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Shi Song Rong, Louis R. Pasquale, Anna Larson, Kinga Maria Bujakowska, Janey L Wiggs; Functional annotation of glaucoma-associated ATXN2 variants using zebrafish. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5157.
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Genome-wide association studies have identified significant associations of common and rare ATXN2 variants with primary open angle glaucoma (POAG). To establish a model system to test the functional effects of potentially pathogenic ATXN2 variants, we characterized the ocular phenotype associated with reduced atxn2 expression in zebrafish.
A translation-blocking (TB) morpholino was used to reduce atxn2 expression by injection into AB strain zebrafish embryos at the 1- to 8-cell stage. Eye size, retinal structures, and visual motor response (VMR) were compared between TB morphants, negative controls (NC), and wild-type (WT) fish at 5 days post fertilization (dpf). We further tested the specificity of the morpholino by co-injecting human ATXN2 mRNA with the mopholino.
Atxn2 expression was assessed at 1-5 dpf, with expression evident initially on day 1 and peak expression at 4 dpf. After injection of an atxn2 TB morpholino, decreased eye size was observed at 5 dpf in comparison with both the body size and head size (i.e., eye-to-body (PTB vs. NC<0.001) and eye-to-head (PTB vs. NC<0.038)). The retina of TB morphants at 5 dpf was also thinner (PTB vs. NC=0.018) with reduced retinal ganglion cell (RGC) count (PTB vs. NC=0.018) and disorganization of the RGC layer. TB morphants had reduced vision as measured by lower activities during the first second following light change in the VMR assay (PTB vs. NC<0.001). Co-injection of human ATXN2 mRNA rescued the eye size and VMR in TB morphants (P< 0.05).
Loss of atxn2 function results in smaller eyes with thinner retina, disorganized RGC layer, and impaired vision in zebrafish larvae. Rescue of these defects using human ATXN2 mRNA supports the specificity of the atxn2 TB morpholino, and provides a system to evaluate functional effects of ATXN2 variants associated with POAG.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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