Generation of transparency and cellular organization in lens explants

The lens grows via the proliferation and differentiation of lens epithelial cells into lens fibres. This differentiation process, thought to be controlled by factors present in the vitreous fluid, generates tightly-packed, parallel-aligned fibre cells that confer transparency to the lens. Using lens epithelial-cell explants we examined how explant orientation and growth factor treatment can affect cellular arrangement and explant transparency. Fibre cell differentiation was induced in lens explants by culturing cells with fibroblast growth factor (FGF) or bovine vitreous. Cell shape and arrangement was investigated using confocal microscopy, electron microscopy, immunofluorescence and in situ hybridization. Explant transparency was measured using light microscopy. Confocal microscopy demonstrated that explant orientation determined cellular arrangement, irrespective of the differentiation stimuli used. In explants where epithelial cells were confined between their normal basement membrane (the lens capsule) and the base of the culture dish, the cells became elongated, thin and parallel-aligned. In contrast, in explants cultured with cells directly exposed to the culture media the cells appeared to be shorter, globular and haphazardly arranged. FGF initiated the differentiation of most lens epithelial cells; however, abnormal cellular morphologies developed with subsequent culture of the cells. As a result, the transparency of these explants decreased with prolonged culture. Interestingly, explants cultured with vitreous (i) did not develop abnormal cellular morphologies, (ii) contained two distinct cell types (retained epithelial cells and newly differentiated fibre cells) and (iii) remained transparent throughout the lengthy culture period. In summary, we have developed a culture system that generates a transparent tissue with a cellular arrangement resembling that of the lens in vivo. We have shown that while FGF and vitreous initiate differentiation within this system, better maintenance of fibre cell integrity, more appropriate regulation of molecular events, and better maintenance of explant transparency was achieved in the presence of vitreous. This system offers an opportunity to further investigate the process of lens fibre cell differentiation as well as a means of better identifying the factors that contribute to the development of tissue transparency in vitro.     

Role of Gas6/Axl signaling in lens epithelial cell proliferation and survival

Axl is a receptor tyrosine kinase that is activated by Gas6, a growth factor that belongs to the vitamin K-dependent protein family. Although Gas6 binding to Axl has been shown to transmit mitogenic and/or antiapoptotic signals to a variety of cell types, the role of the Axl-Gas6 system in normal and pathological lens biology is not known.We demonstrate for the first time that Axl protein is expressed in normal rat and bovine lens and that its ligand, Gas6, is present in bovine aqueous humor. In addition, we have detected tyrosine-phosphorylated Axl in normal rat and bovine lens epithelial tissues. We further show that human recombinant Gas6 is able to act as a growth factor in cultured human lens epithelial cells by activating Axl and then the AKT signaling pathway. Gas6 mediates a survival and anti-apoptotic response in cultured human lens epithelial cells subjected to serum-starvation (48-72hr), or treated with transforming growth factor beta1 (5 ng ml(-1), 48hr) or tumor necrosis alpha (100 ng ml(-1), 48hr), as demonstrated by increased number of viable cells, and decreased DNA condensation or caspase-3 activity. In contrast, Gas6 is not able to block apoptosis induced by staurosporin (1microM, 5-24hr) in human lens epithelial cells.Taken together, these data suggest that the Gas6/Axl signaling plays an important role in the control of lens epithelial cell growth and survival and hence in the maintenance of lens homeostasis.     

Cell lineage analysis of lens epithelial cells induced to differentiate into fibres

