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

Figure 11.9. Diagram illustrating FGF induction of fiber differentiation, including cell elongation and fiber-specific changes in expression of molecular markers.

including the retina, cornea, ciliary body, and iris (Chamberlain and McAvoy, 1997). In situ hybridization studies showed that FGF-1 and FGF-2 mRNAs have a ubiquitous distribution in eye tissues similar to that of their proteins (Lovicu et al., 1997). The distribution of other FGFs has been studied, and so far FGF-12 is the only other family member reportedly expressed in the lens (Hartung et al., 1997). Other FGFs, including 3, 5, 8, 9, 11–13, 15, and 19, are also expressed in the retina at various stages of its development (Govindarajan and Overbeek, 2001; Vogel-Hopker et al., 2000; Xie et al., 1999; Zhao et al., 2001). Thus, a number of FGFs are potentially available to lens cells and could have roles in regulating fiber differentiation during development. However, the wide distribution of FGFs in both anterior and posterior segments of the eye raises the question of how fiber differentiation is spatially restricted to the posterior segment. Normally the only cells that undergo fiber differentiation are those below the lens equator, and this is crucial for maintaining lens polarity during the lifelong process of lens growth.

FGF Induces Different Responses at Different Concentrations. A possible mechanism whereby FGF may influence lens polarity and growth patterns emerged from dose-response studies. Analysis of explants cultured with FGF identified three distinct cellular responses: proliferation, migration, and fiber differentiation. Interestingly, these responses were induced sequentially as the FGF concentration was increased (Fig. 11.10; McAvoy and Chamberlain, 1989). Therefore, the concentration of FGF has the potential to influence the nature of the response of lens epithelial cells both qualitatively and quantitatively.

Investigations into the mechanisms that underlie these dose-dependent responses have focused on identifying the relevant signaling pathways. So far, mitogen-activated protein kinase (MAPK) signaling has received the most attention. It has been shown that FGF induces a dose-dependent activation of ERK-1/2 in lens epithelial cells (Lovicu and McAvoy, 2001). Blocking experiments have shown that activation of ERK is required for FGF-induced lens cell proliferation and fiber differentiation. However, inhibition of ERK signaling can block only the morphological changes associated with FGF-induced lens fiber differentiation and not the synthesis of some of the molecular differentiation markers, such as ß-crystallin.

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Figure 11.10. Diagram illustrating the three different responses to FGF observed in lens epithelial explants in vitro and their sequential stimulation by increasing concentrations. (Adapted from Chamberlain and McAvoy, 1997.)

Other studies have also reported an association between ERK signaling and FGF-mediated mammalian (Chow et al., 1995; Govindarajan and Overbeek, 2001) and avian lens fiber differentiation (Le and Musil, 2001). In addition, the involvement of ERK signaling in fiber differentiation is consistent with the immunohistochemical localization of the phosphorylated (active) forms of ERK-1/2 in the transitional zone, where cells elongate and undergo the earliest morphological changes associated with fiber differentiation (Lovicu and McAvoy, 2001). Taken together, these data indicate that FGF-induced ERK signaling is important for proliferation and the regulation of early morphological events associated with fiber differentiation. However, other FGF-induced signaling pathways required for the process of lens fiber differentiation and maturation remain to be identified.

The FGF Gradient Hypothesis. Given the dose-dependent response to FGF and the highly ordered anteroposterior sequence of proliferation, migration (or displacement), and fiber differentiation in the lens, it has been proposed that these polarized patterns of lens cell behavior are determined by an FGF gradient (Fig. 11.11; Chamberlain and McAvoy, 1997). The ability of growth factors to induce different cellular responses at different concentrations has also been shown in other systems and may involve a general mechanism that underlies the establishment of growth and differentiation patterns within tissues (e.g., see Lillien and Wancio, 1998; Pizette et al., 1996).

In the lens, a number of different approaches have been used to test the FGF gradient hypothesis, such as the analysis of FGF distribution, the analysis of FGF receptor expression, and the generation of transgenic mice with altered patterns of FGF distribution and receptor expression.

