RESUMEN
The otocyst harbors progenitors for most cell types of the mature inner ear. Developmental lineage analyses and gene expression studies suggest that distinct progenitor populations are compartmentalized to discrete axial domains in the early otocyst. Here, we conducted highly parallel quantitative RT-PCR measurements on 382 individual cells from the developing otocyst and neuroblast lineages to assay 96 genes representing established otic markers, signaling-pathway-associated transcripts, and novel otic-specific genes. By applying multivariate cluster, principal component, and network analyses to the data matrix, we were able to readily distinguish the delaminating neuroblasts and to describe progressive states of gene expression in this population at single-cell resolution. It further established a three-dimensional model of the otocyst in which each individual cell can be precisely mapped into spatial expression domains. Our bioinformatic modeling revealed spatial dynamics of different signaling pathways active during early neuroblast development and prosensory domain specification.
Asunto(s)
Oído Interno/citología , Oído Interno/embriología , Células-Madre Neurales/citología , Análisis de la Célula Individual , Transcriptoma , Animales , Embrión de Mamíferos/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Análisis de Componente PrincipalRESUMEN
The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met3-9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor ß (TGFß) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.
Asunto(s)
Cabello/citología , Cabello/crecimiento & desarrollo , Organoides/citología , Células Madre Pluripotentes/citología , Piel/citología , Animales , Ectodermo/citología , Femenino , Cabello/trasplante , Color del Cabello , Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/inervación , Folículo Piloso/trasplante , Cabeza , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Organoides/crecimiento & desarrollo , Organoides/inervación , Organoides/trasplante , RNA-Seq , Análisis de la Célula Individual , Piel/crecimiento & desarrollo , Piel/inervación , Trasplante de PielRESUMEN
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.
Asunto(s)
Pollos , Células Ciliadas Auditivas , Animales , Antibacterianos , Cóclea , Células Ciliadas Auditivas/metabolismo , Mamíferos , ARN/metabolismoRESUMEN
Mechanosensitive sensory hair cells are the linchpin of our senses of hearing and balance. The inability of the mammalian inner ear to regenerate lost hair cells is the major reason for the permanence of hearing loss and certain balance disorders. Here, we present a stepwise guidance protocol starting with mouse embryonic stem and induced pluripotent stem cells, which were directed toward becoming ectoderm capable of responding to otic-inducing growth factors. The resulting otic progenitor cells were subjected to varying differentiation conditions, one of which promoted the organization of the cells into epithelial clusters displaying hair cell-like cells with stereociliary bundles. Bundle-bearing cells in these clusters responded to mechanical stimulation with currents that were reminiscent of immature hair cell transduction currents.
Asunto(s)
Células Madre Embrionarias/citología , Células Ciliadas Auditivas/citología , Células Ciliadas Vestibulares/citología , Células Madre Pluripotentes Inducidas/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Ciliadas Auditivas/fisiología , Células Ciliadas Auditivas/ultraestructura , Células Ciliadas Vestibulares/fisiología , Células Ciliadas Vestibulares/ultraestructura , Mecanotransducción Celular , RatonesRESUMEN
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.
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Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Oído Interno/embriología , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Animales , Linaje de la Célula , Cóclea/embriología , Cóclea/metabolismo , Oído Interno/metabolismo , Células Madre Embrionarias/fisiología , Epitelio/metabolismo , Ganglios Parasimpáticos , Ingeniería Genética/métodos , Humanos , Inmunohistoquímica/métodos , Integrasas , Ratones , Ratones Transgénicos , TamoxifenoRESUMEN
Efficient pluripotent stem cell guidance protocols for the production of human posterior cranial placodes such as the otic placode that gives rise to the inner ear do not exist. Here we use a systematic approach including defined monolayer culture, signaling modulation, and single-cell gene expression analysis to delineate a developmental trajectory for human otic lineage specification in vitro. We found that modulation of bone morphogenetic protein (BMP) and WNT signaling combined with FGF and retinoic acid treatments over the course of 18 days generates cell populations that develop chronological expression of marker genes of non-neural ectoderm, preplacodal ectoderm, and early otic lineage. Gene expression along this differentiation path is distinct from other lineages such as endoderm, mesendoderm, and neural ectoderm. Single-cell analysis exposed the heterogeneity of differentiating cells and allowed discrimination of non-neural ectoderm and otic lineage cells from off-target populations. Pseudotemporal ordering of human embryonic stem cell and induced pluripotent stem cell-derived single-cell gene expression profiles revealed an initially synchronous guidance toward non-neural ectoderm, followed by comparatively asynchronous occurrences of preplacodal and otic marker genes. Positive correlation of marker gene expression between both cell lines and resemblance to mouse embryonic day 10.5 otocyst cells implied reasonable robustness of the guidance protocol. Single-cell trajectory analysis further revealed that otic progenitor cell types are induced in monolayer cultures, but further development appears impeded, likely because of lack of a lineage-stabilizing microenvironment. Our results provide a framework for future exploration of stabilizing microenvironments for efficient differentiation of stem cell-generated human otic cell types.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Oído Interno/citología , Ectodermo/citología , Análisis de la Célula Individual/métodos , Animales , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Células Cultivadas , Oído Interno/embriología , Ectodermo/efectos de los fármacos , Ectodermo/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
BACKGROUND: This is an investigation of complete arterial coronary artery bypass grafting (CACABG) using bilateral internal mammary arteries (IMA) and the T-graft technique either on- or off-pump as a routine approach to treat coronary artery disease. METHODS: Between January 2000 and December 2012, 3,445 patients underwent on-pump (n = 2,216) or off-pump (n = 1,229) CACABG. A 30-day follow-up was performed prospectively, a long-term follow-up by a questionnaire, and coronary angiography in selected patients. RESULTS: End points at 30 days were death, myocardial infarction, stroke, repeat revascularization, renal replacement, reoperation, sternal wound infection and atrial fibrillation. FitzGibbon A patency rates were 89.8 vs. 91.4% (p = 0.464) with consecutive percutaneous coronary intervention in the grafted area of 1.8 vs. 1.1% (p = 0.693) on- vs. off-pump, and no reoperation in the grafted area in both groups. CONCLUSION: CACABG by use of skeletonized bilateral IMA with the T-graft technique performed either on- or off-pump is a safe and effective approach.
