Notch3 in generation and maintenance of the T lineage.

描述（由申请方提供）：胸腺在整个生命过程中产生新的T淋巴细胞，以维持外周稳态和免疫功能。与其他经历稳态分化的组织不同，胸腺不包含自我更新的干细胞或祖细胞，而是依赖于在血液中循环的骨髓来源的祖细胞的不断招募。最早的胸腺内祖细胞是多能的，但值得注意的是，缺乏B谱系潜能，因此不对应于骨髓中任何已知的祖细胞。胸腺内的微环境条件诱导这些多能祖细胞采用T谱系命运，并不对称地分化成多种不同的T谱系。虽然Notch 1（N1）已被证明在T谱系特化中发挥关键作用，但它不足以解释胸腺中产生多个T谱系的复杂过程。结合PHS奖R21 AI 53739，我们试图确定T谱系分化的其他假定调节因子。我们发现的基因之一是Notch 3（N3）。N3基因敲除小鼠已经产生，并被其他人发现明显正常，这一发现在我们的实验室得到了证实。然而，令人惊讶的是，我们发现N3缺乏导致骨髓中T祖细胞活性的进行性、年龄相关性退化。这种表型与N3缺乏（一种遗传突变）相关的人类疾病（CADASIL）一致，其特征是血管平滑肌细胞的年龄相关性（成人发病）变性和复发性中风。我们目前的研究结果表明，N3突变是第一个已知的导致骨髓中T谱系前体细胞年龄相关性变性的遗传缺陷。此外，骨髓中对N3的这种需求的特异性表明，N3可能代表骨髓中长期寻求的T谱系细胞前体的标志物。简而言之，该项目的目标是1）扩展并最终确定我们的发现，即N3是维持骨髓中T祖细胞活性所必需的; 2）鉴定骨髓中表达N3的细胞，并将其谱系潜力与早期胸腺内祖细胞的谱系潜力进行比较（特别是B谱系潜能的存在）; 3）确定N3是否在胸腺和骨髓中具有作用，以及该功能与N1的功能重叠的程度;以及4）鉴定T谱系中N3信号传导的靶点，从而开始确定其分子功能。这些方法包括在不同年龄的小鼠中对骨髓中T谱系潜力的体内和体外测定;使用N3：Cre融合蛋白敲入以条件性激活荧光报告基因，谱系追踪通过N3发信号的骨髓细胞的后代;在体内和体外评估CD 4+阳性细胞中的谱系潜力;在N3缺陷小鼠的中间阶段，胸腺内缺失N3和胸腺内缺失N1;使用N3：Gal 3融合蛋白敲入，评估胸腺和骨髓祖细胞中的N3信号传导活性;以及评估通过N3发出信号的细胞中的基因表达，以及它们在年轻的N3敲除小鼠中的对应物。T淋巴细胞必须在整个生命过程中产生，这一过程始于骨髓中的干细胞，并在胸腺中完成。我们已经发现，一种被称为Notch 3的基因的基因突变导致骨髓启动胸腺产生T淋巴细胞的能力加速下降。Notch 3还与另一种与中风有关的年龄相关的退行性疾病有关。该项目旨在了解Notch 3如何发挥作用以防止免疫系统衰老，并可能进一步了解Notch 3在中风中的作用。

DESCRIPTION (provided by applicant): The thymus produces new T lymphocytes throughout life to maintain peripheral homeostasis and immune function. Unlike other tissues that undergo steady-state differentiation, the thymus contains no self-renewing stem or progenitor cells, and instead depends on constant recruitment of marrow-derived progenitors that circulate in the blood. The earliest intrathymic progenitors are multi-potent, but notably, lack B lineage potential, and thus do not correspond to any known progenitor in the marrow. Microenvironmental conditions inside the thymus induce these multipotent progenitors to adopt the T lineage fate, and to asymmetrically differentiate into multiple different T lineages. While Notch1 (N1) has been shown to play a key role in T lineage specification, it is inadequate to explain the complex process of generating multiple T lineages in the thymus. In conjunction with PHS award R21AI53739, we sought to identify other putative regulators of T lineage differentiation. Among the genes that we identified was Notch3 (N3). N3 knockout mice had already been generated and were found by other to be overtly normal, a finding that was confirmed in our laboratory. Surprisingly, however, we found that N3 deficiency results in a progressive, age-related degeneration of T progenitor activity in bone marrow. This phenotype is consistent with a human disease (CADASIL) associated with N3 deficiency (a heritable mutation), which is characterized by age-related (adult-onset) degeneration of vascular smooth muscle cells and recurrent strokes. Our current findings reveal N3 mutation to be the first known genetic defect leading to age-related degeneration of T lineage precursors in bone marrow. Further, the specificity of this requirement for N3 in marrow suggests that N3 may represent a marker for the long sought-after precursor to T lineage cells in marrow. Concisely stated, the goals of this project are 1) to expand and finalize for publication our finding that N3 is required to maintain T progenitor activity in marrow; 2) to identify N3-expressing cells in marrow, and compare their lineage potentials to those of early intrathymic progenitors (notably, for the presence of B lineage potential); 3) to ascertain whether N3 has a role in the thymus as well as the marrow, and to what extent this function overlaps with that of N1; and 4) to identify the targets of N3 signaling in the T lineage, and thus begin to define its molecular function. The approaches involve in vivo and in vitro assays for T lineage potential in bone marrow in mice at various ages; lineage tracing the progeny of marrow cells that signal through N3, using a N3:Cre fusion protein knock-in to conditionally activate a fluorescent reporter; in vivo and in vitro assessment of lineage potential in the reporter-positive cells; intrathymic deletion of N3, and intrathymic deletion of N1 at intermediate stage in N3-deficient mice; assessment of N3 signaling activity in thymus and marrow progenitors, using a N3:Gal3 fusion protein knock-in; and assessment of gene expression in cells that signal through N3, as well as their counterparts in young N3 knockout mice. T lymphocytes must be produced throughout life, a process that initiates with stem cells in the bone marrow, and is completed in the thymus. We have found that genetic mutation of a gene known as Notch3 results in an accelerated, age-related decline in the ability of the bone marrow to initiate T lymphocyte production by the thymus. Notch3 has also been implicated in another age-related degenerative disorder relating to strokes. This project is aimed at understanding how Notch3 functions to prevent senescence of the immune system, and may provide further insights into the role of Notch3 in strokes as well.