Tympanic border cells are Wnt-responsive and can act as progenitors for postnatal mouse cochlear cells.

Permanent hearing loss is caused by the irreversible damage of cochlear sensory hair cells and nonsensory supporting cells. In the postnatal cochlea, the sensory epithelium is terminally differentiated, whereas tympanic border cells (TBCs) beneath the sensory epithelium are proliferative. The functions of TBCs are poorly characterized. Using an Axin2(lacZ) Wnt reporter mouse, we found transient but robust Wnt signaling and proliferation in TBCs during the first 3 postnatal weeks, when the number of TBCs decreases. In vivo lineage tracing shows that a subset of hair cells and supporting cells is derived postnatally from Axin2-expressing TBCs. In cochlear explants, Wnt agonists stimulated the proliferation of TBCs, whereas Wnt inhibitors suppressed it. In addition, purified Axin2(lacZ) cells were clonogenic and self-renewing in culture in a Wnt-dependent manner, and were able to differentiate into hair cell-like and supporting cell-like cells. Taken together, our data indicate that Axin2-positive TBCs are Wnt responsive and can act as precursors to sensory epithelial cells in the postnatal cochlea.

Transient, afferent input-dependent, postnatal niche for neural progenitor cells in the cochlear nucleus.

In the cochlear nucleus (CN), the first central relay of the auditory pathway, the survival of neurons during the first weeks after birth depends on afferent innervation from the cochlea. Although input-dependent neuron survival has been extensively studied in the CN, neurogenesis has not been evaluated as a possible mechanism of postnatal plasticity. Here we show that new neurons are born in the CN during the critical period of postnatal plasticity. Coincidently, we found a population of neural progenitor cells that are controlled by a complex interplay of Wnt, Notch, and TGFß/BMP signaling, in which low levels of TGFß/BMP signaling are permissive for progenitor proliferation that is promoted by Wnt and Notch activation. We further show that cells with activated Wnt signaling reside in the CN and that these cells have high propensity for neurosphere formation. Cochlear ablation resulted in diminishment of progenitors and Wnt/ß-catenin-active cells, suggesting that the neonatal CN maintains an afferent innervation-dependent population of progenitor cells that display active canonical Wnt signaling.

Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea.

Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic, but it harbors cells that can behave as progenitor cells in vitro, including the ability to form new hair cells. Lgr5, a Wnt target gene, marks distinct supporting cell types in the neonatal cochlea. Here, we tested the hypothesis that Lgr5(+) cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior, Lgr5(+) cells isolated by flow cytometry from neonatal Lgr5(EGFP-CreERT2/+) mice proliferated and formed clonal colonies. After 10 d in culture, new sensory cells formed and displayed specific hair cell markers (myo7a, calretinin, parvalbumin, myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells, Lgr5(+) cells were enriched precursors to myo7a(+) cells, most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely, small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a(+) cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5(+) cells give rise to myo7a(+) hair cells in the neonatal Lgr5(EGFP-CreERT2/+) cochlea. In addition, overexpression of ß-catenin initiated proliferation and led to transient expansion of Lgr5(+) cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development.

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.

Investigating the Minimal Clinically Important Difference for AzBio and CNC Speech Recognition Scores.

OBJECTIVE: To assess the minimal clinically important difference (MCID) values for cochlear implant-related speech recognition scores, which have not been previously reported. STUDY DESIGN: Retrospective cohort. SETTING: Tertiary referral center. PATIENTS: Eight hundred sixty-three adult patients who underwent cochlear implantation between 2009 and 2022. MAIN OUTCOME MEASURES: MCID values for consonant-nucleus-consonant (CNC) word scores and AzBio sentences in quiet and noise scores using distribution-based methods (half-standard deviation, standard error of measurement, Cohen's d, and minimum detectable change). RESULTS: In this cohort, the mean preoperative CNC word score was 13.9% (SD, 15.6). The mean preoperative AzBio sentences in quiet score was 19.1% (SD, 22.1), and the mean preoperative AzBio sentences in noise score was 13.0% (SD, 12.0). The average MCID values of several distribution-based methods for CNC, AzBio in quiet, and AzBio in noise were 7.4%, 9.0%, and 4.9%, respectively. Anchor-based approaches with the Speech, Spatial, and Qualities of hearing patient-reported measure did not have strong classification accuracy across CNC or AzBio in quiet and noise scores (ROC areas under-the-curve ≤0.69), highlighting weak associations between improvements in speech recognition scores and subjective hearing-related abilities. CONCLUSIONS: Our estimation of MCID values for CNC and AzBio in quiet and noise allows for enhanced patient counseling and clinical interpretation of past, current, and future research studies assessing cochlear implant outcomes.

