RESUMO
In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.
Assuntos
Polaridade Celular/fisiologia , Ciliopatias/fisiopatologia , Cóclea/citologia , Animais , Cóclea/metabolismo , Ducto Coclear/citologia , Ducto Coclear/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/citologia , Células Ciliadas Auditivas , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica de Varredura , Fenótipo , Transdução de Sinais/fisiologia , Estereocílios/metabolismoRESUMO
BACKGROUND: In the inner ear Wnt signaling is necessary for proliferation, cell fate determination, growth of the cochlear duct, polarized orientation of stereociliary bundles, differentiation of the periotic mesenchyme, and homeostasis of the stria vascularis. In neonatal tissue Wnt signaling can drive proliferation of cells in the sensory region, suggesting that Wnt signaling could be used to regenerate the sensory epithelium in the damaged adult inner ear. Manipulation of Wnt signaling for regeneration will require an understanding of the dynamics of Wnt pathway gene expression in the ear. We present a comprehensive screen for 84 Wnt signaling related genes across four developmental and postnatal time points. RESULTS: We identified 72 Wnt related genes expressed in the inner ear on embryonic day (E) 12.5, postnatal day (P) 0, P6 and P30. These genes included secreted Wnts, Wnt antagonists, intracellular components of canonical signaling and components of non-canonical signaling/planar cell polarity. CONCLUSION: A large number of Wnt signaling molecules were dynamically expressed during cochlear development and in the early postnatal period, suggesting complex regulation of Wnt transduction. The data revealed several potential key regulators for further study.
Assuntos
Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Via de Sinalização Wnt/genética , Animais , Cóclea/citologia , Cóclea/embriologia , Ducto Coclear/citologia , Ducto Coclear/embriologia , Ducto Coclear/crescimento & desenvolvimento , Ducto Coclear/metabolismo , Espaço Extracelular/metabolismo , Espaço Intracelular/metabolismo , Camundongos , Análise Espaço-Temporal , Proteínas Wnt/antagonistas & inibidoresRESUMO
A study of genes expressed in the developing inner ear identified the bHLH transcription factor Scleraxis (Scx) in the developing cochlea. Previous work has demonstrated an essential role for Scx in the differentiation and development of tendons, ligaments and cells of chondrogenic lineage. Expression in the cochlea has been shown previously, however the functional role for Scx in the cochlea is unknown. Using a Scx-GFP reporter mouse line we examined the spatial and temporal patterns of Scx expression in the developing cochlea between embryonic day 13.5 and postnatal day 25. Embryonically, Scx is expressed broadly throughout the cochlear duct and surrounding mesenchyme and at postnatal ages becomes restricted to the inner hair cells and the interdental cells of the spiral limbus. Deletion of Scx results in hearing impairment indicated by elevated auditory brainstem response (ABR) thresholds and diminished distortion product otoacoustic emission (DPOAE) amplitudes, across a range of frequencies. No changes in either gross cochlear morphology or expression of the Scx target genes Col2A, Bmp4 or Sox9 were observed in Scx(-/-) mutants, suggesting that the auditory defects observed in these animals may be a result of unidentified Scx-dependent processes within the cochlea.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Ducto Coclear/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Ciliadas Auditivas Internas/metabolismo , Organogênese/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Morfogenética Óssea 4/biossíntese , Proteína Morfogenética Óssea 4/genética , Ducto Coclear/citologia , Colágeno Tipo II/biossíntese , Colágeno Tipo II/genética , Células Ciliadas Auditivas Internas/citologia , Camundongos , Camundongos Knockout , Fatores de Transcrição SOX9/biossíntese , Fatores de Transcrição SOX9/genéticaRESUMO
In mammals, auditory information is processed by the hair cells (HCs) located in the cochlea and then rapidly transmitted to the CNS via a specialized cluster of bipolar afferent connections known as the spiral ganglion neurons (SGNs). Although many anatomical aspects of SGNs are well described, the molecular and cellular mechanisms underlying their genesis, how they are precisely arranged along the cochlear duct, and the guidance mechanisms that promote the innervation of their hair cell targets are only now being understood. Building upon foundational studies of neurogenesis and neurotrophins, we review here new concepts and technologies that are helping to enrich our understanding of the development of the nervous system within the inner ear.
