Ординатура / Офтальмология / Английские материалы / Development of the Ocular Lens_Lovicu, Lee Robinson_2004

tips of developing lens fiber cells, extending laterally for a short distance between the fiber cells (Fig. 10.4C; Walker and Menko, unpublished manuscript). In this region of the lens, intense staining for α6 integrin is also detected (Fig. 10.4B). Interestingly, the intermediate filament protein vimentin was similarly localized in the embryonic lens (Walker and Menko, unpublished manuscript; Sandilands et al., 1995). The co-localization pattern for α6 integrin, β4 integrin, and laminin-5 and the close association of vimentin suggest a novel function for α6β4 in the lens, possibly the formation of a unique junction in lens fiber cells and the enablement of their coordinate migration along the anterior epithelium and the posterior capsule.

Studies of the signaling role of α6 integrin in the embryonic lens have shown that this integrin is present in differentiation-specific signaling complexes with the activated IGF-1 receptor (Walker et al., 2002c). In addition, signaling proteins typical of growth factor receptor signaling pathways are recruited to α6 integrin signaling complexes as lens cells differentiate, including Shc (Walker et al., 2002a), Grb2 (Walker et al., 2002a), and activated ERK (Walker et al., 2002c). These results suggest that α6 integrin signaling in the developing lens involves recruitment of growth factor receptors and their downstream effectors.

10.2.6. αV Integrin Receptors

αV integrin forms heterodimers with multiple β subunits (Hynes, 1992). At least one of these receptors, αvβ3, is expressed in the embryonic lens. Both the αV and β3 integrin subunits are associated with lens cells throughout their differentiation from epithelial to fiber cells (Walker et al., 2002c). αVβ3 and α6β1/β4 are the only integrins that persist into the most terminally differentiated regions of the embryonic lens. The αV integrin heterodimers are receptors for multiple ligands, including vitronectin, tenascin, fibronectin and thrombospondin (Hynes, 1992), and regulate such events as migration and apoptosis. As a receptor for tenascin in the lens capsule, αVβ3 integrin is likely to be involved in regulating the migration of lens fiber cells. This possibility is supported by the presence of large αV-containing focal contacts in lens cells grown in culture (Fig. 10.5A; Menko, unpublished manuscript).

10.2.7. Integrins and Lens Disease

When lens cells are plated within a collagen type I gel, they undergo an epithelialmesenchymal transformation that alters their integrin and matrix expression. Under these conditions, lens cells lose polarity, becoming bipolar and spindle shaped. These lens cells down-regulate the differentiation-specific α6 integrin and its ligand laminin while upregulating the mesenchymal cell–specific α5 integrin and its ligand fibronectin (Zuk and Hay, 1994). Cataract formation in transgenic mice that overexpress PAX6(5a) is accompanied by the elevated expression of α5 integrin in the lens fiber cells (Duncan et al., 2000). This increased expression of α5 integrin was correlated with the finding of potential PAX6(5A)-binding sites in the human α5 promoter. The effects on lens cells after plating in collagen gels are reminiscent of changes in lens epithelial cells during postcapsular opacification following cataract surgery. This secondary cataract forms following cataract surgery when the remaining anterior epithelial cells migrate inappropriately along the posterior capsule. The differentiation program of these cells is altered, as evidenced by changes in gene expression, including an altered repertoire of integrins and induction of both collagen type I and α-smooth actin. Blocking the function of the newly expressed or activated integrins

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Figure 10.5. Focal adhesion plaques in chick embryo lens cultures contain both αV integrin and FAK. Chick embryo lens cells were plated in culture and immunostained using antibodies to αV integrin or FAK (focal adhesion kinase). Both were found to be components of large focal adhesion plaques in these well-spread lens epithelial cells.

is likely to lead to a lowered incidence of postcapsular opacification. Since α3β1 integrin has been proposed as a transdominant inhibitor of α5β1 integrin function (Hodivala-Dilke et al., 1998), these results raise the possibility that α3β1 repression of α5β1 function may be critical to the maintenance of cell polarity in the lens epithelium.