In explant cultures, lens epithelial cells grown in unsupplemented medium retain a morphology and packing arrangement similar to that found in the lens in vivo. In this culture system the epithelial cells can be induced to differentiate into fibres by the addition of retina conditioned medium (RCM). RCM also stimulates cell division. In order to trace the fibre cell lineage and to examine the relationship between cell division and fibre differentiation, single epithelial cells in explants from neonatal rat lenses were labelled with fluorescein-isothiocyanate-dextran. Explants were sub-divided into nine squares and one cell per square was injected with fluorescent label via a microcapillary. By marking the positions of labelled cells at 24-hr intervals for 6 days it was shown that most of the epithelial cells moved laterally within the explant. On average, cells in control explants moved about 20 microns day-1. Cells did not move in any particular direction within the explants and often changed direction. RCM stimulated a dramatic increase in migratory activity. There was about a four-fold increase in migratory activity in the first 24-hr interval, then, even with continued exposure to RCM, this activity quickly dropped over the next 2 days to the same levels as found in controls. As in controls, the cells moved in no particular direction and often changed direction. The observation that RCM stimulates cell migration in this explant system raises the possibility of an important role for active cell migration in the lens in situ. After 6 days culture the dimensions of labelled cells were measured using an image analyser. The areas of cells in controls fell within a narrow range from about 60- to 200 microns2. In contrast, explants grown in RCM had a wide range of cell areas from about 120- to 1500 microns2 and a large proportion of the cells showed some degree of elongation. In explants grown in RCM, 27.3% of labelled cells divided and half of these divisions were during the first 24 hr of culture. Overall there were about 9% more divisions recorded in RCM-treated than in control explants. An analysis of sizes of cells after 6 days of culture showed that whether or not cells divided after addition of RCM they showed very similar frequencies of cell sizes. Therefore, proliferating cells in the explants appear to be as capable of elongating and differentiating into fibres as the non-proliferating cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reorganization of centrosomal marker proteins coincides with epithelial cell differentiation in the vertebrate lens

The differentiation of epithelial cells in the vertebrate lens involves a series of changes that includes the degradation of all intracellular organelles and a dramatic elongation of the cells. The latter is accompanied by a substantial remodelling of the cytoskeleton and changes in the distribution of the actin, microtubule and intermediate filament cytoskeletons during lens cell differentiation have been well documented. There have, however, been no studies of microtubule organizing centres (MTOCs) and specifically centrosomes during lens cell differentiation. We have investigated the fate of the centrosomal MTOCs during cellular differentiation in the bovine lens using gamma-tubulin, ninein, centrin 2 and centrin 3 as markers. Our studies show that these markers oscillate between a clear centrosome-based association in epithelial cells and a defocused cluster in lens fibre cells. Our data further reveal a transient loss of signal for the typical centrosomal marker gamma-tubulin as the lens epithelial cells begin to differentiate into lens fibre cells. This marker apparently disappears in the most distal epithelial cells at the lens equator, only to reappear in early lens fibre cells. The changes in gamma-tubulin distribution are mirrored by the other centrosomal markers, centrins 2 and 3 and ninein that also show a similar transient loss of their signals and subsequent clustering at the apical ends of differentiating fibre cells. The transient loss of staining for these centrosomal markers in the most posterior epithelial cells is a distinctive feature that precedes lens cell elongation. The dramatic reorganization of MTOC markers coincides with gap junction reorganization as seen by the loss of connexin 43 (alpha1-connexin) in these lens epithelial cells suggesting that these events mark a significant change preceding subsequent cell elongation and differentiation into fibre cells.     

Search for a functional glucocorticoid receptor in the mammalian lens

Prolonged glucocorticoid treatment of medical conditions such as rheumatoid arthritis or asthma can lead to the formation of a posterior subcapsular cataract as a negative side effect. Currently, the only treatment for this cataract is surgery because very little is known about the mechanism of glucocorticoid action in the mammalian lens. Understanding of a lens glucocorticoid response is essential for the treatment and prevention of a steroid induced cataract. It has been suggested that glucocorticoids exert their effects on the lens indirectly, non-specifically, or through non-classical mechanisms. While these modes of action may contribute to the formation of glucocorticoid induced posterior subcapsular cataract, the finding of a classical, specific, functional lens glucocorticoid receptor suggests that glucocorticoids target lens epithelial cells directly, specifically, and similar to what has been observed in other cells types. This review explores the discovery of the glucocorticoid receptor in humans lens epithelial cells and the lens specific glucocorticoid response. The distinct changes in lens epithelial cell signaling pathways (MAPK and PI3K-AKT) suggest that glucocorticoids modulate several cellular functions and may explain why a lens glucocorticoid response has been difficult to elucidate.     

