RESUMEN
BACKGROUND: The semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. Little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. Zebrafish represent a particularly tractable model system for the study of inner ear development. This is because the ear can be easily visualized during early embryogenesis, and both forward and reverse genetic techniques are available that can be applied to the discovery of novel genes that contribute to proper ear development. We have previously shown that in zebrafish, the calcium sensing molecule neuronal calcium sensor-1 (NCS-1) is required for semicircular canal formation. The function of NCS-1 in regulating semicircular canal formation has not yet been elucidated. RESULTS: We initiated a multistep functional proteomic strategy to identify neuronal calcium sensor-1 (NCS-1) binding partners (NBPs) that contribute to inner ear development in zebrafish. By performing a Y2H screen in combination with literature and database searches, we identified 10 human NBPs. BLAST searches of the zebrafish EST and genomic databases allowed us to clone zebrafish orthologs of each of the human NBPs. By investigating the expression profiles of zebrafish NBP mRNAs, we identified seven that were expressed in the developing inner ear and overlapped with the ncs-1a expression profile. GST pulldown experiments confirmed that selected NBPs interacted with NCS-1, while morpholino-mediated knockdown experiments demonstrated an essential role for arf1, pi4kbeta, dan, and pink1 in semicircular canal formation. CONCLUSION: Based on their functional profiles, the hypothesis is presented that Ncs-1a/Pi4kbeta/Arf1 form a signaling pathway that regulates secretion of molecular components, including Dan and Bmp4, that are required for development of the vestibular apparatus. A second set of NBPs, consisting of Pink1, Hint2, and Slc25a25, are destined for localization in mitochondria. Our findings reveal a novel signalling pathway involved in development of the semicircular canal system, and suggest a previously unrecognized role for NCS-1 in mitochondrial function via its association with several mitochondrial proteins.
Asunto(s)
Oído Interno/embriología , Proteínas Sensoras del Calcio Neuronal/genética , Neuropéptidos/genética , Organogénesis/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Proteínas Portadoras/clasificación , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Oído Interno/metabolismo , Oído Interno/ultraestructura , Embrión no Mamífero , Exocitosis/genética , Exocitosis/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Organogénesis/fisiología , Proteómica/métodos , ARN Mensajero/análisis , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Within the vestibular system of virtually all vertebrate species, gravity and linear acceleration are detected via coupling of calcified masses to the cilia of mechanosensory hair cells. The mammalian ear contains thousands of minute biomineralized particles called otoconia, whereas the inner ear of teleost fish contains three large ear stones called otoliths that serve a similar function. Otoconia and otoliths are composed of calcium carbonate crystals condensed on a core protein lattice. Otoconin-90 (Oc90) is the major matrix protein of mammalian and avian otoconia, while otolith matrix protein (OMP) is the most abundant matrix protein found in the otoliths of teleost fish. We have identified a novel gene, otoc1, which encodes the zebrafish ortholog of Oc90. Expression of otoc1 was detected in the ear between 15 hpf and 72 hpf, and was restricted primarily to the macula and the developing epithelial pillars of the semicircular canals. Expression of otoc1 was also detected in epiphysis, optic stalk, midbrain, diencephalon, flexural organ, and spinal cord. During embryogenesis, expression of otoc1 mRNA preceded the appearance of omp-1 transcripts. Knockdown of otoc1 mRNA translation with antisense morpholinos produced a variety of aberrant otolith phenotypes. Our results suggest that Otoc1 may serve to nucleate calcium carbonate mineralization of aragonitic otoliths.