RESUMEN
BACKGROUND: Understanding the mechanisms that regulate hair cell (HC) differentiation in the organ of Corti (OC) is essential to designing genetic therapies for hearing loss due to HC loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in HC and supporting cell differentiation in the mouse OC. To investigate the genetic landscape regulated by FGF20 signaling in OC progenitors, we employ Translating Ribosome Affinity Purification combined with Next Generation RNA Sequencing (TRAPseq) in the Fgf20 lineage. RESULTS: We show that TRAPseq targeting OC progenitors effectively enriched for RNA from this rare cell population. TRAPseq identified differentially expressed genes (DEGs) downstream of FGF20, including Etv4, Etv5, Etv1, Dusp6, Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, Sall3, and cell cycle regulators such as Cdc20. Analysis of Cdc20 conditional-null mice identified decreased cochlea length, while analysis of Sall1-null and Sall1-ΔZn2-10 mice, which harbor a mutation that causes Townes-Brocks syndrome, identified a decrease in outer hair cell number. CONCLUSIONS: We present two datasets: genes with enriched expression in OC progenitors, and DEGs downstream of FGF20 in the embryonic day 14.5 cochlea. We validate select DEGs via in situ hybridization and in vivo functional studies in mice.
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Factores de Crecimiento de Fibroblastos/fisiología , Órgano Espiral/metabolismo , Ribosomas/metabolismo , Animales , Diferenciación Celular , Factores de Crecimiento de Fibroblastos/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Terapia Genética , Células Ciliadas Auditivas Externas/metabolismo , Audición , Ratones , Ratones Transgénicos , Mutación , Neurogénesis , Órgano Espiral/embriología , Fenotipo , Biosíntesis de Proteínas , Análisis de Secuencia de ARN , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Precise control of organ growth and patterning is executed through a balanced regulation of progenitor self-renewal and differentiation. In the auditory sensory epithelium-the organ of Corti-progenitor cells exit the cell cycle in a coordinated wave between E12.5 and E14.5 before the initiation of sensory receptor cell differentiation, making it a unique system for studying the molecular mechanisms controlling the switch between proliferation and differentiation. Here we identify the Yap/Tead complex as a key regulator of the self-renewal gene network in organ of Corti progenitor cells. We show that Tead transcription factors bind directly to the putative regulatory elements of many stemness- and cell cycle-related genes. We also show that the Tead coactivator protein, Yap, is degraded specifically in the Sox2-positive domain of the cochlear duct, resulting in down-regulation of Tead gene targets. Further, conditional loss of the Yap gene in the inner ear results in the formation of significantly smaller auditory and vestibular sensory epithelia, while conditional overexpression of a constitutively active version of Yap, Yap5SA, is sufficient to prevent cell cycle exit and to prolong sensory tissue growth. We also show that viral gene delivery of Yap5SA in the postnatal inner ear sensory epithelia in vivo drives cell cycle reentry after hair cell loss. Taken together, these data highlight the key role of the Yap/Tead transcription factor complex in maintaining inner ear progenitors during development, and suggest new strategies to induce sensory cell regeneration.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Autorrenovación de las Células , Órgano Espiral/embriología , Órgano Espiral/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Ciclo Celular , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas , Ratones , Órgano Espiral/citología , Unión Proteica , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Células Madre/metabolismo , Factores de Transcripción/genética , Proteínas Señalizadoras YAPRESUMEN
Mammalian hearing requires the development of the organ of Corti, a sensory epithelium comprising unique cell types. The limited number of each of these cell types, combined with their close proximity, has prevented characterization of individual cell types and/or their developmental progression. To examine cochlear development more closely, we transcriptionally profile approximately 30,000 isolated mouse cochlear cells collected at four developmental time points. Here we report on the analysis of those cells including the identification of both known and unknown cell types. Trajectory analysis for OHCs indicates four phases of gene expression while fate mapping of progenitor cells suggests that OHCs and their surrounding supporting cells arise from a distinct (lateral) progenitor pool. Tgfßr1 is identified as being expressed in lateral progenitor cells and a Tgfßr1 antagonist inhibits OHC development. These results provide insights regarding cochlear development and demonstrate the potential value and application of this data set.