Assuntos
Ducto Coclear/fisiologia , Células Ciliadas Auditivas/fisiologia , Fatores de Crescimento Neural/genética , Neurogênese/fisiologia , Células Receptoras Sensoriais/fisiologia , Gânglio Espiral da Cóclea/fisiologia , Animais , Movimento Celular , Ducto Coclear/citologia , Ducto Coclear/crescimento & desenvolvimento , Ducto Coclear/inervação , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/citologia , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mecanotransdução Celular , Fatores de Crescimento Neural/metabolismo , Células Receptoras Sensoriais/citologia , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/crescimento & desenvolvimento , Gânglio Espiral da Cóclea/inervação , Sinapses/fisiologia , Transmissão SinápticaRESUMO
HDR syndrome (also known as Barakat syndrome) is a developmental disorder characterized by hypoparathyroidism, sensorineural deafness and renal disease. Although genetic mapping and subsequent functional studies indicate that GATA3 haplo-insufficiency causes human HDR syndrome, the role of Gata3 in sensorineural deafness and auditory system development is largely unknown. In this study, we show that Gata3 is continuously expressed in the developing mouse inner ear. Conditional knockout of Gata3 in the developing inner ear disrupts the morphogenesis of mouse inner ear, resulting in a disorganized and shortened cochlear duct with significant fewer hair cells and supporting cells. Loss of Gata3 function leads to the failure in the specification of prosensory domain and subsequently, to increased cell death in the cochlear duct. Moreover, though the initial generation of cochleovestibular ganglion (CVG) cells is not affected in Gata3-null mice, spiral ganglion neurons (SGNs) are nearly depleted due to apoptosis. Our results demonstrate the essential role of Gata3 in specifying the prosensory domain in the cochlea and in regulating the survival of SGNs, thus identifying a molecular mechanism underlying human HDR syndrome.
Assuntos
Ducto Coclear/embriologia , Orelha Interna/embriologia , Orelha Interna/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Células Receptoras Sensoriais/fisiologia , Animais , Apoptose , Ducto Coclear/citologia , Ducto Coclear/inervação , Modelos Animais de Doenças , Orelha Interna/inervação , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Humanos , Hipoparatireoidismo/genética , Hipoparatireoidismo/metabolismo , Camundongos , Camundongos Knockout , Nefrose/genética , Nefrose/metabolismo , Gânglio Espiral da Cóclea/fisiologiaRESUMO
Hair cells of the developing mammalian inner ear are progressively defined through cell fate restriction. This process culminates in the expression of the bHLH transcription factor Atoh1, which is necessary for differentiation of hair cells, but not for their specification. Loss of several genes will disrupt ear morphogenesis or arrest of neurosensory epithelia development. We previously showed in null mutants that the loss of the transcription factor, Gata3, results specifically in the loss of all cochlear neurosensory development. Temporal expression of Gata3 is broad from the otic placode stage through the postnatal ear. It therefore remains unclear at which stage in development Gata3 exerts its effect. To better understand the stage specific effects of Gata3, we investigated the role of Gata3 in cochlear neurosensory specification and differentiation utilizing a LoxP targeted Gata3 line and two Cre lines. Foxg1(Cre)â¶Gata3(f/f) mice show recombination of Gata3 around E8.5 but continue to develop a cochlear duct without differentiated hair cells and spiral ganglion neurons. qRT-PCR data show that Atoh1 was down-regulated but not absent in the duct whereas other hair cell specific genes such as Pou4f3 were completely absent. In addition, while Sox2 levels were lower in the Foxg1(Cre):Gata3(f/f) cochlea, Eya1 levels remained normal. We conclude that Eya1 is unable to fully upregulate Atoh1 or Pou4f3, and drive differentiation of hair cells without Gata3. Pax2-Creâ¶Gata3(f/f) mice show a delayed recombination of Gata3 in the ear relative to Foxg1(Cre):Gata3(f/f) . These mice exhibited a cochlear duct containing patches of partially differentiated hair cells and developed only few and incorrectly projecting spiral ganglion neurons. Our conditional deletion studies reveal a major role of Gata3 in the signaling of prosensory genes and in the differentiation of cochlear neurosenory cells. We suggest that Gata3 may act in combination with Eya1, Six1, and Sox2 in cochlear prosensory gene signaling.
