RESUMO
Notch signalling is a major regulator of cell fate decisions and tissue patterning in metazoans. It is best known for its role in lateral inhibition, whereby Notch mediates competitive interactions between cells to limit adoption of a given developmental fate. However, it can also function by lateral induction, a cooperative mode of action that was originally described during the patterning of the Drosophila wing disc and creates boundaries or domains of cells of the same character. In this chapter, we introduce these two signalling modes and explain how they contribute to distinct aspects of the development and regeneration of the vertebrate inner ear, the organ responsible for the perception of sound and head movements. We discuss some of the factors that could influence the context-specific outcomes of Notch signalling in the inner ear and the ongoing efforts to target this pathway for the treatment of hearing loss and vestibular dysfunction.
Assuntos
Diferenciação Celular , Orelha Interna/embriologia , Orelha Interna/fisiologia , Receptores Notch/metabolismo , Regeneração , Transdução de Sinais , Animais , Orelha Interna/citologia , Orelha Interna/metabolismo , Perda Auditiva/metabolismo , Perda Auditiva/fisiopatologia , HumanosRESUMO
The loss of sensory hair cells from the inner ear is a leading cause of hearing and balance disorders. The mammalian ear has a very limited ability to replace lost hair cells, but the inner ears of non-mammalian vertebrates can spontaneously regenerate hair cells after injury. Prior studies have shown that replacement hair cells are derived from epithelial supporting cells and that the differentiation of new hair cells is regulated by the Notch signaling pathway. The present study examined molecular influences on regeneration in the avian utricle, which has a particularly robust regenerative ability. Chicken utricles were placed in organotypic culture and hair cells were lesioned by application of the ototoxic antibiotic streptomycin. Cultures were then allowed to regenerate in vitro for seven days. Some specimens were treated with small molecule inhibitors of γ-secretase or ADAM10, proteases which are essential for transmission of Notch signaling. As expected, treatment with both inhibitors led to increased numbers of replacement hair cells. However, we also found that inhibition of both proteases resulted in increased regenerative proliferation. Subsequent experiments showed that inhibition of γ-secretase or ADAM10 could also trigger proliferation in undamaged utricles. To better understand these phenomena, we used RNA-Seq profiling to characterize changes in gene expression following γ-secretase inhibition. We observed expression patterns that were consistent with Notch pathway inhibition, but we also found that the utricular sensory epithelium contains numerous γ-secretase substrates that might regulate cell cycle entry and possibly supporting cell-to-hair cell conversion. Together, our data suggest multiple roles for γ-secretase and ADAM10 in vestibular hair cell regeneration.
Assuntos
Proteína ADAM10/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Células Ciliadas Vestibulares/citologia , Receptores Notch/metabolismo , Regeneração/fisiologia , Sáculo e Utrículo/crescimento & desenvolvimento , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Embrião de Galinha , Galinhas , Células Epiteliais/fisiologia , Técnicas de Cultura de Órgãos , Sáculo e Utrículo/citologiaRESUMO
Mechanosensory hair cells (HCs) are the primary receptors of our senses of hearing and balance. Elucidation of the transcriptional networks regulating HC fate determination and differentiation is crucial not only to understand inner ear development but also to improve cell replacement therapies for hearing disorders. Here, we show that combined expression of the transcription factors Gfi1, Pou4f3 and Atoh1 can induce direct programming towards HC fate, both during in vitro mouse embryonic stem cell differentiation and following ectopic expression in chick embryonic otic epithelium. Induced HCs (iHCs) express numerous HC-specific markers and exhibit polarized membrane protrusions reminiscent of stereociliary bundles. Transcriptome profiling confirms the progressive establishment of a HC-specific gene signature during in vitro iHC programming. Overall, this work provides a novel approach to achieve robust and highly efficient HC production in vitro, which could be used as a model to study HC development and to drive inner ear HC regeneration.
