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

Autoradiographic studies helped to clarify the origin of the regenerate as well. Labeled cells were followed to determine their fate. Even though both dorsal and ventral irises were labeled, only the dorsal labeled cells were found to give rise to the regenerating lens (Reyer, 1966a, 1971; Eisenberg and Yamada, 1966; Eguchi and Shingai, 1971). Zalik and Scott (1971) went even farther, transplanting dorsal irises labeled in vitro and then grafting them into a host lentectomized eye; indeed, these labeled cells gave rise to a regenerating lens. Ultrastructural studies performed in the 1960s confirmed the early work of Wolff, basically verifying that the iris PECs do undergo depigmentation and eventually give rise to a regenerate (Eguchi, 1963, 1964; Dumont and Yamada, 1972; Yamada and Dumont, 1972; Reyer, 1990a, 1990b).

12.4.2. Is There a Retinal Factor Involved in the Process?

One of the issues that still occupy scientists today is the contribution of the retina to lens regeneration. Knowing that the dorsal iris PECs of several salamander species, such as those belonging to the genara Triturus and Salamandra, are competent to give rise to a regenerating lens, researchers asked why these competent dorsal irises, if implanted, would give rise to lenses in certain circumstances and not in others. If these irises were implanted in the cavities of lentectomized eyes from a species of salamander that lacks the regenerating ability, such as Amblystoma punctatum, lenses would form. On the other hand, a competent dorsal iris, placed in the body cavity of an adult salamander or in a subcutaneous location would not form a lens, regardless of whether the salamander was competent to regenerate its lens or not (Ikeda, 1935, 1936; Stone, 1958a; Reyer, 1953, 1954).

Interestingly, though, if the neural retina was included in these transplants, then lenses would form (Stone, 1958a). So a series of experiments were performed to try to elucidate the role of the neural retina in lens regeneration. One experiment that pinpointed the contribution of the retina to lens regeneration involved removing the neural retina, choroid, and retinal pigment epithelium along with the lens. In this case, no lens regenerated. If only the neural retina and the lens were removed, then the lens would regenerate but only after the neural retina started to regenerate as well (Stone, 1958a). In other experiments, the physical block or separation of the neural retina and the iris prevented any lens formation (Stone, 1958b; Zalokar, 1944). In vitro studies directed by Eguchi (1967) and Yamada et al. (1973), the culturing of iris-corneal complexes or fragments of iris pigment epithelium from the dorsal margin with neural retina supported the growth and differentiation potential of the retinal factor.

Not only was it determined that there existed one or more retinal factors that promoted lens regeneration, but it was shown that this same influence was responsible for lens polarity – the formation of the lens fibers in the posterior region facing the neural retina and of lens epithelial cells in the anterior region facing the cornea (Reyer, 1948; Stone, 1954a, 1954b). It is tempting to speculate that this factor is the same factor that supported the formation of a perfect lens in a regenerating newt limb when dorsal iris explants were transplanted to the blastema (Fig. 12.2). Even the anteroposterior polarity of the lens was maintained, with the anterior (front) part of the lens facing the wound epithelium (Reyer et al., 1973; Ito et al., 1999). The molecules responsible for this activity might include FGFs and FGFRs, because these have been implicated in determining the lens polarity in the developing eye and because the wound epithelium of the regenerating limb is a rich source of FGFs (Caruelle et al., 1989; Chamberlain and McAvoy, 1989; de Iongh and McAvoy, 1993; Lovicu and McAvoy, 1993; Lovicu and Oberbeek, 1998; Robinson et al., 1995a; Robinson et al., 1995b; Robinson et al., 1998; Mullen et al., 1996; Zenjari et al., 1997; Stolen et al., 1997, Stolen and Griep, 2000).

