RESUMO
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.
Assuntos
Órgão Espiral/embriologia , Diferenciação Celular , Separação Celular/métodos , Técnicas de Cocultura , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Órgão Espiral/metabolismo , Receptores de Fator de Crescimento Neural/metabolismoRESUMO
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.
Assuntos
Orelha Interna/citologia , Células-Tronco Neurais/citologia , Órgão Espiral/embriologia , Fatores de Transcrição SOXB1/análise , Animais , Antígenos de Diferenciação/análise , Linhagem da Célula , Orelha Interna/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Genes Reporter , Imageamento Tridimensional , Proteína Jagged-1/biossíntese , Proteína Jagged-1/genética , Proteínas Luminescentes/análise , Camundongos , Camundongos Transgênicos , Órgão Espiral/citologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The V-shaped hair bundles atop auditory hair cells and their uniform orientation are manifestations of epithelial planar cell polarity (PCP) required for proper perception of sound. PCP is regulated at the tissue level by a conserved core Wnt/PCP pathway. However, the hair cell-intrinsic polarity machinery is poorly understood. Recent findings implicate hair cell microtubules in planar polarization of hair cells. To elucidate the microtubule-mediated polarity pathway, we analyzed Lis1 function in the auditory sensory epithelium in the mouse. We show that conditional deletion of Lis1 in developing hair cells causes defects in cytoplasmic dynein and microtubule organization, resulting in planar polarity defects without overt effects on the core PCP pathway. Lis1 ablation during embryonic development results in defects in hair bundle morphology and orientation, cellular organization and junctional nectin localization. We present evidence that Lis1 regulates localized Rac-PAK signaling in embryonic hair cells, probably through microtubule-associated Tiam1, a guanine nucleotide exchange factor for Rac. Lis1 ablation in postnatal hair cells significantly disrupts centrosome anchoring and the normal V-shape of hair bundles, accompanied by defects in the pericentriolar matrix and microtubule organization. Lis1 is also required for proper positioning of the Golgi complex and mitochondria as well as for hair cell survival. Together, our results demonstrate that Lis1 mediates the planar polarity of hair cells through regulation of microtubule organization downstream of the tissue polarity pathway.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Polaridade Celular/fisiologia , Células Ciliadas Auditivas/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Órgão Espiral/embriologia , Transdução de Sinais/fisiologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Animais , Western Blotting , Primers do DNA/genética , Deleção de Genes , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica de Varredura , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Organelas/fisiologia , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células TRESUMO
The canonical Wnt/ß-catenin signaling pathway is known to play crucial roles in organogenesis by regulating both proliferation and differentiation. In the inner ear, this pathway has been shown to regulate the size of the otic placode from which the cochlea will arise; however, direct activity of canonical Wnt signaling as well as its function during cochlear mechanosensory hair cell development had yet to be identified. Using TCF/Lef:H2B-GFP reporter mice and transfection of an independent TCF/Lef reporter construct, we describe the pattern of canonical Wnt activity in the developing mouse cochlea. We show that prior to terminal mitosis, canonical Wnt activity is high in early prosensory cells from which hair cells and support cells will differentiate, and activity becomes reduced as development progresses. Using an in vitro model we demonstrate that Wnt/ß-catenin signaling regulates both proliferation and hair cell differentiation within the developing cochlear duct. Inhibition of Wnt/ß-catenin signaling blocks proliferation during early mitotic phases of development and inhibits hair cell formation in the differentiating organ of Corti. Conversely, activation increases the number of hair cells that differentiate and induces proliferation in prosensory cells, causing an expansion of the Sox2-positive prosensory domain. We further demonstrate that the induced proliferation of Sox2-positive cells may be mediated by the cell cycle regulator cyclin D1. Lastly, we provide evidence that the mitotic Sox2-positive cells are competent to differentiate into hair cells. Combined, our data suggest that Wnt/ß-catenin signaling has a dual function in cochlear development, regulating both proliferation and hair cell differentiation.
