RESUMEN
The cochlea's ability to discriminate sound frequencies is facilitated by a special topography along its longitudinal axis known as tonotopy. Auditory hair cells located at the base of the cochlea respond to high-frequency sounds, whereas hair cells at the apex respond to lower frequencies. Gradual changes in morphological and physiological features along the length of the cochlea determine each region's frequency selectivity, but it remains unclear how tonotopy is established during cochlear development. Recently, sonic hedgehog (SHH) was proposed to initiate the establishment of tonotopy by conferring regional identity to the primordial cochlea. Here, using mouse genetics, we provide in vivo evidence that regional identity in the embryonic cochlea acts as a framework upon which tonotopy-specific properties essential for frequency selectivity in the mature cochlea develop. We found that follistatin (FST) is required for the maintenance of apical cochlear identity, but dispensable for its initial induction. In a fate-mapping analysis, we found that FST promotes expansion of apical cochlear cells, contributing to the formation of the apical cochlear domain. SHH, in contrast, is required both for the induction and maintenance of apical identity. In the absence of FST or SHH, mice produce a short cochlea lacking its apical domain. This results in the loss of apex-specific anatomical and molecular properties and low-frequency-specific hearing loss.
Asunto(s)
Folistatina , Proteínas Hedgehog , Animales , Ratones , Folistatina/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Cóclea/fisiología , Audición/fisiología , Mamíferos/metabolismoRESUMEN
Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming; however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 re-expression increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71's RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation.
Asunto(s)
Cóclea , Células Ciliadas Auditivas , Animales , Humanos , Ratones , Diferenciación Celular/genética , Cóclea/metabolismo , Perfilación de la Expresión Génica , Células Ciliadas Auditivas/metabolismo , Células Madre/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The evolutionarily conserved lethal-7 (let-7) microRNAs (miRNAs) are well-known activators of proliferative quiescence and terminal differentiation. However, in the murine auditory organ, let-7g overexpression delays the differentiation of mechano-sensory hair cells (HCs). To address whether the role of let-7 in auditory-sensory differentiation is conserved among vertebrates, we manipulated let-7 levels within the chicken auditory organ: the basilar papilla. Using a let-7 sponge construct to sequester let-7 miRNAs, we found that endogenous let-7 miRNAs are essential for limiting the self-renewal of HC progenitor cells. Furthermore, let-7b overexpression experiments revealed that, similar to mice, higher than normal let-7 levels slow/delay HC differentiation. Finally, we identify CHD7, a chromatin remodeler, as a candidate for mediating the repressive function of let-7 in HC differentiation and inner ear morphogenesis. Our analysis uncovered an evolutionarily conserved let-7-5p-binding site within the chicken Chd7 gene and its human and murine homologs, and we show that let-7g overexpression in mice limits CHD7 expression in the developing inner ear, retina and brain. Haploinsufficiency of CHD7 in humans causes CHARGE syndrome and attenuation of let-7 function may be an effective method for treating CHD7 deficiency.
Asunto(s)
Proteínas Aviares/biosíntesis , Pollos/metabolismo , ADN Helicasas/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Células Ciliadas Auditivas/metabolismo , MicroARNs/metabolismo , Células Madre/metabolismo , Animales , Proteínas Aviares/genética , Diferenciación Celular , Embrión de Pollo , Pollos/genética , ADN Helicasas/genética , Células Ciliadas Auditivas/citología , Humanos , Ratones , MicroARNs/genética , Células Madre/citologíaRESUMEN
Mechano-sensory hair cells within the inner ear cochlea are essential for the detection of sound. In mammals, cochlear hair cells are only produced during development and their loss, due to disease or trauma, is a leading cause of deafness. In the immature cochlea, prior to the onset of hearing, hair cell loss stimulates neighboring supporting cells to act as hair cell progenitors and produce new hair cells. However, for reasons unknown, such regenerative capacity (plasticity) is lost once supporting cells undergo maturation. Here, we demonstrate that the RNA binding protein LIN28B plays an important role in the production of hair cells by supporting cells and provide evidence that the developmental drop in supporting cell plasticity in the mammalian cochlea is, at least in part, a product of declining LIN28B-mammalian target of rapamycin (mTOR) activity. Employing murine cochlear organoid and explant cultures to model mitotic and nonmitotic mechanisms of hair cell generation, we show that loss of LIN28B function, due to its conditional deletion, or due to overexpression of the antagonistic miRNA let-7g, suppressed Akt-mTOR complex 1 (mTORC1) activity and renders young, immature supporting cells incapable of generating hair cells. Conversely, we found that LIN28B overexpression increased Akt-mTORC1 activity and allowed supporting cells that were undergoing maturation to de-differentiate into progenitor-like cells and to produce hair cells via mitotic and nonmitotic mechanisms. Finally, using the mTORC1 inhibitor rapamycin, we demonstrate that LIN28B promotes supporting cell plasticity in an mTORC1-dependent manner.
