Non-essential role for cilia in coordinating precise alignment of lens fibres.

The primary cilium, a microtubule-based organelle found in most cells, is a centre for mechano-sensing fluid movement and cellular signalling, notably through the Hedgehog pathway. We recently found that each lens fibre cell has an apically situated primary cilium that is polarised to the side of the cell facing the anterior pole of the lens. The direction of polarity is similar in neighbouring cells so that in the global view, lens fibres exhibit planar cell polarity (PCP) along the equatorial-anterior polar axis. Ciliogenesis has been associated with the establishment of PCP, although the exact relationship between PCP and the role of cilia is still controversial. To test the hypothesis that the primary cilia have a role in coordinating the precise alignment/orientation of the fibre cells, IFT88, a key component of the intraflagellar transport (IFT) complex, was removed specifically from the lens at different developmental stages using several lens-specific Cre-expressing mouse lines (MLR10- and LR-Cre). Irrespective of which Cre-line was adopted, both demonstrated that in IFT88-depleted cells, the ciliary axoneme was absent or substantially shortened, confirming the disruption of primary cilia formation. However no obvious histological defects were detected even when IFT88 was removed from the lens placode as early as E9.5. Specifically, the lens fibres aligned/oriented towards the poles to form the characteristic Y-shaped sutures as normal. Consistent with this, in primary lens epithelial explants prepared from these conditional knockout mouse lenses, the basal bodies still showed polarised localisation at the apical surface of elongating cells upon FGF-induced fibre differentiation. We further investigated the lens phenotype in knockouts of Bardet-Biedl Syndrome (BBS) proteins 4 and 8, the components of the BBSome complex which modulate ciliary function. In these BBS4 and 8 knockout lenses, again we found the pattern of the anterior sutures formed by the apical tips of elongating/migrating fibres were comparable to the control lenses. Taken together, these results indicate that primary cilia do not play an essential role in the precise cellular alignment/orientation of fibre cells. Thus, it appears that in the lens cilia are not required to establish PCP.

Atypical Cadherin Fat1 Is Required for Lens Epithelial Cell Polarity and Proliferation but Not for Fiber Differentiation.

PURPOSE: The Fat family of atypical cadherins, originally identified in Drosophila, play diverse roles during embryogenesis and adult tissue maintenance. Among four mammalian members, Fat1 is essential for kidney and muscle organization, and is also essential for eye development; Fat1 knockout causes partial penetrant microphthalmia or anophthalmia. To account for the partial penetrance of the Fat1 phenotype, involvement of Fat4 in eye development was assessed. Lens phenotypes in Fat1 and 4 knockouts were also examined. METHODS: Fat1 and Fat4 mRNA expression was examined by in situ hybridization. Knockout phenotypes of Fat1 and Fat4 were analyzed by hematoxylin and eosin (H&E) and immunofluorescent staining. RESULTS: We found Fat4 knockout did not affect eye induction or enhance severity of Fat1 eye defects. Although Fat1 and Fat4 mRNAs are similarly expressed in the lens epithelial cells, only Fat1 knockout caused a fully penetrant lens epithelial cell defect, which was apparent at embryonic day 14.5 (E14.5). The columnar structure of the lens epithelial cells was disrupted and in some regions cell aggregates were formed. In these multilayered regions, apical cell junctions were fragmented and the apical-basal polarity was lost. EdU incorporation assay also showed enhanced proliferation in the lens epithelial cells. Interestingly, these defects were found mainly in the central zone of the epithelial layer. The lens epithelial cells of the germinative zone maintained their normal morphology and fiber differentiation occurred normally at the equator. CONCLUSIONS: These observations indicate that Fat1 is essential for lens epithelial cell polarity and proliferation but not for terminal differentiation.

Negative regulation of TGFß-induced lens epithelial to mesenchymal transition (EMT) by RTK antagonists.