296 Katia Del Rio-Tsonis and Goro Eguchi

12.4.3. Is There a Lens Factor Involved?

A controversial issue that to date remains unresolved is the type of influence the lens itself has. When a lens is removed and then placed back in the regenerating eye, it exerts a negative influence on the lens regeneration process, and its effect seems to depend on how close the lens is to the dorsal iris. If very close, no regeneration is observed; if placed at some distance from the dorsal iris, then regeneration initiates but is significantly inhibited (Wachs, 1914; Ikeda and Kojima, 1940; Ikeda and Amatatu, 1941; Eguchi, 1961; Dinnean, 1942; Uno, 1943; Stone, 1943; Zalokar, 1944; Takano et al., 1958; Reyer, 1961). To challenge the idea that the lens and its suspensory ligaments act as a physical barrier to the neural factor, dorsal iris transplants were inserted in the anterior chamber, and lenses were either left in situ or removed and then immediately replaced. In this case, no lens regenerates were observed (Wachs, 1914; Stone, 1943, 1952; Reyer, 1961). When dorsal iris transplants were inserted in the vitreous chamber through an incision in the ventral part of the eyecup without disturbing the host lens, regenerates did form, but the regenerates did not behave the same as similar implants where the host lens was removed (Reyer, 1966b). It was concluded that there is a physical barrier factor partially responsible but also that some other factor was influencing the formation of the regenerating lens. It was hypothesized that the lens secretes a substance that either competes with the neural factor or acts as an inhibitory substance specific to the regenerating lens (Zalokar, 1944; Stone and Vultee, 1949; Stone, 1953). Williams and Higginbotham (1975) performed experiments similar to those done by Stone (1966) in order to clarify the role of the intact lens on the lens regeneration process. In these experiments, the ventral iris was replaced by a donor dorsal iris, and the original lens was removed, so that two lenses were created, one from the donor dorsal iris and the other one from the host dorsal iris. When the host lens was removed 53–91 days after the original lenetectomy, it regenerated in the presence of the donor lens, but it was noted that in all cases there was a significant amount of space between the dorsal iris and the donor lens, allowing the neural factor to be available. Eguchi’s (1961) experiments supported the idea that displaced lenses must be at a certain distance from the competent iris to permit regeneration.

12.5. Modern Approaches to Lens Regeneration

One approach to dissecting the molecular pathway involved in the process of lens regeneration has been to analyze molecules known to play a role during vertebrate lens development. Some of these molecules include the eye-determining gene Pax6 (Ton et al., 1991; Grindley et al., 1995; Duncan et al., 2000) and Prox1 (Oliver et al., 1993; Tomarev et al., 1996;

Figure 12.2. (facing page) (See color plate XVIII.) The limb-lens connection. Lens formation in the regenerating limb. (a) A perfectly formed lens after dissociated pigment epithelial cells from the dorsal iris were implanted into the blastema. The lens possesses a normal appearance and normal posteroanterior polarity (direction of the arrow), with the anterior (front) part facing the wound epithelium. In (c), the lens is magnified to show its normal appearance. The lens epithelium in the anterior (A) shows the characteristic cuboidal shape, while in the posterior (P), the cells become elongated and differentiate to lens fibers (lf ). The anteroposterior polarity of lens does not coincide with the anteroposterior axis of the limb. (b) Failure to form a lens after transplantation of dissociated pigment epithelium cells from the ventral iris. The cells have not dedifferentiated and have remained pigmented. (d) magnification of the pigmented aggregate shown in (b). (Courtesy of Dr. M. Okamoto.)

Tomarev, 1997; Wigle et al., 1999), both early molecular markers for lens development. Even though the mechanisms for lens development and lens regeneration are different, it seems that the basic molecular regulators do participate in both.

12.5.1. Expression of Molecules Involved in Lens Development

and Regeneration

12.5.1.1. Role of Homeobox-Containing Genes

During vertebrate lens development, the lens arises from the head ectoderm after receiving inductive signals from the optic vesicle. This region of the head ectoderm seems to be predisposed to become a lens, as it expresses Pax6 even before optic vesicle induction (Li et al., 1994). Tissue recombination experiments in rats have shown that Pax6 expression in the head ectoderm is essential for lens development (Fujiwara et al., 1994), and studies using conditional Pax6 knockouts have shown that Pax6 is autonomously required for lens formation (Ashery-Padan et al., 2000). On the other hand, Prox1 expression is apparent just before the optic vesicle induces the head ectoderm to become the lens placode, the structure that will develop into the lens vesicle (Tomarev et al., 1996). Recently, Wigle et al. (1999) demonstrated that Prox1 knockout mice develop a hollow lens because the mutant lens cells fail to polarize and elongate properly. In addition, both Pax6 and Prox1 are transcription factors that regulate lens crystallin gene expression (Cvekl and Piatigorsky, 1996; Gopal-Srivastava et al., 1996; Tomarev et al., 1996; Duncan et al., 1998; Wigle et al., 1999; Lengler et al., 2001).

