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

Figure 8.8. E2F-1 regulates both S phase entry and apoptosis. Once it is released from pRB suppression due to inactivation of pRB by Cdk phosphorylation or by binding of viral oncogene products such as E7 and T antigen, E2F-1 promotes S phase entry by activating cyclin E expression. Persistent activity of E2F-1 could lead to apoptosis through transcriptionally activating p19ARF, p73, or other unidentified factors.

suggests that there are reduced message levels for numerous β- and γ -crystallins in lenses from Rb null (Liu and Zacksenhaus, 2000) and E7 transgenic mice (Hyde, Potter, and Griep, unpublished data) as compared with nontransgenic control mice.

What is the molecular pathway through which Rb-deficient cells continue past the restriction point into the S phase and undergo apoptosis (see Fig. 8.8 for a summary)? As discussed in section 8.2, it is thought that a key role for pRb is to regulate the transcriptional activation activity of E2F transcription factor family members, leading to repression of E2F targets when pRb is bound to E2Fs. Cyclin E is known to be a key target of E2F1-dependent transcription activation. Thus, the prediction would be that inactivation of pRb results in continued expression of cyclin E in the abnormal lens fibers, which then drives G1-S progression, resulting in continued cell cycle progression. Indeed, cyclin E and a host of other cell cycle regulators whose expression is normally repressed during fiber cell differentiation continue to be expressed or are expressed in expanded patterns at the RNA level in the abnormal Rb-deficient fiber cells. These include cyclins and Cdks (Fromm and Overbeek, 1996), E2Fs 1–3 (McCaffrey et al., 1999; Hyde and Griep, 2002, and unpublished data),

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Rb (H. Pan and A. E. Griep, unpublished data), and p53 (Pan and Griep, 1995). Another target of E2F1 is p19ARF. p19ARF shares coding exons with the CKI pl6INK4a gene but is translated in a different reading frame (thus ARF, alternative reading frame) and expressed through a different promoter (Kamijo et al., 1997; Quelle et al., 1995). p19ARF encodes a polypeptide that stabilizes p53 (Pomerantz et al., 1998; Zhang et al., 1998a). Therefore, it is predicted that lack of Rb leads to apoptosis, at least in part due to stabilization of p53 by the increased expression of p19ARF (Fig. 8.8). Indeed, p19ARF, whose expression is undetectable in normal mouse lenses, is found expressed in E7 transgenic lenses, and its expression is entirely dependent on E2F1 (Hyde and Griep, 2002). These data suggest a pathway that inactivation of pRb leads to up-regulation of E2F1, which in turn leads to the up-regulation of cyclin E and p19ARF. p19ARF then leads to an increase in the level of p53, which results in activation of the expression of the proapoptotic gene Bax (Miyashita and Reed, 1995) and the CKI p21 (El-Diery et al., 1993; Harper et al., 1993; Xiong et al., 1993). In the context of the lens, apoptosis is the end result rather than cell cycle arrest. These data support a model that is consistent with other studies delineating common molecular pathways toward apoptosis.

Results from genetic analyses indicate that much but not all of this predicted model is correct. First, E2F1 appears to be one mediator of the effects of loss of Rb function. In Rb/E2F1 double knockout mice (mice lacking both Rb and E2F1), the proliferative defects are substantially rescued and the apoptotic defects are almost entirely rescued, at least at day 13.5 in embryonic development (Tsai et al., 1998). Rescue of the lens defects in the E7 transgenic mouse by the E2F1 null allele was also substantial (McCaffrey et al., 1999). Consistent with these data, deregulated expression of E2F1 during fiber cell differentiation leads to proliferation and apoptosis in abnormally differentiating fiber cells (Chen et al., 2000). Downstream of E2F1 is p19ARF. In Rb/pINK4a double knockout mice (in which both p16 and p19ARF are mutated), apoptosis but not proliferation defects are partially rescued (Pomerantz et al., 1998), although other studies document that the effect of a specific p19ARF knockout on apoptosis in the Rb null lens is minimal (Tsai et al., 2002). Downstream of p19ARF is p53. A p53 null mutation rescues nearly completely the apoptotic defects of the Rb null mutation at day E13.5 (Morgenbesser et al., 1994). The same p53 mutation provides near complete rescue of E7-induced apoptosis at day E13.5. The patterns and degree of rescue afforded by either the E2F1 null allele or the p53 null allele overlap, suggesting that these genetic factors lie in the same pathway. Finally, consistent with the up-regulation of Bax expression in E7 transgenic lenses, investigation of the effects of a Bax null mutation on E7-induced apoptosis has revealed that the Bax null mutation provides partial rescue to apoptosis in the same spatial regions as the p53 null allele (Nguyen and Griep, unpublished data). Thus, initial analysis of the pathway regulated by Rb seems to support, at least in part, the existingmodels.