An eclectic range of ocular growth factors with differing actions are present within the aqueous and vitreous humors that bathe the lens. Growth factors that exert their actions via receptor tyrosine kinases (RTKs), such as FGF, play a normal regulatory role in lens; whereas other factors, such as TGFß, can lead to an epithelial to mesenchymal transition (EMT) that underlies several forms of cataract. The respective downstream intracellular signaling pathways of these factors are in turn tightly regulated. One level of negative regulation is thought to be through RTK-antagonists, namely, Sprouty (Spry), Sef and Spred that are all expressed in the lens. In this study, we tested these different negative regulators and compared their ability to block TGFß-induced EMT in rat lens epithelial cells. Spred expression within the rodent eye was confirmed using RT-PCR, western blotting and immunofluorescence. Rat lens epithelial explants were used to examine the morphological changes associated with TGFß-induced EMT over 3 days of culture, as well as α-smooth muscle actin (α-sma) immunolabeling. Cells in lens epithelial explants were transfected with either a reporter (EGFP) vector (pLXSG), or with plasmids also coding for different RTK-antagonists (i.e. pLSXG-Spry1, pLSXG-Spry2, pLXSG-Sef, pLSXG-Spred1, pLSXG-Spred2, pLSXG-Spred3), before treating with TGFß for up to 3 days. The percentages of transfected cells that underwent TGFß-induced morphological changes consistent with an EMT were determined using cell counts and validated with a paired two-tailed t-test. Explants transfected with pLXSG demonstrated a distinct transition in cell morphology after TGFß treatment, with ∼60% of the cells undergoing fibrotic-like cell elongation. This percentage was significantly reduced in cells overexpressing the different antagonists, indicative of a block in lens EMT. Of the antagonists tested under these in vitro conditions, Spred1 was the most potent demonstrating the greatest block in TGFß-induced fibrotic cell elongation/EMT. Through the overexpression of RTK-antagonists in lens epithelial cells we have established a novel role for Spry, Spred and Sef as negative regulators of TGFß-induced EMT. Further investigations may help us develop a better understanding of the molecular mechanisms involved in maintaining the integrity of the normal lens epithelium, with these antagonists serving as putative therapeutic agents for prevention of EMT, and hence cataractogenesis.

Sfrp1 and Sfrp2 are not involved in Wnt/ß-catenin signal silencing during lens induction but are required for maintenance of Wnt/ß-catenin signaling in lens epithelial cells.

During eye lens development, regulation of Wnt/ß-catenin signaling is critical for two major processes: initially it must be silent in the lens placode for lens development to proceed, but subsequently it is required for maintenance of the lens epithelium. It is not known how these different phases of Wnt/ß-catenin activity/inactivity are regulated. Secreted frizzled related protein-2 (Sfrp2), a putative Wnt-Fz antagonist, is expressed in lens placode and in lens epithelial cells and has been put forward as a candidate for regional Wnt/ß-catenin pathway regulation. Here we show its closely-related isoform, Sfrp1, has a complimentary pattern of expression in the lens, being absent from the placode and epithelium but expressed in the fibers. As mice with single knockouts of Sfrp1 or Sfrp2 had no defects in lens formation, we examined lenses of Sfrp1 and Sfrp2 double knockout (DKO) mice and showed that they formed lens placode and subsequent lens structures. Consistent with this we did not observe ectopic TCF/Lef activity in lens placode of DKOs. This indicates that Sfrp1 and Sfrp2 individually, or together, do not constitute the putative negative regulator that blocks Wnt/ß-catenin signaling during lens induction. In contrast, Sfrp1 and Sfrp2 appear to have a positive regulatory function because Wnt/ß-catenin signaling in lens epithelial cells was reduced in Sfrp1 and Sfrp2 DKO mice. Lenses that formed in DKO mice were smaller than controls and exhibited a deficient epithelium. Thus Sfrps play a role in lens development, at least in part, by regulating aspects of Wnt/ß-catenin signaling in lens epithelial cells.

Dexamethasone influences FGF-induced responses in lens epithelial explants and promotes the posterior capsule coverage that is a feature of glucocorticoid-induced cataract.

