Program in Stem Cell and Developmental Biology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.
PLoS One. 2011;6(9):e24937. doi: 10.1371/journal.pone.0024937. Epub 2011 Sep 13.
Notch1 (N1) signaling induced by intrathymic Delta-like (DL) ligands is required for T cell lineage commitment as well as self-renewal during "β-selection" of TCRβ⁺CD4⁻CD8⁻ double negative 3 (DN3) T cell progenitors. However, over-expression of the N1 intracellular domain (ICN1) renders N1 activation ligand-independent and drives leukemic transformation during β-selection. DN3 progenitors also express Notch3 (N3) mRNA, and over-expression of ligand-independent mutant N3 (ICN3) influences β-selection and drives T cell leukemogenesis. However, the importance of ligand-activated N3 in promoting β-selection and ICN1-induced T cell leukemogenesis has not been examined. To address these questions we generated mice lacking functional N3. We confirmed that DN3 progenitors express N3 protein using a N3-specific antibody. Surprisingly however, N3-deficient DN3 thymocytes were not defective in generating DP thymocytes under steady state conditions or in more stringent competition assays. To determine if N3 co-operates with N1 to regulate β-selection, we generated N1;N3 compound mutants. However, N3 deficiency did not exacerbate the competitive defect of N1⁺/⁻ DN3 progenitors, demonstrating that N3 does not compensate for limiting N1 during T cell development. Finally, N3 deficiency did not attenuate T cell leukemogenesis induced by conditional expression of ICN1 in DN3 thymocytes. Importantly, we showed that in contrast to N1, N3 has a low binding affinity for DL4, the most abundant intrathymic DL ligand. Thus, despite the profound effects of ectopic ligand-independent N3 activation on T cell development and leukemogenesis, physiologically activated N3 is dispensable for both processes, likely because N3 interacts poorly with intrathymic DL4.
Notch1 (N1) 信号由胸腺内 Delta-like (DL) 配体诱导，对于 T 细胞谱系的承诺以及 TCRβ⁺CD4⁻CD8⁻双阴性 3 (DN3) T 细胞祖细胞的“β选择”期间的自我更新是必需的。然而，N1 细胞内结构域 (ICN1) 的过度表达使 N1 激活配体不依赖，并在β选择期间驱动白血病转化。DN3 祖细胞也表达 Notch3 (N3) mRNA，并且无配体依赖性突变体 N3 (ICN3) 的过度表达影响β选择并驱动 T 细胞白血病发生。然而，激活的 N3 在促进β选择和 ICN1 诱导的 T 细胞白血病发生中的重要性尚未被研究。为了解决这些问题，我们生成了缺乏功能性 N3 的小鼠。我们使用 N3 特异性抗体证实 DN3 祖细胞表达 N3 蛋白。然而，令人惊讶的是，在稳态条件下或更严格的竞争测定中，缺乏 N3 的 DN3 胸腺细胞在产生 DP 胸腺细胞方面没有缺陷。为了确定 N3 是否与 N1 合作调节β选择，我们生成了 N1;N3 复合突变体。然而，N3 缺乏并没有加剧 N1⁺/⁻DN3 祖细胞的竞争缺陷，表明在 T 细胞发育过程中，N3 不会弥补 N1 的限制。最后，N3 缺乏并没有减弱 ICN1 在 DN3 胸腺细胞中条件表达诱导的 T 细胞白血病发生。重要的是，我们表明与 N1 相反，N3 与 DL4（胸腺内最丰富的 DL 配体）的结合亲和力较低。因此，尽管异位无配体依赖性 N3 激活对 T 细胞发育和白血病发生有深远影响，但生理激活的 N3 对于这两个过程都是可有可无的，这可能是因为 N3 与胸腺内 DL4 相互作用不佳。
