RESUMO
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.
Assuntos
Galinhas , Células Ciliadas Auditivas , Animais , Antibacterianos , Cóclea , Células Ciliadas Auditivas/metabolismo , Mamíferos , RNA/metabolismoRESUMO
Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.
Assuntos
Linhagem da Célula/genética , Cóclea/citologia , Estudos de Associação Genética , Mitose , Biossíntese de Proteínas , Regeneração/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Sobrevivência Celular , Células Epiteliais/citologia , Regulação da Expressão Gênica , Integrases/metabolismo , Camundongos , Família Multigênica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
While the mouse has been a productive model for inner ear studies, a lack of highly specific genes and tools has presented challenges. The absence of definitive otic lineage markers and tools is limiting in vitro studies of otic development, where innate cellular heterogeneity and disorganization increase the reliance on lineage-specific markers. To address this challenge in mice and embryonic stem (ES) cells, we targeted the lineage-specific otic gene Fbxo2 with a multicistronic reporter cassette (Venus/Hygro/CreER = VHC). In otic organoids derived from ES cells, Fbxo2VHC specifically delineates otic progenitors and inner ear sensory epithelia. In mice, Venus expression and CreER activity reveal a cochlear developmental gradient, label the prosensory lineage, show enrichment in a subset of type I vestibular hair cells, and expose strong expression in adult cerebellar granule cells. We provide a toolbox of multiple spectrally distinct reporter combinations for studies that require use of fluorescent reporters, hygromycin selection, and conditional Cre-mediated recombination.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Orelha Interna/embriologia , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Animais , Linhagem da Célula , Cóclea/embriologia , Cóclea/metabolismo , Orelha Interna/metabolismo , Células-Tronco Embrionárias/fisiologia , Epitélio/metabolismo , Gânglios Parassimpáticos , Engenharia Genética/métodos , Humanos , Imuno-Histoquímica/métodos , Integrases , Camundongos , Camundongos Transgênicos , TamoxifenoRESUMO
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.
Assuntos
Células Ciliadas Auditivas , Perda Auditiva , Humanos , Animais , Transdução de Sinais/fisiologia , Galinhas/metabolismo , Perda Auditiva/metabolismo , Receptores ErbB/metabolismo , Mamíferos/metabolismoRESUMO
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.
Assuntos
Regeneração , Análise de Célula Única , Transcriptoma , Humanos , Animais , Regeneração/genética , Camundongos , Sáculo e Utrículo/metabolismo , Sáculo e Utrículo/citologia , Neuroma Acústico/genética , Neuroma Acústico/metabolismo , Neuroma Acústico/patologia , Orelha Interna/metabolismo , Orelha Interna/citologia , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/genética , Masculino , Células Ciliadas Vestibulares/metabolismo , Feminino , Perfilação da Expressão GênicaRESUMO
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.
Assuntos
Cóclea , Células-Tronco Neurais , Camundongos , Animais , Células-Tronco Neurais/metabolismo , Neurônios , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
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.
Assuntos
Células Ciliadas Auditivas , Sáculo e Utrículo , Animais , Galinhas , Células Epiteliais , MamíferosRESUMO
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 (<48âh). 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 (6.4â±â9.2 vs 35.6â±â23.7âmin, respectively) as well as a shorter time between pronouncements of death to organ recovery (22.6â±â30.4 vs 64.8â±â22.8âh, 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.
Assuntos
Orelha Interna , Obtenção de Tecidos e Órgãos , Morte Encefálica , Humanos , Seleção de Pacientes , Estudos Retrospectivos , Doadores de TecidosRESUMO
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.
Assuntos
Cóclea/fisiologia , Células Epiteliais/metabolismo , Organoides/metabolismo , Animais , Células Progenitoras Linfoides , CamundongosRESUMO
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.
Assuntos
Galinhas/anatomia & histologia , Cóclea/citologia , Animais , Biomarcadores/metabolismo , Epitélio/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Ciliadas Auditivas Internas/citologia , Células Labirínticas de Suporte/citologia , RNA-Seq , Reprodutibilidade dos Testes , Análise de Célula ÚnicaRESUMO
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.
