XB-ART-40427Dev Neurobiol December 1, 2009; 69 (14): 950-8.
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Complete reconstruction of the retinal laminar structure from a cultured retinal pigment epithelium is triggered by altered tissue interaction and promoted by overlaid extracellular matrices.
The retina regenerates from retinal pigment epithelial (RPE) cells by transdifferentiation in the adult newt and Xenopus laevis when it is surgically removed. This was studied under a novel culture condition, and we succeeded, for the first time, in developing a complete retinal laminar structure from a single epithelial sheet of RPE. We cultured a Xenopus RPE monolayer sheet isolated from the choroid on a filter cup with gels overlaid and found that the retinal tissue structure differentiated with all retinal layers present. In the culture, RPE cells isolated themselves from the culture substratum (filter membrane), migrated, and reattached to the overlaid gel, on which they initiated transdifferentiation. This was exactly the same as observed during in vivo retina regeneration of X. laevis. In contrast, when RPE monolayers were cultured similarly without isolation from the choroid, RPE cells proliferated, but remained pigmented instead of transdifferentiating, indicating that alteration in tissue interaction triggers transdifferentiation. We then examined under the conventional tissue culture condition whether altered RPE-choroid interaction induces Pax6 expression. Pax6 was upregulated in RPE cells soon after they were removed from the choroid, and this expression was not dependent of FGF2. FGF2 administration was needed for RPE cells to maintain Pax6 expression. From the present results, in addition to our previous ones, we propose a two-step mechanism of transdifferentiation: the first step is a reversible process and is initiated by the alteration of the cell-extracellular matrix and/or cell-cell interaction followed by Pax6 upregulation. FGF2 plays a key role in driving RPE cells into the second step, during which they differentiate into retinal stem cells.
PubMed ID: 19701886
Article link: Dev Neurobiol
Species referenced: Xenopus laevis
Genes referenced: fgf2 pax6 rho rpe
Article Images: [+] show captions
|Figure 1 Summary of Gel overlay culture for isolated RPE of Xenopus laevis. The anterior parts were removed from enucleated eyecups, and the retina and sclera were then discarded. The rest of the tissues consisting of RPE and choroid was either cultured on a filter cup or treated with dispase to remove the choroid. They were then cultured on filter cups. Gels, either Matri-Gel or collagen gel, were then overlaid on the tissues. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]|
|Figure 2 Cultures of RPE with choroid and isolated RPE overlaid with Matri-Gel. A: Isolation of RPE from the choroid by dispase treatment. RPE sheets (black arrow) are clearly removed from the choroid. Bruch’ membrane (blue arrow) remains with the choroid. Azan staining. Isolated RPE sheets (B, D, F, H) and RPE with choroid (C, E, G) were cultured on filter membranes. In the culture of RPE with choroid, two layers of RPE are observed as shown in (C, E, G); one is attached to the choroid, and the other to the gel (asterisks). RPE cells are well arranged to form simple epithelial sheets. RPE cells became smaller and more flattened as cultures proceeded, and they did not transdifferentiate into the retina. In the cultures of isolated RPEs (B, D, F, H), an epithelial layer of unpigmented cells is found attached to the gel (asterisk) on Day 14 (D), and clearly distinguishable from the pigmented cell layer. On day 20, the unpigmented layer shows a pseudostratified form and nerve fiberlike processes are seen apposing the gel (blue arrow in F). On day 35, a well-organized, retinalike structure with different layers is observed (H). B–H: Hematoxyline and eosin staining. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]|
|Figure 3 Differentiation of retinal laminar structure and localization of retinal antigens in isolated RPE cultures. Cultures were fixed on days 35–40 and subjected to immunocytochemistry for rhodopsin (A, B), GFAP (C, D), and acetylated tubulin (E, F). Rhodopsin was detected on the outer segmentlike structures. At a higher magnification, numerous rod outer segmentlike structures were seen and were positively stained for rhodopsin (G, H). GFAP-positive structures extend transversely from the inner to the outer limiting membranous structures (arrows in D). Acetylated tubulin-positive fibers are found in the nerve fiber layer and inner plexiform layer (arrows in E and F). (A, C, E) Hematoxyline and eosin staining. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.]|
|Figure 5 A schematic drawing illustrating a two-step model of retinal regeneration. In the first step, alteration in cell-substratum and/or cell–cell interaction may trigger Pax6 expression. FGF2 may drive Pax6-positive RPE cells into the second step, where RPE cells undergo transdifferentiation. The first step is FGF-independent, whereas the second step is FGF-dependent. FGF2, a key signal molecule for transdifferentiation, is effective on Pax6-positive cells, which then undergo transdifferentiation to become retinal stem cells. The first step is a reversible process, whereas the second is irreversible. BM, basement menbrane; RVM, retinal vascular membrane. [Color figure can be viewed in the online issue, which is available at www.interscience. wiley.com.]|