Aberrant spreading of lens epithelial cells along the posterior capsule is the basis for development of glucocorticoid (GC)-induced cataract; the resulting foci of nucleated cells at the posterior pole causing disruptions to normal lens cellular architecture. In this study, rat lens epithelial explants were used to assess the effects of dexamethasone (DEX), a widely used synthetic GC, on FGF2-induced lens cell proliferation and elongation as well as the ability of lens cells to spread and cover the posterior capsule. In the presence of FGF2, DEX significantly promoted lens cell proliferation after 48 h. Cell coverage of the posterior capsule was also enhanced during 5 days culture. In contrast, cell elongation was retarded by the inclusion of DEX. In the absence of FGF2, DEX had no marked effects on any of these cellular processes. Thus, in the presence of FGF2, DEX promoted cell proliferation and posterior capsule coverage but inhibited cell elongation. These results provide insights into the molecular mechanism underlying GC-induced cataract in humans.

Wnt-frizzled signaling is part of an FGF-induced cascade that promotes lens fiber differentiation.

PURPOSE: It is well established that lens fiber differentiation depends on an FGF-initiated growth factor signaling cascade. Given that recent studies indicate Wnt-Frizzled/Planar Cell Polarity (Wnt-Fz/PCP) signaling has a role in coordinating the orientation and alignment of fibers, this study set out to investigate the relationship between this pathway and FGF-induced fiber differentiation. METHODS: Rat lens epithelial explants were cultured with FGF-2. Regulators of Wnt-Fz signaling, secreted frizzled-related protein-1 (Sfrp1), and inhibitor of Wnt production-2 (IWP-2) were applied to assess the role of this pathway in FGF-induced fiber differentiation. A TCF/Lef reporter mouse was used to assess canonical Wnt-Fz/ß-catenin signaling. RESULTS: FGF-induced fiber differentiation was accompanied by upregulation of Wnt-Fz signaling components, Fz3, Fz6, Dishevelled-2 (Dvl2), and Dishevelled-3. During differentiation, Fz and the centrosome/primary cilium translocated to the apical tip/leading edge of similarly polarized groups of cells. Addition of Sfrp1 or IWP-2 to FGF-treated explants inhibited cell elongation and reduced expression of fiber-specific markers, filensin and ß-crystallin. Expression of Wnt-Fz signaling components was also reduced and a significant reduction in the active form of Dvl2 indicated inhibition of the pathway. Analysis of the TCF/Lef reporter mouse showed no evidence of canonical Wnt-Fz/ß-catenin signaling during FGF-induced fiber differentiation. CONCLUSIONS: This study shows that Wnt-Fz signaling is a component of the FGF-initiated cascade that regulates fiber differentiation. The presence of groups of fibers with Fz and centrosome/primary cilium polarized to the leading edge of each cell is consistent with a role for noncanonical Wnt-Fz signaling in coordinating polarized behavior of differentiating fibers.

Tear levels of SFRP1 are significantly reduced in keratoconus patients.

PURPOSE: To measure secreted frizzled-related protein 1 (SFRP1) levels in human tears and to investigate tear SFRP1 as a potential biomarker for keratoconus (KC). METHODS: Tears were collected from control (n = 33) and KC patients (n = 33) using micropipette tubes. Total tear protein was measured using a FluoroProfile Protein Quantification kit. An in-house enzyme-linked immunosorbent assay (ELISA) was developed to measure SFRP1 in control and KC tears. Statistical analyses of age, gender, the association of SFRP1, and total tear protein with KC were conducted. RESULTS: Tear SFRP1 was significantly decreased in KC, compared to age-matched controls (3.41 ng/µl ± 3.12 versus 5.55 ng/µl ± 5.62, respectively; p = 0.039). Conversely, total tear protein was significantly increased in KC, compared to age-matched controls (12.38 µg/µl ± 4.76 versus 9.40 µg/µl ± 3.88, respectively; p = 0.038). The ratio of SFRP1/total tear protein was also found to be significantly decreased in the KC group (p = 0.007). No significant association between tear SFRP1 and total tear protein was detected. CONCLUSIONS: Tear SFRP1 was significantly decreased in age-matched KC versus control patients, and may be further reduced in moderate KC. Tear-SFRP1 levels alone do not provide an obvious biomarker for KC; however, our results provide further evidence that tear-protein profiles are altered in KC, and suggest the involvement of SFRPs in the pathogenesis of KC.

