Role of Retinoic Acid in Lens Regeneration

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1 DEVELOPMENTAL DYNAMICS 219: (2000) BRIEF COMMUNICATION Role of Retinoic Acid in Lens Regeneration PANAGIOTIS A. TSONIS, 1 *MICHAEL T. TROMBLEY, 1 TIMOTHY ROWLAND, 1 ROSHANTHA A.S. CHANDRARATNA, 2 AND KATIA DEL RIO-TSONIS 3 * 1 Laboratory of Molecular Biology, Department of Biology, University of Dayton, Dayton, Ohio 2 Retinoid Research, Department of Chemistry and Biology, Allergan Inc., Irvine, California 3 Department of Zoology, Miami University, Oxford, Ohio Prompted by the actions of retinoids and their receptors in gene regulation, in the developing eye and especially in the lens, we have undertaken adetailed study to examine the effects of retinoids on urodele lens regeneration. First, we examined the effects of exogenous retinoids. It was found that exogenous retinoids had no significant effect on lens regeneration. However, when synthesis of retinoic acid was inhibited by disulfiram, or when the function of the retinoid receptors was impaired by using arar antagonist, the process of lens regeneration was dramatically affected. In the majority of the cases, lens regeneration was inhibited and lens morphogenesis was disrupted. In afew cases, we were also able to observe ectopic lens regeneration from places other than the normal site, which is from the dorsal iris. The most spectacular case was the regeneration of alens from the cornea, an event possible only in premetamorphic frogs. These data show that inhibition of retinoid receptors is paramount for the normal course and distribution of lens regeneration. We havealsoexaminedexpressionofrar-deltaduring lens regeneration. This receptor was expressed highly in the regenerating lens only. Therefore, it seems that this receptor is specific for the regeneration process and consequently such expression correlates well with the effects of RAR inhibition observed in our studies Wiley-Liss, Inc. Key words: newt; lens; regeneration; retinoids INTRODUCTION Lens regeneration in adult amphibia is one of the most spectacular cases of transdifferentiation of aterminally differentiated cell type to another. Indeed, after lens removal, the pigment epithelial cells from the dorsal iris dedifferentiate and then transdifferentiate to lens cells (Tsonis, 1999, 2000a,b). Although in vivo lens regeneration is possible in amphibia and some fishes, pigment epithelium from many animals (including humans) has the capacity for in vitro transdifferentiation to lens cells. Such aproperty of the pigment epithelium has urged many scientists to investigate lens regeneration to identify the factors that are involved and thus delineate the mechanisms of transdifferentiation and lens regeneration. Recently, afew factors have been found to be specifically expressed in the dorsal iris during dedifferentiation of the pigment epithelium and the formation of the lens vesicle. These factors are Pax-6, FGFR-1, and the homeobox-containing Prox-1. (Del Rio-Tsonis et al., 1995, 1997, 1998, 1999).Tofurtherextendandunderstandtheregulative events during lens regeneration we have turned our attentiontosomeotherimportantregulatorsthatseem to be involved with Pax-6, homeobox, and FGF signaling pathways. Acting through their nuclear receptors, retinoids have been implicated in determining the differentiationandmorphogenesisofseveraltissues.thesereceptors when dimerized are transcriptional factors able to activate genes that possess elements in their regulatory regions that can be occupied by them. One of the organs whose development is largely dependent on retinoids and their receptors is the eye. During eye development, retinoic acid is synthesized in the retina and controls the fate of the cells comprising the neural retina. Retinoids are synthesized asymmetrically in the developing retina as has been determined by the expression of the enzymes responsible for their synthesis (McCaffery et al., 1992, 1993; Hyatt et al., 1996; Wagner et al., 2000). The activation of certain retinoic acid receptors (RARs) has been found critical for retina morphogenesis. 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 Grant sponsor: National Institutes of Health; Grant number: EY *Correspondence to: Panagiotis A. Tsonis, Laboratory of Molecular Biology, Department of Biology, University of Dayton, Dayton, OH tsonis@neelix.udayton.edu or Katia Del Rio-Tsonis, Department of Zoology, Miami University, Oxford, OH Received 11 July 2000; Accepted 6September WILEY-LISS, INC.

