A zinc finger transcription factor, ZicL, is a direct activator ofBrachyuryin the notochord specification ofCiona intestinalis

Citations Over TimeTop 10% of 2004 papers

Abstract

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.

Related Papers

• → Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis(1997)476 cited
• → Direct activation of a notochord cis-regulatory module by Brachyury and FoxA in the ascidianCiona intestinalis(2009)50 cited
• → Functional and evolutionary insights from the Ciona notochord transcriptome(2017)49 cited
• → The identification of transcription factors expressed in the notochord of Ciona intestinalis adds new potential players to the brachyury gene regulatory network(2011)47 cited
• → The evolutionarily conserved leprecan gene: Its regulation by Brachyury and its role in the developing Ciona notochord(2009)34 cited