Studies on cell differentiation: inducing capacity of sulfhydryl-containing amino acids on post-nodal pieces of chick...

The inducing capacity of sulfhydryl (SH)-containing amino acids (cysteine and glutathione) on post-nodal pieces (PNPs) of stage 4 chick blastoderms was investigated. PNPs were treated for different lengths of time with chick Ringer's solution (control group) or chick Ringer's solution containing cysteine or glutathione, followed by culturing for 2-10 days on Spratt-Haas agar medium or on the chorioallantoic membrane of 8-day chick embryos. Control PNPs rarely showed differentiation, but those treated with the amino acids for six hours or longer developed structures such as neural tissue, notochord, somite mesoderm, and nephric tubules. The pulsatile tissue was only seen in the PNPs cultured for four days or longer. Two-four hours of treatment was too short to provoke induction in a statistically significant number of PNPs. The highest frequency of induction was noted in those pretreated with glutathione (8 mug/ml) for eight hours, followed by culturing for four days. The magnitude of the inducing capacity and toxicity of the amino acids were concentration dependent: a deleterious effect was observed at 14 mug/ml; the highest frequency of induction occurred at 8 mug/ml, but the frequency decreased as the concentration decreased; at 2 mug/ml all PNPs remained viable, but only a few (9-14%) showed differentiation. The inducing capacity of the amino acids was counteracted by equimolar concentrations of rho-chloromercuribenzoic acid or omega-chloroacetophenone. The effects of glutathione (8 mug/ml) differed from those of Hensen's node grafts in that the former caused sublethal cytolysis and inhibited H-3-uridine uptake in competent ectodermal cells during the first 18 hours of cultivation.