Editor's evaluation: Single-cell transcriptomic profiling of the zebrafish inner ear reveals molecularly distinct hair cell and supporting cell subtypes
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The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.
Danio
Otic vesicle
Lateral line
Morpholino
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Hair cells are specialized mechanosensory receptors in vertebrates that detect and process auditory and vestibular information with remarkable precision, fidelity and efficiency (Schwander et al., 2010). Most of our knowledge about these cells stems from in vitro preparations using isolated tissue, which creates the need for a relatively simple in vivo vertebrate model to study hair cells.
The zebrafish (Danio rerio) is being increasingly used to study the genetic basis of hearing and deafness but also the function and physiology of hair cells (Nicolson, 2005). However, the use of the zebrafish as an in vivo model to study hair cell function is currently limited by our poor understanding of their biophysical properties. The aim of this study was to provide a detailed description of the biophysical properties of zebrafish hair cells both in the lateral line as well as inner ear during early and mature stages of fish development. I have used single cell patch-clamp electrophysiology to measure potassium currents and synaptic transmission in hair cells.
I found that hair cells from the lateral line and inner ear show different current types, the expression of which depends upon the position of the cell within the lateral line neuromast or inner ear macula. Moreover, I found that the abundance of hair cell types in the lateral line changes over time, which potentially reflects adaptations to a changing sensory environment for the fish. The synaptic machinery of the lateral line hair cells is comparable in terms of efficiency to its mammalian counterpart, but less sensitive. Lastly, I have also developed an approach to study hair cell properties in vivo in the juvenile fish.
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Lateral line
Kinocilium
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Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turnover during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However, in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo . This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 days post fertilization (dpf) zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.
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Ototoxicity
Otic vesicle
Diphtheria Toxin
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Background: Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Results : Zebrafish and other non‐mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Conclusions : Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Developmental Dynamics 243:1187–1202, 2014 . © 2014 Wiley Periodicals, Inc.
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Hair cells in the inner ear and lateral lines are mechanosensitive receptor cells whose development and function are tightly regulated. Several transcription factors as well as splicing factors have been identified to play important roles in hair cell development, whereas the role of RNA stability in this process is poorly understood. In the present work, we report that RNA-binding motif protein 24a (Rbm24a) is indispensable for hair cell development in zebrafish. Rbm24a expression is detected in the inner ear as well as lateral line neuromasts. Albeit rbm24a deficient zebrafish do not survive beyond 9 days post fertilization (dpf) due to effects outside of the inner ear, rbm24a deficiency does not affect the early development of inner ear except for delayed otolith formation and semicircular canal fusion. However, hair cell development is severely affected and hair bundle is disorganized in rbm24a mutants. As a result, the auditory and vestibular function of rbm24a mutants are compromised. RNAseq analyses identified several Rbm24a-target mRNAs that are directly bound by Rbm24a and are dysregulated in rbm24a mutants. Among the identified Rbm24a-target genes, lrrc23 , dfna5b , and smpx are particularly interesting as their dysregulation might contribute to the inner ear phenotypes in rbm24a mutants. In conclusion, our data suggest that Rbm24a affects hair cell development in zebrafish through regulating mRNA stability.
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In humans, most hearing loss results from death of hair cells, the mechanosensory receptors of the inner ear. Two goals of current hearing research are to protect hair cells from degeneration and to regenerate new hair cells, replacing those that are lost due to aging, disease, or environmental challenges. One limitation of research in the auditory field has been the relative inaccessibility of the mechanosensory systems in the inner ear. Zebrafish possess hair cells in both their inner ear and their lateral line system that are morphologically and functionally similar to human hair cells. The external location of the mechanosensory hair cells in the lateral line and the ease of in vivo labeling and imaging make the zebrafish lateral line a unique system for the study of hair cell toxicity, protection, and regeneration. This review focuses on the lateral line system as a model for understanding loss and protection of mechanosensory hair cells. We discuss chemical screens to identify compounds that induce hair cell loss and others that protect hair cells from known toxins and the potential application of these screens to human medicine.
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Abstract Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turn over during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo . This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 dpf zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.
Utricle
Ototoxicity
Otic vesicle
Diphtheria Toxin
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Abstract Although hair cells are the sensory receptors of the auditory and vestibular systems in the ears of all vertebrates, hair cell properties are different between non-mammalian vertebrates and mammals. To understand the basic biological properties of hair cells from non-mammalian vertebrates, we examined the transcriptome of adult zebrafish auditory and vestibular hair cells. GFP-labeled hair cells were isolated from inner-ear sensory epithelia of a pou4f3 promoter-driven GAP-GFP line of transgenic zebrafish. One thousand hair cells and 1,000 non-sensory surrounding cells (nsSCs) were separately collected for each biological replicate, using the suction pipette technique. RNA sequencing of three biological replicates for the two cell types was performed and analyzed. Comparisons between hair cells and nsSCs allow identification of enriched genes in hair cells, which may underlie hair cell specialization. Our dataset provides an extensive resource for understanding the molecular mechanisms underlying morphology, function, and pathology of adult zebrafish hair cells. It also establishes a framework for future characterization of genes expressed in hair cells and the study of hair cell evolution.
Kinocilium
Utricle
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Abstract Inner ear hair cells are the fundamental unit of sound and vibration detection in inner ear and lateral line structures. Our understanding of the structure and ultrastructure of hair cells has heretofore relied upon two-dimensional imaging. The development of serial block-face scanning electron microscopy (SBFSEM) changes this paradigm and allows for ultrastructural evaluation of hair cells in three-dimensions. This is the first report of SBFSEM analysis in the myo7aa -/- mutant where we evaluated several attributes of zebrafish hair cells from the inner ear cristae in both wildtype and myo7aa -/- mutant zebrafish through three-dimensional reconstruction. We describe ribbon synapse number, location, and volume, mitochondrial localization, and innervation for individual hair cells. We determined that myo7aa -/- mutant ribbon synapses have a smaller volume and surface area; however, all other hair cell attributes investigated were not significantly different between the mutant and wildtype zebrafish. These findings are critical for the development of therapies for deafness caused by mutations in myo7aa , as this study supports that the necessary hair cell machinery for hearing is largely intact. In addition, the methodology and measurements developed in this study provide a guide for the evaluation of zebrafish hair cells using SBFSEM.
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