10.2.8. Integrin-Associated Focal Adhesion Complexes

Upon integrin engagement by ligand, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, is activated and recruited to integrin complexes, along with cytoskeletal proteins characteristic of the focal adhesion complex (FAC). These FAC proteins include talin, α-actinin, paxillin, and vinculin. Embryonic lens cells express all of these FAC proteins (Menko et al., 1998). However, their expression and distribution is regulated with the state of lens cell differentiation. Talin, α-actinin, and FAK are more highly expressed in lens epithelial cells than in lens fiber cells, while vinculin expression is up-regulated with lens fiber cell differentiation (Menko et al., 1998). Vinculin is found in both cell-matrix adhesion complexes (FACs) and cell-cell adhesion complexes in the lens. The increased expression of vinculin observed in lens fiber cells appears to be related to its association with fiber

cell membranes just after fiber cells detach from the lens capsule (Beebe et al., 2001). The ability of lens cells to organize FACs is best visualized when lens cells are grown in culture and immunostained for a FAC protein such as FAK (Fig. 10.5B; Menko, unpublished manuscript). The FACs are typically linked to the cytoskeleton. Therefore, it is not surprising that many of the FAC proteins expressed by the embryonic lens change their linkage to the actin cytoskeleton as the lens cells differentiate. One such example is paxillin. Although its expression changes little with embryonic lens cell differentiation, it becomes highly linked to the cytoskeleton in the lens fiber cells (Menko et al., 1998). Paxillin’s increased linkage to the cytoskeleton is correlated with increased localization to lateral fiber cell membranes at a time just before fiber cell nuclei and organelles are degraded (Beebe et al., 2001). In contrast to its expression in the embryonic lens, paxillin is down-regulated in the fiber cell region of the adult mouse lens (Duncan et al., 2000). However, constitutive expression of PAX6(5a) as a transgene in the mouse lens results in increased expression of paxillin, along with α5 integrin, in the dysgenic fiber cells (Duncan et al., 2000). Many of the molecular components of FACs, including actin, paxillin, and FAK, have also been identified as components of the unique adhesion structures that form between differentiating lens fiber cells and the posterior capsule, known as basal membrane complexes (BMCs; Bassnett et al., 1999). Formation of the BMCs, which remain associated with the posterior lens capsule following mechanical stripping of the capsule from the lens, is dependent on β1 integrin function.

10.3. Cadherins

Cadherins make up a family of integral membrane proteins that mediate homophilic calciumdependent cell-cell adhesion. They are involved in the cell-sorting events that regulate early mophogenetic developmental processes and in the regulation of cell differentiation (Takeichi, 1991, 1995). Their ability to form functional junctions (adherens junctions) is dependent on their interaction with the actin cytoskeleton, which is mediated by β-catenin or γ -catenin (plakoglobin), both of which associate with the cadherin cytoplasmic domain (Aberle et al., 1996; Gumbiner and McCrea, 1993). These molecules, in turn, associate with α-catenin, which binds to the actin cytoskeleton either directly or through its association with α-actinin. Enhanced tyrosine phosphorylation of these catenins has been shown to result in the blocking of cadherin linkage to the cytoskeleton, thereby inhibiting cadherin function (Takeda et al., 1995; Volberg et al., 1991).

