The lens equator: A platform for molecular machinery that regulates the switch from cell proliferation to differentiation in the vertebrate lens

The vertebrate lens is a transparent, spheroidal tissue, located in the anterior region of the eye that focuses visual images on the retina. During development, surface ectoderm associated with the neural retina invaginates to form the lens vesicle. Cells in the posterior half of the lens vesicle differentiate into primary lens fiber cells, which form the lens fiber core, while cells in the anterior half maintain a proliferative state as a monolayer lens epithelium. After formation of the primary fiber core, lens epithelial cells start to differentiate into lens fiber cells at the interface between the lens epithelium and the primary lens fiber core, which is called the equator. Differentiating lens fiber cells elongate and cover the old lens fiber core, resulting in growth of the lens during development. Thus, lens fiber differentiation is spatially regulated and the equator functions as a platform that regulates the switch from cell proliferation to cell differentiation. Since the 1970s, the mechanism underlying lens fiber cell differentiation has been intensively studied, and several regulatory factors that regulate lens fiber cell differentiation have been identified. In this review, we focus on the lens equator, where these regulatory factors crosstalk and cooperate to regulate lens fiber differentiation. Normally, lens epithelial cells must pass through the equator to start lens fiber differentiation. However, there are reports that when the lens epithelium structure is collapsed, lens fiber cell differentiation occurs without passing the equator. We also discuss a possible mechanism that represses lens fiber cell differentiation in lens epithelium.     

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.     

The mechanism of cell elongation during lens fiber cell differentiation

Lens fiber formation is characterized by extensive cell elongation. Earlier studies have shown that lens cell elongation in vitro can occur in the absence of microtubules and is associated with a proportional increase in cell volume. We have previously suggested that lens fiber cell elongation is directly caused by an increase in cell volume. In this report, lenses from 3- and 6-day-old chicken embryos were three-dimensionally reconstructed from serial sections to provide a measure of cell volume and length during various stages of primary and secondary lens fiber formation. In both cases, cell volume was highly correlated with cell length during lens cell elongation. In addition, during primary lens fiber formation, large intercellular spaces between lens vesicle cells disappeared as these cells began to elongate to form lens fibers. Loss of intercellular spaces would be expected if increasing cell volume were responsible for cell elongation. Finally, results of experiments in which the lens capsule was cut with a fine tungsten needle suggested that the capsule was elastic and normally under tension. These findings were used to formulate a model which accounts for the major events in lens morphogenesis based on (1) the regulation of cell volume, (2) the junctions present between lens cells, and (3) the constraint provided by the elasticity of the lens capsule.     

Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells

Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (n?=?52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (n?=?11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student’s t-test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.     

Leucine zipper domain of HIV-1 gp41 interacted specifically with alpha-catenin

Interactions between viral and cellular proteins could explain the molecular mechanisms behind the viral life cycle of HIV-1. The envelope protein gp41 of HIV-1 specifically interacted with alpha-catenin, not with beta-catenin. This interaction was shown by in vitro protein assay and in vivo transfected cell systems. Microinjection of the DNA expressing HIV-1 gp160 and alpha-catenin, into the HeLa cell, resulted in the colocalization of gp41 and alpha-catenin. Interestingly the noncleavable mutant of gp160 and alpha-catenin were found to be colocalized in the cell membrane. Mapping of the interaction sites between these two proteins revealed that the leucine zipper-like structure, located between the first and second alpha-helix domains from the carboxy terminus of HIV-1 gp41, interacted strongly with the carboxy terminus of alpha-catenin.     

Developmental analysis of the eye lens obsolescence (Elo) gene in the mouse: cell proliferation and Elo gene expression in the aggregation chimera.

