Understanding the role of growth factors in embryonic development: insights from the lens

Growth factors play key roles in influencing cell fate and behaviour during development. The epithelial cells and fibre cells that arise from the lens vesicle during lens morphogenesis are bathed by aqueous and vitreous, respectively. Vitreous has been shown to generate a high level of fibroblast growth factor (FGF) signalling that is required for secondary lens fibre differentiation. However, studies also show that FGF signalling is not sufficient and roles have been identified for transforming growth factor-β and Wnt/Frizzled families in regulating aspects of fibre differentiation. In the case of the epithelium, key roles for Wnt/β-catenin and Notch signalling have been demonstrated in embryonic development, but it is not known if other factors are required for its formation and maintenance. This review provides an overview of current knowledge about growth factor regulation of differentiation and maintenance of lens cells. It also highlights areas that warrant future study.     

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.     

Lens fibre cell differentiation and organelle loss: many paths lead to clarity

The programmed removal of organelles from differentiating lens fibre cells contributes towards lens transparency through formation of an organelle-free zone (OFZ). Disruptions in OFZ formation are accompanied by the persistence of organelles in lens fibre cells and can contribute towards cataract. A great deal of work has gone into elucidating the nature of the mechanisms and signalling pathways involved. It is apparent that multiple, parallel and redundant pathways are involved in this process and that these pathways form interacting networks. Furthermore, it is possible that the pathways can functionally compensate for each other, for example in mouse knockout studies. This makes sense given the importance of lens clarity in an evolutionary context. Apoptosis signalling and proteolytic pathways have been implicated in both lens fibre cell differentiation and organelle loss, including the Bcl-2 and inhibitor of apoptosis families, tumour necrosis factors, p53 and its regulators (such as Mdm2) and proteolytic enzymes, including caspases, cathepsins, calpains and the ubiquitin-proteasome pathway. Ongoing approaches being used to dissect the molecular pathways involved, such as transgenics, lens-specific gene deletion and zebrafish mutants, are discussed here. Finally, some of the remaining unresolved issues and potential areas for future studies are highlighted.     

The lens epithelium: focus on the expression and function of the alpha-crystallin chaperones

Lens epithelial cells are the parental cells responsible for growth and development of the transparent ocular lens. Many elegant investigations into their biology have focused on the factors that initiate and regulate lens epithelial cell differentiation. Because they serve key transport and cell maintenance functions throughout life, and are the primary source of metabolic activity in the lens, mechanisms to maintain lens epithelial cell integrity and survival are critical for lens transparency. The molecular chaperones alpha-crystallins are abundant proteins synthesized in the differentiated lens fiber cell cytoplasm. However, their expression in lens epithelial cells has only been appreciated very recently. Besides their important roles in the refractive and light focusing properties of the lens, alpha-crystallins have been implicated in a number of non-refractive pathways including those involving stress response, apoptosis and cell survival. The most convincing evidence for their importance in the lens epithelium has been shown by studies on the properties of lens epithelial cells from alphaA and alphaB-crystallin gene knockout mice. Novel combination of genetics, cell and molecular biology should lead to a greater understanding of how lens epithelial cells proliferate, differentiate and survive.     

FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling.

Members of the fibroblast growth factor (FGF) family induce lens epithelial cells to undergo cell division and differentiate into fibres; a low dose of FGF can stimulate cell proliferation (but not fibre differentiation), whereas higher doses of FGF are required to induce fibre differentiation. To determine if these cellular events are regulated by the same signalling pathways, we examined the role of mitogen-activated protein kinase (MAPK) signalling in FGF-induced lens cell proliferation and differentiation. We show that FGF induced a dose-dependent activation of extracellular regulated kinase 1/2 (ERK1/2) as early as 15 minutes in culture, with a high (differentiating) dose of FGF stimulating a greater level of ERK phosphorylation than a lower (proliferating) dose. Subsequent blocking experiments using UO126 (a specific inhibitor of ERK activation) showed that activation of ERK is required for FGF-induced lens cell proliferation and fibre differentiation. Interestingly, inhibition of ERK signalling can block the morphological changes associated with FGF-induced lens fibre differentiation; however, it cannot block the synthesis of some of the molecular differentiation markers, namely, beta-crystallin. These findings are consistent with the in vivo distribution of the phosphorylated (active) forms of ERK1/2 in the lens. Taken together, our data indicate that different levels of ERK signalling may be important for the regulation of lens cell proliferation and early morphological events associated with fibre differentiation; however, multiple signalling pathways are likely to be required for the process of lens fibre differentiation and maturation.     