In the mouse embryo lens, E-cadherin is expressed exclusively by lens epithelial cells, disappearing as these cells begin their differentiation (Wigle et al., 1999; Xu et al., 2002). In contrast, expression of N-cadherin, which also is found in lens epithelial cells, is maintained until the fiber cells mature (Xu et al., 2002). While E-cadherin is not expressed in the chick, two other cadherin family members have been identified in the chick embryo lens, with distinctive patterns of expression, N-cadherin (Atreya et al., 1989; Lagunowich and Grunwald, 1989; Volk and Geiger, 1986a, 1986b) and B-cadherin (the chicken homolog of P-cadherin; Leong et al., 2000). In differentiating chick lens cell cultures, the temporal localization of N-cadherin to cell-cell interfaces precedes that of B-cadherin (Leong et al., 2000). α-Catenin, β-catenin, and γ -catenin, all members of the cadherin complexes, are localized to lens cell-cell interfaces (Ferreira-Cornwell et al., 2000). Although N-cadherin is expressed in most regions of the chick embryonic lens, it is reported to be lost from the nuclear fiber region of the lens with age (Atreya et al., 1989; Beebe et al., 2001). This loss may be related to the turnover of N-cadherin through the regulated action of a

258 A. Sue Menko and Janice L. Walker

Figure 10.6. Reorganization of N-cadherin and the actin cytoskeleton accompanies lens cell differentiation in vitro. Cell cultures were established from day 10 chick embryo lenses. The panels represent undifferentiated lens epithelial cells (A, B) and differentiating lens fiber cells (C, D) stained either with an antibody to N-cadherin (A, C) or with phalloidin to detect polymerized actin (B, D). In lens epithelial cells, N-cadherin is organized in adhesion plaque-like structures limited to areas of cell–cell contact, and actin was found in stress fibers that ended at the N-cadherin adhesion plaques. As lens cells differentiated, N-cadherin became concentrated at cell–cell borders. This was paralleled by the reorganization of actin filaments to a cortical distribution.

calcium-activated protease (Maisel and Atreya, 1990) or an increase in its phosphorylation state (Lee et al., 1997; Volberg et al., 1991). In the embryo, the state of tyrosine phosphorylation of N-cadherin is regulated in a stage-specific manner (Lagunowich and Grunwald, 1991). In lens cultures, the formation of cell-cell junctions is prevented by the constitutive expression of the protein tyrosine kinase v-Src (Menko and Boettiger, 1988).

Primary lens epithelial cells in culture first form a well-spread monolayer in which N-cadherin is found in short cell-cell adhesion structures perpendicular to the membrane interface, evocative of a zipper (Fig. 10.6A; Ferreira-Cornwell et al., 2000). The actin cytoskeleton in these cells is organized as stress fibers (Fig. 10.6B). The initiation of lens fiber cell differentiation in culture is characterized by a compaction of the epithelial cell monolayer. This is accompanied by localization of N-cadherin all along cell-cell interfaces (Fig. 10.5C) and reorganization of actin stress fibers to a cortical distribution (Fig. 10.5D; Ferreira-Cornwell et al., 2000). Src is a negative regulator of cadherin junction assembly in the lens. The suppression of Src kinase activity in lens cultures rapidly induces the formation of stable N-cadherin junctions (Walker et al., 2002b). Induction of the cell cycle inhibitors

p27 and p57, withdrawal of lens epithelial cells from the cell cycle, and induction of lens cell differentiation follow cadherin junction assembly (Walker et al., 2002b).

In lens fiber cells, N-cadherin can be found localized specifically to lateral regions of the basal membrane complex, which anchors fiber cells to the capsule and facilitates cell migration (Bassnett et al., 1999; see also chap. 9). It is in these N-cadherin–rich regions that F-actin bundles are seen to insert into regions of the lateral fiber cell membrane. This N-cadherin–actin complex forms a hexagonal lattice along the posterior face of the lens (Bassnett et al., 1999). When chick lens cells are grown in culture in the presence of the N-cadherin function–blocking antibody NCD-2, the formation of normal N-cadherin cellcell junctions is blocked, the cells fail to form a tightly packed monolayer, and differentiation, both morphological and biochemical, is inhibited (Ferreira-Cornwell et al., 2000). Normally, both N-cadherin and B-cadherin linkage to the actin cytoskeleton increases with the chick lens cell differentiation state (Ferreira-Cornwell et al., 2000; Leong et al., 2000). In NCD-2–treated lens cultures, N-cadherin fails to become linked to the actin cytoskeleton and remains dispersed, and filamentous actin is distributed as stress fibers, both features of undifferentiated lens epithelial cells. These effects were paralleled by the suppression of expression of components of the cadherin-catenin complex, β-catenin and α-catenin, suggesting the importance of these proteins in regulating the function of N-cadherin junctions in the differentiating lens.

