Molecular and Cellular Aspects of Amphibian Lens Regeneration

Home , Metamorphosis, Regeneration (biology), SOX3, Xenopus

Progress in Retinal and Eye Research xxx (2010) 1e13

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Progress in Retinal and Eye Research

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Molecular and cellular aspects of amphibian lens regeneration

Jonathan J. Henry a,*, Panagiotis A. Tsonis b,* a Department of Cell and Developmental Biology, University of Illinois, Urbana, IL 61801, USA b Department of Biology and Center for Tissue Regeneration and Engineering, University of Dayton, Dayton, OH 45469-2320, USA

abstract

Lens regeneration among vertebrates is basically restricted to some amphibians. The most notable cases are the ones that occur in premetamorphic frogs and in adult newts. Frogs and newts regenerate their lens in very different ways. In frogs the lens is regenerated by transdifferentiation of the cornea and is limited only to a time before metamorphosis. On the other hand, regeneration in newts is mediated by transdifferentiation of the pigment epithelial cells of the dorsal iris and is possible in adult animals as well. Thus, the study of both systems could provide important information about the process. Molecular tools have been developed in frogs and recently also in newts. Thus, the process has been studied at the molecular and cellular levels. A synthesis describing both systems was long due. In this review we describe the process in both Xenopus and the newt. The known molecular mechanisms are described and compared. Ó 2010 Published by Elsevier Ltd.

Contents

1. Background ...... 00 2. Lens regeneration in Xenopus ...... 00 2.1. Morphological events ...... 00 2.2. Molecular aspects of lens regeneration in Xenopus ...... 00 2.3. Signaling factors involved in Xenopus lens regeneration ...... 00 3. Studies of lens regeneration and development in Xenopus ...... 00 4. Lens regeneration in newts ...... 00 4.1. Morphological events ...... 00 4.2. Gene regulation ...... 00 4.3. Signaling pathways in newt lens regeneration ...... 00 4.4. MicroRNAs ...... 00 4.5. Knock-down technology and gene expression in newts ...... 00 4.6. Transdifferentiation in newts: a model for stem cell differentiation? ...... 00 5. Xenopus and newt lens regeneration: some additional questions ...... 00 Acknowledgements ...... 00 References...... 00

1. Background remarkable because the lens is regenerated by transdifferentiation of terminally differentiated somatic cells. Regeneration of the lens in Among vertebrates the ability of regenerating the eye lens is adult newts was observed ﬁrst by Collucci (1891) and independently mainly restricted to frogs and newts. Such regeneration is also by Wolff (1895), after whom the process is often called Wolfﬁan regeneration. The process of lens regeneration in frogs was characterized more recently by Freeman (1963). There are, however, some * Corresponding authors. E-mail addresses: [email protected] (J.J. Henry), panagiotis.tsonis@notes. basic differences between regeneration in frogs and newts. Frogs can udayton.edu (P.A. Tsonis). normally regenerate the lens during a certain period before

1350-9462/$ e see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.preteyeres.2010.07.002

