Yanrong Shi

Calpain Expression and Activity during Lens Fiber Cell Differentiation

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was αII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.

A role for epha2 in cell migration and refractive organization of the ocular lens.

PURPOSE The Epha2 receptor is a surprisingly abundant component of the membrane proteome of vertebrate lenses. In humans, genetic studies have linked mutations in EPHA2 to inherited and age-related forms of cataract, but the function of Epha2 in the lens is obscure. To gain insights into the role of Epha2, a comparative analysis of lenses from wild-type and Epha2(-/-) mice was performed. METHODS Epha2 distribution was examined using immunocytochemistry and Western blot analysis. Lens optical quality was assessed by laser refractometry. Confocal microscopy was used to analyze cellular phenotypes. RESULTS In wild-type lenses, Epha2 was expressed by lens epithelial cells and elongating fibers but was degraded during the later stages of fiber differentiation. Epha2-null lenses retained their transparency, but two key optical parameters, lens shape and internal composition, were compromised in Epha2(-/-) animals. Epha2-null lenses were smaller and more spherical than age-matched wild-type lenses, and laser refractometry revealed a significant decrease in refractive power of the outer cell layers of mutant lenses. In the absence of Epha2, fiber cells deviated from their normal course and terminated at sutures that were no longer centered on the optical axis. Patterning defects were also noted at the level of individual cells. Wild-type fiber cells had hexagonal cross-sectional profiles with membrane protrusions extending from the cell vertices. In contrast, Epha2(-/-) cells had irregular profiles, and protrusions extended from all membrane surfaces. CONCLUSIONS These studies indicate that Epha2 is not required for transparency but does play an indispensable role in the cytoarchitecture and refractive quality of the lens.

The Penny Pusher: A Cellular Model of Lens Growth

PURPOSE The mechanisms that regulate the number of cells in the lens and, therefore, its size and shape are unknown. We examined the dynamic relationship between proliferative behavior in the epithelial layer and macroscopic lens growth. METHODS The distribution of S-phase cells across the epithelium was visualized by confocal microscopy and cell populations were determined from orthographic projections of the lens surface. RESULTS The number of S-phase cells in the mouse lens epithelium fell exponentially, to an asymptotic value of approximately 200 cells by 6 months. Mitosis became increasingly restricted to a 300-μm-wide swath of equatorial epithelium, the germinative zone (GZ), within which two peaks in labeling index were detected. Postnatally, the cell population increased to approximately 50,000 cells at 4 weeks of age. Thereafter, the number of cells declined, despite continued growth in lens dimensions. This apparently paradoxical observation was explained by a time-dependent increase in the surface area of cells at all locations. The cell biological measurements were incorporated into a physical model, the Penny Pusher. In this simple model, cells were considered to be of a single type, the proliferative behavior of which depended solely on latitude. Simulations using the Penny Pusher predicted the emergence of cell clones and were in good agreement with data obtained from earlier lineage-tracing studies. CONCLUSIONS The Penny Pusher, a simple stochastic model, offers a useful conceptual framework for the investigation of lens growth mechanisms and provides a plausible alternative to growth models that postulate the existence of lens stem cells.

Ocular Phenotype of Fbn2-Null Mice

PURPOSE Fibrillin-2 (Fbn2) is the dominant fibrillin isoform expressed during development of the mouse eye. To test its role in morphogenesis, we examined the ocular phenotype of Fbn2(-/-) mice. METHODS Ocular morphology was assessed by confocal microscopy using antibodies against microfibril components. RESULTS Fbn2(-/-) mice had a high incidence of anterior segment dysgenesis. The iris was the most commonly affected tissue. Complete iridal coloboma was present in 37% of eyes. Dyscoria, corectopia and pseudopolycoria were also common (43% combined incidence). In wild-type (WT) mice, fibrillin-2-rich microfibrils are prominent in the pupillary membrane (PM) during development. In Fbn2-null mice, the absence of Fbn2 was partially compensated for by increased expression of fibrillin-1, although the resulting PM microfibrils were disorganized, compared with WTs. In colobomatous adult Fbn2(-/-) eyes, the PM failed to regress normally, especially beneath the notched region of the iris. Segments of the ciliary body were hypoplastic, and zonular fibers, although relatively plentiful, were unevenly distributed around the lens equator. In regions where the zonular fibers were particularly disturbed, the synchronous differentiation of the underlying lens fiber cells was affected. CONCLUSIONS Fbn2 has an indispensable role in ocular morphogenesis in mice. The high incidence of iris coloboma in Fbn2-null animals implies a previously unsuspected role in optic fissure closure. The observation that fiber cell differentiation was disturbed in Fbn2(-/-) mice raises the possibility that the attachment of zonular fibers to the lens surface may help specify the equatorial margin of the lens epithelium.

Further analysis of the lens phenotype in Lim2-deficient mice.

