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Manipulation of hematopoietic cell development by expression of transcription factors

通过转录因子的表达调控造血细胞发育

基本信息

批准号：
12670301
负责人：
OGAWA Minetaro
金额：
$ 2.18万
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：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670301/
关键词：
transcription factors hematopoietic cells endothelial cells mesoderm embryonic stem cells promoter flow cytometry in vitro differentiation 発生学血管内皮エンハンサーマウス

项目摘要

We have been proposing a hypothesis that the primitive hematopoietic cell lineage diverges directly from lateral mesoderm whereas the definitive hematopoietic cell lineage originates from the endothelial cell lineage which is another descendant of mesoderm. In this project, we analyzed expression and function of transcription factors which are thought to play important roles in hematopoietic cell development by using an in vitro differentiation system of mouse ES cells.We produced ES cell clones which revealed activity of the Gatal and Gata2 genes by expressing a GFP transgene as a marker. Expression of the Gata2 gene was already detected in a fraction of the earliest lateral mesoderm cells developed in a culture of the ES cells (day 3). The Gata2-expressing mesoderm cells purified by FACS from day 4 cultures exclusively contained primitive erythroid colony-forming cells (CFU-EryP) as well as stromal cell-dependent progenitors for definitive hematopoietic cells. On the other hand, expr … More ession of the Gatal gene in mesoderm cells became detectable after 4 days of ES cell differentiation. CFU-EryP was highly enriched in the Gata1-expressing mesoderm cell fraction. Mesoderm cells which have not started expression of the Gata1 gene by day 4 contained high frequency of endothelial cell progenitors. Endothelial cells derived from the progenitors possessed a potential to give rise to definitive hematopoietic cells.We next introduced an estrogen receptor/Cre-mediated inducible Tal1 transgene into an ES cell clone which is deficient for the endogenous Tall gene. By rescuing Tal1 expression in various stages of ES cell differentiation, we demonstrated that mesoderm cells require Tal1 at a period between day 3 and day 4 for the development of not only CFU-EryP but also hematogenic endothelial cells. Furthermore, overexpression of Tal1 in wild type ES cells resulted in an increase of Gata1-expressing mesoderm cells. The results collectively suggest that Tal1 and Gata2 are the prerequisite for the initiation of hematopoietic cell lineages. Less

我们一直提出一种假说，原始的造血细胞谱系直接从外侧中胚层分化而来，而最终的造血细胞谱系起源于中胚层的另一个后代--内皮细胞谱系。在本项目中，我们利用小鼠ES细胞的体外分化系统，分析了被认为在造血细胞发育中起重要作用的转录因子的表达和功能，并通过表达GFP转基因作为标记，获得了显示GATAL和GATA2基因活性的ES细胞克隆。在胚胎干细胞培养(第3天)发育的最早的外侧中胚层细胞的一部分中已经检测到Gata2基因的表达。FACS从第4天纯化的表达Gata2的中胚层细胞只含有原始红系集落形成细胞(CFU-EryP)以及特定造血细胞的基质细胞依赖的祖细胞。另一方面，Expr…ES细胞分化4天后，中胚层细胞中可检测到GATAL基因的进一步表达。CFU-EryP在表达GATA1的中胚层细胞组分中高度浓缩。在第4天还没有开始表达GATA1基因的中胚层细胞含有高频率的内皮细胞前体细胞。接下来，我们将雌激素受体/Cre介导的诱导型TAL1转基因导入内源性TALL基因缺失的ES细胞克隆。通过在ES细胞分化的不同阶段挽救Tal1的表达，我们证明了中胚层细胞在第3天到第4天之间不仅需要Tal1来发育CFU-EryP，而且还需要Tal1来发育造血内皮细胞。此外，在野生型ES细胞中过表达Tal1导致表达GATA1的中胚层细胞增加。这些结果共同表明，Tal1和Gata2是启动造血细胞谱系的先决条件。较少