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Analysis of Timing Control during Neural Differentiation Process in Ascidian 2 Cell Induction System

海鞘2细胞诱导体系中神经分化过程的时序控制分析

基本信息

批准号：
10670049
负责人：
TAKAHASHI Kunitaro
金额：
$ 2.43万
依托单位：
MEIJI PHARMACEUTICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670049/
关键词：
Ascidian Embryo new neural induction cell triplet inward rectifier K+ channel genomic library 5'proximal region GFP reporter gene ホヤ予定神経割球神経形質発現時間的制御因子ギャップ結合 GFP融合蛋白 A3割球発現可視化

项目摘要

Two or three cleavage-arrested embryonic cell system which consisted of presumptive nerve and presumptive notochord blastomeres separated from the ascidian 4 or 8 cell embryo were used for neural induction. It was aimed to study early neuronal differentiation in terms of (1) control mechanism of neural commitment by bFGF receptor type tyrosine kinase and (2) control mechanism of the expression time of the voltage-dependent ion channels by the elimination of the gap junction with the serine threonine protein kinases which existed in the tyrosine kinase downstream. It was hypothesized that the time-control in these induction events was due to the clock action of the phosphorylation cascade early, and of the Ca wave through the gap junction later.New neural induction system which was altogether differentiated to epidermal or neuronal cells depending upon the adhesion and culture conditions was found, when Anterior A3 blastomeres from 4 cell embryos was cultured with two ectodermal a4-2 or … More notochordal A4-1 blastomeres from 8 cell embryos. Since this new induction system was made to be an expression system using the promoter of the ascidian inward rectifier K+ (TuIRKA) channel gene, the condition for the epidermal or neuronal differentiation were precisely examined. The expression level of endogenous TuIRKA was electrophysiologically determined quantitatively against the developmental time. All protein coding region and 5'proximal region of TuIRKA gene were obtained as the result that we cloned the gene from the ascidian tadpole genome DNA library and made the restriction map. It was confirmed that the transcriptional control region was included in the 5' region because the reporter GFP gene was combined with this region, and the transcriptional activity was observed at the early stage of epidermally differentiating cells. The clone which coupled the GFP gap junction fusion protein gene with this region was also made to be forcibly expressed and to delay the appearance of the Na channel to the cell membrane. Less

从海鞘4细胞或8细胞胚胎中分离出两个或三个由假定神经和假定脊索卵裂球组成的卵裂停滞胚胎细胞系统，用于神经诱导。本研究旨在从（1）bFGF受体型酪氨酸激酶对神经定向的调控机制和（2）酪氨酸激酶下游丝氨酸/苏氨酸蛋白激酶通过消除差距连接对电压依赖性离子通道表达时间的调控机制两个方面研究早期神经元分化。将4细胞胚胎的前A3卵裂球与两个外胚层a4-2或a4 - 3细胞共培养，发现了一个新的神经诱导系统，该系统根据粘附和培养条件完全分化为表皮细胞或神经元细胞。 ...更多信息 8细胞胚胎的脊索A4-1卵裂球。由于这种新的诱导系统是使用海鞘内向整流K+（TuIRKA）通道基因的启动子的表达系统，因此精确地检查了表皮或神经元分化的条件。内源性TuIRKA的表达水平的电生理测定定量对发育时间。从海鞘蝌蚪基因组DNA文库中克隆了TuIRKA基因，并制作了限制性内切酶图谱，获得了TuIRKA基因的全部蛋白编码区和5 '端近端区域。证实了转录控制区包含在5'区域中，因为报告GFP基因与该区域结合，并且在表皮分化细胞的早期阶段观察到转录活性。将GFP间隙连接融合蛋白基因与该区域偶联的克隆也被强制表达并延迟Na通道向细胞膜的出现。少