Please cite this article in press as: Henry, J.J., Tsonis, P.A., Molecular and cellular aspects of amphibian lens regeneration, Progress in Retinal and Eye Research (2010), doi:10.1016/j.preteyeres.2010.07.002 2 J.J. Henry, P.A. Tsonis / Progress in Retinal and Eye Research xxx (2010) 1e13 metamorphosis and the source of the regenerated lens is the cornea a new lens will occur in the presence of the original lens provided an epithelium. On the other hand, lens regeneration in newts occurs opening is created that allows for retinal factors to reach the corneal throughout their adult life and is mediated via transdifferentiation of epithelium (Reeve and Wild, 1978; Filoni et al., 1978, 1980; Bosco et al., the dorsal iris pigment epithelial cells (PECs). A few other organisms 1979, 1980). The process of lens regeneration can be recapitulated in can apparently replace the lens via Wolffian lens regeneration, culture (Bosco et al., 1993, 1994, 1997a,b). For example, trans- including cobitid fish (Sato, 1961; Mitashov, 1966). In this review we differentiation will occur in co-cultures of cornea epithelial and neural will outline and compare these processes mainly in both amphibians retina tissues (Fig. 2D). In fact, the efficiency is nearly as good, or even with a focus on the molecular and cellular aspects. In recent years, better than that which occurs in vivo. This represents a very convenient advances in molecular technology and manipulation in these animals system in which to study this process under a more controlled set of has led to important understandingof the process andits relevance to conditions. Direct contact between the cornea epithelium and the regenerative biology in general, and positions these models as neural retina does not appear to be required for lens regeneration to excellent systems in which to undertake further studies. take place, as lens cell differentiation will occur in cultures of corneal epithelium grown in retina-conditioned media (Bosco et al., 1997a). 2. Lens regeneration in Xenopus Together, these findings indicate that the retinal tissue provides diffusible factors sufficient to support lens regeneration. 2.1. Morphological events Through a variety of tissue transplantation experiments, it has been shown that only the larval cornea and the pericorneal Though not as extensive as the forms of regeneration displayed by epidermis, which includes a combined area twice the diameter of certain urodele amphibians (e.g., newts and salamanders), some the eyecup, has the potential to form lens cells in X. laevis (Freeman, species of anurans (frogs) exhibit the capacity to regenerate certain 1963). The competence of this larval tissue to respond to these structures such as the intestine, larval tail (including the spinal cord, signals is established by developmental history, which includes the and muscles), limb structures, and even parts of the eye (including the initial series of early and late embryonic lens inductive interactions retina, and lens; reviewed by Henry, 2003; Slack et al., 2004, 2008; and the presence of the eyecup (Bosco and Filoni, 1992; Cannata Callery, 2006; Tseng and Levin, 2008; Henry et al., 2008; Beck et al., et al., 2003; Arresta et al., 2005). Once competence is established, 2009; Filoni, 2009). The most widely studied species are African the continued presence of the eyecup does not appear to be required frogs of the genus Xenopus.Onespeciesinparticular,Xenopus laevis, to maintain this condition (Cannata et al., 2003), since the eye can be has been used for over 60 years as an experimental model system in removed late during embryogenesis or from the older larvae and the studies of development and regeneration (Kay and Peng, 1991; overlying tissues will still retain the ability to respond if transplanted Tinsley and Kobel, 1996; Gurdon and Hopwood, 2000; Beck and into the vitreous chamber. Slack, 2001; Henry, 2003; Callery, 2006; Henry et al., 2008). More The success and extent of lens regeneration gradually decreases as recently, many researchers have adopted a related species, Xenopus the larvae approach metamorphosis (Freeman, 1963; Filoni et al., tropicalis. The latter has certain advantages that include a shorter 1997). This appears to be due to the increasing rapidity with which generation time and X. tropicalis is a true diploid, in contrast to the the inner corneal endothelium heals back to cover the pupillary allotetraploid condition found in X. laevis. These features and the opening, and mayalso be related to the extent of differentiation of the smaller genome size favored the selection of X. tropicalis for genome cornea epithelium. Experimentally, one can demonstrate that the sequencing, which was been completed by the U.S. Department of capacity to differentiate lens cells is still present in the corneal Energy’s Joint Genome Institute (http://genome.jgi-psf.org/Xentr4/ epithelium throughout this time interval and apparently even after Xentra4.home.html). Given the recent advances in high-throughput metamorphosis, if one exposes this tissue to the key retinal factors via sequencing technology, an effort is being mounted to sequence the transplantation into the vitreous chamber (Freeman and Overton, genome of X. laevis (http://www.xenbase.org/common/), which will 1962; Filoni et al., 1997). Likewise, the factors provided by the serve as an additional resource for molecular level studies, like those neural retina are still present in adult eyes (Bosco and Willems,1992). described below. Researchers have demonstrated that corneal epithelium of post- Species in the genus Xenopus are the only frogs known to regen- metamorphic frogs can undergo transdifferentiation, if implanted erate lenses (reviewed by Henry, 2003; Filoni, 2009). Lens regeneration into the vitreous chamber of either pre- or post-metamorphic frogs in X. laevis was first described by Freeman (1963) and differs signifi- (Freeman and Overton, 1961; Bosco and Willems, 1992; Bosco and cantly from that of Wolffian lens regeneration, which has been well Filoni, 1992; Filoni et al., 1997). More recently, Yoshii et al. (2007) described for newts and salamanders (discussed below). Following observed examples of lens regeneration in adult Xenopus and sug- removal of the original lens in Xenopus tadpoles, the inner layer of the gested that these may arise from small numbers of lens epithelial outer corneal epithelium undergoes a series of transformations to cells that remain in the eye following incomplete removal of the reform a new lens. Freeman sub-divided this process into 5 stages, original lens (though no attempt to trace these cells was made in that which are illustrated in Fig.1. This process has been described as one of study to prove this claim). transdifferentiation (metaplasia) of the corneal epithelium (though see X. tropicalis is also capable of regenerating lenses though the further discussion, below), and shares many similarities to that of success rate is much lower than that found in X. laevis (Fig. 2AeC; embryonic lens formation, but there are also some interesting differ- Henry and Elkins, 2001). In the former species the inner corneal ences (Henry, 2003; discussed below). Lens formation typically occurs endothelium heals more rapidlycompared to that in X. laevis, and this in the cornea epithelium that lies directly over the pupillary opening tends to cut off the critical signaling factors required to support lens and requires secreted factors provided by the neural retina (Freeman, regeneration. Another species, Xenopus borealis does not normally 1963; Filoni et al., 1981, 1982, 1983). Under normal circumstances, regenerate a lens following lentectomy, due to the rapidity with the physical presence of the inner cornea endothelium (derived from which the inner cornea heals back to cover the pupillary opening neural crest) and the lens prevent these factors from reaching the (Filoni et al., 2006). On the other hand, the corneal epithelium of X. cornea epithelium (Freeman,1963; Filoni et al.,1997; Henry and Elkins, borealis is capable of forming a lens if implanted directly into the 2001). Removal of either barrier alone will not initiate lens regenera- vitreous chamber where it can be continuously exposed to the key tion (Bosco et al., 1980; Filoni et al., 1980; Cioni et al., 1982). Further- inducing factors. In contrast, the corneal epithelium of other frog more, experiments reveal that the lens and inner cornea do not species, (e.g., members of the genera Rana, Bufo, Hyla and produce inhibitors of lens regeneration. For instance, regeneration of Disscoglossus) do not appear to have the competence to respond to

Fig. 1. Cornea-lens transdifferentiation in X. laevis. Sections show different stages of lens regeneration following removal of the lens (stages follow the convention of Freeman, 1963). A. Stage 1. During this stage cells of the inner corneal epithelium assume a cuboidal shape within 24 h following lens removal. B. Stage 2. During this stage, cells begin to assume a thickened placodal arrangement. Nuclei of these cells are found to contain one nucleolus (characteristic of lens epithelial cells), rather than two (characteristic of cornea epithelial cells). C. Early stage 3. D. Middle stage 3. E. Late stage 3. During stage 3, a loosely organized aggregate of cells begins to separate from the corneal epithelium. These cells begin to organize with distinct apical-basal polarity to form a lens vesicle. F. Early stage 4. G. Middle stage 4. H. Late stage 4. During stage 4 a definitive lens vesicle is present. Elongated primary lens fiber cells have formed on the side closest to the vitreous chamber. Typically the lens vesicle is separated from the overlying corneal epithelium at this stage. I. Early stage 5. During this stage, secondary lens fiber cells are being added from the lens epithelium at the equatorial zone. Nuclei of the primary fiber cells begin to disappear. This stage is followed by further addition of secondary fiber cells and growth of the lens. ce, corneal epithelium; cn, corneal endothelium; ic, inner layer of the corneal epithelium; ir, iris; oc, outer layer of the corneal epithelium; le, lens epithelium; lf, lens fibers; rl, regenerating lens vesicle. Refer to text for further details. Figure from Henry (2003), after Freeman (1963). Scale bar equals 25 mm. these inductive signals; however, it appears that the eyes of all signals that trigger lens regeneration are either short ranged and/or frog species examined do produce the factors required to support the response is concentration dependant. The initial observations regeneration, as established in xenoplastic (cross-species) trans- made by Freeman (1963) indicate that lens-forming transformations plantation experiments (reviewed by Henry and Elkins, 2001; Henry, initially occur at multiple foci within this region (each reaching 2003 and Filoni et al., 2006). stages 1e2, refer to Fig. 1 for description of these stages), however, As mentioned above, the new lens normallyarises from the tissue only one of these foci eventually forms a lens. The other foci appear that lies directly over the pupillary opening. This suggests that the to regress. Rarely does one see the formation of multiple lenses. This

