Life-long eye lens function requires an appropriate gradient refractive index, biomechanical integrity and transparency. We conducted an extensive study of wild-type mouse lenses 1-30 months of age to define common age-related changes. Biomechanical testing and morphometrics revealed an increase in lens volume and stiffness with age. Lens capsule thickness and peripheral fiber cell widths increased between 2 to 4 months of age but not further, and thus, cannot account for significant age-dependent increases in lens stiffness after 4 months. In lenses from mice older than 12 months, we routinely observed cataracts due to changes in cell structure, with anterior cataracts due to incomplete suture closure and a cortical ring cataract corresponding to a zone of compaction in cortical lens fiber cells. Refractive index measurements showed a rapid growth in peak refractive index between 1 to 6 months of age, and the area of highest refractive index is correlated with increases in lens nucleus size with age. These data provide a comprehensive overview of age-related changes in murine lenses, including lens size, stiffness, nuclear fraction, refractive index, transparency, capsule thickness and cell structure. Our results suggest similarities between murine and primate lenses and provide a baseline for future lens aging studies.

Mutations of the RNA-granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7−/− mice. Early postnatal Tdrd7−/− animals precipitously develop cataract suggesting a global-level breakdown/mis-regulation of key cellular processes. High-throughput RNA-sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal-modulator, HSPB1, among high-priority down-regulated candidates in Tdrd7−/− lens. A protein two-dimensional fluorescence difference in-gel electrophoresis-coupled mass spectrometry screen also identified HSPB1 down-regulation, offering independent support for its importance to Tdrd7−/− cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance and its reduction in Tdrd7−/− lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7−/− cataract. In agreement, scanning electron microscopy revealed abnormal fiber cell morphology in Tdrd7−/− lenses. Further, abnormal phalloidin and WGA staining of Tdrd7−/− fiber cells, particularly those exhibiting nuclear degradation, reveal distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, RNA-immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA-imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in differentiating fiber cells, suggesting that TDRD7-ribonucleoprotein complexes may be involved in optimal build-up of key factors. Finally, Hspb1-knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7’s novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.

The transparent ocular lens in the anterior chamber of the eye is responsible for fine focusing of light onto the retina. The lens is entirely cellular with bulk of the tissue composed of fiber cells, and the anterior hemisphere of the lens is covered by a monolayer of epithelial cells. Lens epithelial cells are important for maintaining fiber cell homeostasis and for continual growth of the lens tissue throughout life. Cataracts, defined as any opacity in the lens, remain the leading cause of blindness in the world. Following cataract surgery, lens epithelial cells can undergo a process of epithelial-to-mesenchymal transition (EMT), leading to secondary cataracts due to posterior capsular opacification (PCO). Since the epithelial cells make up only a small fraction of the lens, specialized techniques are required to study lens epithelial cell biology and pathology. Studies using native lens epithelial cells often require pooling of samples to obtain enough cells to make sufficient samples for traditional molecular biology techniques. Here, we provide detailed protocols that enable the study of native mouse lens epithelial cells, including immunostaining of the native lens epithelium in flat mounts, extraction of RNA and proteins from pairs of lens epithelial monolayers, and isolation of lens epithelial cells for primary culture. These protocols will enable researchers to gain better insight on representative molecular expression and cellular structure of lens epithelial cells. We also provide comparative data between native, primary culture, and immortalized lens epithelial cells and discuss the advantages and disadvantages of each technique presented.

Purpose: Fine focusing of light by the eye lens onto the retina relies on the ability of the lens to change shape during the process of accommodation. Little is known about the cellular structures that regulate elasticity and resilience. We tested whether Eph–ephrin signaling is involved in lens biomechanical properties. Methods: We used confocal microscopy and tissue mechanical testing to examine mouse lenses with genetic disruption of EphA2 or ephrin-A5. Results: Confocal imaging revealed misalignment of the suture between each shell of newly added fiber cells in knockout lenses. Despite having disordered sutures, loss of EphA2 or ephrin-A5 did not affect lens stiffness. Surprisingly, knockout lenses were more resilient and recovered almost completely after load removal. Confocal microscopy and quantitative image analysis from live lenses before, during, and after compression revealed that knockout lenses had misaligned Y-sutures, leading to a change in force distribution during compression. Knockout lenses displayed decreased separation of fiber cell tips at the anterior suture at high loads and had more complete recovery after load removal, which leads to improved whole-lens resiliency. Conclusions: EphA2 and ephrin-A5 are needed for normal patterning of fiber cell tips and the formation of a well-aligned Y-suture with fiber tips stacked on top of previous generations of fiber cells. The misalignment of lens sutures leads to increased resilience after compression. The data suggest that alignment of the Y-suture may constrain the overall elasticity and resilience of the lens.

Purpose: Our studies in mouse eye lenses demonstrate that ephrin-A5 and EphA2 are needed for normal epithelial cells and lens transparency. We sought to determine whether EphA2 and ephrin-A5 are important for lens morphometrics, nucleus formation, and refractive index. Methods: We performed tissue morphometric measurements, electron microscopy, Western blots, and interferometric measurements using an X-ray synchrotron beam source to measure the gradient of refractive index (GRIN) to compare mouse lenses with genetic disruption of EphA2 or ephrin-A5. Results: Morphometric analysis revealed that although there is no change in the overall lens volume, there is a change in lens shape in both EphA2^−/− lenses and ephrin-A5^−/− lenses. Surprisingly, EphA2^−/− lenses had small and soft lens nuclei different from hard lens nuclei of control lenses. SEM images revealed changes in cell morphology of EphA2^−/− fiber cells close to the center of the lens. Inner EphA2^−/− lens fibers had more pronounced tongue-and-groove interdigitations and formed globular membrane morphology only in the deepest layers of the lens nucleus. We did not observe nuclear defects in ephrin-A5^−/− lenses. There was an overall decrease in magnitude of refractive index across EphA2^−/− lenses, which is most pronounced in the nucleus. Conclusions: This work reveals that Eph-ephrin signaling plays a role in fiber cell maturation, nuclear compaction, and lens shape. Loss of EphA2 disrupts the nuclear compaction resulting in a small lens nucleus. Our data suggest that Eph-ephrin signaling may be required for fiber cell membrane reorganization and compaction and for establishing a normal GRIN.

The eye lens is responsible for fine focusing of light onto the retina, and its function relies on tissue transparency and biomechanical properties. Recent studies have demonstrated the importance of Eph-ephrin signaling for the maintenance of life-long lens homeostasis. The binding of Eph receptor tyrosine kinases to ephrin ligands leads to a bidirectional signaling pathway that controls many cellular processes. In particular, dysfunction of the receptor EphA2 or the ligand ephrin-A5 lead to a variety of congenital and age-related cataracts, defined as any opacity in the lens, in human patients. In addition, a wealth of animal studies reveal the unique and overlapping functions of EphA2 and ephrin-A5 in lens cell shape, cell organization and patterning, and overall tissue optical and biomechanical properties. Significant differences in lens phenotypes of mouse models with disrupted EphA2 or ephrin-A5 signaling indicate that genetic modifiers likely affect cataract phenotypes and progression, suggesting a possible reason for the variability of human cataracts due to Eph-ephrin dysfunction. This review summarizes the roles of EphA2 and ephrin-A5 in the lens and suggests future avenues of study.