T lymphocyte-specific gene targeting of Notch receptor glycosyltransferase fringe

NOTCH1是一种Notch受体，对T细胞从淋巴样前体向外发育是必不可少的。例如，Notch1缺陷小鼠完全缺乏T细胞发育。Notch受体及其配体之间的相互作用一般受Notch受体糖基化的影响，哺乳动物中用于糖基化Notch受体的酶是边缘分子，包括三个成员：疯狂边缘、躁狂边缘和自由基边缘。然而，Notch1糖基化在T细胞发育中的作用尚不完全清楚。通过寻找在T细胞发育过程中动态变化其表达的基因，我们发现在胸腺细胞成熟过程中，疯狂条纹(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打靶技术来产生T淋巴细胞-特异性LFG缺陷小鼠。不幸的是，加拿大的另一个小组以我们的项目的方式生产了LFG基因敲除小鼠。最后，我们改变了策略，以实现我们的目标，我们得到了以下结果。利用胎儿胸腺器官培养异位表达LFG的CD4^-CD8^-胸腺细胞可促进未成熟CD8^+T细胞的分化，同时抑制CD4^+CD8^-成熟T细胞的发育。我们发现，LFG通常加强Notch和Notch配体(Delta家族)之间的相互作用，调节T细胞的发育，可能影响Notch1的糖基化。有趣的是，淋巴前体中LFG的高表达通过加强自身(细胞自主)的Notch信号来调节T细胞的发育，并通过在胸腺(非自主)竞争有限数量的Notch配体来抑制邻近前体细胞向T细胞的发展(非自主)。这些结果表明，LFG在设定Notch信号的阈值，以细胞自主和非自主的方式促进T细胞发育方面具有新的作用。

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.