Fig. 2. In vivo and in vitro lens regeneration in living transgenic X. tropicalis and X. laevis larval tissues carrying a transgene encoding the jellyfish green fluorescent protein (GFP) coupled to a g-crystallin enhancer/promoter. Combined light and epifluorescence micrographs show expression of GFP (green) in the regenerating lenses of these examples. A. Eye of transgenic X. tropicalis following removal of the lens. Note absence of differentiated lens cells and GFP in the eye at one week following lens removal. B. Eye at 10 days following lens removal. Note the presence of a small regenerating lens containing GFP expressing cells. C. Eye at 21 days following lens removal. Note the growth of a larger regenerating lens containing GFP expressing cells. D. In vitro transdifferentiation in co-cultures of transgenic X. laevis corneal epithelium and non-transgenic larval eyecup. Note GFP expressing lens cells derived from the transgenic corneal epithelium. pl, pupil; tl, transdifferentiating lens cells. AeC from Henry et al. (2008), after Henry and Elkins (2001). D, from L. Fukui and J. Henry (unpublished data, University of Illinois, Champaign-Urbana). Scale bar equals 200 mm.

Please cite this article in press as: Henry, J.J., Tsonis, P.A., Molecular and cellular aspects of amphibian lens regeneration, Progress in Retinal and Eye Research (2010), doi:10.1016/j.preteyeres.2010.07.002 4 J.J. Henry, P.A. Tsonis / Progress in Retinal and Eye Research xxx (2010) 1e13 suggests that some mechanism may operate to ensure that only one (Pannese et al., 1995; Kablar et al., 1996; Cvekl and Piatigorsky, 1996; lens is ultimately formed, possibly involving some form of lateral Zygar et al.,1998; Schaefer et al.,1999; Cui et al., 2004). As is the case in inhibition (see further discussion below). other organisms, ectopic expression of Pax6 leads to the formation of ectopic eyes and lenses in Xenopus (Altmann et al., 1997; Chow et al., 2.2. Molecular aspects of lens regeneration in Xenopus 1999). Sox3 is a member of the SOX family of SRY testis determining factor family of HMG box transcription factors known to play Patterns of crystallin expression have been characterized during important roles in eye development (Penzel et al., 1997; Furuta and lens regeneration in X. laevis and some interesting differences have Hogan, 1998; Zygar et al., 1998; Ashery-Padan et al., 2000; Koster been found compared to the situation encountered during embry- et al., 2000). Sox proteins play roles in regulating crystallin expres- onic lens formation. The use of antibodies reveals that a- and sion (Uwanogho et al., 1995; Kamachi et al., 1995, 1998, 2001; b-crystallinproteins are detected in both fibercells and lens epithelial Nishiguchi et al., 1998; Chow and Lang, 2001; Ishibashi and Yasuda, cells beginning at Freeman stage 4 of lens regeneration, while 2001). Pitx3 is a paired-like (bicoid) homeodomain containing tran- g-crystallin proteins are apparently restricted only to lens fiber cells scription factor expressed early in presumptive and placodal lens (Campbell,1965, Brahma and McDevitt,1974; Campbell and Truman, ectoderm (Chang et al., 2001; Pommereit et al., 2001). Pitx3 and 1977; Reeve and Wild, 1978; Brahma, 1980; Henry and Mittleman, a related gene Pitx1 are required for normal lens and retinal devel- 1995). During embryonic lens development translation of various opment (Semina et al., 2000; Khosrowshahian et al., 2005; Shi et al., crystallins, however, is restricted to lens fiber cells, beginning around 2005; Medina-Martinez et al., 2009). Another gene, Prox1 encodes stage 29/30 of development (stages of Nieuwkoop and Faber, 1956). a prospero-like transcription factor, which is initially expressed in the The transcription of certain crystallin genes has also been examined lens placode (Oliver et al., 1993; Tomarev et al., 1996, 1998; Schaefer during lens regeneration (Schaefer et al., 1999; Mizuno et al., 1999a; et al., 1999; Chow and Lang, 2001; Reza et al., 2002). Prox1 is impor- Malloch et al., 2009). Again, some interesting differences have been tant for lens fiber cell differentiation (Wigle et al., 1999) and serves to observed compared to the patterns of expression detected during activate crystallin expression (Cvekl and Piatigorsky,1996; Ring et al., embryonic stages. For instance, aA-, and bB1-crystallin transcripts 2000). are first detected in presumptive lens fiber cells of the regenerated Significantly, Otx2, Pax6, Sox3, and Prox1 are all expressed during lens vesicle (middle to late Freeman stage 3), and subsequently only lens regeneration in Xenopus (Schaefer et al., 1999; Mizuno et al., in differentiated lens fiber cells at later stages. g-crystallin transcripts 1999b; Cannata et al., 2003). Furthermore, Henry et al. (2002) are not detected until early Freeman stage 4 of regeneration and only showed that Pax6 is expressed in larval corneal epithelium prior to in lens fiber cells. However, during embryogenesis, aA- bB1- and g- removal of the lens. Following lens removal, Pax6 expression appears crystallin transcripts are detected simultaneously in the lens placode, to be up-regulated in the cornea and regenerating lens (Schaefer and only in differentiated lens fiber cells at later stages of develop- et al., 1999; Mizuno et al., 1999b; Cannata et al., 2003). Gargioli ment. These findings indicate that there are some differences in the et al. (2008) examined the expression of Otx2, Pax6, Sox3, Pitx3, regulation of crystallin expression during regeneration vs. develop- Prox1 and bB1-cry prior to lens removal in Xenopus. These investi- ment of the lens. In contrast, Mizuno et al. (2005) demonstrated that gators noted that only Pax6 is expressed in the larval ectoderm, and the expression of bB1-crystallin during lens regeneration requires further, its expression is restricted to the lentogenic area that includes the same promoter elements as those required during embryonic both the corneal and pericorneal ectoderm. They further demon- lens development, suggesting that elements of a shared regulatory strated that injections of exogenous Pax6 in early embryonic cells (at network appear to be operating in both of these lens-forming the 2-cell stage) trigger ectopic, endogenous Pax6 expression. processes. Presumably this occurred through auto-regulation of Pax6 expres- A large number of transcription factors play key roles in eye and sion, though they did not actually demonstrate that the injected, lens development (Beebe, 1994; Wride, 1996; Cvekl and Piatigorsky, exogenous message was translated. This treatment, however, was 1996; Graw, 1996, 2003; Fini et al., 1997; Oliver and Gruss, 1997; apparently sufficient to impart lentogenic capacity in flank Tomarev, 1997; Filoni et al., 1997; Jean et al., 1998; Kondoh, 1999; epidermis, as assayed by transplanting this tissue inside the vitreous Lupo et al., 2000, 2006; Ogino and Yasuda, 2000; Wawersik and chamber. These findings suggest that pax6 expression is linked with Maas, 2000; Hanson, 2001; Chow and Lang, 2001; Zhang et al., the acquisition of lens regeneration competence in larval ectoderm. 2002; Graw and Loster, 2003; Zuber et al., 2003; Zaghloul et al., Other researchers have described patterns of Pax6 expression as 2005; Adler and Canto-Soler, 2007; Cvekl and Duncan, 2007), being linked with embryonic lens-forming competence in other including Otx2, Pax6, Sox3, Pitx3,andProx1 as well as others. For vertebrate systems (e.g., chicken and rat, Li et al.,1994; Fujiwara et al., instance, Otx2 encodes an orthodenticle-related transcription factor 1994). that contains a bicoid class DNA-binding homeodomain. Otx2 acts as In order to further characterize the molecular level events that a “head-field selector” and is required for the development of the underlie the process of lens regeneration in X. laevis, a subtracted forebrain and midbrain (Chow and Lang, 2001; Ogino et al., 2008). cDNA library enriched for genes expressed during the first four days Otx2 is expressed early in embryonic head ectoderm, including the of this process was prepared (Freeman stages 1e3; Henry et al., 2002; presumptive lens ectoderm (Pannese et al., 1995; Kablar et al., 1996; Malloch et al., 2009). Representative clones were sequenced and the Zygar et al., 1998), and is required for the formation of the lens and information was submitted for BLAST analysis to characterize gene retinal pigmented epithelium in mice (Martinez-Morales et al., 2001). expression during the early stages of lens regeneration. A searchable Otx2 has recently been shown to play an important role in regulating database of information pertaining to each of these sequences is Notch induced FoxE3 expression (a forkhead transcription factor available on-line at www.life.illinois.edu/henry/EST_databases.html. related to Xenopus lens1), which is essential for lens formation A total of 734 unique genes were identified. The assemblage of genes (Kenyon et al., 1999; Ogino et al., 2008). Pax6 represents a central is typical of those associated with metazoan organismal develop- transcriptional regulator of eye development, containing two sepa- ment and includes a high concentration of genes involved in rate DNA binding domains that include a homeodomain and a paired regulating transcription and signal transduction. Groups with more box (Ashery-Padan and Gruss, 2001). Pax6 is expressed early during prevalent representation include transcription factors, proteins development in a relatively broad area of anterior embryonic ecto- involved in RNA synthesis and processing, integral membrane and derm and plays specific roles in eye development and lens cell transmembrane proteins, components of a number of conserved differentiation, including the regulation of crystallin gene expression signaling pathways, proteins involved in protein processing,

