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

As cells continue to progress through G1, pRb becomes further phosphorylated by cyclinCdk complexes (Fig. 8.2). This hyperphosphorylation of pRb leads to the dissociation of pRb from the pRb-E2F-DP complex, thus permitting either the derepression or activation of E2F targets (Dyson, 1998; Mulligan and Jacks, 1998; Nevins, 1998). Clearly, the hyperphosphorylation of pRb at the G1-S transition is important for that transition, and cyclin E is important in facilitating this event. However, there must be other roles for cyclin E in the G1-S transition because cyclin E is required for this transition in Rb null cells (Lukas et al., 1997; Ohtsubo et al., 1995). It is known that cyclin E is required for centrosome duplication (Hinchcliffe et al., 1999; Lacy et al., 1999; Matsumoto et al., 1999) and is involved in the up-regulation of histone transcription via phosphorylation of NPAT, nuclear protein mapped to the AT locus (Ma et al., 2000; Zhao et al., 2000). Thus, the evidence suggests that cyclin E–Cdk2 is the primary kinase involved in the G1-S transition (Dulic et al., 1992; Koff et al., 1992; Ohtsubo et al., 1995; Pagano et al., 1993; van den Heuvel and Harlow, 1993).

The roles of the pRb family members p107 and p130 are less well understood. The p130 protein is thought to play its role primarily in quiescent cells (i.e., cells that are in G0), whereas the p107 protein is thought to play its role primarily in the S phase (Dyson, 1998; Mulligan and Jacks, 1998). The list of targets for the pocket proteins is lengthy. One important family of targets, as mentioned above, is the E2F family of transcription factors, of which there are six members currently identified (Dyson, 1998; Nevins, 1998). Extensive in vitro analysis has led to the view that pRb preferentially binds to and regulates the activity primarily of E2Fs 1–3, p107 preferentially regulates E2F4, and p130 regulates E2Fs 4 and 5 (Bagchi et al., 1991; Cao et al., 1992; Chellappan et al., 1991; Chittenden et al., 1991; Devoto et al., 1992; Sardet et al., 1995; Shirodkar et al., 1992). The activities of pRb, p107, and p130 are regulated by their phosphorylation state, which in turn is regulated by cyclin-Cdk complexes in a cell cycle–dependent fashion.

In the S phase, cyclin A–Cdk2 is the primary cyclin complex, and it is thought to promote DNA synthesis by facilitating origin firing through phosphorylating protein substrates in the origin of replication complexes (Stillman, 1996). Inhibition of cyclin A function prevents DNA synthesis in cultured cells (Pagano et al., 1992). Inhibition of cyclin A’s function through microinjection of antibodies into cultured human fibroblasts also resulted in inhibition of entry to mitosis (Pagano et al., 1992). Given cyclin A’s function in DNA replication and entry to mitosis, it is not unexpected that mouse embryos deficient in cyclin A2 (an ubiquitously expressed form of cyclin A) die early in embryogenesis (Murphy et al., 1997). Deletion of cyclin A1 (germ cell–specific cyclin A) causes a block of spermatogenesis before the first meiotic division (Liu et al., 1998). In addition, cyclin A–Cdk2 inactivates E2F1 (and presumably E2F2 and 3 as well) in the S phase through phosphorylating their heterodimeric partner DP-1, therefore preventing E2Fs from binding DNA (Dynlacht et al., 1994; Krek et al., 1995). This function of cyclin A–Cdk2 cannot be accomplished by cyclin E–Cdk2 kinase activity (Dynlacht et al., 1994), and it is important for orderly S phase progression, as persistent E2F1 activity delays or arrests the S phase and leads to regrowth or apoptosis.

