Abstract
BackgroundThe 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.ResultsWe 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, pi4kβ, dan, and pink1 in semicircular canal formation.ConclusionBased on their functional profiles, the hypothesis is presented that Ncs-1a/Pi4kβ/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.
Highlights
The semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration
Based on their functional properties we chose to pursue DAN, a secreted Bmp antagonist that has previously been shown to play a role in semicircular canal formation [23]; VAMP2, a synaptosomal protein involved in exocytosis [24,25,26]; and the mitochondrial proteins HINT2, SLC25A25 (SCAMC2), and PINK1, a mitochondrial kinase which has been found to be mutated in inherited forms of Parkinson's disease [27]
We show that several new and previously identified neuronal calcium sensor-1 (NCS-1) binding protein (NBP) appear to play an important role in the development of the zebrafish inner ear
Summary
The semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. The semicircular canals, one subdivision of the vestibular system, function as sensors of angular acceleration These structures enhance survival by implementing postural and visual stabilization during motion in a three-dimensional environment [1,2,3]. The zebrafish ortholog of the human deafness gene dfna, was subsequently shown to be necessary for ugdh expression and proper semicircular canal and jaw development [14]. Another zebrafish mutant, gallery, displays only a lateral semicircular canal protrusion and an immature anterior protrusion [15]. The gallery gene has not yet been identified, the defects in this mutant are believed to be caused by overexpression of Bmp (bone morphogenetic protein 4) in the presumptive cristae
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