Please cite this article in press as: Henry, J.J., Tsonis, P.A., Molecular and cellular aspects of amphibian lens regeneration, Progress in Retinal and Eye Research (2010), doi:10.1016/j.preteyeres.2010.07.002 J.J. Henry, P.A. Tsonis / Progress in Retinal and Eye Research xxx (2010) 1e13 5 transport and degradation, proteins involved in matrix remodeling (Furuta and Hogan, 1998; Belecky-Adams et al., 2002; Chow and including a number of matrix metalloproteases (MMPs), regulators of Lang, 2001; Yang, 2004; Esteve and Bovolenta, 2006; Cvekl and the cell cycle, metabolism, apoptosis, inflammation and immune Duncan, 2007; Wordinger and Clark, 2007; Adler and Canto-Soler, responses, proteins involved in chromatin remodeling in addition to 2007). known lens proteins (e.g., crystallins). Using cultures of isolated larval corneal epithelium, Bosco et al. Of the transcription factors identified in this set, a number have (1994, 1997b); demonstrated that addition of FGF1 appears to be not been previously implicated in the process of lens formation; sufficient to trigger lens cell differentiation. Furthermore, Arresta though some, including eyes-absent homolog 2 and Prox1, are clearly et al. (2005) found that a commercial polyclonal antibody that important for eye/lens formation (Malloch et al., 2009). On the other recognizes the FGFR2 receptor (bek variant) labels stage 53 larval hand, elements of several signal transduction cascades are also cornea epithelium but not dorsal head or flank epidermis (stages of present, which have previously been shown to play important roles Nieuwkoop and Faber,1956). In addition, this antibody was found to in lens development, regeneration and differentiation, including the label dorsal head epidermis if this was previously subjected to eyes Bone Morphogenetic Protein (BMP), Delta-Notch, Wnt, FGF, implanted at stage 46 of larval development. These observations link Hedgehog, TGF-b, Rho-Ras and retinoic acid signaling pathways FGFR2 expression with the acquisition of lentogenic capacity, and (Manns and Fritzsch, 1991; Hyuga et al., 1993; Kastner et al., 1994; provide further evidence that FGF signaling may be involved in lens Bosco et al., 1994, 1997b; Kodama and Eguchi, 1995; Graw, 1996; regeneration in Xenopus. The Henry lab (University of Illinois, Cvekl and Piatigorsky, 1996; Li et al., 1997; McDevitt et al., 1997; Del Urbana-Champaign) also has several lines of evidence that FGF Rio-Tsonis et al., 1997, 1998, 2000; Gopal-Srivastava et al., 1998; signaling is functionally required in vivo for lens regeneration in X. Enwright and Grainger, 2000; Wagner et al., 2000; Tsonis et al., laevis (Fukui and Henry, unpublished data). 2000, 2002, 2004; de Iongh et al., 2001; Onuma et al., 2002; In another set of studies, Dr. Caroline Beck’s lab (University of Kawamorita et al., 2002; Stump et al., 2003; Beebe et al., 2004; Ang Otago, New Zealand) has shown that over-expression of Noggin in et al., 2004; Lang, 2004; Lupo et al., 2005; Lovicu and McAvoy, transgenic Xenopus larvae inhibits lens regeneration, suggesting that 2005; Grogg et al., 2005, 2006; Chen et al., 2008). elements of the TGF-b signaling pathways, specifically those Expression patterns for most (703) of the genes identified by involving the Bone Morphogenetic Proteins (BMPs) play an impor- Malloch et al. (2009) were examined during embryonic develop- tant role in this process (Caroline W. Beck, personal communication). ment, 634 of these exhibited some embryonic expression. Signifi- As mentioned above, the early observations of Freeman (1963) cantly, 57.7% of the latter are expressed in developing eye tissues and suggest that multiple, small populations of cells scattered over the nearly half (46.8%) included the developing lens. These findings pupillary opening begin to undergo transdifferentiation (reaching indicate that there are significant similarities, as well as some Freeman stages 1e2) but only one of these foci appears to form the differences, between the processes of lens development and lens news lens. An element of the Delta-Notch signaling pathway known regeneration. Some of the differences may be associated with the to play roles in lateral inhibition and boundaries-inductive mecha- processes of wound healing, as well as cellular dedifferentiation that nisms (Artavanis-Tsakonas et al., 1999; Schweisguth, 2004; Gazave may be associated with lens regeneration (Carinato et al., 2000; et al., 2009) is expressed during lens regeneration (including Mind Henry et al., 2002; Malloch et al., 2009). It will be important to bomb homolog 1, which encodes a E3 ubiquitin protein ligase known decipher the roles of those genes that only appear to be expressed in to regulate Delta-mediated Notch signaling, Malloch et al., 2009). regenerating lenses. Perhaps Delta-Notch signaling plays a role in regulating the devel- Comparison of the genes expressed during lens regeneration in opment of these foci. As argued above, some mechanism may Xenopus with those expressed in other regenerating tissues/organ- operate to ensure that only a single lens is ultimately regenerated. A isms, did not reveal a substantial conserved core of genes that appear similar mechanism could also function in the context of embryonic to underlie different regenerative phenomena (Malloch et al., 2009). lens development given that a larger field of competent tissue is There are, however, some commonalities. For instance, MMPs established early during development than actually generates the appear to be expressed in a number of different regenerating tissues, developing lens. As mentioned above, direct contact between the including regenerating lenses (Yang and Bryant, 1994; Miyazaki cornea epithelium and the retina is not required for lens regenera- et al., 1996; Yang et al., 1999; Kherif et al., 1999; Carinato et al., tion, and this might preclude the involvement of Delta-Notch 2000; Kato et al., 2003; Vinarsky et al., 2005; Odelberg, 2005; signaling in that particular interaction. Malloch et al., 2009). In addition, elements of the stress, inflammation and immune response pathways also appear to be expressed in 3. Studies of lens regeneration and development in Xenopus many regenerating systems, including Xenopus lens regeneration (Lu and Richardson, 1991; Meyer-Franke et al., 1998; Leon et al., As a model vertebrate system, Xenopus offers significant advan- 2000; Patruno et al., 2001; Imokawa and Brockes, 2003; Harty tages for the study of regeneration, which have been extolled in et al., 2003; Imokawa et al., 2004; Mescher and Neff, 2005; a number of recent reviews (Slack et al., 2004, 2008; Callery, 2006; Makino et al., 2005; Kanao and Miyachi, 2006; Brockes and Kumar, Tseng and Levin, 2008; Henry et al., 2008; Beck et al., 2009; Filoni, 2008; Filbin, 2006; Godwin and Brockes, 2006; Mescher et al., 2009). Tremendous molecular and genomic resources exist for this 2007; Pearl et al., 2008; Malloch et al., 2009). These researchers system (Klein et al., 2002, 2006; Carruthers and Stemple, 2006). propose conflicting hypotheses as to whether stress, inflammation Likewise, Xenopus has been used widely in the study of eye devel- and immune response pathways stimulate or inhibit regeneration. opment (Henry et al., 2008). Maintenance of a colony of adult frogs is rather simple and inexpensive, and the adults provide an abundant 2.3. Signaling factors involved in Xenopus lens regeneration supply of embryonic material, which is available year-round. Furthermore, the large eggs and embryos develop externally and The inductive signals that trigger lens regeneration have not yet rapidly. In addition, a vast array of molecular tools are available to been identified. There is some evidence, however, that they may be facilitate studies of gene function in these animals. As previously similar to those that are responsible for embryonic lens induction mentioned, these include the availability of a sequenced genome (Henryand Mittleman,1995; Cannata et al., 2003, 2008; Arresta et al., (http://genome.jgi-psf.org/Xentr4/Xentra4.home.html). The X. tro- 2005). Studies indicate that members of the FGF family and specific picalis genome has a high number of regions with long stretches of BMPs (BMP4, and BMP7) play key roles in eye and lens development synteny with mammalian genomes (each typically a hundred or

