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Genesis
2013 Jun 01;516:410-9. doi: 10.1002/dvg.22378.
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Loss of cell-extracellular matrix interaction triggers retinal regeneration accompanied by Rax and Pax6 activation.
Nabeshima A, Nishibayashi C, Ueda Y, Ogino H, Araki M.
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The whole retina regenerates from retinal pigmented epithelial (RPE) cells by transdifferentiation in the adult newt and Xenopus laevis when it is surgically removed. We produced a transgenic animal line, in which EGFP expression is under the control of Rax pomotor. Using F1 and F2 generations, we analyzed Rax-EGFP expression during retinal regeneration in a tissue culture model. In the culture, 4 zones were distinguished as RPE cells migrating outwards from the periphery of the explant: the explant zone, epithelial zone, transition zone and differentiation zone. Expression of transcription factors such as Pax6 and Rax-EGFP was observed in different zones. Rax-EGFP expression preceded Pax6 expression, and the expression of both genes occurred in RPE cells that had lost contact with the basement membrane facing the choroid. We have developed a new culture method in which RPE tissues are embedded in Matrigel. This method has many advantages over the previous gel-overlay method to reproduce construction of 3D-retinal structures and clearly showed that RPE cells need to be detached from the choroid before entering the regeneration pathway. The present results indicate that the temporal changes in cell-cell and cell-extracellular matrix interactions regulate transdifferentiation.
FIG. 1. Expression of the Rax gene in the transgenic Xenopus F0 founder. A, B: In situ hybridization of the Rax gene in Xenopus embryos at St. 35. In both whole mount and section preparations, Rax was expressed specifically in the neural retina. C, D: Pax6 is also expressed similarly to Rax at this stage of development. E: EGFP expression in transgenic animals at St. 27 (E, F) and St. 31 (G, H). EGFP is local- ized only in the eye region. In H, the neural retina is intensely stained for EGFP and the lens is devoid of staining.
FIG. 2. Xenopus RPE with choroid cultured for 82 days. A: A phase-contrast micrograph of culture on Day 12. Four different zones can be recognized from the cell morphology. In A, the explant zone (asterisk), epithelial zone, transition zone, and differentiation zone are shown from left to right, demarcated by dotted lines. B: Connexin 43 immunoreactivity is found in cells of the epithelial zone. C: Hoechst staining indicates cells become more flattened in the transition zone. D: Double staining for Pax6 (orange color) and connexin 43 (green color) in culture on Day 8. Intense Pax6 localization appears to be found in the nuclei of cells at the transition zone. Asterisk indicates the explant, and the dotted line is the boundary between the explant and the epithelial zone.
FIG. 3. Real time detection of EGFP fluorescence by Rax-promoter activity in cultured RPE cells. A, B: Day 3, (C) Day7, (F) Day 15. In A and B, only a few cells are positive for EGFP within the explant (asterisks), while many cells express EGFP in the epithelial zone. The dotted line shows the boundary of the explant and the epithelial zone. The red arrowheads indicate cells with EGFP fluorescence. In C, cells in the epithelial zone are positive for EGFP fluorescence, while those in the transition zone become mostly negative (red arrowheads). In F, similar observations were obtained in cultures at Day 15, and cells in the transition zone show faint EGFP fluorescence (red arrow- heads). The areas demarcated by the rectangular squares in C and D are shown at a higher magnification in D, E and G, H, respectively.
FIG. 4. EGFP expression in the cultured RPE cells. A, B: Phase contrast (A) and EGFP fluorescence (B) micrographs of the same culture on Day 5. The dotted line demarcates the explant from the epithelial zone. Real time EGFP fluorescence is seen in some of the cells in the explant zone (arrows in B). Most of the cells in the epithelial zone appear to show EGFP fluorescence, while in the transition zone only a few cells have fluorescence. (C, D) Cultures in A were fixed, and sections were made vertically to the membrane filter. They were immunostained for EGFP. EGFP-positive cells are shown by arrowheads. The areas demarcated by rectangular squares in C and D are shown in E-1 to E-3 and F-1 to F-3. E-2 and F-2 show EGFP immunofluorescence, while E-3 and F-3 are Hoechst nuclear staining. Arrowheads in E-1, E-2 and E-3 show identical cell nuclei. Fine dotted lines in E and F indicate the position of membrane filters.
FIG. 5. Neuronal differentiation in Matrigel embedding culture of an explant consisting of RPE and choroid and the effect of Dispase treat- ment. Tissues were treated with Dispase. The explants were then embedded in Matrigel and cultured for 26 days. A: An overview image of the culture. The explant is at the center of the micrograph, and numerous cells and long branching fibers extend out from the periphery of the explant. Fibers often form a bundle (arrow). Phase-contrast micrograph. B: Acetylated tubulin immunocytochemistry shows intensely stained long fibers extending at the periphery of the gel. C, D: No treatment with Dispase, and (E, F) treatment with Dispase for 48 h. (D, F) show images of a higher magnification of C and E, respectively. Black arrows in C and D and white arrows in E and F indicate the positions of Bruch membranes. Arrowheads in F show the nuclei of RPE cells disposed randomly. C: Sections were stained with hematoxylin and eosin.
FIG. 6. Regeneration of neural retinal tissues in Matrigel-embedding culture of RPE and choroid after Dispase treatment. A: Day 5, B: Day 10, C: Day 30. In A, the epithelium has grown to form a stratified type of epithelium, and RPE cells facing the Bruchmembrane (arrow) are more densely pigmented. In C, the unpigmented stratified type of epithelium has become separated from the RPE layer, which is on Bruch membrane (arrow). In C, a well-developed layer of cells is observed, and the ganglion cell layer and the inner plexiform layer (red arrow) can be recognized. A: Sections were stained with hematoxylin and eosin. D: are sections neighboring each other. D: transmis- sion image, (E) acetylated tubulin immunocytochemisty, and (F) rhodopsin immunocytochemistry. Arrows indicate the position of Bruch membrane. G: A section of an explant without Dispase treatment. The arrow demarcates RPE cells from the choroid (asterisk). H, I: Pax6 immunocytochemistry in a Matrigel-embedding culture on Day 3. In H, Pax6-positive cells are located at the upper surface of the RPE. The lower cells close to Bruch membrane (indicated by arrows) are negative for Pax6. I: Hoechst staining of the same section.
