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

regeneration program in the PECs of the ventral iris. This carcinogen has been shown to produce tumors in other systems (Sugimura et al., 1970; Seto et al., 1999). On the other hand, not all carcinogens have this effect. For example, nickel subsulfide, another potent carcinogen, has been tested in this system, with results ranging from inhibition of regeneration to development of tumors in the ventral iris, depending on the concentration (Okamoto, 1987, 1997). The fact that MNNG was able to reprogram the PECs of the ventral iris implies that the regeneration machinery was switched on and a series of genes activated or repressed. It is clear from these experiments that this regenerating system responds differently to the different stimuli, and the fact that the ventral iris can be induced to transdifferentiate in vivo under these extreme conditions indicates that this reaction can be induced in other species.

As previously mentioned, if we disrupt the signal transduction pathway of FGFR-1 or the action or synthesis of retinoids, the regeneration will be modified, even inhibited. On the other hand, if we add extra factors or chemicals, we can observe changes as well. For example, by ectopically placing FGF-1 and FGF-4 into lentectomized eyes, we can affect lens fiber differentiation and polarity (Del Rio-Tsonis et al., 1997).

12.5.3. Differences between Dorsal and Ventral Iris PECs

One important approach to understanding the restriction of the lens regenerative process is to look at the molecules that may be differentially expressed or present in the dorsal versus the ventral iris. As discussed earlier, a few of the molecules involved in lens development also seem to play a role in lens regeneration, and these seemed to be preferentially expressed in the dorsal PECs during regeneration (eg. Pax6, Prox1, FGFR-1).

Imokawa and Eguchi (1992) and Imokawa et al. (1992) generated a monoclonal antibody (2NI-36) that detects an antigen expressed in the ventral iris but not in the dorsal during lens regeneration. If this antibody is used to treat the ventral iris in vitro, and if this treated iris is then transplanted into the eye cavity, a regenerate forms. It has been speculated that 2NI-36 antigen is a glycoprotein that helps to stabilize the differentiated state of the iris cells and that these cells can destabilize and transdifferentiate if the antigen is lost (see also section 7.1.4).

12.6.Lens Regenerative Capacity of Vertebrates

12.6.1.Lens Regenerative Abilities in Amphibians

It is curious that a limited number of species have the gift of lens regeneration. Within vertebrates, the amphibians seemed to be among the fortunate ones. As adult organisms, some urodele amphibians have this amazing capability. Among the species that can replace the missing lens are members of the genera Triturus, Pleurodeles, Salamandra, Salamandrina, Typhlotriton, and Eurycea (Stone, 1967). Stone also found eight species unable to regenerate their lens, including members of the families Ambystomidae and Plethodontidae and even some species of Eurycea. The fact that two closely related species do not share the same regenerative capabilities raises the issue of evolutionary conservation and selective pressures to keep or lose such an ability. Anuran amphibians such as Xenopus laevis can regenerate their lens but only premetamorphosis. In addition, the mode of regeneration differs from that of the salamanders. In these animals, the lens regenerates from the inner cell layer of the outer cornea (Freeman, 1963).

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12.6.2. Lens Regenerative Abilities in Other Vertebrates

Some species of adult fish can regenerate their lens via transdifferentiation of the dorsal iris, similarly to the preselected salamanders (Sato, 1961; Mitashov, 1966). Again, here we can pose the question of how evolutionary pressures selected completely different organisms from different classes of vertebrates to share regenerative abilities.

Within other classes of vertebrates, chicks have been reported to be able to replace their lenses upon removal during a small window during their development. It is not clear if these embryos actually regenerate their lenses via the transdifferentiation of the PECs of the iris or if regeneration results from the induction of the still competent ectoderm by the optic vesicle (van Deth, 1940; McKeehan, 1961; Genis´ -Galvez,´ 1962; Niazi, 1967; Wedlock and McCallion, 1968). On the other hand, rabbits and cats have been reported to be able to regenerate their lenses upon removal, but only if the anterior and posterior capsular bags are left intact. The regenerates seem to be the end result of the growth of lens epithelial cells left in the capsular bags (Gwon et al., 1989; Gwon et al., 1990; Gwon et al., 1992; Gwon et al., 1993a; Gwon et al., 1993b).

12.7. Transdifferentiation of PECs as the Basis of Lens Regeneration

Although lens regeneration from the dorsal iris in the newt has convincingly been shown histologically (as previously mentioned), the ultimate proof of such cell type conversion has come from in vitro studies.