Models of epithelial histogenesis

Epithelial tissues emerge from coordinated sequences of cell renewal, specialization and assembly. Like corresponding immature tissues, adult epithelial tissues are provided by stem cells which are responsible for tissue homeostasis. Advances in epithelial histogenesis has permitted to clarify several aspects related to stem cell identification and dynamics and to understand how stem cells interact with their environment, the so-called stem cell niche. The development and maintenance of epithelial tissues involves epithelial-mesenchymal signalling pathways and cell-matrix interactions which control target nuclear factors and genes. The tooth germ is a prototype for such inductive tissue interactions and provides a powerful experimental system for the study of genetic pathways during development. Clonogenic epithelial cells isolated from developing as well mature epithelial tissues has been used to engineer epithelial tissue-equivalents, e.g. epidermal constructs, that are used in clinical practise and biomedical research. Information on molecular mechanisms which regulate epithelial histogenesis, including the role of specific growth/differentiation factors and cognate receptors, is essential to improve epithelial tissue engineering.     

Cadherins in the mammary gland and the melanocyte lineage

Cadherins are transmembrane glycoproteins involved in cell-cell adhesion, signalling, proliferation and differentiation. In this review, we have focused upon in vivo cadherin expression and function in two different biological systems, the mammary gland epithelium and the melanocyte lineage. Development of the mammary gland represents a paradigm of in situ epithelial differentiation and the melanocyte lineage of a model of non-epithelial (or mesenchymal) cell differentiation where cells migrate extensively from their site of origin towards the skin compartment. In the mammary epithelium, the predominantly expressed cadherin is E-cadherin, a cell surface molecule that directs morphogenesis and maintenance of the epithelial structure. In the melanocyte lineage, the expression of a number of cadherins is strictly spatiotemporally regulated during development and adult life. The specific functions mediated by this very dynamic cadherin expression are not yet known and their characterisation represents a challenge for the future.     

A cell polarity protein aPKCλ is required for eye lens formation and growth

The organisation of individual cells into a functional three-dimensional tissue is still a major question in developmental biology. Modulation of epithelial cell shape is a critical driving force in forming tissues. This is well illustrated in the eye lens where epithelial cells elongate extensively during their differentiation into fibre cells. It is at the lens equator that epithelial cells elongate along their apical-basal axis. During this process the elongating epithelial cells and their earliest fibre cell derivatives remain anchored at their apical tips, forming a discrete region or modiolus, which we term the lens fulcrum. How this is achieved has received scant attention and is little understood. Here, we show that conditional depletion of aPKCλ, a central effector of the PAR polarity complex, disrupts the apical junctions in elongating epithelial cells so that the lens fulcrum fails to form. This results in disorganised fibre cell alignment that then causes cataract. Interestingly, aPKCλ depletion also promotes epithelial-mesenchymal transition of the lens epithelial cells, reducing their proliferation, leading ultimately to a small lens and microphthalmia. These observations indicate that aPKCλ, a regulator of polarity and apical junctions, is required for development of a lens that is the correct size and shape.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

Spatial and temporal expression of Wnt and Dickkopf genes during murine lens development

Recent studies indicate a role for Wnt signalling in regulating lens cell differentiation (Stump et al., 2003). To further our understanding of this, we investigated the expression patterns of Wnts and Wnt signalling regulators, the Dickkopfs (Dkks), during murine lens development. In situ hybridisation showed that Wnt5a, Wnt5b, Wnt7a, Wnt7b, Wnt8a and Wnt8b genes are expressed throughout the early lens primordia. At embryonic day 14.5 (E14.5), Wnt5a, Wnt5b, Wnt7a, Wnt8a and Wnt8b are reduced in the primary fibres, whereas Wnt7b remains strongly expressed. This trend persists up to E15.5. At later embryonic stages, Wnt expression is predominantly localised to the epithelium and elongating cells at the lens equator. As fibre differentiation progresses, Wnt expression becomes undetectable in the cells of the lens cortex. The one exception is Wnt7b, which continues to be weakly expressed in cortical fibres. This pattern of expression continues through to early postnatal stages. However, by postnatal day 21 (P21), expression of all Wnts is distinctly weaker in the central lens epithelium compared with the equatorial region. This is most notable for Wnt5a, which is barely detectable in the central lens epithelium at P21. Dkk1, Dkk2 and Dkk3 have similar patterns of expression to each other and to the majority of the Wnts during lens development. This study shows that multiple Wnt and Dkk genes are expressed during lens development. Expression is predominantly in the epithelial compartment but is also associated, particularly in the case of Wnt7b, with early events in fibre differentiation.     