Differential Distribution of FGF in the Ocular Media. In vivo, the anteroposterior patterns of lens cell behavior correlate with the distribution of the ocular media. The anterior region of the lens is bathed by aqueous, while the region of the lens below the equator (where fibers differentiate) is bathed by vitreous. To test the hypothesis that the ocular

Figure 11.11. Diagram indicating how anteroposterior patterns of lens cell behavior may be determined by a gradient of FGF stimulation. In the mature lens, the epithelium (epi) can be divided into two main zones, the central epithelium (CE) and the germinative zone (GZ), which extends to the equator (EQ). Immediately posterior to the equator is the transitional zone (TZ). These zones coincide with compartments defined by the anatomy of the eye: Central epithelial cells are exposed to aqueous of the anterior chamber; germinative zone cells are exposed to aqueous of the posterior chamber, which is demarcated by the ciliary body (cb) and iris (ir); the transitional zone extends into the compartment that contains the vitreous. The cellular behaviors indicated are observed both in vivo and in lens epithelial explants cultured with increasing concentrations of FGF. (Adapted from Chamberlain and McAvoy, 1997.)

media influence the differentiated fate of lens cells, epithelial explants were cultured in either aqueous or vitreous. In explants exposed to vitreous, the cells became elongated and multilayered and acquired morphological and molecular markers of fiber cells. In contrast, the cells in explants cultured with aqueous maintained an epithelial phenotype (Fig. 11.12; Lovicu et al., 1995).

Thus, vitreous induced a fiber differentiation response but aqueous did not. Analysis by SDS-PAGE and Western blotting showed that FGF-1 and FGF-2 are present in both media but that more of these FGF isoforms are recovered from vitreous than from aqueous. By fractionating vitreous and testing fractions by FGF ELISA (using antibodies specific for FGF-1 and FGF-2) and biological assay, it was shown that a large proportion of the fiber-differentiating activity of vitreous is FGF associated (Schulz et al., 1993). However, several fractions, containing about 16% of the total activity, appeared to have virtually no FGF. This observation indicates that factors other than FGF-1 and FGF-2 are present in the ocular media and have fiber-differentiating activity. Such factors may be unrelated to FGFs but may also have the ability to activate signaling pathways involved in the differentiation process. Alternatively, these molecules may be other members of the FGF family. Of the ones that have been studied, FGFs 3, 5, 8, 9, 12, and 15 have been detected in the eye. Given that FGFs 3, 8, and 9 (among other FGFs; see Lovicu and Overbeek, 1998) have been shown to induce premature fiber differentiation in the lens epithelium in transgenic

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Figure 11.12. Transmission electron micrographs of neonatal rat lens epithelial explants cultured with ocular media for five days. (A) In the presence of aqueous, cells remained in a monolayer on the lens capsule (Ca) and retained a cuboidal epithelial morphology. (B) In the presence of vitreous, explants thickened, and many cells showed early fiber elongation characteristic of cells in the transitional zone (see Fig. 11.5B). The inset in B also shows that a capsulelike matrix forms on the exposed surface of the elongating cells. Fresh bovine aqueous and vitreous were diluted with an equal volume of culture medium before use. Scale bar: 5 µm. (Adapted from Lovicu et al., 1995.)

mice (see below), it is possible that a number of FGFs may work together to maintain a fiber-differentiating environment in the vitreous.

A higher concentration of FGF in the vitreous than in the aqueous may help to ensure that fiber differentiation is restricted to the posterior compartment; however, the ability of the vitreous to promote fiber differentiation may not be due solely to a relatively high concentration of FGF. Other factors present in the vitreous may positively modulate

the effects of FGF. Possible modulators include extracellular molecules such as heparan sulphate (HS) and heparan sulphate proteoglycan (HSPG), which are known to stabilize FGF, and cell surface HSPGs, which appear to be involved in presenting the ligand to the high-affinity tyrosine kinase receptors (Ornitz and Itoh, 2001). There is very little information available about such molecules in the aqueous and vitreous and how they could influence FGF activity and bioavailability. However, some anteroposterior differences in HSPGs have been reported in the lens capsule. For example, there appears to be a higher HSPG:core protein ratio in the posterior capsule than in the anterior capsule (Schulz et al., 1997). It is not known if this influences FGF activity in relation to lens cells, but it is interesting that greater FGF biological activity and reactivity resides in the posterior than in the anterior capsule (de Iongh and McAvoy, 1992; Lovicu and McAvoy, 1993; Schulz et al., 1993). Clearly, much more needs to be done to investigate the complex interactions between FGF and extracellular matrix molecules such as HS and HSPG in the ocular media and capsule in order to understand how they influence patterns of lens cell behavior.