Sprouty is a negative regulator of transforming growth factor ß-induced epithelial-to-mesenchymal transition and cataract.

Fibrosis affects an extensive range of organs and is increasingly acknowledged as a major component of many chronic disorders. It is now well accepted that the elevated expression of certain inflammatory cell-derived cytokines, especially transforming growth factor ß (TGFß), is involved in the epithelial-to-mesenchymal transition (EMT) leading to the pathogenesis of a diverse range of fibrotic diseases. In lens, aberrant TGFß signaling has been shown to induce EMT leading to cataract formation. Sproutys (Sprys) are negative feedback regulators of receptor tyrosine kinase (RTK)-signaling pathways in many vertebrate systems, and in this study we showed that they are important in the murine lens for promoting the lens epithelial cell phenotype. Conditional deletion of Spry1 and Spry2 specifically from the lens leads to an aberrant increase in RTK-mediated extracellular signal-regulated kinase 1/2 phosphorylation and, surprisingly, elevated TGFß-related signaling in lens epithelial cells, leading to an EMT and subsequent cataract formation. Conversely, increased Spry overexpression in lens cells can suppress not only TGFß-induced signaling, but also the accompanying EMT and cataract formation. On the basis of these findings, we propose that a better understanding of the relationship between Spry and TGFß signaling will not only elucidate the etiology of lens pathology, but will also lead to the development of treatments for other fibrotic-related diseases associated with TGFß-induced EMT.

Analysis of PCP defects in mammalian eye lens.

Multicellular tissues and organs often show planar cell polarity (PCP) where the constituent cells align along an axis to form coordinated patterns. Mammalian eye lenses are mainly comprised of epithelial-derived fibre cells, which exhibit highly ordered alignment that is regulated by PCP signaling. Each fibre cell has an apically situated primary cilium and in most cases this is polarized towards the lens anterior pole. Here we describe how to visualize the global cellular alignment of lens fibre cells by examining the suture pattern that is formed by the tips of fibres meeting at the anterior pole. We also describe a method for whole mount preparation, which allows observation of the polarized distribution of primary cilia at the apical surface of lens fibres. Given its relative simplicity, at least in cellular terms, and its requirement for a high degree of precision in cellular alignment and orientation, we predict that the lens will be an excellent model system to help elucidate the role of cilia and PCP components in the development of three-dimensional organization in tissues and organs.

Planar cell polarity in the mammalian eye lens.

The major role of the eye lens is to transmit and focus images onto the retina. For this function, the lens needs to develop and maintain the correct shape, notably, the precise curvature and high-level order and organization of its elements. The lens is mainly comprised of highly elongated fiber cells with hexagonal cross-sectional profiles that facilitate regular packing. Collectively, they form concentrically arranged layers around the anterior-posterior polar axis, and their convex curvature contributes to the spheroidal shape of the lens. Although the lens has been a popular system for developmental studies, little is known about the mechanism(s) that underlies the development of its exquisite three-dimensional cellular architecture. In this review, we will describe our recent work, which shows how planar cell polarity (PCP) operates in lens and contributes to its morphogenesis. We believe that the lens will be a useful model system to study PCP in general and gain insights into mechanisms that generate high-level cellular order during development.

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.

Sef is a negative regulator of fiber cell differentiation in the ocular lens.

Growth factor signaling, mediated via receptor tyrosine kinases (RTKs), needs to be tightly regulated in many developmental systems to ensure a physiologically appropriate biological outcome. At one level this regulation may involve spatially and temporally ordered patterns of expression of specific RTK signaling antagonists, such as Sef (similar expression to fgfs). Growth factors, notably FGFs, play important roles in development of the vertebrate ocular lens. FGF induces lens cell proliferation and differentiation at progressively higher concentrations and there is compelling evidence that a gradient of FGF signaling in the eye determines lens polarity and growth patterns. We have recently identified the presence of Sef in the lens, with strongest expression in the epithelial cells. Given the important role for FGFs in lens developmental biology, we employed transgenic mouse strategies to determine if Sef could be involved in regulating lens cell behaviour. Over-expressing Sef specifically in the lens of transgenic mice led to impaired lens and eye development that resulted in microphthalmia. Sef inhibited primary lens fiber cell elongation and differentiation, as well as increased apoptosis, consistent with a block in FGFR-mediated signaling during lens morphogenesis. These results are consistent with growth factor antagonists, such as Sef, being important negative regulators of growth factor signaling. Moreover, the lens provides a useful paradigm as to how opposing gradients of a growth factor and its antagonist could work together to determine and stabilise tissue patterning during development and growth.