Assuntos
Cóclea/embriologia , Células Ciliadas Auditivas/metabolismo , Órgão Espiral/embriologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Cóclea/citologia , Cóclea/metabolismo , Ciclina D1/metabolismo , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Órgão Espiral/metabolismo , Organogênese , Fatores de Transcrição SOXB1/metabolismoRESUMO
Sensorineural hearing loss results from damage to the hair cells of the organ of Corti and is irreversible in mammals. While hair cell regeneration may prove to be the ideal therapy after hearing loss, prevention of initial hair cell loss could provide even more benefit at a lower cost. Previous studies have shown that the deletion of Atoh1 results in embryonic loss of hair cells while the absence of Barhl1, Gfi1, and Pou4f3 leads to the progressive loss of hair cells in newborn mice. We recently reported that in the early embryonic absence of N-Myc (using Pax2-Cre), hair cells in the organ of Corti develop and remain until at least seven days after birth, with subsequent progressive loss. Thus, N-Myc plays a role in hair cell viability; however, it is unclear if this is due to its early expression in hair cell precursors and throughout the growing otocyst as it functions through proliferation or its late expression exclusively in differentiated hair cells. Furthermore, the related family member L-Myc is mostly co-expressed in the ear, including in differentiated hair cells, but its function has not been studied and could be partially redundant to N-Myc. To test for a long-term function of the Mycs in differentiated hair cells, we generated nine unique genotypes knocking out N-Myc and/or L-Myc after initial formation of hair cells using the well-characterized Atoh1-Cre. We tested functionality of the auditory and vestibular systems at both P21 and four months of age and under the administration of the ototoxic drug cisplatin. We conclude that neither N-Myc nor L-Myc is likely to play important roles in long-term hair cell maintenance. Therefore, it is likely that the late-onset loss of hair cells resulting from early deletion of the Mycs leads to an unsustainable developmental defect.
Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Órgão Espiral/embriologia , Órgão Espiral/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Embrião de Mamíferos , Células Ciliadas Auditivas Internas/citologia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Microscopia Confocal , Órgão Espiral/metabolismoRESUMO
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
Atonal homolog1 (Atoh1) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO) mouse line using Tg(Atoh1-cre), in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to "self-terminate" its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Células Ciliadas Auditivas/metabolismo , Órgão Espiral/metabolismo , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sobrevivência Celular/genética , Cóclea/embriologia , Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/ultraestrutura , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Órgão Espiral/embriologia , Órgão Espiral/crescimento & desenvolvimento , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estereocílios/genética , Estereocílios/metabolismo , Fator de Transcrição Brn-3C/genética , Fator de Transcrição Brn-3C/metabolismoRESUMO
Gene-based therapeutics are being developed as novel treatments for genetic hearing loss. One roadblock to effective gene therapy is the identification of vectors which will safely deliver therapeutics to targeted cells. The cellular heterogeneity that exists within the cochlea makes viral tropism a vital consideration for effective inner ear gene therapy. There are compelling reasons to identify a viral vector with tropism for organ of Corti supporting cells. Supporting cells are the primary expression site of connexin 26 gap junction proteins that are mutated in the most common form of congenital genetic deafness (DFNB1). Supporting cells are also primary targets for inducing hair cell regeneration. Since many genetic forms of deafness are congenital it is necessary to administer gene transfer-based therapeutics prior to the onset of significant hearing loss. We have used transuterine microinjection of the fetal murine otocyst to investigate viral tropism in the developing inner ear. For the first time we have characterized viral tropism for supporting cells following in utero delivery to their progenitors. We report the inner ear tropism and potential ototoxicity of three previously untested vectors: early-generation adenovirus (Ad5.CMV.GFP), advanced-generation adenovirus (Adf.11D) and bovine adeno-associated virus (BAAV.CMV.GFP). Adenovirus showed robust tropism for organ of Corti supporting cells throughout the cochlea but induced increased ABR thresholds indicating ototoxicity. BAAV also showed tropism for organ of Corti supporting cells, with preferential transduction toward the cochlear apex. Additionally, BAAV readily transduced spiral ganglion neurons. Importantly, the BAAV-injected ears exhibited normal hearing at 5 weeks of age when compared to non-injected ears. Our results support the use of BAAV for safe and efficient targeting of supporting cell progenitors in the developing murine inner ear.