Differential Distribution of FGF Receptors. Cellular responses to FGF are mediated via a family of four receptor genes (FGFR1–4; Bansal, 2002; Ornitz and Itoh, 2001). Lens cells have been shown to express three of the FGFR genes: FGFR1, FGFR2, and FGFR3. Each receptor has its own unique pattern of expression, but overall there is a substantial increase in receptor expression below the lens equator (McAvoy et al., 1999). Such an anteroposterior gradient in receptor expression could contribute to an anteroposterior gradient of FGF signaling in the lens.

Transgenic and Knockout Studies. Studies with transgenic mice provide strong support for the “FGF gradient hypothesis.” Through use of the α-crystallin promoter, transgenic mice were generated that express high levels of various FGFs specifically in the lens. In these transgenic mice, overexpression of FGF-1, FGF-3, FGF-4, FGF-5, FGF-7, FGF-8, or FGF-9 induced the anterior epithelial cells to undergo premature fiber differentiation (Fig. 11.13; Lovicu and Overbeek, 1998; Robinson et al., 1995a; Robinson et al., 1998). They lost their typical cuboidal epithelial morphology, became elongated, and accumulated ß-crystallin, which is normally restricted to the fiber mass. In most cases the lens lost its characteristic cellular polarity. In studies using this transgenic model, FGF-2 is the only member of the FGF family reported so far that did not induce differentiation in the epithelium (Stolen et al., 1997). Furthermore, in studies using a similar transgenic approach, the process of lens fiber differentiation was impaired by the overexpression of either a signaling-defective, inhibitory FGFR (Chow et al., 1995; Robinson et al., 1995b; Stolen and Griep, 2000) or a specific secreted FGF receptor (Govindarajan and Overbeek, 2001).

Results from these transgenic studies lend strong support to the hypothesis that in the normal situation an anteroposterior FGF gradient is involved in regulating fiber differentiation and determining lens polarity. For example, the simplest interpretation of the FGF overexpression studies is that the normal ocular FGF gradient is destroyed. Hence, the anterior epithelial cells are exposed to higher than normal levels of FGF and consequently undergo an inappropriate fiber differentiation response. The transgenic studies also show that most of the FGFs tested exhibit a capacity to induce fiber differentiation. This is consistent with the observation that lens cells express three of the four FGF receptor genes, thereby gaining

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Figure 11.13. (See color plate XVII.)Induction of fiber differentiation by FGF-4 in transgenic mice. Sections of embryonic day 15.5 nontransgenic (A, C) and transgenic (B, D) eyes, either stained with haematoxylin and eosin (A, B) or immunolabeled for ß-crystallin expression (C, D). In transgenic mice, anterior lens epithelial cells undergo extensive elongation (B, double arrow), differentiating into fibers, as shown by the expression of ß-crystallin (D, double arrow). In nontransgenic mice, the lens maintains its distinct polarity with a monolayer of cuboidal epithelial cells (A, le, asterisk), overlying a full complement of fiber cells (lf), reactive for ß-crystallin. Characteristically, ß-crystallin is not expressed in the anterior lens epithelium (C, le, asterisk). The small arrowheads in C and D define the anterior surface of the lens. Scale bar: 60 µm. (Adapted from Lovicu and Overbeek, 1998.)

the capacity to bind a wide range of FGF family members. However, it is not clear yet which of the 23 currently known FGFs are involved in this process in the normal lens.

In this context it is also important to note that, although FGF-1 and FGF-2 are present in the ocular environment and appear to provide a major component of the fiber-differentiating activity of vitreous humor, studies with adult mice made homozygous null for both FGF-1 and FGF-2 show no defect in lens or eye development (Miller et al., 2000). This result clearly shows that neither FGF-1 nor FGF-2, alone or in combination, is necessary for lens fiber differentiation. The same applies to FGF-9, as mice made homozygous null for the FGF-9 gene show no lens phenotype (unpublished data), although a retinal phenotype was reported (Zhao et al., 2001). As more FGFs are tested for their activity in lens fiber differentiation, we may identify an FGF or FGFs that are critical for lens fiber differentiation. However, perhaps it is more likely that redundancy exists in the system and that several FGFs are responsible for fiber differentiation. In this scenario, the absence of one or a couple of FGFs would have little or no effect, as the remaining FGFs would compensate and maintain normal function. The definitive test for the critical role of FGFs in fiber differentiation awaits the results from experiments in which all FGF signaling has been blocked, such as in mice with lenses that are null for all FGF receptors.

