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Joseph Fogerty, Kyle M Patterson, Brian D Perkins; Differential effects of mutations in the miR183/96/182 cluster on zebrafish sensory tissues. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3570. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The miR183/96/182 cluster consists of three paralogous miRNAs located in a short intergenic region that is highly conserved across metazoans. They account for nearly 70 percent of all expressed miRNAs in the retina, and are also expressed in inner ear hair cells. Several animal models of retinitis pigmentosa show reduced expression of this cluster, suggesting that its dysregulation contributes to retinal disease. The seed sequences of all three miRNAs are highly conserved, with those of miR96 and miR182 being identical, suggesting functional redundancy. The purpose of this study is to determine the scope of functional redundancy among these miRNAs within the zebrafish retina.
The CRISPR/Cas9 system was used to generate mutations in miR183, miR96, and miR182 in zebrafish. In vitro luciferase assays were used to test function and target specificity of wild-type and mutant miRNA alleles. Mutants were evaluated by immunostaining for photoreceptor-specific markers and by plastic histology at 5 dpf and 4 months.
We generated mutations at each miRNA locus individually and confirmed that these mutations inhibited miRNA targeting. Wild-type miRNAs targeted only their cognate sequences, indicating a capacity for functional autonomy. Homozygous mutants at any single miRNA locus were viable, fertile, and lacked retinal phenotypes. A miR183-/-; miR96-/- double mutant was similarly unaffected. High-dosage CRISPRs targeting miR182 in the miR183-/-; miR96-/- double mutant yielded a cohort of fish that developed abnormal swimming patterns, indicating a malfunctioning vestibular system, but the retinas of these animals remained normal at 4 months.
Prior studies in a mouse miR96 mutant suggest that the abnormal swimming patterns are likely a result of hair cell degeneration. While miRNA target specificity was very high when tested in vitro, the observation that only mutations at multiple loci were sufficient to generate an abnormal phenotype suggests that they may function cooperatively in vivo. The lack of a retinal phenotype despite the clear vestibular defect indicates that the targets of these miRNAs are likely highly tissue specific. Further development and analysis of compound mutations within this cluster as well as identification of target mRNAs will help us to understand its neuroprotective properties.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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