Direct activation by Ets and Zic is required for initial expression of the Brachyury gene in the ascidian notochord

Extrinsic fibroblast growth factor (FGF) signal and intrinsic factors that determine the response of the signal-receiving blastomeres to FGF regulate mesoderm patterning in embryos of the ascidian Halocynthia roretzi. To investigate how cells integrate information from extrinsic and intrinsic inputs, we examined Brachyury (Hr-Bra) promoter activity in the early embryo. Hr-Bra, which encodes a key transcription factor for notochord development, is expressed exclusively in notochord precursors in a manner dependent on the FGF-MEK-MAPK-Ets signaling pathway and on the intrinsic factors Zic and FoxA. Reporter gene expression driven by the 900-bp upstream region of the Hr-Bra promoter was detected as early as the 110-cell stage in notochord precursors by in situ hybridization with a LacZ probe. Deletion analysis combined with MEK inhibitor treatment demonstrated that the - 598/- 499 region carries FGF-responsiveness. Electrophoretic mobility shift assay identified three Ets-binding sites in this region that were required for promoter activity. Further deletion analysis conducted by injecting eggs with reporter constructs at higher concentration suggested that the - 398/- 289 region also has enhancer activity, although ectopic reporter expression was detected in nerve cord and endoderm precursors. The - 398/- 289 region has a Zic-binding site that was also essential for the enhancer activity. These results indicate that Ets- and Zic-binding sites are critical for the initiation of Hr-Bra expression. In conclusion, information from both extrinsic and intrinsic factors is integrated at the level of enhancer of the target gene by direct binding of the transcription factors to the enhancer region.