11.2.1.3. TGFß Superfamily

Bmps. In a previous section, we summarized how Bmp signaling participates in lens induction. Recent evidence indicates that Bmp signaling pathways continue to play a role as the lens matures. A number of observations have suggested that Bmp signaling is important for fiber cell maturation. Inhibition of Bmp activity with recombinant noggin (Zimmerman et al., 1996) in explants of the mouse eye (Faber et al., 2002) or in noggin retrovirus-infected chick eyes (Belecky-Adams et al., 1997) has resulted in reduced elongation of lens fiber cells. A chick lens epithelial explant assay was also used to show that fiber cell elongation activity present in vitreous could be inhibited by noggin (Belecky-Adams et al., 1997) and furthermore that vitreous depleted of noggin-binding factors could have its fiber cell elongation activity restored by the addition of Bmps (Belecky-Adams et al., 1997). Transgenic mice expressing an inhibitory, dominant-negative form of the Bmp receptor Alk6 also showed reduced fiber cell elongation, although this was restricted to the ventronasal quadrant of the lens vesicle (Faber et al., 2002). Finally, both mouse (Faber et al., 2002) and chick (Belecky-Adams et al., 1997) lens vesicles showed the anticipated nuclear pattern of immunoreactivity with antibodies (Korchynskyi et al., 1999) to the active, phosphorylated form of the Bmp signaling proteins Smad1, 5, and 8 (Massague, 1998). Combined, these data indicate that the Bmp signaling machinery is active in the lens at stages when fiber cell elongation is initiated and suggest that Bmp pathways may be evolutionarily conserved and important for the regulation of lens polarity.

TGFß. Members of the TGFß growth family and their receptors are expressed in the lens, and in vitro studies show that TGFß induces pathological changes in the lens epithelium (de Iongh et al., 2001a). However, recent studies with transgenic mice also indicate other important functions for TGFß in fiber differentiation (de Iongh et al., 2001b). Expression of a mutant TGFß type II receptor transgene under the control of the αA- crystallin promoter results in severe degeneration of fiber cells in the inner cortex. In these “dominant-negative” TGFß receptor mice, lens fiber differentiation is disrupted in the inner cortex at about the stage of early nuclear condensation in the lens bow. These fibers first become swollen, then disintegrate. Thus, inhibition of TGFß receptor signaling disturbs fiber cell maturation and/or maintenance, suggesting an important role for TGFß signaling in coordinating events in fiber differentiation. At this time, however, it is not clear which specific event or events this signaling pathway regulates.

11.2.1.4. Other Growth Factors in Fiber Differentiation

PDGF. Members of this family of growth factors are expressed near the lens (Reneker and Overbeek, 1996). Transgenic mice that overexpressed PDGF-A specifically in the lens displayed visible ocular abnormalities early in development (E15), including lenticular defects such as lens enlargement and the appearance of cataracts (Reneker and Overbeek, 1996). The lens epithelium was observed to be multilayered, consisting of a surface epithelial monolayer and an underlying subepithelium. A considerable increase in DNA synthesis was detected in the epithelium of these transgenic lenses compared with the wild type (see later). The transgenic lenses also exhibited areas of cells in both the subepithelial and surface epithelial layers that expressed fiber-specific ß-crystallin. Hence, the overexpression of PDGF-A caused some cells to undergo at least some aspects of fiber cell differentiation. However, it was not clear in this transgenic study if the phenotype was due to the influence of PDGF on fiber differentiation or if it was a result of secondary effects

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induced by disturbing patterns of PDGF signaling. Results from explant studies support the latter possibility. In epithelial explants, PDGF is unable to induce fiber differentiation on its own but can potentiate the fiber-differentiating activity of FGF (Kok et al., 2002). It is noteworthy that PDGFR-α expression is lost early in the transitional zone in the process of fiber differentiation (Reneker and Overbeek, 1996); therefore, any influence that PDGF may have on fiber differentiation is likely to be restricted to early stages of the process.

