Actinomycin D: Effect on induced metamorphosis in the Mexican axolotl

Abstract

Actinomycin D was used to inhibit the action of TS in the induction of metamorphic changes in the Mexican axolotl. AmD was administered before and after (in hours) T3 injection as follows: (1) -48; ( 2 ) -24; ( 3 ) -8; (4) 0; ( 5 ) +24; ( 6 ) +48; ( 7 ) $72; ( 8 ) +120; ( 9 ) +168. Five pg AmD administered alone was not lethal. However the majority of the animals injected with T3 and Am D died within 30 to 33 days. All experiments were at 20°C. Suppression of metamorphic change was greater in group 2 than 1 and greater in groups 4 and 5 than 6. Group 3 showed maximum inhibitory effects of AmD. Very little inhibition was seen in groups 7 through 9. Gill filament resorption and sloughing of skin could not be inhibited completely in any of the groups. Exophthalmia was seen in all except group 3. Least changes occurred in the main gill rachis and the fins. When Am D was administered to hypophysectomized axolotls eight hours prior to T3 there was complete gill filament resorption before any change was observed in controls. Also skin sloughing and exophthalmia were precocious in Am D and T3 injected hypophysectomized axol o t l~. The observations above indicate variable responses in the metamorphosing tissues of the axolotl to AmD. Hence utmost caution i s necessary in the interpretation of any of the inhibitory effects of Am D at the cellular level.This report presents evidence that SUSceptible tissues of the Mexican axolotl differ in their responses to 3,5,3'-triiodo-L-thyronine ( T3) induced metamorphosis under the inhibitory action of actinomycin D (Am D) and that in hypophysectomized animals at least one of the changes is accelerated.Recently (Prahlad and DeLanney, '65) questions were raised concerning the mode of action of T3 in the induction of metamorphic changes. Tata ('66b) has shown that an essential step in the biological action of thyroid hormone is regulation of synthesis of nuclear and cytoplasmic RNA and (Tata, '66a) that RNA and protein synthesis is required for induced regression of the tadpole tail in organ culture. He has also shown ('66b) that T3 stimulates, by 30-40% at 45 hours, the Mnei/ammonium sulphate-activated RNA polymerase whose product is DNA-like.In order to elucidate the mode of action of T3 in the induction of metamorphic changes in the neotenic axolotl it appeared logical to use actinomycin D. This antibiotic has been shown to inhibit DNAdependent RNA-polymerase activity (Reich and Goldberg, '64) by specifically binding to guanine containing sites on helical DNA J. EXP. ZOOL., 165: 425-432.and thus preventing the action of RNApolymerase. Recently such specificity has been claimed by Gellert et al. ('65), and Dingman and Sporn ('65). The only study dealing with the effect of actinomycin D on thyroid hormone induced metamorphic changes in the axolotl is that of Zalta and Beetschen ('65). Their procedure does not permit any conclusions as to the temporal relationships regarding the inhibitory action of Am D on TI induced changes. EXPERIMENTALIn this study both dark and white axo...