Establishment of the loss-of-function experimental system using the maxizyme in the ascidian embryo.

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A neural tube is one of the distinguishing characteristics of chordate. In order to analyze the molecular mechanism which underlying neural tube formation in the ascidian embryo, we focused on the RNA-binding protein genes, CiMsi and CiHu.CiMsi is expressed in the presumptive neural tube and mesenchyme cells, while CiHu is expressed in the presumptive mesenchyme cells. Overexpression of CiMsi caused ectopic neural differentiation. On the other hand, disruption of CiMsi function by the morpholino antisense oligo nucleotides (MO) resulted in the disordered cell-movement of the neural tube precursor cells.CiHu gene function was analyzed in the PC-12, rat pheochromocytoma cell line. It can differentiate into neurons by the NGF stimulation and the transformation with mouse HuD gene. When the full-length CiHu construct was introduced into PC-12, CiHu protein was localized in the nucleus and the cells did not differentiate into neurons. When the deletion CiHu construct, which lacked nuclear localization signal, was transformed, deletion CiHu protein was localized to the cytoplasm, and the PC-12 showed the neurite outgrowth. This result revealed the conserved neurite-inducing activity of ascidian CiHu and mouse HuD. While the protein structure responsible for the subcellular localization was different among them. This is the important clue to understanding what kind of change in the amino acid sequence caused the alteration of the developmental system and brought the evolutional change from urochordates to vertebrates.More precise understanding of the CiMsi and CiHu gene functions in the ascidian neural tube formation will provide us an insight how chordates obtained the neural tube, and how vertebrates evolved the tubular brain and dorsal hollow nerve chord.