EGF. Members of the EGF family of growth factors and their receptors have been detected in the lenses of a number of species (Weng et al., 1997). Experimental studies have also indicated that EGF signaling may be involved in regulating proliferation and differentiation of lens cells in various species, including birds and mammals. EGF has been shown to have an effect on proliferation in organ cultures as well as cell cultures (Reddan and Wilson-Dziedzic, 1983), and the appearance of lentoids is more frequent in long-term cultures treated with EGF (Ibaraki et al, 1995). Whether EGF directly influences differentiation is still not clear, as cell cultures invariably include serum. However, recent studies with chick lenses indicate that EGFR signaling occurs in fresh annular pad cells, and cultures of these cells respond to TGF-α by up-regulating filensin, an early marker for fiber differentiation (Ireland and Mrock, 2000). This up-regulation was further increased when cells were costimulated with cAMP analogs. Taken together, these results indicate that EGFR signaling may have a role in regulating aspects of the fiber differentiation response.

11.2.1.5. Overview

Thus, the picture that is emerging from growth factor signaling studies is that the process of lens fiber differentiation and maturation may depend on a combination of growth factor– induced signaling pathways. Evidence from in vitro and in vivo studies indicate that key roles may be played by FGFs and the TGFß superfamily. Other factors that can influence the behavior of lens cells in vitro may also have roles in different aspects of this process, and there are indications that their involvement may occur in a temporal sequence. For example, if PDGF plays a role, it is likely to have its effects early in the process, simply because fiber differentiation involves the loss of PDGF receptors. In this context, it is important to note that, so far, the only factors shown to be sufficient for initiating fiber differentiation when overexpressed are the FGFs. When members of the Bmp (Hung et al., 2002), TGFß (Srinivasan et al., 1998), and IGF (Shirke et al., 2001) families are overexpressed, they fail to elicit premature fiber differentiation of lens epithelial cells into fiber cells, although they are clearly functional, as they have other effects. This in no way excludes members of these growth factor families from having key roles in fiber differentiation; rather it indicates that their roles are likely to be downstream of initiation of the process. The observation that FGF is sufficient to initiate fiber differentiation from epithelial cells in vivo and in vitro indicates that FGF signaling is at the top of a hierarchy of growth factor signaling, at least for secondary fiber differentiation. The FGF gradient hypothesis proposes that fiber differentiation is initiated by FGF once cells shift below the lens equator and enter the vitreous environment. Propagation and progression of the process may then depend on a cascade of signaling events brought about by other exogenous (and probably also endogenous) growth factors, such as members of the TGFß superfamily (Fig. 11.16). An important goal for future research will

be to further test this hypothesis and provide more detailed information on the growth factor signaling pathways involved and their complex interactions.

11.2.2. Factors Involved in Epithelial Differentiation, Growth,

and Maintenance

Whilst a lot of effort has been spent in identifying factors that control fiber differentiation, little attention has been given to the other major lens polarity question: what factors control the formation, growth, and maintenance of the epithelial monolayer? Indeed, the Coulombres’ observation (1963) that a new epithelial layer formed anteriorly in the inverted chicken lens provides strong evidence that epithelial-promoting factors predominate in the anterior environment (Fig. 11.7). In addition to FGF, a number of other growth factors that are present in the anterior segment and have been shown to influence the behavior of lens epithelial cells, particularly proliferation, have been studied in various degrees of detail. RA and its naturally occurring retinoid analogs make up another class of compounds that regulate gene expression in lens cells and appear to have important roles in the maintenance of the epithelial monolayer.

11.2.2.1. Mitogenic Factors

PDGF. Platelet-derived growth factor (PDGF) stimulates proliferation of cultured bovine lens epithelial cells (Wunderlich and Knorr, 1994) and chick (Hyatt and Beebe, 1993; Potts et al., 1994; Potts et al., 1998) and rat (Kok et al., 2002) lens epithelial explants. During murine embryonic development, the expression of PDGF-A and its receptor, PDGFR-α, correlates with the proliferative activity of the lens epithelium (Reneker and Overbeek, 1996). During postnatal development, the expression of PDGF-A becomes restricted to the iris epithelium and ciliary body, which are in close apposition to the lens epithelial cells of the germinative zone. Expression of PDGFR-α is restricted to the lens epithelium at all ages and becomes localized to the epithelial cells of the germinative zone during postnatal development. Hence, both the ligand and its receptor are localized to the region where lens epithelial cells proliferate. In line with a mitogenic role for PDGF, transgenic mice that overexpressed PDGF-A specifically in the lens displayed a considerable increase in DNA synthesis in the epithelium compared with the wild type. The lens epithelium became multilayered and consisted of a surface epithelial monolayer and an underlying subepithelium (Reneker and Overbeek, 1996).