However, a more thorough analysis of mice phenotypes suggests that additional pathways may be activated by dysregulation of Rb function. For example, comparing the phenotypes of Rb/E2F1 double null mice and E7/E2F1 null mice shows that the rescue of E7-induced proliferation and apoptosis defects by the E2F1 null mutation was not as great as the rescue of the Rb null–induced defects (Tsai et al., 1998; McCaffrey et al., 1999). More recent observations indicate that at least one of these other downstream targets is E2F3 (Saavedra et al., 2002; Ziebold et al., 2001). Second, the p19ARF null mutation was at best only partially capable of rescuing the Rb null–induced defects, indicating that there must be other factors involved in regulating p53-dependent apoptosis. Third, while the p53 null mutation nearly

completely rescues Rb null–induced and E7-induced apoptosis at day E13.5, at later times in embryogenesis this allele only affords partial protection against E7-induced apoptosis. This result indicates that apoptosis occurs through both p53-dependent and p53-independent pathways that are temporally regulated during development (Pan and Griep, 1995). These p53-dependent and p53-independent pathways toward apoptosis are also spatially distinct (Pan and Griep, 1995). Interestingly, a second oncoprotein from HPV-16, E6, also was able to rescue E7-induced apoptosis when coexpressed with E7 in the lenses of transgenic mice. The rescue afforded by the expression of this oncoprotein was greater than the p53 mutation alone, indicating that both p53-dependent and p53-independent apoptosis was blocked (Pan and Griep, 1994, 1995). The p53-dependent component of E6’s activities is due presumably to E6’s ability to target p53 for degradation via ubiquitin-dependent proteolysis (Huibregtse et al., 1991; Scheffner et al., 1990), and recent data support this hypothesis (M. M. Nguyen and A. E. Griep, unpublished data). Fourth, the model predicts that the rescue of apoptosis by the E2F1 null mutation should be similar to that of the p53 null mutation. However, the pattern of rescue by the p53 null allele is more restricted than that of the E2F1 null allele, suggesting that E2F1 affects both p53-dependent and p53-independent apoptosis (McCaffrey et al., 1999). Finally, the model predicts that rescue afforded by the Bax null mutation should be similar to that of the p53 null mutation. However, rescue by the Bax null mutation, while similar, does not appear to be as complete (M. M. Nguyen and A. E. Griep, unpublished data). This suggests that Bax is a relevant downstream target of p53 in mediating p53-dependent apoptosis in the lens; however, it is not the only one. Thus, although to the first approximation the data confirm that the predicted pathway is responsible for the proliferative and apoptotic defects of Rb inactivation, extensive analysis suggests that the story is not quite so simple.

There are several possible explanations for these apparent discrepancies between the actual data and the proposed model. First, the fact that E2F1 appears to affect apoptosis through pathways in addition to the p53 pathway comes from the recent work of Irwin et al. (2000). This group has shown that E2F1 can induce apoptosis through the p73 (a homolog of p53) pathway (Irwin et al., 2000), which might also be involved in the apoptosis in the lens invoked by lack of Rb (Fig. 8.8). Second, in considering the extent to which the E7 transgenic lens system and the Rb null lens system are equivalent, it is important to recognize that E7 has the capacity to inactivate the pRb family members p107 and p130. At least, p107 is present in complexes with E2F in fiber cells (Rampalli et al., 1998). In the retina, it is clear that, in the absence of Rb, p107 acts to partially suppress the effects of this mutation, as the Rb/p107 null phenotype is more severe than the Rb null phenotype alone (Robanus-Maandag et al., 1998). By analogy, in the lens, inactivating both family members may lead to a more severe phenotype than inactivation of pRb alone. Alternatively, the differences between the effects of E7 and the Rb null mutation could be accounted for by differences in the ages of the embryos examined. Because the Rb null mutation leads to embryonic death shortly after day 13.5 in embryogenesis (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992), it was not possible to determine the extent to which E2F1 or p53 null mutations would affect Rb null–induced proliferation and apoptosis at later time points. There is evidence that the Rb null mutation can lead to both p53-dependent and p53-independent apoptosis (Macleod et al., 1996). On the other hand, there is evidence to the contrary. When the early lethal effects of the Rb null mutation are rescued by expression of a Rb minigene, lens defects still persist. In this situation, the apoptosis in the lens appears to be entirely p53 dependent (Liu and Zacksenhaus, 2000). Further analysis will be required

