Protocol for in vivo elimination of avian auditory hair cells, multiplexed mRNA detection, immunohistochemistry, and S-phase labeling.

The avian inner ear can naturally regenerate sensory hair cells and is therefore an ideal candidate for investigating mechanisms leading to hair cell regeneration and functional recovery. Here, we present a surgical protocol for eliminating auditory hair cells via sisomicin injection into the lateral semicircular canal. We describe steps for multiplex mRNA detection in chicken basilar papilla and utricle sections. We then detail procedures for integrating immunohistochemistry for concurrent mRNA and protein visualization, complemented by S-phase labeling with EdU. For complete details on the use and execution of this protocol, please refer to Benkafadar et al., Benkafadar et al., Sato et al., Janesick et al., Scheibinger et al.1,2,3,4,5.

Single-cell transcriptomic atlas reveals increased regeneration in diseased human inner ear balance organs.

Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.

Murine cochlear damage models in the context of hair cell regeneration research.

Understanding the complex pathologies associated with hearing loss is a significant motivation for conducting inner ear research. Lifelong exposure to loud noise, ototoxic drugs, genetic diversity, sex, and aging collectively contribute to human hearing loss. Replicating this pathology in research animals is challenging because hearing impairment has varied causes and different manifestations. A central aspect, however, is the loss of sensory hair cells and the inability of the mammalian cochlea to replace them. Researching therapeutic strategies to rekindle regenerative cochlear capacity, therefore, requires the generation of animal models in which cochlear hair cells are eliminated. This review discusses different approaches to ablate cochlear hair cells in adult mice. We inventoried the cochlear cyto- and histo-pathology caused by acoustic overstimulation, systemic and locally applied drugs, and various genetic tools. The focus is not to prescribe a perfect damage model but to highlight the limitations and advantages of existing approaches and identify areas for further refinement of damage models for use in regenerative studies.

Hyperosmotic Sisomicin Infusion: A Mouse Model for Hearing Loss.

Hearing impairment arises from the loss of either type of cochlear sensory hair cells. Inner hair cells act as primary sound transducers, while outer hair cells enhance sound-induced vibrations within the organ of Corti. Established models, such as systemic administration of ototoxic aminoglycosides, yield inconsistent and variable hair cell death in mice. Overcoming this limitation, we developed a method involving surgical delivery of a hyperosmotic sisomicin solution into the posterior semicircular canal of adult mice. This procedure induced rapid and synchronous apoptotic demise of outer hair cells within 14 hours, leading to irreversible hearing loss. The combination of sisomicin and hyperosmotic stress caused consistent and synergistic ototoxic damage. Inner hair cells remained intact until three days post-treatment, after which deterioration in structure and number was observed, culminating in cell loss by day seven. This robust animal model provides a valuable tool for otoregenerative research, facilitating single-cell and omics-based studies toward exploring preclinical therapeutic strategies.

Hair cell regeneration, reinnervation, and restoration of hearing thresholds in the avian hearing organ.

Hearing starts, at the cellular level, with mechanoelectrical transduction by sensory hair cells. Sound information is then transmitted via afferent synaptic connections with auditory neurons. Frequency information is encoded by the location of hair cells along the cochlear duct. Loss of hair cells, synapses, or auditory neurons leads to permanent hearing loss in mammals. Birds, in contrast, regenerate auditory hair cells and functionally recover from hearing loss. Here, we characterized regeneration and reinnervation in sisomicin-deafened chickens and found that afferent neurons contact regenerated hair cells at the tips of basal projections. In contrast to development, synaptic specializations are established at these locations distant from the hair cells' bodies. The protrusions then contracted as regenerated hair cells matured and became functional 2 weeks post-deafening. We found that auditory thresholds recovered after 4-5 weeks. We interpret the regeneration-specific synaptic reestablishment as a location-preserving process that might be needed to maintain tonotopic fidelity.

An essential signaling cascade for avian auditory hair cell regeneration.