Growth factor signaling in vitreous humor-induced lens fiber differentiation.

PURPOSE. Although some of the factors and signaling pathways that are involved in induction of fiber differentiation have been defined, such as FGF-mediated MAPK/ERK and PI3-K/Akt signaling, the factors in the vitreous that regulate this differentiation process in vivo have yet to be identified. The purpose of this study was to better understand the role of growth factors in vitreous that regulate this process by further characterizing the signaling pathways involved in lens fiber differentiation. METHODS. Rat lens epithelial explants were used to compare the ability of vitreous, IGF-1, PDGF-A, EGF, and FGF-2 to stimulate the phosphorylation of ERK1/2 and Akt leading to fiber differentiation, in the presence or absence of selective receptor tyrosine kinase (RTK) inhibitors. RESULTS. Similar to vitreous, FGF induced a sustained ERK1/2 signaling profile, unlike IGF, PDGF, and EGF, which induced a more transient (shorter) activation of ERK1/2. For Akt activation, IGF was the only factor that induced a profile similar to vitreous. IGF, PDGF, and EGF potentiated the effects of a low dose of FGF on lens fiber differentiation by extending the duration of ERK1/2 phosphorylation. In the presence of selective RTK inhibitors, although the sustained vitreous-induced ERK1/2 signaling profile and subsequent fiber differentiation was perturbed, the results also showed that, although prolonged ERK1/2 phosphorylation was necessary, it was not sufficient for fiber differentiation to proceed. CONCLUSIONS. These results are consistent with FGF's being the key growth factor involved in vitreous-induced signaling leading to lens fiber differentiation; however, they also indicate that other vitreal growth factors such as IGF may be involved in fine-tuning ERK1/2- and Akt-phosphorylation to the level that is necessary for initiation and/or maintenance of lens fiber differentiation in vivo.

Secreted frizzled-related protein disrupts PCP in eye lens fiber cells that have polarised primary cilia.

Planar cell polarity (PCP) signaling polarises cells along tissue axes. Although pathways involved are becoming better understood, outstanding issues include; (i) existence/identity of cues that orchestrate global polarisation in tissues, and (ii) the generality of the link between polarisation of primary cilia and asymmetric localisation of PCP proteins. Mammalian lenses are mainly comprised of epithelial-derived fiber cells. Concentrically arranged fibers are precisely aligned as they elongate along the anterior-posterior axis and orientate towards lens poles where they meet fibers from other segments to form characteristic sutures. We show that lens exhibits PCP, with each fiber cell having an apically situated cilium and in most cases this is polarised towards the anterior pole. Frizzled and other PCP proteins are also asymmetrically localised along the equatorial-anterior axis. Mutations in core PCP genes Van Gogh-like 2 and Celsr1 perturb oriented fiber alignment and suture formation. Suppression of the PCP pathway by overexpressing Sfrp2 shows that whilst local groups of fibers are often similarly oriented, they lack global orientation; consequently when local groups of fibers with different orientations meet they form multiple, small, ectopic suture-like configurations. This indicates that this extracellular inhibitor disrupts a global polarising signal that utilises a PCP-mediated mechanism to coordinate the global alignment and orientation of fibers to lens poles.

NHS-A isoform of the NHS gene is a novel interactor of ZO-1.

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.

MAPK/ERK1/2 and PI3-kinase signalling pathways are required for vitreous-induced lens fibre cell differentiation.

