Newt Retinal Regeneration Research Articles

Implications of a Multi-Step Trigger of Retinal Regeneration in the Adult Newt.

The newt is an amazing four-limbed vertebrate that can regenerate various body parts including the retina. In this animal, when the neural retina (NR) is removed from the eye by surgery (retinectomy), both the NR and the retinal pigment epithelium (RPE) eventually regenerate through the process of reprogramming and proliferation of RPE cells. Thus far, we have pursued the onset mechanism of adult newt retinal regeneration. In this study, using an in vitro system, we found that both mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK and β-catenin were involved in cell cycle re-entry of RPE cells. MEK-ERK signaling activity in RPE cells was strengthened by retinectomy, and nuclear translocation of β-catenin in RPE cells was induced by attenuation of cell–cell contact, which was promoted by incision of the RPE or its treatment with ethylene glycol tetraacetic acid (EGTA). EGTA is a Ca2+ chelator that disrupts cadherin-mediated cell–cell adhesion. Reinforcement of MEK-ERK signaling activity was a prerequisite for nuclear translocation of β-catenin. These results suggest that retinectomy followed by attenuation of cell–cell contact may trigger cell cycle re-entry of RPE cells. This study, together with our previous findings concerning the proliferation and multipotency of adult newt RPE cells, provides insight into the mechanism of the multi-step trigger in which the onset of retinal regeneration in the adult newt is rigorously controlled.

Hematological- and Neurological-Expressed Sequence 1 Gene Products in Progenitor Cells during Newt Retinal Development

Urodele amphibians such as Japanese common newts have a remarkable ability to regenerate their injured neural retina, even as adults. We found that hematological- and neurological-expressed sequence 1 (Hn1) gene was induced in depigmented retinal pigment epithelial (RPE) cells, and its expression was maintained at later stages of newt retinal regeneration. In this study, we investigated the distribution of the HN1 protein, the product of the Hn1 gene, in the developing retinas. Our immunohistochemical analyses suggested that the HN1 protein was highly expressed in an immature retina, and the subcellular localization changed during this retinogenesis as observed in newt retinal regeneration. We also found that the expression of Hn1 gene was not induced in mouse after retinal removal. Our results showed that Hn1 gene can be useful for detection of undifferentiated and dedifferentiated cells during both newt retinal development and regeneration.

Immunohistochemical analysis of Musashi-1 expression during retinal regeneration of adult newt

The adult newt retinal regeneration is an ideal model for studying retinal regeneration by transdifferentiation of the retinal pigment epithelium (RPE) cells. Accumulated evidence suggests that the RNA-binding protein Musashi-1 (Msi1) is expressed in mature photoreceptors and RPE cells as well as in retinal stem/progenitor cells, being essential for vision. We have been investigating whether Msi1 is also essential for retinal regeneration. In the last paper [K. Susaki, J. Kaneko, Y. Yamano, K. Nakamura, W. Inami, T. Yoshikawa, Y. Ozawa, S. Shibata, O. Matsuzaki, H. Okano, C. Chiba, Musashi-1, an RNA-binding protein, is indispensable for survival of photoreceptors. Exp. Eye Res. (in press)], we showed that the expression profiles of Msi1 transcripts and protein isoforms change during retinal regeneration. In the current report, we show by immunohistochemistry that Msi1 is expressed in transdifferentiating cells or cells of regenerating retinal tissues. Upon retinectomy, Msi1 protein, which is expressed in the nuclei of intact (stage E-0) RPE cells, changed its subcellular localization, being expressed in both the nucleus and cytoplasm of the RPE-derived stem-like cells at stage E-1. As the retinal rudiment/regenerating retina (rR) and renewing RPE (rRPE) are specified from the stem-like cell population (stage E-2), Msi1 expression was maintained or up-regulated in the rR, while down-regulated in the rRPE. During further retinal regeneration, Msi1 expression was decreased in association with cell differentiation, except for photoreceptors and RPE cells whose Msi1 expression increased as they differentiate. Thus, Msi1 is likely to be regulated at various cellular events during retinal regeneration, implying that Msi1 may have multi-functions in retinal regeneration. All together, it is probable that Msi1 is one of the essential factors that need to be regulated in retinal regeneration.

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.

Intraocular implantation of DNA-transfected retinal pigment epithelium cells: a new approach for analyzing molecular functions in the newt retinal regeneration

Adult newts can regenerate their entire retinas, even after surgical removal of the neural retina (retinectomy), through transdifferentiation of the retinal pigment epithelium (RPE) cells. To develop a new experimental system for analyzing molecular functions during retinal regeneration of adult newts, we attempted to deliver a foreign gene into RPE cells of retina-less eye-cups in vitro. Here we used pCS2mt-GFP as a reporter construct, and selected Polyfect as a transfection reagent. DNA-transfection appeared to be restricted to the RPE cells of retina-less eye-cups and its efficiency was 0.1–0.2%. We tried to implant RPE-choroid tissue containing DNA-transfected RPE cells into the eye of a host animal. The tissue was placed into the posterior eye-chamber immediately after retinectomy so that the implanted RPE tissue was facing the cornea (i.e., normal orientation). The implant and host RPE regenerated one continuous hybrid neural retina. Ocular sections after 60 days of implantation showed that a small number of cells in the regenerating retina were intensely stained with an anti-GFP antibody. Some of those cells were believed to be retinal cells such as ganglion cells, amacrine cells and photoreceptors. The GFP-positive cells in the hybrid regenerating retina could represent clones derived from a single RPE cell. These results indicate that this experimental system could become useful in the study of adult newt 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.

