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

神经管是脊索的显著特征之一。为了分析海鞘胚胎神经管形成的分子机制，我们重点研究了RNA结合蛋白基因CiMsi和CiHu.CiMsi在推测的神经管和间充质细胞中表达，而CiHu在推测的间充质细胞中表达.CiMsi的过度表达导致异位神经分化。另一方面，通过吗啡反义寡核苷酸(MO)干扰CiMsi功能，导致神经管前体细胞运动紊乱。经NGF刺激和转化小鼠HUD基因后，可向神经元分化。当将全长CiHu结构引入PC-12后，CiHu蛋白定位于细胞核，细胞不能分化为神经元。当缺乏核定位信号的缺失CiHu构建体经转化后，缺失的CiHu蛋白定位于细胞质中，PC-12细胞出现突起生长。这一结果揭示了海鞘和小鼠HUD具有保守的轴突诱导活性。而负责亚细胞定位的蛋白质结构不同。这是理解氨基酸序列的什么样的变化导致发育系统的改变，并带来从尾索动物到脊椎动物的进化变化的重要线索。更准确地了解CiMsi和CiHu基因在海鞘神经管形成中的功能将为我们深入了解脊椎动物是如何获得神经管的，以及脊椎动物是如何进化出管状脑和背部中空神经索的。

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.