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J. Phillips, J. L. Chiem, M. Westerfield; Zebrafish Usher Genes Are Necessary for Retinal Cell Function and Survival. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4494.
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The molecular identification of proteins involved in Usher Syndrome has shed new light on the disease pathology, and generated many new questions about the functions of these proteins in normal hearing and vision. Genetic analysis with zebrafish is fast, easy, relatively inexpensive and very sensititive. Thus, we can use zebrafish to great advantage to complement the data emerging from mouse and human studies and make rapid progress toward a more complete understanding of Usher syndrome.
We used TBLASTN searches to locate zebrafish orthologues of all known human USH genes, and analyzed sequence similarity to establish homology within the functional domains of the genes. We amplified transcripts of ush1c, ush2a and ush2c from adult zebrafish retinas and used cRNA probes to characterize their expression patterns in larval tissues. We injected antisense morpholino oligonucleotides designed to knock down expression of these genes in developing embryos and larvae. TUNEL labeling and vital staining of apoptotic cells were used to assay retinal cell death, and antibodies to double cones and to synaptic markers were used to characterize the structural integrity of the retina. We assayed visual function in live larvae with the optokinetic response test.
All three genes are expressed in the neural retina and sensory hair cells of the ear and lateral line from neurogenesis stages through early larval stages. Transcripts are also present in the adult retina. Knock-down of zebrafish Ush1c, Ush2a and Ush2c by morpholino injection results in swimming and balance defects in young larvae similar to those seen in ush1b (myo7a) mutant fish. We observe reduced visual function and increased cell death in the retina within the first week of development. Cone photoreceptor integrity is slightly disrupted in the Ush2a morpholino-injected animals. Pre-and post-synaptic proteins in morpholino-injected animals at the photoreceptor synapses are abnormally localized, appearing patchy and poorly aligned.
Zebrafish USH orthologues are expressed in tissues consistent with the human disease pathology and at time points that suggest roles in development as well as maintenance of sensory cells. Depletion of functional Ush1c (Harmonin), Ush2a (Usherin) or Ush2c (VLGR1) proteins result in poor visual function and retinal cell death, although these defects are not coupled with a pronounced change in photoreceptor morphology. Photoreceptor synapses are perturbed in morpholino injected animals, suggesting that some Usher proteins may be important for establishing or maintaining synaptic integrity.
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