Cyclin B starts to accumulate in the S phase but is sequestered in the cytoplasm throughout S and G2 (Bailly et al., 1992; Ookata et al., 1992; Pines and Hunter, 1991). At the onset of mitosis, cyclin B is translocated into the nucleus, where it binds cdc2 and eventually leads to its activation. Activation of cdc2 marks the beginning of mitosis. Thus, cyclin B is called the mitotic cyclin and cdc2 the mitotic Cdk. Cyclin B–cdc2 is historically known as maturation-promoting factor (MPF), and it induces the maturation of Xenopus oocytes (Lohka et al., 1988; Masui and Markert, 1971; Newport and Kirschner, 1984). To prevent

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premature mitosis, cyclin B–cdc2 complex is held inactive by inhibitory phosphorylation on Thr-14 and Tyr-15 residues of cdc2 protein, which are abruptly removed by phosphatase cdc25 at the end of G2 (Kumagai and Dunphy, 1992). The activity of cdc2 triggers entry into prophase and eventually results in the assembly of spindles and metaphase plate in uncharacterized ways. To exit mitosis, cyclin B must be destroyed through an ubiquitinmediated proteolysis pathway (see below). Ubiquitination of cyclin B is accomplished by the anaphase-promoting complex (APC; King et al., 1994). Destruction of cyclin B is important in reestablishing the G1 state in daughter cells.

A large body of evidence suggests that the critical functions of D-type cyclins in cell cycle control are to inactivate pRb by activating Cdk4 or 6 and to sequester p27KIP1 (Sherr, 1996), leading to activation of cyclin E. Indeed, when cells enter the cell cycle after growth factor exposure, D-type cyclins are expressed earlier than cyclin E and are expressed differentially in different cell types (Matsushime et al., 1991). Inhibition of the function of D-type cyclins has minimal effect on cell cycle progression in cells lacking pRb (Lukas et al., 1995; Medema et al., 1995). With cyclin E downstream of D-type cyclins, D-type cyclins become the sensors linking mitogenic stimuli and the cell cycle machinery (Pines, 1995; Sherr and Roberts, 1995). The intracellular signaling pathways that regulate D-type cyclin expression are multiple, mostly involving Ras and Myc (Leone et al., 1997).

Acting in opposition to cyclins are the cyclin-dependent kinase inhibitors (CKIs). Currently, two classes of CKIs have been identified in mammals, the p21CIP1 and p16INK4 families, which differ in structure, mechanism of inhibition, and specificity. The p16INK4 family (inhibitor of Cdk4) consists of ankyrin repeat proteins, including p15, p16, p18, and p19 (Harper and Elledge, 1996), that bind to and inhibit Cdk4 and Cdk6 kinases. The p21CIP1 family of CKIs, including p21CIP1 (p21), p27KIP1 (p27), and p57KIP2 (p57), inhibit all Cdks involved in the G1-S transition. The fact that there are so many genes encoding Cdk inhibitors reflects the challenge an organism faces in putting a brake on proliferation during development and tumorigenesis. Compared with cyclins, however, little is known about factors or extracellular stimuli that would induce expression of these negative cell cycle regulators.

Many key cell cycle regulators are also subject to ubiquitin-mediated proteolysis, making the processes they regulate irreversible. Ubiquitin-mediated proteolysis is a major protein degradation pathway in eukaryotic cells (Ciechanover et al., 2000). Proteins destined for degradation are tagged with the highly conserved protein ubiquitin. Multiple units of ubiquitin are added onto a protein substrate, producing polyubiquitin chains, which are recognized by the 26S proteasome – the protease that destroys the ubiquitin-tagged proteins (Voges et al., 1999). Polyubiquitination is the rate-limiting step in this process, which involves a cascade of ubiquitin transfer reactions requiring three components. First, an ubiquitinactivating enzyme (El) activates ubiquitin, that is transferred to the second component, the ubiquitin-conjugating enzyme (E2). The third component, E3, acts as an adapter between the E2 and the substrate by contacting both of them simultaneously. Specificity (as to which substrate to ubiquitinate) is largely provided by E3. Therefore, E3 is called the ubiquitin ligase. There are three major E3 classes: the HECT-domain proteins, the anaphase-promoting complex (APC), and the SKP1/CUL1/F-box (SCF) ubiquitin ligases (Harper and Elledge, 1999). In SCF-mediated ubiquitination, an F-box protein targets a substrate to the ligase. There are many different F-box proteins, reflecting the need of a cell to degrade a variety of proteins. F-box proteins usually recognize substrates that have been phosphorylated. Thus, phosphorylation provides a signal for degradation (Harper and Elledge, 1999; Montagnoli et al., 1999). A number of cell cycle regulators are degraded through the SCF pathway,