During lens regeneration in the newt, the lens is replaced by the PECs of the dorsal iris. These cells are derived from the neural ectoderm, not the head surface ectoderm, as for normal lens development. These cells will transdifferentiate into lens cells and replace the lost lens. Even though, in regeneration and development, the origin of the lenses differs, as does the process of their formation, Pax6 and Prox1 expression seems to be common to both. Soon after lentectomy, Pax6 was found to be expressed in both the ventral and a dorsal iris PECs, but Pax6 expression becomes restricted to the dorsal PECs at a stage where dedifferentiation is apparent (Fig. 12.3; Mizuno et al., 1999). This gene continues to be expressed in the lens vesicle and eventually becomes restricted to the lens epithelial layer of the regenerating lens (Del Rio-Tsonis et al., 1995; Mizuno et al., 1999). Pax6 is expressed in the adult intact newt eye; however, its expression is not evident in the axolotl, a urodele unable to regenerate the lens (Del Rio-Tsonis et al., 1995). Prox1 has been found to be specifically expressed and regulated in the pigment epithelium of the adult newt dorsal iris and in the dorsal iris during lens regeneration (Fig. 12.4; Del Rio-Tsonis et al., 1999; Mizuno et al., 1999). Such expression patterns suggest a role for these two genes in regeneration-competent PECs. Functional studies are currently being undertaken to clarify the role of these molecules.

12.5.1.2. Role of Fibroblast Growth Factors

Another set of molecules involved in lens development and lens regeneration consists of FGFs and their receptors. FGF signaling is essential for the early inductive events taking place during the lens placode stage (Faber et al., 2001). In addition, it has been proposed that FGFs determine the polarity of the lens (Lovicu and McAvoy, 1993; Schulz et al., 1993; Govindarajan and Overbeek, 2001). It has been shown that FGF-1 is present as a gradient in the vertebrate eyeball, with higher concentration needed for fiber differentiation in the

300 Katia Del Rio-Tsonis and Goro Eguchi

Figure 12.4. Regulation of Prox-1 protein during the process of lens regeneration in the newt Notophthalmus viridecens. Expression via Western blot analysis. Lane 1, extracts from ventral iris of the regenerating eye; lane 2, extracts isolated from the dorsal iris of the regenerating eye; lane 3, extracts from the ventral iris of the intact eye; lane 4, extracts from the dorsal intact eye. (From Del Rio-Tsonis et al., 1999.)

posterior chamber and lower concentration in the anterior, where the lens epithelial cells are proliferating (Chamberlain and McAvoy, 1989; McAvoy and Chamberlain, 1989; McAvoy et al., 1991; Le and Musil, 2001). A series of mice transgenic for FGFs and their receptors have been generated, and they show that when these molecules are misexpressed in the eye, the normal development and differentiation of the lens is disrupted (Robinson et al., 1995a; Robinson et al., 1995b, Robinson et al., 1998; Stolen et al., 1997; Stolen and Griep, 2000; Lovicu and Overbeek, 1998; Govindarajan and Overbeek, 2001).

In newt lens regeneration, FGF-1, FGFR-1, FGFR-2, and FGFR-3 have been shown to be expressed in the dedifferentiating cells of the regenerating lens vesicle as well as in the subsequent stages of lens fiber differentiation (Del Rio-Tsonis et al., 1997; Del Rio-Tsonis et al., 1998; McDevitt et al., 1997). However, only FGFR-1 product seems to be present specifically in the dorsal iris during dedifferentiation (Fig. 12.5). Functional studies in which the function of FGFR-1 was blocked using an inhibitor supported its role in regulating lens regeneration (Fig. 12.6). The results showed inhibition of lens regeneration and lens fiber differentiation (Del Rio-Tsonis et al., 1998). In addition, as in cases of FGF transgenic mice, exogenous FGFs were capable of inducing similar abnormalities in the regenerating lens (Del Rio-Tsonis et al., 1997). These abnormalities included vacuolated lens, double lens formation, and lenses with abnormal polarity.