Hearing loss is a chronic disease affecting millions of people worldwide, yet no restorative treatment options are available. Although non-mammalian species can regenerate their auditory sensory hair cells, mammals cannot. Birds retain facultative stem cells known as supporting cells that engage in proliferative regeneration when surrounding hair cells die. Here, we investigated gene expression changes in chicken supporting cells during auditory hair cell death. This identified a pathway involving the receptor F2RL1, HBEGF, EGFR, and ERK signaling. We propose a cascade starting with the proteolytic activation of F2RL1, followed by matrix-metalloprotease-mediated HBEGF shedding, and culminating in EGFR-mediated ERK signaling. Each component of this cascade is essential for supporting cell S-phase entry in vivo and is integral for hair cell regeneration. Furthermore, STAT3-phosphorylation converges with this signaling toward upregulation of transcription factors ATF3, FOSL2, and CREM. Our findings could provide a basis for designing treatments for hearing and balance disorders.

Transcriptional dynamics of delaminating neuroblasts in the mouse otic vesicle.

An abundance of research has recently highlighted the susceptibility of cochleovestibular ganglion (CVG) neurons to noise damage and aging in the adult cochlea, resulting in hearing deficits. Furthering our understanding of the transcriptional cascades that contribute to CVG development may provide insight into how these cells can be regenerated to treat inner ear dysfunction. Here we perform a high-depth single-cell RNA sequencing analysis of the E10.5 otic vesicle and its surrounding tissues, including CVG precursor neuroblasts and emerging CVG neurons. Clustering and trajectory analysis of otic-lineage cells reveals otic markers and the changes in gene expression that occur from neuroblast delamination toward the development of the CVG. This dataset provides a valuable resource for further identifying the mechanisms associated with CVG development from neurosensory competent cells within the otic vesicle.

Fbxo2CreERT2: A new model for targeting cells in the neonatal and mature inner ear.

The mammalian inner ear contains six sensory patches that allow detection of auditory stimuli as well as movement and balance. Much research has focused on the organ of Corti, the sensory organ of the cochlea that detects sound. Unfortunately, these cells are difficult to access in vivo, especially in the mature animal, but the development of genetically modified mouse models, including Cre/Lox mice, has improved the ability to label, purify or manipulate these cells. Here, we describe a new tamoxifen-inducible CreER mouse line, the Fbxo2CreERT2 mouse, that can be used to specifically manipulate cells throughout the cochlear duct of the neonatal and mature cochlear epithelium. In vestibular sensory epithelia, Fbxo2CreERT2-mediated recombination occurs in many hair cells and more rarely in supporting cells of neonatal and adult mice, with a higher rate of Fbxo2CreERT2 induction in type 1 versus type 2 hair cells in adults. Fbxo2CreERT2 mice, therefore, are a new tool for the specific manipulation of epithelial cells of the inner ear and targeted manipulation of vestibular type 1 hair cells.

Cell-type identity of the avian utricle.

The avian utricle, a vestibular organ of the inner ear, displays turnover of sensory hair cells throughout life. This is in sharp contrast to the mammalian utricle, which shows limited regenerative capacity. Here, we use single-cell RNA sequencing to identify distinct marker genes for the different sensory hair cell subtypes of the chicken utricle, which we validated in situ. We provide markers for spatially distinct supporting cell populations and identify two transitional cell populations of dedifferentiating supporting cells and developing hair cells. Trajectory reconstruction resulted in an inventory of gene expression dynamics of natural hair cell generation in the avian utricle.

Avian auditory hair cell regeneration is accompanied by JAK/STAT-dependent expression of immune-related genes in supporting cells.

The avian hearing organ is the basilar papilla that, in sharp contrast to the mammalian cochlea, can regenerate sensory hair cells and thereby recover from deafness within weeks. The mechanisms that trigger, sustain and terminate the regenerative response in vivo are largely unknown. Here, we profile the changes in gene expression in the chicken basilar papilla after aminoglycoside antibiotic-induced hair cell loss using RNA-sequencing. We identified changes in gene expression of a group of immune-related genes and confirmed with single-cell RNA-sequencing that these changes occur in supporting cells. In situ hybridization was used to further validate these findings. We determined that the JAK/STAT signaling pathway is essential for upregulation of the damage-response genes in supporting cells during the second day after induction of hair cell loss. Four days after ototoxic damage, we identified newly regenerated, nascent auditory hair cells that express genes linked to termination of the JAK/STAT signaling response. The robust, transient expression of immune-related genes in supporting cells suggests a potential functional involvement of JAK/STAT signaling in sensory hair cell regeneration.

Surgical Approach for Rapid and Minimally Traumatic Recovery of Human Inner Ear Tissues From Deceased Organ Donors.