Please cite this article in press as: Henry, J.J., Tsonis, P.A., Molecular and cellular aspects of amphibian lens regeneration, Progress in Retinal and Eye Research (2010), doi:10.1016/j.preteyeres.2010.07.002 6 J.J. Henry, P.A. Tsonis / Progress in Retinal and Eye Research xxx (2010) 1e13 more genes). Hence, it is evolutionarily well positioned to identify tropicalis biology and genomics (e.g, “Xenbase,” www.xenbase.org conserved elements in vertebrate genomes (e.g., Ogino et al., 2008). and see also “Xine,” http://blumberg.bio.uci.edu/xine/index.htm). A It is a relatively simple matter to carry out both gain- and loss-of- long-standing course taught at Cold Spring Harbor covers topics function analyses to examine the necessity and sufficiency of specific related to the cell and developmental biology of Xenopus, and the genes. For example morpholinos may be used to knockdown trans- Embryology and Physiology Courses taught at the Marine Biological lation of specificmessages(Ekker, 2000; Corey and Abrams, 2001; Nutt Laboratory also cover Xenopus biology, training future generations of et al., 2001; Heasman, 2002; Gene Tools, LLC, Philomath, OR; www. scientists in the use of this system. These resources continue to gene-tools.com). Affymetrix produces micro-arrays to study gene position Xenopus as a key system for the study of regeneration and expression in both X. laevis and X. tropicalis (www.affymetrix.com). A development for many years to come. reverse genetics/TILLING resource (Stemple, 2004)attheSanger Centre (Cambridge, UK) has recently led to the identification of the first 4. Lens regeneration in newts null mutation in the Rx gene (a key homeobox regulator of eye formation, S. Carruthers, D. Stemple and R. Grainger. University of 4.1. Morphological events Virgina, Unpublished, see also Mathers et al., 1997) and other muta- tions are in the initial stages of characterization. In the case of the newt, lens regeneration involves trans- The relative ease with which one can prepare transgenic frogs is differentiation of the pigmented epithelial cells of the iris (PECs). a tremendous benefit and facilitates molecular functional studies at Following removal of the lens, the PECs re-enter the cell cycle, more advanced stages of larval development, which is essential for dedifferentiate, and lose their characteristic pigmentation. In vivo, studying regenerative phenomenon (Kroll and Amaya,1996; Amaya regeneration occurs from the dorsal iris population of PECs. Despite and Kroll, 1999; Amaya et al., 1998; Huang et al., 1999; Marsh- the apparent similarity between the dorsal and ventral PECs, the Armstrong et al., 1999; Offield et al., 2000; Sparrow et al., 2000; ability to regenerate through transdifferentiation under normal Hirsch et al., 2002; Ogino et al., 2006; Pan et al., 2006; Slack et al., conditions, belongs exclusively to the dorsal PECs. Interestingly, 2008: Beck et al., 2009). For instance, transgenic frogs carrying however, culturing of both dorsal and ventral PECs results in lentoid various genes under the control of the heat-shock (hsp70) promoter body formation (Del Rio-Tsonis and Tsonis, 2003; Tsonis and Del (see Wheeler et al., 2000) have been used to study tail and limb Rio-Tsonis, 2004). This means that the ventral PECs have the regeneration (Beck and Slack, 2001; Beck et al., 2003, 2006; Slack potential for transdifferentiation, but this is not permitted in vivo. et al., 2004; Lin and Slack, 2008). Beck et al. (2003, 2006), see also Early events of dedifferentiation occur through day 8 of the Slack et al. (2004) investigated the activity of the BMP and Notch regeneration process. Cell proliferation is initiated by day 4 post- signaling pathways during spinal cord and muscle regeneration in lentectomy. Visible dedifferentiation and loss of pigment from the the tail and limb, akin to the unpublished study by Beck’s lab that PECs begins by day 8. By day 10 of the process, a lens vesicle has examined lens regeneration (as described above). Knocking down formed, which represents the precursor to what will become a fully either BMP or Notch inhibited regeneration while activation of differentiated lens. After the lens vesicle has formed, the posterior either promoted regeneration. These experiments showed further cells start to elongate, express crystallins and differentiate to lens that BMP acts upstream of Notch in spinal cord regeneration, but fibers. The anterior cells become lens epithelium (Fig. 3)(Eguchi, these two pathways appeared to act independently in the case of 1963, 1964). muscle regeneration. Lin and Slack (2008) also used transgenic frogs Once lens differentiation has started the process is remarkably to show that FGF and Wnt signaling are important in Xenopus tail similar to lens development (as far as the sequential appearance of regeneration by expressing the Wnt inhibitor dkk1 and a dominant the different crystallins is concerned). Indeed, studies using anti- negative (truncated) form of an FGF receptor under the control of the bodies to crystallins or cDNA probes have conclusively shown that hsp70 heat-shock promoter. there is a parallel of their synthesis during development and regen- Tissues isolated from transgenic frogs carrying a GFP reporter eration. For instance, aA, bB1- and g-crystallins appeared all at the driven by the cytomegalovirus (CMV) promoter have been used in same time at the ventral-posterior portion of the lens vesicle (Sato transplantation experiments to follow the fates of cells that stage IVeV; nearly 10e12 days post-lentectomy (Yamada, 1977)), contribute to regenerated tissues in the tail (Gargioli and Slack, with g-crystallin (in contrast with the other two) being specific for 2004). This study demonstrated that notochord and spinal cord the lens fibers and not the lens epithelial cells (Mizuno et al., 2002). both regenerated from prexisting notochord and spinal cord stump tissues, respectively. However, muscles arose from a population of satellite “stem” cells, rather than from pre-existing myofibers. Chen 4.2. Gene regulation et al. (2006) showed further that this population of satellite cells specifically express Pax7. Using a similar approach, Lin et al. (2007) Focusing on the possible mechanisms of genetic regulation, it showed that melanophores do not arise from a pre-existing pop- would only make sense to hypothesize that different genes are at play ulation of unpigmented, melanophores precursors during tail in the dorsal and ventral iris creating two very different populations regeneration. The cre/lox system has also been employed to of cells, one that regenerates and one that does not, respectively. generate transgenic frogs that express yellow fluorescent protein as There are of course, many genes and proteins to consider in the a muscle specific lineage marker (under control of the cardiac actin regeneration process. As previously mentioned, a master regulator promoter) to show that differentiated muscle cells do not undergo gene in eye development, Pax6 is found in both the dorsal and ventral transdifferentiation to form other cell types during tail regeneration iris during Wolffian lens regeneration (Del Rio-Tsonis et al., 1995; (Ryffel et al., 2003). Transgenic X. tropicalis lines carrying a g1- Madhavan et al., 2006). The same is true for Six3, a partner of Pax6 crystallin promoter driving lens GFP expression have proven to be and by itself sufficient to induce lens when ectopically expressed in very useful for the study of lens development and regeneration frogs and fish (Oliver et al., 1996; Altmann et al., 1997). Prox1 is (Offield et al., 2000; Henry and Elkins, 2001, see Fig. 2AeC). Labs, a transcriptional regulator of crystallin synthesis and is also including our own, are now applying these transgenic approaches to expressed just before cells of the vesicle elongate and express crys- study lens regeneration. tallins (Del Rio-Tsonis et al., 1999). Interestingly these genes are also Finally, tremendous NIH-funded community web resources are expressed in the ventral iris tissues. This finding was unexpected and available that provide valuable information on X. laevis and X. it means that the ventral iris does initially undergo similar events as