Assuntos
Adenoviridae/genética , Surdez/terapia , Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Células Labirínticas de Suporte/virologia , Órgão Espiral/virologia , Tropismo Viral , Estimulação Acústica , Animais , Audiometria de Tons Puros , Limiar Auditivo , Surdez/genética , Surdez/fisiopatologia , Potenciais Evocados Auditivos do Tronco Encefálico , Técnicas de Transferência de Genes/efeitos adversos , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Idade Gestacional , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células Labirínticas de Suporte/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microinjeções , Microscopia de Fluorescência , Órgão Espiral/embriologia , Órgão Espiral/metabolismo , Órgão Espiral/fisiopatologia , Células-Tronco/virologia , Transdução GenéticaRESUMO
Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27(Kip1) Cdk inhibitor, Ink4 proteins, or Rb. Here, we use tamoxifen-inducible mouse models to delete p27(Kip1) in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27(Kip1) even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27(Kip1) expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27(Kip1) expression in animals with established pituitary tumors, in an inducible "knock-on" model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27(Kip1) did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27(Kip1) expression in the maintenance of cellular quiescence and terminal differentiation.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Neoplasias Experimentais/metabolismo , Órgão Espiral/metabolismo , Adeno-Hipófise Parte Intermédia/metabolismo , Animais , Animais Recém-Nascidos , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular , Galinhas , Inibidor de Quinase Dependente de Ciclina p27/genética , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Órgão Espiral/citologia , Órgão Espiral/embriologia , Organogênese , Adeno-Hipófise Parte Intermédia/embriologia , Adeno-Hipófise Parte Intermédia/patologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Tamoxifeno/farmacologiaRESUMO
Directing the orientation of cells in three dimensions is a fundamental aspect of many of the processes underlying the generation of the appropriate shape and function of tissues and organs during embryonic development. In an epithelium, this requires not only the establishment of apicobasal polarity, but also cell arrangement in a specific direction in the plane of the cell sheet. The molecular pathway central to regulating this planar cell polarity (PCP) was originally discovered in the fruit fly Drosophila melanogaster and has more recently been shown to act in a highly analogous way in vertebrates, involving a strongly overlapping set of genes. Mutant studies and molecular analyses have led to insights into the role of ordered planar cell polarity in the development of a wide variety of organs and tissues. In this review, we give an overview of recent developments in the study of planar polarity signaling in vertebrates.
Assuntos
Polaridade Celular/fisiologia , Vertebrados/embriologia , Animais , Polaridade Celular/genética , Drosophila , Humanos , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Órgão Espiral/citologia , Órgão Espiral/embriologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Vertebrados/crescimento & desenvolvimento , Vertebrados/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
PURPOSE OF REVIEW: A significant proportion of hearing loss and deafness is caused by defects in the structure or function of cells within the organ of Corti. Identification of the molecular factors that regulate the development of this structure should provide valuable insights regarding inner ear formation and the signaling pathways that underlie congenital auditory deficits. In addition, targeted modulation of these same factors could be developed as therapies for hair cell regeneration. RECENT FINDINGS: Results from experiments using transgenic and mutant mice, as well as in-vitro techniques, have identified genes and signaling pathways that are required to either specify unique auditory cell types, such as hair cells or supporting cells, or to generate the highly ordered cellular pattern that is characteristic for the organ of Corti. In particular, the hedgehog and fibroblast growth factor signaling pathways modulate the formation of the progenitor cells that will give rise to the organ of Corti. SRY-box containing gene 2, a transcription factor that is required for the formation of the cochlear progenitor cell population, has paradoxically been shown to also act as an inhibitor of hair cell development. Finally, the motor protein myosin II regulates extension of the organ of Corti and the alignment of hair cells and supporting cells into ordered rows. SUMMARY: A better understanding of the signaling pathways that direct different aspects of cochlear development, such as specific of cell fates or cellular patterning, offers the potential to identify new pathways or molecules that could be targeted for therapeutic interventions.