OBJECTIVE: To develop a surgical approach for rapid and minimally traumatic recovery of inner ear tissue from human organ and tissue donors to provide fresh tissue for use in inner ear research. STUDY DESIGN: Exploration of novel surgical methodology and evaluation of the steps necessary for obtaining specimens from donors during the procurement of organs for transplantation. SETTING: Donor procurement locations across multiple local hospitals and tissue processing at the microsurgical temporal bone laboratory. PATIENTS TISSUE SOURCE: Human organ and tissue donors. INTERVENTIONS: Dissection and procurement of the inner ear tissue. MAIN OUTCOME MEASURES: Development of rapid and minimally traumatic inner ear tissue recovery. Primarily, establishing an efficient process which includes collaboration with transplant network, implementing a consent protocol, developing and training an on-call recovery team, and designing a portable surgical kit suitable for use in a variety of settings. RESULTS: The extraction procedure is described in three consecutive steps: the trans-canal exposure, the approach to the vestibule with extraction of the vestibular organs; and the approach to extract inner ear tissues from the cochlear duct. CONCLUSIONS: Organ and tissue donors are a promising and underutilized resource of inner ear organs for purposes of research and future translational studies. Using our modified technique through the trans-canal/trans-otic approach, we were able to extract tissues of the vestibular and auditory end organs in a timely manner.

Selection Criteria Optimal for Recovery of Inner Ear Tissues From Deceased Organ Donors.

OBJECTIVE: To identify optimal conditions for recovering viable inner ear tissues from deceased organ donors. SETTING: Tertiary recovery hospitals and Donor Network West Organ Recovery Center. INTERVENTIONS: Recovering bilateral inner ear tissues and immunohistological analysis. MAIN OUTCOME MEASURES: Immunohistochemical analysis of utricles from human organ donors after brain death (DBD) or donors after cardiac death (DCD). RESULTS: Vestibular tissues from 21 organ donors (39 ears) were recovered. Of these, 18 donors (33 utricles) were examined by immunofluorescence. The sensory epithelium was present in seven utricles (two from DBD and five from DCD). Relative to DBD utricles, DCD organs more commonly displayed dense populations of hair cells and supporting cells. Relative to DBD, DCD had significantly shorter postmortem interval time to tissue recovery (<48 h). Compared to donors with no sensory epithelium, donors with intact and viable sensory epithelium (both DCD and DBD) had significantly shorter lag time to resuscitation prior to hospital admission (6.4 ±â€Š9.2 vs 35.6 ±â€Š23.7 min, respectively) as well as a shorter time between pronouncements of death to organ recovery (22.6 ±â€Š30.4 vs 64.8 ±â€Š22.8 h, respectively). CONCLUSIONS: Organ donors are a novel resource for bilateral inner ear organs. Selecting tissue donors within defined parameters can optimize the quality of recovered inner ear tissues, thereby facilitating future research investigating sensory and nonsensory cells.

Towards Insect-Friendly Road Lighting-A Transdisciplinary Multi-Stakeholder Approach Involving Citizen Scientists.

(1) The project "Tatort Streetlight" implements an insect-friendly road light design in a four year before-after, control-impact (BACI) approach involving citizen scientists. It will broaden the stakeholder interests from solely anthropogenic perspectives to include the welfare of insects and ecosystems. Motivated by the detrimental impacts of road lighting systems on insects, the project aims to find solutions to reduce the insect attraction and habitat fragmentation resulting from roadway illumination. (2) The citizen science approach invites stakeholders to take part and join forces for the development of a sustainable and environmentally friendly road lighting solution. Here, we describe the project strategy, stakeholder participation and motivation, and how the effects of the alternative road luminaire and lighting design can be evaluated. (3) The study compares the changes in (a) insect behavior, (b) night sky brightness, and (c) stakeholder participation and awareness. For this purpose, different experimental areas and stakeholders in four communities in Germany are identified. (4) The project transfers knowledge of adverse effects of improperly managed road illumination and interacts with various stakeholders to develop a new road lighting system that will consider the well-being of street users, local residents, and insects.

Fluorescent in situ mRNA detection in the adult mouse cochlea.

Ossification and the delicateness of the cochlear duct make histologic assessments of the mature cochlea a challenging endeavor. Treatments to soften the bone facilitate sectioning and dissection of the cochlear duct but limit in situ mRNA detection in such specimens. Here, we provide a protocol for in situ mRNA detection using hybridization chain reaction in whole-mount preparations of the adult mouse cochlea. We show examples for multi-probe detection of different mRNAs and describe combination of this method with conventional immunohistochemistry.