Assuntos
Reprogramação Celular , Células Ciliadas Auditivas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Forma Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Embrião de Galinha , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Fluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Reporter , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/efeitos dos fármacos , Camundongos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcriptoma/genética , Tretinoína/farmacologiaRESUMO
Hearing relies on the mechanosensory inner and outer hair cells (OHCs) of the organ of Corti, which convert mechanical deflections of their actin-rich stereociliary bundles into electrochemical signals. Several actin-associated proteins are essential for stereocilia formation and maintenance, and their absence leads to deafness. One of the most abundant actin-bundling proteins of stereocilia is plastin 1, but its function has never been directly assessed. Here, we found that plastin 1 knock-out (Pls1 KO) mice have a moderate and progressive form of hearing loss across all frequencies. Auditory hair cells developed normally in Pls1 KO, but in young adult animals, the stereocilia of inner hair cells were reduced in width and length. The stereocilia of OHCs were comparatively less affected; however, they also showed signs of degeneration in ageing mice. The hair bundle stiffness and the acquisition of the electrophysiological properties of hair cells were unaffected by the absence of plastin 1, except for a significant change in the adaptation properties, but not the size of the mechanoelectrical transducer currents. These results show that in contrast to other actin-bundling proteins such as espin, harmonin or Eps8, plastin 1 is dispensable for the initial formation of stereocilia. However, the progressive hearing loss and morphological defects of hair cells in adult Pls1 KO mice point at a specific role for plastin 1 in the preservation of adult stereocilia and optimal hearing. Hence, mutations in the human PLS1 gene may be associated with relatively mild and progressive forms of hearing loss.
Assuntos
Células Ciliadas Auditivas Internas/patologia , Perda Auditiva/fisiopatologia , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Estereocílios/patologia , Fatores Etários , Animais , Perda Auditiva/genética , Perda Auditiva/patologia , Humanos , Camundongos , Camundongos Knockout , MutaçãoRESUMO
The formation of the salt-and-pepper mosaic of hair cells and supporting cells in the sensory epithelia of the inner ear is regulated by Notch signalling and lateral inhibition, but the dynamics of this process and precise mode of action of delta-like 1 (Dll1) in this context are unclear. Here, we transfected the chicken inner ear with a fluorescent reporter that includes elements of the mammalian Hes5 promoter to monitor Notch activity in the developing sensory patches. The Hes5 reporter was active in proliferating cells and supporting cells, and Dll1 expression was highest in prospective hair cells with low levels of Notch activity, which occasionally contacted more differentiated hair cells. To investigate Dll1 functions we used constructs in which Dll1 expression was either constitutive, regulated by the Hes5 promoter, or induced by doxycycline. In support of the standard lateral inhibition model, both continuous and Hes5-regulated expression of Dll1 promoted hair cell differentiation cell-autonomously (in cis) and inhibited hair cell formation in trans. However, some hair cells formed despite contacting Dll1-overexpressing cells, suggesting that some progenitor cells are insensitive to lateral inhibition. This is not due to the cis-inhibition of Notch activity by Dll1 itself, as induction of Dll1 did not cell-autonomously reduce the activity of the Hes5 reporter in progenitor and supporting cells. Altogether, our results show that Dll1 functions primarily in trans to regulate hair cell production but also that additional mechanisms operate downstream of lateral inhibition to eliminate patterning errors in the sensory epithelia of the inner ear.
Assuntos
Orelha Interna/embriologia , Orelha Interna/metabolismo , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Embrião de Galinha , Doxiciclina/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
The Notch and Wnt signalling pathways play key roles in the formation of inner ear sensory organs, but little is known about their transcriptional effectors and targets in this context. Here, we perturbed Notch and Wnt activities in the embryonic chicken otic vesicle using pharmacological treatment or in ovo electroporation of plasmid DNA, and used RNA-Seq to analyse the resulting changes in gene expression. Compared to pharmacological treatments, in ovo electroporation changed the expression of fewer genes, a likely consequence of the variability and mosaicism of transfection. The pharmacological inhibition of Notch activity induced a rapid change in the expression of known effectors of this pathway and genes associated with neurogenesis, consistent with a switch towards an otic neurosensory fate. The Wnt datasets contained many genes associated with a neurosensory biological function, confirming the importance of this pathway for neurosensory specification in the otocyst. Finally, the results of a preliminary gain-of-function screening of selected transcription factors and Wnt signalling components suggest that the endogenous programs of otic neurosensory specification are very robust, and in general unaffected by the overexpression of a single factor. Altogether this work provides new insights into the effectors and candidate targets of the Notch and Wnt pathways in the early developing inner ear and could serve as a useful reference for future functional genomics experiments in the embryonic avian inner ear.
