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.

从发育受阻的梭鱼8-细胞胚胎中分离出的前动物卵裂球，包括假定的神经区，在分离培养时分化为表皮型，但当与假定的脊索型A41卵裂球接触或用蛋白水解酶处理时，诱导分化为神经型。应用这种简化的神经诱导模型，用荧光探针对钙离子浓度进行图像分析，并对神经元特异性离子通道的转录和表达进行定量分析，以阐明诱导的细胞内机制。到目前为止，4-L卵裂球内的钙离子浓度与诱导没有明显的正相关。A和C两种激活剂应用于分离的A-1&1&gt；卵裂球，但没有发现神经诱导的迹象。然而，碱性成纤维细胞生长因子，一种酪氨酸脱氢酶受体的激活剂，确实诱导了神经型分化。通过对细胞间隙连接通透性的光学测量，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诱导神经分化或表皮分化所致。通过电生理学或核糖核酸酶保护实验测定Na通道和异常整流钾通道的表达和转录起始期。结果发现，前者在诱导后即开始转录，后者在诱导后立即停止转录。为了证实上述正常胚胎的上述变化，在8-细胞期分别向推测的神经细胞A_2&gt；4-2&gt；和脊索A_1&gt；卵裂球注入不同的荧光细胞谱系标记，并在假定的细胞期的诱导期用共聚焦显微镜观察两个结构域之间的接触。

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.