12.7.1. Iris PECs as the Actual Regeneration Cells

In experiments done in the 1970s, the dorsal and ventral iris PECs of newt eyes have been separately dissociated and cultured in vitro. After a long lag of more than two weeks, iris PECs started to grow vigorously. The actively growing cells gradually lost melanosomes and formed a typical monolayer sheet. From the progeny of nonpigmented epithelial cells in such monolayer cell sheets, three-dimensional transparent structures expressing lens-specific molecules developed within 40 days of culturing. No significant difference was observed between the dorsal and ventral PECs (Fig. 12.9; Eguchi et al., 1974). These findings had been confirmed by clonal analysis. More than 15% of the clonal colonies grown from singly dissociated dorsal or ventral iris PECs differentiated into lentoids with lens specificities (Abe and Eguchi, 1977). These experiments showed that the iris PECs can transdifferentiate in vitro and thus be the source of the lens epithelial cells that will reorganize the new forming lens.

12.7.2. Lens-Forming Potential of the Iris PE as a Whole

Application of MNNG during the process of lens regeneration in the newt (Eguchi and Watanabe, 1973) suggested that cell-cell and cell-substrate adhesion and communication were reduced throughout the iris pigment epithelium (PE); supernumerary lens regeneration was induced at different regions of the iris, the ventral iris in addition to the dorsal marginal region, where regeneration usually takes place (Fig. 12.8). These findings also suggest that normally the tissue architecture at the dorsal marginal PE must be unstable enough to react to micro environmental changes caused by lentectomy. Therefore, changes in the ventral iris PECs in cell culture conditions were also studied and compared with those in the dorsal iris

Figure 12.9. In vitro lens transdifferentiation of the newt iris PECs. The dorsal and ventral PE (DPE and VPE, respectively) are separated from the stromal tissue (S), dissociated, and cultured. Lentoids expressing lens specificities differentiate from populations of dedifferentiated PECs (dePEC) in cultures of both dorsal and ventral iris PECs. m, melanosome discharged from PECs. (From Eguchi, 1998.)

PECs (Eguchi et al., 1974). It has been clearly demonstrated that the iris PECs as a whole conserve the capacity to transdifferentiate to lens cells (Fig. 12.9) and that the iris PECs are the source of the cells that replace the lens (Eguchi and Shingai, 1971; Eguchi et al., 1974; Abe and Eguchi, 1977). In addition, it has been strongly suggested by these studies that the tissue architecture of the ventral iris PECs in situ must be much more stable than that of the dorsal iris PECs, but its destabilization can be induced by artificial dissociation and long-term culturing.

12.7.3. Lens Transdifferentiation in Higher Vertebrates PE

There is a possibility that PECs of chick embryos may also transdifferentiate to lens cells postlentectomy, since the lens is thought to be replaced either from the rudiment of the iris epithelium or from the still competent ectoderm during a restricted period of their development (van Deth, 1940; Genis´ -Galvez,´ 1962). Recently, it has been established that iris PECs from a one-day-old chick can readily transdifferentiate to lens cells in vitro. This cell type can provide a very useful cell culture system for studying transdifferentiation from iris PECs to lens cells (Kosaka et al., 1998). In addition, well-differentiated retina PECs dissociated from eyes of older chick embryos have been cultured in vitro (Eguchi and Okada, 1973). Retina PECs of chick embryos, maintained in successive culturing, eventually develop into lentoids expressing lens-specific molecules. Direct evidence of lens transdifferentiation in chick embryo retinal PECs was established by clonal analysis, and the results of this study led to the establishment of an effective cell culture system that provides a pure population of cells at each step of lens transdifferentiation, particularly multipotent dedifferentiated PECs (Itoh and Eguchi, 1986a, 1986b). This cell culture system has facilitated the analysis of the cellular and molecular mechanisms of transdifferentiation. In vitro cell culture studies have been extended to other higher vertebrates, and lens transdifferentiation of iris or retina PECs in conditions of cell culture has been confirmed in the following: Japanese quail (Coturnix), mouse (Mus), bovine (Bos), and ape (Macaca) (Eguchi, unpublished research). Lens transdifferentiation has been confirmed even in human iris and retina PECs dissociated from adult (80-year-old and 22-year-old) and fetal (16-week-old) eyes (Eguchi, 1988, 1993, 1998; Tsonis et al., 2001). In the case of PECs from the 80-year-old donor, singly dissociated PECs were seeded at clonal cell density. Many of these single PECs grew vigorously, rapidly losing their phenotype and formed clonal colonies consisting of

