RESUMEN
The cochlea uses two types of mechanosensory cell to detect sounds. A single row of inner hair cells (IHCs) synapse onto neurons to transmit sensory information to the brain, and three rows of outer hair cells (OHCs) selectively amplify auditory inputs1. So far, two transcription factors have been implicated in the specific differentiation of OHCs, whereas, to our knowledge, none has been identified in the differentiation of IHCs2-4. One such transcription factor for OHCs, INSM1, acts during a crucial embryonic period to consolidate the OHC fate, preventing OHCs from transdifferentiating into IHCs2. In the absence of INSM1, embryonic OHCs misexpress a core set of IHC-specific genes, which we predict are involved in IHC differentiation. Here we find that one of these genes, Tbx2, is a master regulator of IHC versus OHC differentiation in mice. Ablation of Tbx2 in embryonic IHCs results in their development as OHCs, expressing early OHC markers such as Insm1 and eventually becoming completely mature OHCs in the position of IHCs. Furthermore, Tbx2 is epistatic to Insm1: in the absence of both genes, cochleae generate only OHCs, which suggests that TBX2 is necessary for the abnormal transdifferentiation of INSM1-deficient OHCs into IHCs, as well as for normal IHC differentiation. Ablation of Tbx2 in postnatal, largely differentiated IHCs makes them transdifferentiate directly into OHCs, replacing IHC features with those of mature and not embryonic OHCs. Finally, ectopic expression of Tbx2 in OHCs results in their transdifferentiation into IHCs. Hence, Tbx2 is both necessary and sufficient to make IHCs distinct from OHCs and maintain this difference throughout development.
Asunto(s)
Diferenciación Celular , Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas Externas , Animales , Diferenciación Celular/genética , Cóclea/citología , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Externas/citología , Ratones , Proteínas de Dominio T BoxRESUMEN
In multicellular tissues, the size and shape of cells are intricately linked with their physiological functions. In the vertebrate auditory organ, the neurosensory epithelium develops as a mosaic of sensory hair cells (HCs), and their glial-like supporting cells, which have distinct morphologies and functional properties at different frequency positions along its tonotopic long axis. In the chick cochlea, the basilar papilla (BP), proximal (high-frequency) HCs, are larger than their distal (low-frequency) counterparts, a morphological feature essential for sound perception. Mitochondrial dynamics, which constitute the equilibrium between fusion and fission, regulate differentiation and functional refinement across a variety of cell types. We investigate this as a potential mechanism for regulating the shape of developing HCs. Using live imaging in intact BP explants, we identify distinct remodelling of mitochondrial networks in proximal compared with distal HCs. Manipulating mitochondrial dynamics in developing HCs alters their normal morphology along the proximal-distal (tonotopic) axis. Inhibition of the mitochondrial fusion machinery decreased proximal HC surface area, whereas promotion of fusion increased the distal HC surface area. We identify mitochondrial dynamics as a key regulator of HC morphology in developing inner ear epithelia.
Asunto(s)
Cóclea , Células Ciliadas Auditivas , Mitocondrias , Dinámicas Mitocondriales , Animales , Cóclea/embriología , Cóclea/citología , Cóclea/crecimiento & desarrollo , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Mitocondrias/metabolismo , Embrión de Pollo , Forma de la Célula , Pollos , Diferenciación CelularRESUMEN
Intercellular signaling mediated by evolutionarily conserved planar cell polarity (PCP) proteins aligns cell polarity along the tissue plane and drives polarized cell behaviors during tissue morphogenesis. Accumulating evidence indicates that the vertebrate PCP pathway is regulated by noncanonical, ß-catenin-independent Wnt signaling; however, the signaling components and mechanisms are incompletely understood. In the mouse hearing organ, both PCP and noncanonical Wnt (ncWnt) signaling are required in the developing auditory sensory epithelium to control cochlear duct elongation and planar polarity of resident sensory hair cells (HCs), including the shape and orientation of the stereociliary hair bundle essential for sound detection. We have recently discovered a Wnt/G-protein/PI3K pathway that coordinates HC planar polarity and intercellular PCP signaling. Here, we identify Wnt7b as a ncWnt ligand acting in concert with Wnt5a to promote tissue elongation in diverse developmental processes. In the cochlea, Wnt5a and Wnt7b are redundantly required for cochlear duct coiling and elongation, HC planar polarity, and asymmetric localization of core PCP proteins Fzd6 and Dvl2. Mechanistically, Wnt5a/Wnt7b-mediated ncWnt signaling promotes membrane recruitment of Daple, a nonreceptor guanine nucleotide exchange factor for Gαi, and activates PI3K/AKT and ERK signaling, which promote asymmetric Fzd6 localization. Thus, ncWnt and PCP signaling pathways have distinct mutant phenotypes and signaling components, suggesting that they act as separate, parallel pathways with nonoverlapping functions in cochlear morphogenesis. NcWnt signaling drives tissue elongation and reinforces intercellular PCP signaling by regulating the trafficking of PCP-specific Frizzled receptors.
