Loss of ephrin-A5 function disrupts lens fiber cell packing and leads to cataract

Abstract

Cell-cell interactions organize lens fiber cells into highly ordered structures to maintain transparency. However, signals regulating such interactions have not been well characterized. We report here that ephrin-A5, a ligand of the Eph receptor tyrosine kinases, plays a key role in lens fiber cell shape and cell-cell interactions. Lens fiber cells in mice lacking ephrin-A5 function appear rounded and irregular in cross-section, in contrast to their normal hexagonal appearance in WT lenses. Cataracts eventually develop in 87% of ephrin-A5 KO mice. We further demonstrate that ephrin-A5 interacts with the EphA2 receptor to regulate the adherens junction complex by enhancing recruitment of beta-catenin to N-cadherin. These results indicate that the Eph receptors and their ligands are critical regulators of lens development and maintenance.

Fig. 1.

Development of cataracts in ephrin-A5^−/− mice. (A and B) Slit-lamp images of adult WT (A) and ephrin-A5^−/− (B) mouse lenses. (C and D) Scheimpflug images of adult WT (C) and ephrin-A5^−/− (D) mouse lenses. (E–J) Sections (5 μm thick) of the WT (E and H) and mutant (F, G, I, and J) lens. (H, I, and J) Higher magnification images of the bow region of images shown in E, F, and G, respectively. Unusually large fiber cells (arrow) and vacuoles (arrowhead) were observed in mutant lenses. (Scale bar in E, 500 μm; in H, 100 μm.)

Fig. 2.

Loss of cell shape control in ephrin-A5^−/− lens. (A) Lens fiber cell morphology of P6 WT and ephrin-A5^−/− lenses. The lens sections (5 μm thick) were stained with H&E. Notable differences in cell size, shape, and packing organization were observed in the mutant lens. (B) Lens fiber cell morphology of P21 WT and ephrin-A5^−/− lenses. Cross-sections of WT and mutant lenses were prepared (10 μm thick) and stained with Alexa Fluor 546-phalloidin to delineate cell morphology. (C) Quantification of the length-to-width ratio in WT and ephrin-A5^−/− lenses. *Significant difference at P < 0.05 (t test). (Scale bar in A, 40 μm; in B, 5 μm.)

Fig. 3.

Change in N-cadherin localization in ephrin-A5^−/− lens. (A) Altered patterns of expression of N-cadherin and the gap junction protein ZO-1 in ephrin-A5^−/− lenses. P21 WT and ephrin-A5^−/− lens cryosections were prepared (10 μm thick) and stained with anti–N-cadherin and anti–ZO-1 antibodies. (B) Fractions of N-cadherin signals detected in the cytoplasm in P21 WT and ephrin-A5^−/− lenses. The fractions were obtained by dividing the fluorescent signals in the cytoplasm by the signals of the entire cell. Cell boundaries are defined by staining with Alexa Fluor 546-phalloidin. *Significant at P < 0.05 (t test). (Scale bar in A, 5 μm.)

Fig. 4.

Both EphA2 and ephrin-A ligands are expressed at the cell junctions. (A) Phalloidin staining of WT lens shows lens fiber cell organization. (B and C) WT transverse sections of P21 lenses stained with anti-EphA2 and EphA3-Fc, respectively. Low-magnification images demonstrate that both EphA2 and A-ephrins are normally expressed at higher levels in the subcortical region. (D) EphA3-Fc staining on ephrin-A5^−/− lens sections. Staining was mostly lost on mutant lenses indicating that the subcortical signals were a result of ephrin-A5 expression. (B′–D′) High-magnification confocal images of B–D. Note that WT EphA2 receptor (B′) and ephrin-A5 (C′) expression is the highest at the cell–cell junctions. (E) WT control without primary antibody. Images were collected with equal exposure times. Arrows in A denote the subcortical (sc) lens fiber region for A–E. (Scale bar in top left, 20 μm; top right, 5 μm.)

Fig. 5.

Activation of EphA2 promotes recruitment of β-catenin to N-cadherin. (A) Ephrin-A5 activates the EphA2 receptor in 293T cells. (B) Reduction of EphA2 phosphorylation in ephrin-A5^−/− P6 lenses. (C) EphA2 and N-cadherin co-localize on the P6 WT lens fiber cell membrane. Note the increased cytoplasmic N-cadherin staining in the mutant lens. Arrows indicate vertices where EphA2 and N-cadherin show strong co-localization. Arrowheads denote extracellular space between the fiber cells in the mutant lens, which are devoid of cytoplasmic N-cadherin staining. (D) Double immunofluorescence staining of P21 lens fiber cells with anti-EphA2 and anti–β-catenin antibodies. Arrows in the upper panels indicate where strong EphA2 and β-catenin co-localization was observed in the WT lenses. Arrowhead in the upper left panel indicates some of the EphA2 expression is not co-localized with beta-catenin. Arrowheads in the lower panels show spaces between the lens fiber cells. (E) Ephrin-A5 stimulation and EphA2 transfection promote the recruitment of β-catenin to N-cadherin. (F) EphA2 co-immunoprecipitates with β-catenin. (Scale bars in C and D, 5 μm.)