Assuntos
Ducto Coclear/citologia , Fator de Transcrição GATA3/metabolismo , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Animais , Fator de Transcrição GATA3/genética , CamundongosRESUMO
The neuropeptide somatostatin (SST) exerts several important physiological actions in the adult central nervous system through interactions with membrane-bound receptors. Transient expression of SST and its receptors has been described in several brain areas during early ontogeny. It is therefore believed that SST may play a role in neural maturation. The present study provides the first evidence for the developmental expression of SST receptors in the mammalian cochlea, emphasizing their possible roles in cochlear maturation. In the developing mouse cochlea, cells immunoreactive to somatostatin receptor 1 (SSTR1) and somatostatin receptor 2 (SSTR2) were located in the embryonic cochlear duct on Kolliker's organ as early as embryonic day (E) 14 (E14). At E17, the expression of both receptors was high and already located at the hair cells and supporting cells along the length of the cochlear duct, which have become arranged into the characteristic pattern for the organ of Corti (OC) at this stage. At birth, SSTR1- and SSTR2-containing cells were only localized in the OC. In general, immunoreactivity for both receptors increased in the mouse cochlea from postnatal day (P) 0 (P0) to P10; the majority of immunostained cells were inner hair cells, outer hair cells, and supporting cells. Finally, a peak in the mRNA and protein expression of both receptors is present near the time when they respond to physiological hearing (i.e., hearing of airborne sound) at P14. At P21, SSTR1 and SSTR2 levels decrease dramatically. A similar developmental pattern was observed for SSTR1 and SSTR2 mRNA, suggesting that the expression of the SSTR1 and SSTR2 genes is controlled at the transcriptional level throughout development. In addition, we observed reduced levels of phospho-Akt and total Akt in SSTR1 knockout and SSTR1/SSTR2 double-knockout mice compared with wild-type mice. We know from previous studies that Akt is involved in hair cell survival. Taken together, the dynamic nature of SSTR1 and SSTR2 expression at a time of major developmental changes in the cochlea suggests that SSTR1 and SSTR2 (and possibly other members of this family) are involved in the maturation of the mammalian cochlea.
Assuntos
Cóclea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/biossíntese , Receptores de Somatostatina/biossíntese , Animais , Cóclea/embriologia , Cóclea/crescimento & desenvolvimento , Ducto Coclear/citologia , Ducto Coclear/embriologia , Ducto Coclear/crescimento & desenvolvimento , Ducto Coclear/metabolismo , Células Epiteliais/metabolismo , Feminino , Idade Gestacional , Células Ciliadas Auditivas/metabolismo , Audição/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Órgão Espiral/citologia , Órgão Espiral/embriologia , Órgão Espiral/crescimento & desenvolvimento , Órgão Espiral/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Somatostatina/deficiência , Receptores de Somatostatina/genética , Transcrição GênicaRESUMO
The mammalian cochlea offers a unique opportunity to study the effects of planar cell polarity signaling during vertebrate development. First, convergence and extension play a role in outgrowth and cellular patterning within the duct, and second, hair cell stereociliary bundles are uniformly oriented towards the lateral edge of the duct. Defects in convergence and extension are manifested as a shortening of the cochlea duct and/or changes in cellular patterning, which can be quantified following dissection from mouse mutants or observed directly using an in vitro outgrowth assay. Changes in stereociliary bundle orientation can be observed and quantitated using either fluorescent tags or scanning electron microscopy (SEM) to visualize individual bundles. The high degree of regularity in many aspects of cochlear anatomy, including cellular patterning and stereociliary bundle orientation, makes it possible to detect subtle changes in the development of PCP in response to either genetic or molecular perturbations.