Asunto(s)
Polaridad Celular , Proteínas Wnt , Vía de Señalización Wnt , Proteína Wnt-5a , Animales , Polaridad Celular/fisiología , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Proteína Wnt-5a/metabolismo , Proteína Wnt-5a/genética , Ratones , Vía de Señalización Wnt/fisiología , Cóclea/metabolismo , Cóclea/citología , Cóclea/crecimiento & desarrollo , Células Ciliadas Auditivas/metabolismo , Receptores Frizzled/metabolismo , Receptores Frizzled/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , MorfogénesisRESUMEN
The mouse auditory organ cochlea contains two types of sound receptors: inner hair cells (IHCs) and outer hair cells (OHCs). Tbx2 is expressed in IHCs but repressed in OHCs, and neonatal OHCs that misexpress Tbx2 transdifferentiate into IHC-like cells. However, the extent of this switch from OHCs to IHC-like cells and the underlying molecular mechanism remain poorly understood. Furthermore, whether Tbx2 can transform fully mature adult OHCs into IHC-like cells is unknown. Here, our single-cell transcriptomic analysis revealed that in neonatal OHCs misexpressing Tbx2, 85.6% of IHC genes, including Slc17a8, are upregulated, but only 38.6% of OHC genes, including Ikzf2 and Slc26a5, are downregulated. This suggests that Tbx2 cannot fully reprogram neonatal OHCs into IHCs. Moreover, Tbx2 also failed to completely reprogram cochlear progenitors into IHCs. Lastly, restoring Ikzf2 expression alleviated the abnormalities detected in Tbx2+ OHCs, which supports the notion that Ikzf2 repression by Tbx2 contributes to the transdifferentiation of OHCs into IHC-like cells. Our study evaluates the effects of ectopic Tbx2 expression on OHC lineage development at distinct stages of either male or female mice and provides molecular insights into how Tbx2 disrupts the gene expression profile of OHCs. This research also lays the groundwork for future studies on OHC regeneration.
Asunto(s)
Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas Externas , Proteínas de Dominio T Box , Animales , Proteínas de Dominio T Box/metabolismo , Proteínas de Dominio T Box/genética , Ratones , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Femenino , Animales Recién Nacidos , Transdiferenciación Celular/fisiología , Transdiferenciación Celular/genética , Masculino , Cóclea/metabolismo , Cóclea/citología , Ratones Endogámicos C57BLRESUMEN
The cochlea encodes sound pressures varying over six orders of magnitude by collective operation of functionally diverse spiral ganglion neurons (SGNs). The mechanisms enabling this functional diversity remain elusive. Here, we asked whether the sound intensity information, contained in the receptor potential of the presynaptic inner hair cell (IHC), is fractionated via heterogeneous synapses. We studied the transfer function of individual IHC synapses by combining patch-clamp recordings with dual-color Rhod-FF and iGluSnFR imaging of presynaptic Ca2+ signals and glutamate release. Synapses differed in the voltage dependence of release: Those residing at the IHC' pillar side activated at more hyperpolarized potentials and typically showed tight control of release by few Ca2+ channels. We conclude that heterogeneity of voltage dependence and release site coupling of Ca2+ channels among the synapses varies synaptic transfer within individual IHCs and, thereby, likely contributes to the functional diversity of SGNs. The mechanism reported here might serve sensory cells and neurons more generally to diversify signaling even in close-by synapses.
