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H. Vastinsalo, J. B. Phillips, E.-M. Sankila, M. Westerfield; Expression of Usher Syndrome Type 3 Gene (clrn1) in Zebrafish Sensory Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4134.
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© ARVO (1962-2015); The Authors (2016-present)
Usher syndrome (USH) is the most common form of combined blindness and deafness. Usher syndrome type 3 (USH3) is the rarest subtype, caused by mutations in Clarin 1 (CLRN1). CLRN1 protein has four predicted transmembrane regions and is known to localize in the plasma membrane when expressed in cell cultures. The function of CLRN1 in the retina and the cochlea is unknown. In this study, we investigate the expression and localization of clrn1 in zebrafish to study the function of Clrn1 in hearing, balance and sight.
Expression of the primary zebrafish clrn1 variant was studied in sectioned zebrafish adult and larval eyes and ears by in situ hybridization. Clrn1 protein localization was studied with an antibody specific to zebrafish Clrn1. Morpholino oligonucleotide (MO) was used to knockdown clrn1 expression and RT-PCR was used to identify altered clrn1 splicing in MO-treated larvae.
In situ hybridization showed that zebrafish clrn1 is expressed in hair cells of the larval ear and neuromasts. In larval retina, in situ expression was concentrated in the inner nuclear layer (INL), with lower expression levels detected in the photoreceptors. In adult retina, clrn1 expression was found in photoreceptors and in the INL. The Clrn1 antibody showed similar localization with Clrn1 localized to photoreceptors and a subset of cells in the INL at both larval and adult stages. RT-PCR confirmed the efficacy of MO injection.
Zebrafish clrn1 expression and protein localization in sensory hair cells was consistent with the reported findings of Clrn1 expression in mouse cochlea. In the retina, Clrn1 localization has not been reported previously, and our results demonstrate that Clrn1 is present both in the adult INL and photoreceptors. Our studies show that both clrn1 RNA and protein are present in the zebrafish larval ear and eye, and thus we can knock down Clrn1 protein expression to study the function of Clrn1 in sensory cell development.
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