Assuntos
Polaridade Celular , Cóclea/citologia , Animais , Cóclea/metabolismo , Ducto Coclear/citologia , Ducto Coclear/metabolismo , Dissecação , Embrião de Mamíferos/citologia , Feminino , Corantes Fluorescentes , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica de Varredura , Gravidez , Estereocílios/metabolismo , Osso Temporal/cirurgia , Fixação de TecidosRESUMO
The water channel aquaporin-4 (AQP4) is expressed in the cochlea and is essential for normal hearing. Unlike other AQPs, multiple isoforms of AQP4 have been reported in diverse tissues, three of which, M1, M23, and Mz, function as water channels. In addition, these protein isoforms are found in higher order complexes. Morphologically these higher order complexes correspond to orthogonal arrays of particles (OAPs) that are found in cell membranes by freeze fracture analysis. Using RT-PCR, quantitative PCR and blue-native PAGE immunoblots we identified all functional AQP4 isoforms -M1, M23, and Mz- and the formation of higher-order complexes in the organ of Corti of the rat. Complementary freeze-fracture studies revealed OAPs distributed in the lateral and basal membrane domains of the cochlear duct supporting cells, specifically Hensen's cells and outer sulcus cells. The unique inter- and intracellular heterogeneity in size, density and shape of OAPs suggests exceptional physiological requirements for the maintenance of water homeostasis during auditory sensory transduction in the cochlea.
Assuntos
Aquaporina 4/metabolismo , Cóclea/metabolismo , Animais , Aquaporina 4/genética , Cóclea/citologia , Ducto Coclear/citologia , Ducto Coclear/metabolismo , Eletroforese em Gel Bidimensional , Técnica de Fratura por Congelamento , Órgão Espiral/citologia , Órgão Espiral/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de RNA/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Endocytosis plays an important role in cell function and the activation and propagation of signaling pathways. Signaling occurs on endocytic pathways and signaling endosomes, and endocytosis is subjected to high-order regulation by cellular signaling mechanisms. Marginal cells showed active endocytosis of microperoxidase (MPO) via the clathrin-independent pathway. We examined the signaling pathway that regulates MPO endocytosis in marginal cells using specific inhibitors and activators of signaling molecules. The results showed that pertussis toxin - which inhibits the ribosylation of G-protein-coupled receptor - did not affect MPO endocytosis, but Clostridium botulinum C3 toxin - which induces RhoA inactivation resulting in extracellular-signal-related kinase inactivation - inhibited MPO endocytosis. The main endocytotic pathway of MPO did not depend on the Rho-associated protein kinase molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade.
Assuntos
Ducto Coclear/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Peroxidases/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , ADP Ribose Transferases/farmacologia , Animais , Toxinas Botulínicas/farmacologia , Membrana Celular/enzimologia , Ducto Coclear/citologia , Endocitose/fisiologia , Endolinfa/metabolismo , Cobaias , Toxina Pertussis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Humans and other mammals are highly susceptible to permanent hearing and balance deficits due to an inability to regenerate sensory hair cells lost to inner ear trauma. In contrast, nonmammalian vertebrates, such as birds, robustly regenerate replacement hair cells and restore hearing and balance functions to near-normal levels. There is considerable interest in understanding the cellular mechanisms responsible for this difference in regenerative capacity. Here we report on involvement of the TGFbeta superfamily type II activin receptors, Acvr2a and Acvr2b, in regulating proliferation in mature avian auditory sensory epithelium. Cultured, posthatch avian auditory sensory epithelium treated with Acvr2a and Acvr2b inhibitors shows decreased proliferation of support cells, the cell type that gives rise to new hair cells. Conversely, addition of activin A, an Acvr2a/b ligand, potentiates support cell proliferation. Neither treatment (inhibitor or ligand) affected hair cell survival, suggesting a specific effect of Acvr2a/b signaling on support cell mitogenicity. Using immunocytochemistry, Acvr2a, Acvr2b, and downstream Smad effector proteins were differentially localized in avian and mammalian auditory sensory epithelia. Collectively, these data suggest that signaling through Acvr2a/b promotes support cell proliferation in mature avian auditory sensory epithelium and that this signaling pathway may be incomplete, or actively blocked, in the adult mammalian ear.
