Adult newts can regenerate their entire retina through transdifferentiation of the retinal pigment epithelium (RPE). The objective of this study was to redescribe the retina regeneration process by means of modern biological techniques. We report two different antibodies (RPENo.112 and MAB5428) that recognize the newt homolog of RPE65, which is involved in the visual cycle and exclusively label the RPE cell-layer in the adult newt eye. We analyzed the process of retinal regeneration by immunohistochemistry and immunoblotting and propose that this process should be divided into nine stages. We found that the RPE65 protein is present in the RPE-derived new retinal rudiment at 14 days postoperative (po) and in the regenerating retinas at the 3-4 cell stage (19 days po). These observations suggest that certain characteristics of RPE cells overlap with those of retinal stem/progenitor cells during the period of transdifferentiation. However, RPE65 protein was not detected in either retinal stem/progenitor cells in the ciliary marginal zone (CMZ) of adult eyes or in neuroepithelium present during retina development, where it was first detected in differentiated RPE. Moreover, the gene expression of RPE65 was drastically downregulated in the early phase of transdifferentiation (by 10 days po), while those of Connexin43 and Pax-6, both expressed in regenerating retinas, were differently upregulated. These observations suggest that the RPE65 protein in the RPE-derived retinal rudiment may represent the remainder after protein degradation or discharge rather than newly synthesized protein.

Adult newts can regenerate their entire retinas through transdifferentiation of the retinal pigment epithelium (RPE) cells. As yet, however, underlying molecular mechanisms remain virtually unknown. On the other hand, in embryonic/larval vertebrates, an MEK [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] pathway activated by fibroblast growth factor-2 (FGF2) is suggested to be involved in the induction of transdifferentiation of the RPE into a neural retina. Therefore, we examined using culture systems whether the FGF2/MEK pathway is also involved in the adult newt RPE transdifferentiation. Here we show that the adult newt RPE cells can switch to neural cells expressing pan-retinal-neuron (PRN) markers such as acetylated tubulin, and that an MEK pathway is essential for the induction of this process, whereas FGF2 seems an unlikely primary induction factor. In addition, we show by immunohistochemistry that the PRN markers are not expressed until the 1-3 cells thick regenerating retina, which contains retinal progenitor cells, appears. Our current results suggest that the activation of an MEK pathway in RPE cells might be involved in the induction process of retinal regeneration in the adult newt, however if this is the case, we must assume complementary mechanisms that repress the MEK-mediated misexpression of PRN markers in the initial process of transdifferentiation.

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