Decreased membrane permeability to potassium is responsible for the cell volume increase that drives lens fiber cell elongation

Differentiation of lens epithelial cells into fiber cells involves an increase in cell volume which previously was proposed to be the direct cause of the extensive cell elongation which accompanies fiber formation. In this study we have continued to investigate the mechanism underlying cell elongation by defining the minimum nutrient and ion requirements of elongating cells, measuring potassium and sodium fluxes in stimulated and unstimulated lens epithelia, and determining the effects of several pharmacological agents on elongation and ion transport. We have shown that elongation will occur in a basic salt/glucose solution with Insulin-like growth factor I/somatomedin-C stimulation. Neither sodium nor any metabolite appears to be the osmotically active species which drives the increase in cell volume. However, potassium efflux rate coefficient was 47% lower in differentiating cells than in unstimulated cells, whereas potassium uptake rates and ouabain effects were similar. Cells did not elongate in potassium-free medium nor in the presence of several drugs which prevent the accumulation of intracellular potassium or hinder osmotic water flux. Unstimulated cells elongated, however, with the application of quinidine, a drug known to block potassium channels. We propose that stimulation of lens epithelial cells with an insulin-like growth factor signals the closure of a certain population of potassium channels. As a result, potassium efflux from the differentiating cells slows while active potassium uptake continues at a constant rate. Potassium then accumulates within the cell causing water influx, an increase in cell volume, and cell elongation.     

Volume regulation in lens epithelial cells and differentiating lens fiber cells

Previous studies from our laboratory have led us to conclude that lens cell elongation is caused by an increase in cell volume. This volume increase results from an increase in the potassium content of the cells due to decreased potassium efflux. In contrast, an increase in the volume of most cells triggers a regulatory volume decrease (RVD) that is usually mediated by increased potassium efflux. For this reason, chicken embryo lens epithelial cells were tested to see whether they were capable of typical cell volume regulation. Changes in cell volume during lens fiber differentiation were first estimated by 3H2O water uptake. Cell water increased in proportion to cell length in elongating lens cells. Treatment of epithelial cells cultured in basal medium with dilute or concentrated medium, or with medium containing 50 mM sucrose, resulted in typical volume regulatory responses. Cells lost or gained volume in response to osmotic stress, then returned to their previous volume. In addition, the elongation and increase in cell volume that accompanies lens fiber cell differentiation occurred normally in either hypo- or hypertonic media. This observation showed that the activation of mechanisms to compensate for osmotic stress did not interfere with the increase in volume that accompanies elongation. The ability of elongating cells to volume regulate was also tested. Lens epithelial cells were stimulated to elongate by exposure to embryonic vitreous humor, then challenged with hypotonic medium. These elongating cells regulated their volume as effectively as unstimulated cells. Therefore, cells that were increasing their volume due to reduced potassium efflux could adjust their volume in response to osmotic stress, presumably by increasing potassium efflux. This suggests that the changes in potassium efflux that occur during differentiation and RVD are regulated by distinct mechanisms.     

Rac1 GTPase-deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival

Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell–cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover.     

Formation of intercellular gas space in the diaphragm during the development of aerenchyma in the leaf petiole of Sagittaria trifolia

The development of aerenchyma in the petiole of Sagittaria trifolia L. was studied by means of light-microscopy, scanning electron microscope, transmission electron microscope and immunofluorescence, focusing on the formation of intercellular spaces in diaphragms and its relationship with the organization of cortical microtubule arrays. A complex and organized honeycomb-like schizogenous aerenchyma formed by cylinders and vascular diaphragms was observed in the petiole of S. trifolia at different developmental stages. Cell division was the primary factor contributing to the increased volume of air spaces at early stages, while cell enlargement became the primary factor at later stages. The cortical microtubules localize at the sites where intercellular spaces and the secondary cell walls will be formed or deposited during the formation of intercellular spaces by the separation of diaphragm cells. Cortical microtubules were observed at the boundary of diaphragm cells and the fringes of intercellular spaces at later developmental stages where cell expansion occurs rapidly. These observations support the hypothesis that reorganization of cortical microtubule arrays might be related to the formation of air spaces in diaphragms and are involved in the deposition of secondary cell walls.     

