Neural differentiation in cleavage‐arrested ascidian blastomeres induced by a proteolytic enzyme.
Neural differentiation in cleavage‐arrested ascidian blastomeres induced by a proteolytic enzyme.
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蛋白水解酶诱导的分裂停滞的海鞘卵裂球的神经分化。
DOI：
10.1113/jphysiol.1993.sp019594
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Kunitaro Takahashi
中科院分区：
文献类型：
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作者：
H. Okado;Kunitaro Takahashi
1. 如先前报道，从海鞘、Halocynthia roretzi 或 aurantium 的 8 细胞胚胎中分离出的外胚层 a4-2 卵裂球，并在卵裂停滞条件下培养，总是分化为表皮表型，在细胞表面显示出持久的 Ca(2+) 依赖性动作电位和/或被膜。 a4-2 卵裂球与脊索中胚层卵裂球 A4-1 接触，分化为神经表型，其特征是快速 Na(+) 依赖性尖峰。在对照胚胎的 32 细胞阶段，用 > 0.003% 枯草杆菌蛋白酶处理从 H. aurantium 胚胎分离的 a4-2 卵裂球 60 分钟时，会分化为类似的神经表型。对细胞接触诱导和蛋白水解酶诱导进行了比较，结果表明它们在几个方面是相似的。 2. 当使用丝氨酸蛋白酶枯草杆菌蛋白酶作为神经诱导剂时，a4-2 卵裂球的神经能力（以诱导 Na+ 尖峰的百分比频率来衡量）在 32 细胞阶段后增加，在原肠胚阶段下降。神经能力的时间进程与接触 A4-1 卵裂球的时间进程相同。 3. 使用枯草杆菌蛋白酶作为神经诱导剂，并通过与来自 8 细胞胚胎的 A4-1 卵裂球接触，检查了从 16 细胞胚胎分离的四种不同外胚层卵裂球的神经能力。对于两种类型的诱导，前卵裂球的能力均高于后卵裂球。沿前后轴神经能力的这种区域差异与正常发育期间神经细胞谱系的预期平行，即在其衍生物中具有更多神经谱系细胞的卵裂球表现出更高的能力。 4. 链霉菌枯草杆菌蛋白酶抑制剂 SSI (0.1%) 是枯草杆菌蛋白酶型丝氨酸蛋白酶的特异性蛋白酶抑制剂，通过与脊索中胚层卵裂球 A4-1 接触，显着抑制 (50%) 外胚层卵裂球 a4-2 的神经诱导。 5. 莫能菌素、布雷菲德菌素 A 和巴弗洛霉素 A1 均影响分泌过程，抑制脊索中胚层卵裂球 A4-1 的神经诱导能力。 6. 这些结果允许这样的假设：从产生脊索中胚层的卵裂球分泌的蛋白酶诱导外胚层卵裂球分化为神经细胞类型。
1. As previously reported, ectodermal a4‐2 blastomeres isolated from 8‐cell embryos of the ascidian, Halocynthia roretzi or aurantium, and cultured under conditions of cleavage arrest always differentiated into an epidermal phenotype, showing long‐lasting Ca(2+)‐dependent action potentials and/or tunic on the cell surface. a4‐2 blastomeres contacted by a chordamesodermal blastomere, A4‐1, differentiated into a neural phenotype, characterized by fast Na(+)‐dependent spikes. Differentiation to a similar neural phenotype occurred when isolated a4‐2 blastomeres from H. aurantium embryos were treated with > 0.003% subtilisin for 60 min at the 32‐cell stage of the control embryo. Comparisons between induction by cell contact and induction by proteolytic enzymes were made and showed them to be similar in several respects. 2. When the serine protease, subtilisin, was used as the neural inducer, neural competence of a4‐2 blastomeres, measured as the percentage frequency of the induction of Na+ spikes, increased after the 32‐cell stage and decreased during the gastrula stage. The time course of the neural competence was the same as that for contact with the A4‐1 blastomere. 3. The neural competence of four different ectodermal blastomeres isolated from the 16‐cell embryo was also examined using subtilisin as a neural inducer, and by contact with the A4‐1 blastomere from the 8‐cell embryo. The competence was higher in anterior blastomeres than in posterior blastomeres for both types of induction. This regional difference in neural competence along the antero‐posterior axis paralleled that expected from neural cell lineage during normal development, i.e. blastomeres with more cells of neural lineage among their derivatives showed higher competence. 4. Streptomyces subtilisin inhibitor, SSI (0.1%), a specific protease inhibitor for subtilisin‐type serine proteases, significantly suppressed (50%) neural induction of the ectodermal blastomere, a4‐2, by contact with the chordamesodermal blastomere, A4‐1. 5. Monensin, brefeldin A and bafilomycin A1, all of which affect secretory processes, suppressed the neural inducing ability of the chordamesodermal blastomere, A4‐1. 6. These results permit the hypothesis that a protease secreted from the chordamesoderm‐generating blastomere induces the ectodermal blastomere to differentiate into neural cell type.