including cyclins D and E, E2Fl, and p27, in which a F-box protein called SKP2 is required (Montagnoli et al., 1999; Nakayama et al., 2000). The C-terminal QT domain of p27 contains a critical threonine residue, which when phosphorylated by Cdk2–cyclin E leads to p27 degradation (Montagnoli et al., 1999).

Thus, at present considerable detail on the mechanisms of cell cycle regulation has been gleaned from in vitro studies. This body of knowledge has guided studies to determine how the cell cycle is controlled at the molecular level in the lens. After briefly reviewing the classical work on the patterns of cell proliferation in the developing lens, this chapter discusses how mechanisms of cell cycle control in the lens are similar to or different from the paradigm set forth by these in vitro analyses.

8.3. Cellular Proliferation in the Lens

This section reviews some of the seminal findings that have elucidated the patterns of cell proliferation in the developing vertebrate lens.

By day E11.0–E11.5 in mouse embryogenesis, the developing lens has entered the lens vesicle stage. Although work has demonstrated that all cells within this vesicle have the same developmental potential, the position of any such cell with respect to the developing optic cup dictates its fate in subsequent cellular differentiation stages of lens development. Cells in the posterior, in close proximity to the optic cup, are destined to become postmitotic and differentiate into the primary fiber cells, whereas cells in the anterior, directly opposite the optic cup, are destined to retain their epithelial character and proliferative capacity. After the initial differentiation of the primary fiber cells that fill the lens vesicle, the continued growth of the lens depends on the proliferation of lens epithelial cells and their subsequent differentiation into fiber cells. Because other chapters in this text discuss at length the early embryonic development of the lens (chaps. 1 and 2) and the nature of the environment that influences cells in the posterior as compared with those in the anterior (chaps. 2 and 11), this chapter focuses on the cell cycle machinery that permits the lens to maintain distinct populations of cells with differing proliferative characteristics from the lens vesicle stage forward.

One of the reasons that the lens is such an ideal in vivo model system for studying cell cycle control is that the program of differentiation is very well characterized at the histological level. This histological characterization includes the mapping of regions of high and low proliferative activity in the lens as a function of developmental age and placing this information in the context of cellular morphology and patterns of crystallin gene expression (Fig. 8.4). In the 1960s, a number of investigators, including Hanna and O’Brien (1961), Mikulicich and Young (1963), and Modak et al. (1968), used incorporation of tritiated thymidine into newly synthesized DNA and autoradiographic techniques to map the regions of the chick, rat, and mouse lens where cells are actively undergoing DNA synthesis. The measurements of DNA synthesis were combined with measurements of mitotic activity to determine cell cycle times for cells in different regions of the lens. These studies revealed that DNA synthesis and mitosis occur throughout the lens pit and the forming lens vesicle. However, as the vesicle stage proceeds to the beginnings of elongation of the primary fibers, regions of highest DNA synthesis become localized more anteriorly. Thus, DNA synthesis and mitosis become restricted to the anterior epithelium. As development proceeds in late embryos and especially postnatally, a distinct compartmentalization of regions with a high proliferative index and regions with substantially lower proliferative index arises within the epithelium. In young postnatal rats (birth to 35 days), the regions of highest proliferative

index become focused in narrow bands of approximately 50 cells located just anterior to the equator, a region known as the proliferation or germinative zone. The estimated cell cycle time is shortest in this region of high proliferative index and much longer in the central epithelium. With increasing age, cell cycle time increases throughout the epithelium.

