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wim Quint, kirke tadema, erik de vrieze, rachel Lukowicz, sanne broekman, Beerend Winkelman, melanie hoevenaars, Martijn Gruiter, Erwin van Wijk, Frank Schaeffel, Magda Meester-Smoor, Adam Miller, rob willemsen, Caroline C. W. Klaver, Adriana Iglesias; Loss of Gap Junction Delta-2 (GJD2) gene orthologs leads to refractive error in zebrafish. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2878.
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SNPs close to GJD2, a gene encoding a gap junction protein, have been consistently found to influence the risk of refractive error (RE) and myopia. To functionally investigate the pathophysiological link between GJD2/Cx36 and RE, we evaluated biometrical, optical, and electrophysiological changes in gjd2 deficient zebrafish eyes.
Here, we studied depletion of two zebrafish GJD2 orthologues, gjd2a (Cx35.5) and gjd2b (Cx35.1). The expression profiles of both genes were assessed by IHC and single-cell RNA-seq. Axial length (AL) was measured by SD-Optical Coherence Tomography (SD-OCT) (n=40) at 1.5-3 months post fertilization (mpf). RE was measured between 1.5-9mpf (n=20). Cataracts were visualized by SD-OCT and differential interference contrast microscopy. A linear mixed model was used to evaluate differences between mutant and WT control fish for AL, RE and lens opacity. Electroretinograms (ERGs) were recorded in 2.5 mpf fish (n=22) and differential B-wave amplitudes analyzed by one-way ANOVA.
Our expression studies showed that both gjd2a (Cx35.5) and gjd2b (Cx35.1) are expressed in the fish retina; only Cx35.1 (gjd2b) is also expressed in the lens.Depletion of gjd2a led to hyperopia, characterized by decreased AL (~50µm at 1.5-3mpf, P<0.001) and corresponding ascending RE relative to WT (~+2D at 1.5mpf to ~+8D at 9mpf, P<0.001). ERG showed a decreased B-wave amplitude in gjd2a mutants (p<0.001).We found no alterations in axial length in the gjd2b mutants between 1.5-3mpf. However, we observed a progressive and severe nuclear cataract affecting 100% of the eyes at 6mpf (~28% increase in opacity, p<0.001). The cataract was accompanied by a myopic shift in refractive status (~-5D at 1.5mpf to ~-10D at 9mpf, p<0.001) and late-onset increase in AL (~77um at 6mpf, P<0.001).
This work in zebrafish showed that both gjd2a (Cx35.5) and gjd2b (Cx35.1) cause changes in refractive status, thereby providing the first evidence for a functional role of GJD2 in refraction. Both mutants showed biometrical and optical alterations, interestingly, in opposite directions. The absence of Cx35.5 led to hyperopia and a significant electrophysiological defect, whereas the absence of Cx35.1 resulted in a severe opacification of the lens and, as a result, a myopic defocus and late-onset axial elongation. We conclude that aberrant electrical gap junction signaling leads to defective emmetropization.
This is a 2021 ARVO Annual Meeting abstract.
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