Tissue interaction between the retinal pigment epithelium and the choroid triggers retinal regeneration of the newt Cynops pyrrhogaster

Complete retinal regeneration in adult animals occurs only in certain urodele amphibians, in which the retinal pigmented epithelial cells (RPE) undergo transdifferentiation to produce all cell types constituting the neural retina. A similar mechanism also appears to be involved in retinal regeneration in the embryonic stage of some other species, but the nature of this mechanism has not yet been elucidated. The organ culture model of retinal regeneration is a useful experimental system and we previously reported RPE transdifferentiation of the newt under this condition. Here, we show that cultured RPE cells proliferate and differentiate into neurons when cultured with the choroid attached to the RPE, but they did not exhibit any morphological changes when cultured alone following removal of the choroid. This finding indicates that the tissue interactions between the RPE and the choroid are essential for the former to proliferate. This tissue interaction appears to be mediated by diffusible factors, because the choroid could affect RPE cells even when the two tissues were separated by a membrane filter. RPE transdifferentiation under the organotypic culture condition was abolished by a MEK (ERK kinase) inhibitor, U0126, but was partially suppressed by an FGF receptor inhibitor, SU5402, suggesting that FGF signaling pathway has a central role in the transdifferentiation. While IGF-1 alone had no effect on isolated RPE, combination of FGF-2 and IGF-1 stimulated RPE cell transdifferentiation similar to the results obtained in organ-cultured RPE and choroid. RT-PCR revealed that gene expression of both FGF-2 and IGF-1 is up-regulated following removal of the retina. Thus, we show for the first time that the choroid plays an essential role in newt retinal regeneration, opening a new avenue for understanding the molecular mechanisms underlying retinal regeneration.     

Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: A study of retinal regeneration in a novel animal model

Our research group has extensively studied retinal regeneration in adult Xenopus laevis. However, X. laevis does not represent a suitable model for multigenerational genetics and genomic approaches. Instead, Xenopus tropicalis is considered as the ideal model for these studies, although little is known about retinal regeneration in X. tropicalis. In the present study, we showed that a complete retina regenerates at approximately 30 days after whole retinal removal. The regenerating retina was derived from the stem/progenitor cells in the ciliary marginal zone (CMZ), indicating a novel mode of vertebrate retinal regeneration, which has not been previously reported. In a previous study, we showed that in X. laevis, retinal regeneration occurs primarily through the transdifferentiation of retinal pigmented epithelial (RPE) cells. RPE cells migrate to the retinal vascular membrane and reform a new epithelium, which then differentiates into the retina. In X. tropicalis, RPE cells also migrated to the vascular membrane, but transdifferentiation was not evident. Using two tissue culture models of RPE tissues, it was shown that in X. laevis RPE culture neuronal differentiation and reconstruction of the retinal three‐dimensional (3‐D) structure were clearly observed, while in X. tropicalis RPE culture neither ßIII tubulin‐positive cells nor 3‐D retinal structure were seen. These results indicate that the two Xenopus species are excellent models to clarify the cellular and molecular mechanisms of retinal regeneration, as these animals have contrasting modes of regeneration; one mode primarily involves RPE cells and the other mode involves stem/progenitor cells in the CMZ. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 739–756, 2014     

Upregulation of matrix metalloproteinase triggers transdifferentiation of retinal pigmented epithelial cells in Xenopus laevis: A Link between inflammatory response and regeneration

In adult Xenopus eyes, when the whole retina is removed, retinal pigmented epithelial (RPE) cells become activated to be retinal stem cells and regenerate the whole retina. In the present study, using a tissue culture model, it was examined whether upregulation of matrix metalloproteinases (Mmps) triggers retinal regeneration. Soon after retinal removal, Xmmp9 and Xmmp18 were strongly upregulated in the tissues of the RPE and the choroid. In the culture, Mmp expression in the RPE cells corresponded with their migration from the choroid. A potent MMP inhibitor, 1,10‐PNTL, suppressed RPE cell migration, proliferation, and formation of an epithelial structure in vitro. The mechanism involved in upregulation of Mmps was further investigated. After retinal removal, inflammatory cytokine genes, IL‐1β and TNF‐α , were upregulated both in vivo and in vitro. When the inflammation inhibitors dexamethasone or Withaferin A were applied in vitro, RPE cell migration was severely affected, suppressing transdifferentiation. These results demonstrate that Mmps play a pivotal role in retinal regeneration, and suggest that inflammatory cytokines trigger Mmp upregulation, indicating a direct link between the inflammatory reaction and retinal regeneration. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1086–1100, 2017     

