Roles of Wnt/β-catenin/TCF signal transduction pathway in early T cell development

Wnt/β-catenin/TCF信号转导通路在早期T细胞发育中的作用

• 批准号: 10670299
• 负责人: FUJIMOTO Shinji
• 金额: $ 1.86万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670299/
• 关键词: murine early T cell development; Wnt; Frizzled 3; dominant negative form; retroviral vector; signal transduction; シグナル伝達経路; ドミナントネガティプフォーム; T前駆細胞; 造血系前駆細胞; TCF; LEF-1; Frizzled3; β カテニン; RT-PCR

Wnt/β-catenin/TCF signaling is important for murine early T cell development. However, our understanding of each transmitting molecule is incomplete. As the result of this study, a member of the Frizzled (Fz) family, Fz3, to which secreted Wnt protein binds, is revealed to be one of key factors in the pathway. I have also established a method efficiently transducing gene into murine hematopoietic progenitors.The expression level of Fz genes in fetal and adult thymus was examined by RT-PCR.We found that Fz3 is exclusively transcribed in fetal thymocytes, and that the highest expression is observed at the developmental stage after which rearrangement of T cell receptor (TCR) β chain gene occurs. In order to investigate the function of Fz3 during early T cell development, a dominant negative form retaining only Fz3 extracellular domain (sFz3) was forced to express in hematopoietic progenitors and the cells were cultured under the conditions suitable for T cell development. For the transduction, a retroviral vector with GFP gene as a marker was used. The number of cells recovered after the culture was far less than that obtained from the vector-transduced sample. Surprisingly, immature CD3^-CD4^-CD8^-CD44^-CD25^+ T cells still existed in the sFz3-transduced sample. Additionally, Fz3 is not expressed in TCR^+ T cells. Taken together, my data strongly suggest that the signal mediated by Fz3 is indispensable for differentiation and proliferation of T progenitors which rearranged TCR β but not α chain gene, and that the signal is partly responsible for multiplying more primitive T progenitors with unrearranged TCR β chain loci. In the future, Wnt molecules that bind to Fz3 in the T progenitors should be determined. In addition, analysis of target genes regulated by the Fz3 signaling will be required to comprehend the molecular mechanisms of early T cell development.

Wnt/β-catenin/TCF信号传导对于小鼠早期T细胞发育是重要的。然而，我们对每一个传输分子的理解是不完整的。作为本研究的结果，卷曲（Fz）家族的成员，Fz 3，分泌的Wnt蛋白结合，被揭示是该途径中的关键因子之一。本研究还建立了一种将Fz基因导入小鼠造血祖细胞的方法，用RT-PCR方法检测了Fz基因在胎儿胸腺和成年胸腺中的表达，发现Fz 3基因只在胎儿胸腺细胞中转录，在发育阶段表达最高，之后T细胞受体（TCR）β链基因发生重排。为了研究Fz 3在早期T细胞发育过程中的功能，迫使仅保留Fz 3胞外结构域的显性阴性形式（sFz 3）在造血祖细胞中表达，并在适合T细胞发育的条件下培养细胞。对于转导，使用具有GFP基因作为标记的逆转录病毒载体。培养后回收的细胞数远少于从载体转导的样品中获得的细胞数。令人惊讶的是，未成熟的CD 3 ^-CD 4 ^-CD 8 ^-CD 44 ^-CD 25 ^+ T细胞仍然存在于sFz 3转导的样品中。此外，Fz 3在TCR^+ T细胞中不表达。综上所述，我们的数据强烈地表明，Fz 3介导的信号是T祖细胞分化和增殖所必需的，其重排了TCR β而不是α链基因，并且该信号部分地负责增殖更多具有未重排的TCR β链位点的原始T祖细胞。将来，应该确定与T祖细胞中Fz 3结合的Wnt分子。此外，需要对Fz 3信号转导调控的靶基因进行分析，以理解早期T细胞发育的分子机制。

项目成果

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Kawamoto, Hiroshi：“小鼠胎儿肝脏中 T 细胞祖细胞的出现早于 B 细胞祖细胞”（待发表）。

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Ikawa, Tomokatsu：“单个祖细胞揭示了小鼠胎儿胸腺中常见 T/自然杀伤 (NK) 祖细胞对单能 T 和 NK 祖细胞的承诺”，实验医学杂志 190・11（1999）。

Kawamoto,H: "T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver."Immunity. 12. 441-450 (2000)

Kawamoto, H：“在小鼠胎儿肝脏中，T 细胞祖细胞比 B 细胞祖细胞出现得更早。”免疫。

Ohmura, K., Kawamoto, H., Fujimoto, S., Ozaki, S., Nakao, K.and Kastura, Y.: "Emergence of T, B, and myeloid lineage-committed as well as multipotent hematopoietic progenitors in the aortagonad-mesonephros region of day 10 fetuses of the mouse."J.lmmunol.

Ohmura, K.、Kawamoto, H.、Fujimoto, S.、Ozaki, S.、Nakao, K. 和 Kastura, Y.：“T、B 和骨髓谱系定向以及多能造血祖细胞的出现

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