Loss of Gap Junction Delta-2 (GJD2) gene orthologs leads to refractive error in zebrafish

Abstract

Purpose : 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.

Methods : 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.

Results : 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).

Conclusions : 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.