In the 1970s, McAvoy (1978a) mapped the positions of high and low mitotic activity within the lens from embryonic and postnatal rats with respect to the aqueous and vitreous humors, cell morphology, and the expression pattern of crystallin genes. Consistent with earlier studies, he observed that, with increasing age of the animal, proliferating cells become progressively more localized to the germinative or proliferation zone and that these cells exhibit the shortest cell cycle time. McAvoy further established that α-crystallins first appear in the lens pit of the rat at day 12 in embryogenesis, β-crystallins are not detected until cells had stopped dividing and begun elongating, and γ -crystallins are not detected until even later in the elongation process. Much later, Zwaan determined that, in the mouse, α- crystallins first appear in the lens cup in actively cycling cells and do not lead to withdrawal from the cell cycle (Zwaan, 1983).

Many studies suggested that within the anterior of the eye, especially in the equatorial regions, proliferation factors were present that supported cell division in cells of the proliferation zone, whereas in the posterior of the eye, factors were present that supported elongation of cells in the elongation zone. Other studies compared the proliferative behavior of cells in the central epithelium to those in the proliferation zone. In vivo, the cycling cells in the central epithelium of the postnatal rodent become progressively fewer with increasing age. When cells from this region from 10to 15-day-old rats are explanted into tissue culture and maintained in serum-free medium, there is a large boost in the number of proliferating cells. However, when cells from 3-day-old rats are placed in culture, typically there is very little boost in proliferation (McAvoy and McDonald, 1984). These results suggest that cells in the central epithelium withdraw from the cell cycle into a quiescent G0 phase and that the proportion of cells in quiescence increases greatly between 3 and 10 days of age. The results also suggest that the reduced capacity of cells in the anterior epithelium to proliferate may be due at least in part to diminishing environmental stimuli, such as the possible decrease in the availability of proliferation factors as these cells become further removed from their presumed source, the ciliary body. Alternatively, the factors inhibiting proliferation in vivo may be absent in this tissue culture system.

Collectively, these studies demonstrated that the pattern of growth and differentiation in the lens results in the compartmentalization of cells with differing proliferative capacities (Fig. 8.4). Cells in the central epithelium, which have a cuboidal morphology and lens epithelial gene expression patterns, exhibit substantial proliferative capacity in early embryos but lose this capacity in vivo as the animal ages. Cells in the proliferation zone, which have a cuboidal morphology and lens epithelial gene expression patterns, exhibit high mitotic activity. Cells in the transition zone (i.e., cells posterior to the lens equator) exhibit low mitotic activity and begin to show morphology and gene expression patterns consistent with early fiber cell differentiation. The cells that make up the bulk of the fiber cell compartment exhibit no mitotic activity, a highly elongated morphology and expression of fiber cell-specific markers such as β- and γ -crystallins. By mapping the mitotic activity within the lens, these studies, combined with the known morphology and pattern of activation of differentiationspecific crystallin gene expression, provided the reference for subsequent studies to address how at the molecular level cell proliferation and differentiation are regulated in the lens.

With this knowledge in hand, many investigators set out to identify growth factors that would stimulate lens epithelial cell proliferation in vitro using explants from chick and

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rat. The candidate proliferation factors included factors in embryo-derived serum (Hyatt and Beebe, 1993), EGF (Redden and Wilson-Dziedzic, 1983), PDGF (Brewitt and Clark, 1988), insulin-like growth factor (Redden and Dziedzic, 1982), and certain concentrations of fibroblast growth factors (McAvoy and Chamberlain, 1989). More recently, the activities of many of these factors in regulating lens epithelial cell growth in vivo have been examined. As the focus of this chapter is on the intracellular cell cycle machinery that regulates cell proliferation and differentiation, we direct your attention to chapter 11 for a full accounting of growth factor regulation of lens cell growth and differentiation.