Asunto(s)
Células Ciliadas Auditivas/fisiología , Células Laberínticas de Soporte/metabolismo , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Genotipo , Ratones , MicroARNs/genética , Organoides , Proteínas de Unión al ARN/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important role in the specification of the prosensory region, which gives rise to sound-sensing hair cells and neighboring supporting cells (SCs). While JAG1's expression is maintained in SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we demonstrate that JAG1 is essential for the formation and maintenance of Hensen's cells, a highly specialized SC subtype located at the edge of the auditory epithelium. Using Sox2CreERT2/+::Jag1loxP/loxP mice of both genders, we show that Jag1 deletion at the onset of differentiation, at embryonic day 14.5, disrupted Hensen's cell formation. Similar loss of Hensen's cells was observed when Jag1 was deleted after Hensen's cell formation at postnatal day (P) 0/P1 and fate-mapping analysis revealed that in the absence of Jag1, some Hensen's cells die, but others convert into neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved in mitochondrial function and protein synthesis were downregulated in the sensory epithelium of P0 cochlea lacking Jag1 Finally, using Fgfr3-iCreERT2 ::Jag1loxP/loxP mice to delete Jag1 at P0, we observed a similar loss of Hensen's cells and found that adult Jag1 mutant mice have hearing deficits at the low-frequency range.SIGNIFICANCE STATEMENT Hensen's cells play an essential role in the development and homeostasis of the cochlea. Defects in the biophysical or functional properties of Hensen's cells have been linked to auditory dysfunction and hearing loss. Despite their importance, surprisingly little is known about the molecular mechanisms that guide their development. Morphologic and fate-mapping analyses in our study revealed that, in the absence of the Notch ligand JAGGED1, Hensen's cells died or converted into Claudius cells, which are specialized epithelium-like cells outside the sensory epithelium. Confirming a link between JAGGED1 and cell survival, transcriptional profiling showed that JAGGED1 maintains genes critical for mitochondrial function and tissue homeostasis. Finally, auditory phenotyping revealed that JAGGED1's function in supporting cells is necessary for low-frequency hearing.
Asunto(s)
Cóclea/metabolismo , Proteína Jagged-1/metabolismo , Células Laberínticas de Soporte/fisiología , Animales , Supervivencia Celular , Cóclea/citología , Cóclea/crecimiento & desarrollo , Regulación hacia Abajo , Potenciales Evocados Auditivos del Tronco Encefálico , Femenino , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Proteína Jagged-1/genética , Masculino , Ratones , Ratones Noqueados , Embarazo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
The vertebrate inner ear houses highly specialized sensory organs, tuned to detect and encode sound, head motion and gravity. Gene expression programs under the control of transcription factors orchestrate the formation and specialization of the non-sensory inner ear labyrinth and its sensory constituents. More recently, epigenetic factors and non-coding RNAs emerged as an additional layer of gene regulation, both in inner ear development and disease. In this review, we provide an overview on how epigenetic modifications and non-coding RNAs, in particular microRNAs (miRNAs), influence gene expression and summarize recent discoveries that highlight their critical role in the proper formation of the inner ear labyrinth and its sensory organs. Finally, we discuss recent insights into how epigenetic factors and miRNAs may facilitate, or in the case of mammals, restrict inner ear sensory hair cell regeneration.