Aqueous humour- and growth factor-induced lens cell proliferation is dependent on MAPK/ERK1/2 and Akt/PI3-K signalling

The aqueous humour of the eye is a rich source of growth factors, many of which have been shown to be lens cell mitogens; however, the identity of the endogenous mitogen(s) for lens cells is still unknown. As a first approach to identify the mechanisms by which these aqueous humour-derived growth factors induce lens cell proliferation, the present study set out to examine MAPK/ERK1/2 and PI3-K/Akt signalling associated with lens cell proliferation. Using a lens explant system, we examined the effects of different lens mitogens (aqueous humour, FGF, PDGF, IGF and EGF) using 5'-2'-bromo-deoxyuridine incorporation. In addition, we adopted immunolabelling techniques to compare the roles that the ERK1/2 and PI3-K signalling pathways play in regulating lens cell proliferation. We showed that the aqueous humour, and all the other growth factors examined, could activate ERK1/2 and PI3-K/Akt signalling. By targeting these pathways using specific pharmacological inhibitors, we were able to show that both ERK1/2 and PI3-K signalling are required for growth factor-induced lens cell proliferation, and that there was a strong correlation between the spatial distribution of proliferating cells in lens explants with ERK1/2 labelling. Furthermore, our blocking studies confirmed that PI3-K/Akt signalling can act upstream of ERK1/2, potentiating ERK1/2 phosphorylation in growth factor-induced lens cell proliferation. A better understanding of the signalling pathways required for aqueous humour-induced lens cell proliferation may ultimately allow us to identify the mitogen(s) that are important for regulating lens cell proliferation in situ.     

Cultured chicken embryo lens cells resemble differentiating fiber cells in vivo and contain two kinetic pools of connexin56

The lens is an avascular organ in which gap junctions play a pivotal role for cell physiology and transparency. Here we evaluate a lens culture system as a model for studies of lens gap junction dynamics. In culture, chicken embryo lens cells initially form a monolayer of epithelial cells. Subsequently, the epithelial cells differentiate into lentoids, birefringent multicellular structures composed of fiber-like cells. We examined the cultures for the expression of cellular markers and lens fiber specific proteins using immunofluorescence and immunoblot analysis. We also determined the half-life of connexin56 (Cx56), a fiber-specific gap junction protein. All lens cells in culture expressed actin, endoplasmic reticulum proteins and N-cadherin. Only lentoid cells expressed the lens fiber connexins, Cx45.6 and Cx56. Cx56 localized at appositional membranes and did not co-localize with endoplasmic reticulum proteins or N-cadherin. Two pools of Cx56 were detected in these cultures, one with a half-life of a few hours and the other with a half-life of days. The two pools contained phosphorylated forms of Cx56 of different apparent molecular weights. These results suggest that lens cells in culture can be used as a model for the study of lens biology. They also suggest that phosphorylation of Cx56 might be regulating the stability of the protein.     

Reduced epithelial adhesion after extended contact lens wear correlates with reduced hemidesmosome density in cat cornea.

Reduced epithelial adhesion in cat corneas after continuous wear of thick hydrogel contact lenses has been reported previously. To investigate the mechanism(s) underlying this observed loss of epithelial adhesion further, the corneas of both eyes of cats that had worn low-oxygen-transmissible thick parallel-design hydrogel contact lenses only in one eye for 8-121 days were examined using both light and transmission electron microscopy (TEM). Contact lens wear induced many changes in the epithelium, including a decrease in the number of cell layers and appearance of cuboidal rather than columnar basal cell shapes. In addition, TEM revealed that the number of hemidesmosomes (HDs) per micrometer of basement membrane was reduced significantly after contact lens wear. Anchoring fibrils in lens-wearing corneas appeared normal, and the reduction in epithelial adhesion occurred without obvious epithelial edema. Decreased epithelial adhesion after contact lens wear appears to be directly related to the reduced numbers of HDs. Possible reasons for decreased HD density, such as loss of basal cell shape and chronic epithelial hypoxia after contact lens wear, are discussed.     

Sequential structural response of lens epithelium to retina-conditioned medium

When lens epithelium is cultured with retina-conditioned medium, many of the cells undergo fibre differentiation within 6-10 days. Here we report the temporal sequence of structural events that characterize this change in an organ culture model system. Within an hour of exposure to conditioned medium, some cells withdrew from the epithelial monolayer and began migrating over stationary cells still attached to the capsule. By 24 hr, migratory activity was largely responsible for causing the explant to become multilayered, increasing its thickness while at the same time reducing its surface area. Control cells attached to each other along their lateral boundaries through interdigitations of microvilli. After BRCM treatment, microvilli flattened out and the membranes had a crinkled appearance. Eventually, as the cultured cells developed into fibres, membranes straightened and developed knob and socket junctions and large numbers of their organelles underwent degradation within autophagic vacuoles. Nucleoli began to enlarge by 16 hr and by the time cells had been exposed for 24 hr, some nucleoli were enlarged to seven-fold their original area, as measured on electron micrographs. This nucleolar change was followed over the next few days by a gradual increase in cytoplasmic protein, and cells became plump or elongated. The ultrastructural changes that we observed in culture are similar to those that can be seen in the intact lens. Such fidelity of change indicates that this cultured lens explant system is an excellent model for experimental intervention and analysis of the processes involved in terminal lens fibre differentiation.     

