Yagi Kasumi, Satou Yutaka, Satoh Nori
Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Development. 2004 Mar;131(6):1279-88. doi: 10.1242/dev.01011.
In ascidian embryos, Brachyury is expressed exclusively in blastomeres of the notochord lineage and play an essential role in the notochord cell differentiation. The genetic cascade leading to the transcriptional activation of Brachyury in A-line notochord cells of Ciona embryos begins with maternally provided beta-catenin, which is essential for endodermal cell specification. beta-catenin directly activates zygotic expression of a forkhead transcription factor gene, FoxD, at the 16-cell stage, which in turn somehow activates a zinc finger transcription factor gene, ZicL, at the 32-cell stage, and then Brachyury at the 64-cell stage. One of the key questions to be answered is whether ZicL functions as a direct activator of Brachyury transcription, and this was addressed in the present study. A fusion protein was constructed in which a zinc finger domain of Ciona ZicL was connected to the C-terminus of GST. Extensive series of PCR-assisted binding site selection assays and electrophoretic mobility shift assays demonstrated that the most plausible recognition sequence of Ciona ZicL was CCCGCTGTG. We found the elements CACAGCTGG (complementary sequence: CCAGCTGTG) at -123 and CCAGCTGTG at -168 bp upstream of the putative transcription start site of Ci-Bra in a previously identified basal enhancer of this gene. In vitro binding assays indicated that the ZicL fusion protein binds to these elements efficiently. A fusion gene construct in which lacZ was fused with the upstream sequence of Ci-Bra showed the reporter gene expression exclusively in notochord cells when the construct was introduced into fertilized eggs. In contrast, fusion constructs with mutated ZicL-binding-elements failed to show the reporter expression. In addition, suppression of Ci-ZicL abolished the reporter gene expression, while ectopic and/or overexpression of Ci-ZicL resulted in ectopic reporter expression in non-notochord cells. These results provide evidence that ZicL directly activates Brachyury, leading to specification and subsequent differentiation of notochord cells.
在海鞘胚胎中，短尾相关蛋白（Brachyury）仅在脊索谱系的卵裂球中表达，并在脊索细胞分化中发挥重要作用。在玻璃海鞘胚胎A系脊索细胞中，导致Brachyury转录激活的基因级联反应始于母体提供的β-连环蛋白，它对内胚层细胞特化至关重要。β-连环蛋白在16细胞期直接激活一个叉头转录因子基因FoxD的合子表达，FoxD继而在32细胞期以某种方式激活一个锌指转录因子基因ZicL，然后在64细胞期激活Brachyury。有待回答的关键问题之一是ZicL是否作为Brachyury转录的直接激活因子发挥作用，本研究对此进行了探讨。构建了一种融合蛋白，其中玻璃海鞘ZicL的锌指结构域连接到GST的C末端。一系列广泛的PCR辅助结合位点筛选试验和电泳迁移率变动分析表明，玻璃海鞘ZicL最可能的识别序列是CCCGCTGTG。我们在该基因先前鉴定的基础增强子中，在Ci-Bra假定转录起始位点上游-123 bp处发现了元件CACAGCTGG（互补序列：CCAGCTGTG），在-168 bp处发现了CCAGCTGTG。体外结合试验表明，ZicL融合蛋白能有效结合这些元件。当将一个lacZ与Ci-Bra上游序列融合的融合基因构建体导入受精卵时，报告基因仅在脊索细胞中表达。相反，具有突变的ZicL结合元件的融合构建体未能显示报告基因表达。此外，抑制Ci-ZicL消除了报告基因表达，而Ci-ZicL的异位和/或过表达导致报告基因在非脊索细胞中异位表达。这些结果证明ZicL直接激活Brachyury，导致脊索细胞的特化和随后的分化。