2 RETINOIC ACID IN LENS REGENERATION 589 (Manns and Fritzsch, 1991). In addition, retinoids/receptors activate expression of crystallins, including alphab-crystallin, a protein characterizing terminal differentiation of the lens (Gopal-Srivastava et al., 1998). The retinoid receptors activate expression of alphabcrystallin gene by means of the lens-specific regulatory regions found in its promoter, same regions used by Pax-6 to activate alphab-crystallin. Indeed, RA signaling in the developing eye is dependent on Pax-6. In mice carrying a Pax-6 mutation, retinoid signaling in the eye is decreased and the lens anlage cannot respond to exogenous RA (Enwright and Grainer, 2000). Pax-6 is an eye and lens determining master gene and mutations can disrupt lens morphology as well (Ton et al., 1991; Duncan et al., 2000). These properties of retinoids and their receptors during eye and lens development as well as involvement of retinoids in regulation of genes that might be important for lens regeneration have prompted us to examine their role in amphibian lens regeneration. Indeed our results indicate a role of retinoids/receptors in lens regeneration, which is consistent with their role in the developing eye. TABLE 1. Effects of Various Treatments on Newt Lens Regeneration Treatment Total eyes treated Number of lenses affected Control 31 0 All-trans-retinoic acid cis-retinoic acid 30 0 Retinol palmitate 27 0 Disulfiram a RAR antagonist b a Three cases of dedifferentiation and vesicle formation in the ventral iris. b Three cases of ectopic lens formation. TABLE 2. Breakdown of Antagonist and Disulfiram Treatments on Newt Eyes Treatment Total eyes treated Number of lenses affected Antagonist soln (22 M) 16 8 Antagonist soln (50 M) antagonist bd (10 mg/ml) antagonist bds (10 mg/ml) antagonist bd (20 mg/ml) 10 5 a 2 antagonist bds (20 mg/ml) 9 6 Disulfiram solution Disulfiram bd (50 mg/ml) 9 3 Disulfiram bd (225 mg/ml) 5 3 a Three cases of ectopic lens regeneration. RESULTS AND DISCUSSION Lens regeneration in the adult newt begins with dedifferentiation and proliferation of dorsal iris pigment epithelium cells (PECs). By dedifferentiation, we mean the loss of characteristics that define the pigment epithelial cells, such as pigmentation. Dedifferentiation initiates molecular events, such as re-entering the cell cycle, which are necessary for cell proliferation and the subsequent regeneration of the lens. At approximately 10 days postlentectomy, a lens vesicle is formed from the depigmented dorsal PECs. Around 12 to 16 days postlentectomy, the internal layer of the lens vesicle thickens and synthesis of crystallins begins. This event marks the beginning of primary lens fiber differentiation. During days 15 to 19, proliferation and depigmentation of PECs slows down. In the internal layer, the lens fiber complex is formed and in the margin of the external layers nondividing secondary lens fibers appear. By 18 to 20 days, the PECs have stopped proliferating and the lens fibers continue to accumulate crystallins. Lens regeneration is considered complete by day Therefore, lens regeneration is possible by transdifferentiation, which is the differentiation of one cell type from another (in this case lens cells from pigment epithelial cells). The process of dedifferentiation and transdifferentiation has been proven beyond any doubt in this system. When single PEC cells are placed in culture these processes can be observed. As the PECs proliferate, they become depigmented and then transdifferentiate to lens cells (Eguchi et al., 1974). Therefore, although in many other regenerative tissues stem cells may play a role, such a possibility is very low for lens regeneration. We have found that treating newts with exogenous retinoids after lentectomy has no effect on the regenerating lens. All-trans-retinoic acid, 9-cis-retinoic acid (injections), and retinol palmitate (solution) all failed to interfere with the regeneration of the lens in newt eye, nor were able to elicit lens regeneration from any other site in the eye (Table 1). Treatment of lentectomized eyes with beads soaked in retinoids or agonists to RAR and RXR resulted in no effects as well (not shown). To determine whether retinoic acid and RAR are required for normal lens regeneration, we performed experiments aiming in depriving the lentectomized eye of RA. In one series of experiments we blocked the synthesis of retinoic acid with disulfiram. Disulfiram has been shown to inhibit the action of retinal aldehyde dehydrogenase (Vallari and Pietruszko, 1982), thus preventing the synthesis of retinoic acid. Disulfiram has been shown to inhibit the synthesis of retinoic acid from retinaldehyde in the chick, mouse, and rat retina at concentrations between 1 and 10 M (McCaffery et al., 1992, 1993). Whereas at 20 days postlentectomy, the control newts had almost completely regenerated lenses, in newts treated with 10 M disulfiram, we found inhibition or retardation of lens regeneration in over 50% (19 of 35) of cases (Tables 1, 2). Figure 1 shows a lens inhibited by disulfiram. Interestingly, in some cases, we observed dedifferentiation and vesicle formation in the ventral iris as well that was comparable to that in the dorsal iris (Fig. 1). A second series of experiments involved blocking the action of retinoic acid receptors (RARs). To accomplish

3 590 TSONIS ET AL. Figure 1. Figure 2.

