Lin Ya-ping, Ouchi Yasuo, Satoh Shinya, Watanabe Sumiko
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Invest Ophthalmol Vis Sci. 2009 Jan;50(1):68-74. doi: 10.1167/iovs.07-1619. Epub 2008 Aug 21.
The transcription factor Sox2 plays important roles in both human and mouse retinal development. Although loss-of-function mutations in Sox2 have been studied in mice, gain-of-function experiments in the neural retina have been lacking.
The detailed expression pattern of Sox2 in the developing mouse retina was examined by immunohistochemistry. Then, Sox2 was expressed in a retinal explant culture prepared from E17 mouse embryos by retrovirus-mediated gene transfer to examine its role in retinal development. In addition, shRNA was used to suppress Sox2 in a retinal explant using a retrovirus-mediated system.
Sox2 was expressed throughout the neuroblastic layer in the embryonic retina, but only in the inner nuclear layer in the mature retina. Double immunostaining revealed that Sox2 was expressed in Müller glial cells and in a subset of amacrine cells. Forced expression of Sox2 in a mouse retinal explant culture resulted in the dramatic accumulation of amacrine cells in the inner nuclear layer; in addition, cells expressing amacrine cell markers were also found on the innermost side of the outer nuclear layer. The expression of Pax6, which plays an important role in amacrine cell differentiation, was observed in the Sox2-expressing cells, and Sox2 activated the Pax6 promoter to drive luciferase expression in Y79 cells. A decrease in retinal progenitor cell proliferation accompanied these effects. The suppression of Sox2 expression by shRNA resulted in a decreased number of cells in the inner nuclear layer.
Therefore, ectopic Sox2 expression can induce amacrine cells in the mouse retina from stage E17 onward, possibly by facilitating cell cycle exit.
转录因子Sox2在人类和小鼠视网膜发育中均发挥重要作用。尽管已在小鼠中研究了Sox2的功能丧失突变，但在神经视网膜中的功能获得实验仍较为缺乏。
通过免疫组织化学检查Sox2在发育中小鼠视网膜中的详细表达模式。然后，通过逆转录病毒介导的基因转移，在由E17小鼠胚胎制备的视网膜外植体培养物中表达Sox2，以研究其在视网膜发育中的作用。此外，使用逆转录病毒介导的系统，利用短发夹RNA（shRNA）抑制视网膜外植体中的Sox2表达。
Sox2在胚胎视网膜的整个神经母细胞层中表达，但在成熟视网膜中仅在内核层中表达。双重免疫染色显示，Sox2在Müller胶质细胞和一部分无长突细胞中表达。在小鼠视网膜外植体培养物中强制表达Sox2导致内核层中无长突细胞大量积聚；此外，在外核层的最内侧也发现了表达无长突细胞标志物的细胞。在表达Sox2的细胞中观察到在无长突细胞分化中起重要作用的Pax6的表达，并且Sox2激活Pax6启动子以驱动Y79细胞中的荧光素酶表达。这些效应伴随着视网膜祖细胞增殖的减少。shRNA抑制Sox2表达导致内核层中的细胞数量减少。
因此，异位表达Sox2可能从E17期起在小鼠视网膜中诱导无长突细胞，可能是通过促进细胞周期退出实现的。
