EphA2 and Src regulate equatorial cell morphogenesis during lens development

Abstract

High refractive index and transparency of the eye lens require uniformly shaped and precisely aligned lens fiber cells. During lens development, equatorial epithelial cells undergo cell-to-cell alignment to form meridional rows of hexagonal cells. The mechanism that controls this morphogenesis from randomly packed cuboidal epithelial cells to highly organized hexagonal fiber cells remains unknown. In Epha2(-/-) mouse lenses, equatorial epithelial cells fail to form precisely aligned meridional rows; moreover, the lens fulcrum, where the apical tips of elongating epithelial cells constrict to form an anchor point before fiber cell differentiation and elongation at the equator, is disrupted. Phosphorylated Src-Y424 and cortactin-Y466, actin and EphA2 cluster at the vertices of wild-type hexagonal epithelial cells in organized meridional rows. However, phosphorylated Src and phosphorylated cortactin are not detected in disorganized Epha2(-/-) cells with altered F-actin distribution. E-cadherin junctions, which are normally located at the basal-lateral ends of equatorial epithelial cells and are diminished in newly differentiating fiber cells, become widely distributed in the apical, lateral and basal sides of epithelial cells and persist in differentiating fiber cells in Epha2(-/-) lenses. Src(-/-) equatorial epithelial cells also fail to form precisely aligned meridional rows and lens fulcrum. These results indicate that EphA2/Src signaling is essential for the formation of the lens fulcrum. EphA2 also regulates Src/cortactin/F-actin complexes at the vertices of hexagonal equatorial cells for cell-to-cell alignment. This mechanistic information explains how EphA2 mutations lead to disorganized lens cells that subsequently contribute to altered refractive index and cataracts in humans and mice.

Keywords: Cataracts; Eph; Ephrin; Lens.

Fig. 1.

Confocal images of the equatorial region of P21 GFP⁺ wild-type and Epha2^-/- mouse lenses. (A-D) The wild-type (WT) lens shows the typical mosaic GFP expression pattern in the equatorial epithelial cells (A), with hexagonal cells organized into meridional rows (below the dashed line, arrowheads) and straight and organized peripheral differentiating fiber cells (C). By contrast, equatorial epithelial cells in the Epha2^-/- lens lack organized meridional rows (B, arrows), and underlying fiber cells are wavy and disorganized (D). (E,F) Three-dimensional reconstruction of z-stacks in the anterior-posterior view through the WT lens reveals organized fiber cells in the bow region (E), whereas a comparable reconstruction through the Epha2^-/- lens demonstrates disorganization of peripheral fiber cells with variable cell width (F). The WT lens fulcrum is indicated by the arrowhead (E); in the Epha2^-/- lens, multiple points of apical constriction are observed (F, arrowheads). (G,H) Reconstruction of z-stacks through the transverse view shows that WT fiber cells are neatly organized into straight rows (G), whereas Epha2^-/- fiber cells are misaligned (H). The epithelial cells are on the left in G and H.

Fig. 2.

EphA2 and β-actin distribution in WT and Epha2^-/- equatorial epithelial cells. Double immunolabeling of β-actin (red) and EphA2 (green) with DAPI staining (blue, nuclei) of equatorial lens epithelial cells from lens capsule flat-mounts of P21 WT and Epha2^-/- mice. When equatorial cells organize into meridional rows, β-actin and EphA2 are enriched and colocalize at the vertices of hexagonal WT epithelial cells (arrowheads). By contrast, β-actin forms abnormal aggregates on the membranes of Epha2^-/- epithelial cells that fail to pack into organized meridional rows (arrows). Antibody specificity is demonstrated by the lack of EphA2 staining in Epha2^-/- cells.

Fig. 3.

E-cadherin and total cortactin distribution in WT and Epha2^-/- equatorial epithelial cells. (A) Double immunolabeling of E-cadherin (green) and β-actin (red) with DAPI staining (blue, nuclei) of equatorial lens epithelial cells from lens capsule flat-mounts of P21 WT and Epha2^-/- mice. Confocal images reveal colocalization of β-actin and E-cadherin at the cell membranes of hexagonal epithelial cells in the WT sample. β-actin is enriched at the cell vertices of WT equatorial epithelial cells (arrowheads). However, there is only weak E-cadherin staining at the membranes of the Epha2^-/- disorganized equatorial epithelial cells. β-actin (red) forms numerous abnormal aggregates on the membranes of Epha2^-/- cells (arrows). (B) Double immunolabeling of EphA2 (green) and total cortactin (red) with DAPI staining (blue) of equatorial lens epithelial cells from lens capsule flat-mounts of P21 WT and Epha2^-/- mice. EphA2 and total cortactin are enriched and colocalize at the vertices of hexagonal WT epithelial cells packed into organized meridional rows (arrowheads). By contrast, staining for total cortactin shows abnormal clusters at the cell membranes of disorganized equatorial Epha2^-/- epithelial cells (arrows).