Asunto(s)
Puente de Arteria Coronaria/mortalidad , Puente de Arteria Coronaria/métodos , Enfermedad de la Arteria Coronaria/cirugía , Arterias Mamarias/cirugía , Anciano , Angiografía Coronaria , Puente de Arteria Coronaria/efectos adversos , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Femenino , Estudios de Seguimiento , Alemania , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/epidemiología , Intervención Coronaria Percutánea , Reoperación/estadística & datos numéricos , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Cóclea/crecimiento & desarrollo , Cóclea/fisiología , Oído Medio/citología , Células Madre/fisiología , Vía de Señalización Wnt/fisiología , Animales , Animales Recién Nacidos , Proteína Axina/genética , Proteína Axina/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Pollos , Cóclea/citología , Oído Medio/metabolismo , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/fisiología , Ratones , Ratones Transgénicos , Modelos Biológicos , Células Madre/citología , Células Madre/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
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.
Asunto(s)
Núcleo Coclear/fisiología , Células-Madre Neurales/fisiología , Animales , Proliferación Celular , Núcleo Coclear/citología , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/citología , NeurogénesisRESUMEN
Single cell trajectory analysis is a computational approach that orders cells along a pseudotime axis. This temporal modeling approach allows the characterization of transitional processes such as lineage development, response to insult, and tissue regeneration. The concept can also be applied to resolve spatial organization of cells within the originating tissue. Known as temporal and spatial transcriptomics, respectively, these methods belong to the most powerful analytical techniques for quantitative gene expression data currently available. Here, we discuss three different approaches: principal component analysis, the 'Monocle' algorithm, and self-organizing maps. We use a previously published qRT-PCR dataset of single neuroblast cells isolated from the developing mouse inner ear to highlight the basic features of the three methods and their individual limitations, as well as the distinct advantages that make them useful for research on the inner ear. The complex developmental morphogenesis of the inner ear and its specific challenges such as the paucity of cells as well as important open questions such as sensory hair cell regeneration render this organ a prime target for single cell trajectory analysis strategies.
Asunto(s)
Cóclea/embriología , Oído Interno/embriología , Regeneración/fisiología , Análisis de la Célula Individual/métodos , HumanosRESUMEN
BACKGROUND: Cisplatin is a widely used chemotherapeutic agent that can also cause ototoxic injury. One potential treatment for cisplatin-induced hearing loss involves the activation of endogenous inner ear stem cells, which may then produce replacement hair cells. In this series of experiments, we examined the effects of cisplatin exposure on both hair cells and resident stem cells of the mouse inner ear. RESULTS: Treatment for 24 hr with 10 µM cisplatin caused significant loss of hair cells in the mouse utricle, but such damage was not evident until 4 days after the cisplatin exposure. In addition to killing hair cells, cisplatin treatment also disrupted the actin cytoskeleton in remaining supporting cells, and led to increased histone H2AX phosphorylation within the sensory epithelia. Finally, treatment with 10 µM cisplatin appeared to have direct toxic effects on resident stem cells in the mouse utricle. Exposure to cisplatin blocked the proliferation of isolated stem cells and prevented sphere formation when those cells were maintained in suspension culture. CONCLUSION: The results suggest that inner ear stem cells may be injured during cisplatin ototoxicity, thus limiting their ability to mediate sensory repair.
