Notch3 in generation and maintenance of the T lineage.

项目摘要 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，而是依赖于恒定的 在血液中循环的骨髓来源的祖细胞的募集。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，并不对称地分化为 多种不同的T细胞谱系Notch 1（N1）在T细胞系中起关键作用， 规范，它不足以解释产生多个T谱系的复杂过程 在胸腺中。结合PHS裁决R21 AI 53739，我们试图确定其他推定的 调节T细胞分化。我们发现的基因之一是Notch 3（N3）。N3 基因敲除小鼠已经产生，其他人发现它们明显正常， 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 表型与N3缺乏相关的人类疾病（CADASIL）一致（a 遗传性突变），其特征是年龄相关的（成年发病）血管变性， 平滑肌细胞和复发性中风我们目前的研究结果表明，N3突变是 第一个已知的遗传缺陷导致骨中T谱系前体的年龄相关性退化 骨髓此外，骨髓中对N3的这种需求的特异性表明，N3可能 代表了骨髓中T细胞前体的标志物。简明地 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祖细胞活性所需; 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 存在B谱系潜力）; 3）确定N3是否在胸腺中以及在 骨髓，以及这种功能与N1的功能在多大程度上重叠;以及4）识别 T谱系中N3信号传导的靶点，并因此开始定义其分子功能。的 方法包括在体内和体外测定小鼠骨髓中的T谱系潜能， 不同的年龄;谱系追踪通过N3信号的骨髓细胞的后代，使用 N3：Cre融合蛋白敲入以条件性激活荧光报告基因;体内和体外 评估β-受体阳性细胞的谱系潜力;胸腺内缺失N3， N3缺陷小鼠中期胸腺内N1缺失; N3评估 使用N3：Gal 3融合蛋白敲入，在胸腺和骨髓祖细胞中的信号传导活性; 和评估通过N3信号的细胞中的基因表达，以及它们的对应物 在年轻的N3基因敲除小鼠中。

PROJECT SUMMARY 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.