The formation of N-cadherin junctions is necessary for the formation of functional gap junctions, mediators of intercellular communication, between lens fiber cells (Frenzel and Johnson, 1996). While N-cadherin does not co-localize with the lens gap junctional protein connexin 56 in lens fiber cells formed in culture (Berthoud et al., 1999), temporal localization of B-cadherin closely parallels that of connexin 56 (Leong et al., 2000).

10.4. Other Lens Cell Adhesion Molecules

N-CAM is a calcium-independent homotypic cell-cell adhesion molecule that functions in cell adhesion, cell signaling, and neurite outgrowth (Beggs et al., 1994; Beggs et al., 1997; Kolkova et al., 2000; Maness et al., 1996; Walsh and Doherty, 1997). It is expressed by lens epithelial cells, and its expression decreases with lens fiber cell differentiation (Watanabe et al., 1989). N-CAM expression in the lens is also isoform specific. Whereas N-CAM 140 is the predominant isoform expressed in lens epithelial cells, N-CAM 120 is found principally in lens fiber cells (Katar et al., 1993). Glycosylation of N-CAM in the lens is regulated with developmental age. In embryonic lens epithelial cells, N-CAM has less sialic acid than in epithelial cells of the adult lens. Increased quantity of sialic acid in N-CAM is correlated with decreased adhesion (Watanabe et al., 1992). When N-CAM function is blocked in lens epithelial cell explants, the cells appear thinner and contain less mature gap junctions (Watanabe et al., 1989). These results suggest a role for N-CAM in lens differentiation, possibly through the regulation of fiber cell gap junctions or adherens junctions.

Galectin-3, a member of the galactin family of lectins, is a secreted β-galactoside– binding protein with adhesive properties whose expression is developmentally regulated (Perillo et al., 1998). It localizes to the plasma membrane of lens fiber cells, where it is associated as a peripheral protein. In the lens fiber cells, the junction-forming intrinsic fiber cell membrane protein MP20 has been identified as a candidate ligand for galectin-3 (Gonen et al., 2000). GRIFIN, a protein related to the galectins but missing β-galactoside–binding activity, is expressed exclusively in the lens, where it is localized to the cell-cell interfaces of lens fiber cells (Ogden et al., 1998).

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10.5. Summary

For more than a century now, scientists have been trying to unravel how cells differentiate into distinct cell types. The process of differentiation is controlled by an elaborate plan and is influenced by intrinsic as well as extrinsic factors. Clearly, there is epigenetic control of lens cell differentiation, since environmental factors can infuence the outcome of both lens cell differentiation and development.

In this chapter we have reviewed matrix molecules and adhesion receptors that are implicated in the process of regulating the differentiation of a lens epithelial cell into a lens fiber cell. They are all expressed in distinct spatiotemporal patterns. Some of these molecules probably regulate lens cell proliferation while others appear to be involved in signaling the initiation of lens cell differentiation or the distinct morphogenetic changes that accompany the formation of the lens fiber cells. It is evident that we can no longer consider just one set of molecules to be the critical regulators of lens cell differentiation and development; rather we must determine how the complex interplay of molecules determines the differentiation state of cells in the lens.

