Abstract
Purpose :
miR-183, miR-96, and miR-182 are expressed from a single locus and constitute the majority of microRNAs in the retina. Various animal models have demonstrated that these miRNAs are critical for retinal homeostasis. Because these miRNAs share significant sequence homology, we hypothesized that they may be redundant. To test this, we generated single, double, and triple miRNA mutant zebrafish and examined their retinal structure and function.
Methods :
We used Crispr/Cas9 to generate mutations in mir-183, mir-96, and mir-182 in all combinations. miRNA and candidate downstream gene expression was evaluated by quantitative PCR. Retinal structure was evaluated by immunohistochemistry at multiple developmental time points from 5 days post fertilization to adulthood, and retinal function was measured by analyzing the optokinetic response (OKR). Confocal microscopy was used to image sensory neurons the ears of larval fish.
Results :
Targeted miRNA expression was lost in all cases, ruling out the possibility of compensation by gene duplication. Animals missing one miRNA from the cluster were normal. mir-183/96 double mutants had normal retinal structure at all timepoints but developed abnormal swimming behavior by 12 months, consistent with late-onset vestibular malfunction. miR-183/96/182 triple knockout (miR-183 TKO) fish developed a vestibular defect as juveniles that was lethal within 2 months. Confocal imaging confirmed hair cell disorganization in miR-183 TKO mutant animals. All mutant lines had a normal OKR at 5dpf. Cacna2d1a, a predicted target of all three miRNAs, was upregulated in retina from mir-182 and mir-183 mutants.
Conclusions :
Using zebrafish as a model system, we have performed the most extensive genetic analysis of the mir-183/96/182 cluster to date. Loss of the cluster, either whole or in part, was inconsequential to retinal development within the age range studied, although early lethality of miR-183 TKO mutants precluded analyses in aged animals. However, miR-183 TKO mutants did exhibit significant defects in the structure and function of the zebrafish ear. Loss of vestibular function at different timepoints in mir-183/96 double mutants and mir-183 TKO mutants suggests that the three miRNAs cooperate additively to support vestibular hair cells, in that progressive loss of miRNAs from the cluster accelerates the development of this phenotype.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.