Fig. 3. The process of lens regeneration in newts. Lens formation from the dorsal iris during regeneration shown through scanning electron microscopy. (A) Day 10 after lentectomy, a lens vesicle (grey) forms at the dorsal margin of the iris. (B) Day 14, elongation and further lens development as differentiation of lens fibers occurs at the posterior part (arrow) of the vesicle. (C) Day 20 of lens reformation. The anterior portion has become the lens epithelium (colored yellow). In contrast, lens fiber cells are forming in the posterior region. (D) Day 30, regeneration has concluded. the dorsal iris. In fact there may be a general inhibitory effect that collaborate with the Wnt pathway. When explants of dorsal iris were prevents further regeneration from taking place. treated with FGF2 in the presence of Wnt inhibitors, the action of To gain more insights about gene expression patterns, a micro- FGF2 was inhibited. Combined addition of FGF2 and Wnt3a was in array analysis with newt cDNA was utilized and it revealed that gene fact able to induce lens transdifferentiation of ventral explants as signatures in the dorsal and ventral iris are in fact very similar well (Hayashi et al., 2006). (Makarev et al., 2007). Even genes responsible for tissue remodeling, Interestingly, research on another signaling pathway, the Bone such as collagenase and cathepsin are present and up-regulated in Morphogenetic Protein pathway (BMP), revealed that its inhibition both dorsal and ventral irises during regeneration. Surprisingly allows the ventral iris to transdifferentiate into lens. Importantly, expression levels for some of these genes are even higher in the such induction was also observed when ventral PECs were trans- ventral iris. It is not unreasonable to suggest, given these unexpected fected with Six-3 and treated with retinoic acid (Grogg et al., 2005). results on gene expression, that we might encounter novel regula- These manipulations of signaling pathways and regulatory genes tory events during newt regenerative processes (Makarev et al., clearly indicate that induction of the ventral iris is possible and such 2007). EST analysis from irises 8 days after lentectomy, when observations might open newavenues in experimenting with higher dedifferentiation of PECs is ongoing, has also revealed very impor- animals that are unable to regenerate eye tissues. tant information. An initial set of 10,449 cDNA sequence reads were obtained. The total length of these sequences was 9.85 Mbp that was reduced to 4.82 Mbp by assembling overlapping reads using CAP3 4.4. MicroRNAs software (Huang and Madan,1999). This assembly was composed of 4725 unique sequences (1368 contigs and 3357 singlets). Of 1219 MicroRNAs, or miRNAs are short RNAs, about 22 nucleotides long, contigs and 3357 singlets, 780 contigs and 1666 singlets were which can bind to complementary sequences of RNA and subse- annotated by Blast2GO (Conesa et al., 2005). The 15 most frequently quently prevent mRNA translation. miRNAs may have multiple counted biological process terms, cellular component terms, and binding sequences due to their short size and can block translation molecular functions were reported (Maki et al., 2010). It is inter- on a wide scale. As such they might be involved in the transition from esting to note here that when compared with a same analysis one cell type to another as we see during regeneration. Cloning of performed during Xenopus lens regeneration the most frequent GO newt miRNA from the eye has pinpointed differential regulation in annotations were very similar (Table 1). GO (Gene Ontology) is used both dorsal and ventral iris. Some of the targets of the cloned miR- to describe the function of a gene as it pertains to processes and NAs were predicted to be FGFR2, and Sox9 (for miR-124a), Pax3, place in the cell. There are 3 independent sets of ontologies, the chordin, and TGFbR1 for let7b (Makarev et al., 2006). Based on this, molecular function (or molecular component, e.g. DNA binding), the further study of the role of miRNA regulation in regeneration biological process (e.g. transcription) and the cellular component (or compartment) where the gene product can be found (e.g. nucleus). Table 1 The 5 most frequent GO identified in Xenopus and Newt.

