Araki M (2007), Regeneration of the amphibian retina: role of t...

XB-ART-35432

Dev Growth Differ 2007 Feb 01;492:109-20. doi: 10.1111/j.1440-169X.2007.00911.x.

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

Araki M .

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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.

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Species referenced: Xenopus laevis
Genes referenced: fgf2 pax6 rpe rpe65 tbx2

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	Fig. 1. Development of the eye. (A) Transverse section of the chick proencephalon at the 10 somite stage. The optic vesicle makes contact with different types of tissues at the dorsal (a), distal (b) and ventral (c) regions. These are the dorsal mesenchyme derived from the cephalic neural crest, the surface epidermis and ventral mesenchyme derived from the procaudal mesoderm, respectively. (B) Schematic drawings of transversal images of the developing eye. The optic vesicle neuroepithelium is subdivided into the dorsal and ventral regions, which later develop into the retinal pigmented epithelium (RPE) and neural retina, respectively.
	Fig. 2. Newt retinal regeneration. Retinal regeneration at different stages after the surgical removal of the retina is shown in the light micrographs from (A) to (H). The retinal pigment epithelial (RPE) cells appear to be irregularly arranged at day 5 (arrow in E), which then become more epithelial at day 10 (arrow in F). At the same time, RPE cells become depigmented. Then, a multistratified epithelium is formed (large arrow in G) and one pigmented single epithelium is also observed (small arrow in G). About 1 month later, a well stratified retinal tissue and a RPE have developed (arrow in H). Hematoxylin and eosin staining. BrdU labeling study indicates that at day 4 only a few cells in the RPE layer are labeled, while at day 5 most of the RPE cells have become labeled (arrow in J). (I and J are from Ikegami et al. 2002).
	Fig. 3. In vitro neural cell differentiation from newt retinal pigmented epithelium (RPE) cells. (A–C) RPE with the choroid are cultured on membrane filters. Cells migrate out from the explant shown at the left hand side in (A) and extend long processes as seen in (B). These cells are positively stained for various neuronspecific cell markers such as Syntaxin (C). (D–F) Singly isolated RPE alone are cultured. (D) RPE culture at day 5 and (E) the same culture at day 30. No cell proliferation or neural differentiation could be seen. When the same RPE tissue is cultured with fibroblast growth factor (FGF)2 plus insulinlike growth factor (IGF)1, extensive neural differentiation is observed (F). (G, H) RPE attaches firmly to the choroid as seen in (G), and after dispase treatment RPE (asterisk in H) can be clearly separated from the choroid (from Mitsuda et al. 2005).
	Fig. 4. Intraocular transplantation of cultured retinal pigmented epithelium (RPE). RPE with the adhering tissues (the choroid and the sclera) was cultured for 10 days and then transplanted into the newt eye chamber. A part of Figure 5A is shown at higher magnification in (B). Well defined structures of RPE (black arrow) and developing retina are observed. The yellow arrow indicates the Bruch membrane. Ch, choroids; S, sclera (from Mitsuda et al. 2005).
	Fig. 5. Retinal regeneration in Xenopus laevis. (A–C) Light micrographs of eyes at day 15, 20 and 30 after retinectomy. At day 30, a well defined structure of the retina has regenerated. The lens has also regenerated. (D, E) Intact eye. A part of Figure (D) is shown at a higher magnification in (E). The black arrow indicates the retinal vascular membrane consisting of the basement membrane and capillaries and this structure plays a crucial role in retina regeneration. The red arrow indicates the retinal pigmented epithelium (RPE) layer.
	Fig. 6. A newly formed retinal pigmented epithelium (RPE) layer on the retinal vascular membrane (RVM). (A, B) The same tissue was simultaneously stained for RPE65, a specific marker for RPE and Pax6. The newly formed layer (arrows) is positively stained for both antibodies. The original RPE layer (asterisks) also becomes positive for Pax6. (C, D) Light micrographs of regenerating retina at day 10 and 20. A flat pigmented epithelium is observed on RVM (arrows in C) which vigorously proliferates to form a stratified epithelium as seen in (D). In this stratified epithelium pigmented cells are still found at the basal side (arrows) and some RPE cells extend toward the newly formed layer (arrowheads) (from Yoshii et al. 2007).
	Fig. 7. Schematic diagram of retinal regeneration in Xenopus laevis (a) and the newt (b). (a) Upper part in (A) shows retinectomized eye cavity and the lower shows an intact one. Cells from two origins regenerate the retina; ciliary marginal cells and the retinal pigmented epithelial cells. The ciliary marginal zone (CMZ) partially remains after retinectomy with the present surgical procedure and CMZ stem cells initiate migration on the retinal vascular membrane (RVM) to the posterior direction. (B) At the same time, some of the retinal pigmented epithelium (RPE) cells leave the RPE layer, migrate and attach to the RVM, where they form a new RPE layer, as indicated in (C). Numerous capillaries (indicated as C) are seen in RVM. RPE cells on the RVM proliferate and transdifferentiate to neural retinal precursor cells (D, E). RPE cells that were positively stained for RPE65 are shown by brown colored nuclei or pigmented granules in the cytoplasm (from Yoshii et al. 2007). (b) In the newt retina regeneration, RPE cells become more loosely adhered to each other soon after the retinectomy. At about day 5, cells initiate proliferation and become depigmented and show a more well packed epithelial structure. RPE cells do not leave the epithelium and within the epithelium cells at the most basal side (attaching to the Bruch’s membrane) become pigmented. More apically located cells now form a stratified epithelial structure. IML, inner limiting membrane.
	Fig. 8. Transdifferentiation of retinal pigmented epithelium (RPE) into the retinal tissue in Silver quail homozygote. (A) Eye cups of day 15 homozygote embryos. At the left hand side of the figure is an eye of a Silver embryo and at the right is a wild type embryo. Both are left eyes. A circular non-pigmented area is seen in the Silver embryo at the nasal side from the pectene. (B) Micrograph of 8-day embryonic homozygote eye. The region indicated by an arrow shows a thickening of RPE epithelium and has already transdifferentiated to the retinal fate. (C) In a 13-day embryo, transdifferentiated neural retina shows well-organized cell layers with a reversed polarity. The original retina begins to degenerate. An arrowhead shows scleral cartilage. (D, E) Culture of RPE sheets from Silver homozygote embryos. Tissues were removed from day 3 embryos and the RPE sheet was separated from the neighboring tissue, the choroid and then cultured for 9–10 days on filter membranes in the absence (D) or presence of fibroblast growth factor (FGF)2 (E). In (D) the epithelial sheet remains densely pigmented and does not transdifferentiate. In the presence of FGF2 (E), RPE cells proliferate, depigment and then transdifferentiate into iodopsin-immunoreactive cone cells.