Changing protein patterns during lens cell aging in vitro

Changes in biosynthesis of lens proteins upon culturing have been studied by one- and two-dimensional gel electrophoretic techniques. In primary cells still growing on the capsule, αB₂-crystallin is synthesized in a relatively high amount next to the main cytoskeletal constituents actin, tubulin and vimentin. In addition, a minor amount of βB_p seems to be synthesized too. When the cells grow off the capsule, α-crystallin synthesis diminishes. β-Crystallin synthesis continues at a low rate in cells growing on plastic or in cells forming ‘lentoid bodies’. When the cells are subcultured, the synthesis of actin and vimentin becomes more pronounced, while tubulin synthesis is no longer detectable after three transfes The relative amount of vimentin decreases, as compared to actin, during aging and elongation of the cells. When the cells have been transferred ten times and have started to elongate, a 55 kDa protein doublet differing from tubulin is observed in the two-dimensional gel patterns. We observed that elongation of lens cells in culture is accompanied by an increase in the synthesis of a polypeptide of the 26 kDa region. Furthermore, a major glycoprotein is found in the 130 kDa region, but overall glycosylation of proteins seems to decrease during lens cell elongation in vitro.     

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)     

Morphologic study of ventricular trabeculation in the embryonic chick heart

This paper presents a morphologic study of ventricular trabeculation in chick embryo hearts between days 2 and 5 of incubation. Trabeculation appears to be the expression of three closely interrelated events: the formation of endocardial outgrowths that eventually invade the myocardium; the development of large intercellular spaces between the myocytes, and the decrease in thickness of the cardiac jelly. Endocardial cells present morphologic differences between trabeculated and nontrabeculated areas of the ventricular region. The elongation of the endocardial cells in the endocardial outgrowths and the presence of mitoses suggest that the endocardium grows out by means of an increase in cell number and by redistribution and elongation of the preexisting endocardial cells. The intercellular spaces of the myocardium appear filled with abundant extracellular material. It is suggested that the continuous synthesis of extracellular material by the myocytes may increase the hydrostatic pressure within the myocardium, inducing the formation and the enlargement of these intercellular spaces. The development and later rupture of endocardium-covered cords is described here. These cords are made up of a core of cardiac jelly material revested by endocardium. The cords may be engaged in the removal of substantial amounts of cardiac jelly during the formation of the trabeculae.     

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.     

Transient appearance of and regional differences in apical cell surface materials during early morphogenesis of the chicken lens

Summary Apical cell surface materials were analysed with staining and lectin histochemistry in the chicken lens, from the earliest stages of lens morphogenesis through the completion of primary fibre cell elongation. Acidic materials were found to accumulate on the apical cell surface of the presumptive lens fibres from the mid cup stage through the early stages of lens vesicle formation, peaking just before lens fibre cell elongation. These materials labelled strongly with concanavalin A, but not with soybean lectin. By the completion of fibre cell elongation, these materials were gone. Conversely, the apical surface of the future lens epithelial cells demonstrated neutral materials, which were also largely removed by the completion of primary fibre cell elongation. These materials labelled with both concanavalin A and soybean lectin. The identity of these materials is not known, but their location prior to and during chicken lens morphogenesis suggests that they may be involved in establishing polarity during elongation of the primary lens fibre cells.     

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.     

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.     

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation

Previous studies showed that the retina produces factors that promote the differentiation of lens fiber cells, and identified members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as potential fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the presence of BMP receptors in chicken embryo lenses. We have now observed that phosphorylated SMAD1, an indicator of signaling through BMP receptors, localizes to the nuclei of elongating lens fiber cells. Transduction of chicken embryo retinas and/or lenses with constructs expressing noggin, a secreted protein that binds BMPs and prevents their interactions with their receptors, delayed lens fiber cell elongation and increased cell death in the lens epithelium. In an in vitro explant system, in which chicken embryo or adult bovine vitreous humor stimulates chicken embryo lens epithelial cells to elongate into fiber-like cells, these effects were inhibited by noggin-containing conditioned medium, or by recombinant noggin. BMP2, 4, or 7 were able to reverse the inhibition caused by noggin. Lens cell elongation in epithelial explants was stimulated by treatment with FGF1 or FGF2, alone or in combination with BMP2, but not to the same extent as vitreous humor. These data indicate that BMPs participate in the differentiation of lens fiber cells, along with at least one additional, and still unknown factor.     