Assuntos
Galinhas/genética , Células Ciliadas Auditivas/patologia , Transcriptoma/genética , Aminoglicosídeos/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Canais Semicirculares/efeitos dos fármacos , Canais Semicirculares/metabolismo , Sisomicina/administração & dosagem , Sisomicina/farmacologia , Fatores de Tempo , Transcriptoma/efeitos dos fármacosRESUMO
Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/fisiologia , Células Ciliadas Vestibulares/fisiologia , Proteoma/análise , Animais , Embrião de Galinha , Expressão Gênica , Células Ciliadas Auditivas/química , Células Ciliadas Vestibulares/química , Espectrometria de Massas , ProteômicaRESUMO
Hearing loss can develop as a consequence of primary auditory neuron degeneration. These neurons are present within the spiral ganglion of the inner ear and co-exist with glial cells that assist in neuronal maintenance and function. There are limited interventions for individuals with hearing impairment, hence novel biological solutions must be explored. Regenerative strategies can benefit from in vitro methods to examine the long-term culture of purified cell populations. The culturing of neuronal, glial, and non-neuronal, non-glial cell types in both neonatal and adult mice is presented along with the whole-organ explant culture of the spiral ganglion. High yields of spiral ganglion glial and non-glial cells were cultured from both neonatal and adult mice. Dissociated spiral ganglion cells from Sox2-EGFP mice were sorted based on EGFP expression using fluorescence activated cell sorting. The EGFP+ fraction included purified glial populations, whereas the EGFP- fraction contained non-glial cells. Purified glial cells could be reprogrammed into induced neurons displaying neuronal markers and morphology at a higher efficiency than non-glial cells. Previous studies have only allowed for the short-term culturing of spiral ganglion cell populations and have placed emphasis on neonatal cells. There has also been a lack of methods able to cultivate pure cell populations. Here, the coupling of transgenic mouse lines, fluorescence activated cell sorting and advanced culture conditions allow cultivation and characterization of neuronal, glial and non-neuronal, non-glial cells from the spiral ganglion. These techniques are used to demonstrate that different spiral ganglion cell subtypes (glial vs. non-glial) display different competencies for direct neuronal reprogramming.
RESUMO
This article was updated to correct a formatting error in Table 1.
RESUMO
In this study, we present a systematic characterization of hair cell loss and regeneration in the chicken utricle in vivo. A single unilateral surgical delivery of streptomycin caused robust decline of hair cell numbers in striolar as well as extrastriolar regions, which in the striola was detected very early, 6 h post-insult. During the initial 12 h of damage response, we observed global repression of DNA replication, in contrast to the natural, mitotic hair cell production in undamaged control utricles. Regeneration of hair cells in striolar and extrastriolar regions occurred via high rates of asymmetric supporting cell divisions, accompanied by delayed replenishment by symmetric division. While asymmetric division of supporting cells is the main regenerative response to aminoglycoside damage, the detection of symmetric divisions supports the concept of direct transdifferentiation where supporting cells need to be replenished after their phenotypic conversion into new hair cells. Supporting cell divisions appear to be well coordinated because total supporting cell numbers throughout the regenerative process were invariant, despite the initial large-scale loss of hair cells. We conclude that a single ototoxic drug application provides an experimental framework to study the precise onset and timing of utricle hair cell regeneration in vivo. Our findings indicate that initial triggers and signaling events occur already within a few hours after aminoglycoside exposure. Direct transdifferentiation and asymmetric division of supporting cells to generate new hair cells subsequently happen largely in parallel and persist for several days.
Assuntos
Aminoglicosídeos/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Sáculo e Utrículo/efeitos dos fármacos , Animais , Galinhas , Replicação do DNA/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas/fisiologia , Regeneração/fisiologiaRESUMO
This article was updated to correct a formatting error in Table 1.
RESUMO
Protruding from the apical surface of inner ear sensory cells, hair bundles carry out mechanotransduction. Bundle growth involves sequential and overlapping cellular processes, which are concealed within gene expression profiles of individual cells. To dissect such processes, we developed CellTrails, a tool for uncovering, analyzing, and visualizing single-cell gene-expression dynamics. Utilizing quantitative gene-expression data for key bundle proteins from single cells of the developing chick utricle, we reconstructed de novo a bifurcating trajectory that spanned from progenitor cells to mature striolar and extrastriolar hair cells. Extraction and alignment of developmental trails and association of pseudotime with bundle length measurements linked expression dynamics of individual genes with bundle growth stages. Differential trail analysis revealed high-resolution dynamics of transcripts that control striolar and extrastriolar bundle development, including those that encode proteins that regulate [Ca2+]i or mediate crosslinking and lengthening of actin filaments.
Assuntos
Células Ciliadas Auditivas/citologia , Morfogênese/genética , Software , Transcrição Gênica , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Diferenciação Celular , Galinhas , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/ultraestrutura , Camundongos , Sáculo e Utrículo/citologia , Fatores de TempoRESUMO
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.