Chikafumi Chiba

The retinal pigment epithelium: An important player of retinal disorders and regeneration

The retinal pigment epithelium (RPE) is a partner of the neural retina and is indispensable for vision. In humans, proliferation and transformation (cell-type switching) of RPE cells after a traumatic injury of the neural retina causes a retinal disorder leading to loss of vision. In contrast, in certain adult amphibians such as Xenopus laevis and the newt, a similar process in RPE cells leads to regeneration of the entire retina. In this review, on the basis of accumulating evidence in basic biology and medical sciences, similarities and differences between these RPE-mediated retinal disorders and regeneration in adult vertebrates are highlighted, providing a connection to future research that should be designed to establish clues for the treatment of pathogenesis caused by RPE while promoting RPE-mediated retinal regeneration in a patients eyes.

Visual cycle protein RPE65 persists in new retinal cells during retinal regeneration of adult newt

Adult newts can regenerate their entire retina through transdifferentiation of the retinal pigment epithelium (RPE). The objective of this study was to redescribe the retina regeneration process by means of modern biological techniques. We report two different antibodies (RPE‐No.112 and MAB5428) that recognize the newt homolog of RPE65, which is involved in the visual cycle and exclusively label the RPE cell‐layer in the adult newt eye. We analyzed the process of retinal regeneration by immunohistochemistry and immunoblotting and propose that this process should be divided into nine stages. We found that the RPE65 protein is present in the RPE‐derived new retinal rudiment at 14 days postoperative (po) and in the regenerating retinas at the 3–4 cell stage (19 days po). These observations suggest that certain characteristics of RPE cells overlap with those of retinal stem/progenitor cells during the period of transdifferentiation. However, RPE65 protein was not detected in either retinal stem/progenitor cells in the ciliary marginal zone (CMZ) of adult eyes or in neuroepithelium present during retina development, where it was first detected in differentiated RPE. Moreover, the gene expression of RPE65 was drastically downregulated in the early phase of transdifferentiation (by 10 days po), while those of Connexin43 and Pax‐6, both expressed in regenerating retinas, were differently upregulated. These observations suggest that the RPE65 protein in the RPE‐derived retinal rudiment may represent the remainder after protein degradation or discharge rather than newly synthesized protein. J. Comp. Neurol. 40:391–407, 2006.

Musashi-1, an RNA-binding protein, is indispensable for survival of photoreceptors

Musashi-1 (Msi1), an RNA-binding protein (RBP), has been postulated to play important roles in the maintenance of the stem-cell state, differentiation, and tumorigenesis. However, the expression and function of Msi1 in differentiated cells remain obscure. Here we show that Msi1 is expressed in mature photoreceptors and retinal pigment epithelium (RPE) cells, and is indispensable for the survival of photoreceptors. We found in the adult newt eye that Msi1 is expressed in all photoreceptors and RPE cells as well as in the retinal stem/progenitor cells in the ciliary marginal zone (CMZ). We found in the analyses of the newt normal and regenerating retinas that the expression profiles of the Msi1 transcripts and protein isoforms in the photoreceptors are different from those in the retinal stem/progenitor cells. Furthermore, we found that all photoreceptors and RPE cells of the adult mice also express Msi1, and that Msi1 knockout (Msi1-KO) results in degeneration of photoreceptors and a lack of a visual cycle protein RPE65 in the microvilli of RPE cells. Taken together, our current results demonstrate that the expression of Msi1 in mature photoreceptors and RPE cells is evolutionarily conserved, and that Msi1 bears essential functions for vision. Considering such an Msi1-KO phenotype in the retina, it is now reasonable to address whether defects of the Msi1 functions are responsible for inherited retinal diseases. Studying the regulation of Msi1 and the target RNAs of Msi1 in photoreceptors and RPE cells might contribute to fundamental and clinical studies of retinal degeneration.

Expressing exogenous genes in newts by transgenesis

The great regenerative abilities of newts provide the impetus for studies at the molecular level. However, efficient methods for gene regulation have historically been quite limited. Here we describe a protocol for transgenically expressing exogenous genes in the newt Cynops pyrrhogaster. This method is simple: a reaction mixture of I-SceI meganuclease and a plasmid DNA carrying a transgene cassette flanked by the enzyme recognition sites is directly injected into fertilized eggs. The protocol achieves a high efficiency of transgenesis, comparable to protocols used in other animal systems, and it provides a practical number of transgenic newts (∼20% of injected embryos) that survive beyond metamorphosis and that can be applied to regenerative studies. The entire protocol for obtaining transgenic adult newts takes 4–5 months.