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Figure 8.9. Cell cycle and differentiation. When cells receive differentiation signals, they activate expression of CKIs through the hypothesized transcription factor (TF1) to arrest the cell cycle. A second hypothesized transcription factor (TF2) activates the transcriptional program of differentiation, which may require the presence of active pRb. TF1 and TF2 could be the same transcription factor. In addition, it is possible that CKIs contribute directly to the differentiation process.

to determine if E7 is affecting p107 function and if this activity contributes to the overall phenotype.

In addition to defects in cell cycle regulation, Rb null, E7 transgenic, and SV40 truncT antigen transgenic mice show abnormal levels of differentiation specific marker proteins such as β- and γ -crystallins. This differentiation defect in Rb null lenses could be a result of deregulated cell cycle progression. In this model, the capacity to show morphological and biochemical traits of the differentiated fiber cell would be dependent on cell cycle withdrawal. In an alternative model, however, the effects of inactivating Rb on the expression of differentiation-specific genes could be attributable to the independent activity of pRb (Fig. 8.9). The latter alternative is possible given that pRb has been shown to interact with transcription factors that are critical regulators of differentiation. These factors include MyoD in skeletal muscle cells (Gu et al., 1993), c-Jun in keratinocytes (Nead et al., 1998), and CCAAT/enhancer-binding proteins (C/EBPs) in adipocytes (Chen et al., 1996). Additionally, this transcriptional coactivator function of pRb is separable from its ability to suppress E2F target genes (Sellers et al., 1998). It remains to be determined if pRb plays a direct role in the differentiation of lens fiber cells, and if so, by which means. However, it is known that pRb can bind to Pax6 in vitro and in embryonic chick lens (Cvekl et al., 1999), suggesting that it is possible that pRb-Pax6 interaction may be important for optimal Pax6-dependent transcriptional regulation. Alternatively, pRb could interact with c-Maf, whose absence in the lens is correlated with decreased mRNA levels of the differentiation-specific crystallins (Ring et al., 2000).

Given the fact that pRb is essential in lens development and is inactivated by G1 Cdks, what then keeps pRb in its hypophosphorylated active state once the lens fiber cells start to differentiate? The expression patterns of cell cycle genes indicate the presence of D-type cyclins in lens fiber cells, which could be balanced by the expression of a Cdk inhibitor. Indeed, if this balance is tipped off by forced expression of G1 cyclins and Cdks as transgenes in the lens, then overproliferation of lens fiber cells, presumably along with pRb inactivation, ensued (Lahoz et al., 1999). The effect of expression of these transgenes is dramatized if combined with the loss of the Cdk inhibitor p57KIP2 (Lahoz et al., 1999). p57KIP2 has been

suggested to be the main effector that down-regulates Cdk activity to activate pRb, based on its high levels of expression in cells that are beginning to differentiate in the equator region of the lens (Lovicu and McAvoy, 1999; Matsuoka et al., 1995; Zhang et al., 1998). However, loss of p57KIP2 in mice causes only a slight increase in the proliferation rates of these cells, much less severe than what has been observed in mice lacking Rb, suggesting that other mechanisms are complementing p57KIP2 loss (Zhang et al., 1997). Analysis of mice doubly null for p27KIP1 and p57KIP2 indicates that p27KIP1 plays a redundant role with p57KIP2 in the development of the lens (Zhang et al., 1998). Loss of p27KIP1 alone does not cause lens abnormalities, but it has a dramatic effect on the proliferation and differentiation of lens fiber cells when combined with p57KIP2 deficiency (Zhang et al., 1998). The phenotypes of p27KIP1/p57KIP2 double null lenses bear strong similarities to those of Rb null lenses, indicating these two CKIs cooperatively activate pRb in the differentiation of lens fiber cells.