Asunto(s)
Epigénesis Genética , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodominio/genética , MicroARNs/genética , Organogénesis/genética , Factores de Transcripción Otx/genética , Animales , Diferenciación Celular , Embrión de Pollo , Cromatina/química , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , MicroARNs/metabolismo , Factores de Transcripción Otx/metabolismo , Regeneración/genéticaRESUMEN
Proper tissue development requires strict coordination of proliferation, growth, and differentiation. Strict coordination is particularly important for the auditory sensory epithelium, where deviations from the normal spatial and temporal pattern of auditory progenitor cell (prosensory cell) proliferation and differentiation result in abnormal cellular organization and, thus, auditory dysfunction. The molecular mechanisms involved in the timing and coordination of auditory prosensory proliferation and differentiation are poorly understood. Here we identify the RNA-binding protein LIN28B as a critical regulator of developmental timing in the murine cochlea. We show that Lin28b and its opposing let-7 miRNAs are differentially expressed in the auditory sensory lineage, with Lin28b being highly expressed in undifferentiated prosensory cells and let-7 miRNAs being highly expressed in their progeny-hair cells (HCs) and supporting cells (SCs). Using recently developed transgenic mouse models for LIN28B and let-7g, we demonstrate that prolonged LIN28B expression delays prosensory cell cycle withdrawal and differentiation, resulting in HC and SC patterning and maturation defects. Surprisingly, let-7g overexpression, although capable of inducing premature prosensory cell cycle exit, failed to induce premature HC differentiation, suggesting that LIN28B's functional role in the timing of differentiation uses let-7 independent mechanisms. Finally, we demonstrate that overexpression of LIN28B or let-7g can significantly alter the postnatal production of HCs in response to Notch inhibition; LIN28B has a positive effect on HC production, whereas let-7 antagonizes this process. Together, these results implicate a key role for the LIN28B/let-7 axis in regulating postnatal SC plasticity.
Asunto(s)
Cóclea/embriología , Cóclea/metabolismo , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario , Mamíferos/embriología , Mamíferos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Ciclo Celular/genética , Diferenciación Celular/genética , Linaje de la Célula , Proteínas de Unión al ADN/genética , Desarrollo Embrionario/genética , Epitelio/embriología , Epitelio/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Células Laberínticas de Soporte/citología , Células Laberínticas de Soporte/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Unión al ARN/genética , Receptores Notch/metabolismo , Transducción de Señal/genética , Factores de TiempoRESUMEN
Mechano-sensory hair cells (HCs), housed in the inner ear cochlea, are critical for the perception of sound. In the mammalian cochlea, differentiation of HCs occurs in a striking basal-to-apical and medial-to-lateral gradient, which is thought to ensure correct patterning and proper function of the auditory sensory epithelium. Recent studies have revealed that Hedgehog signaling opposes HC differentiation and is critical for the establishment of the graded pattern of auditory HC differentiation. However, how Hedgehog signaling interferes with HC differentiation is unknown. Here, we provide evidence that in the murine cochlea, Hey1 and Hey2 control the spatiotemporal pattern of HC differentiation downstream of Hedgehog signaling. It has been recently shown that HEY1 and HEY2, two highly redundant HES-related transcriptional repressors, are highly expressed in supporting cell (SC) and HC progenitors (prosensory cells), but their prosensory function remained untested. Using a conditional double knock-out strategy, we demonstrate that prosensory cells form and proliferate properly in the absence of Hey1 and Hey2 but differentiate prematurely because of precocious upregulation of the pro-HC factor Atoh1. Moreover, we demonstrate that prosensory-specific expression of Hey1 and Hey2 and its subsequent graded downregulation is controlled by Hedgehog signaling in a largely FGFR-dependent manner. In summary, our study reveals a critical role for Hey1 and Hey2 in prosensory cell maintenance and identifies Hedgehog signaling as a novel upstream regulator of their prosensory function in the mammalian cochlea. The regulatory mechanism described here might be a broadly applied mechanism for controlling progenitor behavior in the central and peripheral nervous system.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas de Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Proteínas Hedgehog/fisiología , Proteínas Represoras/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Ciclo Celular/genética , Diferenciación Celular/genética , Proliferación Celular , Cóclea/citología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Ratones Noqueados , Ratones Transgénicos , Proteínas Represoras/genética , Transducción de Señal/fisiología , Análisis Espacio-Temporal , Técnicas de Cultivo de TejidosRESUMEN
Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming, however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 reactivation increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71â™s RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation.
RESUMEN
Sensory hair cells of the mammalian organ of Corti in the inner ear do not regenerate when lost as a consequence of injury, disease, or age-related deafness. This contrasts with other vertebrates such as birds, where the death of hair cells causes surrounding supporting cells to re-enter the cell cycle and give rise to both new hair cells and supporting cells. It is not clear whether the lack of mammalian hair cell regeneration is due to an intrinsic inability of supporting cells to divide and differentiate or to an absence or blockade of regenerative signals. Here we show that post-mitotic supporting cells purified from the postnatal mouse cochlea retain the ability to divide and trans-differentiate into new hair cells in culture. Furthermore, we show that age-dependent changes in supporting cell proliferative capacity are due in part to changes in the ability to downregulate the cyclin-dependent kinase inhibitor p27(Kip1) (also known as Cdkn1b). These results indicate that postnatal mammalian supporting cells are potential targets for therapeutic manipulation.
