T lymphocyte-specific gene targeting of Notch receptor glycosyltransferase fringe

Notch1是Notch受体之一，在T细胞从淋巴前体细胞发育过程中起着重要作用。例如，notch1缺陷小鼠完全缺乏T细胞发育。Notch受体与其配体之间的相互作用通常受Notch受体糖基化的影响，而将其糖基化的酶在哺乳动物中包括三种成员：疯子、躁狂和激进边缘。然而，Notch1糖基化在T细胞发育过程中的作用尚未完全了解。通过寻找在T细胞发育过程中动态改变表达的基因，我们发现在胸腺细胞成熟过程中，lunatic fringe （Lfg）表现出不同的表达水平。为了阐明Lfg在T细胞分化中的作用，我们尝试使用条件基因靶向技术产生T淋巴细胞特异性Lfg缺陷小鼠。不幸的是，加拿大的另一个研究小组用我们项目的方式培育出了Lfg基因敲除小鼠。最后，我们改变了我们的策略来实现我们的目标，我们得到了以下结果。通过胎儿胸腺器官培养，Lfg异位表达的CD4^-CD8^-胸腺细胞的培养增强了未成熟CD8^+ T细胞的分化，同时抑制了CD4^+CD8^-成熟T细胞的发育。我们发现通常加强Notch与Notch配体（Delta家族）之间相互作用的Lfg调控T细胞发育可能影响Notch1的糖基化。有趣的是，Lfg在淋巴样前体中的高表达通过加强自身Notch信号传导来调节T细胞的发育（细胞自主），并通过胸腺中有限数量的Notch配体竞争来抑制来自邻近前体的T细胞发育（非自主）。这些结果表明，Lfg具有新的作用，可以设置Notch信号通路的阈值，以细胞自主和非自主的方式促进T细胞的发育。

Notch1, one of Notch receptors, is essential for T cell development from lymphoid precursor. For instance, Notch1-deficient mice completely lack T cell development. Interactions between Notch receptors and their ligands are generally affected by glycosylation of Notch receptors, and enzymes to glycosylate them are fringes including three members in mammals : lunatic, manic and radical fringes. However, the role of glycosylation of Notch1 during T cell development has not been fully understood. By searching genes that dynamically change their expression during T cell development, we found that lunatic fringe (Lfg) showed different expression levels during the maturation process of thymocytes. To clarify an involvement of Lfg in T cell differentiation, we attempted to generate T lymphocyte-specifically Lfg-deficient mice using conditional gene targeting technology. Unfortunately the other group in Canada has produced the Lfg knockout mice in the way of our project. Finally, we changed our strategy to address our aim and we got the following results. The culture of CD4^-CD8^- thymocytes in which Lfg was ectopically expressed by using fetal thymic organ culture enhanced differentiation of immature CD8^+ T cells while inhibiting CD4^+CD8^- mature T-cell development. we found that Lfg that generally strengthens the interaction between Notch and Notch ligands (Delta family) regulates T cell development probably affecting the glycosylation of Notch1. Interestingly, the high expression of Lfg in lymphoid precursor regulates T cell development by strengthening Notch signaling of their own (cell autonomous) and inhibits the development toward T cells from neighboring precursors by competing limited amounts of Notch ligands in the thymus (non-autonomous). These results indicate that Lfg has novel roles to set the threshold of Notch signaling to promote T cell development in a cell autonomous and non-autonomous manner.