However, two significant differences exist between the phenotypes of the Rb null versus p27KIP1/p57KIP2 double null mutants. First, the extent of overproliferation as assessed by BrdU incorporation appears to be significantly greater in p27KIP1/p57KIP2 mutants than in Rb mutants. This may reflect the fact that these two CKIs function not only upstream of pRb by blocking Cyclin D–Cdk4 activity but also downstream of pRb by blocking cyclin E–Cdk2-mediated S-phase entry. Alternatively, the increase in Cdk activity due to CKI loss may result in inactivation of additional pRb family members such as p130 and p107, leading to a more severe proliferation defect than Rb loss alone. Thus, proliferation of lens fiber cells lacking pRb may be limited due to the action of p27 and p57 on Cdks other than Cdk4 and Cdk6. The second major difference is that the rates of apoptosis in CKI-deficient lenses are much lower than for Rb-deficient lenses and are similar to the rates seen in Rb/p53 double mutant lenses. pRb is required to establish the transcriptional program that brings about differentiation of multiple cell types but has also been shown to inhibit apoptosis during myoblast differentiation and in other situations (Wang, 1997). Thus, low rates of apoptosis in p27KIP1/p57KIP2 double mutant lenses may reflect an antiapoptotic role for pRb. If the absence of p27KIP1 and p57KIP2 results in the inactivation of pRb to such an extent that it phenocopies the differentiation defect of Rb null mutant lenses, why the difference in apoptosis rates? There are several plausible explanations for this difference. First is that pRb could have an antiapoptotic function that is not regulated by Cdk phosphorylation and therefore would not be altered by CKI loss. Second is that, even in the absence of the CKIs, there may be residual pRb activity such that the apoptosis-inhibiting functions of pRb are largely intact. Even in the absence of CKIs, there is likely to be residual regulation of pRb if Cdk activity is still cyclical. In contrast, an Rb null cell would constitutively derepress all pRb-regulated genes such as E2F1, an apoptosis-inducing gene (Kowalik et al., 1995; Qin et al., 1994; Shan and Lee, 1994), and might display a more severe phenotype for this reason. Third, it is also possible that CKI mutant cells have higher Cdk activity levels, which prematurely inactivate E2F1 function (Dynlacht et al., 1994; Krek et al., 1994), thereby balancing the apoptotic-inducing consequences of inactivating pRb. The fact that the apoptosis rates of p27KIP1/p57KIP2 double mutants are similar to the rates observed in Rb/p53 double mutant mice (Morgenbesser et al., 1994) is consistent with interfering with E2F1 function; apoptosis caused by Rb loss is partially mediated by E2F1 (Tsai et al., 1998) and E2F1-mediated apoptosis is argued to be p53-dependent (DeGregori et al., 1997; Qin et al., 1994).

In sum, our knowledge of the mechanism through which cell cycle withdrawal is achieved during fiber cell differentiation has increased greatly over the past decade. This rapid expansion in our knowledge has resulted from in vivo genetic analysis of the function of

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components of the cell cycle machinery. Yet, despite our gains, many questions remain about the mechanism of cell cycle regulation in the lens and its coupling to differentiation.

8.6. Regulation of Proliferation in the Lens Epithelium

As discussed in section 8.3, the lens epithelium is a monolayer of cells that have proliferative capacity. Yet the cells within this layer differ in their proliferative characteristics, depending on their position relative to the ciliary process. In contrast to our rather extensive knowledge of how withdrawal from the cell cycle is achieved at the time of fiber cell differentiation, at present our knowledge of the mechanisms of cell cycle regulation in the epithelium is rather limited. We do, however, have a a basic understanding of the expression patterns and activities of many cell cycle genes, as discussed in section 8.4. The data indicate that in general the expected players are expressed and that the regulatory circuits are likely to be complex. As it was for the mechanisms of cell cycle withdrawal during differentiation, genetic studies are essential for determining the roles of specific factors or families of factors. Only recently, with the identification of molecular reagents that target the expression of transgenes to the epithelium, is progress beginning to be made on this important question. This section describes our current understanding, based on genetic studies, of cell cycle genes important for controlling cell proliferation in the epithelium.