Proliferation of lens epithelial cells on the Acrysof intraocular lens: clinical and histological features of a case

Postoperative deposits on the surface of intraocular lenses (IOLs) have been demonstrated in vivo for many IOL material types. The Acrysof acrylic lens develops a granular material extending from the capsulorhexis margin onto the IOL's anterior surface during the first 4 weeks after surgery in a significant proportion of cases. Complete resolution of this membrane occurs by 3 months in almost all cases. The case is presented of an 81‐year‐old woman who had a persisting membrane covering the IOL surface at 6 months. This membrane was removed surgically and pathology showed the constituent cells to be of lens epithelial cell origin.     

Changes in three types of ubiquitin mRNA and ubiquitin-protein conjugate levels during lens development

Ubiquitin is a small, highly conserved protein that covalently attaches to other proteins to form a unique branched protein structure. The best characterized function of this post-translational modification is to mark the modified protein for degradation by the proteasome. To investigate whether ubiquitin genes are regulated in lens development, the authors analyzed the levels of three ubiquitin mRNAs (UbA(52), UbB and UbC) in freshly dissected fiber and epithelial cells, and in epithelial explants induced to differentiate ex vivo. Explants, comprising the capsule and adherent epithelial cells, were dissected from lenses of 3 day old Sprague Dawley rats and cultured +/-bFGF to induce differentiation. Quantitative competitive RT-PCR was used to determine the mRNA levels in fresh and cultured cells. UbA(52), UbB and UbC mRNAs were 3.2 (P < 0.0001), 5.0 (P < 0.0001) and 6.8 (P < 0.0001) fold higher, respectively, in freshly dissected epithelial cells than in differentiated fiber cells. Immunological spot assays showed that ubiquitin protein is over two fold as high in rat pup lens epithelial cells as in fiber cells. The ubiquitin protein in fiber cells of adult rat is lower than that in adult epithelium and in pup fiber cells, indicating that ubiquitin content further decreased during lens fiber maturation. Western blots showed a greater amount of protein-conjugated ubiquitin (MW > 81 kD) in epithelial cells than in fiber cells, demonstrating a parallel pattern between the expression of ubiquitin mRNA, the level of ubiquitin protein and the level of conjugates in the cells. Epithelial cell explant cultures permit study of cells initiating differentiation. In contrast to fully differentiated fiber cells, explant cultures induced to initiate differentiation underwent differential up-regulation of ubiquitin gene expression. UbA(52) and UbB mRNA levels in +bFGF (differentiating) explant cultures were 2.6 (P < 0.001) and 1.4 (P < 0.001) fold higher, respectively, than those of -bFGF cultures. UbC mRNA content was similar in explants cultured with or without bFGF. Dissection of the isolated epithelial cells into regions representing distinct populations gave results consistent with this observation of the explant results. UbA(52), UbB and UbC mRNAs are 2.0, 2.2 and 1.76 fold higher, respectively, in the peripheral (initiating differentiation) than in the central (undifferentiated) region of epithelial cells. These results together indicate that UbA(52) and UbB mRNAs are transiently increased during the initiation and early stages of differentiation. However, UbC mRNA appears to be relatively unaffected at the earliest stage in this differentiation model and may have a different distribution than UbA(52) and UbB in the anterior lens cells. These data are consistent with an important role for ubiquitin during the early stages of lens differentiation. The selective expression indicates that the three genes have specific differentiation related functions.     