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Cóclea/citología , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas/citología , Órgano Espiral/citología , Animales , Células Cultivadas , Cóclea/embriología , Cóclea/crecimiento & desarrollo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Ratones , Órgano Espiral/embriología , Órgano Espiral/crecimiento & desarrollo , Análisis de la Célula Individual/métodos , Factores de TiempoRESUMEN
During cochlear development, hair cells (HCs) and supporting cells differentiate in the prosensory domain to form the organ of Corti, but how one row of inner HCs (IHCs) and three rows of outer HCs (OHCs) are organized is not well understood. Here, we investigated the process of HC induction by monitoring Atoh1 expression in cochlear explants of Atoh1-EGFP knock-in mouse embryos and showed that only the cells that express Atoh1 over a certain threshold are selected for HC fate determination. HC induction initially occurs at the medial edge of the prosensory domain to form IHCs and subsequently at the lateral edge to form OHCs, while Hedgehog signaling maintains a space between IHCs and OHCs, leading to formation of the tunnel of Corti. These results reveal dynamic Atoh1 expression in HC fate control and suggest that multi-directional signals regulate OHC induction, thereby organizing the prototype of the organ of Corti.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Cóclea/embriología , Células Ciliadas Auditivas/citología , Animales , Tipificación del Cuerpo , Proteína Morfogenética Ósea 4/fisiología , Diferenciación Celular , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/fisiología , Proteínas Hedgehog/fisiología , Imagenología Tridimensional , Ratones , Microscopía Fluorescente , Microscopía por Video , Órgano Espiral/embriología , Receptores Notch/fisiología , Transducción de SeñalRESUMEN
Damage or loss of auditory hair cells leads to irreversible sensorineural hearing loss in human, thus regeneration of these cells to reconstruct auditory sensory epithelium holds the promise for the treatment of deafness. Regulatory factors involved in the development of auditory sensory epithelium play crucial roles in hair cell regeneration and hearing restoration. Here, we first focus on the transcription factor Atoh1 which is critical for hair cell development and regeneration, and comprehensively summarize the current understanding of the protein structure, target binding motif, developmental expression pattern, functional role, and upstream and downstream regulatory mechanism of Atoh1 in the context of controlling the cell fate commitment to hair cells or transdifferentiation from supporting cells. We also discuss cellular context dependency of Atoh1 in hair cell induction which should be taken into consideration when using Atoh1 gene therapy for hair cell regeneration. Next, we review the roles of Gfi1, Pou4f3, and Barhl1 in hair cell maturation and maintenance, and suggest that manipulation of these genes and their downstream targets will be helpful for the generation of functional hair cells with long-term viability. Finally, we provide an overview of the interplay between Notch, Wnt, Shh, and FGF signaling pathways during auditory sensory epithelium development. By analyzing crosstalk between these pathways, we suggest that combination of Wnt signaling activation with Hey1 and Hey2 inhibition will be crucial for hair cell regeneration and hearing restoration. Furthermore, this review highlights the importance of deeper understanding of the cellular context for hair cell development and the interconnection between these key regulators in developing new strategies to treat sensorineural hearing loss.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Oído Interno/metabolismo , Regulación del Desarrollo de la Expresión Génica , Órgano Espiral/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Oído Interno/embriología , Oído Interno/crecimiento & desarrollo , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Órgano Espiral/embriología , Órgano Espiral/crecimiento & desarrollo , Factor de Transcripción Brn-3C/genética , Factor de Transcripción Brn-3C/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Class III Semaphorin (Sema) secreted ligands are known to repel neurites expressing Neuropilin (Nrp) and/or Plexin (Plxn) receptors. There is, however, a growing body of literature supporting that Sema signaling also has alternative roles in development such as synaptogenesis, boundary formation, and vasculogenesis. To evaluate