This study investigates the primary effect of the eye lens obsolescence (Elo) gene of the mouse. Morphological features of the Elo lens were defined as follows: (1) deficient elongation of lens fiber cells, (2) morphological abnormality of nuclei of lens fiber cells, (3) lack of eosinophilic granules in the central fiber cells and (4) rupture of lens capsule in the posterior region. We have immunohistologically examined, by means of an in vivo BrdU incorporation system, whether or not the Elo gene regulates cell proliferation during lens development. The lens fiber cells were morphologically abnormal in day 13 embryonic Elo lens. However, there were no significant differences in morphology or cell proliferation between normal and Elo lens epithelium until day 14 of gestation. After day 15, the total cell number in the Elo lens epithelium was significantly less than that in the normal, but the total numbers of S-phase cells in the two genotypes were not significantly different. The ratio of the total S-phase cell number to the total number of lens epithelial cells may be affected by the developmental stage, but not directly by the genotype. The genotype, however, may be having a direct influence at later ages because malformation of Elo lens fiber cells must cause reduction of the total number of lens epithelial cells in older embryos. Although, at 30 days old, Elo lens cells were externally extruded through the ruptured capsule into the vitreous cavity, BrdU-labelled lens epithelial cells were detectable. To investigate whether the Elo lens phenotype is determined by its own genotype or by its cellular environment, we produced aggregation chimeras between C3H-Elo/+(C/C) and BALB/c(c/c). Most lenses of BALB/c dominant chimeras were oval in shape without the ruptured lens capsule. However, they were opaque in the center and slightly smaller in size than normal. The lenses of C3H-Elo/+ dominant chimeras were morphologically similar to the Elo lens. Although normal nuclei were regularly arranged in the anterior region, Elo-type nuclei were located in the posterior region. Immunohistological staining by using anti-C3H strain-specific antibody demonstrated that the lens fiber cells with abnormal nuclei were derived only from C3H-Elo/+, not from BALB/c. These observations suggest that the primary effect of the Elo gene in the developing lens may be specific to the fiber cell differentiation rather than to the cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)     

THE DIFFERENTIATING ABILITY OF RAT LENS EPITHELIAL CELLS IN CELL CULTURE

Dissociated cells of lens epithelia of adult rats were monolayerly cultured in vitro. After about 15–20 days' period of active cell growth, such characteristic structures that correspond to "lentoid bodies" described previously in chick cultures were formed. These structures consisted of elongated cells, ultrastructural profile of which was similar with lens fiber. The presence of gamma-crystallin, a marker molecule specific to mature lens fiber, was confirmed in these elongated cells by means of fluorescent antibody technique. The differentiation of lens fiber in vitro was also recognized in clones originating from single lens epithelial cells cultured at very low cell density.     

alpha6 Integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching.

The developing chicken embryo lens provides a unique model for examining the relationship between alpha6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that alpha6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell-cell interfaces of the differentiating cortical lens fiber cells. Both alpha6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of alpha6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens alpha6 integrin was uniquely localized along the cell-cell borders of the differentiating fiber cells, similar to beta1. alpha6beta4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of alpha6A and alpha6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that alpha6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with alpha6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that alpha6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of alpha6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the alpha6A isoform remained high until the cells became terminally differentiated. alpha6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of alpha6B relative to alpha6A provides a developmental switch in the process of lens fiber cell differentiation.     

Contributions by members of the TGFbeta superfamily to lens development

Members of the TGFbeta superfamily of growth and differentiation factors, including the TGFbeta, BMP, activin and nodal families, play important signaling roles throughout development. This paper summarizes some of the functions of these ligands in lens development. Targeted deletion of the genes encoding one of the BMP receptors, Alk3 (BMP receptor-1A), showed that signaling through this receptor is essential for normal lens development. Lenses lacking Alk3 were smaller than normal, with thin epithelial layers. The fiber cells of Alk3 null lenses became vacuolated and degenerated within the first week after birth. Lenses lacking Alk3 function were surrounded by abnormal mesenchymal cells, suggesting that the lenses provided inappropriate signals to surrounding tissues. Lens epithelial and fiber cells contained endosomes that were associated with activated (phosphorylated) SMAD1 and SMAD2. Endosomal localization of pSMAD1 was reduced in the absence of Alk3 signaling. The presence of pSMAD2 in lens fiber cell nuclei and the observation that the activin antagonist follistatin inhibited lens cell elongation suggested that an activin-like molecule participates in lens fiber cell differentiation. Lenses deficient in type II TGFbeta receptors were clear and had fiber cells of normal morphology. This suggests that TGFbeta signaling is not essential for the normal differentiation of lens fiber cells. The targeted deletion of single or multiple receptors of the TGFbeta superfamily in the lens should further characterize the role of these signaling molecules in lens development. This approach may also provide a useful way to define the downstream pathways that are activated by these receptors during the development of the lens and other tissues.     