Proteoglycans, specifically heparan sulphate proteoglycans, can bind to many FGF molecules at a time enhancing the ability of FGFs to bind to their receptors resulting in FGFR dimerization (Spivak-Kroizman et al., 1994). It is interesting to note that during lens regeneration a sequential loss of cell surface molecules, including proteoglycans, takes place (Zalik and Scott, 1973; Ortiz et al., 1992). This disappearance may affect the availability of FGFs for activating their receptors or their mode of action in such activation. Further research in this area is needed in order to establish clear relationships between key cell surface changes and other events taking place during the dedifferentiation process.

12.5.1.3. Role of Retinoids

Retinoids and their receptors are another set of important players in eye development and also in axis determination during many developmental processes. During eye development, retinoic acid is synthesized in the retina and controls the fate of the cells composing the neural retina (McCaffery et al., 1992; McCaffery et al., 1993; Hyatt et al., 1996a; Wagner et al., 2000). In mice lacking retinoid receptors, the ventral iris is not developed (Kastner et al., 1994). In addition to these roles in retina development, exogenous retinoic acid has been implicated in the induction of ectopic lens differentiation during eye development (Manns

Figure 12.6. Effects on inhibition of FGFR-1 signaling during the process of lens regeneration in the newt Notophthalmus viridecens. To try to establish the role of FGFR-1 in lens regeneration, an FGFR-1–specific inhibitor was used. This inhibitor binds to FGFR-1 to produce a conformational change, which blocks the function of FGFR-1 in the signal transduction pathway. (A) Twenty day lens regenerate showing normal lens formation. (B–D) FGFR-1 inhibitor–treated eyes; lens regeneration did not advance beyond the dedifferentiation stage. No lens or fiber differentiation could be observed. (D) The most advanced stage of regeneration was a small lens vesicle, comparable to Sato stage IV, that did not show any clear primary fiber differentiation. (From Del Rio-Tsonis et al., 1998.)

and Fritzsch, 1991). Retinoid receptors also regulate expression of crystallins, including αB, E, and F crystallins (Gopal-Srivastava et al., 1998; Kralova et al., 2002).

Retinoic acid has also been shown to be important during newt lens regeneration. If the synthesis of retinoic acid is inhibited by disulfiram, a compound that can prevent its synthesis (Vallari and Pietruszko, 1982; Maden, 1997), or if the function of the retinoid receptors is impaired using a retinoic acid receptor antagonist, the process of lens regeneration can be dramatically affected (Fig. 12.7). In the majority of the cases, lens regeneration from the dorsal iris is inhibited (Figs. 12.7B, C). In a few cases, ectopic lens regeneration from places

Figure 12.7. (See color plate XX.) Effects of inhibiting RA signaling on lens regeneration in the newt Notophthalmus viridecens. (A) A section through a normal lens (control) regenerated 20 days postlentectomy (10×). Note the lens regenerated from the dorsal iris (di). vi, ventral iris; c, cornea; le, lens epithelium (large arrowhead; the small arrowheads indicate the differentiation of lens fibers [elongated cells] in the posterior part of the lens); a, anterior part of the eye; p, posterior. (B) Lens regeneration in an eye treated with disulfiram. Note the inhibition of the regenerating lens (rl) at the tip of the dorsal iris. The tip of the ventral iris (vi) also shows some degree of dedifferentiation. Also, detachment of the retina (r) can be observed (20×). (C–H) Lens regeneration in eyes treated with RAR antagonist. (C) Inhibition of lens regeneration from the dorsal iris. The regenerating lens (rl) has not advanced beyond the initial stages of dedifferentiation (20×). (D) Retardation and abnormalities of the regenerating lens (arrowhead). The lens is oblong and positioned more dorsally and posteriorly than a normal regenerating lens (20×). di, dorsal iris. (E and F) A case of lens regeneration from the cornea. In E, note the continuity between the regenerating lens (arrowhead) and the cornea (c). In F, we can observe the differentiation of elongated primary lens fibers (arrowheads) in the posterior part of the lens, which is a dominant feature of lens morphology (20×). (G) A case of lens regeneration in the ventral part of the eye. The regenerating lens is clearly associated with the ventral iris (vi) and not the dorsal iris (di) (10×). (H) A case of double lens regeneration from the dorsal iris. Note the small lens vesicle (arrowhead) at the tip of the dorsal iris (di) (at the correct location) and the abnormal regenerating lens (rl) dorsal and posterior to the dorsal iris (20×). All eyes were examined 20 days postlentectomy. (From Tsonis et al., 2000.)