4.3. Signaling pathways in newt lens regeneration GO Xenopus Newt Biological Process Regulation of transcription, Regulation of transcription, A number of cell signaling pathways have been implicated in the DNA dependent DNA dependent process of lens regeneration in the newt. Members of the hedgehog Transcription Signal transduction signaling pathway are also expressed during the regeneration Transport Ribosome biogenesis process; Sonic hedgehog (Shh) and Indian hedgehog (Ihh)onlybeing Signal transduction Translation Multicellular organismal Electron transport expressed in the regenerating and developing lens, and no longer development expressed once the lens is fully reformed. When one interferes with Cellular Nucleus Cytoplasm this signaling pathway, the overall regeneration process is inhibited. Compartment Membrane Nucleus Also, cell proliferation rates are decreased and differentiation in the Cytoplasm Integral to membrane regenerating lens suffers (Tsonis et al., 2004). Retinoic acid receptors Integral to membrane Transcription factor complex are also expressed in the lens during regeneration and their inhibition Intracellular Extracellular space might account for aberrant lens formation (Tsonis et al., 2000, 2002). Molecular Protein binding Protein binding Exogenous FGFs have been shown to elicit regeneration of Component Metal ion binding ATP binding a second lens from the dorsal iris (Del Rio-Tsonis et al.,1997; Hayashi DNA binding Zinc ion binding Nucleotide binding RNA binding et al., 2004). It has been suggested that this action of FGFs is medi- Zinc ion binding DNA binding ated via an induction of cell proliferation. The FGF pathway seems to

Fig. 4. Pax6 morpholino treatment inhibits crystallin expression. Morpholinos were injected at day 10 after lentectomy, and animals were collected for assay at day 13 after lentectomy. (A and F) Lens vesicles from untreated control eyes express a- and b-crystallin (a-cry or b-cry). (B and G) Eyes treated with control morpholino (C-Mo) also express a and b crystallin. (D and I) Lens vesicles from eyes treated with Pax6 morpholino (Pax6-Mo) do not show any a-orb-crystallin expression. (C, E, H, and J) a-orb-crystallin staining only from B and D (C and E) and b-crystallin staining only from G and I (H and J).