Assuntos
Ativinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Ducto Coclear/citologia , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Receptores de Activinas Tipo II/metabolismo , Ativinas/genética , Ativinas/metabolismo , Animais , Animais Recém-Nascidos , Bromodesoxiuridina/metabolismo , Contagem de Células/métodos , Embrião de Galinha , Galinhas , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio/anatomia & histologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Células Labirínticas de Suporte/metabolismo , Camundongos , Técnicas de Cultura de Órgãos , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismoRESUMO
CONCLUSION: Megalin immunoreactivity was observed in kidney proximal tubule cells, vestibular dark cells, and epithelial cells of the endolymphatic sac. Endocytic mechanisms appear to differ between the endolymphatic sac and proximal tubule cells. We speculate that megalin is secreted by a certain type of cell into the endolymphatic space, and is then absorbed from the endolymphatic space by another type of cell to maintain endolymphatic sac homeostasis. OBJECTIVES: We previously detected megalin immunoreactivity in the rat cochlear duct. Megalin may be involved in endocytosis in the vestibular organ and endolymphatic sac. To examine this possibility, we extended our immunocytochemical investigation to the rat inner ear cells with special attention to vestibular dark cells and endolymphatic sac. MATERIALS AND METHODS: We observed immunoreactivity of megalin under light and electron microscopy. The primary antibody was rabbit polyclonal antibody that had been raised against rat immunoaffinity-purified megalin. RESULTS: The luminal membrane and subapical area of dark cells in the semicircular canal were immunolabeled. The stainable substance in the endolymphatic space was strongly stained. The cytoplasm of epithelial cells was also stained in various patterns.
Assuntos
Saco Endolinfático/citologia , Células Epiteliais/química , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/análise , Vestíbulo do Labirinto/citologia , Animais , Ducto Coclear/química , Ducto Coclear/citologia , Endocitose , Saco Endolinfático/química , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Imuno-Histoquímica , Rim/química , Masculino , Microscopia Eletrônica de Transmissão , Ratos , Ratos Wistar , Vestíbulo do Labirinto/químicaRESUMO
Immunoreactivity for the facilitated glucose transporter 1 (GLUT-1) has been found in the cochlear stria vascularis, but whether the strial marginal cells are immunopositive for GLUT-1 remains uncertain. To determine the cellular localization of GLUT-1 and to clarify the glucose pathway in the stria vascularis of rats and guinea pigs, immunohistochemistry was performed on sections, dissociated cells, and whole-tissue preparations. Immunoreactivity for GLUT-1 in sections was observed in the basal side of the strial tissue and in capillaries in both rats and guinea pigs. However, the distribution of the positive signals within the guinea pig strial tissue was more diffuse than that in rats. Immunostaining of dissociated guinea pig strial cells revealed GLUT-1 in the basal cells and capillary endothelial cells, but not in the marginal cells. These results indicated that GLUT-1 was not expressed in the marginal cells, and that another isoform of GLUT was probably expressed in these cells. Three-dimensional observation of whole-tissue preparations demonstrated that cytoplasmic prolongations from basal cells extended upward to the apical surface of the stria vascularis from rats and guinea pigs, and that the marginal cells were surrounded by these protrusions. We speculate that these upward extensions of basal cells have been interpreted as basal infoldings of marginal cells in previous reports from other groups. The three-dimensional relationship between marginal cells and basal cells might contribute to the transcellular glucose pathway from perilymph to intrastrial space.