Asunto(s)
Antineoplásicos/efectos adversos , Cisplatino/efectos adversos , Oído Interno/efectos de los fármacos , Oído Interno/embriología , Células Madre Embrionarias/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Oído Interno/citología , Células Madre Embrionarias/fisiología , Femenino , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/fisiología , Ratones , Ratones Endogámicos C57BL , Embarazo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Sáculo y Utrículo/citología , Sáculo y Utrículo/efectos de los fármacos , Sáculo y Utrículo/embriologíaRESUMEN
In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.
Asunto(s)
Nervios Periféricos/crecimiento & desarrollo , Nervios Periféricos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Nervio Coclear/anomalías , Nervio Coclear/metabolismo , Nervio Coclear/ultraestructura , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Proteína P0 de la Mielina/genética , Proteína P0 de la Mielina/metabolismo , Factor 6 de Transcripción de Unión a Octámeros/genética , Factor 6 de Transcripción de Unión a Octámeros/metabolismo , Nervios Periféricos/patología , Nervios Periféricos/ultraestructura , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Schwann/metabolismoRESUMEN
Losing either type of cochlear sensory hair cells leads to hearing impairment. Inner hair cells act as primary mechanoelectrical transducers, while outer hair cells enhance sound-induced vibrations within the organ of Corti. Established inner ear damage 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 h, leading to irreversible hearing loss. The combination of sisomicin and hyperosmotic stress caused consistent and synergistic ototoxic damage. Inner hair cells remained until three days post-treatment, after which deterioration in structure and number was observed, culminating in a complete hair 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.
Asunto(s)
Modelos Animales de Enfermedad , Pérdida Auditiva , Animales , Ratones , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/patología , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/patología , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/patología , Apoptosis/efectos de los fármacos , Aminoglicósidos/administración & dosificación , Aminoglicósidos/efectos adversos , Aminoglicósidos/toxicidad , Presión OsmóticaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Cóclea , Modelos Animales de Enfermedad , Células Ciliadas Auditivas , Regeneración , Animales , Células Ciliadas Auditivas/patología , Células Ciliadas Auditivas/metabolismo , Ratones , Cóclea/patología , Cóclea/fisiopatología , Humanos , Audición , Pérdida Auditiva Provocada por Ruido/fisiopatología , Pérdida Auditiva Provocada por Ruido/patología , Pérdida Auditiva/patología , Pérdida Auditiva/fisiopatología , Estimulación AcústicaRESUMEN
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.
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Pollos , Pérdida Auditiva , Animales , Células Ciliadas Auditivas/fisiología , Audición , Sonido , MamíferosRESUMEN
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.
Asunto(s)
Pollos , Células Ciliadas Auditivas , Inmunohistoquímica , ARN Mensajero , Animales , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mensajero/análisis , Inmunohistoquímica/métodos , Células Ciliadas Auditivas/metabolismoRESUMEN
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.
Asunto(s)
Células Ciliadas Auditivas , Pérdida Auditiva , Humanos , Animales , Transducción de Señal/fisiología , Pollos/metabolismo , Pérdida Auditiva/metabolismo , Receptores ErbB/metabolismo , Mamíferos/metabolismoRESUMEN
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.
Asunto(s)
Regeneración , Análisis de la Célula Individual , Transcriptoma , Humanos , Animales , Regeneración/genética , Ratones , Sáculo y Utrículo/metabolismo , Sáculo y Utrículo/citología , Neuroma Acústico/genética , Neuroma Acústico/metabolismo , Neuroma Acústico/patología , Oído Interno/metabolismo , Oído Interno/citología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Masculino , Células Ciliadas Vestibulares/metabolismo , Femenino , Perfilación de la Expresión GénicaRESUMEN
The transient receptor potential channels TRPML2 and TRPML3 (MCOLN2 and MCOLN3) are nonselective cation channels. They are widely expressed in mammals. However, little is known about their physiological function(s) and activation mechanism(s). TRPML3 can be activated or rather de-inhibited by exposing it first to sodium-free extracellular solution and subsequently to high extracellular sodium. TRPML3 can also be activated by a variety of small chemical compounds identified in a high throughput screen and is inhibited by low pH. Furthermore, it was found that TRPML3 is constitutively active in low or no sodium-containing extracellular solution. This constitutive activity is independent of the intracellular presence of sodium, and whole-cell current densities are similar with pipette solutions containing cesium, potassium, or sodium. Here, we present mutagenesis data generated based on the hypothesis that negatively charged amino acids in the extracellular loops of TRPML3 may interfere with the observed sodium inhibition. We systematically mutated negatively charged amino acids in the first and second extracellular loops and found that mutating Glu-361 in the second loop has a significant impact on the sodium-mediated block of TRPML3. We further demonstrate that the TRPML3-related cation channel TRPML2 is also activated by lowering the extracellular sodium concentration as well as by a subset of small chemical compounds that were previously identified as activators of TRPML3, thus confirming the functional activity of TRPML2 at the plasma membrane and suggesting similar gating mechanisms for both TRPML channels.