Obvious candidates as regulators of the cell cycle in these cells are pRb and/or its family members p107 and p130. With some cells in the epithelium dividing only rarely, at least in postnatal animals, while others are actively cycling, it might be expected that the entire pocket protein family and all of its target E2F transcription factors would be important. Results from recent studies exploiting gene knockout and transgenic approaches that disrupt the function of pRb family members are consistent with this prediction. The first piece of evidence comes from analysis of the lens in mice rendered Rb deficient by gene-targeting strategies. Analysis of the lenses in these Rb null embryos failed to identify changes in the proliferative characteristics of the epithelial cells (Morgenbesser et al., 1994). However, because Rb null embryos die early in lens differentiation, it is possible that Rb is required in the epithelium at later stages of lens differentiation. Because of the strong desire of developmental biologists to study the role of Rb at later stages in embryogenesis, several groups have generated Rb null rescue chimeras or transgenic mice with Rb minigenes that permit analysis at later embryonic time points. Analysis of lens phenotypes in these animals also failed to identify alterations in epithelial cell proliferation characteristics that might suggest disruption of cell cycle control (Liegeois et al., 1996; Liu and Zacksenhaus, 2000; Maandag et al., 1994; Williams et al., 1994). Thus to date, a specific effect of Rb mutation in the lens epithelium has not been reported. Likewise, there appears to be no defects in the lens in p107 or p130 null mice (Cobrinik et al., 1996; Lee et al., 1996). It is possible that in the epithelium Rb family members may have redundant or compensatory functions that could be revealed by analysis of double or triple knockout mice. Due to early embryonic lethality, it was not possible to assess whether Rb/p107 double null mice would have a phenotype in the lens epithelium (Lee et al., 1996). However, it was possible to examine lens phenotypes in p107 null, p130 null, and the double null mice. No phenotypes have been reported (Cobrinik et al., 1996), suggesting at least that the loss of function of these combinations of pocket proteins alone is not sufficient to dysregulate cell cycle control in the epithelium. Loss of all three Rb family members has recently been shown to be necessary for abrogating the G1 checkpoint in cultured mouse embryonic fibroblasts (Dannenberg et al., 2000; Sage et al., 2000), and pRb and p107/p130 appear to be required for regulation

of different sets of genes (Hurford et al., 1997). Together, these data suggest that if the pocket proteins are essential regulators of lens epithelial cell growth, all of the pocket protein family members might be required.

If the pocket protein members are capable of providing redundant or compensatory functions in the epithelium, it is possible that transgenic expression in the epithelium of a DNA tumor virus oncoprotein that interferes with the function of all pocket proteins might be informative. It has been reported that transgenic mice in which the expression of HPV-16 E7 is regulated by the human keratin 14 develop cataracts (Herber et al., 1996). Recent analysis of the lens phenotype in these mice indicates that the transgenes are expressed postnatally in the epithelium and transition zone and that this expression is associated with increased numbers of proliferating cells throughout the epithelium (Nguyen et al., 2002). The capacity of E7 to induce high levels of proliferation in the lens epithelium in these mice is attributable to its capacity to regulate pocket protein activity, as mutations that abrogate E7’s regulation of pRb and its family members also abrogate the lens phenotype (Nguyen et al., 2002). Thus, in contrast to the differentiating lens cell, where pRb function is necessary and sufficient for cell cycle withdrawal, it appears that all pRb family members may be required for maintaining cell cycle control in the epithelium.

Given the complex pattern of cell proliferation in the lens epithelium (see section 8.3), it is possible that multiple factors and/or pathways contribute to the regulation of cell cycle control in this layer. Interestingly, expression of the other HPV-16 oncogene, E6, in the lens epithelium in transgenic mice also leads to cataract formation (Song et al., 1999), and transgene expression was correlated with high levels of proliferation throughout the epithelium (Nguyen et al., 2002). The mechanism through which E6 induced high levels of proliferation appears to be its targeting of PDZ proteins such as the mouse homologs of Drosophila proteins Discs-large (DLG) and Scribble rather than its targeting of p53 (Song et al., 1999; Nguyen et al., 2002, Nguyen et al., 2003).

How is expression of cell cycle genes regulated in the lens epithelium? The answer is very much unknown at this point in time. It is known that certain growth factors such as PDGF stimulate the proliferation of cultured lens epithelial cells (Brewitt and Clark, 1988) and that overexpression of PDGF (Reneker and Overbeek, 1996) and IGF1 (Shirke et al., 2001) in transgenic mice lead to increased levels of proliferating cells in the epithelium. Also, preliminary investigations suggest that constitutive expression of activated H-ras in the epithelium can lead to heightened levels of proliferation in these cells (L. Reneker, personal communication). Given these data and the known connections between the ras- raf-MAPK pathway and the expression of cyclins (Lundberg and Weinberg, 1999), it would seem quite likely that a growth factor–mediated pathway would be operational in the lens. Initial studies are indeed now beginning to shed light on the important players in cell cycle control in the epithelium, but overall the mechanisms of cell cycle control in the epithelium remain to be elucidated.