Fig. 4.

EphA2, phosphorylated cortactin, β-actin and phosphorylated Src distribution in WT and Epha2^-/- equatorial epithelial cells. (A) Triple immunolabeling of EphA2 (green), phosphorylated cortactin-Y466 (cortactin-pY466, red) and β-actin (purple) with DAPI staining (blue, nuclei) of equatorial lens epithelial cells from lens capsule flat-mounts of P21 WT and Epha2^-/- mice. In hexagonal WT equatorial epithelial cells, EphA2, cortactin-pY466 and β-actin are localized at the cell membrane and are enriched and colocalize at cell vertices (arrowheads). By contrast, only punctate staining signals for cortactin-pY466 can be found in the disorganized Epha2^-/- epithelial cells. (B) Similarly, double immunolabeling of phosphorylated Src-Y424 (Src-pY424, red) and EphA2 (green) reveals that both proteins are enriched and colocalize at the vertices of hexagonal WT epithelial cells (arrowheads). However, in the disorganized Epha2^-/- epithelial cells, only random punctate signals from Src-pY424 are observed.

Fig. 5.

EphA2, phosphorylated cortactin and phosphorylated Src staining in P14 WT and Epha2^-/- frozen lens sections. EphA2 (left) and cortactin-pY466 (middle) are present in WT epithelial and fiber cells, and Src-pY424 (right) signal is predominant in WT epithelial cells. EphA2 and Src-pY424 are enriched at the lens fulcrum (arrowheads). By contrast, Epha2^-/- lens epithelial and fiber cells have only weak cortactin-pY466 and Src-pY424 staining.

Fig. 6.

E-cadherin and phalloidin staining of P14 WT and Epha2^-/- frozen lens sections. Double immunolabeling of E-cadherin (green) and with phalloidin (F-actin, red) in frozen lens sections demonstrates that E-cadherin and F-actin are highly enriched only at the lens fulcrum (arrowheads) in the WT lens section, but form multiple aggregates in the Epha2^-/- lens section (open arrowheads). Abnormal E-cadherin signal is observed in Epha2^-/- lens fiber cells (arrows).

Fig. 7.

Wheat germ agglutinin and phalloidin staining of P21 WT, Src^-/- and Epha2^-/- lenses and frozen sections. (A) Confocal images of the equatorial region of P21 whole fixed lenses labeled with wheat germ agglutinin (WGA, red) and stained with DAPI show that the WT lens has aligned meridional rows (left, arrows), whereas both Epha2^-/- and Src^-/- lenses have disorganized meridional epithelial cells (middle and right, arrows). (B) Confocal images of the lens equator where epithelial cells transition to fiber cells show that the lens fulcrum (black arrow on far left) is strongly stained by WGA (left, arrows, red) in the WT lens. By contrast, Epha2^-/- and Src^-/- lenses lack a distinct lens fulcrum and have WGA-stained aggregates (middle and right, arrows) at the boundary between epithelial and fiber cells. In the WT lens, peripheral lens fibers are neatly aligned (left, arrowheads), and, similarly, Src^-/- lens fibers appear aligned as well (right, arrowheads). No obvious fiber cell boundaries are visible in the Epha2^-/- lens (middle). (C) Phalloidin staining of the WT lens section shows that F-actin is enriched at the lens fulcrum (arrowhead). However, in the Epha2^-/- and Src^-/- lens sections, there are multiple F-actin aggregates rather than a distinct lens fulcrum (open arrowheads).

Fig. 8.

Model for EphA2 functions in the organization of hexagonal equatorial epithelial cells into meridional rows. Hexagonal equatorial epithelial cells are packed into organized meridional rows at the lens fulcrum. On the basal-lateral sides of equatorial epithelial cells, EphA2 is likely to interact with an ephrin ligand to cluster at the vertices of the hexagonal cells. EphA2 phosphorylates Src, which consequently activates cortactin to trigger actin to cluster at cell vertices. This signaling cascade and cell adhesion events lead to the organization of equatorial epithelial cells into meridional rows, which is probably a prerequisite for fiber cell organization. On the apical side of equatorial epithelial cells, EphA2 clustering phosphorylates and activates Src to recruit E-cadherin and actin to form the lens fulcrum, which serves as an anchor point for the transition from epithelial cells to fiber cells.