Notch3 in generation and maintenance of the T lineage.

描述（申请人提供）：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，而是依赖于血液循环中不断募集的骨髓源祖细胞。最早的胸腺内祖细胞是多能的，但值得注意的是，缺乏B系潜能，因此不对应于任何已知的骨髓祖细胞。胸腺内的微环境条件诱导这些多能祖细胞采用T系命运，并不对称分化为多个不同的T系。虽然Notch1 （N1）已被证明在T谱系规范中起关键作用，但它不足以解释胸腺中产生多个T谱系的复杂过程。结合PHS award R21AI53739，我们试图确定其他可能的T谱系分化调节因子。在我们鉴定的基因中有Notch3 （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、与年龄相关的变性。这种表型与一种与N3缺乏（一种遗传性突变）相关的人类疾病（CADASIL）一致，其特征是与年龄相关（成人发病）的血管平滑肌细胞变性和复发性中风。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系前体年龄相关变性的遗传缺陷。此外，骨髓中对N3的特异性需求表明，N3可能是骨髓中T系细胞长期追求的前体的标记物。简而言之，该项目的目标是：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谱系潜力的存在）；3)确定N3在胸腺和骨髓中是否也有作用，这种作用与N1在多大程度上重叠；4)确定T谱系中N3信号的靶点，从而开始确定其分子功能。这些方法包括在体内和体外测定不同年龄小鼠骨髓中的T系电位；谱系追踪通过N3信号的骨髓细胞后代，使用N3:Cre融合蛋白敲入有条件地激活荧光报告基因；报告阳性细胞的体内和体外谱系电位评估；N3缺陷小鼠胸腺内N3缺失和中期胸腺内N1缺失；利用N3:Gal3融合蛋白敲入评估胸腺和骨髓祖细胞中N3信号活性；并评估通过N3发出信号的细胞中的基因表达，以及在年轻的N3敲除小鼠中的对应细胞。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，Notch3基因的基因突变会导致骨髓启动胸腺T淋巴细胞生产能力的加速、与年龄相关的下降。Notch3也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Notch3如何防止免疫系统衰老，并可能进一步了解Notch3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.