8.7. Significance of Understanding Cell Cycle Control for Clinical Issues

The transparency of the lens depends on the formation and maintenance of its precise cellular structure. The formation of this structure in turn depends on the precise control of cell growth and differentiation throughout the life of the animal. When the lens structure breaks down, loss of transparency, or cataract, arises. As cataract is the leading cause of blindness worldwide, it is considered to be a major public health problem. The most common treatment for cataract today, surgical removal of the lens fiber mass while leaving the lens capsule and

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epithelium behind, leads to secondary cataracts in a high proportion of individuals. These secondary cataracts referred to as posterior subcapsular cataracts (PCOs), affect 50% of the population of cataract patients, children and adults alike. As the condition is caused by the proliferation and migration of the remaining lens epithelial cells posteriorly, with concomitant aberrant differentiation (Apple, 1992; Beebe, 1992; Kappelhof and Vrensen, 1992), understanding the factors that control cell proliferation is critical for our future ability to develop new strategies for effectively preventing, delaying, or treating cataract.

This chapter has focused on the many recent endeavors to understand the mechanism of cell cycle control, primarily through the analysis of animal models. Although the absolute changes in the rate at which the lens grows differ between humans and rodents, the general pattern of greater growth rate in embryos than in adults is conserved (Bron et al., 2000; van Heyningen, 1976). As discussed in this chapter and at length in other chapters in this text, it is thought that the proliferative and differentiative characteristics of the lens epithelial cells are influenced by their environment, but the exact nature of that environment is not understood. These uncertainties notwithstanding, the lens is most perfectly modeled in vivo by the intact lens itself. Therefore, the normal processes of growth and differentiation at various times in the lifespan of an animal, be it embryonic or postnatal, can provide models to elucidate how cataract may form. Animal models in which lens fibers are ablated but epithelial cells remain behind in an adult animal (Breitman et al., 1989; Landel et al., 1988; Pan and Griep, 1994) offer a way of mimicking the lens after cataract surgery. Using these models, it should be possible to learn more about the mechanisms that stimulate cell proliferation under abnormal conditions and compare them to how cell proliferation and differentiation are regulated during normal lens differentiation. From these comparisons may emerge new ideas for how to repress aberrant cell proliferation and its effects in the lens.

8.8. Key Questions for Future Investigation

As we have detailed in this chapter, a great deal has been learned about the mechanism through which the cell cycle is regulated in the lens, including withdrawal from the cell cycle at the time of fiber cell differentiation. Yet despite these advances, much remains to be learned. Among the many questions that remain to be answered about how cell cycle control is maintained, a few are appropriate to raise in the conclusion of this chapter. That lens cells are compartmentalized into domains with distinct proliferative activity has been known for decades. Yet very little is known at the mechanistic level about how this compartmentalization is achieved in vivo. This is especially true for the epithelium, which is subdivided into regions with distinct proliferative behavior that in addition is influenced by age. Ultimately, models of cell cycle control in the epithelium must account for how cells in the central epithelium are regulated differently than cells in the proliferation zone. What are the intracellular cell cycle components that are required and what is the function of each? What extracellular signals regulate the expression and activity of the cell cycle genes and through which intracellular signaling pathways are these signals mediated? And what are the direct targets of these factors? Although we have some initial clues as to some of these key regulators, much remains to determine if initial hypotheses are in fact correct.

Many of these same questions can also be asked of the differentiating fiber cells. Although we have a much clearer picture of what the key factors are for differentiating cells than for undifferentiated cells, many questions remain. What are the signal transduction pathways that regulate the expression and activity of key cell cycle genes? What are the

relevant in vivo targets of key cell cycle factors such as pRb? What is the role of other pRb family members, if any, in lens differentiation? How are cell cycle withdrawal, fiber cell elongation, and differentiation-specific changes in gene expression coordinately regulated? What are the mechanisms through which aging affects cell cycle regulation? These are but a few of the many questions that need to answered if we are to understand how lens growth and differentiation are achieved and, ultimately, how these factors may impact cataract formation. The study of genetically manipulated animal models, in combination with advances in genomics and proteomics, should provide tools to answer these and many more questions about how the cell cycle is regulated in the lens.