these options during inner ear development, we used in situ hybridization or immunohistochemistry to map the expression of Sema3D, Sema3F, Nrp1, Nrp2, and PlxnA1 in the chicken (Gallus gallus) inner ear from embryonic day (E)5-E10. The resulting expression patterns in either the otic epithelium or its surrounding mesenchyme suggest that Sema signaling could be involved in each of the varied functions reported for other tissues. Sema3D expression flanking the sensory tissue in vestibular organs suggests that it may repel Nrp2- and PlxnA1-expressing neurites of the vestibular ganglion away from nonsensory epithelia, thus channeling them into the sensory domains at E5-E8. Expression of Sema signaling genes in the sensory hair cells of both the auditory and vestibular organs on E8-E10 may implicate Sema signaling in synaptogenesis. In the nonsensory regions of the cochlea, Sema3D in the future tegmentum vasculosum opposes Nrp1 and PlxnA1 in the future cuboidal cells; the abutment of ligand and receptors in adjacent domains may enforce or maintain the boundary between them. In the mesenchyme, Nrp1 colocalized with capillary-rich tissue. Sema3D immediately flanks this Nrp1-expressing tissue, suggesting a role in endothelial cell migration towards the inner ear. In summary, Sema signaling may play multiple roles in the developing inner ear.
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Pollos/metabolismo , Oído Interno/metabolismo , Neuropilina-1/biosíntesis , Semaforinas/biosíntesis , Animales , Movimiento Celular , Embrión de Pollo , Oído Interno/embriología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Neurogénesis , Neuropilina-1/genética , Órgano Espiral/embriología , Órgano Espiral/metabolismo , Semaforinas/genética , Transducción de Señal , Organismos Libres de Patógenos Específicos , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismo , Proteínas Wnt/farmacología , Proteínas Wnt/fisiologíaRESUMEN
Sensory hair cells located in the organ of Corti are essential for cochlear mechanosensation. Their loss is irreversible in humans resulting in permanent hearing loss. The development of therapeutic interventions for hearing loss requires fundamental knowledge about similarities and potential differences between animal models and human development as well as the establishment of human cell based-assays. Here we analyze gene and protein expression of the developing human inner ear in a temporal window spanning from week 8 to 12 post conception, when cochlear hair cells become specified. Utilizing surface markers for the cochlear prosensory domain, namely EPCAM and CD271, we purify postmitotic hair cell progenitors that, when placed in culture in three-dimensional organoids, regain proliferative potential and eventually differentiate to hair cell-like cells in vitro. These results provide a foundation for comparative studies with otic cells generated from human pluripotent stem cells and for establishing novel platforms for drug validation.
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Órgano Espiral/embriología , Diferenciación Celular , Separación Celular/métodos , Técnicas de Cocultivo , Molécula de Adhesión Celular Epitelial/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Órgano Espiral/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismoRESUMEN
Developmental remodeling of the sensory epithelium of the cochlea is required for the formation of an elongated, tonotopically organized auditory organ, but the cellular processes that mediate these events are largely unknown. We used both morphological assessments of cellular rearrangements and time-lapse imaging to visualize cochlear remodeling in mouse. Analysis of cell redistribution showed that the cochlea extends through a combination of radial intercalation and cell growth. Live imaging demonstrated that concomitant cellular intercalation results in a brief period of epithelial convergence, although subsequent changes in cell size lead to medial-lateral spreading. Supporting cells, which retain contact with the basement membrane, exhibit biased protrusive activity and directed movement along the axis of extension. By contrast, hair cells lose contact with the basement membrane, but contribute to continued outgrowth through increased cell size. Regulation of cellular protrusions, movement and intercalation within the cochlea all require myosin II. These results establish, for the first time, many of the cellular processes that drive the distribution of sensory cells along the tonotopic axis of the cochlea.