4 RETINOIC ACID IN LENS REGENERATION 591 this, we used the RAR antagonist (Allergan). This compound has been shown to be a high affinity antagonist of RA-induced function of all three RAR subtypes (alpha, beta, and gamma). Pooling results from all delivering methods used, approximately 60% (43 of 71) of cases showed inhibition, retardation, or abnormal morphogenesis of the lens (Tables 1, 2). Altering the concentration or number of beads did not produce a significant difference in results. Changing the concentration of the antagonist in solution did produce an effect, however. At 22 M, the lens was effected in 50% (8 of 16) of cases, whereas at 50 M the lens was affected in 75% (9 of 12) of cases (Table 2). Such affected regenerated lenses are shown in Figure 1. However, the most interesting result of the antagonist treatments involved the site at which lens regeneration occurred. In some cases, we observed ectopic lens regeneration (Fig. 1). The most spectacular was a case of lens regeneration from the cornea (Fig. 1E,F) a process possible only in premetamorphic frogs (Filoni et al., 1997). Despite that these cases of ectopic lens regeneration are few, they definitely are very significant and suggest that RARs are involved in the different modes of lens regeneration occurring in different animals. The reader should bear in mind that such an ectopic regeneration can never occur in adult newts, and it has never been observed under normal circumstances. In our laboratory and over the past few years, we have used several hundreds control regenerating eyes that have received a bead soaked in DMSO, without seeing any effect on lens regeneration. In this context the effect of beads soaked in antagonist cannot be an artifact. Also after each lentectomy, the lens is checked macroscopically for any part (lens epithelium) left behind. Any suspicious cases are never used. But the best argument that the effects of RAR antagonist are real and specific is that all ectopic cases (3 of 10) were resulted with a particular treatment (administration of one bead soaked in 20 mg/ml solution). In all cases of ectopic lens regeneration, the normal regeneration from the dorsal iris was inhibited, crediting the arguments against any artifact. Therefore, these results clearly implicate retinoids and their receptors in the control of lens regeneration, possibly by a concentration-specific mechanism. It is possible that PE cells capable of transdifferentiation are very sensitive to a particular concentration of retinoids or RARs or susceptible within a specific time window. The different modes of regeneration might be controlled by the differential use of retinoid isoforms and their receptor dimer choice. Obviously, surpassing a concentration threshold would have no effect on these mechanisms and perhaps that is why we did not observe any effect with the exogenous retinoids. However, lowering the concentration of retinoids and, therefore, reducing the number of functional receptors, can result in impairment of lens regeneration from the dorsal iris and trigger it from other places. This could suggest the existence of retinoid gradients in the eye. Such gradients have been shown in mice (McCaffery et al., 1992), but nothing is known about them in newts and their role in lens regeneration. Alternatively, it could be that when antagonists are bound to RARs, they keep the receptors in the conformation that can interact with co-repressors. This, in turn, would change the number or receptors that can be occupied by agonists (Soderstrom et al., 1997; Perissi et al., 1999). In all, our results provide the first concrete evidence of a role of retinoids in lens regeneration and strongly suggest that the use of retinoids/receptors might elucidate the mechanisms involved in the different types of lens regeneration (e.g., newt vs. frog) and its absence in other vertebrates. The above data clearly allude that RARs are expressed in the regenerating newt lens. Therefore, an important corroboration would be to show expression of Fig. 1. A: A section through a normal lens (control) regenerated 20 days post lentectomy (10 ). Note that 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 F: A case of lens regeneration from the cornea. E: Note the continuity between the regenerating lens (arrowhead) and the cornea (c). F: We can observe the differentiation of elongated primary lens fibers in the posterior part of the lens, which is a dominant feature of lens morphology (arrowheads) (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. Fig. 2. Expression of RAR-delta during lens regeneration. A: A section through an eye with an intact lens. No expression is evident in the lens (4 ). B: A section through the retina of an intact eye. Some weak expression of RAR-delta is detected in the ganglion cells (g) (20 ). C: A section through a 10-day regenerating lens. Expression can be seen in the dedifferentiating dorsal iris as the lens vesicle is formed (arrowhead) (20 ). D: A section through a 15-day regenerating lens. Again, the expression is evident in the regenerating lens (20 ). E: A section though a regenerating lens 20 days postlentectomy. Note high expression in the lens epithelium and lens fibers (20 ). F: A section through a 15-day regenerating lens hybridized with the sense probe (negative control). Note the absence of any staining (20 ). G,H: Sections through the ventral iris corresponding to the same stages as in C and D. Even though the cells are obscured by the heavy pigmentation, not much purple color comparable to the dorsal counterparts (indicating expression) is apparent.