Asunto(s)
Diferenciación Celular , Cóclea/citología , Células Ciliadas Auditivas Internas/citología , Animales , Ciclo Celular , Células Cultivadas , Técnicas de Cocultivo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Citometría de Flujo , Ratones , Órgano Espiral/citología , Receptor de Factor de Crecimiento Nervioso/biosíntesisRESUMEN
Hair cell (HC) loss within the inner ear cochlea is a leading cause for deafness in humans. Before the onset of hearing, immature supporting cells (SCs) in neonatal mice have some limited capacity for HC regeneration. Here, we show that in organoid culture, transient activation of the progenitor-specific RNA binding protein LIN28B and Activin antagonist follistatin (FST) enhances regenerative competence of maturing/mature cochlear SCs by reprogramming them into progenitor-like cells. Transcriptome profiling and mechanistic studies reveal that LIN28B drives SC reprogramming, while FST is required to counterbalance hyperactivation of transforming growth factor-ß-type signaling by LIN28B. Last, we show that LIN28B and FST coactivation enhances spontaneous cochlear HC regeneration in neonatal mice and that LIN28B may be part of an endogenous repair mechanism that primes SCs for HC regeneration. These findings indicate that SC dedifferentiation is critical for HC regeneration and identify LIN28B and FST as main regulators.
Asunto(s)
Reprogramación Celular , Folistatina , Animales , Reprogramación Celular/genética , Cóclea/metabolismo , Folistatina/genética , Folistatina/metabolismo , Células Ciliadas Auditivas/metabolismo , Ratones , Regeneración/genéticaRESUMEN
The mammalian auditory sensory epithelium has one of the most stereotyped cellular patterns known in vertebrates. Mechano-sensory hair cells are arranged in precise rows, with one row of inner and three rows of outer hair cells spanning the length of the spiral-shaped sensory epithelium. Aiding such precise cellular patterning, differentiation of the auditory sensory epithelium is precisely timed and follows a steep longitudinal gradient. The molecular signals that promote auditory sensory differentiation and instruct its graded pattern are largely unknown. Here, we identify Activin A and its antagonist follistatin as key regulators of hair cell differentiation and show, using mouse genetic approaches, that a local gradient of Activin A signaling within the auditory sensory epithelium times the longitudinal gradient of hair cell differentiation. Furthermore, we provide evidence that Activin-type signaling regulates a radial gradient of terminal mitosis within the auditory sensory epithelium, which constitutes a novel mechanism for limiting the number of inner hair cells being produced.
Asunto(s)
Activinas/metabolismo , Diferenciación Celular , Cóclea/citología , Folistatina/metabolismo , Células Ciliadas Auditivas/citología , Animales , Ciclo Celular , Células Ciliadas Auditivas/metabolismo , Ratones , Transducción de SeñalRESUMEN
Sensory hair cells of the auditory organ are generated during embryogenesis and remain postmitotic throughout life. Previous work has shown that inactivation of the cyclin-dependent kinase inhibitor (CKI) p19(Ink4d) leads to progressive hearing loss attributable to inappropriate DNA replication and subsequent apoptosis of hair cells. Here we show the synergistic action of another CKI, p21(Cip1), on cell cycle reactivation. The codeletion of p19(Ink4d) and p21(Cip1) triggered profuse S-phase entry of auditory hair cells during a restricted period in early postnatal life, leading to the transient appearance of supernumerary hair cells. In addition, we show that aberrant cell cycle reentry leads to activation of a DNA damage response pathway in these cells, followed by p53-mediated apoptosis. The majority of hair cells were absent in adult cochleas. These data, together with the demonstration of changing expression patterns of multiple CKIs in auditory hair cells during the stages of early postnatal maturation, show that the maintenance of the postmitotic state is an active, tissue-specific process, cooperatively regulated by several CKIs, and is critical for the lifelong survival of these sensory cells.