Activin signaling limits the competence for retinal regeneration from the pigmented epithelium

Regeneration of the retina in amphibians is initiated by the transdifferentiation of the retinal pigmented epithelium (RPE) into neural progenitors. A similar process occurs in the early embryonic chick, but the RPE soon loses this ability. The factors that limit the competence of RPE cells to regenerate neural retina are not understood; however, factors normally involved in the development of the eye (i.e. FGF and Pax6) have also been implicated in transdifferentiation. Therefore, we tested whether activin, a TGFbeta family signaling protein shown to be important in RPE development, contributes to the loss in competence of the RPE to regenerate retina. We have found that addition of activin blocks regeneration from the RPE, even during stages when the cells are competent. Conversely, a small molecule inhibitor of the activin/TGFbeta/nodal receptors can delay, and even reverse, the developmental restriction in FGF-stimulated neural retinal regeneration.     

Neural cell differentiation from retinal pigment epithelial cells of the newt: An organ culture model for the urodele retinal regeneration

Transdifferentiation from retinal pigment epithelium (RPE) to neural retina (NR) was studied under a new culture system as an experimental model for newt retinal regeneration. Adult newt RPEs were organ cultured with surrounding connective tissues, such as the choroid and sclera, on a filter membrane. Around day 7 in vitro, lightly pigmented “neuron‐like cells” with neuritic processes were found migrating out from the explant onto the filter membrane. Their number gradually increased day by day. BrdU‐labeling study showed that RPE cells initiated to proliferate under the culture condition on day 4 in vitro, temporally correlating to the time course of retinal regeneration in vivo. Histological observations of cultured explants showed that proliferating RPE cells did not form the stratified structure typically observed in the NR but they rather migrated out from the explants. Neuronal differentiation was examined by immunohistochemical detection of various neuron‐specific proteins; HPC‐1 (syntaxin), GABA, serotonin, rhodopsin, and acetylated tubulin. Immunoreactive cells for these proteins always possessed fine and long neurite‐like processes. Numerous lightly pigmented cells with neuron‐like morphology showed HPC‐1 immunoreactivity. Fibroblast growth factor‐2 (FGF‐2), known as a potent factor for the transdifferentiation of ocular tissues in various vertebrates, substantially increased the numbers of both neuron‐like cells and HPC‐1‐like immunoreactive cells in a dose‐dependent manner. These results indicate that our culture method ensures neural differentiation of newt RPE cells in vitro and provides, for the first time, a suitable in vitro experimental model system for studying tissue‐intrinsic factors responsible for newt retinal regeneration. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 209–220, 2002; DOI 10.1002/neu.10031     

Epithelia-mesenchyme interaction plays an essential role in transdifferentiation of retinal pigment epithelium of silver mutant quail: localization of FGF and related molecules and aberrant migration pattern of neural crest cells during eye rudiment formation

Homozygotes of the quail silver mutation, which have plumage color changes, also display a unique phenotype in the eye: during early embryonic development, the retinal pigment epithelium (RPE) spontaneously transdifferentiates into neural retinal tissue. Mitf is considered to be the responsible gene and to function similarly to the mouse microphthalmia mutation, and tissue interaction between RPE and surrounding mesenchymal tissue in organ culture has been shown to be essential for the initiation of the transdifferentiation process in which fibroblast growth factor (FGF) signaling is involved. The immunohistochemical results of the present study show that laminin and heparan sulfate proteoglycan, both acting as cofactors for FGF binding, are localized in the area of transdifferentiation of silver embryos much more abundantly than in wild-type embryos. More intense immunohistochemical staining with FGF-1 antibody, but not with FGF-2 antibody, is also found in the neural retina, RPE, and choroidal tissue of silver embryos than in wild-type embryos. HNK-1 immunohistochemistry revealed that clusters of HNK-1-positive cells (presumptive migrating neural crest cells) are frequently located around the developing eyes and in the posterior region of the silver embryonic eye. Finally, chick-quail chimerical eyes were made by grafting silver quail optic vesicles to chicken host embryos: in most cases, no transdifferentiation occurs in the silver RPE, but in a few cases, transdifferentiation occurs where silver quail cells predominate in the choroid tissue. These observations together with our previous in vitro study indicate that the silver mutation affects not only RPE cells but also cephalic neural crest cells, which migrate to the eye rudiment, and that these crest cells play an essential role in the transdifferentiation of RPE, possibly by modifying the FGF signaling pathway. The precise molecular mechanism involved in RPE-neural crest cell interaction is still unknown, and the quail silver mutation is considered to be a good experimental model for studying the role of neural crest cells in vertebrate eye development.     

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.     

MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: Is FGF2 an induction factor?

Adult newts can regenerate their entire retinas through transdifferentiation of the retinal pigment epithelium (RPE) cells. As yet, however, underlying molecular mechanisms remain virtually unknown. On the other hand, in embryonic/larval vertebrates, an MEK [mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) kinase] pathway activated by fibroblast growth factor‐2 (FGF2) is suggested to be involved in the induction of transdifferentiation of the RPE into a neural retina. Therefore, we examined using culture systems whether the FGF2/MEK pathway is also involved in the adult newt RPE transdifferentiation. Here we show that the adult newt RPE cells can switch to neural cells expressing pan‐retinal‐neuron (PRN) markers such as acetylated tubulin, and that an MEK pathway is essential for the induction of this process, whereas FGF2 seems an unlikely primary induction factor. In addition, we show by immunohistochemistry that the PRN markers are not expressed until the 1–3 cells thick regenerating retina, which contains retinal progenitor cells, appears. Our current results suggest that the activation of an MEK pathway in RPE cells might be involved in the induction process of retinal regeneration in the adult newt, however if this is the case, we must assume complementary mechanisms that repress the MEK‐mediated misexpression of PRN markers in the initial process of transdifferentiation.     

Expression of regulatory genes Px6, Otx2, Six3, and FGF2 during newt retina regeneration

Molecular-genetic mechanisms of regeneration of adult newt (Pleurodeles waltl) retina were studied. For the first time, a comparative analysis of the expression of regulatory genes Pax6, Otx2, and Six3 and FGF2 genes encoding signal molecules was performed in the normal retinal pigment epithelium (RPE) and retina and at successive stages of retina regeneration. Cell differentiation types were determined using genetic markers of cell differentiation in the RPE (RPE65) and the retina (βII-tubulin and Rho). Activation of the expression of neurospecific genes Pax6 and Six3 and the growth factor gene FGF2 and suppression of activation of the regulatory gene Otx2 and the RPE65 were observed at the stage of multipotent neuroblast formation in the regenerating retina. The expression of genes Pax6, Six3, and Fgf2 was retained at a later stage of retina regeneration at which the expression of retinal differentiation markers, the genes encoding β II-tubulin (βII-tubulin) and rhodopsin (Rho), was also detected. We assume that the above regulatory genes are multifunctional and control not only transdifferentiation of RPE cells (the key stage of retina regeneration) but also differentiation of regenerating retina cells. The results of this study, demonstrating coexpression of Pax6, Six3, Fgf2, βII-tubulin, and Rho genes, provide indirect evidence for the interaction of regulatory and specific genes during retina regeneration.     

Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in the silver mutation of the Japanese quail

The neural retina and retinal pigment epithelium (RPE) diverge from the optic vesicle during early embryonic development. They originate from different portions of the optic vesicle, the more distal part developing as the neural retina and the proximal part as RPE. As the distal part appears to make contact with the epidermis and the proximal part faces mesenchymal tissues, these two portions would encounter different environmental signals. In the present study, an attempt has been made to investigate the significance of interactions between the RPE and mesenchymal tissues that derive from neural crest cells, using a unique quail mutant silver (B/B) as the experimental model. The silver mutation is considered to affect neural crest-derived tissues, including the epidermal melanocytes. The homozygotes of the silver mutation have abnormal eyes, with double neural retinal layers, as a result of aberrant differentation of RPE to form a new neural retina. Retinal pigment epithelium was removed from early embryonic eyes (before the process began) and cultured to see whether it expressed any phenotype characteristic of neural retinal cells. When RPE of the B/B mutant was cultured with surrounding mesenchymal tissue, neural retinal cells were differentiated that expressed markers of amacrine, cone or rod cells. When isolated RPE of the B/B mutant was cultured alone, it acquired pigmentation and did not show any property characteristic of neural retinal cells. The RPE of wild type quail always differentiated to pigment epithelial cells. In the presence of either acidic fibroblast growth factor (aFGF) or basic FGF (bFGF), the RPE of the B/B mutant differentiated to neural retinal cells in the absence of mesenchymal tissue, but the RPE of wild type embryos only did so in the presence of 10–40 times as much aFGF or bFGF. These observations indicate that genes responsible for the B/B mutation are expressed in the RPE as well as in those cells that have a role in the differentiation of neural crest cells. They further suggest that development of the neural retina and RPE is regulated by some soluble factor(s) that is derived from or localized in the surrounding embryonic mesenchyme and other ocular tissues, and that FGF may be among possible candidates.     

MEK mediates in vitro neural transdifferentiation of the adult newt retinal pigment epithelium cells: Is FGF2 an induction factor?

