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J. B. Phillips, J. J. Lentz, K. Owens, K. Khanobdee, S. Sampath, B. J. Keats, D. Raible, D. S. Williams, M. Westerfield; Analysis of Zebrafish Ush1c (Harmonin) in Sensory Cell Function and Synaptic Integrity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3070.
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Usher syndrome is a clinically and genetically heterogeneous form of inherited deaf-blindness. Approximately 6% of congenitally deaf children are affected and will undergo progressive vision loss by the first or second decade of life. Of the 9 identified Usher proteins, the multi-PDZ domain protein Harmonin may serve as a major scaffolding molecule. We are investigating the functional role of Harmonin in retinal and mechanosensory hair cells.
We cloned zebrafish ush1c, which encodes Harmonin, and obtained sequence for three isoforms. We used in situ hybridization and a Harmonin antibody to visualize gene expression and protein localization, respectively. Morpholino oligonucleotides were used to knock down protein levels in developing embryos and larvae, and visual and auditory defects were detected through functional and histological assays.
Ush1c is expressed in the neural retina and the otic epithelium from the earliest time at which these cell types are specified. Ush1c isoform A is enriched in bipolar cells in the larval retina, whereas isoforms B and C are expressed in all retinal cell layers at all stages examined. The Harmonin antibody localizes to photoreceptors, bipolar cells, and retinal ganglion cells, as well as to the OPL. Hair cells of the inner ear and lateral line are also labeled with this antibody. Knock-down of Ush1c by morpholinos results in impaired balance and reduced function and viability of retinal and hair cells. Synaptic markers in the outer plexiform layer are mislocalized in morpholino-injected animals, and examination of ultrastructure reveals reduced numbers of mature synaptic connections between cones and second order neurons.
Harmonin has a conserved role in vertebrate vision and hearing, and depletion of Harmonin results in reduced visual and auditory function. These studies provide the first evidence of a synaptic defect due the presence of an abnormal Usher protein. Our data also indicate a role for Harmonin in the inner retina.
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