8.4. Expression Patterns of Cell Cycle Regulatory Genes in the Developing Lens

As the first essential step toward understanding the mechanism of cell cycle regulation in the lens, many studies were performed to characterize expression patterns of cell cycle genes, primarily using the mouse and rat as model systems. In some instances, the analysis was carried out at the level of mRNA expression, while other studies sought to identify the proteins and their activities. From these efforts is emerging a good picture of the expression domains of the critical players. This section summarizes our current understanding of the expression patterns of cyclins, Cdks, CKIs, Rb family members, and E2Fs in the lens (see Table 8.2 for a summary).

In the developing mouse lens, cyclin A is expressed in the lens epithelial cells but not in the postmitotic fiber cells (Fromm and Overbeek, 1996; Hyde and Griep, 2002). Cyclin B is expressed in both epithelium and fiber cells (Gao et al., 1995). D-type cyclins are expressed throughout the lens, and the level of D2 is highest in the epithelium (Fromm and Overbeek, 1996; Gao and Zelenka, 1997; Geng et al., 1999; Zhang et al., 1998). Low levels of cyclin E message are detected (Lahoz et al., 1999). Although the localization of cyclin E within the lens has not been determined, it would not be unreasonable to predict that its expression is localized to the epithelium, as this is the compartment in which cycling cells are located. Several Cdks (Cdk2, Cdk4 and Cdc2) are detected at the mRNA and protein levels in the epithelium (Gao et al., 1995). Cdk4 is also present in the equatorial region, where cells stop dividing and start differentiation (Fromm and Overbeek, 1996). Cdc2 is found in the fiber cells (Gao et al., 1995; He et al., 1998). Cdk2-associated kinase activity is detected in epithelial cells but not in fiber cells (Gao et al., 1999). Other Cdks that are

Table 8.2. Expression Domains of Cell Cycle Regulators

in the Lens

*Presumed to be epithelial specific due to very low expression level and function in cycling cells.

#Present in anterior epithelium at much lower level than in equatorial and transition zones.

Figure 8.5. (See color plate XIII.) Expression of Cdk inhibitor genes during lens development. In situ hybridization was performed on transverse sections through the region of the eye of an E15.5 mouse embryo using antisense probes for p57 (A) and p27 (B). Arrows in A indicate cells in the equatorial zone of the lens, which express high levels of p57. p27 is expressed at a low level throughout the lens and at a higher level in the retina (r).

not usually associated with cell cycle regulation, such as Cdk5, Cdk7, and Cdk8, are also expressed in the lens and presumably play different roles (Gao et al., 1999). The expression patterns of these positive cell cycle regulators are generally consistent with the fact that cell proliferation in the lens is restricted to the epithelial cells. However, cyclin B and cdc2 are expressed in fiber cells, and furthermore cyclin B–cdc2 complexes with kinase activity have been detected in fiber cells at specific developmental time points. These data suggest that these factors, which are normally considered to be involved only in cell growth, may also play a role in cell differentiation (He et al., 1998).

Many CKIs are expressed in the lens. The expression patterns of these factors are consistent with a role in counteracting the positive effects of cyclin-Cdk complexes on cell cycle progression. The CKI p16 (Pan and Griep, unpublished data) is expressed in the epithelium. Two members of the p21 family, p27KIP1 and p57KIP2 (Zhang et al., 1998), are expressed in the lens in distinct but overlapping patterns (Fig. 8.5). The p27 message seems to be present in all fiber cells in developing mouse lens. p57 mRNA is found both in the epithelium and in fiber cells; however, interestingly, p57 transcripts are most abundant in the equatorial region (Lovicu and McAvoy, 1999; Zhang et al., 1998). The founding member of this family of Cdk inhibitors, p21CIP1, is not expressed in the lens at detectable levels (Zhang et al., 1998). The role of these CKIs presumably is to regulate the activity of various cyclin-Cdk complexes. Most notably, cyclin D–Cdk4 complexes would be expected to be inactivated in fiber cells. Indeed, Cdk4 activity is not found in rat fiber cells, and coimmunoprecipitation experiments have confirmed that p57 is bound to Cdk4 in these cells (Gao et al., 1999). Cdk4-p57 complexes are found in epithelial cells as well, suggesting a role for this CKI in maintaining normal cell cycling in the epithelium.