Adult newts can regenerate their entire retinas through transdifferentiation of the retinal pigment epithelium (RPE) cells. As yet, however, underlying molecular mechanisms remain virtually unknown. On the other hand, in embryonic/larval vertebrates, an MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] pathway activated by fibroblast growth factor-2 (FGF2) is suggested to be involved in the induction of transdifferentiation of the RPE into a neural retina. Therefore, we examined using culture systems whether the FGF2/MEK pathway is also involved in the adult newt RPE transdifferentiation. Here we show that the adult newt RPE cells can switch to neural cells expressing pan-retinal-neuron (PRN) markers such as acetylated tubulin, and that an MEK pathway is essential for the induction of this process, whereas FGF2 seems an unlikely primary induction factor. In addition, we show by immunohistochemistry that the PRN markers are not expressed until the 1-3 cells thick regenerating retina, which contains retinal progenitor cells, appears. Our current results suggest that the activation of an MEK pathway in RPE cells might be involved in the induction process of retinal regeneration in the adult newt, however if this is the case, we must assume complementary mechanisms that repress the MEK-mediated misexpression of PRN markers in the initial process of transdifferentiation.     

Alpha melanocyte stimulating hormone (alpha-MSH) enhances eicosanoid production by bovine retinal pigment epithelium.

Explants of bovine eyes consisting of retina, with its underlying choroid and sclera (termed retinal explants) were maintained in organ culture in the absence or presence of alpha-melanocyte stimulating hormone (alpha-MSH) for up to 19 days. The conditioned media was collected twice a week and assayed for the following eicosanoids, prostaglandin E2 (PGE2) and prostacyclin. The addition of alpha-MSH to the incubation media resulted in a 1.5 fold enhancement in the production of both PGE2 and prostacyclin. This stimulatory effect diminished after 11 days. Additionally, the three tissue components comprising the retinal explants i.e. 1. neural retina 2. retinal pigment epithelium (RPE) with its underlying vascular layer (choroid) and 3. scleral tissue were separated and incubated in the presence or absence of alpha-MSH. Hormone treatment caused an enhanced eicosanoid production by RPE tissue alone, while its production by the neuronal retina and sclera was reduced or unaffected respectively. This demonstrates that the RPE layer is the source for the alpha-MSH induced eicosanoid production observed in the whole retinal explant. Our findings demonstrate, for the first time that alpha-MSH can stimulate prostaglandin production by RPE maintained in organ culture.     

Neural cell differentiation from retinal pigment epithelial cells of the newt: an organ culture model for the urodele retinal regeneration.

Transdifferentiation from retinal pigment epithelium (RPE) to neural retina (NR) was studied under a new culture system as an experimental model for newt retinal regeneration. Adult newt RPEs were organ cultured with surrounding connective tissues, such as the choroid and sclera, on a filter membrane. Around day 7 in vitro, lightly pigmented "neuron-like cells" with neuritic processes were found migrating out from the explant onto the filter membrane. Their number gradually increased day by day. BrdU-labeling study showed that RPE cells initiated to proliferate under the culture condition on day 4 in vitro, temporally correlating to the time course of retinal regeneration in vivo. Histological observations of cultured explants showed that proliferating RPE cells did not form the stratified structure typically observed in the NR but they rather migrated out from the explants. Neuronal differentiation was examined by immunohistochemical detection of various neuron-specific proteins; HPC-1 (syntaxin), GABA, serotonin, rhodopsin, and acetylated tubulin. Immunoreactive cells for these proteins always possessed fine and long neurite-like processes. Numerous lightly pigmented cells with neuron-like morphology showed HPC-1 immunoreactivity. Fibroblast growth factor-2 (FGF-2), known as a potent factor for the transdifferentiation of ocular tissues in various vertebrates, substantially increased the numbers of both neuron-like cells and HPC-1-like immunoreactive cells in a dose-dependent manner. These results indicate that our culture method ensures neural differentiation of newt RPE cells in vitro and provides, for the first time, a suitable in vitro experimental model system for studying tissue-intrinsic factors responsible for newt retinal regeneration.     

