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Nodal regulates neural tube formation in the Ciona intestinalis embryo

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Abstract

Overexpression of a lefty orthologue, Ci-lefty, caused a failure of neural tube closure in the protochordate ascidian Ciona intestinalis. The body bent dorsally, and anterior–posterior elongation was inhibited. A similar phenotype was observed in embryos treated with SB431542, an inhibitor of Nodal receptors, suggesting that Ci-Lefty antagonized Nodal signaling as reported in other deuterostome species. Overexpression of Ci-nodal also resulted in a similar phenotype, suggesting that a correct quantity and/or a spatial restriction of Nodal signaling are important for the neural tube to form. In addition to known Ci-Nodal target genes, orthologues of Zic (Ci-ZicL) and cdx (Ci-cdx) were activated by Ci-Nodal. Expression of a dominant negative Ci-cdx caused defects in neural tube formation similar to those obtained on treatment with SB431542 or overexpression of Ci-lefty. A regulatory cascade composed of Ci-Nodal, Ci-ZicL, and Ci-Cdx may play an important role in neural tube formation in the Ciona embryo.

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Acknowledgements

We thank Nori Satoh and Kazuko Hirayama at Kyoto University and Zenji Imoto at the Usa Marine Biological Institute of Kochi University for providing animals. We thank Hidetoshi Saiga at Tokyo Metropolitan University for kindly providing the plasmid containing EnR. We are also grateful to You Katsuyama for valuable advice. This work was supported by MEXT Japan. K. M. was supported by the Sasakawa Scientific Research Grant from The Japan Science Society.

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  1. Department of Materials Science, Kochi University, 2-5-1 Akebono-cho, Kochi-shi, Kochi, 780-8520, Japan

    Kaoru Mita & Shigeki Fujiwara

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  1. Kaoru Mita
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  2. Shigeki Fujiwara
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Correspondence to Kaoru Mita.

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Communicated by: N. Satoh

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Supplementary Fig. S1

Characterization of a lefty/antivin orthologue in C. intestinalis. (a) The amino acid sequence predicted from the Ci-lefty cDNA is aligned with other Lefty protein sequences obtained from mouse (m), zebrafish (z), and the sea urchin Paracentrotus lividus (Pl). The putative signal peptide region is indicated by a bar. Cysteine residues conserved among Lefty proteins are shown by green shade. An arrowhead indicates the position where cysteine is conserved in other TGF-β superfamily proteins but not in Lefty proteins. Putative protein product of Ci-lefty consists of 385 amino acids. A characteristic configuration of seven cysteine residues, called a cysteine knot, is conserved among TGF-β superfamily members (Kingsley 1994). Among the family, only the Lefty/Antivin group lacks the fourth cysteine residue (Thisse and Thisse 1999; Juan and Hamada 2001). A predicted amino acid sequence of Ci-Lefty showed the Lefty/Antivin-type cysteine knot. The N-terminal signal peptide sequence was observed in the Lefty proteins, including Ci-Lefty. (b) A phylogenetic tree of TGF-β superfamily proteins constructed by the maximum likelihood method (Felsenstein 1981), using full-length amino acid sequences. Zebrafish and mouse TGF-β proteins were defined as an outgroup. Ci, C. intestinalis; m, mouse; Pl, P. lividus; z, zebrafish. Orthologues of lefty formed a monophyletic cluster. Within the cluster, the branching pattern was consistent with widely accepted phylogenetic relationships of deuterostome groups. Paralogous subgroups in vertebrates (e.g. Lefty1 and Lefty2 in the mouse) diverged within the vertebrate clade. (GIF 43 kb)

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Mita, K., Fujiwara, S. Nodal regulates neural tube formation in the Ciona intestinalis embryo. Dev Genes Evol 217, 593–601 (2007). https://doi.org/10.1007/s00427-007-0168-x

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