Asunto(s)
Apoptosis/fisiología , Cóclea/patología , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Genes cdc , Células Ciliadas Auditivas/patología , Fase S , Animales , Animales Recién Nacidos , Animales Lactantes , Recuento de Células , Cóclea/crecimiento & desarrollo , Cruzamientos Genéticos , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/biosíntesis , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN , Reparación del ADN/genética , Progresión de la Enfermedad , Células Ciliadas Auditivas/metabolismo , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitosis , Especificidad de Órganos , Órgano Espiral/patología , Transfección , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
The cyclin-dependent kinase inhibitor p21/WAF1/CIP1 is an important regulator of cell cycle progression, senescence, and differentiation. Genotoxic stress leads to activation of the tumor suppressor p53 and subsequently to induction of p21 expression. Here we show that the tumor suppressor p53 cooperates with the transcription factor Sp1 in the activation of the p21 promoter, whereas histone deacetylase 1 (HDAC1) counteracts p53-induced transcription from the p21 gene. The p53 protein binds directly to the C terminus of Sp1, a domain which was previously shown to be required for the interaction with HDAC1. Induction of p53 in response to DNA-damaging agents resulted in the formation of p53-Sp1 complexes and simultaneous dissociation of HDAC1 from the C terminus of Sp1. Chromatin immunoprecipitation experiments demonstrated the association of HDAC1 with the p21 gene in proliferating cells. Genotoxic stress led to recruitment of p53, reduced binding of HDAC1, and hyperacetylation of core histones at the p21 promoter. Our findings show that the deacetylase HDAC1 acts as an antagonist of the tumor suppressor p53 in the regulation of the cyclin-dependent kinase inhibitor p21 and provide a basis for understanding the function of histone deacetylase inhibitors as antitumor drugs.
Asunto(s)
Ciclinas/genética , Histona Desacetilasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Antineoplásicos/farmacología , Sitios de Unión , Unión Competitiva , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Daño del ADN , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Histona Desacetilasa 1 , Inhibidores de Histona Desacetilasas , Humanos , Proteínas Gestacionales/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/metabolismo , TransfecciónRESUMEN
Expression of the cyclin-dependent kinase inhibitor p27(Kip1) defines a post-mitotic population of cells in the embryonic mammalian cochlea that constitutes the nascent organ of Corti. Here, we describe techniques to purify these precursors using a transgenic p27/GFP reporter and fluorescence activated cell sorting (FACS). We demonstrate that these cells express other markers of the sensory lineage, such as Sox2, and when placed in dissociated cell culture differentiate as hair cells and supporting cells. The purified sensory progenitors thus obtained provide a means of studying the process of hair cell and supporting cell differentiation in vitro, as well as providing a means of analyzing the molecular and physiological properties of this unique population of cells.
Asunto(s)
Cóclea/citología , Cóclea/embriología , Células Ciliadas Auditivas/fisiología , Células Laberínticas de Soporte/fisiología , Células Madre/fisiología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Embrión de Mamíferos , Femenino , Citometría de Flujo/métodos , Proteínas Fluorescentes Verdes/biosíntesis , Masculino , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2´-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.
Asunto(s)
Embrión de Pollo , Electroporación/métodos , Transfección/métodos , Animales , Técnicas de Transferencia de Gen , Vectores Genéticos , PlásmidosRESUMEN
The auditory sensory epithelium, composed of mechano-sensory hair cells (HCs) and highly specialized glial-like supporting cells (SCs), is critical for our ability to detect sound. SCs provide structural and functional support to HCs and play an essential role in cochlear development, homeostasis and repair. Despite their importance, however, surprisingly little is known about the molecular mechanisms guiding SC differentiation. Here, we provide evidence that in addition to its well-characterized inhibitory function, canonical Notch signaling plays a positive, instructive role in the differentiation of SCs. Using γ-secretase inhibitor DAPT to acutely block canonical Notch signaling, we identified a cohort of Notch-regulated SC-specific genes, with diverse functions in cell signaling, cell differentiation, neuronal innervation and synaptogenesis. We validated the newly identified Notch-regulated genes in vivo using genetic gain (Emx2(Cre/+); Rosa26(N1ICD/+)) and loss-of-function approaches (Emx2(Cre/+); Rosa26(DnMAML1/+)). Furthermore, we demonstrate that Notch over-activation in the differentiating murine cochlea (Emx2(Cre/+); Rosa26(N1ICD/+)) actively promotes a SC-specific gene expression program. Finally, we show that outer SCs -so called Deiters' cells are selectively lost by prolonged reduction (Emx2(Cre/+); Rosa26(DnMAML1/+/+)) or abolishment of canonical Notch signaling (Fgfr3-iCreER; Rbpj(-/Δ)), indicating a critical role for Notch signaling in Deiters' cell development.
