A definite number of aphidicolin-sensitive cell-cyclic events are required for acetylcholinesterase development in the presumptive muscle cells of the ascidian embryos.

In order to determine whether or not a crucial number of DNA replications are prerequisite for cellular differentiation, we have studied development of a tissue-specific enzyme, muscle acetylcholinesterase (AChE) in the presumptive muscle cells of cleavage-arrested ascidian embryos. Embryos were cleavage-arrested with cytochalasin B (an inhibitor of cytokinesis) and aphidicolin (an inhibitor of DNA synthesis). The 64-cell-stage embryos which had been permanently cleavage-arrested with cytochalasin B developed AChE in all the eight presumptive muscle cells, but the same stage embryos which had been prevented from undergoing further divisions by simultaneous treatment with aphidicolin and cytochalasin did not produce AChE at all. Cytochalasin-arrested 76-cell-stage embryos were able to differentiate AChE in the ten presumptive muscle cells, while aphidicolin-cytochalasin-arrested 76-cell stages in as many as four cells. The early gastrulae which had been arrested with cytochalasin B produced AChE in all the sixteen presumptive muscle cells, while the same stage embryos arrested with aphidicolin and cytochalasin in as many as twelve cells. Cytochalasin-arrested late gastrulae developed AChE in twenty blastomeres, while aphidicolin-cytochalasin-arrested late gastrulae in eighteen cells. The presumptive muscle cells at these four stages consist of three different (seventh, eighth, and ninth) generations, and the relative positions of the blastomeres in the cleavage-arrested embryos remained fixed. Judging from the relative positions of the blastomeres, the AChE-producing cells in aphidicolin-cytochalasin-arrested embryos were always at eighth or ninth generation, while those with no AChE activity were certainly at seventh generation. Based on these findings it was supposed that aphidicolin-sensitive cell-cycle events, presumably DNA replication, are closely associated with AChE development and that the eighth cleavage cycle may be 'quantal' for the histospecific enzyme development.