The expression of nonchordate deuterostome Brachyury genes in the ascidian Ciona embryo can promote the differentiation of extra notochord cells

The notochord is a structure present in all chordates and its development requires the transcription of Brachyury. While previous studies have shown that Brachyury is essential for notochord formation in vertebrate embryos, this gene is also expressed during the embryogenesis of nonchordate deuterostomes, hemichordates and echinoderms. Here we report that nonchordate deuterostome Brachyury genes can trigger the differentiation of extra notochord cells when these genes are ectopically expressed in ascidian embryos. The 2.6 kb upstream region of fork head gene (Ci-fkh) of Ciona intestinalis promotes the tissue-specific expression of a reporter gene in endoderm, notochord and nerve cord. By taking advantage of this promoter, we misexpressed the Brachyury gene of ascidian (Ci-Bra), cephalochordate amphioxus (Am(Bb)Bra2), hemichordate acorn worm (PfBra), and echinoderm sea urchin (SpBra), in Ciona embryos. The misexpression of not only the chordate Brachyury, but also the nonchordate deuterostome Brachyury, resulted in the transformation of presumptive endodermal cells into notochord cells. This was confirmed by in situ hybridization experiments using four different notochord-specific probes from Ciona that have different temporal expression patterns. RT-PCR analyses indicated that Ci-Bra was not upregulated by the product of Am(Bb)Bra2, PfBra or SpBra. In situ hybridization showed no ectopic expression of Ci-Bra in the manipulated embryos. These results suggest that the introduction of nonchordate deuterostome Brachyury genes into ascidian embryos can trigger the differentiation of notochord cells in ascidian embryos. Evolutionary alteration in the genetic circuitry, especially downstream of this transcription factor, seems critical for the evolution of notochord and chordate body plan.