Newt forelimb regeneration blastemas in vitro: cellular response to explanation and effects of various...

Macromolecular synthesis was studied in cultured newt forelimb blastemas. Late bud stage blastemas were explanted into organ culture and thus denervated. When the response of the blastemas to explantation was examined, it was found that a peak in 3H-leucine incorporation occurred at 2 hr of incubation, and a similar peak in 3H-thymidine incorporation occurred at 3-4 hr. After these initial increases, both parameters declined to levels lower than those determined at the time of explantation. The peaks in macromolecular synthesis reported here in vitro are similar to those observed in blastemas in vivo after denervation (Singer, '74). In the second part of the study, the ability of various growth-promoting substances to stimulate DNA synthesis in cultured blastemas was investigated. Increasing concentrations of fetal bovine serum caused increased levels of 3H-thymidine incorporation, with maximal stimulation at 30% serum. Newt brain extract (NBVE) was as effective as additional serum, with optimal stimulation at 100 micrograms total NBE protein per ml culture medium. Fibroblast growth factor (FGF), derived from brain tissue, at a concentration of 10 ng/ml was as effective as both 30% serum and the optimal concentration of NBE. Epidermal growth factor (EGF) produced a maximal effect at 1 ng/ml. Neither nerve growth factor nor the platelet-derived growth factor were stimulatory. Bovine insulin was highly active in stimulating DNA synthesis at concentrations from 1-10 micrograms/ml. The requirement of nerves for blastemal development, as well as the possible roles of the various growth-promoting substances in this process, are discussed.