Mapping the Universe of Eph Receptor and Ephrin Ligand Transcripts in Epithelial and Fiber Cells of the Eye Lens

Abstract

The eye lens is a transparent, ellipsoid organ in the anterior chamber of the eye that is required for fine focusing of light onto the retina to transmit a clear image. Cataracts, defined as any opacity in the lens, remains the leading cause of blindness in the world. Recent studies in humans and mice indicate that Eph–ephrin bidirectional signaling is important for maintaining lens transparency. Specifically, mutations and polymorphisms in the EphA2 receptor and the ephrin-A5 ligand have been linked to congenital and age-related cataracts. It is unclear what other variants of Ephs and ephrins are expressed in the lens or whether there is preferential expression in epithelial vs. fiber cells. We performed a detailed analysis of Eph receptor and ephrin ligand mRNA transcripts in whole mouse lenses, epithelial cell fractions, and fiber cell fractions using a new RNA isolation method. We compared control samples with EphA2 knockout (KO) and ephrin-A5 KO samples. Our results revealed the presence of transcripts for 12 out of 14 Eph receptors and 8 out of 8 ephrin ligands in various fractions of lens cells. Using specific primer sets, RT-PCR, and sequencing, we verified the variant of each gene that is expressed, and we found two epithelial-cell-specific genes. Surprisingly, we also identified one Eph receptor variant that is expressed in KO lens fibers but is absent from control lens fibers. We also identified one low expression ephrin variant that is only expressed in ephrin-A5 control samples. These results indicate that the lens expresses almost all Ephs and ephrins, and there may be many receptor–ligand pairs that play a role in lens homeostasis.