Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Dev Neurobiol
2009 Dec 01;6914:950-8. doi: 10.1002/dneu.20745.
Show Gene links
Show Anatomy links
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.
Kuriyama F
,
Ueda Y
,
Araki M
.
???displayArticle.abstract???
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.
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.]
