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Studies on differentiation induced by cell-cell interaction with optical-image analysis - Intracellular mechanism of neural induction in the isolated cleavage-arrested blastomeres from the early Halocynthia embryo.

利用光学图像分析研究细胞间相互作用诱导的分化——早期盐藻胚胎分离的卵裂停滞卵裂球中神经诱导的细胞内机制。

基本信息

批准号：
02404021
负责人：
TAKAHASHI Kunitaro
金额：
$ 14.53万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The anterior-animal a_<4-2> blastomere isolated from the cleavage-arrested Halocynthia 8-cell embryo, which includes the presumptive neural domain, differentiated into epidermal type when cultured in isolation, but was Induced to differentiate into neural type when contacted with a presumptive notochordal A41 blastomere or treated with proteolytic enzymes. Using this simplified neuralinduction model, the image-analysis of Ca ion concentration with fluorescent probes and the quantitative analysis of the transcription and the expression of neural specific ion channels were performed in order to clarify the intracellular mechanisms of the induction. So far Ca ion concentration within the a_<4-l> blastomere showed no positive correlation with the induction. Both activators for A-kinase and C-kinase were applied on the isolated a_<4-1> blastomere but no indication of neural induction was found. However, basic FGF, an activator of tyrosine klnase receptors, did induce neural type differentiation. The down or up-regulation of the intercellular gap junction was found as a result of inductive neural or epidermal differentiation of the contacted two blastomeres respectively by optical measurement of the junctional permeability. The initiation periods of expression and transcription of Na channels and anomalous rectifier K channels were determined electrophysiologically or by RNAse protection assay with the cloned DNA probe. It was found that the transcription of the former initiated just after the induction and that of the latter was stopped at the time. In order to confirm above changes in the normal embryos the presumptive neural a_<4-2> and notochordal A_<4-1> blastomeres were injected with differently fluorescent cell-lineage markers respectively at the stage of 8-cell and the contact between two domains was observed at supposed induction period of the 64-cell stage with a confocal microscope.

从<4-2>分裂停滞的盐爪草8细胞胚胎中分离出的前体动物a_卵裂球，在分离培养时可分化为表皮型，但在与脊索A41卵裂球接触或用蛋白水解酶处理时，可被诱导分化为神经型。利用这个简化的神经诱导模型，用荧光探针对Ca ~（2+）浓度进行图像分析，并对神经特异性离子通道的转录和表达进行定量分析，以阐明神经诱导的细胞内机制。到目前为止，a_卵裂球内Ca ~（2+）浓度<4-l>与诱导率无明显正相关。A激酶和C激酶激活剂<4-1>均未诱导出神经细胞，而酪氨酸激酶受体激活剂碱性成纤维细胞生长因子（basicFGF）可诱导神经细胞分化。通过光学测量细胞间缝隙连接的通透性，发现细胞间缝隙连接的下调或上调分别是由于接触的两个卵裂球诱导神经或表皮分化的结果。通过电生理或RNA酶保护试验，用克隆的DNA探针测定钠通道和异常整流钾通道表达和转录的起始期。结果发现，前者的转录启动后，立即诱导，后者的时间停止。为了证实上述变化，在正常胚胎中，<4-2><4-1>在8-细胞期将不同荧光细胞系标记物分别注射到假定的神经A_2和脊索A_2卵裂球中，并在假定的64-细胞期诱导期用共聚焦显微镜观察两个区域之间的接触。