Retinal regeneration after injury induced by gamma-ray irradiation during early embryogenesis in medaka, <i>Oryzias latipes</i>

Zebrafish, a small fish model, exhibits a multipotent ability for retinal regeneration after damage throughout its lifetime. Compared with zebrafish, birds and mammals exhibit such a regenerative capacity only during the embryonic period, and this capacity decreases with age. In medaka, another small fish model that has also been used extensively in biological research, the retina’s inner nuclear layer (INL) failed to regenerate after injury in the hatchling at eight days postfertilization (dpf). We characterized the regenerative process of the embryonic retina when the retinal injury occurred during the early embryonic period in medaka. We employed a 10 Gy dose of gamma-ray irradiation to initiate retinal injury in medaka embryos at 3 dpf and performed histopathological analyses up to 21 dpf. One day after irradiation, numerous apoptotic neurons were observed in the INL; however, these neurons were rarely observed in the ciliary marginal zone and the photoreceptor layer. Numerous pyknotic cells were clustered in the irradiated retina until two days after irradiation. These disappeared four days after irradiation, but the abnormal bridging structures between the INL and ganglion cell layer (GCL) were present until 11 days after irradiation, and the neural layers were completely regenerated 18 days after irradiation. After gamma-ray irradiation, the spindle-like Müller glial cells in the INL became rounder but did not lose their ability to express SOX2. Irradiated retina at 3 dpf of medaka embryos could be completely regenerated at 18 days after irradiation (21 dpf), although the abnormal layer structures bridging the INL and GCL were transiently formed in the retinas of all the irradiated embryos. Four days after irradiation, embryonic medaka Müller glia were reduced in number but maintained SOX2 expression as in nonirradiated embryos. This finding contrasts with previous reports that 8 dpf medaka larvae could not fully regenerate damaged retinas because of loss of SOX2 expression.作为小型模式生物的斑马鱼（Zebrafish）在其整个生命周期中，受损后均具备视网膜再生的多能能力。与斑马鱼相比，鸟类与哺乳类仅在胚胎期具备此类再生能力，且该能力随年龄增长逐渐衰退。青鳉（medaka）是另一类被广泛应用于生物研究的小型模式生物，其受精后8天（eight days postfertilization, dpf）的孵化幼体视网膜内核层（inner nuclear layer, INL）在受损后无法完成再生。本研究对青鳉早期胚胎阶段发生视网膜损伤时的胚胎视网膜再生过程进行了表征。我们采用10 Gy剂量的γ射线辐照（gamma-ray irradiation），在受精后3天（3 dpf）的青鳉胚胎中诱导视网膜损伤，并在受精后21 dpf前开展组织病理学分析（histopathological analyses）。辐照后1天，内核层中可见大量凋亡神经元（apoptotic neurons），但此类神经元在睫状缘区（ciliary marginal zone）与光感受器层（photoreceptor layer）中极少被观测到。直至辐照后2天，辐照视网膜中仍可见大量固缩细胞（pyknotic cells）聚集；辐照后4天，此类固缩细胞消失，但内核层与神经节细胞层（ganglion cell layer, GCL）之间的异常桥接结构可持续至辐照后11天，而神经各层直至辐照后18天才完全完成再生。γ射线辐照后，内核层中呈梭形的米勒胶质细胞（Müller glial cells）形态变圆，但并未丧失表达SOX2的能力。尽管所有辐照胚胎的视网膜中均一过性形成了内核层与神经节细胞层之间的异常层状结构，但在受精后3 dpf接受辐照的青鳉胚胎，其视网膜可在辐照后18天（即21 dpf）完全再生。辐照后4天，青鳉胚胎的米勒胶质细胞数量有所减少，但仍如未辐照胚胎一般维持SOX2的表达。这一发现与既往研究结论相悖：既往研究表明，受精后8 dpf的青鳉幼体因丧失SOX2表达能力，无法完全修复受损视网膜。