Evolutionary alterations of the minimal promoter for notochord-specific Brachyury expression in ascidian embryos

The Brachyury genes of two divergent ascidians, As-T of Halocynthia roretzi and Ci-Bra of Ciona intestinalis, are expressed exclusively in notochord precursor cells. A previous study showed that the notochord-specific expression of Ci-Bra is controlled by a minimal promoter that is composed of three distinct regions: a region responsible for repression of expression in non-notochord mesoderm cells, a region for activation of expression in notochord cells, and a region for activation of expression in non-notochord mesoderm cells, distal to proximal to the transcription initiation site, respectively. We examined various deletion constructs of the As-T/lacZ fusion gene and demonstrate that a module between -289 and -250 bp of the 5'-flanking region is responsible for notochord-specific expression of the reporter gene. Gel-shift assays suggested the binding of nuclear protein(s) to this module. The 5'-flanking region of As-T contains a potential T-binding motif (-ACCTAGGT-) around -160 bp. Deletion of this motif from the p(-289)As-T/lacZ diminished the reporter gene expression. In addition, coinjection of p(-289)As-T/lacZ and synthetic As-T mRNA resulted in ectopic expression of lacZ in non-notochord cells, suggesting that the T-binding motif is responsible for autoactivation of the gene. These findings revealed striking differences between the minimal promoters of As-T and Ci-Bra so far revealed, with respect to their notochord-specific expression. Furthermore, reciprocal injections of reporter gene constructs, namely As-T/lacZ into Ciona eggs and Ci-Bra/lacZ into Halocynthia eggs, suggest alterations in the cis-regulatory elements and trans-activation factors that have occurred during evolution of the two ascidian species.