Murine cochlear cell sorting and cell-type-specific organoid culture.

Neonatal mouse cochlear duct cells can proliferate and grow in vitro into inner ear organoids. Distinctive cochlear duct cell types have different organoid formation capacities. Here, we provide a flow cytometric cell-sorting method that allows the subsequent culture of individual cochlear cell populations. For the efficient culture of the sorted cells, we provide protocols for growing free-floating inner ear organoids, the adherence of organoids to a substrate, and the expansion of organoid-derived inner ear colonies. For complete details on the use and execution of this protocol, please refer to Kubota et al. (2021).

Spatiotemporal dynamics of inner ear sensory and non-sensory cells revealed by single-cell transcriptomics.

The utricle is a vestibular sensory organ that requires mechanosensitive hair cells to detect linear acceleration. In neonatal mice, new hair cells are derived from non-sensory supporting cells, yet cell type diversity and mechanisms of cell addition remain poorly characterized. Here, we perform computational analyses on single-cell transcriptomes to categorize cell types and resolve 14 individual sensory and non-sensory subtypes. Along the periphery of the sensory epithelium, we uncover distinct groups of transitional epithelial cells, marked by Islr, Cnmd, and Enpep expression. By reconstructing de novo trajectories and gene dynamics, we show that as the utricle expands, Islr+ transitional epithelial cells exhibit a dynamic and proliferative phase to generate new supporting cells, followed by coordinated differentiation into hair cells. Taken together, our study reveals a sequential and coordinated process by which non-sensory epithelial cells contribute to growth of the postnatal mouse sensory epithelium.

Cell-type identity of the avian cochlea.

In contrast to mammals, birds recover naturally from acquired hearing loss, which makes them an ideal model for inner ear regeneration research. Here, we present a validated single-cell RNA sequencing resource of the avian cochlea. We describe specific markers for three distinct types of sensory hair cells, including a previously unknown subgroup, which we call superior tall hair cells. We identify markers for the supporting cells associated with tall hair cells, which represent the facultative stem cells of the avian inner ear. Likewise, we present markers for supporting cells that are located below the short cochlear hair cells. We further infer spatial expression gradients of hair cell genes along the tonotopic axis of the cochlea. This resource advances neurobiology, comparative biology, and regenerative medicine by providing a basis for comparative studies with non-regenerating mammalian cochleae and for longitudinal studies of the regenerating avian cochlea.

Transcriptomic characterization of dying hair cells in the avian cochlea.

Sensory hair cells are prone to apoptosis caused by various drugs including aminoglycoside antibiotics. In mammals, this vulnerability results in permanent hearing loss because lost hair cells are not regenerated. Conversely, hair cells regenerate in birds, making the avian inner ear an exquisite model for studying ototoxicity and regeneration. Here, we use single-cell RNA sequencing and trajectory analysis on control and dying hair cells after aminoglycoside treatment. Interestingly, the two major subtypes of avian cochlear hair cells, tall and short hair cells, respond differently. Dying short hair cells show a noticeable transient upregulation of many more genes than tall hair cells. The most prominent gene group identified is associated with potassium ion conductances, suggesting distinct physiological differences. Moreover, the dynamic characterization of >15,000 genes expressed in tall and short avian hair cells during their apoptotic demise comprises a resource for further investigations toward mammalian hair cell protection and hair cell regeneration.

Greater epithelial ridge cells are the principal organoid-forming progenitors of the mouse cochlea.

In mammals, hearing loss is irreversible due to the lack of regenerative potential of non-sensory cochlear cells. Neonatal cochlear cells, however, can grow into organoids that harbor sensory epithelial cells, including hair cells and supporting cells. Here, we purify different cochlear cell types from neonatal mice, validate the composition of the different groups with single-cell RNA sequencing (RNA-seq), and assess the various groups' potential to grow into inner ear organoids. We find that the greater epithelial ridge (GER), a transient cell population that disappears during post-natal cochlear maturation, harbors the most potent organoid-forming cells. We identified three distinct GER cell groups that correlate with a specific spatial distribution of marker genes. Organoid formation was synergistically enhanced when the cells were cultured at increasing density. This effect is not due to diffusible signals but requires direct cell-to-cell contact. Our findings improve the development of cell-based assays to study culture-generated inner ear cell types.