Cellphysiological Analysis on Inductive Differentiation using Isolated Early Embryonic Cell-Roles of Inducer Receptor, Gap Junction, Retinoic Acid

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1) On anterior animal blastomeres isolated from cleavage-arrested Halocynthia 8-cell embryos, the neural inducing effect of bFGF in the concentration range from 10 to 100 ng/ml was confirmed with expression of Na channels as the marker. The neural inducing activity was identical with those by a protease, subtilisin, and by cell-contact in terms of developmental-time dependency and channel expression, indicating existence of a common neural inducer receptor. Cloning of receptor-tyrosine-kinase homologs from the cDNA libraries of Halocynthia unfertilized eggs and 100-cell embryos was carried out by PCR and 12 clones were obtained. Among those clones pTK6, pTK7, pTK9 and pTK11 were highly homologous to those of mammalian FGF receptors.2) Gap junctions between the 2 cell system separated from the cleavage-arrested Halocynthia 8-cell embryo increased or disappeared dependently upon either autonomous epidermal or induced neural differentiation respectively. The disappearance of gap junction … More after neural induction was inhibited and delayd by kinase inhibitor, K252a. Concomitantly the expression of Na and K channels was also delayd. However, in the case of the neurally induced and afterward separated single blastomere no delay of channel expression was observed. It was concluded that K252a specifically inhibited the gap-junctional disappearance and thereby delayd the channel expression. It is suggested that the disappearance of gap junction is caused by the phosphorylation as a result of neural induction while it can control the timing of the functional expression of neural differentiation.3) Na channel gene TuNa I was cloned and demonstrated to be transcribed specifically in neurons within the neural tissue of Halocynthia tadpole larvae by whole-mount in-situ hybridization. The transcription was also observed in the cleavage-arrested embryos. Further the cell-contact dependent transcription was confirmed by using the 2 cell neural induction system separated from the cleavage-arrested Halocynthia 8-cell embryo. Less