Assuntos
Transportador de Glucose Tipo 1/metabolismo , Glucose/metabolismo , Estria Vascular/metabolismo , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Transporte Biológico , Cóclea/citologia , Cóclea/metabolismo , Ducto Coclear/citologia , Ducto Coclear/metabolismo , Endolinfa/metabolismo , Células Endoteliais/metabolismo , Transportador de Glucose Tipo 1/imunologia , Cobaias , Imuno-Histoquímica , Microscopia Confocal , Modelos Biológicos , Perilinfa/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos , Ratos Endogâmicos BN , Estria Vascular/citologiaRESUMO
The organ of Corti, the sensory epithelium of the mammalian cochlea, develops from a subset of cells located along the dorsal side (referred to as the floor) of the cochlear duct. Over the course of embryonic development, cells within the developing organ of Corti become committed to develop as each of the unique cell types within the organ, including inner and outer hair cells, and at least four different types of supporting cells. Moreover, these different cell types are subsequently arranged into a highly rigorous cellular mosaic that includes the formation of ordered rows of both hair cells and supporting cells. The events that regulate both the location of the organ of Corti within the cochlear duct, the specification of each cell type and cellular patterning remain poorly understood. However, recent results have significantly improved our understanding of the molecular, genetic and cellular factors that mediate some of the decisions required for the development of this structure. In this review I will present an overview of cochlear development and then discuss some of the most recent and enlightening results regarding the molecular mechanism underlying the formation of this remarkable structure.
Assuntos
Diferenciação Celular/fisiologia , Órgão Espiral/citologia , Órgão Espiral/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ducto Coclear/citologia , Ducto Coclear/embriologia , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/citologia , Células Labirínticas de Suporte/citologia , Modelos Biológicos , Órgão Espiral/embriologia , Órgão Espiral/metabolismo , Receptor Notch1/metabolismo , Transdução de SinaisRESUMO
During vertebrate inner ear development, compartmentalization of the auditory and vestibular apparatuses along two axes depends on the patterning of transcription factors expressed in a region-specific manner. Although most of the patterning is regulated by extrinsic signals, it is not known how Nkx5.1 and Msx1 are patterned. We focus on Dan, the founding member of the Cerberus/Dan gene family that encodes BMP antagonists, and describe its function in morphogenesis and patterning. First, we confirmed that Dan is expressed in the dorso-medial region of the otic vesicle that corresponds to the presumptive endolymphatic duct and sac (ed/es). Second, we used siRNA knockdown to demonstrate that depletion of Dan induced both a severe reduction in the size of the ed/es and moderate deformities of the semicircular canals and cochlear duct. Depletion of Dan also caused suppression of Nkx5.1 in the dorso-lateral region, suppression of Msx1 in the dorso-medial region, and ectopic induction of Nkx5.1 and Msx1 in the ventro-medial region. Most of these phenotypes also appeared following misexpression of the constitutively active form of BMP receptor type Ib. Thus, Dan is required for the normal morphogenesis of the inner ear and, by inhibiting BMP signaling, for the patterning of the transcription factors Nkx5.1 and Msx1.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Ducto Coclear/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Organogênese , Inibidores de Proteases/metabolismo , Proteínas/fisiologia , Transdução de Sinais , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião de Galinha , Ducto Coclear/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica , Organogênese/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/genéticaRESUMO
