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.

从Halocynthia 8细胞胚胎中分离出的前动物a_<4-2>囊胚，其中包括假定的神经结构域，在分离培养时分化为表皮型，但在与假定的脊索A41囊胚接触或用蛋白水解酶处理时诱导分化为神经型。利用该简化的神经诱导模型，利用荧光探针对Ca离子浓度进行图像分析，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。目前为止，a_<4- 1>卵裂球内的钙离子浓度与诱导没有正相关。a激酶和c激酶激活剂均应用于离体a_<4-1>卵裂球，但未发现神经诱导的迹象。然而，碱性FGF，酪氨酸klnase受体的激活剂，确实诱导神经类型分化。通过光学测量细胞间隙连接的通透性，发现细胞间隙连接的下调或上调分别是由于接触的两个卵裂球的神经或表皮诱导分化所致。通过电生理或RNAse保护实验测定Na通道和异常整流K通道的表达和转录起始时间。研究发现，前者的转录恰好在诱导后开始，后者的转录恰好在诱导后停止。为了证实上述变化在正常胚胎中是否存在，我们分别在8细胞期给推定的神经a_<4-2>和脊索a_< 4-1>卵裂球注射不同的荧光细胞谱系标记，并在推定的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。

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.