Asunto(s)
Tronco Encefálico/fisiología , Calcio/metabolismo , Cóclea/fisiología , Potenciales Evocados Auditivos del Tronco Encefálico , Células Ciliadas Auditivas Internas/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Tronco Encefálico/citología , Cóclea/citología , Proteínas de Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células Ciliadas Auditivas Internas/citología , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Cochlear hair cells (HCs) in the inner ear are responsible for sound detection. For HC fate specification, the master transcription factor Atoh1 is both necessary and sufficient. Atoh1 expression is dynamic and tightly regulated during development, but the cis-regulatory elements mediating this regulation remain unresolved. Unexpectedly, we found that deleting the only recognized Atoh1 enhancer, defined here as Eh1, failed to impair HC development. By using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we discovered two additional Atoh1 enhancers: Eh2 and Eh3. Notably, Eh2 deletion was sufficient for impairing HC development, and concurrent deletion of Eh1 and Eh2 or all three enhancers resulted in nearly complete absence of HCs. Lastly, we showed that Atoh1 binds to all three enhancers, consistent with its autoregulatory function. Our findings reveal that the cooperative action of three distinct enhancers underpins effective Atoh1 regulation during HC development, indicating potential therapeutic approaches for HC regeneration.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Oído Interno , Elementos de Facilitación Genéticos , Células Ciliadas Auditivas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular , Cóclea/citología , Oído Interno/citología , Células Ciliadas Auditivas/fisiologíaRESUMEN
Dync1li1, a subunit of cytoplasmic dynein 1, is reported to play important roles in intracellular retrograde transport in many tissues. However, the roles of Dync1li1 in the mammalian cochlea remain uninvestigated. Here we first studied the expression pattern of Dync1li1 in the mouse cochlea and found that Dync1li1 is highly expressed in hair cells (HCs) in both neonatal and adult mice cochlea. Next, we used Dync1li1 knockout (KO) mice to investigate its effects on hearing and found that deletion of Dync1li1 leads to early onset of progressive HC loss via apoptosis and to subsequent hearing loss. Further studies revealed that loss of Dync1li1 destabilizes dynein and alters the normal function of dynein. In addition, Dync1li1 KO results in a thinner Golgi apparatus and the accumulation of LC3+ autophagic vacuoles, which triggers HC apoptosis. We also knocked down Dync1li1 in the OC1 cells and found that the number of autophagosomes were significantly increased while the number of autolysosomes were decreased, which suggested that Dync1li1 knockdown leads to impaired transportation of autophagosomes to lysosomes and therefore the accumulation of autophagosomes results in HC apoptosis. Our findings demonstrate that Dync1li1 plays important roles in HC survival through the regulation of autophagosome transportation.
Asunto(s)
Autofagosomas , Dineínas Citoplasmáticas , Células Ciliadas Auditivas , Animales , Apoptosis/fisiología , Autofagosomas/metabolismo , Cóclea/citología , Cóclea/metabolismo , Dineínas Citoplasmáticas/metabolismo , Dineínas/metabolismo , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , RatonesRESUMEN
The sensory epithelium of the cochlea, the organ of Corti, has complex cytoarchitecture consisting of mechanosensory hair cells intercalated by epithelial support cells. The support cells provide important trophic and structural support to the hair cells. Thus, the support cells must be stiff yet compliant enough to withstand and modulate vibrations to the hair cells. Once the sensory cells are properly patterned, the support cells undergo significant remodeling from a simple epithelium into a structurally rigid epithelium with fluid-filled spaces in the murine cochlea. Cell adhesion molecules such as cadherins are necessary for sorting and connecting cells in an intact epithelium. To create the fluid-filled spaces, cell adhesion properties of adjoining cell membranes between cells must change to allow the formation of spaces within an epithelium. However, the dynamic localization of cadherins has not been properly analyzed as these spaces are formed. There are three cadherins that are reported to be expressed during the first postnatal week of development when the tunnel of Corti forms in the cochlea. In this study, we characterize the dynamic localization of cadherins that are associated with cytoskeletal remodeling at the contacting membranes of the inner and outer pillar cells flanking the tunnel of Corti.