RESUMO
Epithelial homeostasis is essential for sensory transduction in the auditory and vestibular organs of the inner ear, but how it is maintained during trauma is poorly understood. To examine potential repair mechanisms, we expressed ß-actin-enhanced green fluorescent protein (EGFP) in the chick inner ear and used live-cell imaging to study how sensory epithelia responded during aminoglycoside-induced hair cell trauma. We found that glial-like supporting cells used two independent mechanisms to rapidly eliminate dying hair cells. Supporting cells assembled an actin cable at the luminal surface that extended around the pericuticular junction and constricted to excise the stereocilia bundle and cuticular plate from the hair cell soma. Hair bundle excision could occur within 3 min of actin-cable formation. After bundle excision, typically with a delay of up to 2-3 h, supporting cells engulfed and phagocytosed the remaining bundle-less hair cell. Dual-channel recordings with ß-actin-EGFP and vital dyes revealed phagocytosis was concurrent with loss of hair cell integrity. We conclude that supporting cells repaired the epithelial barrier before hair cell plasmalemmal integrity was lost and that supporting cell activity was closely linked to hair cell death. Treatment with the Rho-kinase inhibitor Y-27632 did not prevent bundle excision but prolonged phagocytic engulfment and resulted in hair cell corpses accumulating within the epithelium. Our data show that supporting cells not only maintain epithelial integrity during trauma but suggest they may also be an integral part of the hair cell death process itself.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/fisiologia , Células Ciliadas Auditivas/fisiologia , Células Labirínticas de Suporte/citologia , Células Labirínticas de Suporte/fisiologia , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Aminoglicosídeos/toxicidade , Animais , Comunicação Celular/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Galinhas , Cílios/fisiologia , Cílios/ultraestrutura , Cóclea/citologia , Cóclea/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Ciliadas Auditivas/citologia , Homeostase/fisiologia , Neurotoxinas/toxicidade , Técnicas de Cultura de Órgãos , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Regeneração/fisiologiaRESUMO
The auditory and vestibular organs of the inner ear and the neurons that innervate them originate from Sox2-positive and Notch-active neurosensory domains specified at early stages of otic development. Sox2 is initially present throughout the otic placode and otocyst, and then it becomes progressively restricted to a ventro-medial domain. Using gain- and loss-of-function approaches in the chicken otocyst, we show that these early changes in Sox2 expression are regulated in a dose-dependent manner by Wnt/beta-catenin signalling. Both high and very low levels of Wnt activity repress Sox2 and neurosensory competence. However, intermediate levels allow the maintenance of Sox2 expression and sensory organ formation. We propose that a dorso-ventral (high-to-low) gradient and wave of Wnt activity initiated at the dorsal rim of the otic placode progressively restricts Sox2 and Notch activity to the ventral half of the otocyst, thereby positioning the neurosensory competent domains in the inner ear.
Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Orelha Interna/embriologia , Regulação da Expressão Gênica , Fatores de Transcrição SOXB1/genética , Via de Sinalização Wnt , Animais , Proteínas Aviárias/metabolismo , Embrião de Galinha/embriologia , Galinhas/metabolismo , Orelha Interna/metabolismo , Mutação com Ganho de Função , Mutação com Perda de Função , Fatores de Transcrição SOXB1/metabolismoRESUMO
The mammalian inner ear has a limited capacity to regenerate its mechanosensory hair cells. This lack of regenerative capacity underlies the high incidence of age-related hearing loss in humans. In contrast, non-mammalian vertebrates can form new hair cells when damage occurs, a mechanism that depends on re-activation of expression of the pro-hair cell transcription factor Atoh1. Here, we show that members of the E2F transcription factor family, known to play a key role in cell cycle progression, regulate the expression of Atoh1. E2F1 activates chicken Atoh1 by directly interacting with a cis-regulatory region distal to the avian Atoh1 gene. E2F does not activate mouse Atoh1 gene expression, since this regulatory element is absent in mammals. We also show that E2F1 expression changes dynamically in the chicken auditory epithelium during ototoxic damage and hair cell regeneration. Therefore, we propose a model in which the mitotic regeneration of non-mammalian hair cells is due to E2F1-mediated activation of Atoh1 expression, a mechanism which has been lost in mammals.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator de Transcrição E2F1/metabolismo , Células Ciliadas Auditivas/fisiologia , Regeneração , Animais , Linhagem Celular , Embrião de Galinha , Regulação da Expressão Gênica , CamundongosRESUMO
Unlike mammals, birds regenerate auditory hair cells (HCs) after injury. During regeneration, mature non-sensory supporting cells (SCs) leave quiescence and convert into HCs, through non-mitotic or mitotic mechanisms. During embryogenesis, Notch ligands from nascent HCs exert lateral inhibition, restricting HC production. Here, we examined whether Notch signaling (1) is needed in mature birds to maintain the HC/SC pattern in the undamaged auditory epithelium or (2) governs SC behavior once HCs are injured. We show that Notch pathway genes are transcribed in the mature undamaged epithelium, and after HC injury, their transcription is upregulated in the region of highest mitotic activity. In vitro treatment with DAPT, an inhibitor of Notch activity, had no effect on SCs in the undamaged epithelium. Following HC damage, DAPT had no direct effect on SC division. However, after damage, DAPT caused excessive regeneration of HCs at the expense of SCs, through both mitotic and non-mitotic mechanisms. Conversely, overexpression of activated Notch in SCs after damage caused them to maintain their phenotype and inhibited HC regeneration. Therefore, signaling through Notch is not required for SC quiescence in the healthy epithelium or to initiate HC regeneration after damage. Rather, Notch prevents SCs from regenerating excessive HCs after damage.
Assuntos
Galinhas/fisiologia , Células Ciliadas Auditivas/citologia , Receptores Notch/fisiologia , Regeneração/fisiologia , Células-Tronco/citologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Dipeptídeos/farmacologia , Epitélio/fisiologia , Células Ciliadas Auditivas/fisiologia , Células Labirínticas de Suporte/citologia , Células Labirínticas de Suporte/fisiologia , Mitose/fisiologia , Células-Tronco/fisiologia , Técnicas de Cultura de TecidosRESUMO
The mechanisms of formation of the distinct sensory organs of the inner ear and the non-sensory domains that separate them are still unclear. Here, we show that several sensory patches arise by progressive segregation from a common prosensory domain in the embryonic chicken and mouse otocyst. This process is regulated by mutually antagonistic signals: Notch signalling and Lmx1a. Notch-mediated lateral induction promotes prosensory fate. Some of the early Notch-active cells, however, are normally diverted from this fate and increasing lateral induction produces misshapen or fused sensory organs in the chick. Conversely Lmx1a (or cLmx1b in the chick) allows sensory organ segregation by antagonizing lateral induction and promoting commitment to the non-sensory fate. Our findings highlight the dynamic nature of sensory patch formation and the labile character of the sensory-competent progenitors, which could have facilitated the emergence of new inner ear organs and their functional diversification in the course of evolution.