5 592 TSONIS ET AL. RARs in the newt eye. To address this question, we used a newt RAR-delta probe. This particular gene was selected because it has been shown to be instrumental for the effects of retinoic acid on limb regeneration in the newt (Pecorino et al., 1996). The in situ hybridization showed some very interesting results. RAR-delta is expressed in very low levels in the intact newt eye. Most of this expression is restricted in the ganglion cell layer, whereas the lens does not show any significant level above background. However, as the pigment epithelial cells dedifferentiate and start forming a vesicle, the receptor is expressed. Its expression reaches higher levels as the vesicle grows, with highest when lens fibers differentiate (Fig. 2). These results indicate that RAR-delta is important for the transdifferentiation process as well for the differentiation of lens fibers and can explain why inhibition of RAR receptors can affect the regenerating lens. The clear difference in expression patterns with the intact lens further strengthens the role of this receptor in the regeneration of the lens. This is the first transcription factor that seems to be specifically regulated in the regenerating lens when compared with the intact lens. Such a unique regulation could become an important tool in understanding the mechanisms involved in lens regeneration. EXPERIMENTAL PROCEDURES Animal Operations Newts (Notophthalmus viridescens) were lentectomized under general anesthesia by using 1% 3-aminobenzoic acid ethyl ester (Sigma). Eyes were collected at different stages of lens regeneration, fixed, embedded in paraffin, and processed for either histologic staining or in situ hybridization. Treatment With Exogenous Retinoids Newts were treated with 9-cis-retinoic acid and alltrans-retinoic acid at 100 g/g body weight by injection at 4 days postlentectomy and by immersing them in solution of 10 units/ml retinol palmitate for 5 days immediately after lentectomy. We used these concentrations based on the effects they elicit during limb regeneration (Tsonis, 1996). Newts were also treated with beads soaked in 9-cis-retinoic acid, all-trans-retinoic acid, an RAR agonist (AGN , Allergan), and an RXR agonist (AGN , Allergan). Beads were soaked in 10 or 20 mg/ml solution of these substances in DMSO. We used the AG1-X2 formate beads ( mesh) from Bio-Rad. Treatment With Disulfiram and RAR Antagonist Newts were treated with disulfiram, a compound that prevents the synthesis of retinoic acid (Vallari and Pietruszko, 1982; Maden, 1997). Disulfiram was administered in 10 M solution by first dissolving it in DMSO and then adding it directly to the water. Experimental animals were kept in disulfiram continuously after lentectomy, and control animals were kept in the same concentration of DMSO. Disulfiram was also administered by bead. Beads were soaked in DMSO overnight. Experimental beads were soaked overnight in either 50 mg/ml or 225 mg/ml of disulfiram in DMSO. Newts were also treated with a potent RAR-specific antagonist (193109, Allergan) (Standenev et al., 1996) by several different methods. Experimental beads were soaked overnight in the RAR antagonist at either 10 or 20 mg/ml in DMSO. Beads were then placed into the center of the eye immediately after lentectomy. Some newts received an additional bead inserted ventrally and posteriorly to the original cut 11 days postlentectomy. Control newts received DMSO-soaked beads. Newts were also treated by immersing them into a solution of the antagonist. The antagonist was dissolved in DMSO and was added to the water at either 22 Mor50 M. The newts in solution were treated for 24 hours once every 3 days up to 25 days postlentectomy. Newt RAR-delta Probe A newt probe for RAR-delta (homologue of mammalian gamma1) (Pecorino et al., 1996) was a gift from Dr. Jeremy P. Brockes (University College London). The probe was transcribed with T3 and T7 RNA polymerases to create sense and antisense transcripts and labeled with digoxigenin-11 UTP. The probes were then hydrolyzed with alkali and used for in situ hybridization with sections of regenerating lens taken at different times after lentectomy. In Situ Hybridization This technique was performed as described extensively in other studies (Del Rio-Tsonis et al., 1998, 1999). ACKNOWLEDGMENT P.A.T. received support from the National Institutes of Health. REFERENCES Del Rio-Tsonis K, Washabaugh CH, Tsonis PA Expression of pax-6 during urodele eye development and lens regeneration. 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