Dominant white spotting W/W(v) and W(v)/W(v) mice are well-known mutants that lack strial intermediate cells in their cochlea and manifest hereditary sensorineural hearing loss. We recently reported marked thickening of and IgG deposition on the basement membrane of strial capillaries in W/W(v) mutant mice, similar to observations made in aged animals and in animals with autoimmune sensorineural hearing loss. The present study aimed to clarify the age-dependent changes in these pathological findings of strial capillaries in the W/W(v) mice. Male WBB6F1 +/+ and dominant white spotting W/W(v) mutant mice were sacrificed by transcardiac perfusion with paraformaldehyde solution. The cochlear ducts were isolated and subjected to light- and electron-microscopy, immunohistochemistry, immunoelectron microscopy. Alternatively, lanthanum chloride tracer examination in the isolated cochlear ducts was performed in order to compare the permeability of the strial capillaries between +/+ and W/W(v) mice. In the W/W(v) mice, thickening of and IgG deposition on the basement membrane of strial capillaries were observed as early as 1 week after birth and became more noticeable with age. Deposited IgG was preferentially localized to the thickened basement membrane and was also observed in partially the intercellular space between adjacent of endothelial cells. In addition, pinocytotic vesicles both in the apical and basal lesions of such cells also showed IgG deposition. Lanthanum chloride was retained along apical plasma membrane of the endothelial cells in the +/+ mice but penetrated through the endothelial layer in the W/W(v) mice. These results indicate that active transport via pinocytotic vesicles as well as increased permeability of strial capillaries in the W/W(v) mice occur in the early stage after birth, resulting in the morphological alterations in the strial capillaries of these mice.
Assuntos
Capilares/patologia , Ducto Coclear/metabolismo , Genes Dominantes , Estria Vascular/patologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Membrana Basal/metabolismo , Capilares/metabolismo , Capilares/ultraestrutura , Membrana Celular/metabolismo , Ducto Coclear/citologia , Ducto Coclear/ultraestrutura , Células Endoteliais/ultraestrutura , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Lantânio , Masculino , Camundongos , Camundongos Mutantes , Microscopia Eletrônica , Microscopia Imunoeletrônica , Permeabilidade , Estria Vascular/metabolismo , Estria Vascular/ultraestruturaRESUMO
Nuclear factor-kappa B (NF-kB) transcriptional activity is induced by numerous stimuli. To identify tissues exhibiting NF-kB transcriptional activity during development, we analyzed transgenic reporter mice that express beta-galactosidase from an NF-kB-responsive element. We report that NF-kB activation is widespread and present in numerous epithelial structures and within vasculature. Several regions of the developing central nervous system, including the roof plate and floor plate of the midbrain, show prominent NF-kB activation. To assess the role of the TRAF6 adaptor protein in developmental NF-kB activity, we analyzed NF-kB activation in reporter mice rendered null for TRAF6. Deletion of TRAF6 resulted in the loss of NF-kB activity in epithelia, in vasculature, and in roof and floor plate but had no effect on NF-kB activity developing telencephalon, choroid plexus, cochlear canal, and thymus. These data indicate that NF-kB transcriptional activity is present in a broad range of structures during development and that TRAF6 plays a critical role mediating developmental NF-kB activation in many but not all tissues.
Assuntos
Vasos Sanguíneos/metabolismo , Sistema Nervoso Central/metabolismo , Epitélio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/embriologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Plexo Corióideo/citologia , Plexo Corióideo/embriologia , Plexo Corióideo/metabolismo , Ducto Coclear/citologia , Ducto Coclear/embriologia , Ducto Coclear/metabolismo , Epitélio/embriologia , Mesencéfalo/citologia , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Telencéfalo/citologia , Telencéfalo/embriologia , Telencéfalo/metabolismo , Timo/citologia , Timo/embriologia , Timo/metabolismo , beta-Galactosidase/metabolismo , Quinase Induzida por NF-kappaBRESUMO
The sensory epithelium of the mammalian cochlea, the organ of Corti, is comprised of ordered rows of cells, including inner and outer hair cells. Recent results suggest that physical changes in the overall size and shape of the cochlear duct, including possible convergence and extension, could play a role in the development of this pattern. To examine this hypothesis, changes in cell size and distribution were determined for different regions of the cochlea duct during embryonic development. In addition, changes in the spatial distribution of sensory precursor cells were determined at different developmental time points based on expression of p27kip1. Unique changes in luminal surface area, cell density, and number of cell contacts were observed for each region of the duct. Moreover, the spatial distribution of p27kip1-positive cells changed from short and broad early in development, to long and narrow. These results are consistent with the hypothesis that convergence and extension plays a role in cellular patterning within the organ of Corti.