Assuntos
Orelha Interna/anatomia & histologia , Regulação da Expressão Gênica no Desenvolvimento , Organogênese , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Galinhas , Orelha Interna/embriologia , Orelha Interna/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Notch/genéticaRESUMO
The Notch signaling pathway controls differentiation of hair cells and supporting cells in the vertebrate inner ear. Here, we have investigated whether Numb, a known regulator of Notch activity in Drosophila, is involved in this process in the embryonic chick. The chicken homolog of Numb is expressed throughout the otocyst at early stages of development and is concentrated at the basal pole of the cells. It is asymmetrically allocated at some cell divisions, as in Drosophila, suggesting that it could act as a determinant inherited by one of the two daughter cells and favoring adoption of a hair-cell fate. To test the implication of Numb in hair cell fate decisions and the regulation of Notch signaling, we used different methods to overexpress Numb at different stages of inner ear development. We found that sustained or late Numb overexpression does not promote hair cell differentiation, and Numb does not prevent the reception of Notch signaling. Surprisingly, none of the Numb-overexpressing cells differentiated into hair cells, suggesting that high levels of Numb protein could interfere with intracellular processes essential for hair cell survival. However, when Numb was overexpressed early and more transiently during ear development, no effect on hair cell formation was seen. These results suggest that in the inner ear at least, Numb does not significantly repress Notch activity and that its asymmetric distribution in dividing precursor cells does not govern the choice between hair cell and supporting cell fates.
RESUMO
Joubert syndrome (JBTS) is a severe recessive neurodevelopmental ciliopathy which can affect several organ systems. Mutations in known JBTS genes account for approximately half of the cases. By homozygosity mapping and whole-exome sequencing, we identified a novel locus, JBTS23, with a homozygous splice site mutation in KIAA0586 (alias TALPID3), a known lethal ciliopathy locus in model organisms. Truncating KIAA0586 mutations were identified in two additional patients with JBTS. One mutation, c.428delG (p.Arg143Lysfs*4), is unexpectedly common in the general population and may be a major contributor to JBTS. We demonstrate KIAA0586 protein localization at the basal body in human and mouse photoreceptors, as is common for JBTS proteins, and also in pericentriolar locations. We show that loss of TALPID3 (KIAA0586) function in animal models causes abnormal tissue polarity, centrosome length and orientation, and centriolar satellites. We propose that JBTS and other ciliopathies may in part result from cell polarity defects.
Assuntos
Proteínas de Ciclo Celular/genética , Polaridade Celular , Centrossomo/metabolismo , Cerebelo/anormalidades , Mutação , Retina/anormalidades , Anormalidades Múltiplas/genética , Animais , Modelos Animais de Doenças , Anormalidades do Olho/genética , Humanos , Doenças Renais Císticas/genética , CamundongosRESUMO
Hair cell losses in the mammalian cochlea following an ototoxic insult are irreversible. However, past studies have shown that amikacin treatment in rat cochleae resulted in the transient presence of atypical Deiters' cells (ACs) in the damaged organ of Corti. These ACs arise through a transformation of Deiters' cells, which produce, at their apical pole, densely packed microvilli reminiscent of early-differentiating stereociliary bundles. The ACs do not, however, express typical hair cell markers such as parvalbumin or calbindin. The present study was designed to determine whether specific growth factors could influence the survival and differentiation of these ACs and stimulate hair cell regeneration processes in vitro. Apical-medial segments of organ of Corti of juvenile amikacin-treated rats were established as organotypic cultures, and the effects of epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), transforming growth factor-alpha (TGFalpha), and retinoic acid were studied using morphological and molecular approaches. Our results indicate that TGFalpha supports the survival of the damaged organ of Corti and influences ACs differentiation in vitro, possibly acting through reorganization of the actin cytoskeleton. These effects could be directly mediated through activation of the EGF receptor, which is expressed by supporting cells in the mature organ of Corti. TGFalpha does not, however, allow the ACs to progress towards a hair cell phenotype.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Degeneração Neural/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Neurotoxinas/toxicidade , Fator de Crescimento Transformador alfa/farmacologia , Actinas/efeitos dos fármacos , Actinas/metabolismo , Amicacina/toxicidade , Animais , Antibacterianos/toxicidade , Bromodesoxiuridina , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestrutura , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Regeneração Nervosa/fisiologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Fator de Crescimento Transformador alfa/metabolismo , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
The functioning of the mammalian cochlea is entirely based on its mechanical properties, which are supported by a highly complex tissue architecture resulting from the precise arrangement of sensory hair cells and non-sensory supporting cells. Growing evidence indicates that evolutionary conserved signaling pathways are involved in inner ear development and in the differentiation of its diverse cell types. We investigated whether members of the Wnt and Frizzled gene families, which play key roles in a wide variety of cellular and developmental processes, are expressed in the postnatal rat cochlea. A PCR screening of a rat cochlea cDNA library performed with degenerate primers allowed us to isolate five members of the Wnt gene family (RWnt-2B, -4, -5A, -5B, and -7A) and six members of the Frizzled gene family (Rfz1, Rfz2, Rfz3, Rfz4, Rfz6, Rfz9). In situ hybridization and immunocytochemistry experiments demonstrated that RWnt-4, -5B, -7A have distinct, although partly overlapping, expression patterns in the juvenile rat cochlea. These results suggest that the Wnt-Frizzled signaling pathway could be involved in several aspects of late cochlear differentiation and/or auditory function.