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Figure 11.1. (A–F) Sections through the developing eyes of E10–14 rat embryos stained with haematoxylin and phloxine. (A) At E10, bilateral outgrowths from the developing brain form the optic vesicles (ov). (B) In early E11 embryos, the optic vesicle is closely associated with a region of head ectoderm that is destined to form a lens. (C) In the late E11 embryo, both the ectoderm and the neuroectoderm are thickened along the region of close proximity, forming the lens placode (lp) and retinal disc (rd), respectively. (D) Invagination of the lens placode and the optic vesicle at E12 leads to the formation of the lens pit/vesicle (lv) and the optic cup (oc), respectively. (E) By E13, the lens vesicle has completely closed and detached from the optic cup (oc). The posterior lens vesicle cells elongate to form primary lens fiber cells, leading to narrowing of the vesicle lumen. (F) By E14, the lens vesicle lumen has disappeared, and the primary lens fibers (lf) are in contact with the anterior lens vesicle cells, which form the epithelium (e). Vitreous humor and hyaloid vasculature forms between the developing lens and retina. The inner layer of the optic cup will form the neural retina (nr). Scale bars: (A), 50 µm; (B, C), 75 µm; (D–F), 100 µm. (Adapted from de Iongh and McAvoy, 1993.)

species, removal of the optic vesicle primordium resulted in no lens formation. Later experiments, in which other species developed lenses in the absence of the optic vesicle (Mencl, 1903), indicated a more complex situation. Embryological manipulations performed by a number of groups, including the Grainger Laboratory (Grainger et al., 1992), have indicated that lens induction is a multistage process that probably involves multiple types of inductive signaling. For example, it is possible to define stages of lens competence and “lens-forming bias” that are clearly distinct. The idea that lens induction is a multistage process is beginning to receive support from molecular genetic analysis. For example, there are two phases of Pax6 expression in the lens lineage (see below). Gene expression in these phases is distinctly regulated, but both are required for lens development. The embryological manipulations that have led to the multistage model of lens induction are dealt with in detail in chapter 2 of this volume.

In the mouse, the optic vesicle is essential for lens induction. This is illustrated by explant experiments in which recombination of optic vesicle and presumptive lens ectoderm

indicates that the optic vesicle is only dispensable for lens development after approximately 9.5 days of embryonic development (E9.5; Furuta and Hogan, 1998). Induced mutations in genes that are expressed exclusively in the neural component of the eye also illustrate this point. For example, Lhx2 null mice form optic vesicles but do not form lenses (Porter et al., 1997), while Rx null mice do not form optic vesicles and also do not form lenses (Mathers et al., 1997). These and other observations have motivated those interested in lens induction in the mouse to focus their efforts on understanding the mediators of the optic vesicle–presumptive lens interaction.

11.1.1. Transcription Factors Involved in Lens Induction

A number of transcription factors have activities that are critical for normal eye development. In many cases, transcription factor gene expression has been used as a molecular indicator of the activity of a growth factor signaling pathway that lies genetically upstream. From this point of view, to understand how growth factor signaling pathways are integrated during lens development, we must first describe how the involved transcription factors participate.

11.1.1.1. Pax6

Pax6 is a transcription factor that contains both a paired domain and a homeodomain (Walther and Gruss, 1991). The phenotypes of both mice and humans with null mutations in the Pax6 gene indicate that this transcription factor has a critical role in eye development. Heterozygosity in either species leads to microphthalmia, iris and optic nerve hypoplasia (Hill et al., 1991; Hogan et al., 1986; Jordan et al., 1992), and in some circumstances the persistent lens stalk that manifests as Peters’ anomaly (Hanson et al., 1994). Pax6 homozygosity leads to major craniofacial defects, including the absence of eyes (Hill et al., 1991; Hogan et al., 1986). In the developmental context, Pax6Sey/Sey mice display an arrest of eye development after the optic vesicles have formed. This manifests as an absence of formation of the lens placode and a failure of morphological patterning in the optic vesicle (Grindley et al., 1995; Hogan et al., 1986).