Asunto(s)
Cadherinas , Cóclea , Animales , Cadherinas/metabolismo , Ratones , Epitelio/metabolismo , Cóclea/metabolismo , Cóclea/crecimiento & desarrollo , Cóclea/citología , Órgano Espiral/metabolismo , Órgano Espiral/citología , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/citología , Adhesión Celular/fisiologíaRESUMEN
During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via ß-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.
Asunto(s)
Cóclea/embriología , Epitelio/metabolismo , Células Madre Mesenquimatosas/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Desdiferenciación Celular , Diferenciación Celular , Proliferación Celular , Cóclea/citología , Cóclea/crecimiento & desarrollo , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Proteínas Wnt , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Linaje de la Célula/genética , Cóclea/citología , Estudios de Asociación Genética , Mitosis , Biosíntesis de Proteínas , Regeneración/genética , Células Madre/citología , Células Madre/metabolismo , Animales , Diferenciación Celular , Supervivencia Celular , Células Epiteliales/citología , Regulación de la Expresión Génica , Integrasas/metabolismo , Ratones , Familia de Multigenes , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Planar cell polarity (PCP) is critical for morphogenesis in metazoans. PCP in vertebrates regulates stereocilia alignment in neurosensory cells of the cochlea and closure of the neural tube through convergence and extension movements (CE). Noncanonical Wnt morphogens regulate PCP and CE in vertebrates, but the molecular mechanisms remain unclear. Smurfs are ubiquitin ligases that regulate signaling, cell polarity and motility through spatiotemporally restricted ubiquitination of diverse substrates. Here, we report an unexpected role for Smurfs in controlling PCP and CE. Mice mutant for Smurf1 and Smurf2 display PCP defects in the cochlea and CE defects that include a failure to close the neural tube. Further, we show that Smurfs engage in a noncanonical Wnt signaling pathway that targets the core PCP protein Prickle1 for ubiquitin-mediated degradation. Our work thus uncovers ubiquitin ligases in a mechanistic link between noncanonical Wnt signaling and PCP/CE.
Asunto(s)
Polaridad Celular , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Portadoras/metabolismo , Movimiento Celular , Cóclea/citología , Cóclea/embriología , Proteínas Dishevelled , Proteínas con Dominio LIM , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Tubo Neural/embriología , Defectos del Tubo Neural/embriología , Fosfoproteínas/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMEN
Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein-RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.
Asunto(s)
Proteínas Asociadas a CRISPR/administración & dosificación , Edición Génica/métodos , Genes Dominantes/genética , Terapia Genética/métodos , Pérdida Auditiva/genética , Estimulación Acústica , Alelos , Animales , Animales Recién Nacidos , Umbral Auditivo , Secuencia de Bases , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/uso terapéutico , Sistemas CRISPR-Cas , Supervivencia Celular , Cóclea/citología , Cóclea/metabolismo , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico , Femenino , Fibroblastos , Células Ciliadas Auditivas/citología , Pérdida Auditiva/fisiopatología , Pérdida Auditiva/prevención & control , Humanos , Liposomas , Masculino , Proteínas de la Membrana/genética , Ratones , Reflejo de SobresaltoRESUMEN
The ability of pigeons to sense geomagnetic fields has been conclusively established despite a notable lack of determination of the underlying biophysical mechanisms. Quasi-spherical iron organelles previously termed "cuticulosomes" in the cochlea of pigeons have potential relevance to magnetoreception due to their location and iron composition; however, data regarding the magnetic susceptibility of these structures are currently limited. Here quantum magnetic imaging techniques are applied to characterize the magnetic properties of individual iron cuticulosomes in situ. The stray magnetic fields emanating from cuticulosomes are mapped and compared to a detailed analytical model to provide an estimate of the magnetic susceptibility of the individual particles. The images reveal the presence of superparamagnetic and ferrimagnetic domains within individual cuticulosomes and magnetic susceptibilities within the range 0.029 to 0.22. These results provide insights into the elusive physiological roles of cuticulosomes. The susceptibilities measured are not consistent with a torque-based model of magnetoreception, placing iron storage and stereocilia stabilization as the two leading putative cuticulosome functions. This work establishes quantum magnetic imaging as an important tool to complement the existing array of techniques used to screen for potential magnetic particle-based magnetoreceptor candidates.