Expression pattern of a newt Notch homologue in regenerating newt retina

We isolated part of a newt Notch homologue, N-Notch, from regenerating newt retina. The spatio-temporal pattern of N-Notch expression was studied by in situ hybridization at different stages of newt retinal regeneration. Proliferating cells were confirmed by the injection of bromodeoxyuridine (BrdU). In the early stage of regeneration, when the retina was one to two cells thick, all proliferating retinal progenitors expressed N-Notch. As the thickness of the retina increased with regeneration, N-Notch expression decreased in BrdU-positive cells on the vitreal side of the retina. Subsequently, presumptive retinal ganglion cells that were BrdU-negative cells appeared at the vitreal edge of the regenerating retina. These differentiating cells did not express N-Notch. Later, N-Notch expression decreased in the BrdU-positive cells on the scleral surface of the retina. Subsequently, presumptive photoreceptor cells that were BrdU-negative cells appeared in this region. These differentiating cells also did not express N-Notch. The proliferating retinal progenitors ceased expressing N-Notch and then stopped dividing during the differentiation of ganglion cells and photoreceptor cells. It was found that retinal regeneration involves the expression of an important developmental signaling molecule, Notch, in retinal progenitors and the expression of Notch ceased as cell differentiation proceeded during retinal regeneration.

Expression of a newt Notch homologue and cell differentiation during newt retinal regeneration

Expression of a newt Notch homologue and cell differentiation during newt retinal regeneration

Time course of appearance of GABA and GABA receptors during retinal regeneration in the adult newt

Appearance and maturation of the GABA (γ-aminobutyric acid) system during newt retinal regeneration were studied by electrophysiological, immunohistochemical, and biochemical techniques. (1) Responses to GABA appeared in neurons dissociaed from regenerating retinae before the segregation of the plexiform layers; whereas (2) GABA immunoreactivity appeared at sites of the presumptive horizontal cell and amacrine cell layers at the beginning of theegregation f these layers. During subsequent regeneration, GABA-immunoreactive cells at the amacrine cell layer increased in number and extended lateral processes, forming a GABA-immunoreactive inner plexiform layer. Also GABA immunoreactivity increased in the region of the outer plexiform layer, but not their somata which showed decreased GABA immunoreactivity. (3) GABA synthesis in the retina increased significantly at the beginning of the segregation of the plexiform layers. These results suggest that the increase of GABA synthesis during retinal regeneration correlates well with the development of GABA-immunoreactive cells and that functional GABA receptors appear earlier than increased GABA synthesis.

Remodeling processes during neural retinal regeneration in adult urodeles: an immunohistochemical survey.

Dynamic features of neural retina regeneration in the adult newt Pleurodeles waltl were analyzed using immunohistochemical studies. Antibody to Glial Fibrillary Acidic Protein (GFAP) was used as a marker of the retinal glial supportive system in order to obtain an overview of the retinal reorganization pattern. Unexpectedly, retinal progenitor cells displayed GFAP staining, as did later Müller glial processes and astrocytes supporting ganglional axons. To study changes of plasticity during retinal restoration, the expression patterns of highly- (PSA) and weakly-sialylated N-CAM were examined by double staining. In the retina of adult newts, a sustained expression of total-N-CAM and PSA-N-CAM was detected. However, while an intense distribution of N-CAM was observed throughout the retina, PSA labeling was especially seen in the outer retinal layers. During retinal regeneration, similar widespread staining patterns were observed with the two antibodies, but labeling appeared higher with anti-total-N-CAM antibody than with anti-PSA-N-CAM antibody. On the other hand, tenascin (Tn) expression was analyzed for the first time during retinal regeneration. At the early stages, brightly stained matrix fibers of abundant Tn accumulating in the eye cavity were seen close to the retinal rudiment cells, which suggested that Tn was secreted from these cells. Tn expression was seen nearly throughout the retinal regenerate during neurite migration and then became restricted to the plexiform layers. In the light of the functions attributed to N-CAM and Tn in histogenetic events, the putative roles played by these morphoregulatory molecules in adult newt retinal regeneration were discussed.

Appearance and maturation of responses of solitary spiking cells to excitatory and inhibitory amino acids during newt retinal regeneration

Appearance and maturation of responses of solitary spiking cells to excitatory and inhibitory amino acids during newt retinal regeneration