Duration of ERK1/2 phosphorylation induced by FGF or ocular media determines lens cell fate

The ocular environment is important for the establishment and maintenance of lens growth patterns and polarity. In the anterior chamber of the eye, the aqueous humour regulates lens epithelial cell proliferation whereas in the posterior, the vitreous humour regulates the differentiation of the lens cells into fiber cells. Members of the fibroblast growth factor (FGF) growth factor family have been shown to induce lens epithelial cells to undergo cell division and differentiate into fibers, with a low dose of FGF able to induce cell proliferation (but not fiber differentiation), and higher doses required to induce fiber differentiation. Both these cellular events have been shown to be regulated by the MAPK/ERK1/2 signalling pathway. In the present study, to better understand the contribution of ERK1/2 signalling in regulating lens cell proliferation and differentiation, we characterized the ERK1/2 signalling profiles induced by different doses of FGF, and compared these to those induced by the different ocular media. Here, we show that FGF induced a dose-dependent sustained activation of ERK1/2, with both a high (fiber differentiating) dose of FGF and vitreous, stimulating and maintaining a prolonged (up to 18 hr) ERK1/2 phosphorylation profile. In contrast, a lower (proliferating) dose of FGF, and aqueous, stimulated ERK1/2 phosphorylation for only up to 6 hr. If we selectively reduce the 18 hr ERK1/2 phosphorylation profile induced by vitreous to 6 hr, by specifically blocking FGF receptor signalling, the vitreous now fails to induce lens fiber differentiation but retains the ability to induce lens cell proliferation. These findings not only provide insights into the important role that FGF plays in the different ocular media that bathe the lens, but enlighten us on some of the putative molecular mechanisms by which one specific growth factor, in this case FGF, can elicit a different cellular response in the same cell type.     

The synthesis and localization of crystallins in different cell compartments of the crystalline lens in adult frogs: immunoautoradiographic and immunofluorescent research

The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (Mikha?lov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.     

Sef and Sprouty expression in the developing ocular lens: implications for regulating lens cell proliferation and differentiation

In many developmental systems, growth factor signalling must be temporally and spatially regulated, and this is commonly achieved by growth factor antagonists. Here, we describe the expression patterns of newly identified growth factor inhibitors, Sprouty and Sef, in the developing ocular lens. Sprouty and Sef are both expressed in the lens throughout embryogenesis, and become restricted to the lens epithelium, indicating that lens cell proliferation and fibre differentiation may be tightly regulated by such antagonists. Future studies will be aimed at understanding how these negative regulatory molecules modulate growth factor-induced signalling pathways and cellular processes in the lens.     

Sef and Sprouty expression in the developing ocular lens: Implications for regulating lens cell proliferation and differentiation

In many developmental systems, growth factor signalling must be temporally and spatially regulated, and this is commonly achieved by growth factor antagonists. Here, we describe the expression patterns of newly identified growth factor inhibitors, Sprouty and Sef, in the developing ocular lens. Sprouty and Sef are both expressed in the lens throughout embryogenesis, and become restricted to the lens epithelium, indicating that lens cell proliferation and fibre differentiation may be tightly regulated by such antagonists. Future studies will be aimed at understanding how these negative regulatory molecules modulate growth factor-induced signalling pathways and cellular processes in the lens.     

Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals

Cataract is a major ocular disease that causes blindness in many developing countries of the world. It is well established that various factors such as oxidative stress, UV, and other toxic agents can induce both in vivo and in vitro cataract formation. However, a common cellular basis for this induction has not been previously recognized. The present study of lens epithelial cell viability suggests such a general mechanism. When lens epithelial cells from a group of 20 cataract patients 12 to 94 years old were analyzed by terminal deoxynucleotidyl transferase (TdT) labeling and DNA fragmentation assays, it was found that all of these patients had apoptotic epithelial cells ranging from 4.4 to 41.8%. By contrast, in eight normal human lenses of comparable age, very few apoptotic epithelial cells were observed. We suggest that cataract patients may have deficient defense systems against factors such as oxidative stress and UV at the onset of the disease. Such stress can trigger lens epithelial cell apoptosis that then may initiate cataract development. To test this hypothesis, it is also demonstrated here that hydrogen peroxide at concentrations previously found in some cataract patients induces both lens epithelial cell apoptosis and cortical opacity. Moreover, the temporal and spatial distribution of induced apoptotic lens epithelial cells precedes development of lens opacification. These results suggest that lens epithelial cell apoptosis may be a common cellular basis for initiation of noncongenital cataract formation.     

Cytoskeletal and contractile structures in bovine lens cell differentiation

Bovine lenses from animals of different ages were separated into two epithelial sections, a cortical region and the lens nucleus. Both the 10000 g supernatant fraction and pellet of these sections were analysed by electrophoresis in SDS-containing polyacrylamide gels. When comparing total protein patterns of the cytoskeletal preparations from the different parts of lenses of different ages a decrease in the amount of vimentin, the protein subunit of lens intermediate-sized filaments (IF), was observed upon lens cell differentiation and aging. Amounts of monomeric (G) and filamentous (F) actin in the different stages of lens cell differentiation were quantitated using the DNase I inhibition technique. A significant increase in the relative amount of F-actin was observed upon fibre cell formation. A slight, but significant increase in the total amount of actin relative to the total amount of cellular protein was observed when passing from the central part of the lens epithelium to the epithelial cells in the elongation zone. In the fibre cells the amount of total actin decreased from cortex to nucleus. A possible function of microfilament-assembly in the process of lens cell differentiation is suggested.