Digital Otoscopy With Computer-Aided Composite Image Generation: Impact on the Correct Diagnosis, Confidence, and Time.

OBJECTIVE: This study investigated the comparative performance of ear, nose, and throat (ENT) physicians in correctly detecting ear abnormalities when reviewing digital otoscopy imaging using 3 different visualization methods, including computer-assisted composite images called "SelectStitch," single video frame "Still" images, and video clips. The study also explored clinicians' diagnostic confidence levels and the time to make a diagnosis. STUDY DESIGN: Clinician diagnostic reader study. SETTING: Online diagnostic survey of ENT physicians. METHODS: Nine ENT physicians reviewed digital otoscopy examinations from 86 ears with various diagnoses (normal, perforation, retraction, middle ear effusion, tympanosclerosis). Otoscopy examinations used artificial-intelligence (AI)-based computer-aided composite image generation from a video clip (SelectStitch), manually selected best still frame from a video clip (Still), or the entire video clip. Statistical analyses included comparisons of ability to detect correct diagnosis, confidence levels, and diagnosis times. RESULTS: The ENT physicians' ability to detect ear abnormalities (33.2%-68.7%) varied depending on the pathologies. SelectStitch and Still images were not statistically different in detecting abnormalities (P > .50), but both were different from Video (P < .01). However, the performance improvement observed with Videos came at the cost of significantly longer time to determining the diagnosis. The level of confidence in the diagnosis was positively associated with correct diagnoses, but varied by particular pathology. CONCLUSION: This study explores the potential of computer-assisted techniques like SelectStitch in enhancing otoscopic diagnoses and time-saving, which could benefit telemedicine settings. Comparable performance between computer-generated and manually selected images suggests the potential of AI algorithms for otoscopy applications.

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.

RNA Sequencing and Gene Ontology Analysis in Acute Invasive Fungal Sinusitis.

BACKGROUND: Acute invasive fungal sinusitis (AIFS) is an aggressive and dangerous disease of the paranasal sinuses with high morbidity and mortality. The immune response at the level of the nasal mucosa, the site of entry, has not been previously evaluated. OBJECTIVE: To evaluate differential gene expression in the sinonasal mucosa of AIFS patients as compared to control patients using RNA sequencing. METHODS: Sinonasal tissue samples were prospectively obtained from consenting patients undergoing surgery between November, 2020 and November, 2021. RNA extraction and sequencing were performed and differential expression was analyzed to detect transcriptional differences between patient groups. RESULTS: Tissue samples were collected from 4 patients with active AIFS diagnoses, 2 patients with recovered AIFS, 1 patient with a diagnosis of non-invasive fungal ball, and 4 healthy controls. 255 genes were differentially expressed in AIFS patients as compared to control patients. Specific Gene Ontology (GO) biological processes that were identified as differentially expressed in AIFS patients as compared to controls included the following: 1. GO:0007155 (cell adhesion), 2. GO:0030199 (collagen fibril organization) and 3. GO:0001525 (angiogenesis). CONCLUSION: Transcriptional differences were noted between AIFS and control patients in sinonasal tissue samples. Future work is necessary to determine causes of the differential gene expressions between AIFS and control patients, specifically those who are immunosuppressed, or with preexisting non-invasive forms of fungal sinusitis, to guide treatment and prevention strategies.

TAD evolutionary and functional characterization reveals diversity in mammalian TAD boundary properties and function.

Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find 14% of all human TAD boundaries to be shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons compared to species-specific boundaries. CRISPR-Cas9 knockouts of an ultraconserved boundary in a mouse model lead to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in the upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations and showcases the functional importance of TAD evolution.

TAD Evolutionary and functional characterization reveals diversity in mammalian TAD boundary properties and function.

Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species, and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find that only 14% of all human TAD boundaries are shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons, compared to species-specific boundaries. CRISPR-Cas9 knockouts of two ultraconserved boundaries in mouse models leads to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations, and showcase the functional importance of TAD evolution.

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.

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.

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.

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.

American Neurotology Society, American Otological Society, and American Academy of Otolaryngology - Head and Neck Foundation Guide to Enhance Otologic and Neurotologic Care During the COVID-19 Pandemic.

