RESUMEN
Lack of sensory hair cell (HC) regeneration in mammalian adults is a major contributor to hearing loss. In contrast, the neonatal mouse cochlea retains a transient capacity for regeneration, and forced Wnt activation in neonatal stages promotes supporting cell (SC) proliferation and induction of ectopic HCs. We currently know little about the temporal pattern and underlying mechanism of this age-dependent regenerative response. Using an in vitro model, we show that Wnt activation promotes SC proliferation following birth, but prior to postnatal day (P) 5. This age-dependent decline in proliferation occurs despite evidence that the Wnt pathway is postnatally active and can be further enhanced by Wnt stimulators. Using an in vivo mouse model and RNA sequencing, we show that proliferation in the early neonatal cochlea is correlated with a unique transcriptional response that diminishes with age. Furthermore, we find that augmenting Wnt signaling through the neonatal stages extends the window for HC induction in response to Notch signaling inhibition. Our results suggest that the downstream transcriptional response to Wnt activation, in part, underlies the regenerative capacity of the mammalian cochlea.
Asunto(s)
Cóclea/fisiología , Mamíferos/fisiología , Regeneración/genética , Transcripción Genética , Vía de Señalización Wnt/genética , Animales , Animales Recién Nacidos , Proliferación Celular , Transdiferenciación Celular , Embrión de Mamíferos/citología , Epitelio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Células Laberínticas de Soporte/citología , Células Laberínticas de Soporte/metabolismo , Masculino , Ratones , Estabilidad Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción TCF/metabolismo , beta Catenina/metabolismoRESUMEN
Here we present spatio-temporal localization of Kremen1, a transmembrane receptor, in the mammalian cochlea, and investigate its role in the formation of sensory organs in mammal and fish model organisms. We show that Kremen1 is expressed in prosensory cells during cochlear development and in supporting cells of the adult mouse cochlea. Based on this expression pattern, we investigated whether Kremen1 functions to modulate cell fate decisions in the prosensory domain of the developing cochlea. We used gain and loss-of-function experiments to show that Kremen1 is sufficient to bias cells towards supporting cell fate, and is implicated in suppression of hair cell formation. In addition to our findings in the mouse cochlea, we examined the effects of over expression and loss of Kremen1 in the zebrafish lateral line. In agreement with our mouse data, we show that over expression of Kremen1 has a negative effect on the number of mechanosensory cells that form in the zebrafish neuromasts, and that fish lacking Kremen1 protein develop more hair cells per neuromast compared to wild type fish. Collectively, these data support an inhibitory role for Kremen1 in hair cell fate specification.
Asunto(s)
Cóclea/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/metabolismo , Sistema de la Línea Lateral/metabolismo , Proteínas de la Membrana/genética , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Cóclea/embriología , Cóclea/crecimiento & desarrollo , Sistema de la Línea Lateral/embriología , Sistema de la Línea Lateral/crecimiento & desarrollo , Mecanorreceptores/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Neurogénesis/genética , Interferencia de ARN , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: In the inner ear Wnt signaling is necessary for proliferation, cell fate determination, growth of the cochlear duct, polarized orientation of stereociliary bundles, differentiation of the periotic mesenchyme, and homeostasis of the stria vascularis. In neonatal tissue Wnt signaling can drive proliferation of cells in the sensory region, suggesting that Wnt signaling could be used to regenerate the sensory epithelium in the damaged adult inner ear. Manipulation of Wnt signaling for regeneration will require an understanding of the dynamics of Wnt pathway gene expression in the ear. We present a comprehensive screen for 84 Wnt signaling related genes across four developmental and postnatal time points. RESULTS: We identified 72 Wnt related genes expressed in the inner ear on embryonic day (E) 12.5, postnatal day (P) 0, P6 and P30. These genes included secreted Wnts, Wnt antagonists, intracellular components of canonical signaling and components of non-canonical signaling/planar cell polarity. CONCLUSION: A large number of Wnt signaling molecules were dynamically expressed during cochlear development and in the early postnatal period, suggesting complex regulation of Wnt transduction. The data revealed several potential key regulators for further study.
