Transdifferentiation Of Retinal Pigment Epithelium Research Articles

Retina regeneration: lessons from vertebrates.

Unlike mammals, vertebrates such as fishes and frogs exhibit remarkable tissue regeneration including the central nervous system. Retina being part of the central nervous system has attracted the interest of several research groups to explore its regenerative ability in different vertebrate models including mice. Fishes and frogs completely restore the size, shape and tissue structure of an injured retina. Several studies have unraveled molecular mechanisms underlying retina regeneration. In teleosts, soon after injury, the Müller glial cells of the retina reprogram to form a proliferating population of Müller glia-derived progenitor cells capable of differentiating into various neural cell types and Müller glia. In amphibians, the transdifferentiation of retinal pigment epithelium and differentiation of ciliary marginal zone cells contribute to retina regeneration. In chicks and mice, supplementation with external growth factors or genetic modifications cause a partial regenerative response in the damaged retina. The initiation of retina regeneration is achieved through sequential orchestration of gene expression through controlled modulations in the genetic and epigenetic landscape of the progenitor cells. Several developmental biology pathways are turned on during the Müller glia reprogramming, retinal pigment epithelium transdifferentiation and ciliary marginal zone differentiation. Further, several tumorigenic pathways and gene expression events also contribute to the complete regeneration cascade of events. In this review, we address the various retinal injury paradigms and subsequent gene expression events governed in different vertebrate species. Further, we compared how vertebrates such as teleost fishes and amphibians can achieve excellent regenerative responses in the retina compared with their mammalian counterparts.

Spatial and temporal regulation of Wnt/β-catenin signaling is essential for development of the retinal pigment epithelium

Wnt/beta-catenin signaling is highly active in the dorsal retinal pigment epithelium (RPE) during eye development. To study the role of Wnt/beta-catenin signaling in the RPE development we used a conditional Cre/loxP system in mice to inactivate or ectopically activate Wnt/beta-catenin signaling in the RPE. Inactivation of Wnt/beta-catenin signaling results in transdifferentiation of RPE to neural retina (NR) as documented by downregulation of RPE-specific markers Mitf and Otx2 and ectopic expression of NR-specific markers Chx10 and Rx, respectively. In contrast, ectopic activation of Wnt/beta-catenin signaling results in the disruption of the RPE patterning, indicating that precise spatial and temporal regulation of Wnt/beta-catenin signaling is required for normal RPE development. Using chromatin immunoprecipitation (ChIP) and reporter gene assays we provide evidence that Otx2 and RPE-specific isoform of Mitf, Mitf-H, are direct transcriptional targets of Wnt/beta-catenin signaling. Combined, our data suggest that Wnt/beta-catenin signaling plays an essential role in development of RPE by maintaining or inducing expression of Mitf and Otx2.

β-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression

The retinal pigment epithelium (RPE) consists of a monolayer of cuboidal, pigmented cells that is located between the retina and the choroid. The RPE is vital for growth and function of the vertebrate eye and improper development results in congenital defects, such as microphthalmia or anophthalmia, or a change of cell fate into neural retina called transdifferentiation. The transcription factors microphthalmia-associated transcription factor (Mitf) and orthodenticle homolog 2 (Otx2) are crucial for RPE development and function; however, very little is known about their regulation. Here, by using a Wnt-responsive reporter, we show that the Wnt/beta-catenin pathway is activated in the differentiating mouse RPE. Cre-mediated, RPE-specific disruption of beta-catenin after the onset of RPE specification causes severe defects, resulting in microphthalmia with coloboma, disturbed lamination, and mislocalization of adherens junction proteins. Upon beta-catenin deletion, the RPE transforms into a multilayered tissue in which the expression of Mitf and Otx2 is downregulated, while retina-specific gene expression is induced, which results in the transdifferentiation of RPE into retina. Chromatin immunoprecipitation (ChIP) and luciferase assays indicate that beta-catenin binds near to and activates potential TCF/LEF sites in the Mitf and Otx2 enhancers. We conclude that Wnt/beta-catenin signaling is required for differentiation of the RPE by directly regulating the expression of Mitf and Otx2. Our study is the first to show that an extracellular signaling pathway directly regulates the expression of RPE-specific genes such as Mitf and Otx2, and elucidates a new role for the Wnt/beta-catenin pathway in organ formation and development.

Regeneration of the amphibian retina: Role of tissue interaction and related signaling molecules on RPE transdifferentiation

Regeneration of eye tissue is one of the classic subjects in developmental biology and it is now being vigorously studied to reveal the cellular and molecular mechanisms involved. Although many experimental animal models have been studied, there may be a common basic mechanism that governs retinal regeneration. This can also control ocular development, suggesting the existence of a common principle between the development and regeneration of eye tissues. This notion is now becoming more widely accepted by recent studies on the genetic regulation of ocular development. Retinal regeneration can take place in a variety of vertebrates including fish, amphibians and birds. The newt, however, has been considered to be the sole animal that can regenerate the whole retina after the complete removal of the retina. We recently discovered that the anuran amphibian also retains a similar ability in the mature stage, suggesting the possibility that such a potential could be found in other animal species. In the present review article, retinal regeneration of amphibians (the newt and Xenopus laevis) and avian embryos are described, with a particular focus on transdifferentiation of retinal pigmented epithelium. One of the recent progresses in this field is the availability of tissue culture methods to analyze the initial process of transdifferentiation, and this enables us to compare the proliferation and neural differentiation of retinal pigmented epithelial cells from various animal species under the same conditions. It was revealed that tissue interactions between the retinal pigmented epithelium and underlying connective tissues (the choroid) play a substantial role in transdifferentiation and that this is mediated by a diffusible signal such as fibroblast growth factor 2. We propose that tissue interaction, particularly mesenchyme-neuroepithelial interaction, is considered to play a fundamental role both in retinal development and regeneration.