Assuntos
Órgão Espiral/embriologia , Animais , Contagem de Células , Proteínas de Ciclo Celular/metabolismo , Ducto Coclear/citologia , Ducto Coclear/embriologia , Inibidor de Quinase Dependente de Ciclina p27 , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Camundongos , Morfogênese , Órgão Espiral/citologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
Effects of a single local dose of gentamicin upon sensory and nonsensory cells throughout the cochlea were assessed by changes in immunostaining patterns for a broad array of functionally important proteins. Cytochemical changes in hair cells, spiral ganglion cells, and cells of the stria vascularis, spiral ligament, and spiral limbus were found beginning 4 days post administration. The extent of changes in immunostaining varied with survival time and with cell type and was not always commensurate with the degree to which individual cell types accumulated gentamicin. Outer hair cells, types I and II fibrocytes of the spiral ligament, and fibrocytes in the spiral limbus showed marked decreases in immunostaining for a number of constituents. In contrast, inner hair cells, type III fibrocytes and root cells of the spiral ligament, cells of the stria vascularis, and interdental cells in the spiral limbus showed less dramatic decreases, and in some cases they showed increases in immunostaining. Results indicate that, in addition to damaging sensory cells, local application of gentamicin results in widespread and disparate disruptions of a variety of cochlear cell types. Only in the case of ganglion cells was it apparent that the changes in nonsensory cells were secondary to loss or damage of hair cells. These results indicate that malfunction of the ear following gentamicin treatment is widespread and far more complex than simple loss of sensory elements. The results have implications for efforts directed toward detecting, preventing, and treating toxic effects of aminoglycosides upon the inner ear.
Assuntos
Cóclea/efeitos dos fármacos , Cóclea/metabolismo , Gentamicinas/farmacologia , Animais , Cóclea/citologia , Cóclea/inervação , Ducto Coclear/citologia , Ducto Coclear/metabolismo , Cobaias , Imuno-Histoquímica/métodos , Terminações Nervosas/citologia , Terminações Nervosas/metabolismo , Órgão Espiral/metabolismo , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/metabolismo , Lâmina Espiral/citologia , Lâmina Espiral/metabolismo , Coloração e Rotulagem , Estria Vascular/citologia , Estria Vascular/metabolismoRESUMO
The role of apoptosis and proliferation in maintenance of cochlear lateral wall cells was examined. The methods employed for detection of apoptosis were the Hoechst fluorescence stain and TUNEL (TdT-mediated dUTP-biotin nick-end-labeling) assay, and proliferations were 5-bromo-2'-deoxyuridine (BrdU) incorporation and presence of the proliferating cell nuclear antigen. The incidence of apoptosis in the strial marginal cell was 50% greater (32.9+/-3.7%) than strial intermediate and basal cells but similar to spiral ligament cells. Although division of marginal strial cells was rarely detected, a significant number of proliferating cells in the remaining stria vascularis and spiral ligament were observed. These data implied that replacement of marginal cells arose elsewhere and could be followed by a BrdU-deoxythymidine pulse-chase study. At 2 h post injection, nuclear BrdU in marginal cells was not detected; however, by 24 h post injection, 20-25% of marginal cell nuclei were BrdU-positive. These observations are consistent with the hypothesis that marginal cells were replaced by underlying cells. Cell migration appears to be an important mechanism for preserving the function and structure of the stria vascularis.