: This combined American Neurotology Society, American Otological Society, and American Academy of Otolaryngology - Head and Neck Surgery Foundation document aims to provide guidance during the coronavirus disease of 2019 (COVID-19) on 1) "priority" of care for otologic and neurotologic patients in the office and operating room, and 2) optimal utilization of personal protective equipment. Given the paucity of evidence to inform otologic and neurotologic best practices during COVID-19, the recommendations herein are based on relevant peer-reviewed articles, the Centers for Disease Control and Prevention COVID-19 guidelines, United States and international hospital policies, and expert opinion. The suggestions presented here are not meant to be definitive, and best practices will undoubtedly change with increasing knowledge and high-quality data related to COVID-19. Interpretation of this guidance document is dependent on local factors including prevalence of COVID-19 in the surgeons' local community. This is not intended to set a standard of care, and should not supersede the clinician's best judgement when managing specific clinical concerns and/or regional conditions.Access to otologic and neurotologic care during and after the COVID-19 pandemic is dependent upon adequate protection of physicians, audiologists, and ancillary support staff. Otolaryngologists and associated staff are at high risk for COVID-19 disease transmission based on close contact with mucosal surfaces of the upper aerodigestive tract during diagnostic evaluation and therapeutic procedures. While many otologic and neurotologic conditions are not imminently life threatening, they have a major impact on communication, daily functioning, and quality of life. In addition, progression of disease and delay in treatment can result in cranial nerve deficits, intracranial and life-threatening complications, and/or irreversible consequences. In this regard, many otologic and neurotologic conditions should rightfully be considered "urgent," and almost all require timely attention to permit optimal outcomes. It is reasonable to proceed with otologic and neurotologic clinic visits and operative cases based on input from expert opinion of otologic care providers, clinic/hospital administration, infection prevention and control specialists, and local and state public health leaders. Significant regional variations in COVID-19 prevalence exist; therefore, physicians working with local municipalities are best suited to make determinations on the appropriateness and timing of otologic and neurotologic care.

Genetic analysis of posterior medial barrel subfield (PMBSF) size in somatosensory cortex (SI) in recombinant inbred strains of mice.

BACKGROUND: Quantitative trait locus (QTL) mapping is an important tool for identifying potential candidate genes linked to complex traits. QTL mapping has been used to identify genes associated with cytoarchitecture, cell number, brain size, and brain volume. Previously, QTL mapping was utilized to examine variation of barrel field size in the somatosensory cortex in a limited number of recombinant inbred (RI) strains of mice. In order to further elucidate the underlying natural variation in mouse primary somatosensory cortex, we measured the size of the posterior medial barrel subfield (PMBSF), associated with the representation of the large mystacial vibrissae, in an expanded sample set that included 42 BXD RI strains, two parental strains (C57BL/6J and DBA/2J), and one F1 strain (B6D2F1). Cytochrome oxidase labeling was used to visualize barrels within the PMBSF. RESULTS: We observed a 33% difference between the largest and smallest BXD RI strains with continuous variation in-between. Using QTL linkage analysis from WebQTL, we generated linkage maps of raw total PMBSF and brain weight adjusted total PMBSF areas. After removing the effects of brain weight, we detected a suggestive QTL (likelihood ratio statistic [LRS]: 14.20) on the proximal arm of chromosome 4. Candidate genes under the suggestive QTL peak for PMBSF area were selected based on the number of single nucleotide polymorphisms (SNPs) present and the biological relevance of each gene. Among the candidate genes are Car8 and Rab2. More importantly, mRNA expression profiles obtained using GeneNetwork indicated a strong correlation between total PMBSF area and two genes (Adcy1 and Gap43) known to be important in mouse cortex development. GAP43 has been shown to be critical during neurodevelopment of the somatosensory cortex, while knockout Adcy1 mice have disrupted barrel field patterns. CONCLUSION: We detected a novel suggestive QTL on chromosome 4 that is linked to PMBSF size. The present study is an important step towards identifying genes underlying the size and possible development of cortical structures.

Genetic analysis of tongue size and taste papillae number and size in recombinant inbred strains of mice.