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Movimiento Celular , Cóclea/embriología , Regulación del Desarrollo de la Expresión Génica , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tipificación del Cuerpo , Proliferación Celular , Tamaño de la Célula , Femenino , Genotipo , Células Ciliadas Auditivas/citología , Homocigoto , Mamíferos , Ratones , Miosina Tipo II/metabolismo , Órgano Espiral/embriología , Factores de Transcripción SOXB1/genética , Imagen de Lapso de TiempoRESUMEN
Vestibular hair cells of the inner ear are specialized receptors that detect mechanical stimuli from gravity and motion via the deflection of a polarized bundle of stereocilia located on their apical cell surfaces. The orientation of stereociliary bundles is coordinated between neighboring cells by core PCP proteins including the large adhesive G-protein coupled receptor Celsr1. We show that mice lacking Celsr1 have vestibular behavioral phenotypes including circling. In addition, we show that Celsr1 is asymmetrically distributed at cell boundaries between hair cells and neighboring supporting cells in the developing vestibular and auditory sensory epithelia. In the absence of Celsr1 the stereociliary bundles of vestibular hair cells are misoriented relative to their neighbors, a phenotype that is greatest in the cristae of the semicircular canals. Since horizontal semi-circular canal defects lead to circling in other mutant mouse lines, we propose that this PCP phenotype is the cellular basis of the circling behavior in Celsr1 mutants.
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Polaridad Celular , Oído Interno/citología , Oído Interno/embriología , Células Ciliadas Vestibulares/citología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Conducta Animal , Oído Interno/metabolismo , Epitelio/metabolismo , Eliminación de Gen , Ratones Noqueados , Órgano Espiral/citología , Órgano Espiral/embriología , Órgano Espiral/metabolismo , Fenotipo , Transducción de Señal , Estereocilios/metabolismoRESUMEN
The signals that induce the organ of Corti and define its boundaries in the cochlea are poorly understood. We show that two Notch modifiers, Lfng and Mfng, are transiently expressed precisely at the neural boundary of the organ of Corti. Cre-Lox fate mapping shows this region gives rise to inner hair cells and their associated inner phalangeal cells. Mutation of Lfng and Mfng disrupts this boundary, producing unexpected duplications of inner hair cells and inner phalangeal cells. This phenotype is mimicked by other mouse mutants or pharmacological treatments that lower but not abolish Notch signaling. However, strong disruption of Notch signaling causes a very different result, generating many ectopic hair cells at the expense of inner phalangeal cells. Our results show that Notch signaling is finely calibrated in the cochlea to produce precisely tuned levels of signaling that first set the boundary of the organ of Corti and later regulate hair cell development.
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Glicosiltransferasas/metabolismo , Órgano Espiral/embriología , Proteínas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Glucosiltransferasas , Glicosiltransferasas/genética , Ratones , Mutación , Proteínas/genéticaRESUMEN
The transcription factor Sox2 is both necessary and sufficient for the generation of sensory regions of the inner ear. It regulates expression of the Notch ligand Jag1 in prosensory progenitors, which signal to neighboring cells to up-regulate Sox2 and sustain prosensory identity. However, the expression pattern of Sox2 in the early inner ear is very broad, suggesting that Sox2-expressing progenitors form a wide variety of cell types in addition to generating the sensory regions of the ear. We used Sox2-CreER mice to follow the fates of Sox2-expressing cells at different stages in ear development. We find that Sox2-expressing cells in the early otocyst give rise to large numbers of non-sensory structures throughout the inner ear, and that Sox2 only becomes a truly prosensory marker at embryonic day (E)11.5. Our fate map reveals the organ of Corti derives from a central domain on the medial side of the otocyst and shows that a significant amount of the organ of Corti derives from a Sox2-negative population in this region.