Evidence for Notch signaling involvement in retinal regeneration of adult newt

Involvement of Notch signaling in retinal regeneration by transdifferentiation of pigment epithelium cells was investigated using the adult newt Cynops pyrrhogaster. During retinal regeneration, cells expressing Notch-1 first appeared in the regenerating retina one to two cells thick (stage E-3) originated from the retinal pigment epithelium (RPE) cells, and increased in number as the regenerating retina increased in thickness. Notch-1 expression was decreased in the central retina in association with cell differentiation and became restricted to the peripheral retina. Administration of a Notch signaling blocker DAPT resulted in the appearance of a cluster of neurons, earlier than in normal regeneration, along the regenerating retina 1-3 cells thick (stage E-3 to I-1). Immunoblot analysis suggested that DAPT could perturb the processing of Notch-1. Similar results were obtained in the newt embryonic retinal development. These results suggest that the Notch-1 signaling system may be reset to regulate neurogenesis during retinal regeneration. However, PCR analysis revealed that the adult newt RPE cells express Hes-1, neurogenin1 and sometimes Delta-1 Hes-1, neurogenin1 and sometimes Delta-1 all of which are differently regulated in association with retinal regeneration, implying that Notch signaling might also be involved early in the process of transdifferentiation.

A developmentally regulated switch from stem cells to dedifferentiation for limb muscle regeneration in newts

The newt, a urodele amphibian, is able to repeatedly regenerate its limbs throughout its lifespan, whereas other amphibians deteriorate or lose their ability to regenerate limbs after metamorphosis. It remains to be determined whether such an exceptional ability of the newt is either attributed to a strategy, which controls regeneration in larvae, or on a novel one invented by the newt after metamorphosis. Here we report that the newt switches the cellular mechanism for limb regeneration from a stem/progenitor-based mechanism (larval mode) to a dedifferentiation-based one (adult mode) as it transits beyond metamorphosis. We demonstrate that larval newts use stem/progenitor cells such as satellite cells for new muscle in a regenerated limb, whereas metamorphosed newts recruit muscle fibre cells in the stump for the same purpose. We conclude that the newt has evolved novel strategies to secure its regenerative ability of the limbs after metamorphosis.

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.

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

Appearance and maturation of the GABA (gamma-aminobutyric acid) system during newt retinal regeneration were studied by electrophysiological, immunohistochemical, and biochemical techniques. (1) Responses to GABA appeared in neurons dissociated 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 the segregation of 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.

MEK-ERK and heparin-susceptible signaling pathways are involved in cell-cycle entry of the wound edge retinal pigment epithelium cells in the adult newt

The onset mechanism of proliferation in mitotically quiescent retinal pigment epithelium (RPE) cells is still obscure in humans and newts, although it can be a clinical target for manipulating both retinal diseases and regeneration. To address this issue, we investigated factors or signaling pathways involved in the first cell‐cycle entry of RPE cells upon retinal injury using a newt retina‐less eye‐cup culture system in which the cells around the wound edge of the RPE exclusively enter the cell cycle. We found that MEK–ERK signaling is necessary for their cell‐cycle entry, and signaling pathways whose activities can be modulated by heparin, such as Wnt‐, Shh‐, and thrombin‐mediated pathways, are capable of regulating the cell‐cycle entry. Furthermore, we found that the cells inside the RPE have low proliferation competence even in the presence of serum, suggesting inversely that a loss of cell‐to‐cell contact would allow the cells to enter the cell cycle.

Gap junctional coupling between progenitor cells of regenerating retina in the adult newt.

Gap junctional coupling between progenitor cells of regenerating retina in the adult newt was examined by a slice-patch technique. Retinal slices at the early regeneration stage comprised one to two layers of cells with mitotic activity, progenitor cells. These cells were initially voltage-clamped at a holding potential of -80 mV, near their resting potentials, and stepped to either hyperpolarizing or depolarizing test potentials under suppression of voltage-gated membrane currents. About half the cells showed passively flowing currents that reversed polarity around their resting potentials. The currents often exhibited a voltage- and time-dependent decline. As the difference between the test potential and resting potential increased, the time until the current decreased to the steady-state level became shorter and the amount of steady-state current decreased. Thus, the overall current profile was almost symmetrical about the current at the resting potential. Input resistance estimated from the initial peak of the currents was significantly smaller than that expected in isolated progenitor cells. In a high-K(+) solution, which decreased the resting potential to around 0 mV, the symmetrical current profile was also obtained, but only when the membrane potential was held at 0 mV before the voltage steps. These observations suggest that the current was driven and modulated by the junctional potential difference between the clamping cell and its neighbors. In addition, we examined effects of uncoupling agents on the currents. A gap junction channel blocker, halothane, suppressed the currents almost completely, indicating that the currents are predominantly gap junctional currents. Furthermore, injection of biocytin into the current-recorded cells revealed tracer coupling. These results demonstrate that progenitor cells of regenerating retina couple with each other via gap junctions, and suggest the presence of their cytoplasmic communication during early retinal regeneration.