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In the present study we report that basic fibroblast growth factor (bFGF, FGF-2) promotes the transdifferentiation of Xenopus laevis larval retinal pigment epithelium (RPE) into neural retina. Using specific antibodies we have examined the cellular composition of the regenerated retinal tissue. Our results show that, in addition to retinal neurons and photoreceptors, glial cells were also regenerated from the transdifferentiated RPE. These results were specific to FGF-2, since other factors that were tested, including acidic FGF (aFGF, FGF-1), epidermal growth factor (EGF), laminin, ECL, and Matrigel, exhibited no activity in inducing retinal regeneration. These results are the first in amphibians demonstrating the functional role of FGF-2 in inducing RPE transdifferentiation. Transplantation studies were carried out to investigate retinal regeneration from the RPE in an in vivo environment. Sheets of RPE implanted into the lens-less eyes of larval hosts transformed into neurons and glial cells only when under the influence of host retinal factors. In contrast, no retinal transdifferentiation occurred if the RPE was implanted into the enucleated orbit. Taken together, these results show that the amphibian RPE is capable of transdifferentiation into neuronal and glial cell-phenotypes and implicate FGF-2 as an important factor in inducing retinal regeneration in vitro.
Fig. 1. Photomicrographs of tissue sections from RPE explants treated for 14 DIV with FGF-2 (20 ng/ml). A,C: Differential interference contrast (DIC) images of the sectioned RPE explants. B,D: The corresponding fluorescence images. A,B: XAR-1 immunoreactivity of the RPE cells. The asterisk indicates a region of newly generated, nonpigmented cells within the RPE explant. C,D: XAN-1 labeling of neuroepithelial cells and neurons throughout the regenerating RPE explant. Abbreviation: RPE, Retinal pigment epithelium. Scale
bars 5 100 µm.
Fig. 2. Photomicrographs of tissue sections from control RPE explants after 21 DIV. A,C,E: DIC
photomicrographs of the sectioned RPE explants. B,D,F: The corresponding fluorescence images. A,B: XAR-1
labeling of the RPE cells of the explant. C,D: The control RPE explants were not labeled with the XAN-1
antibody. E,F: The control RPE explants were not labeled with the anti-GFAP antibody. The asterisk in E
indicates a central region within the RPE explant devoid of cells. Scale bars 5 100 µm.
Fig. 3. Photomicrographs of tissue sections from RPE explants
treated for 21 DIV with FGF-2 (20 ng/ml). A,C,E: DIC photomicrographs of
the sectioned RPE explants. B,D,F: The corresponding fluorescence
images. A,B: XAN-1 labeling of RPE regenerated neural tissue. C–F:
XAP-1 labeling of photoreceptor-like cells regenerated from the RPE. E,F:
Higher magnification images of C and D, respectively. The inset in E is a higher magnification image from the region marked by the arrow. In the
inset, the asterisks indicate three XAP-1 immunoreactive cells that
morphologically resemble immature photoreceptors. The arrows in F
illustrate XAP-1-IR processes within the regenerated retina. Abbreviation:
RPE, retinal pigment epithelium. Scale bars for A,B,E,F 5 50 µm and for
C,D 5 100 µm.
Fig. 4. Basic FGF-induced regeneration of retinal glia. Photomicrographs
of tissue sections from RPE explants treated for 21 DIV with
FGF-2 (20 ng/ml). A,C,E: DIC photomicrographs of the sectioned RPE
explants. B,D,F: The corresponding fluorescence images. A,B: AntiGFAP
labeling of glia cells. C–F: Anti-VIM labeling of glial cells. E,F: higher magnification images of C and D, respectively. The arrows in F
illustrate anti-VIM-IR within radially oriented cells resembling Mu¨ ller glial
cells. Abbreviation: RPE, Retinal pigment epithelium. Scale bars for
A–D 5 100 µm and for E and F 5 50 µm.
Fig. 5. Photomicrographs of tissue sections of Xenopus RPE transplants
following transdifferentiation within the lens-less eyes of tadpole
hosts. The transplanted RPE is marked by the asterisk. A,D,F: DIC
photomicrographs of the sectioned RPE transplants. B,C,E,G: The
corresponding fluorescence images. A–C: XAN-1 labeling of transplanted
RPE and host retina. The arrow in B and C indicates XAN-1-IR in the
non-pigmented tissue which transdifferentiated from the transplanted RPE. D–G: Anti-VIM labeling of transplanted RPE after 28 DIV. F,G:
Higher magnification images of the RPE transplant in D and E, respectively.
The arrowheads in G show examples of anti-VIM-labeled Mu¨ ller
cells within the host retina. The arrows in E and G indicate anti-VIM-IR within
the transdifferentiated RPE. Abbreviations: RPE, Retinal pigment epithelium;
INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell
layer. Scale bars for A,B,D,E 5 10 µm; C 5 30 µm; F,G 5 50 µm.