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Oído Interno/citología , Células-Madre Neurales/citología , Órgano Espiral/embriología , Factores de Transcripción SOXB1/análisis , Animales , Antígenos de Diferenciación/análisis , Linaje de la Célula , Oído Interno/embriología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Genes Reporteros , Imagenología Tridimensional , Proteína Jagged-1/biosíntesis , Proteína Jagged-1/genética , Proteínas Luminiscentes/análisis , Ratones , Ratones Transgénicos , Órgano Espiral/citología , Receptores Notch/fisiología , Transducción de Señal/fisiologíaRESUMEN
Determination of cell fate within the prosensory domain of the developing cochlear duct relies on the temporal and spatial regulation of the bHLH transcription factor Atoh1. Auditory hair cells and supporting cells arise in a wave of differentiation that patterns them into discrete rows mediated by Notch-dependent lateral inhibition. However, the mechanism responsible for selecting sensory cells from within the prosensory competence domain remains poorly understood. We show in mice that rather than being upregulated in rows of cells, Atoh1 is subject to transcriptional activation in groups of prosensory cells, and that highly conserved sites for Hes/Hey repressor binding in the Atoh1 promoter are needed to select the hair cell and supporting cell fate. During perinatal supporting cell transdifferentiation, which is a model of hair cell regeneration, we show that derepression is sufficient to induce Atoh1 expression, suggesting a mechanism for priming the 3' Atoh1 autoregulatory enhancer needed for hair cell expression.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas de Ciclo Celular/fisiología , Linaje de la Célula , Cóclea/embriología , Células Ciliadas Auditivas/fisiología , Proteínas de Homeodominio/fisiología , Órgano Espiral/fisiología , Proteínas Represoras/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sitios de Unión , Diferenciación Celular , Transdiferenciación Celular , Cóclea/fisiología , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Mutación , Órgano Espiral/embriología , Regiones Promotoras Genéticas , Transducción de Señal , Factor de Transcripción HES-1RESUMEN
We describe the development of the human inner ear with the invagination of the otic vesicle at 4 weeks gestation (WG), the growth of the semicircular canals from 5 WG, and the elongation and coiling of the cochlea at 10 WG. As the membranous labyrinth takes shape, there is a concomitant development of the sensory neuroepithelia and their associated structures within. This review details the growth and differentiation of the vestibular and auditory neuroepithelia, including synaptogenesis, the expression of stereocilia and kinocilia, and innervation of hair cells by afferent and efferent nerve fibres. Along with development of essential sensory structures we outline the formation of crucial accessory structures of the vestibular system - the cupula and otolithic membrane and otoconia as well as the three cochlea compartments and the tectorial membrane. Recent molecular studies have elaborated on classical anatomical studies to characterize the development of prosensory and sensory regions of the fetal human cochlea using the transcription factors, PAX2, MAF-B, SOX2, and SOX9. Further advances are being made with recent physiological studies that are beginning to describe when hair cells become functionally active during human gestation. This article is part of a Special Issue entitled
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Oído Interno/embriología , Células Ciliadas Auditivas/fisiología , Vestíbulo del Laberinto/embriología , Animales , Diferenciación Celular , Linaje de la Célula , Cóclea/embriología , Humanos , Ratones , Neuronas/metabolismo , Órgano Espiral/embriología , Membrana Otolítica/embriología , Factor de Transcripción PAX2/metabolismo , Regeneración , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción SOXB1/metabolismo , Canales Semicirculares/embriología , Sinapsis/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Vestíbulo del Laberinto/metabolismoRESUMEN
A key function of the thyroid hormone receptor ß (Thrb) gene is in the development of auditory function. However, the roles of the 2 receptor isoforms, TRß1 and TRß2, expressed by the Thrb gene are unclear, and it is unknown whether these isoforms promote the maintenance as well as development of hearing. We investigated the function of TRß1 in mice with a Thrb(b1) reporter allele that expresses ß-galactosidase instead of TRß1. In the immature cochlea, ß-galactosidase was detected in the greater epithelial ridge, sensory hair cells, spiral ligament, and spiral ganglion and in adulthood, at low levels in the hair cells, support cells and root cells of the outer sulcus. Although deletion of all TRß isoforms causes severe, early-onset deafness, deletion of TRß1 or TRß2 individually caused no obvious hearing loss in juvenile mice. However, over subsequent months, TRß1 deficiency resulted in progressive loss of hearing and loss of hair cells. TRß1-deficient mice had minimal changes in serum thyroid hormone and thyrotropin levels, indicating that hormonal imbalances were unlikely to cause hearing loss. The results suggest mutually shared roles for TRß1 and TRß2 in cochlear development and an unexpected requirement for TRß1 in the maintenance of hearing in adulthood.