Asunto(s)
Células Laberínticas de Soporte/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Recuento de Células , Muerte Celular , Diferenciación Celular/genética , Cóclea/citología , Cóclea/embriología , Cóclea/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Células Laberínticas de Soporte/citología , Ratones , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidad de Órganos/genética , Fenotipo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The signals that induce the organ of Corti and define its boundaries in the cochlea are poorly understood. We show that two Notch modifiers, Lfng and Mfng, are transiently expressed precisely at the neural boundary of the organ of Corti. Cre-Lox fate mapping shows this region gives rise to inner hair cells and their associated inner phalangeal cells. Mutation of Lfng and Mfng disrupts this boundary, producing unexpected duplications of inner hair cells and inner phalangeal cells. This phenotype is mimicked by other mouse mutants or pharmacological treatments that lower but not abolish Notch signaling. However, strong disruption of Notch signaling causes a very different result, generating many ectopic hair cells at the expense of inner phalangeal cells. Our results show that Notch signaling is finely calibrated in the cochlea to produce precisely tuned levels of signaling that first set the boundary of the organ of Corti and later regulate hair cell development.
Asunto(s)
Glicosiltransferasas/metabolismo , Órgano Espiral/embriología , Proteínas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Glucosiltransferasas , Glicosiltransferasas/genética , Ratones , Mutación , Proteínas/genéticaRESUMEN
Math1 is a bHLH transcription factor expressed in neural progenitor cells in multiple regions of the nervous system. Previously we identified a Math1 enhancer that directs expression of reporter genes in a Math1 specific pattern [Development 127 (2000) 1185]. We have used a portion of this enhancer to drive expression of a nuclear GFP reporter in the Math1 lineage in transgenic mice. In this transgenic mouse strain, GFP is expressed in Math1 domains in the (1). developing spinal cord in progenitors to dI1 dorsal interneurons, (2). granule-cell progenitors in the developing cerebellum, (3). Merkel cells in the skin, and (4). hair cells in the developing vestibular and auditory systems. Furthermore, non-Math1 related expression is detected that is likely due to the absence of inhibitory regulatory sequences from the transgene. These expression domains include (1). the apical ectodermal ridge in developing limbs, (2). post-mitotic cells in the developing cortex and spinal cord, (3). the dentate gyrus, (4). retina, and (5). olfactory epithelium. Because GFP marks specific neuronal cell types in living tissue, this transgenic strain is a powerful tool for future studies on the development and electrophysiological properties of distinct cell types in the central nervous system and in sensory systems.
Asunto(s)
Cerebelo/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas Luminiscentes/genética , Factores de Transcripción/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cerebelo/metabolismo , Femenino , Proteínas Fluorescentes Verdes , Hibridación in Situ , Interneuronas/metabolismo , Operón Lac , Proteínas Luminiscentes/metabolismo , Células de Merkel/citología , Células de Merkel/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Piel/citología , Piel/metabolismo , Médula Espinal/embriología , Médula Espinal/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In mammals, auditory hair cells are generated only during embryonic development and loss or damage to hair cells is permanent. However, in non-mammalian vertebrate species, such as birds, neighboring glia-like supporting cells regenerate auditory hair cells by both mitotic and non-mitotic mechanisms. Based on work in intact cochlear tissue, it is thought that Notch signaling might restrict supporting cell plasticity in the mammalian cochlea. However, it is unresolved how Notch signaling functions in the hair cell-damaged cochlea and the molecular and cellular changes induced in supporting cells in response to hair cell trauma are poorly understood. Here we show that gentamicin-induced hair cell loss in early postnatal mouse cochlear tissue induces rapid morphological changes in supporting cells, which facilitate the sealing of gaps left by dying hair cells. Moreover, we provide evidence that Notch signaling is active in the hair cell damaged cochlea and identify Hes1, Hey1, Hey2, HeyL, and Sox2 as targets and potential Notch effectors of this hair cell-independent mechanism of Notch signaling. Using Cre/loxP based labeling system we demonstrate that inhibition of Notch signaling with a γ- secretase inhibitor (GSI) results in the trans-differentiation of supporting cells into hair cell-like cells. Moreover, we show that these hair cell-like cells, generated by supporting cells have molecular, cellular, and basic electrophysiological properties similar to immature hair cells rather than supporting cells. Lastly, we show that the vast majority of these newly generated hair cell-like cells express the outer hair cell specific motor protein prestin.