Assuntos
Órgão Espiral/citologia , Órgão Espiral/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Diferenciação Celular/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Proteínas Hedgehog/fisiologia , Camundongos , Camundongos Transgênicos , Miosina Tipo II/fisiologia , Síndrome de Pallister-Hall/genética , Fatores de Transcrição SOXB1/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Inhibitors of differentiation (Id) play an essential role in the neurogenesis of the central nervous system. However, the expression and function of Id in the development of cochlear sensory epithelial cells have yet to be elucidated. In this study, we demonstrate the Id1 gene was expressed in the rapidly growing otocyst on embryonic day 12 (E12) and in the organ of Corti, spiral ganglions, and stria vascularis on postnatal day 1 (P1) by cellular and molecular biologic techniques. Knockdown of the Id1 gene with short interfering RNA (siRNA) in a cochlear sensory epithelial cell line (OC1) significantly reduced its proliferation, whereas overexpression of Id1 in OC1 significantly increased the proliferation of OC1, suggesting a role of Id1 in the development of cochlear sensory epithelial cells. The proliferative action of Id1 on OC1 was mediated by nuclear factor-kappaB (NF-kappaB) and cyclin D1 (a downstream molecule of NF-kappaB). Blockage of the NF-kappaB activity with pyrrolidine dithiocarbamate (PDTC) or enhancement of the NF-kappaB activity with p65 (a subunit of NF-kappaB) in OC1 significantly inhibited or increased, respectively, the cell proliferation and transcription of cyclin D1 induced by Id1. Truncation of the NF-kappaB binding site in the cyclin D1 promoter fully abrogated the transcription of cyclin D1, suggesting that the cyclin D1 transcription is dependent on NF-kappaB. We concluded from this study that Id1 induces the proliferation of OC1 via the NF-kappaB/cyclin D1 pathway.
Assuntos
Cóclea/citologia , Ciclina D1/metabolismo , Proteína 1 Inibidora de Diferenciação/genética , NF-kappa B/metabolismo , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Divisão Celular , Cóclea/embriologia , Replicação do DNA , Regulação da Expressão Gênica no Desenvolvimento , Órgão Espiral/citologia , Órgão Espiral/embriologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Fase S , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/embriologiaRESUMO
The basic helix-loop-helix (bHLH) transcription factor Math1 (also called Atoh1) is both necessary and sufficient for hair cell development in the mammalian cochlea (Bermingham et al., 1999; Zheng and Gao, 2000). Previous studies have demonstrated that a dynamic pattern of Math1 expression plays a key role in regulating the number and position of mechanosensory hair cells. However, the factors that regulate the temporal and spatial expression of Math1 within the cochlea are unknown. The bHLH-related inhibitors of differentiation and DNA binding (Id) proteins are known to negatively regulate many bHLH transcription factors, including Math1, in a number of different systems. Therefore, Id proteins are good candidates for regulating Math1 in the cochlea. Results from PCR and in situ hybridization indicate that Id1, Id2, and Id3 are expressed within the cochlear duct in a pattern that is consistent with a role in regulation of hair cell development. In particular, expression of Ids and Math1 overlapped in cochlear progenitor cells before cellular differentiation, but a specific downregulation of Id expression was observed in individual cells that differentiated as hair cells. In addition, progenitor cells in which the expression of Ids was maintained during the time period for hair cell differentiation were inhibited from developing as hair cells. These results indicate a key role for Ids in the regulation of expression of Math1 and hair cell differentiation in the developing cochlea.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/citologia , Proteína 1 Inibidora de Diferenciação/fisiologia , Proteína 2 Inibidora de Diferenciação/fisiologia , Proteínas Inibidoras de Diferenciação/fisiologia , Órgão Espiral/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , DNA Complementar/genética , Eletroporação , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Genes Reporter , Células Ciliadas Auditivas/metabolismo , Proteína 1 Inibidora de Diferenciação/biossíntese , Proteína 1 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/biossíntese , Proteína 2 Inibidora de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/biossíntese , Proteínas Inibidoras de Diferenciação/genética , Camundongos , Técnicas de Cultura de Órgãos , Órgão Espiral/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