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Envejecimiento , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva/genética , Receptores beta de Hormona Tiroidea/genética , Animales , Cóclea/embriología , Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/patología , Pérdida Auditiva/sangre , Pérdida Auditiva/metabolismo , Inmunohistoquímica , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Órgano Espiral/embriología , Órgano Espiral/crecimiento & desarrollo , Órgano Espiral/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores beta de Hormona Tiroidea/metabolismo , Hormonas Tiroideas/sangre , Tirotropina/sangre , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Atoh1, a basic helix-loop-helix (bHLH) transcription factor (TF), is essential for the differentiation of hair cells (HCs), mechanotransducers that convert sound into auditory signals in the mammalian organ of Corti (OC). Previous work demonstrated that replacing mouse Atoh1 with the fly ortholog atonal rescues HC differentiation, indicating functional replacement by other bHLH genes. However, replacing Atoh1 with Neurog1 resulted in reduced HC differentiation compared with transient Atoh1 expression in a 'self-terminating' Atoh1 conditional null mouse (Atoh1-Cre; Atoh1(f/f)). We now show that combining Neurog1 in one allele with removal of floxed Atoh1 in a self-terminating conditional mutant (Atoh1-Cre; Atoh1(f/kiNeurog1)) mouse results in significantly more differentiated inner HCs and outer HCs that have a prolonged longevity of 9 months compared with Atoh1 self-terminating littermates. Stereocilia bundles are partially disorganized, disoriented and not HC type specific. Replacement of Atoh1 with Neurog1 maintains limited expression of Pou4f3 and Barhl1 and rescues HCs quantitatively, but not qualitatively. OC patterning and supporting cell differentiation are also partially disrupted. Diffusible factors involved in patterning are reduced (Fgf8) and factors involved in cell-cell interactions are affected (Jag1, Hes5). Despite the presence of many HCs with stereocilia these mice are deaf, possibly owing to HC and OC patterning defects. This study provides a novel approach to disrupt OC development through modulating the HC-specific intracellular TF network. The resulting disorganized OC indicates that normally differentiated HCs act as 'self-organizers' for OC development and that Atoh1 plays a crucial role to initiate HC stereocilia differentiation independently of HC viability.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Células Ciliadas Auditivas/fisiología , Proteínas del Tejido Nervioso/metabolismo , Órgano Espiral/embriología , Animales , Técnicas de Sustitución del Gen , Inmunohistoquímica , Hibridación in Situ , Ratones , Microscopía Electrónica de Rastreo , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The miR-183 family consists of 3 related microRNAs (miR-183, miR-96, miR-182) that are required to complete maturation of primary sensory cells in the mammalian inner ear. Because the level of these microRNAs is not uniform across hair cell subtypes in the murine cochlea, the question arises as to whether hair cell phenotypes are influenced by microRNA expression levels. To address this, we used the chicken embryo to study expression and misexpression of this gene family. By in situ hybridization, expression of all 3 microRNAs is robust in immature hair cells of both auditory and vestibular organs and is present in the statoacoustic ganglion. The auditory organ, called the basilar papilla, shows a weak radial gradient (highest on the neural side) in prosensory cells near the base on embryonic day 7. About nine days later, the basilar papilla also displays a longitudinal gradient (highest in apical hair cells) for the 3 microRNAs. Tol2-mediated gene delivery was used to ask whether cell phenotypes are malleable when the prosensory epithelium was forced to overexpress the miR-183 family. The expression plasmid included EGFP as a reporter located upstream of an intron carrying the microRNA genes. The vectors were electroporated into the otic cup/vesicle, resulting in strong co-expression of EGFP and the miR-183 family that persisted for at least 2 weeks. This manipulation did not generate ectopic hair cells in non-sensory territories of the cochlear duct, although within the basilar papilla, hair cells were over-represented relative to supporting cells. There was no evidence for a change in hair cell phenotypes, such as short-to-tall, or basal-to-apical hair cell features. Therefore, while increasing expression of the miR-183 family was sufficient to influence cell lineage decisions, it did not redirect the differentiation of hair cells towards alternative radial or longitudinal phenotypes.