308 Katia Del Rio-Tsonis and Goro Eguchi

Figure 12.10. Potential of aged human retinal PECs to transdifferentiate into lens cells. (a) Clonal growth (l–4) and generation of lentoids (L) actively expressing lens specificities (5) from a single PEC derived from an 80-year-old donor (l–3: bar = 20 µm; 4: bar = 50 µm, 5: bar = 150 µm). (b) Expression of human lens crystallin genes αA- and β-crystallin by cultured cells differentiating into lentoids. Both αA- and β-crystallin gene products are obviously detected only in transdifferentiated lens cells generating lentoids (tLC) (shown in a5) but never in both dedifferentiated cells (dePEC) and redifferentiated cells (rePEC) – dePEC cells that reverted to the pigmented phenotype.

dedifferentiated cells (Fig. 12.10al–4). In many of these clonal colonies, differentiation of lentoids expressing lens-specific molecules was observed within 10 days (Fig. 12.10a and b; Eguchi, 1988, 1993, 1998). The same results were obtained from cell culture analysis of PECs of both a 22-year-old adult donor and a 16-week-old fetus. Thus it has been concluded that the potential for lens transdifferentiation of PECs is conserved even in humans throughout life.

12.7.4. Molecular Events and Factors Regulating Lens

Transdifferentiation of PECs

12.7.4.1. Cell Type–Specific Gene Expression

Using the cell culture system of chick embryonic PECs (Itoh and Eguchi, 1986b), transcription patterns for the following genes have been analyzed: PEC-specific MMP115 (Mochii et al., 1988a; Mochii et al., 1988b; Mochii et al., 1991) and pP344 (Iio et al., 1994; Kobayashi, Agata, and Eguchi, unpublished research); the genes for lens cell–specific αA-, βA3/Al-, βB1-, βB2-, γ -, and δ-crystallin; and the chick myc, erb-B, and ras protooncogenes (Agata et al., 1993). Expression analysis clearly indicates that the PEC-specific genes are transiently repressed at the stage of dedifferentiation. In contrast, transcription of crystallin genes is activated upon induction of transdifferentiation of PECs into lens cells (Fig. 12.11a and b). Lens transdifferentiation of PECs proceeds through a stable intermediate state in which neither lens cell–specific nor PEC-specific genes are expressed.

Figure 12.11. Results of gene expression analysis by Northern blotting during lens transdifferentiation of retinal PECs from older chick embryos. (a) Expression of the PEC specific gene pP344 and of the melanosomal matrix protein gene MMP115 is observed only in redifferentiated PECs (rePEC). Five micrograms of Poly(A)+ mRNA were hybridized with pP344, MMP115, and β-actin probes. (b) Expression of lens cell–specific crystallin genes is observed only in transdifferentiated lens cells (tLC). Ten micrograms of poly(A)+ mRNA from redifferentiated PECs (rePEC), dedifferentiated PECs (dePEC), and transdifferentiated PECs (tLC) were used. (c) High level of expression of the c-myc proto-oncogene was observed in dedifferentiated PECs. Five micrograms each of poly(A)+ mRNA from well-differentiated PECs and dedifferentiated PECs and 0.5 µg of poly(A)+ mRNA from transdifferentiated lens cells maintained in culture for either one (tLC1) or two (tLC2) weeks were used. (From Agata et a1., 1993.)

Dedifferentiated PECs, which are vigorously growing, consume glucose instead of oxygen and lose the property of contact inhibition of growth (Eguchi and Itoh, 1982; Itoh and Eguchi, 1982) in addition to losing gap junctional cell-cell communication (Kodama and Eguchi, 1994). Such properties resemble those of neoplastic cells. Expression of c-myc increases significantly in dedifferentiated PECs (Fig. 12.11c), whereas other oncogenes are not activated (Agata et al., 1993), suggesting that increased expression of the c-myc oncogene is associated with the high growth potential of dedifferentiated PECs.

12.7.4.2. Growth Factors

Through careful testing of FGFs purified from bovine brain, it has been confirmed that both FGF-1 and FGF-2 dramatically enhance lens transdifferentiation of well-differentiated PECs from older chick embryos (Hyuga et al., 1993), strongly suggesting that FGFs must be among the essential factors regulating lens transdifferentiation. This possibility has been supported by studies on the regulatory functions of FGFs in lens regeneration in the newt in vivo (Figs. 12.5 and 12.6; Del Rio-Tsonis et al., 1997; Del Rio-Tsonis et al., 1998; McDevitt et al., 1997). It has recently been found that epidermal growth factor (EGF) is also essential for lens transdifferentiation of one-day-old chick iris PECs, which are much more stable than embryonic PECs. Transdifferentiation of PECs can be dramatically enhanced by EGF in the presence of FGF (Kosaka, Mochii, and Eguchi, unpublished research).