Lens epithelial cells withdraw from the cell cycle to differentiate into secondary fibre cells in response to vitreal factors. Fibroblast growth factor (FGF) in the vitreous has been shown to induce lens fibre differentiation in vivo and in vitro through the activation of defined intracellular signalling, namely via MAPK/ERK1/2 and PI3-K/Akt pathways. To better understand the role of these growth factor-activated signalling pathways in lens fibre differentiation, FGF- and vitreous-induced lens fibre differentiation was examined in primary rat lens epithelial cell explants. The induction of cell elongation and fibre specific beta- and gamma-crystallin expression in lens explants was accompanied by distinct phosphorylation profiles for ERK1/2 and Akt. Using selective inhibitors (U0126 and LY294002) in blocking studies, these pathways were shown to be required for different aspects of lens fibre differentiation. Furthermore, a short 'pulse' treatment of explants with FGF showed that the activation of ERK1/2 over 24 h was not sufficient for the progression of lens fibre differentiation and that cyclic ERK1/2 phosphorylation was required throughout the extended differentiation process. In conclusion, these results support a key role for both ERK1/2 and PI3-kinase/Akt signalling pathways in FGF- and vitreous-induced lens fibre differentiation.

Growth factors involved in aqueous humour-induced lens cell proliferation.

Lens epithelial cell proliferation is regulated by growth factors in the aqueous humour of the eye. Although the lens fibre cell-differentiating factors are well defined, the factors in aqueous that promote lens cell proliferation are not. Mitogens present in aqueous primarily signal through the MAPK/ERK and PI3-K/Akt pathways. By characterising the signalling pathways involved in lens cell proliferation, we aim to identify the factors in aqueous that regulate this process in vivo. Using rat lens epithelial explants, 5'-2'-bromo-deoxyuridine and H(3)-thymidine incorporation were used to compare the effects of aqueous, insulin-like growth factor (IGF-1), platelet-derived growth factor (PDGF-A), epidermal growth factor (EGF) and fibroblast growth factor (FGF-2) on lens cell proliferation. Western blotting was employed to characterise ERK1/2 and Akt signalling induced by these mitogens. The above assays were also repeated in the presence of selective receptor inhibitors. Similar to aqueous, FGF induced a sustained ERK1/2 signalling profile (up to 6 h), unlike IGF, PDGF and EGF that induced a transient activation of ERK1/2. In the presence of a FGF receptor (FGFR) inhibitor, the sustained aqueous-induced ERK1/2 signalling profile was perturbed, resembling the transient IGF-, PDGF- or EGF-induced profile. In the presence of other growth factor receptor inhibitors, aqueous maintained its sustained, 6 h, ERK1/2 signalling profile, although ERK1/2 phosphorylation at earlier time periods was reduced. No one-specific receptor inhibitor could block aqueous-induced lens cell proliferation; however, combinations of inhibitors could, providing FGFR signalling was blocked. Multiple growth factors are likely to regulate lens cell proliferation in vivo, with a key role for FGF in aqueous-induced signalling and lens cell proliferation.

Wnt signaling is required for organization of the lens fiber cell cytoskeleton and development of lens three-dimensional architecture.

How an organ develops its characteristic shape is a major issue. This is particularly critical for the eye lens as its function depends on having appropriately ordered three-dimensional cellular architecture. Recent in vitro studies indicate that Wnt signaling plays key roles in regulating morphological events in FGF-induced fiber cell differentiation in the mammalian lens. To further investigate this the Wnt signaling antagonist, secreted frizzled-related protein 2 (Sfrp2), was overexpressed in lens fiber cells of transgenic mice. In these mice fiber cell elongation was attenuated and individual fibers exhibited irregular shapes and consequently did not align or pack regularly; microtubules, microfilaments and intermediate filaments were clearly disordered in these fibers. Furthermore, a striking feature of transgenic lenses was that fibers did not develop the convex curvature typically seen in normal lenses. This appears to be related to a lack of protrusive processes that are required for directed migratory activity at their apical and basal tips as well as for the formation of interlocking processes along their lateral margins. Components of the Wnt/Planar Cell Polarity (PCP) pathway were downregulated or inhibited. Taken together this supports a role for Wnt/PCP signaling in orchestrating the complex organization and dynamics of the fiber cell cytoskeleton.

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.