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Expresión Génica , MicroARNs/metabolismo , Órgano Espiral/metabolismo , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Diferenciación Celular , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Células Ciliadas Auditivas/metabolismo , Humanos , MicroARNs/genética , Órgano Espiral/citología , Órgano Espiral/embriología , Interferencia de ARN , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/embriología , Ganglio Espiral de la Cóclea/metabolismoRESUMEN
Ciliopathies are a group of developmental disorders that manifest with multi-organ anomalies. Mutations in TMEM67 (MKS3) cause a range of human ciliopathies, including Meckel-Gruber and Joubert syndromes. In this study we describe multi-organ developmental abnormalities in the Tmem67(tm1Dgen/H1) knockout mouse that closely resemble those seen in Wnt5a and Ror2 knockout mice. These include pulmonary hypoplasia, ventricular septal defects, shortening of the body longitudinal axis, limb abnormalities, and cochlear hair cell stereociliary bundle orientation and basal body/kinocilium positioning defects. The basal body/kinocilium complex was often uncoupled from the hair bundle, suggesting aberrant basal body migration, although planar cell polarity and apical planar asymmetry in the organ of Corti were normal. TMEM67 (meckelin) is essential for phosphorylation of the non-canonical Wnt receptor ROR2 (receptor-tyrosine-kinase-like orphan receptor 2) upon stimulation with Wnt5a-conditioned medium. ROR2 also colocalises and interacts with TMEM67 at the ciliary transition zone. Additionally, the extracellular N-terminal domain of TMEM67 preferentially binds to Wnt5a in an in vitro binding assay. Cultured lungs of Tmem67 mutant mice failed to respond to stimulation of epithelial branching morphogenesis by Wnt5a. Wnt5a also inhibited both the Shh and canonical Wnt/ß-catenin signalling pathways in wild-type embryonic lung. Pulmonary hypoplasia phenotypes, including loss of correct epithelial branching morphogenesis and cell polarity, were rescued by stimulating the non-canonical Wnt pathway downstream of the Wnt5a-TMEM67-ROR2 axis by activating RhoA. We propose that TMEM67 is a receptor that has a main role in non-canonical Wnt signalling, mediated by Wnt5a and ROR2, and normally represses Shh signalling. Downstream therapeutic targeting of the Wnt5a-TMEM67-ROR2 axis might, therefore, reduce or prevent pulmonary hypoplasia in ciliopathies and other congenital conditions.
Asunto(s)
Tipificación del Cuerpo , Trastornos de la Motilidad Ciliar/metabolismo , Encefalocele/metabolismo , Epitelio/embriología , Proteínas de la Membrana/metabolismo , Morfogénesis , Enfermedades Renales Poliquísticas/metabolismo , Vía de Señalización Wnt , Animales , Animales Recién Nacidos , Diferenciación Celular , Polaridad Celular , Cilios/metabolismo , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Epitelio/metabolismo , Células HEK293 , Humanos , Pulmón/embriología , Pulmón/metabolismo , Proteínas de la Membrana/deficiencia , Ratones , Mutación/genética , Órgano Espiral/anomalías , Órgano Espiral/embriología , Órgano Espiral/patología , Fenotipo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Retinitis Pigmentosa , Estereocilios/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5a , beta Catenina/metabolismoRESUMEN
Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development(1-8).