Assuntos
Diferenciação Celular/genética , Cóclea/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Neurotransmissores/genética , Transdução de Sinais/genética , Proteínas de Peixe-Zebra , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Cóclea/citologia , Cóclea/metabolismo , DNA Complementar/análise , DNA Complementar/genética , Receptores Frizzled , Biblioteca Gênica , Testes Genéticos , Glicoproteínas/genética , Glicoproteínas/metabolismo , Imuno-Histoquímica , Células Labirínticas de Suporte/citologia , Células Labirínticas de Suporte/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Receptores Acoplados a Proteínas G , Receptores de Neurotransmissores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt , Proteína Wnt4RESUMO
During embryonic development, hair cells and support cells in the sensory epithelia of the inner ear derive from progenitors that express Sox2, a member of the SoxB1 family of transcription factors. Sox2 is essential for sensory specification, but high levels of Sox2 expression appear to inhibit hair cell differentiation, suggesting that factors regulating Sox2 activity could be critical for both processes. Antagonistic interactions between SoxB1 and SoxB2 factors are known to regulate cell differentiation in neural tissue, which led us to investigate the potential roles of the SoxB2 member Sox21 during chicken inner ear development. Sox21 is normally expressed by sensory progenitors within vestibular and auditory regions of the early embryonic chicken inner ear. At later stages, Sox21 is differentially expressed in the vestibular and auditory organs. Sox21 is restricted to the support cell layer of the auditory epithelium, while it is enriched in the hair cell layer of the vestibular organs. To test Sox21 function, we used two temporally distinct gain-of-function approaches. Sustained over-expression of Sox21 from early developmental stages prevented prosensory specification, and abolished the formation of both hair cells and support cells. However, later induction of Sox21 expression at the time of hair cell formation in organotypic cultures of vestibular epithelia inhibited endogenous Sox2 expression and Notch activity, and biased progenitor cells towards a hair cell fate. Interestingly, Sox21 did not promote hair cell differentiation in the immature auditory epithelium, which fits with the expression of endogenous Sox21 within mature support cells in this tissue. These results suggest that interactions among endogenous SoxB family transcription factors may regulate sensory cell formation in the inner ear, but in a context-dependent manner.
