NEURAL DIFFERENTIATION IN CLEAVAGE-ARRESTED ASCIDIAN BLASTOMERES INDUCED BY A PROTEOLYTIC-ENZYME

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 Ca2+-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.