Asunto(s)
Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Regulación del Desarrollo de la Expresión Génica , Vía de Señalización Wnt/genética , Animales , Cóclea/citología , Cóclea/embriología , Conducto Coclear/citología , Conducto Coclear/embriología , Conducto Coclear/crecimiento & desarrollo , Conducto Coclear/metabolismo , Espacio Extracelular/metabolismo , Espacio Intracelular/metabolismo , Ratones , Análisis Espacio-Temporal , Proteínas Wnt/antagonistas & inhibidoresRESUMEN
BACKGROUND INFORMATION: The vertebrate basic helix-loop-helix transcription factor Atoh1 is essential for maturation and survival of mechanosensory hair cells of the inner ear, neurogenesis, differentiation of the intestine, homeostasis of the colon and is implicated in cancer progression. Given that mutations in Atoh1 are detected in malignant tumours, study of functionally different Atoh1 alleles and homologues might yield useful avenues for investigation. The predicted sequence of chicken Atoh1 (cAtoh1) has large regions of dissimilarity to that of mammalian Atoh1 homologues. We hypothesise that cAtoh1 might have intrinsic functional differences to mammalian Atoh1. RESULTS: In this study, we cloned and sequenced the full open reading frame of cAtoh1. In overexpression experiments, we show that this sequence is sufficient to generate a cAtoh1 protein capable of inducing hair cell markers when expressed in nonsensory regions of the developing inner ear, and that morpholino-mediated knock-down using a section of the sequence 5' to the start codon inhibits differentiation of hair cells in the chicken basilar papilla. Furthermore, we compare the behaviour of cAtoh1 and human Atoh1 (hAtoh1) in embryonic mouse cochlear explants, showing that cAtoh1 is a potent inducer of hair cell differentiation and that it can overcome Sox2-mediated repression of hair cell differentiation more effectively than hAtoh1. CONCLUSIONS: cAtoh1 is both necessary and sufficient for avian mechanosensory hair cell differentiation. The non-conserved regions of the cAtoh1 coding region have functional consequences on its behaviour.
Asunto(s)
Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Pollos/genética , Secuencia de Aminoácidos , Animales , Proteínas Aviares/química , Secuencia de Bases , Biomarcadores/metabolismo , Diferenciación Celular , Clonación Molecular , Cóclea/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/metabolismo , Humanos , Células Laberínticas de Soporte/metabolismo , Mamíferos/metabolismo , Ratones , Datos de Secuencia Molecular , Peso Molecular , Factores de Transcripción SOXB1/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.
Asunto(s)
Cóclea/embriología , Microscopía Fluorescente/métodos , Imagen de Lapso de Tiempo/métodos , Animales , Cóclea/química , Cóclea/metabolismo , Ratones , Factores de Transcripción SOXB1/análisis , Factores de Transcripción SOXB1/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
The development of hair cells in the auditory system can be separated into steps; first, the establishment of progenitors for the sensory epithelium, and second, the differentiation of hair cells. Although the differentiation of hair cells is known to require the expression of basic helix-loop-helix transcription factor, Atoh1, the control of cell proliferation in the region of the developing cochlea that will ultimately become the sensory epithelium and the cues that initiate Atoh1 expression remain obscure. We assessed the role of Wnt/ß-catenin in both steps in gain- and loss-of-function models in mice. The canonical Wnt pathway mediator, ß-catenin, controls the expression of Atoh1. Knock-out of ß-catenin inhibited hair-cell, as well as pillar-cell, differentiation from sensory progenitors but was not required to maintain a hair-cell fate once specified. Constitutive activation of ß-catenin expanded sensory progenitors by inducing additional cell division and resulted in the differentiation of extra hair cells. Our data demonstrate that ß-catenin plays a role in cell division and differentiation in the cochlear sensory epithelium.