Further analysis of the lens of ephrin-A5-/- mice: development of postnatal defects

Abstract

Purpose: The cells of the mammalian lens must be carefully organized and regulated to maintain clarity. Recent studies have identified the Eph receptor ligand ephrin-A5 as a major contributor to lens development, as mice lacking ephrin-A5 develop abnormal lenses, resulting in cataracts. As a follow-up to our previous study on the cataracts observed in ephrin-A5(-/-) animals, we have further examined the morphological and molecular changes in the ephrin-A5(-/-) lens.

Methods: Wild-type and ephrin-A5(-/-) eyes at various ages were fixed, sectioned, and examined using histological techniques. Protein expression and localization were determined using immunohistochemistry and western blot analysis.

Results: Lens abnormalities in the ephrin-A5(-/-) animals are observed at postnatal stages, with lens opacity occurring by postnatal day 21. Structural defects in the lens are first observed in the outer lens fiber cell region where cells in the ephrin-A5(-/-) lens are severely disorganized. Ephrin-A5 and the Eph receptor EphA2 are expressed during early ocular development and continue to be expressed into postnatal stages. The cataracts in the ephrin-A5(-/-) mutants occur regardless of the presence of the CP49 mutation.

Conclusions: In this follow-up study, we have uncovered additional details explicating the mechanisms underlying ephrin-A5 function in the lens. Furthermore, elucidation of the expression of ephrin-A5 and the Eph receptor EphA2 in the lens supports a fundamental role for this receptor-ligand complex in lens development. These observations, in concert with our previous study, strongly suggest that ephrin-A5 has a critical role in postnatal lens fiber organization to maintain lens transparency.

Figure 1

Disruptions in the gross morphology of the ephrin-A5^-/- lens appear in postnatal stages. A: Postnatal day 7 (P7) and P14 ephrin-A5 mutant lenses appear grossly normal. However, by P21 opacity becomes quite prominent in the ephrin-A5^-/- lens while the wild-type lens remains transparent. Scale bar in top left panel=500 μm. B: While the lens sizes are comparable at P7 and P14 (p>0.05, n=12 lenses per group), the ephrin-A5^-/- lens becomes significantly smaller than the wild-type counterpart at P21 (p<0.05, n=12 lenses per group). C: Weights of the wild-type and ephrin-A5^-/- lenses are comparable at each of the stages. n=12 lenses per group. D: No differences are observed in posterior suture formation between both groups as both groups show the classical Y-suture formation. However, fiber cells appear more disorganized in the ephrin-A5^-/- lenses. Scale bar=50 μm.

Figure 2

Low magnification posterior suture analysis shows no disruption of Y-shaped suture formation in the ephrin-A5^-/- lens. A-F: Wild-type lenses (A-C) display a Y-shaped suture structure with highly organized and packed fiber cells. Ephrin-A5^-/- lenses (D-F) also display a Y-shaped suture structure similar to wild-types. However, fiber cell organization and packing are disrupted in these lenses. Scale bar=500 μm.

Figure 3

Deformations in the structure of ephrin-A5^-/- lenses occur in postnatal eyes. A-F: Embryonic development of wild-type (WT; A-C) and ephrin-A5^-/- (D-F) lenses appear similar, with no abnormalities observed in the early ephrin-A5^-/- lens. Scale bars in mm. G-L: While wild-type lenses (G-I) show no deformities in postnatal stages, ephrin-A5^-/- lenses (J-L) display noticeable lens deficits by P21 with the presence of vacuoles around the lens bow (compare H and K, see arrows). The deficits become progressively more severe, as larger vacuoles and complete posterior lens rupture is observed by P60 (Compare I and L, see arrow). Scale bars in mm.

Figure 4

Distinct alterations in cell shape are observed in the ephrin-A5^-/- lens fiber cell layers. A-F: Wild-type (A-C) and ephrin-A5^-/- (D-F) P21 lenses are labeled for ZO-1 (A and D) and β-Catenin (B and E) to delineate cell borders or to distinguish distinct lens fiber areas. Disruptions in fiber cell organization are observed in the ephrin-A5^-/- lens. Scale bar = 100 μm. G-I: Disorganization of the fiber cells in the ephrin-A5^-/- lens are observed in all fiber cell regions, including the cortical (G), subcortical (H), and central (I) areas. Scale bar=50 μm.

Figure 5

Expression of Aquaporin 0 in the ephrin-A5^-/- lens is observed along the cell membranes. A and B: In both wild-type (A) and ephrin-A5^-/- (B) lenses, Aquaporin 0 (AQP0) is found throughout the membranes of mature fiber cells. Scale bar=200 μm.

Figure 6

The lens epithelial regions appear undisturbed in ephrin-A5^-/- animals. A-H: No distinct differences in cellular morphology or adherens junction protein expression of β-catenin (A-D) and E-cadherin (E-H) are observed between the wild-type (A and B, E and F) and ephrin-A5^-/- (C and D, G and H) lenses in these regions. Scale bar=20 μm.

Figure 7

The status of CP49 does not affect ephrin-A5^-/- cataract formation. A-D: Ephrin-A5^+/+ lenses with the CP49 mutation (Ephrin-A5^+/+;CP49^-/-) appear transparent (A, lens is denoted by dotted line), while ephrin-A5^-/- lenses, regardless of the status of CP49, display cataract formation (B-D). Lens deformations were observed in 100% of the ephrin-A5^-/-;CP49^+/+ lenses (n=4), 73% of the ephrin-A5^-/-;CP49^+/- lenses (n=11), and 83% of the ephrin-A5^-/-;CP49^-/- lenses (n=6).

Figure 8

Ephrin-A5 is expressed extensively within the developing eye. A-E: EphA5-AP staining shows significant expression of ephrin-A ligand in the wild-type eye as early as E12 and persists through postnatal ages. Scale bars are in micrometers. F-J: Little to no EphA5-AP staining is observed in the ephrin-A5^-/- eye indicating that ephrin-A5 is the major ephrin-A ligand expressed in the eye. Scale bars are in micrometers.

Figure 9

Ephrin-A5 expression is observed in several parts of the developing eye. A and E: EphA5-AP staining is observed in the lens epithelium (le), lens bow (lb), cornea (c), and ciliary body (cb) in the E14 wild-type eye while absent in the ephrin-A5^-/- animal. B and F: Expression of ephrin-A ligands are maintained in the wild-type at P0 though in lower levels in comparison with earlier embryonic stages, while remaining absent in the ephrin-A5^-/- eye. C and G: Ephrin-A ligand expression is observed in the lens bow region of P7 wild-type mice while absent in ephrin-A5^-/- mice at the same age. D and H: High levels of ephrin-A ligand is observed in the cornea of wild-type mice at P7 and not present in ephrin-A5^-/- mice at the same age. Scale bars are in micrometers.

Figure 10

EphA2 is expressed throughout the developing lens. A-F: EphA2LacZ/+ tissue was reacted with X-Gal to detect EphA2 expression in the developing eye. Staining is observed in the E11 lens and found throughout lens development in subsequent embryonic and postnatal stages. Scale bars are in micrometers. G and H: At E14, expression is observed in the lens fiber cells near the bow and lens epithelium (G). Extensive expression is also observed and near the junction between fiber cells and epithelium (H). I and J: At P7, EphA2 is expressed in the outer lens fiber cell regions (I) as well as in the lens epithelium (J, epithelium is delineated by white dotted line). Scale bars are in mictrometers.