The pulling,pushing and fusing of lens fibers

Lens development and differentiation are intricate and complex processes characterized by distinct molecular and morphological changes. The growth of a transparent lens involves proliferation of the epithelial cells and their subsequent differentiation into secondary fiber cells. Prior to differentiation, epithelial cells at the lens equator exit from the cell cycle and elongate into long, ribbon-like cells. Fiber cell elongation takes place bidirectionally as fiber tips migrate both anteriorly and posteriorly along the apical surface of the epithelium and inner surface of the capsule, respectively. The differentiating fiber cells move inward from the periphery to the center of the lens on a continuous basis as the lens grows throughout life. Finally, when fiber cells reach the center or suture line, their basal and apical tips detach from the epithelium and capsule, respectively, and interlock with cells from the opposite direction of the lens and form the suture line. Further, symmetric packing of fiber cells and degradation of most of the cellular organelle during fiber cell terminal differentiation are crucial for lens transparency. These sequential events are presumed to depend on cytoskeletal dynamics and cell adhesive interactions; however, our knowledge of regulation of lens fiber cell cytosketal reorganization, cell adhesive interactions and mechanotransduction, and their role in lens morphogenesis and function is limited at present. Recent biochemical and molecular studies have targeted cytoskeletal signaling proteins, including Rho GTPases, Abl kinase interacting proteins, cell adhesion molecules, myosin II, Src kinase and phosphoinositide 3-kinase in the developing chicken and mouse lens and characterized components of the fiber cell basal membrane complex. These studies have begun to unravel the vital role of cytoskeletal proteins and their regulatory pathways in control of lens morphogenesis, fiber cell elongation, migration, differentiation, survival and mechanical properties.     

Lens Fiber Cell Differentiation and Denucleation Are Disrupted through Expression of the N-Terminal Nuclear Receptor Box of Ncoa6 and Result in p53-dependent and p53-independent Apoptosis

Nuclear receptor coactivator 6 (NCOA6) is a multifunctional protein implicated in embryonic development, cell survival, and homeostasis. An 81-amino acid fragment, dnNCOA6, containing the N-terminal nuclear receptor box (LXXLL motif) of NCOA6, acts as a dominant-negative (dn) inhibitor of NCOA6. Here, we expressed dnNCOA6 in postmitotic transgenic mouse lens fiber cells. The transgenic lenses showed reduced growth; a wide spectrum of lens fiber cell differentiation defects, including reduced expression of γ-crystallins; and cataract formation. Those lens fiber cells entered an alternate proapoptotic pathway, and the denucleation (karyolysis) process was stalled. Activation of caspase-3 at embryonic day (E)13.5 was followed by double-strand breaks (DSBs) formation monitored via a biomarker, γ-H2AX. Intense terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) signals were found at E16.5. Thus, a window of ∼72 h between these events suggested prolonged though incomplete apoptosis in the lens fiber cell compartment that preserved nuclei in its cells. Genetic experiments showed that the apoptotic-like processes in the transgenic lens were both p53-dependent and p53-independent. Lens-specific deletion of Ncoa6 also resulted in disrupted lens fiber cell differentiation. Our data demonstrate a cell-autonomous role of Ncoa6 in lens fiber cell differentiation and suggest novel insights into the process of lens fiber cell denucleation and apoptosis.     

Unfolded Protein Response (UPR) is activated during normal lens development

The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2α were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis.     

Changes in fibronectin staining in the human lens during ageing and cataractogenesis

The content and localization of fibronectin, an extracellular glycoprotein, in the serial sections of lenses of normal human donors and cataractous patients of different ages were determined by the indirect immunoperoxidase staining technique. This was followed by the evaluation with quantitative morphometric analysis. It was shown that fibronectin was present in the area of cell contacts as single deposits of faint orange-brown stained material in the lens samples of young donors. The fibronectin level was raised in lens sections from aged donors. Its accumulation was detected mostly within the spaces of the lens fiber cells. At different stages of cataractogenesis a dramatic decrease of the fibronectin content was detected in the lens sections obtained from patients of different ages. A new linear spectrophotometric technique was developed for evaluation of the lens transparency, to correlate the lens opacity with corresponding histological data obtained from the immunostaining technique. Morphological studies performed further suggested that the lens fiber cell plasma membrane structures were deteriorated. This was observed as changes of fibronectin staining in the lens sections at different periods of human ageing and cataract development. It is concluded that a decrease of fibronectin staining in the human lens is an indication for the structural damage of the lens fiber cell plasma membranes during ageing and cataractogenesis.     

Functional role for stable microtubules in lens fiber cell elongation

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.     

Periaxin is required for hexagonal geometry and membrane organization of mature lens fibers

Transparency of the ocular lens depends on symmetric packing and membrane organization of highly elongated hexagonal fiber cells. These cells possess an extensive, well-ordered cortical cytoskeleton to maintain cell shape and to anchor membrane components. Periaxin (Prx), a PDZ domain protein involved in myelin sheath stabilization, is also a component of adhaerens plaques in lens fiber cells. Here we show that Prx is expressed in lens fibers and exhibits maturation dependent redistribution, clustering discretely at the tricellular junctions in mature fiber cells. Prx exists in a macromolecular complex with proteins involved in membrane organization including ankyrin-B, spectrin, NrCAM, filensin, ezrin and desmoyokin. Importantly, Prx knockout mouse lenses were found to be softer and more easily deformed than normal lenses, revealing disruptions in fiber cell hexagonal packing, membrane skeleton and membrane stability. These observations suggest a key role for Prx in maturation, packing, and membrane organization of lens fiber cells. Hence, there may be functional parallels between the roles of Prx in membrane stabilization of the myelin sheath and the lens fiber cell.     