Assuntos
Orelha Interna/embriologia , Fatores de Transcrição SOXB2/biossíntese , Animais , Embrião de Galinha , Orelha Interna/citologia , Orelha Interna/metabolismo , Eletroporação , Genes Reporter , Imuno-Histoquímica , Hibridização In Situ , Fatores de Transcrição SOXB2/fisiologiaRESUMO
The vertebrate inner ear is composed of several specialized epithelia containing mechanosensory "hair" cells, sensitive to sound and head movements. In mammals, the loss of hair cells for example during aging or after noise trauma is irreversible and results in permanent sensory deficits. By contrast, avian, fish, and amphibians can efficiently regenerate lost hair cells following trauma. The chicken inner ear is a classic model system to investigate the cellular and molecular mechanisms of inner ear development and regeneration, yet it suffered until recently from a relative lack of flexible tools for genetic studies. With the introduction of in ovo electroporation and of Tol2 transposon vectors for gene transfer in avian cells, the field of experimental possibilities has now expanded significantly in this model. Here we provide a general protocol for in ovo electroporation of the chicken otic placode and illustrate how this approach, combined with Tol2 vectors, can be used to drive long-term and inducible gene expression in the embryonic chicken inner ear. This method will be particularly useful to investigate the function of candidate genes regulating progenitor cell behavior and sensory cell differentiation in the inner ear.
Assuntos
Galinhas , Elementos de DNA Transponíveis/genética , Orelha Interna/embriologia , Orelha Interna/fisiologia , Eletroporação/métodos , Regeneração/genética , Transfecção/métodos , Animais , Orelha Interna/citologia , Orelha Interna/metabolismo , Ectoderma/embriologia , Ectoderma/metabolismo , Vetores Genéticos/genética , Transfecção/instrumentaçãoRESUMO
Notch signalling is well-known to mediate lateral inhibition in inner ear sensory patches, so as to generate a balanced mixture of sensory hair cells and supporting cells. Recently, however, we have found that ectopic Notch activity at an early stage can induce the formation of ectopic sensory patches. This suggests that Notch activity may have two different functions in normal ear development, acting first to promote the formation of the prosensory patches, and then later to regulate hair-cell production within the patches. The Notch ligand Serrate1 (Jag1 in mouse and humans) is expressed in the patches from an early stage and may provide Notch activation during the prosensory phase. Here, we test whether Notch signalling is actually required for prosensory patch development. When we block Notch activation in the chick embryo using the gamma-secretase inhibitor DAPT, we see a complete loss of prosensory epithelial cells in the anterior otocyst, where they are diverted into a neuroblast fate via failure of Delta1-dependent lateral inhibition. The cells of the posterior prosensory patch remain epithelial, but expression of Sox2 and Bmp4 is drastically reduced. Expression of Serrate1 here is initially almost normal, but subsequently regresses. The patches of sensory hair cells that eventually develop are few and small. We suggest that, in normal development, factors other than Notch activity initiate Serrate1 expression. Serrate1, by activating Notch, then drives the expression of Sox2 and Bmp4, as well as expression of the Serrate1 gene itself. The positive feedback maintains Notch activation and thereby preserves and perhaps extends the prosensory state, leading eventually to the development of normal sensory patches.
Assuntos
Orelha Interna/citologia , Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas Externas , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Embrião de Galinha , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Orelha Interna/embriologia , Embrião não Mamífero , Inibidores Enzimáticos/farmacologia , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/embriologia , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/citologia , Células Ciliadas Auditivas Externas/embriologia , Células Ciliadas Auditivas Externas/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Jagged-1 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Técnicas de Cultura de Órgãos , Receptores Notch/antagonistas & inibidores , Fatores de Transcrição SOXB1 , Proteínas Serrate-Jagged , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triglicerídeos/farmacologia , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/farmacologiaRESUMO
Lateral inhibition mediated by Notch is thought to generate the mosaic of hair cells and supporting cells in the inner ear, but the effects of the activated Notch protein itself have never been directly tested. We have explored the role of Notch signalling by transiently overexpressing activated Notch (NICD) in the chick otocyst. We saw two contrasting consequences, depending on the time and site of gene misexpression: (1) inhibition of hair-cell differentiation within a sensory patch; and (2) induction of ectopic sensory patches. We infer that Notch signalling has at least two functions during inner ear development. Initially, Notch activity can drive cells to adopt a prosensory character, defining future sensory patches. Subsequently, Notch signalling within each such patch mediates lateral inhibition, restricting the proportion of cells that differentiate as hair cells so as to generate the fine-grained mixture of hair cells and supporting cells.