Transgenic Xenopus laevis with the ef1-α promoter as an experimental tool for amphibian retinal regeneration study

Complete retinal regeneration occurs after the removal of the whole tissue in mature Xenopus laevis, as well as in the newt. Here, we produced F1 and F2 lines of transgenic X. laevis containing an EGFP gene under a translation elongation factor 1‐α (ef1‐α) promoter and investigated how the gene is reactivated in retinal pigmented epithelial (RPE) cells when the neural retina (NR) is removed. The results showed that EGFP expression is reduced in the adult ocular tissues of nonmanipulated transgenic animals, and EGFP‐expressing cells are occasionally found heterogeneously in the lens, NR and RPE tissues. During retinal regeneration, the EGFP gene is reactivated in the RPE and ciliary marginal cells. Transgenic animals were also used for a transplant study because of the genetic marker of the donor tissue. Transplanted RPE clearly transdifferentiated to regenerate the retina in the ocular chamber. This study is, to our knowledge, the first report of a transgenic study of amphibian retinal regeneration, and the approach is promising for future molecular analyses. genesis 50:642–650, 2012. © 2012 Wiley Periodicals, Inc.     

A possible role for the vascular membrane in retinal regeneration in Rana catesbienna tadpoles

We have studied the process of retinal regeneration in Rana catesbienna tadpoles using a recently developed monoclonal antibody (2D3) directed against frog neurons and germinitive neuroepithelium. We have found that, following retinal degeneration induced by devascularization, new retina is generated in the posterior eye from transdifferentiating pigment epithelial (RPE) cells and in the anterior eye from increased proliferation at the normal growth zone in the ora serrata. This demonstrates that the anuran retina regenerates in a manner similar to that observed previously in urodeles. In addition, the use of MAb-2D3 has allowed us to study the process of RPE transdifferentiation more accurately than was previously possible, and consequently we have found a high degree of association of migratory pigment cells with the retinal vascular membrane at the time of the initial RPE transdifferentiation to retinal neuroblasts.     

Formation of retinal pigment epithelium in vitro by transdifferentiation of neural retina cells.

Chick embryonic neural retina (NR) dedifferentiates in culture and can transdifferentiate spontaneously into retinal pigment epithelium (RPE). Both, primary RPE and transdifferentiated RPE (RPEt), are characterized by pigmentation, expression of RPE-specific protein, eRPEAG and lack of expression of the neural cell adhesion molecule, NCAM. In contrast, NR cells are unpigmented and express NCAM but not eRPE(AG). Functionally, both primary RPE and the RPEt cells display a pH(i) response to bFGF, which is different from that of the NR. We used these characteristics to distinguish cell types in primary cultures of chick NR and follow the changes in phenotype that occur during transdifferentiation. We show that the RPEt forms as small "islands" in the packed regions of the primary, "mother" NR cell sheets, in a stochastic process. Because of a small number of cells involved in the initiation of the transdifferentiation we refer to it as a "leader effect" to contrast it with the "community effect" which requires many competent cells to be present in a group to be able to respond to an inductive signal. The RPEt then expands centrifugally and underneath the surrounding NR sheet. To determine if the RPEt maintains its identity in isolation while displaying the RPE-typical phenotypic plasticity, we explanted the islands of RPEt and treated half of them with bFGF. The untreated RPEt maintained its closely packed, polygonal pigmented phenotype but the bFGF-treated RPEt transdifferentiated into a non-pigmented, NR-like phenotype, indicating that RPEt encompasses the full differentiation repertoire of native RPE.     

Neural retinal regeneration in the anuran amphibian Xenopus laevis post-metamorphosis: transdifferentiation of retinal pigmented epithelium regenerates the neural retina

In urodele amphibians like the newt, complete retina and lens regeneration occurs throughout their lives. In contrast, anuran amphibians retain this capacity only in the larval stage and quickly lose it during metamorphosis. It is believed that they are unable to regenerate these tissues after metamorphosis. However, contrary to this generally accepted notion, here we report that both the neural retina (NR) and lens regenerate following the surgical removal of these tissues in the anuran amphibian, Xenopus laevis, even in the mature animal. The NR regenerated both from the retinal pigment epithelial (RPE) cells by transdifferentiation and from the stem cells in the ciliary marginal zone (CMZ) by differentiation. In the early stage of NR regeneration (5-10 days post operation), RPE cells appeared to delaminate from the RPE layer and adhere to the remaining retinal vascular membrane. Thereafter, they underwent transdifferentiation to regenerate the NR layer. An in vitro culture study also revealed that RPE cells differentiated into neurons and that this was accelerated by the presence of FGF-2 and IGF-1. The source of the regenerating lens appeared to be remaining lens epithelium, suggesting that this is a kind of repair process rather than regeneration. Thus, we show for the first time that anuran amphibians retain the capacity for retinal regeneration after metamorphosis, similarly to urodeles, but that the mode of regeneration differs between the two orders. Our study provides a new tool for the molecular analysis of regulatory mechanisms involved in retinal and lens regeneration by providing an alternative animal model to the newt, the only other experimental model.