Beta1-integrin signaling is essential for lens fiber survival

Abstract

Integrins have been proposed to play a major role in lens morphogenesis. To determine the role of beta1-integrin and its down-stream signaling partner, integrin linked kinase (ILK), in lens morphogenesis, eyes of WT mice and mice with a nestin-linked conditional knockout of beta1-integrin or ILK were analyzed for defects in lens development. Mice, lacking the genes encoding the beta1-integrin subunit (Itgb1) or ILK (Ilk), showed a perinatal degeneration of the lens. Early signs of lens degeneration included vacuolization, random distribution of lens cell nuclei, disrupted fiber morphology and attenuation and separation of the lens capsule. The phenotype became progressively more severe during the first postnatal week eventually leading to the complete loss of the lens. A more severe phenotype was observed in ILK mutants at similar stages. Eyes from embryonic day 13 beta1-integrin-mutant embryos showed no obvious signs of lens degeneration, indicating that mutant lens develops normally until peri-recombination. Our findings suggest that beta1-integrins and ILK cooperate to control lens cell survival and link lens fibers to the surrounding extracellular matrix. The assembly and integrity of the lens capsule also appears to be reliant on integrin signaling within lens fibers. Extrapolation of these results indicates a novel role of integrins in lens cell-cell adhesions as well as a potential role in the pathogenesis of congenital cataracts.

Keywords: ILK; basement membrane; eye development; integrins; lens development.

Figure 1

β1-integrin expression in control and mutant lens. Side-by-side comparison of a P1 control (A) and mutant (B) lens cross-section confirmed that β1-integrin expression (red) in the P1 mutant lens was nearly eliminated. The nuclear counter-stain (green) showed the abnormal location of nuclei in the posterior pole of the mutant lens (B). Western blots confirmed that β1-integrin (C) and nestin (D) were present in the developing P1 lens of control mice. β1-integrin was detectable in the membrane (M) but not the cytoplasmic fraction (Cyto) of the lens, nestin was detected in both fractions. The arrows point towards the anterior region of the lens and are aligned along the lens midline.

Figure 2

Ocular histology of β1-mutant and control mice. Cross-sections of control (A, F) and β1-mutant mice (B–E,G,H) at P1 (A–E) and P60 (F–H) showed early and late defects of the lens of β1 integrin-defective mice. Higher magnification of the selected areas in (B and G) are shown in (C and H) respectively. Initial stages of lens degeneration were indicated by lens fiber vacuolization in P1 mutants (B–E) however ciliary body and anterior epithelial structures remained normal (C). By P60, normal lens structures (F; control) were absent in the mutant mice (G). Note the fusion of iris and cornea indicating a loss of the anterior chamber. Remaining lens capsule strands could be seen next to the iris and ciliary body (H). Abbreviations: AE, anterior epithelium; L, lens; V, vitreous; R, retina; I, iris; C, cornea; LC, lens capsule. The red arrows indicate the ciliary body structures.

Figure 3

Nuclear localization, lens fiber morphology and lens capsule structure in E13 control (A, C) and mutant (B, D) specimens. Cross-sections were stained for phalloidin (green; lens fiber morphology) and nuclei (red; A, B), or stained for collagen IV (red; lens capsule) and nuclei (green; C, D). In both mutant and control E13 lenses, normal nuclear distribution is found in both the anterior epithelium and posterior lens fibers (A–D). The lens fiber morphology (A, B) and lens capsule structure (C, D) was similarly normal in both controls and mutants at E13. Arrows point to the anterior pole of the lenses. Abbreviations: AE, anterior epithelium; BV, blood vessels; C, cornea; LC, lens capsule.

Figure 4

Lens capsule and lens fiber disruption at P1. Cross-sections of control (A, C) and mutant lenses (B, D) were stained for either phalloidin (green; and nuclei — red; A, B), or collagen IV (red; and nuclei — green; C, D) to show lens fiber morphology and lens capsule structure (respectively). In P1 control lenses (A, C), a monolayer of cells formed the anterior lens epithelium. The lens fiber nuclei were neatly arranged around the equator of the lens, and the lens capsule was well delineated and continuous. In the P1 β1-mutant lenses (B, D) nuclei were randomly distributed throughout the lens and the lens capsule appears fragmented. The anterior epithelium, however, appeared fairly normal. Arrows point to the anterior pole of the lenses. Abbreviations: 1°, primary lens fibers; 2°, secondary lens fibers; AE, anterior epithelium; BV, blood vessels; C, cornea; LC, lens capsule; PS, posterior seam.

Figure 5

Electron micrographs showing defects in the lens capsule of P1 β1-mutant mice. The lens capsule in P1 control mice was between 2 and 3 μm thick and the lens fibers were tightly attached to the capsule (A). The lens capsule from the posterior pole of a normal lens (C) was thinner than the capsule at the equatorial and anterior pole of the lens. In the mutant mice (B, D), the lens capsule showed signs of disintegration and lens fibers detachment (B) Macrophages were often seen in close proximity of the degenerating lens. The capsule from the posterior pole of the mutant lens was unusually thin (D) as compared to the lens capsule from a corresponding site of a control eye (C). Abbreviations: BV, blood vessel; M, macrophage; LC, lens capsule; V, vitreous; LF, lens fibers.

Figure 6

ILK-mutant and control ocular histology. Cross-section of both eyes of a P10 ILK-mutant mouse (A–D), showed massive degeneration of the lens. High power micrographs of the selected areas in (A) and (C) are shown in (B) and (D), respectively. In the eye shown in (C), the lens ruptured and was ejected through the cornea. Eyes from P60 ILK-mutant mice were consistently aphakic (E). A high power view of the selected area is shown in (F). ILK-mutants also displayed a fusion of the iris and cornea (B, D, F) compared to the nicely separated structures in the P60 control (G). A few threads of lens capsule were evident in the vitreous cavity (F). The red arrows points to the ciliary body. Abbreviations: C, cornea; I, iris; L, lens; V, vitreous; R, retina; LC, lens capsule.

Figure 7

Electron micrographs showed defects in the lens capsule of P10 ILK-mutant mice. In the equatorial parts of the lens (A, E), the lens capsule was thick and resembled control eyes (A), yet in many areas the capsule invaded the lens tissue (E). Posterior to these regions, thinning of the lens capsule was seen accompanied by a cloud of lens capsule debris (B). In the posterior pole of the lens (C, D, F), the lens capsule was either very thin (C) or entirely absent (D, F). In areas, there was a gross separation of the lens capsule and lens fibers (C). Degeneration of adjacent lens fibers was frequently seen (C, D), as well as macrophage ingestion of degenerating lens particles (F) and vitreal invaginations of the lens (D). Abbreviations: V, vitreous; LC, lens capsule; LF, lens fibers; M, macrophage.

Figure 8

Apoptosis in control and ILK mutant eyes. TUNEL staining in a P7 control mouse eye (A) revealed no apoptosis in the lens. Apoptosis was found in lens fragments of P7 ILK mutants (B). These fragments were confirmed to be lens remnants through immuno-staining for α-crystallin (C). P7 ILK retina (D) exhibited low levels of tonic apoptosis similar to control retina (A). The ciliary bodies (arrows) of controls and mutants (A, D) showed no apoptosis. Crystallin staining (E) of control eye in (A) reveals robust staining of lens (lens region indicated by “*” in A). Abbreviations: C, cornea; L, lens; V, vitreous; R, retina.