Asunto(s)
Cóclea/diagnóstico por imagen , Columbidae/fisiología , Diagnóstico por Imagen/métodos , Hierro , Magnetismo , Orgánulos , Animales , Cóclea/citología , Diagnóstico por Imagen/instrumentación , Campos Magnéticos , Fenómenos Físicos , Materiales InteligentesRESUMEN
Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation.
Asunto(s)
Aciltransferasas/fisiología , Calcio/metabolismo , Exocitosis/fisiología , Células Ciliadas Auditivas/fisiología , Terminales Presinápticos/fisiología , Sinapsis/fisiología , Vesículas Sinápticas/fisiología , Animales , Cóclea/citología , Cóclea/fisiología , Endocitosis , Femenino , Células Ciliadas Auditivas/citología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transmisión SinápticaRESUMEN
The cochlea is an important sensory organ for both balance and sound perception, and the formation of the cochlea is a complex developmental process. The development of the mouse cochlea begins on embryonic day (E)9 and continues until postnatal day (P)21 when the hearing system is considered mature. Small extracellular vesicles (sEVs), with a diameter ranging from 30 to 200 nm, have been considered a significant medium for information communication in both physiological and pathological processes. However, there are no studies exploring the role of sEVs in the development of the cochlea. Here, we isolated tissue-derived sEVs from the cochleae of FVB mice at P3, P7, P14, and P21 by ultracentrifugation. These sEVs were first characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Next, we used small RNA-seq and mass spectrometry to characterize the microRNA transcriptomes and proteomes of cochlear sEVs from mice at different ages. Many microRNAs and proteins were discovered to be related to inner ear development, anatomical structure development, and auditory nervous system development. These results all suggest that sEVs exist in the cochlea and are likely to be essential for the normal development of the auditory system. Our findings provide many sEV microRNA and protein targets for future studies of the roles of cochlear sEVs.
Asunto(s)
Cóclea/metabolismo , Vesículas Extracelulares/metabolismo , MicroARNs/metabolismo , Proteoma/análisis , Transcriptoma , Animales , Cromatografía Líquida de Alta Presión , Cóclea/citología , Ontología de Genes , Ratones , MicroARNs/genética , Proteómica/métodos , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
Age-related hearing impairment (ARHI), one of the most common medical conditions, is strongly heritable, yet its genetic causes remain largely unknown. We conducted a meta-analysis of GWAS summary statistics from multiple hearing-related traits in the UK Biobank (n = up to 330,759) and identified 31 genome-wide significant risk loci for self-reported hearing difficulty (p < 5x10-8), of which eight have not been reported previously in the peer-reviewed literature. We investigated the regulatory and cell specific expression for these loci by generating mRNA-seq, ATAC-seq, and single-cell RNA-seq from cells in the mouse cochlea. Risk-associated genes were most strongly enriched for expression in cochlear epithelial cells, as well as for genes related to sensory perception and known Mendelian deafness genes, supporting their relevance to auditory function. Regions of the human genome homologous to open chromatin in epithelial cells from the mouse were strongly enriched for heritable risk for hearing difficulty, even after adjusting for baseline effects of evolutionary conservation and cell-type non-specific regulatory regions. Epigenomic and statistical fine-mapping most strongly supported 50 putative risk genes. Of these, 39 were expressed robustly in mouse cochlea and 16 were enriched specifically in sensory hair cells. These results reveal new risk loci and risk genes for hearing difficulty and suggest an important role for altered gene regulation in the cochlear sensory epithelium.
