Notch3 in generation and maintenance of the T lineage.

描述(申请人提供)：胸腺在一生中产生新的T淋巴细胞，以维持外周动态平衡和免疫功能。与其他经历稳定分化的组织不同，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持续招募。最早的胸腺内祖细胞是多能的，但值得注意的是，缺乏B系谱系潜力，因此与骨髓中任何已知的祖细胞都不对应。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，并不对称地分化为多个不同的T谱系。虽然Notch1(N1)已被证明在T谱系规范中起关键作用，但它不足以解释在胸腺中产生多个T谱系的复杂过程。结合PHS奖R21AI53739，我们试图确定T细胞系分化的其他假定调节因子。在我们鉴定的基因中，有Notch3(N3)。已经产生了N3基因敲除小鼠，并被其他人发现是明显正常的，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缺乏会导致骨髓中T祖细胞活性的进行性、与年龄相关的退化。这种表型与一种与N3缺乏症(一种可遗传突变)相关的人类疾病(CADASIL)相一致，N3缺乏症的特征是与年龄相关的(成人发病)血管平滑肌细胞变性和反复发作的中风。我们目前的发现显示，N3突变是第一个已知的导致骨髓中T细胞前体细胞年龄相关退化的基因缺陷。此外，骨髓中对N3的这一要求的特异性表明，N3可能代表着骨髓中长期被追捧的T细胞前体的一个标记。简而言之，这个项目的目标是：1)扩大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，并将它们的谱系潜力与早期胸腺内祖细胞的谱系潜力进行比较(特别是对于B谱系的存在)；3)确定N3是否在胸腺和骨髓中具有作用，以及该功能在多大程度上与N1的作用重叠；以及4)确定N3信号在T谱系中的靶点，从而开始定义其分子功能。这些方法包括：体内和体外分析不同年龄小鼠骨髓中的T细胞谱系潜力；利用N3：Cre融合蛋白敲入有条件地激活荧光报告基因，追踪通过N3信号传递信号的骨髓细胞的后代；在体内和体外评估报告阳性细胞的谱系潜力；N3基因缺陷小鼠的胸腺内缺失N3，并在中期阶段删除N1；使用N3：Gal3融合蛋白敲除来评估胸腺和骨髓前体细胞中N3信号的活性；以及评估通过N3信号传递信号的细胞以及年轻N3基因敲除小鼠中相应细胞的基因表达。T淋巴细胞必须在一生中产生，这一过程始于骨髓中的干细胞，并在胸腺中完成。我们发现，一种名为Notch3的基因突变会导致骨髓启动胸腺产生T淋巴细胞的能力加速、与年龄相关的下降。NOTCH3还与另一种与中风有关的年龄相关退行性疾病有关。该项目旨在了解Notch3是如何防止免疫系统衰老的，并可能为进一步了解Notch3在中风中的作用提供依据。

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.