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Electroporación/métodos , Técnicas de Transferencia de Gen , Órgano Espiral/fisiología , Técnicas de Cultivo de Tejidos/métodos , Animales , Femenino , Ratones , Ratones Transgénicos , Órgano Espiral/citología , Órgano Espiral/embriología , EmbarazoRESUMEN
The mammalian inner ear develops from a placodal thickening into a complex labyrinth of ducts with five sensory organs specialized to detect position and movement in space. The mammalian ear also develops a spiraled cochlear duct containing the auditory organ, the organ of Corti (OC), specialized to translate sound into hearing. Development of the OC from a uniform sheet of ectoderm requires unparalleled precision in the topological developmental engineering of four different general cell types, namely sensory neurons, hair cells, supporting cells, and general otic epithelium, into a mosaic of ten distinctly recognizable cell types in and around the OC, each with a unique distribution. Moreover, the OC receives unique innervation by ear-derived spiral ganglion afferents and brainstem-derived motor neurons as efferents and requires neural-crest-derived Schwann cells to form myelin and neural-crest-derived cells to induce the stria vascularis. This transformation of a sheet of cells into a complicated interdigitating set of cells necessitates the orchestrated expression of multiple transcription factors that enable the cellular transformation from ectoderm into neurosensory cells forming the spiral ganglion neurons (SGNs), while simultaneously transforming the flat epithelium into a tube, the cochlear duct, housing the OC. In addition to the cellular and conformational changes forming the cochlear duct with the OC, changes in the surrounding periotic mesenchyme form passageways for sound to stimulate the OC. We review molecular developmental data, generated predominantly in mice, in order to integrate the well-described expression changes of transcription factors and their actions, as revealed in mutants, in the formation of SGNs and OC in the correct position and orientation with suitable innervation. Understanding the molecular basis of these developmental changes leading to the formation of the mammalian OC and highlighting the gaps in our knowledge might guide in vivo attempts to regenerate this most complicated cellular mosaic of the mammalian body for the reconstitution of hearing in a rapidly growing population of aging people suffering from hearing loss.
Asunto(s)
Oído Interno/embriología , Ectodermo/embriología , Órgano Espiral/embriología , Ganglio Espiral de la Cóclea/embriología , Animales , Oído Interno/crecimiento & desarrollo , Humanos , RatonesRESUMEN
BACKGROUND: Inner ear morphogenesis is tightly regulated by the temporally and spatially coordinated action of signaling ligands and their receptors. Ligand-receptor interactions are influenced by heparan sulfate proteoglycans (HSPGs), cell surface molecules that consist of glycosaminoglycan chains bound to a protein core. Diversity in the sulfation pattern within glycosaminoglycan chains creates binding sites for numerous cell signaling factors, whose activities and distribution are modified by their association with HSPGs. RESULTS: Here we describe the expression patterns of two extracellular 6-O-endosulfatases, Sulf1 and Sulf2, whose activity modifies the 6-O-sulfation pattern of HSPGs. We use in situ hybridization to determine the temporal and spatial distribution of transcripts during the development of the chick and mouse inner ear. We also use immunocytochemistry to determine the cellular localization of Sulf1 and Sulf2 within the sensory epithelia. Furthermore, we analyze the organ of Corti in Sulf1/Sulf2 double knockout mice and describe an increase in the number of mechanosensory hair cells. CONCLUSIONS: Our results suggest that the tuning of intracellular signaling, mediated by Sulf activity, plays an important role in the development of the inner ear.