Asunto(s)
Cóclea/citología , Sitios Genéticos , Predisposición Genética a la Enfermedad , Pérdida Auditiva/genética , Adulto , Animales , Bancos de Muestras Biológicas , Cromatina/genética , Estudios de Cohortes , Epigenoma , Células Epiteliales/fisiología , Femenino , Estudio de Asociación del Genoma Completo , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/fisiología , Humanos , Ratones Endogámicos ICR , Ratones Endogámicos , Polimorfismo de Nucleótido Simple , Análisis de la Célula Individual , Reino UnidoRESUMEN
Gentamicin is a potent broad-spectrum aminoglycoside antibiotic whose use is hampered by ototoxic side-effects. Hospital gentamicin is a mixture of five gentamicin C-subtypes and several impurities of various ranges of nonexact concentrations. We developed a purification strategy enabling assaying of individual C-subtypes and impurities for ototoxicity and antimicrobial activity. We found that C-subtypes displayed broad and potent in vitro antimicrobial activities comparable to the hospital gentamicin mixture. In contrast, they showed different degrees of ototoxicity in cochlear explants, with gentamicin C2b being the least and gentamicin C2 the most ototoxic. Structure-activity relationships identified sites in the C4'-C6' region on ring I that reduced ototoxicity while preserving antimicrobial activity, thus identifying targets for future drug design and mechanisms for hair cell toxicity. Structure-activity relationship data suggested and electrophysiological data showed that the C-subtypes both bind and permeate the hair cell mechanotransducer channel, with the stronger the binding the less ototoxic the compound. Finally, both individual and reformulated mixtures of C-subtypes demonstrated decreased ototoxicity while maintaining antimicrobial activity, thereby serving as a proof-of-concept of drug reformulation to minimizing ototoxicity of gentamicin in patients.
Asunto(s)
Antibacterianos/farmacología , Cóclea/efectos de los fármacos , Gentamicinas/efectos adversos , Gentamicinas/química , Gentamicinas/farmacología , Animales , Antibacterianos/efectos adversos , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Cóclea/citología , Contaminación de Medicamentos , Gentamicinas/aislamiento & purificación , Células Ciliadas Auditivas/efectos de los fármacos , Hospitales , Canales Iónicos/metabolismo , Mecanotransducción Celular/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Ratas Sprague-Dawley , Sisomicina/farmacología , Relación Estructura-ActividadRESUMEN
P2X7 receptors (P2X7Rs) are associated with numerous pathophysiological mechanisms, and this promotes them as therapeutic targets for certain neurodegenerative conditions. However, the identity of P2X7R-expressing cells in the nervous system remains contentious. Here, we examined P2X7R functionality in auditory nerve cells from rodents of either sex, and determined their functional and anatomic expression pattern. In whole-cell recordings from rat spiral ganglion cultures, the purinergic agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) activated desensitizing currents in spiral ganglion neurons (SGNs) but non-desensitizing currents in glia that were blocked by P2X7R-specific antagonists. In imaging experiments, BzATP gated sustained Ca2+ entry into glial cells. BzATP-gated uptake of the fluorescent dye YO-PRO-1 was reduced and slowed by P2X7R-specific antagonists. In rats, P2X7Rs were immuno-localized predominantly within satellite glial cells (SGCs) and Schwann cells (SCs). P2X7R expression was not detected in the portion of the auditory nerve within the central nervous system. Mouse models allowed further exploration of the distribution of cochlear P2X7Rs. In GENSAT reporter mice, EGFP expression driven via the P2rx7 promoter was evident in SGCs and SCs but was undetectable in SGNs. A second transgenic model showed a comparable cellular distribution of EGFP-tagged P2X7Rs. In wild-type mice the discrete glial expression was confirmed using a P2X7-specific nanobody construct. Our study shows that P2X7Rs are expressed by peripheral glial cells, rather than by afferent neurons. Description of functional signatures and cellular distributions of these enigmatic proteins in the peripheral nervous system (PNS) will help our understanding of ATP-dependent effects contributing to hearing loss and other sensory neuropathies.SIGNIFICANCE STATEMENT P2X7 receptors (P2X7Rs) have been the subject of much scrutiny in recent years. They have been promoted as therapeutic targets in a number of diseases of the nervous system, yet the specific cellular location of these receptors remains the subject of intense debate. In the auditory nerve, connecting the inner ear to the brainstem, we show these multimodal ATP-gated channels localize exclusively to peripheral glial cells rather than the sensory neurons, and are not evident in central glia. Physiologic responses in the peripheral glia display classical hallmarks of P2X7R activation, including the formation of ion-permeable and also macromolecule-permeable pores. These qualities suggest these proteins could contribute to glial-mediated inflammatory processes in the auditory periphery under pathologic disease states.