Recently, it has been shown that differentiated mammalian cells, 1999; Mizuno et al., 1999a, 2005; Henry et al., 2002; Henry, 2003; including human, can also be reprogrammed to become induced Cannata et al., 2003; Malloch et al., 2009; Filoni, 2009). Answers to pluripotent stem cells (iPSCs) that can subsequently differentiate to these questions will require careful molecular characterization and different tissues. This reprogramming is mediated by inducing the comparison of the genes expressed in these tissues and the changes expression of four factors, Oct4, Sox2, c-myc and Klf4. Another factor, that occur during the processes of lens regeneration and nanog, seems to be also important (Takahashi and Yamanaka, 2006; development. Takahashi et al., 2007; Okita et al., 2007; Wernig et al., 2007). All It is unclear whether the larval cornea or the adult iris contains these are transcriptional factors expressed in embryonic stem cells. populations of specialized somatic stem cells. For instance, it is The critical question that arises from these studies is: Does reprog- known in other vertebrates that there exists a population of limbal ramming that mediates the formation iPSCs share any similarities to stem cells that supports repair of the cornea epithelium (Buck, reprogramming during regeneration in newts? A recent study 1985; Cotsarelis et al., 1989; Huang and Tseng, 1991; Auran et al., examined expression of these stem cell pluripotency-maintaining 1995; Lavker et al., 1998; Pellegrini et al., 1999; Tseng and Sun, factors during newt lens regeneration (Maki et al., 2009). Interest- 1999). We have looked for a population of slowly dividing stem ingly, there was significant regulation of three of the factors that we cells (BrdU-retaining-cells) in Xenopus and have not found any examined, Sox2, Klf4 and c-myc. Oct4 and nanog were not detected in evidence that these exist in the limbal region. Rather, these these tissues beyond the levels of negative control (-RT), however, preliminary investigations revealed that dividing cells are seen they were expressed in control ovarian tissues (meaning that the scattered throughout the cornea epithelium (see also Freeman, newt does have these genes). 1963). Hence, it is not clear if there is a specialized subpopulation During lens regeneration, Sox2 and Klf4 were up-regulated of stem cells within the cornea that support regeneration. Likewise during the very early stages of regeneration (day 2), while c-myc it is not believed at the present time that the newt iris contains showed a peak of expression at day 8. Day 2 marks an early response a stem cell population. Rather, all the evidence testifies for a clear to lens removal and is expected to be characterized by events that case of transdifferentiation. However, more studies on cell pop- may prepare pre-existing tissues for reprogramming and cell cycle ulations using advanced techniques will eventually clear this issue. re-entry. In fact, cell proliferation is not detected until day 4. Those Though the regenerated lenses appear to be normal on the basis rapid responses to lens removal prior to cell cycle re-entry are of morphological and histological criteria, no attempt has been similar to those observed for nucleostemin (Maki et al., 2007). c-myc made to examine lens function or visual acuity following regener- showed quite opposite patterns to Sox2 and Klf4. It was highly ation in either Xenopus or newts. In addition, it is unclear whether expressed at day 8, which correlated with the establishment of the other associated structures are also fully regenerated, such as the lens vesicle, but without major differences between dorsal and muscles that reposition the lens, etc. ventral iris. It should be noted here that the regeneration-incom- Finally, we do not understand what factors may prevent lens petent ventral iris does show activity such as cell cycle re-entry and regeneration from occurring in other vertebrates, including humans. gene expression, which must be suppressed later (Grogg et al., 2005; Do all vertebrates possess a similar conserved suite of genes that Madhavan et al., 2006). Therefore, expression differences between could be active in these processes? Some evidence suggests that dorsal and ventral iris might be correlated with these events. there might be species specific differences that underlie some The specific stage-related regulation of Sox2, Klf4 and c-myc,and regenerative phenomena (e.g., limb regeneration in newts, Kumar the absence of Oct4 and nanogexpression might indicatewhy the newt et al., 2007; Brockes and Kumar, 2008; Garza-Garcia et al., in cells do not become pluripotent. Manipulating these factors in the press). In the case of lens regeneration, barriers may be present to newt will open new avenues in the field and should be of enormous ensure that abnormal, supernumerary lenses do not form within the importance to compare or contrast the biology of regeneration in eye that would prevent regeneration from occurring. This could classical models and in stem cells. involve the production of specific inhibitors of lens regeneration, or it is possible that key inductive signals are either not present or the 5. Xenopus and newt lens regeneration: some additional receptors or downstream elements of the signal cascade are missing questions in eye tissues. Once we understand the factors required to support lens regeneration, and the reasons why some vertebrates are unable While there have been many advances in our understanding of to regenerate these structures, it may be possible to “restore” this the cellular and molecular mechanisms that control lens regenera- capacity, even in humans, as a therapeutic approach to treat injured tion in Xenopus, and newts, a number of key questions still need to be or diseased lenses. addressed in these systems, in addition to those already raised above. The process of “transdifferentiation” is described as one that takes a population of differentiated cells though an initial process of Acknowledgements cellular dedifferentiation, followed by re-differentiation along an altered trajectory (e.g., Eguchi and Kodama, 1993; Burke and Tosh, The authors’ research is supported by NIH/NEI grant EY09844 2005). 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