a unique hair cell mitochondrial phenotype that includes (1) a high mitochondrial volume, and (2) specific mitochondrial architecture: multiple small mitochondria apically, and a reticular mitochondrial network basally. This phenotype develops gradually over the lifetime of the hair cell. Disrupting this mitochondrial phenotype with a mutation in opa1 impacts mitochondrial health and function. While hair cell activity is not required for the high mitochondrial volume, it shapes the mitochondrial architecture, with mechanotransduction necessary for all patterning, and synaptic transmission necessary for development of mitochondrial networks. These results demonstrate the high degree to which hair cells regulate their mitochondria for optimal physiology, and provide new insights into mitochondrial deafness.

cell biology↗

bioRxiv2022

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.

neuroscience↗

bioRxiv2022

Cachd1 is a novel Frizzled- and LRP6-interacting protein required for neurons to acquire left-right asymmetric character

Neurons on left and right sides of the nervous system frequently show asymmetric properties but how these differences arise is poorly understood. Through a forward genetic screen in zebrafish, we find that loss of function of the transmembrane protein Cachd1 results in right-sided habenula neurons adopting left-sided character. Cachd1 is expressed in habenula neuron progenitors, functions symmetrically downstream of asymmetric environmental signals that determine laterality and influences timing of the normally left-right asymmetric patterns of neurogenesis. Unbiased screening for Cachd1 partners identified the Wnt co-receptor Frizzled7 and further biochemical and structural analysis revealed Cachd1 can bind simultaneously to Fzd proteins and Lrp6, bridging between these two Wnt co-receptors. Consistent with these structural studies, lrp6 mutant zebrafish show symmetric habenulae with left-sided character and epistasis experiments with other Wnt pathway genes support an in vivo role for Cachd1 in modulating Wnt pathway activity in the brain. Together, these studies identify Cachd1 as a conserved novel Wnt-receptor interacting protein with roles in regulating neurogenesis and neuronal identity.

developmental biology↗

bioRxiv2020

Chloroquine Kills Hair Cells in Zebrafish Lateral Line and Murine Cochlear Cultures: Implications for Ototoxicity

Hearing and balance deficits have been reported during and following treatment with the antimalarial drug chloroquine. However, experimental work examining the direct actions of chloroquine on mechanoreceptive hair cells in common experimental models is lacking. This study examines the effects of chloroquine on hair cells using two common experimental models: the zebrafish lateral line and neonatal mouse cochlear cultures. Zebrafish larvae were exposed to varying concentrations of chloroquine phosphate or hydroxychloroquine for 1 hr or 24 hr, and hair cells assessed by antibody staining. A significant, dose-dependent reduction in the number of surviving hair cells was seen across conditions for both exposure periods. Hydroxycholroquine showed similar toxicity. In mouse cochlear cultures, chloroquine damage was specific to outer hair cells in tissue from the cochlear basal turn, consistent with susceptibility to other ototoxic agents. These findings suggest a need for future studies employing hearing and balance monitoring during exposure to chloroquine and related compounds, particularly with interest in these compounds as therapeutics against viral infections including coronavirus.

neuroscience↗