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Cóclea/metabolismo , Nervio Coclear/metabolismo , Audición/fisiología , Neuroglía/metabolismo , Receptores Purinérgicos P2X7/biosíntesis , Animales , Cóclea/química , Cóclea/citología , Nervio Coclear/química , Nervio Coclear/citología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroglía/química , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X7/análisis , RoedoresRESUMEN
Epigenetic regulation of gene transcription by chromatin remodeling proteins has recently emerged as an important contributing factor in inner ear development. Pathogenic variants in CHD7, the gene encoding Chromodomain Helicase DNA binding protein 7, cause CHARGE syndrome, which presents with malformations in the developing ear. Chd7 is broadly expressed in the developing mouse otocyst and mature auditory epithelium, yet the pathogenic effects of Chd7 loss in the cochlea are not well understood. Here we characterized cochlear epithelial phenotypes in mice with deletion of Chd7 throughout the otocyst (using Foxg1Cre/+ and Pax2Cre), in the otic mesenchyme (using TCre), in hair cells (using Atoh1Cre), in developing neuroblasts (using NgnCre), or in spiral ganglion neurons (using ShhCre/+). Pan-otic deletion of Chd7 resulted in shortened cochleae with aberrant projections and axonal looping, disorganized, supernumerary hair cells at the apical turn and a narrowed epithelium with missing hair cells in the middle region. Deletion of Chd7 in the otic mesenchyme had no effect on overall cochlear morphology. Loss of Chd7 in hair cells did not disrupt their formation or organization of the auditory epithelium. Similarly, absence of Chd7 in spiral ganglion neurons had no effect on axonal projections. In contrast, deletion of Chd7 in developing neuroblasts led to smaller spiral ganglia and disorganized cochlear neurites. Together, these observations reveal dosage-, tissue-, and time-sensitive cell autonomous roles for Chd7 in cochlear elongation and cochlear neuron organization, with minimal functions for Chd7 in hair cells. These studies provide novel information about roles for Chd7 in development of auditory neurons.
Asunto(s)
Tipificación del Cuerpo , Cóclea/embriología , Proteínas de Unión al ADN/fisiología , Animales , Cóclea/citología , Cóclea/inervación , Proteínas de Unión al ADN/genética , Eliminación de Gen , Células Ciliadas Auditivas/fisiología , Ratones , Ratones Noqueados , Morfogénesis/genética , Morfogénesis/fisiología , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/embriologíaRESUMEN
The transcription factor sex determining region Y-box 2 (SOX2) is required for the formation of hair cells and supporting cells in the inner ear and is a widely used sensory marker. Paradoxically, we demonstrate via fate mapping that, initially, SOX2 primarily marks nonsensory progenitors in the mouse cochlea, and is not specific to all sensory regions until late otic vesicle stages. SOX2 fate mapping reveals an apical-to-basal gradient of SOX2 expression in the sensory region of the cochlea, reflecting the pattern of cell cycle exit. To understand SOX2 function, we undertook a timed-deletion approach, revealing that early loss of SOX2 severely impaired morphological development of the ear, whereas later deletions resulted in sensory disruptions. During otocyst stages, SOX2 shifted dramatically from a lateral to medial domain over 24-48â h, reflecting the nonsensory-to-sensory switch observed by fate mapping. Early loss or gain of SOX2 function led to changes in otic epithelial volume and progenitor proliferation, impacting growth and morphological development of the ear. Our study demonstrates a novel role for SOX2 in early otic morphological development, and provides insights into the temporal and spatial patterns of sensory specification in the inner ear.