Alpha-smooth muscle actin expression in cultured lens epithelial cells

PURPOSE: Lens epithelial cells transdifferentiate to myofibroblasts during the formation of anterior subcapsular cataracts and secondary cataracts. One of the defining characteristics of myofibroblasts is the expression of alpha-smooth muscle actin (alpha-SMA). This study investigated some of the factors that influence alpha-SMA expression in lens epithelial cells. METHODS: Bovine, rabbit, and human lens epithelial explants or cells were cultured with or without serum. Immunohistochemistry and immunoblotting were used to detect and quantitate alpha-SMA expression. RESULTS: Cells from all species studied expressed alpha-SMA in primary explant culture with or without serum. Immunostaining for alpha-SMA first appeared in a diffuse granular pattern, then accumulated at the cell cortex, and eventually was detected along stress fibers. When lens epithelial cells migrated onto cell-free regions of the capsule or were transferred to a plastic culture dish, alpha-SMA expression increased significantly. Expression of alpha-SMA positively correlated with cell size and cell migration. CONCLUSIONS: Expression of alpha-SMA is a common feature of cultured mammalian lens epithelial cells. Because alpha-SMA expression occurred without the addition of exogenous factors, the fibrosis seen in anterior subcapsular cataracts or secondary cataracts may reflect the intrinsic properties of lens epithelial cells. Interaction between lens epithelial cells and their substratum appears to be an important regulator of myofibroblast formation. Understanding the factors that regulate alpha-SMA expression in lens epithelial cells could lead to the development of methods for preventing secondary cataracts and anterior subcapsular cataracts.     

Aberrant lens fiber differentiation in anterior subcapsular cataract formation: a process dependent on reduced levels of Pax6

PURPOSE: TGFbeta can induce development in lenses of opaque subcapsular fibrotic plaques that have many features of human subcapsular cataracts. To understand further the events associated with the onset and progression of TGFbeta-induced cataract, several different models for anterior subcapsular cataract (ASC) were used and characterized. METHODS: Anterior subcapsular plaques were induced in rat lenses cultured with TGFbeta and in transgenic mice overexpressing TGFbeta in the lens. ASC was also examined in lenses of mice haploinsufficient for Pax6, as well as in human biopsy specimens. Immunofluorescence and in situ hybridization labeling were used to examine changes in patterns of gene expression associated with cataract formation in these models. RESULTS: Examination of TGFbeta-induced cataract in transgenic mice established that the subcapsular plaques are composed of a heterogenous cell population: a population of myofibroblastic cells as well as a population of lens-fiber-like cells. Further support for phenotypic change comes from the observation that the cells in these plaques no longer expressed lens epithelial markers, such as Pax6 and Connexin43. Subsequent examination of human biopsy specimens of ASC, as well as lenses from Pax6-deficient mice, showed that the anterior subcapsular plaques in both cases were also composed of a heterogenous population of cells. In contrast, anterior subcapsular plaques that developed in vitro in response to TGFbeta did not have this same cellular heterogeneity, as no fiber-like cells were present. CONCLUSIONS: These findings suggest that in vivo, during TGFbeta-induced cataract formation, some lens epithelial cells transform into myofibroblastic cells, whereas others differentiate into fiber cells. As this pathologic change is accompanied by altered expression of genes characteristic of the normal lens epithelial cell phenotype and as lenses from Pax6-deficient mice exhibit development of anterior subcapsular plaques closely resembling those induced by TGFbeta in transgenic mice, the authors propose that a reduction in Pax6 levels may be essential for this pathologic process to progress. Furthermore, it is clear from these in vitro studies that TGFbeta alone cannot reproduce the same morphologic and molecular changes associated with ASC formation in vivo, indicating that additional molecule(s) in the eye are important in this process.     

Nuclear pyknosis during lens fibre differentiation in epithelial explants

In our culture system, lens epithelial cells differentiate into fibres under the influence of fibre differentiation factor (FDF) from neural retinas. FDF stimulates DNA synthesis and cell migration within 24 hours. After 2 days we detect increased alpha-crystallin synthesis and, after 4 and 6 days, beta- and gamma-crystallin synthesis, respectively. In this study, a quantitative analysis of DNA levels using the DAPI method shows increases in DNA/explant up to 2 days of culture. This is followed by a substantial drop in DNA/explant by 10 days. The early rise in DNA levels corresponds with the period of cell division stimulated by FDF. The drop in DNA levels corresponds with a significant increase in the proportion of pyknotic nuclei in the explants. Electron microscopy shows pyknotic nuclei in differentiated fibre cells. Since nuclear pyknosis is a normal event during terminal fibre differentiation in vivo, this study confirms that our explant system faithfully reproduces events in fibre differentiation.     

