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

从海鞘4或8细胞胚胎中分离出两个或三个卵裂停滞的胚胎细胞系统，由假定神经和假定脊索裂球组成，用于神经诱导。本研究旨在从以下两个方面研究早期神经元分化：(1)碱性成纤维细胞生长因子受体型酪氨酸激酶对神经元连接的调控机制；(2)通过消除与下游酪氨酸激酶中的丝氨酸苏氨酸蛋白激酶的缝隙连接来控制电压依赖性离子通道的表达时间的机制。这些诱导事件的时间控制是由于早期磷酸化级联和后期通过缝隙连接的钙波的时钟作用。当4个细胞胚胎的前部A3分裂球与两个外胚层A4-2或…培养时，发现了新的神经诱导系统，该系统完全分化为表皮细胞或神经细胞，取决于黏附和培养条件更多的节索状A4-1裂球来自8个细胞胚胎。由于这个新的诱导系统是利用海鞘内向整流钾通道(TuIRKA)基因启动子的表达系统，所以对表皮或神经元分化的条件进行了精确的检测。电生理定量测定内源TuIRKA的表达水平与发育时间的关系。从海鞘蝌蚪基因组DNA文库中克隆了TuIRKA基因的全长蛋白编码区和5‘端序列，并绘制了限制性内切酶图谱。证实转录控制区位于5‘区，因为报告基因GFP与该区域结合，并在表皮分化细胞的早期观察到转录活性。将GFP缝隙连接融合蛋白基因与该区域偶联的克隆也被强制表达，并延迟了细胞膜上Na通道的出现。较少

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