COUP-TFs (chicken ovalbumin upstream promoter-transcription factors) are orphan members of the nuclear receptor superfamily of ligand-activated receptors for which their ligands have not been identified. The two mammalian proteins, COUP-TFI and COUP-TFII, share 80% sequence identity and regulate many aspects of mammalian development and differentiation. In this report, we systemically examined the temporal and spatial expression of COUP-TFI and COUP-TFII transcripts and, for the first time, their protein during development and functional maturation of the cochlea. Both COUP-TFI and COUP-TFII were expressed early in the developing otic vesicle. COUP-TFI expression correlated with the differentiation of hair cells and support cells in the organ of Corti, whereas COUP-TFII expression was down-regulated with differentiation of the organ of Corti. Furthermore, we report for the first time, that the generally nuclear COUP-TFI receptor protein was localized in the cytoplasm of maturing hair cells and pillar cells. Collectively, although COUP-TFI and COUP-TFII are homologues, the expression of each orphan receptor has a restricted and dynamic expression during cochlea development particularly during patterning and differentiation of the cochlear structures.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Orelha Interna/embriologia , Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Órgão Espiral/embriologia , Receptores de Esteroides/biossíntese , Fatores de Transcrição/biossíntese , Animais , Fator I de Transcrição COUP , Fator II de Transcrição COUP , Fatores de Transcrição COUP , Diferenciação Celular , Núcleo Celular/metabolismo , Cóclea/embriologia , Genótipo , Células Ciliadas Auditivas , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Modelos Anatômicos , Órgão Espiral/metabolismo , Fatores de TempoAssuntos
Proliferação de Células , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/fisiologia , Proteína do Retinoblastoma/fisiologia , Animais , Apoptose , Contagem de Células , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Diferenciação Celular , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p27 , Surdez/terapia , Orelha Interna/citologia , Orelha Interna/embriologia , Orelha Interna/fisiologia , Regulação da Expressão Gênica , Camundongos , Mitose , Órgão Espiral/citologia , Órgão Espiral/embriologia , Órgão Espiral/fisiologia , Proteína do Retinoblastoma/genética , Sáculo e Utrículo/citologia , Sáculo e Utrículo/embriologia , Sáculo e Utrículo/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
The expression of Slo channels (alpha subunits of BK channels) was investigated in the developing mouse cochlea using a polyclonal antibody against the C-terminal part of the protein (residues 1098-1196). The first BK channel immunoreactivity was observed in the cochlea at E18, where it was localized within the cytoplasm of cells lining the area of the organ of Corti and the spiral ganglion. There was an increase of immunoreactivity in all cells bordering the scala media (supporting and hair cells of the organ of Corti, the stria vascularis and the Reissner's membrane) in the following stages (postnatal day [P] 0 and P6). From P12 to adult, a strong membranous labeling, increasing with age, appeared in inner hair cells. The distribution of BK channels was mainly observed as dense elongated plaques localized in the lateral membrane below the cuticular plate. In addition, a more discrete immunolabeling for BK channels, as punctuated dots, was observed in the synaptic area of inner hair cells. This dual localization of BK channels within inner hair cells was confirmed by a different technique using a fluorescently labeled high-affinity ligand of these channels: IbTX-D19C-Alexa488. We demonstrated under patch clamp experiments that this fluorescent toxin conserved its native property, i.e. to reversibly inhibit BK currents in isolated inner hair cells. The fluorescent toxin, both in living or fixed tissues, also showed a preferential binding to mature inner hair cells with a similar subcellular distribution described above using immunocytochemical technique. Overall, our present results confirm the appearance of membranous BK channels around P12 in mouse inner hair cells, an age at which the auditory system becomes functional. The expression of BK channels in mature inner hair cells, near the site of mechanical-transduction, might serve to limit receptor potential attenuation due to the space constant, and thus permitting these sensory cells to function as fast and sensitive transducers.