PURPOSE Lim2 (MP20) is the second most abundant integral protein of lens fiber cell membranes. A comparative analysis was performed of wild-type and Lim2-deficient (Lim2(Gt/Gt)) mouse lenses, to better define the anatomic and physiologic roles of Lim2. METHODS Scanning electron microscopy (SEM) and confocal microscopy were used to assess the contribution of Lim2 to lens tissue architecture. Differentiation-dependent changes in cytoskeletal composition were identified by mass spectrometry and immunoblot analysis. The effects on cell-cell communication were quantified using impedance analysis. RESULTS Lim2-null lenses were grossly normal. At the cellular level, however, subtle structural alterations were evident. Confocal microscopy and SEM analysis revealed that cortical Lim2(Gt/Gt) fiber cells lacked the undulating morphology that characterized wild-type fiber cells. On SDS-PAGE analysis the composition of cortical fiber cells from wild-type and Lim2-null lenses appeared similar. However, marked disparities were evident in samples prepared from the lens core of the two genotypes. Several cytoskeletal proteins that were abundant in wild-type core fiber cells were diminished in the cores of Lim2(Gt/Gt) lenses. Electrophysiological measurements indicated a small decrease in the membrane potential of Lim2(Gt/Gt) lenses and a two-fold increase in the effective intracellular resistivity. In the lens core, this may have reflected decreased expression levels of the gap junction protein connexin 46 (Cx46). In contrast, increased resistivity in the outer cell layers of Lim2(Gt/Gt) lenses could not be attributed to decreased connexin expression and may reflect the absence of cell fusions in Lim2(Gt/Gt) lenses. CONCLUSIONS Comparative analysis of wild-type and Lim2-deficient lenses has implicated Lim2 in maintenance of cytoskeletal integrity, cell morphology, and intercellular communication.

A stochastic model of eye lens growth

The size and shape of the ocular lens must be controlled with precision if light is to be focused sharply on the retina. The lifelong growth of the lens depends on the production of cells in the anterior epithelium. At the lens equator, epithelial cells differentiate into fiber cells, which are added to the surface of the existing fiber cell mass, increasing its volume and area. We developed a stochastic model relating the rates of cell proliferation and death in various regions of the lens epithelium to deposition of fiber cells and radial lens growth. Epithelial population dynamics were modeled as a branching process with emigration and immigration between proliferative zones. Numerical simulations were in agreement with empirical measurements and demonstrated that, operating within the strict confines of lens geometry, a stochastic growth engine can produce the smooth and precise growth necessary for lens function.

A full lifespan model of vertebrate lens growth

The mathematical determinants of vertebrate organ growth have yet to be elucidated fully. Here, we utilized empirical measurements and a dynamic branching process-based model to examine the growth of a simple organ system, the mouse lens, from E14.5 until the end of life. Our stochastic model used difference equations to model immigration and emigration between zones of the lens epithelium and included some deterministic elements, such as cellular footprint area. We found that the epithelial cell cycle was shortened significantly in the embryo, facilitating the rapid growth that marks early lens development. As development progressed, epithelial cell division becomes non-uniform and four zones, each with a characteristic proliferation rate, could be discerned. Adjustment of two model parameters, proliferation rate and rate of change in cellular footprint area, was sufficient to specify all growth trajectories. Modelling suggested that the direction of cellular migration across zonal boundaries was sensitive to footprint area, a phenomenon that may isolate specific cell populations. Model runs consisted of more than 1000 iterations, in each of which the stochastic behaviour of thousands of cells was followed. Nevertheless, sequential runs were almost superimposable. This remarkable degree of precision was attributed, in part, to the presence of non-mitotic flanking regions, which constituted a path by which epithelial cells could escape the growth process. Spatial modelling suggested that clonal clusters of about 50 cells are produced during migration and that transit times lengthen significantly at later stages, findings with implications for the formation of certain types of cataract.

Ocular Phenotype of Fbn2-Null MiceOcular Phenotype of Fbn2-Null Mice

PURPOSE Fibrillin-2 (Fbn2) is the dominant fibrillin isoform expressed during development of the mouse eye. To test its role in morphogenesis, we examined the ocular phenotype of Fbn2(-/-) mice. METHODS Ocular morphology was assessed by confocal microscopy using antibodies against microfibril components. RESULTS Fbn2(-/-) mice had a high incidence of anterior segment dysgenesis. The iris was the most commonly affected tissue. Complete iridal coloboma was present in 37% of eyes. Dyscoria, corectopia and pseudopolycoria were also common (43% combined incidence). In wild-type (WT) mice, fibrillin-2-rich microfibrils are prominent in the pupillary membrane (PM) during development. In Fbn2-null mice, the absence of Fbn2 was partially compensated for by increased expression of fibrillin-1, although the resulting PM microfibrils were disorganized, compared with WTs. In colobomatous adult Fbn2(-/-) eyes, the PM failed to regress normally, especially beneath the notched region of the iris. Segments of the ciliary body were hypoplastic, and zonular fibers, although relatively plentiful, were unevenly distributed around the lens equator. In regions where the zonular fibers were particularly disturbed, the synchronous differentiation of the underlying lens fiber cells was affected. CONCLUSIONS Fbn2 has an indispensable role in ocular morphogenesis in mice. The high incidence of iris coloboma in Fbn2-null animals implies a previously unsuspected role in optic fissure closure. The observation that fiber cell differentiation was disturbed in Fbn2(-/-) mice raises the possibility that the attachment of zonular fibers to the lens surface may help specify the equatorial margin of the lens epithelium.