Evaluation of function-related corneal endothelial markers in TAZ deficient mice over time

Abstract

Purpose : Mice deficient in Wwtr1, the gene that encodes transcriptional co-activator with PDZ-binding motif (TAZ), show softer Descemet’s membrane and decreased corneal endothelial cell (CEnC) density reminiscent of late-onset Fuchs’ endothelial dystrophy in humans. The purpose of this study was to evaluate the expression of proteins critical to barrier and pump functions of CEnC in wildtype (WT) and homozygous TAZ (Wwtr1^-/-) and heterozygous TAZ deficient (Wwtr1^+/-) mice over time.

Methods : At least 3 corneas from different animals were collected for each genotype from various age groups (2, 6, 12, and 16 months old). After fixation, corneas were dissected from enucleated globes. Corneal flatmounts were sectioned into several pieces and incubated with primary antibodies against zonula occludens-1 (ZO-1) and Na/K-ATPase separately for evaluation of barrier and pump function of corneal endothelium, respectively. Subsequently, tissues were incubated with fluorophore conjugated secondary antibodies and nuclei were counterstained with DAPI. Whole-mount corneal tissues were mounted endothelial side up on a slide and were imaged using a fluorescent microscope with 20X and 40X objectives.

Results : In WT mice, ZO-1 staining demonstrated a typical hexagonal pattern with a zig-zag distribution along the cell border while Na/K-ATPase demonstrated expected finger-like projections along the basolateral membrane. The Wwtr1^+/- mice showed an expression pattern similar to WT for both markers over time. However, the Wwtr1^-/- exhibited different patterns of expression although high variability existed between animals and regions of the cornea. Specifically, ZO-1 staining was disrupted or absent resulting in partial loss of hexagonal appearance from 2 months old and Na/K-ATPase staining had a more diffuse and lobulated staining pattern lining the cell membrane from 6 months old in Wwtr1^-/- mice when compared with WT controls.

Conclusions : Data demonstrated that homozygous TAZ deficiency caused partial loss of ZO-1 expression and altered immunolabeling of Na/K-ATPase in the corneal endothelium of mice. These findings could potentially affect the barrier and pump function and further study is needed to explain the pathophysiologic mechanism and clinical significance for these findings.

This is a 2021 ARVO Annual Meeting abstract.