Assuntos
Diferenciação Celular/fisiologia , Cóclea/embriologia , Cóclea/crescimento & desenvolvimento , Células Ciliadas Auditivas Internas/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Membrana Celular/metabolismo , Cóclea/citologia , Corantes Fluorescentes , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Audição/fisiologia , Hidrazinas , Canais de Potássio Ativados por Cálcio de Condutância Alta , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurotoxinas/farmacologia , Órgão Espiral/citologia , Órgão Espiral/embriologia , Órgão Espiral/crescimento & desenvolvimento , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Membranas Sinápticas/efeitos dos fármacos , Membranas Sinápticas/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Conditionally immortal cell lines were established from the ventral otocyst of the Immortomouse at embryonic day 10.5 and selected to represent precursors of auditory sensory neural and epithelial cells. Selection was based upon dissection, tissue-specific markers, and expression of the transcription factor GATA3. Two cell lines expressed GATA3 but possessed intrinsically different genetic programs under differentiating conditions. US/VOT-E36 represented epithelial progenitors with potential to differentiate into sensory and nonsensory epithelial cells. US/VOT-N33 represented migrating neuroblasts. Under differentiating conditions in vitro the cell lines expressed very different gene expression profiles. Expression of several cell- and tissue-specific markers, including the transcription factors Pax2, GATA3, and NeuroD, differed between the cell lines in a pattern consistent with that observed between their counterparts in vivo. We suggest that these and other conditionally immortal cell lines can be used to study transient events in development against different backgrounds of cell competence.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Órgão Espiral/citologia , Órgão Espiral/embriologia , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/embriologia , Acetilcolina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Vias Auditivas/citologia , Vias Auditivas/embriologia , Vias Auditivas/fisiologia , Cálcio/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular Transformada , Epitélio/embriologia , Feminino , Perfilação da Expressão Gênica , Canais de Potássio KCNQ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Órgão Espiral/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Gravidez , Gânglio Espiral da Cóclea/fisiologia , Células-Tronco/citologiaRESUMO
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
Hair cells, the sensory receptors of auditory and vestibular systems, use a transducer apparatus that renders them remarkably sensitive to mechanical displacement as minute as 1 nm. To study the embryonic development of the transducer apparatus in hair cells of the chick auditory papilla, we examined hair cells that have been labeled with N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridiniumdibromide, which has been shown to permeate the transducer channels. In addition, mechanotransduction currents were recorded directly using whole-cell patch-clamp techniques. The structure of the hair bundle was examined using scanning electron microscopy, and immunofluorescence labeling for myosin 1c, myosin 7a, and plasma membrane Ca2+ ATPase 2 was studied to determine the developmental expression of these proteins in embryonic chick papillas. We demonstrate that the transducer apparatus is assembled jointly at embryonic day 11 (E11) of the developing chick basilar papilla. The resting open probability of the transducer channels was high at E12 (approximately 0.5) and remained substantially elevated at E14-16; it then declined to the mature value of approximately 0.15 at E21. The displacement sensitivity of the transduction apparatus, the gating force, increased from E12 to E21. Although the expression of different components of the transducer apparatus and the transduction current peaked at approximately E14-16, marked refinement occurred beyond E16. For example, myosin 1c appeared diffusely localized in hair bundles from E12 to E16, but subsequently consolidated into punctate pattern. The fine temporal and precise spatial assembly of the transducer apparatus likely contributes toward the exquisite sensitivity of the transduction ensemble.