The fate of dividing cells during lens morphogenesis, differentiation and growth

Early in development, the ocular lens establishes its distinctive architecture, and this is maintained throughout life as the lens continues to grow. This growth is tightly regulated through the proliferation of the lens epithelial cells and their subsequent differentiation into specialized elongated fiber cells. Although much work has been carried out to define these patterns of growth, very little has been reported on the detailed fate and kinetics of lens cells during embryogenesis. Using BrdU-incorporation, the present study has attempted to follow the fate of lens cells that have undergone at least one round of DNA synthesis during the early stages of lens morphogenesis. Results from this work have confirmed that the rate of lens cell proliferation and new fiber cell differentiation progressively slows as the lens differentiates and grows. In addition, these studies have shown that early in lens development, not all DNA synthesis is restricted to the lens epithelium, with some elongating fiber cells retaining the ability to undergo DNA synthesis. Adopting this system we have also been able to place the initiation of secondary fiber cell differentiation in the mouse lens by E12.5, concomitant with the loss of the lens vesicle lumen by the elongating primary fiber cells. Overall, this study has allowed us to revisit some of the mechanisms involved in early lens development, has provided us with insights into the fate of cells during this rapid phase of murine lens growth, and has provided a novel method to study the rate of new fiber cell differentiation over a defined period of lens development and growth.     

晶状体上皮细胞凋亡调控基因与白内障的研究

白内障是最常见的致盲眼病之一,晶状体上皮细胞的凋亡是除先天性白内障以外的所有类型白内障发生的共同细胞学基础。近些年的研究表明:多种调控基因参与了晶状体上皮细胞凋亡。我们将近年来晶状体上皮细胞凋亡调控基因与白内障的研究情况做一综述。     

Regional mechanical properties and stress analysis of the human anterior lens capsule

The lens capsule of the eye functions, in part, as a deformable support through which the ciliary body applies tractions that can alter lens curvature and corresponding refractive power during the process of accommodation. Although it has long been recognized that characterization of the mechanical properties of the lens capsule is fundamental to understanding this physiologic process as well as clinical interventions, prior data have been limited by one-dimensional testing of excised specimens despite the existence of multiaxial loading in vivo. In this paper, we employ a novel experimental approach to study in situ the regional, multiaxial mechanical behavior of both normal and diabetic human anterior lens capsules. Furthermore, we use these data to calculate material parameters in a nonlinear stress-strain relation via a custom sub-domain inverse finite element method (FEM). These parameters are then used to predict capsular stresses in response to imposed loads using a forward FEM model. Our results for both normal and diabetic human eyes show that the anterior lens capsule exhibits a nonlinear pseudoelastic behavior over finite strains that is typical of soft tissues, and that strains are principal relative to meridional and circumferential directions. Experimental data and parameter estimation suggest further that the capsule is regionally anisotropic, with the circumferential direction becoming increasingly stiffer than the meridional direction towards the equator. Although both normal and diabetic lens capsules exhibited these general characteristic behaviors, diabetic capsules were significantly stiffer at each distension. Finally, the forward FEM model predicted a nearly uniform, equibiaxial stress field during normalcy that will be perturbed by cataract surgery. Such mechanical perturbations may be an underlying modulator of the sustained errant epithelial cell behavior that is observed well after cataract surgery and may ultimately contribute to opacification of the posterior lens capsule.     

Structural analysis of lens epithelial explants induced to differentiate into fibres by fibroblast growth factor (FGF)

Recently we identified fibroblast growth factor (FGF), which is present in significant amounts in neural retinas, as a potent inducer of lens fibre differentiation in our epithelial explant cultures. Fibre differentiation was assessed by synthesis of fibre specific, proteins, beta- and gamma-crystallins, and by cell elongation. However, to establish whether FGF induced the dramatic structural changes characteristic of fibre differentiation we carried out an ultrastructural analysis. In this study epithelial explants exposed to either the acidic or basic form of FGF were shown to undergo the structural changes characteristic of fibre differentiation in the intact lens. These include: (i) cell elongation, (ii) a reduction in cytoplasmic organelles, (iii) the formation of specialized cell-cell junctions, including finger-like processes and fingerprints, ball and socket junctions, tongue-like flaps and imprints, and gap junctions, and (iv) nuclear pyknosis. This shows that FGF faithfully reproduces structural events associated with fibre differentiation as well as the molecular events reported previously, thus providing further evidence that FGF in the eye is important for the control of normal lens fibre differentiation.