On the 'clock' mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis. II. Evidence for association of the clock with the cycle of DNA replication.

Acetylcholinesterase (AChE) is a tissue-specific enzyme of the muscle cells of ascidian embryos and its synthesis begins at the neurula stage. Embryos which had been permanently cleavage-arrested with cytochalasin B could develop AChE activity. The time of first AChE occurrence in embryos which had been arrested in the 32-cell stage with cytochalasin was about the same as in normal embryos. The nucleus in the cell of cytochalasin-arrested embryos divided in good synchrony with that of normal embryos. Embryos which had been continuously arrested with colchicine could also produce AChE activity at nearly the same time as did normal embryos. In the cell of colchicine-arrested embryos normal nuclear divisions did not occur, but the cell showed repeated cycles of nuclear envelope breakdown and nuclear envelope reformation in almost parallel with cell cycles of normal embryos. The cell of colchicine-arrested embryos incorporated [3H]thymidine. Aphidicolin, a specific inhibitor of DNA synthesis, prevented cleavages of ascidian eggs. Embryos which had been permanently arrested with aphidicolin in the cleavage stages up to the 64-cell stage did not develop AChE activity, while embryos which had been treated with it from the 76-cell stage onwards were found to be able to differentiate AChE activity. Based on these findings it was proposed that DNA replication is prerequisite for development of the histospecific protein and that the cycle of DNA replication is closely associated with the clock mechanism which is determining the time of initiation of the enzyme development.