As noted, one important family of target proteins for cyclin-Cdk complexes is the pRb family. All three members of this family are expressed in the lens epithelium, and pRb and p107 are expressed in fiber cells (Rampalli et al., 1998; Pan and Griep, unpublished data). In the neonatal mouse lens, Rb transcripts are most abundant in the equatorial region (Pan and Griep, unpublished data), a pattern reminiscent of p57 expression. Interestingly, p130 protein appears to be degraded through a ubiquitin-dependent process in fiber cells

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(Rampalli et al., 1998). Members of the E2F family of transcription factors are expressed in the lens. In the epithelium, transcripts for E2Fs 1–5 are found, while in the fiber cells, transcripts for E2Fs 1, 3, and 5 are found (Pan, 1995; Rampalli et al., 1998). Presumably, one important role for the pocket proteins would be to regulate the activities of the E2F family members. This would imply that complexes of pocket proteins and E2F family members should be present. Indeed, complexes of E2Fs and pRb and p107 have been detected in lens epithelial and fiber cells, although the particular E2F member or members in these complexes have not been determined. Whether or not p130 is complexed with E2Fs in the epithelial cells also has not been determined.

How is the expression of cell cycle genes regulated in the developing lens? Certainly their expression must be controlled by the genetic program that directs lens development. At present, this genetic program is far from clear. Very little is known about how the expression of positive cell cycle regulators (e.g., cyclins) is regulated. Some regulators of CKI expression, which are transcription factors that regulate expression of cell cycle genes either directly or indirectly, may be involved. For example, the transcription factor Prox1 (see chap. 3) is required for the expression of both p27 and p57 in the lens (Wigle et al., 1999), and among other defects, lack of Prox1 leads to disrupted cell cycle regulation in the lens, similar to the disrupted regulation seen in mice lacking both p27 and p57 (Wigle et al., 1999). Whether Prox1 directly activates the transcription of these two CKIs or does so indirectly via its downstream target genes has yet to be determined. Another possible candidate is the c-Maf gene product (Ring et al., 2000; also see chap. 3). c-Maf deficiency results in lens phenotypes similar to the ones caused by Prox1 inactivation, but the expression of Prox1 in c-Maf null lenses is grossly normal, suggesting that c-Maf could be downstream of Prox1. How is expression of these transcription factors regulated? Presumably growth factor signaling pathways impact on their expression. This question is addressed in chapter 11.

With the comprehensive picture of the expression patterns of cell cycle factors in the lens, it is possible to formulate hypotheses as to how cell cycle control is achieved in the undifferentiated cells of the lens as well as in cells that are embarking on differentiation. The expression patterns suggest that when lens cells differentiate, factors such as pRb and p57 may play seminal roles. They further suggest that the components of cell cycle control set forth earlier in this chapter for undifferentiated proliferating populations of cells may be applicable, at least in part, to the control of cell proliferation in the lens epithelium. The application of technologies to manipulate gene function in the developing mouse lens, such as transgenic and knockout approaches, has provided avenues for testing these hypotheses.

8.5. Cell Cycle Regulation during Fiber Cell Differentiation

Differentiated lens fibers are postmitotic, highly elongated cells that lack subcellular organelles. Intuitively, it would seem that continued cell division would be incompatible with this terminal phenotype. Thus it would appear logical that cell cycle withdrawal and differentiation would be coupled mechanistically, at least at some level. How do lens fiber cells coordinate cell cycle withdrawal and differentiation? The first factors whose requirements for differentiation were tested specifically in this process were pRb and p57, in accordance with the data reviewed above (see sections 8.2 and 8.4). The experimental strategies used to address these hypotheses involved transgenic and gene disruption approaches. This section describes the results of genetic analysis of cell cycle gene function in lens fiber cell differentiation.