Quantitative trait loci (QTLs) analysis has been used to examine natural variation of phenotypes in the mouse somatosensory cortex, hippocampus, cerebellum, and amygdala. QTL analysis has also been utilized to map and identify genes underlying anatomical features such as muscle, organ, and body weights. However, this methodology has not been previously applied to identification of anatomical structures related to gustatory phenotypes. In this study, we used QTL analysis to map and characterize genes underlying tongue size, papillae number, and papillae area. In a set of 43 BXD recombinant inbred (RI) mice (n = 111) and 2 parental strains (C57BL/6J and DBA/2J; n = 7), we measured tongue length, width, and weight. In a subset of 23 BXD RI mice and the parental mice, we measured filiform and fungiform papillae number and fungiform papillae area. Using QTL linkage analysis (through WebQTL), we detected 2 significant and noninteracting QTLs influencing tongue length on chromosomes 5 and 7. We also found a significant QTL on chromosome 19 underlying fungiform papillae area and a suggestive QTL on chromosome 2 linked to fungiform papillae number. From these QTLs, we identified a number of candidate genes within the QTL intervals that include SRY-box containing gene, nebulin-related anchoring protein, and actin-binding LIM protein 1. This study is an important first step in identifying genetic factors underlying tongue size, papillae size, and papillae number using QTL analysis.

A functional role for Anopheles gambiae Arrestin1 in olfactory signal transduction.

Insect sensory arrestins act to desensitize visual and olfactory signal transduction pathways, as evidenced by the phenotypic effects of mutations in the genes encoding both Arr1 and Arr2 in Drosophila melanogaster. To assess whether such arrestins play similar roles in other, more medically relevant dipterans, we examined the ability of Anopheles gambiae sensory arrestin homologs AgArr1 and AgArr2 to rescue phenotypes associated with an olfactory deficit observed in D. melanogaster arrestin mutants. Of these, only AgArr1 facilitated significant phenotypic rescue of the corresponding Drosophila arr mutant olfactory phenotype, consistent with the view that functional orthology is shared by these Arr1 homologs. These results represent the first step in the functional characterization of AgArr1, which is highly expressed in olfactory appendages of An. gambiae in which it is likely to play an essential role in olfactory signal transduction. In addition to providing insight into the common elements of the peripheral olfactory system of dipterans, this work validates the importance of AgArr1 as a potential target for novel anti-malaria strategies that focus on olfactory-based behaviors in An. gambiae.

Early postnatal alcohol exposure reduced the size of vibrissal barrel field in rat somatosensory cortex (SI) but did not disrupt barrel field organization.

Prenatal alcohol exposure (PAE) has been shown to alter the somatosensory cortex in both human and animal studies. In rodents, PAE reduced the size, but not the pattern of the posteromedial barrel subfield (PMBSF) associated with the representation of the whiskers, in newborn, juvenile, and adult rats. However, the PMBSF is not present at birth, but rather first appears in the middle of the first postnatal week during the brain-growth spurt period. These findings raise questions whether early postnatal alcohol exposure might disrupt both barrel field pattern and size, questions that were investigated in the present study. Newborn Sprague-Dawley rats were assigned into alcohol (Alc), nutritional gastric control (GC), and suckle control (SC) groups on postnatal day 4 (P4). Rat pups in Alc and GC were artificially fed with alcohol and maltose-dextrin dissolved in milk, respectively, via an implant gastrostomy tube, from P4 to P9. Pups in the Alc group received alcohol (6.0 g/kg) in milk, while the GC controls received isocaloric equivalent maltose-dextrin dissolved in milk. Pups in the SC group remained with their mothers and breast fed throughout the experimental period. On P10, pups in each group were weighed, sacrificed, and their brains removed and weighed. Cortical hemispheres were separated, weighed, flattened, sectioned tangentially, stained with cytochrome oxidase, and PMBSF measured. The sizes of barrels and the interbarrel septal region within PMBSF, as well as body and brain weights were compared between the three groups. The sizes of PMSBF barrel and septal areas were significantly smaller (P<.01) in Alc group compared to controls, while the PMBSF barrel pattern remained unaltered. Body, whole-brain, forebrain, and hemisphere weights were significantly reduced (P<.01) in Alc pups compared to control groups. GC and SC groups did not differ significantly in all dependent variables, except body weight at P9 and P10 (P<.01). These results suggest that postnatal alcohol exposure, like prenatal exposure, significantly influenced the size of the barrel field, but not barrel field pattern formation, indicating that barrel field pattern formation consolidated prior to P4. These results are important for understanding sensorimotor deficits reported in children suffering from fetal alcohol spectrum disorder (FASD).