Direct exposure of mouse embryonic limb-buds to 5-bromodeoxyuridine in vitro and its effect on chondrogenesis: increasing resistance to the analog at successive stages of development

Abstract

The inhibitory effect of 5-bromodeoxyuridine (BudR) - an analog of thymidine - on embryonic mouse limb-buds was studied in vitro employing an organ-culture system. The effect was found to be dose-related and also depended on the developmental stage of the donor embryos. Limbs at an early stage development (early 11 th-day embryos, somite stage 26-29) were extremely sensitive to the analog. Treatment with low levels (2-4 mug/ml) and for a relatively short period of time in cluture (2-3 days) completely and irreversibly suppressed chondrogenesis in these explants. Limbs from older embryos (somite stage 40 and up) were found to be much less sensitive to the inhibitory effect of the drug; a prolonged exposure to a much higher dose (100-150 mug/ml) resulted in an incomplete suppression of chondrogesesis. Only a 20% inhibition was observed in the cultures of limbs from mid-13th-day mouse embryos. After continuous growth in vitro, the limbs became progressively resistent to the analog and towards the end of the culture period had become refractory to the drug. The time of complete insensitivity appeared earlier in the cures of the limbs taken from older embryos than in the explants of youngerlimbs. These studies show that as limbs continue to differentiate in vivo or in vitro, they become increasingly resistent to the inhibitory effect of BudR in at least as far as the effect on the process of chondrogenesis is concerned. It is suggested that the relative sensitivity or insensitivity to the inhibitory effect of BudR may prove to be a useful parameter in evaluating the developmental stage of an organ.