Figure 8.6. (See color plate XIV.) Inactivation of pRb by HPV-16 E7. Initial phosphorylation of pRb by cyclin D–cdk4/6 complexes normally generates functional, hypophosphorylated pRb that is able to bind to E2F and create a complex capable of repressing transcription of E2F target genes. The E7 oncoprotein from HPV-16 can bind to pRb, thus sequestering pRb from E2F. This leaves free E2F-DP complexes able to activate transcription of E2F targets.

The knowledge that pRb is an important regulator of the cell cycle in vitro prompted investigators to ask if pRb is required for cell cycle withdrawal during fiber cell differentiation. To this end, transgenic mice expressing the E7 oncoprotein from human papillomavirus type 16 (Pan and Griep, 1994) or a C-terminally truncated large T antigen from SV40 (Fromm et al., 1994) under the direction of the murine αA-crystallin promoter were generated. The αA-crystallin promoter would direct expression of linked genes to the lens cells at the time of their initial differentiation. As shown in Figure 8.6, the E7 oncoprotein has the capacity to bind to and inactivate the hypophosphorylated form of pRb (Dyson et al., 1989; Munger et al., 1989). The C-terminally truncated SV40 large T antigen similarly binds to pRb (DeCaprio et al., 1988). Mice expressing either oncoprotein display profound defects in the differentiation of lens fiber cells (Figure 8.7). Most notably, cells in the transition zone fail to withdraw from the cell cycle and fail to elongate. Instead, these abnormal cells showed a high propensity for apoptosis (Fromm et al., 1994; Pan and Griep, 1994). These defects likely are due to inactivation of pRb (or pRb family function) given that mutations in E7 that abrogate its ability to bind pRb also abrogate the phenotype (Pan and Griep, 1994). Consistent with this interpretation, germline mutation of Rb leads to a similar lens phenotype (Morgenbesser et al., 1994). Although activation of the expression of β- and γ -crystallins does occur in these models, immunohistochemistry suggests that the levels of these proteins are abnormally low (Morgenbesser et al., 1994). More recent evidence

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Figure 8.7. (See color plate XV.) Impact of HPV-16 E7 expression on lens fiber cell differentiation in transgenic mice. Paraffin-embedded sections from eyes of neonatal nontransgenic (A, C, E) or E7 transgenic (B, D, F) mice were subjected to histological analysis using hematoxylin and eosin staining (A, B), analysis of DNA synthesis using BrdU incorporation (C, D), or analysis of apoptosis (E, F) using the TUNEL assay. BrdU incorporation was detected immmunohistochemically with diaminobenzidine, and the section was counterstained with hematoxylin, resulting in brown-stained positive nuclei and purple-stained negative nuclei. TUNEL-labeled nuclei were identified using an FITC-conjugated secondary antibody, and sections were counterstained with propidium iodide, resulting in green-stained positive nuclei and red-stained negative nuclei when viewed using FICT and Texas Red filters, respectively. Note the small, disorganized vacuolated appearance of the fiber cell compartment of the E7 transgenic lens (compare A and B). Note the presence of BrdU positive nuclei, indicative of active DNA synthesis, within the fiber cell compartment of the E7 transgenic lens only (compare C and D) and the concomitant appearance of TUNEL positive cells throughout the fiber cell compartment, indicative of apoptosis (compare E and F). e, lens epithelial cells; f, lens fiber cells; r, retina; black arrows (C and D), brown-staining, BrdU-positive nuclei; white arrow (F), green, TUNEL-positive apoptotic nuclei in fiber cell compartment. In all panels, the anterior of the lens is at the top.