Crystal structure of the M-fragment of alpha-catenin: implications for modulation of cell adhesion.

The cytoskeletal protein alpha-catenin, which shares structural similarity with vinculin, is required for cadherin-mediated cell adhesion, and functions to modulate cell adhesive strength and to link the cadherins to the actin-based cytoskeleton. Here we describe the crystal structure of a region of alpha-catenin (residues 377-633) termed the M-fragment. The M-fragment is composed of a tandem repeat of two antiparallel four-helix bundles of virtually identical architectures that are related in structure to the dimerization domain of alpha-catenin and the tail region of vinculin. These results suggest that alpha-catenin is composed of repeating antiparallel helical domains. The region of alpha-catenin previously defined as an adhesion modulation domain corresponds to the C-terminal four-helix bundle of the M-fragment, and in the crystal lattice these domains exist as dimers. Evidence for dimerization of the M-fragment of alpha-catenin in solution was detected by chemical cross-linking experiments. The tendency of the adhesion modulation domain to form dimers may explain its biological activity of promoting cell-cell adhesiveness by inducing lateral dimerization of the associated cadherin molecule.     

Tropomodulin 1 constrains fiber cell geometry during elongation and maturation in the lens cortex

Lens fiber cells exhibit a high degree of hexagonal packing geometry, determined partly by tropomodulin 1 (Tmod1), which stabilizes the spectrin-actin network on lens fiber cell membranes. To ascertain whether Tmod1 is required during epithelial cell differentiation to fiber cells or during fiber cell elongation and maturation, the authors quantified the extent of fiber cell disorder in the Tmod1-null lens and determined locations of disorder by confocal microscopy and computational image analysis. First, nearest neighbor analysis of fiber cell geometry in Tmod1-null lenses showed that disorder is confined to focal patches. Second, differentiating epithelial cells at the equator aligned into ordered meridional rows in Tmod1-null lenses, with disordered patches first observed in elongating fiber cells. Third, as fiber cells were displaced inward in Tmod1-null lenses, total disordered area increased due to increased sizes (but not numbers) of individual disordered patches. The authors conclude that Tmod1 is required first to coordinate fiber cell shapes and interactions during tip migration and elongation and second to stabilize ordered fiber cell geometry during maturation in the lens cortex. An unstable spectrin-actin network without Tmod1 may result in imbalanced forces along membranes, leading to fiber cell rearrangements during elongation, followed by propagation of disorder as fiber cells mature.     

Ras signaling is essential for lens cell proliferation and lens growth during development

The vertebrate ocular lens is a simple and continuously growing tissue. Growth factor-mediated receptor tyrosine kinases (RTKs) are believed to be required for lens cell proliferation, differentiation and survival. The signaling pathways downstream of the RTKs remain to be elucidated. Here, we demonstrate the important role of Ras in lens development by expressing a dominant-negative form of Ras (dn-Ras) in the lens of transgenic mice. We show that lens in the transgenic mice was smaller and lens growth was severely inhibited as compared to the wild-type lens. However, the lens shape, polarity and transparency appeared normal in the transgenic mice. Further analysis showed that cell proliferation is inhibited in the dn-Ras lens. For example, the percentage of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in epithelial layer was about 2- to 3-fold lower in the transgenic lens than in the wild-type lens, implying that Ras activity is required for normal cell proliferation during lens development. We also found a small number of apoptotic cells in both epithelial and fiber compartment of the transgenic lens, suggesting that Ras also plays a role in cell survival. Interestingly, although there was a delay in primary fiber cell differentiation, secondary fiber cell differentiation was not significantly affected in the transgenic mice. For example, the expression of beta- and gamma-crystallins, the marker proteins for fiber differentiation, was not changed in the transgenic mice. Biochemical analysis indicated that ERK activity, but not Akt activity, was significantly reduced in the dn-Ras transgenic lenses. Overall, our data imply that the RTK-Ras-ERK signaling pathway is essential for cell proliferation and, to a lesser extent, for cell survival, but not for crystallin gene expression during fiber differentiation. Thus, some of the fiber differentiation processes are likely mediated by RTK-dependent but Ras-independent pathways.