Notch3 in generation and maintenance of the T lineage.

项目总结 胸腺在一生中会产生新的T淋巴细胞，以维持外周内环境的稳定 和免疫功能。与经历稳定分化的其他组织不同，胸腺 不包含自我更新的干细胞或祖细胞，取而代之的是依赖于 招募在血液中循环的骨髓来源的祖细胞。最早的胸腺内 祖细胞是多能的，但值得注意的是，缺乏B血统潜力，因此与 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血统命运，并不对称地分化为 多个不同的T细胞系。而Notch1(N1)已被证明在T血统中发挥关键作用 规范，这不足以解释生成多个T谱系的复杂过程 在胸腺里。结合PHS奖R21AI53739，我们试图确定其他假定的 T细胞系分化的调节因子。在我们鉴定的基因中，有Notch3(N3)。N3 已经产生了基因敲除小鼠，并被其他人发现是明显正常的，一种 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、与年龄相关的退化。这 表型与一种与N3缺乏(A)相关的人类疾病(CADASIL)一致 可遗传突变)，其特征是与年龄相关的(成人发病)血管变性 平滑肌细胞和反复中风。我们目前的发现表明，N3突变是 首次发现导致骨骼中T前体细胞年龄相关性退变的基因缺陷 骨髓。此外，骨髓中N3的这种要求的特异性表明N3可能 代表了骨髓中长期被追捧的T细胞前体的标志。简明扼要 如上所述，这个项目的目标是1)扩展和最终确定我们的发现，即N3是 需要维持骨髓中T祖细胞的活性；2)鉴定骨髓中表达N3的细胞， 并将其与早期胸腺内祖细胞的谱系潜力进行比较(值得注意的是，对于 存在B谱系潜力)；3)确定N3是否在胸腺以及 骨髓，以及这一功能在多大程度上与N1的功能重叠；以及4)识别 N3信号在T谱系中的靶点，从而开始定义其分子功能。这个 方法包括体内和体外检测小鼠骨髓T细胞系潜能。 不同年龄段；追踪通过N3信号的骨髓细胞后代的谱系，使用 N3：Cre融合蛋白敲入有条件地激活荧光报告；体内和体外 评估报告阳性细胞的谱系潜力；胸腺内N3的缺失，以及 N3基因缺陷小鼠中期胸腺内N1基因缺失；N3基因的评估 胸腺和骨髓祖细胞中的信号活性，使用N3：Gal3融合蛋白敲入； 以及评估通过N3和它们的对应物发出信号的细胞中的基因表达 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.