310 Katia Del Rio-Tsonis and Goro Eguchi

12.7.4.3. Role of the Extracellular Matrix (ECM)

It has been clearly demonstrated that collagen substrate can repress dedifferentiation and lens transdifferentiation in a culture of chick embryonic PECs (Yasuda, 1979). The first half of the process of transdifferentiation in PECs in vitro, regarded as the dedifferentiation of PECs, is marked by a decrease in the adhesiveness of these cells to type I or type II collagen. Northern blot analysis showed that α3, α6, α8, and β1 integrins were transcribed at similar levels in PECs and dedifferentiated PECs. However, β1 integrin, which is found in a high proportion in integrin heterodimers and localizes at focal contact sites in PECs, was lost from those contact sites when they dedifferentiated, although the overall protein level was not changed. When an anti–β1 integrin antibody was added to the PECs in culture, a marked decrease in cell-substrate adhesiveness took place, followed by gradual changes in both morphology and gene expression patterns similar to those observed during dedifferentiation of PECs. β1 Integrin becomes phosphorylated at the onset of dedifferentiation, and this results in loss of its localization at the focal contact sites in the dedifferentiated PECs (Mazaki et al., 1996). In addition, the tyrosine residue of proteins such as paxillin, p125FAK, tensin, and p130Cas, which are involved in signal transduction, was phosphorylated in differentiated PECs, whereas no tyrosine phosphorylation of tensin and p130Cas was confirmed in dedifferentiated PECs (Eguchi, unpublished research). Based on these findings, it is possible to postulate that integrin stimulation in differentiated PECs induces actin fiber organization and that mitosis is regulated through protein tyrosine phosphorylation and cytoskeletal changes. In contrast, mitosis of dedifferentiated PECs must be regulated by other signal transducers such as members of the Src kinase family, since integrin does not mediate cell-ECM adhesion in dedifferentiated PECs (Fig. 12.12). In conclusion, an appropriate distribution of β1 integrin might be essential for maintaining the differentiated state of PECs through cell-ECM adhesion. The transdifferentiation potential of PECs must be completely repressed by some unknown mechanism in the normal eye in situ. A search for molecules responsible for the stabilization of the tissue architecture of the PECs was initiated, and a molecule designated as 2NI-36 was

Figure 12.12. Illustration of the hypothesis that integrins control the organization of actin fiber, cytoskeletal complexes, and mitosis in PECs (left) but not in dedifferentiated PECs (right), where signal transduction might be controlled by a member of the Src kinase family. (From Eguchi, 1998.)

identified as a candidate (Eguchi, 1988; Imokawa and Eguchi, 1992; Imokawa et al., 1992). This molecule cannot be detected in newt iris and retina PECs that are actively growing in vitro, but it does become detectable when PECs begin to organize epithelial structures.

12.8. Future Prospects

The fact that pigment epithelial cells from anywhere in the eyecup are capable of transdifferentiating to lens cells in culture, without any species restrictions, renders this a potential system for clinical applications. Even PECs from aged humans have this capability (Eguchi, 1988, 1993, 1998; Tsonis et al., 2001). This suggests that if we can find the molecular switches that turn on this transdifferentiation in vitro as well as in vivo, we will have at hand the power to regenerate lenses in higher vertebrates, including humans.

It is important to apply state-of-the-art technology to elucidate these molecular switches. The use of techniques such as in vitro transfection of ventral iris cells to introduce potential transdifferentiation regulators and the subsequent implantation of these transgenic aggregated cells into host lentectomized newt eyes could elucidate the molecular mechanisms behind the regeneration process (Hayashi et al., 2001; Hayashi et al., 2002). The use of techniques, including morpholino (Heasman, 2002) and RNAi (Zhou et al., 2002; Scherr et al., 2003) technology, to block the activity of putative essential regeneration molecules should also be a priority. Pinpointing the molecules responsible for these molecular switches will contribute not only to the field of eye research but to the investigation of many other systems in need of regenerative capacities. On the other hand, the basic knowledge obtained from this research will have an impact on our understanding of cancer growth, since it seems that both regeneration and cancer growth share similar initial activities and must share similar regulators early in the game. In addition, because in regeneration the ultimate result is an organized tissue, we can learn how this regenerating, “provisionally destabilized” tissue organizes itself and avoids awry growth and lack of controlled differentiation to form a copy of the original missing part.

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