A number of experiments have indicated that Pax6 is necessary and sufficient for development of the lens. In explant culture, recombination of wild-type presumptive lens epithelium and optic vesicle leads to development of the lens (Fujiwara et al., 1994). If the optic vesicle in such a recombination experiment is derived from Pax6Sey/Sey mice, lens development also occurs. By contrast, if presumptive lens epithelium is taken from Pax6Sey/Sey mice, lens development is prevented. This indicates an autonomous requirement for Pax6 in the presumptive lens region. Generation of wild-type/Pax6Sey/Sey chimeric mice leads to a similar conclusion, as Pax6Sey/Sey cells do not contribute to developing lens structures either at the placodal stage (Collinson et al., 2000) or as the lens matures (Quinn et al., 1996). Finally, conditional deletion of Pax6 using a Cre recombinase-based strategy also indicates that Pax6 is required for lens development (Ashery-Padan et al., 2000).

In Drosophila, the Pax6 homolog eyeless has the remarkable ability to induce ectopic eyes when misexpressed (Halder et al., 1995). An ectopic eye formation response has also been observed in vertebrates when Pax6 is misexpressed. Specifically, in Xenopus laevis, injection of Pax6-encoding RNA early in development resulted in the formation of eyes with all the major features of the mature structure, including retinal neurons and lens (Chow et al., 1999). A modification in the experimental protocol led to a dramatic increase in the frequency with which isolated ectopic lenses would form (Altmann et al., 1997). Many

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Figure 11.2. A model for the genetic pathways regulating lens induction. The arrows indicate genetic interactions, except in the case of the Meis transcription factor–enhancer physical complex. At the apex of the hierarchy is the preplacodal phase of Pax6 expression (Pax6pre-placode). It is understood that the later phase of Pax6 expression in the lens placode (Pax6placode) is dependent upon earlier activity of Pax6. Pax6placode is apparently regulated by at least two enhancers, represented by thick gray bars underneath the bifurcated arrow points on the schematic of the Pax6 gene (exons, thin black vertical bars). One is the socalled ectoderm enhancer (abbreviated EE), and the other is the SIMO element, located in the final intron of the adjacent gene, AK000505, which is transcribed in the opposite direction to Pax6. The Meis transcription factors are likely to regulate the expression of Pax6 directly by binding to the ectoderm enhancer and possibly to the SIMO element. The similar phenotypes (reduced lens lineage proliferation and lens vesicle closure and separation failure) of the dysgenetic lens (FoxE3) and Pax6 EE/ EE (homozygous targeted deletion of the ectoderm enhancer) mutant mice suggest that FoxE3 might participate in this pathway. Lack of expression of the FoxE3 gene in Small eye (Pax6 mutant) homozygous mice and ectoderm enhancer deletion mice indicates that FoxE3 occupies a downstream position.

of these lenses had all the features of a normal lens, including an epithelial cell layer, differentiated fiber cells, and the normal polarized morphology. This indicates that, in the context of the Xenopus embryo, Pax6 is sufficient for lens formation.

A simple analysis of Pax6 gene expression demonstrates that there are multiple phases during development of the lens; this analysis has also established the basis for a genetic pathway for lens induction (Treisman and Lang, 2002). The Pax6Sey-1Neu allele is a point mutation that prevents the formation of a protein product but allows gene transcription (Grindley et al., 1997). Assessment of Pax6 expression throughout eye development indicates that the early head ectoderm phase of Pax6 expression is unaffected by the absence of functional transcription factor but that the later, placodal phase of expression does not occur (Grindley et al., 1995). This indicates that there are two phases of Pax6 expression and that the later phase (corresponding to the lens placode) is dependent upon functional Pax6 in the head ectoderm (Fig. 11.2).

At least two transcriptional enhancers have been identified that mediate Pax6 expression in the presumptive lens ectoderm. The first of these has been designated the “ectoderm