Alpha8 Connexin Regulates the Fiber–to–Epithelial Cell Contact During Lens Development

Abstract

Abstract: : Purpose: It has been hypothesized that the epithelium–to–fiber gap junctions are essential for the lens growth and for maintaining lens transparency. However, studies of α1, α3 and α8 connexin knockouts did not provide definitive evidences for supporting such hypothesis. We have analyzed the lenses of connexin α8–S50P and α8–R205G point mutant mice to evaluate this hypothesis and to study the regulation of gap junctions during lens development. Methods: The α8–S50P and α8–R205G mutant lenses with or without endogenous α3 connexin at different age were examined by morphological and biochemical methods. Results: Immunostaining verifies that both α8–S50P and α8–R205G mutant subunits are unable to form normal gap junctions in the lens fibers. Western blotting shows that the expression of either α8–S50P or α8–R205G mutant subunit causes a reduction of the phosphorylated endogenous α3 connexin. Homozygous α8–S50P and α8–R205G mutant mice develop severe microphthalmia and smaller lenses; histology shows insufficient elongation, swelling, and degeneration in the differentiating fibers at lens bow region. Surprisingly, these defects are rescued in the absence of the endogenous α3 connexin. In addition, primary elongated fibers are unable to form normal contacts with anterior epithelium in heterozygous α8–S50P mutant lenses at embryonic 12.5 dpc (days postconception), and a large cystic lumen develops between epithelium and the underlying fibers at 15.5 dpc while no lumen is observed in the α8 (–/–) and the α3 (–/–) α8 (–/–) double mutant lenses. Conclusions: These results suggest that the phenotypes of differentiating fibers in both α8–S50P and α8–R205G mutant lenses are depend on the presence of the endogenous α3 connexin. We hypothesize that these two mutations produce gain of function α8 mutant subunits when interact with the endogenous α3 subunits. Moreover, one allele of α8–S50P mutation is sufficient to disrupt the stable contact formation between the primary fibers and the anterior epithelial cells. It explains why posterior differentiating fibers undergo elongation defect, swelling, and degeneration. This study provides first direct evidence for the importance of α8 connexin in modulating the fiber–to–epithelial cell contacts during lens development.

Keywords: gap junctions/coupling • cell-cell communication • cataract