Mice that, through gene targeting, lack PDGFR-α have apparently normal lenses (Soriano, 1997), and as reported by Potts et al. (1998), they show normal levels of cell proliferation. Potts et al. argue that, whilst the results from various studies show that PDGF can regulate proliferation in lens epithelial cells under various conditions, the lack of phenotype in PDGFR-α−/− mice indicates that PDGF is probably not involved in maintaining lens cell proliferation in vivo. However, another interpretation is that this result may reflect redundancy in the system; for example, another growth factor, such as FGF, could compensate for the absence of PDGF-α signaling. FGF has been shown to induce a proliferative response in lens epithelial cells (McAvoy and Chamberlain, 1989), and it is expressed in the lens and in tissues near the lens, with particularly high levels of expression near the lens equator and in the neighboring ciliary body and iris (de Iongh and McAvoy, 1993; Lovicu et al., 1997). Therefore, bearing in mind the localization of PDGF and FGF to this region, it is likely that lens cells in the germinative zone are exposed to both PDGF and FGF in vivo (and very

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likely to other mitogens; see below). Since the combination of these growth factors in vitro (Kok et al., 2002) had an additive effect on lens epithelial proliferation, both may contribute to the formation and maintenance of the germinative zone in this region in vivo.

Insulin/IGF. The insulin/IGF family has also been studied in a variety of experimental systems. Insulin and IGF have been shown to stimulate proliferation of cultured lens cells (Reddan and Wilson-Dziedzic, 1983). IGF-1 and IGF-2 induced DNA synthesis in lens epithelial explants, and combining them with FGF synergistically enhanced their effect (Liu et al., 1996). IGF-1 and insulin receptors are expressed in mammalian lens epithelial cells, and IGFs and various binding proteins have been detected in the ocular media of various species (Chamberlain and McAvoy, 1997). Transgenic mice in which IGF-1 was overexpressed from the αA-crystallin promoter showed no premature differentiation of lens epithelial cells but rather an apparent expansion of the epithelial compartment toward the posterior pole (Shirke et al., 2001). This was accompanied by increased proliferation in the germinative zone and its expansion posteriorly. This study also showed that, in the wild type, IGF-1 is expressed in the epithelial cells at the lens equator. Taken together with the presence of IGF-1 receptors in the lens epithelium, these studies indicate that IGFs, as well as other mitogenic factors (see above), may influence spatial patterns of cell proliferation in the lens. A local domain of endogenous IGF-1 stimulation at the lens equator may provide a cue that defines the extent of the germinative zone and the location of the transitional zone.

Other Factors. Other factors that may be involved in regulating cell proliferation in the lens include the EGF/TGFα and HGF growth factor families. EGF and EGF receptors have been detected in the lens of a number of species (Weng et al., 1997), and the effect of EGF on proliferation has been shown in organ explants as well as cell cultures (Reddan and Wilson-Dziedzic, 1983). Expression of HGF and its receptor c-met has been reported in lens epithelial cells of a number of species (Fleming et al., 1998; Weng et al., 1997), and among its other effects, it has been shown to stimulate DNA synthesis (Wormstone et al., 2000).

11.2.2.2. Inhibitory Factors

TGFß. In addition to the mitogenic factors described above, other growth factors may act as negative regulators of lens growth. For example, the ability of TGFß to inhibit the growth of various cell types is now well established (Akhurst and Derynck, 2001). Moreover, TGFß has been shown to inhibit serum-stimulated cell proliferation in passaged bovine lens epithelial cells. Antibody-blocking experiments also indicate that TGFß in aqueous can inhibit lens cell proliferation (Kurosaka and Nagamoto, 1994). In situ hybridization studies showed that TGFß1 and TGFß2 but not TGFß3 mRNA is expressed in the lens during embryonic and postnatal development (Gordon-Thomson et al., 1998) and furthermore that proteins for all three TGFß isoforms are localized in the lens (Gordon-Thomson et al., 1998; Pelton et al., 1991). Both TGFß type I and type II receptors are required for TGFß signaling, but only the type I receptor is expressed in the epithelium throughout development. However, postnatally the type II receptor also appears in the epithelium, coincident with the development of the competence of lens epithelial cells to respond to TGFß (de Iongh et al., 2001a). As TGFß is potentially available to lens cells in situ, suppression of cell proliferation by TGFß may have a role in maintaining mitotic quiescence in the central lens epithelium postnatally.