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

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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