Diverse gap junctions modulate distinct mechanisms for fiber cell formation during lens development and cataractogenesis

Different mutations of alpha3 connexin (Cx46 or Gja8) and alpha8 connexin (Cx50 or Gja8), subunits of lens gap junction channels, cause a variety of cataracts via unknown mechanisms. We identified a dominant cataractous mouse line (L1), caused by a missense alpha8 connexin mutation that resulted in the expression of alpha8-S50P mutant proteins. Histology studies showed that primary lens fiber cells failed to fully elongate in heterozygous alpha8(S50P/+) embryonic lenses, but not in homozygous alpha8(S50P/S50P), alpha8-/- and alpha3-/- alpha8-/- mutant embryonic lenses. We hypothesized that alpha8-S50P mutant subunits interacted with wild-type alpha3 or alpha8, or with both subunits to affect fiber cell formation. We found that the combination of mutant alpha8-S50P and wild-type alpha8 subunits specifically inhibited the elongation of primary fiber cells, while the combination of alpha8-S50P and wild-type alpha3 subunits disrupted the formation of secondary fiber cells. Thus, this work provides the first in vivo evidence that distinct mechanisms, modulated by diverse gap junctions, control the formation of primary and secondary fiber cells during lens development. This explains why and how different connexin mutations lead to a variety of cataracts. The principle of this explanation can also be applied to mutations of other connexin isoforms that cause different diseases in other organs.     

Phytosterol content and the campesterol:sitosterol ratio influence cotton fiber development: role of phytosterols in cell elongation

Phytosterols play an important role in plant growth and development, including cell division, cell elongation, embryogenesis, cellulose biosynthesis, and cell wall formation. Cotton fiber, which undergoes synchronous cell elongation and a large amount of cellulose synthesis, is an ideal model for the study of plant cell elongation and cell wall biogenesis. The role of phytosterols in fiber growth was investigated by treating the fibers with tridemorph, a sterol biosynthetic inhibitor. The inhibition of phytosterol biosynthesis resulted in an apparent suppression of fiber elongation in vitro or in planta. The determination of phytosterol quantity indicated that sitosterol and campesterol were the major phytosterols in cotton fibers; moreover, higher concentrations of these phytosterols were observed during the period of rapid elongation of fibers. Furthermore, the decrease and increase in campesterol:sitosterol ratio was associated with the increase and decease in speed of elongation, respectively, during the elongation stage. The increase in the ratio was associated with the transition from cell elongation to secondary cell wall synthesis. In addition, a number of phytosterol biosynthetic genes were down-regulated in the short fibers of ligon lintless-1 mutant, compared to its near-isogenic wild-type TM-1. These results demonstrated that phytosterols play a crucial role in cotton fiber development, and particularly in fiber elongation.     

Integrin α5/fibronectin1 and focal adhesion kinase are required for lens fiber morphogenesis in zebrafish

Lens fiber formation and morphogenesis requires a precise orchestration of cell– extracellular matrix (ECM) and cell–cell adhesive changes in order for a lens epithelial cell to adopt a lens fiber fate, morphology, and migratory ability. The cell–ECM interactions that mediate these processes are largely unknown, and here we demonstrate that fibronectin1 (Fn1), an ECM component, and integrin α5, its cellular binding partner, are required in the zebrafish lens for fiber morphogenesis. Mutations compromising either of these proteins lead to cataracts, characterized by defects in fiber adhesion, elongation, and packing. Loss of integrin α5/Fn1 does not affect the fate or viability of lens epithelial cells, nor does it affect the expression of differentiation markers expressed in lens fibers, although nucleus degradation is compromised. Analysis of the intracellular mediators of integrin α5/Fn1 activity focal adhesion kinase (FAK) and integrin-linked kinase (ILK) reveals that FAK, but not ILK, is also required for lens fiber morphogenesis. These results support a model in which lens fiber cells use integrin α5 to migrate along a Fn-containing substrate on the apical side of the lens epithelium and on the posterior lens capsule, likely activating an intracellular signaling cascade mediated by FAK in order to orchestrate the cytoskeletal changes in lens fibers that facilitate elongation, migration, and compaction.     

Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation

The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in lenses lacking all three Fgfrs. These included lack of fiber cell elongation, abnormal proliferation in prospective lens fiber cells, reduced expression of the cell cycle inhibitors p27^kip1 and p57^kip2, increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and α-, β- and γ-crystallins. Therefore, while signaling by FGF receptors is essential for lens fiber differentiation, different FGF receptors function redundantly.     

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells’ native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens¹. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.