Chiba Chikafumi, Hoshino Akika, Nakamura Kenta, Susaki Kanako, Yamano Yuka, Kaneko Yuko, Kuwata Osamu, Maruo Fumiaki, Saito Takehiko
Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
J Comp Neurol. 2006 Apr 1;495(4):391-407. doi: 10.1002/cne.20880.
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 (RPE-No.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.
成年蝾螈能够通过视网膜色素上皮（RPE）的转分化来再生其整个视网膜。本研究的目的是借助现代生物技术重新描述视网膜再生过程。我们报告了两种不同的抗体（RPE-No.112和MAB5428），它们能识别RPE65的蝾螈同源物，RPE65参与视觉循环，且仅标记成年蝾螈眼中的RPE细胞层。我们通过免疫组织化学和免疫印迹分析了视网膜再生过程，并提出该过程应分为九个阶段。我们发现，术后14天（po），RPE65蛋白存在于RPE衍生的新视网膜原基中，在3-4细胞阶段（术后19天）存在于再生视网膜中。这些观察结果表明，在转分化期间，RPE细胞的某些特征与视网膜干细胞/祖细胞的特征重叠。然而，在成年眼睫状边缘区（CMZ）的视网膜干细胞/祖细胞或视网膜发育过程中出现的神经上皮中均未检测到RPE65蛋白，RPE65蛋白最初是在分化的RPE中检测到的。此外，在转分化早期（术后10天），RPE65的基因表达急剧下调，而在再生视网膜中均有表达的连接蛋白43和Pax-6的基因表达则有不同程度的上调。这些观察结果表明，RPE衍生的视网膜原基中的RPE65蛋白可能代表蛋白质降解或排出后的剩余物，而非新合成的蛋白质。