Retinoids control anterior and dorsal properties in the developing forebrain

We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina.

Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6.

The transcription factors Pax2 and Pax6 are co-expressed in the entire optic vesicle (OV) prior and concomitant with the establishment of distinct neuroretinal, retinal, pigmented-epithelial and optic-stalk progenitor domains, suggesting redundant functions during retinal determination. Pax2; Pax6 compound mutants display a dose-dependent reduction in the expression of the melanocyte determinant Mitf, accompanied by transdifferentiation of retinal pigmented epithelium (RPE) into neuroretina (NR) in Pax2(-/-); Pax6(+/-) embryos, which strongly resembles the phenotype of Mitf-null mutants. In Pax2(-/-); Pax6(-/-) OVs Mitf fails to be expressed and NR markers occupy the area that usually represents the Mitf(+) RPE domain. Furthermore, both, Pax2 and Pax6 bind to and activate a MITF RPE-promoter element in vitro, whereas prolonged expression of Pax6 in the Pax2-positive optic stalk leads to ectopic Mitf expression and RPE differentiation in vivo. Together, these results demonstrate that the redundant activities of Pax2 and Pax6 direct the determination of RPE, potentially by directly controlling the expression of RPE determinants.

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.

Expression of an array of photoreceptor genes in chick embryonic retinal pigment epithelium cell cultures under the induction of neuroD

Coaxing plastic, non-neuronal cells to transdifferentiate into a particular type of neurons might have clinical applications. Previously we reported that neuroD induces transdifferentiation of retinal pigment epithelium (RPE) cells derived from day-6 chick embryos into cells that resemble young photoreceptor cells. These cells also express visinin, a gene expressed early during cone photoreceptor differentiation. Further characterization showed that the transdifferentiated cells express a number of photoreceptor genes, including interphotoreceptor retinoid binding protein, the α-subunit of phosphodiesterase, and opsin genes encoding rhodopsin, the red, the green, and the blue visual pigments. Our data demonstrate that neuroD can reprogram RPE to become photoreceptor cells with substantial differentiation, and suggest the possibility of generating photoreceptor cells from RPE using neuroD as a molecular trigger.

Expression of gicerin, a cell adhesion molecule, in the abnormal retina in silver plumage color mutation of Japanese quail ( Coturnix japonica)

Silver plumage color mutant ( B/B) quail has an abnormal retina characterizing the transdifferentiation of retinal pigment epithelium (RPE) following the retinal separation in the early developmental stage. In the present study; (i) the expression of gicerin, an immunoglobulin-superfamily cell adhesion molecule, was examined in the retina of B/B quail. In the wild-type quail, gicerin protein was enriched in the apical membrane (facing the neural retina, NR) of RPE cells on embryonic day (E) 4 and then appeared also in NR cells from E5. However, in the B/B retina, no gicerin expression was found in the transdifferentiation area of RPE prior to the retinal separation. (ii) In addition to this, microinjection of anti-gicerin polyclonal antibody into the eyeball of wild-type quail on E3 caused the retinal separation and induced the transdifferentiation of RPE into new NR. These observations suggest that the decrease of gicerin expression might participate in the retinal separation and RPE-transdifferentiation in B/B quail.

Basic fibroblast growth factor (FGF-2) induced transdifferentiation of retinal pigment epithelium: generation of retinal neurons and glia.

In the present study we report that basic fibroblast growth factor (bFGF, FGF-2) promotes the transdifferentiation of Xenopus laevis larval retinal pigment epithelium (RPE) into neural retina. Using specific antibodies we have examined the cellular composition of the regenerated retinal tissue. Our results show that, in addition to retinal neurons and photoreceptors, glial cells were also regenerated from the transdifferentiated RPE. These results were specific to FGF-2, since other factors that were tested, including acidic FGF (aFGF, FGF-1), epidermal growth factor (EGF), laminin, ECL, and Matrigel, exhibited no activity in inducing retinal regeneration. These results are the first in amphibians demonstrating the functional role of FGF-2 in inducing RPE transdifferentiation. Transplantation studies were carried out to investigate retinal regeneration from the RPE in an in vivo environment. Sheets of RPE implanted into the lens-less eyes of larval hosts transformed into neurons and glial cells only when under the influence of host retinal factors. In contrast, no retinal transdifferentiation occurred if the RPE was implanted into the enucleated orbit. Taken together, these results show that the amphibian RPE is capable of transdifferentiation into neuronal and glial cell-phenotypes and implicate FGF-2 as an important factor in inducing retinal regeneration in vitro.

Transdifferentiation of Pigmented Epithelium Induced by the Influence of Lens Epithelium in Frogs

The influence of lens epithelium (LE) of adult frogs on the character of transdifferentiation of retinal pigmented epithelium (RPE) of adult frogs and tadpoles of Rana temporaria has been studied. After a period of intense proliferation RPE cultured in vivo in contact with LE in the tadpole orbit almost exclusively transforms into retina. RPE precultivated in vitro in contact with LE for three days in protein-free medium does not manifest cell divisions and mostly transdifferentiates into lentoids. The problem of the relative significance of inducing determinants and the role of activation or inhibition of proliferation in transdifferentiation is discussed.