Ecdysone-inducible functions of larval fat bodies in Drosophila

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Abstract
Late in the 3rd instar larval stage of D. melanogaster, the titer of the steroid hormone ecdysone sharply increases. This increase is blocked in the temperature-sensitive mutant ecd1 after a temperature shift from 20.degree. C to 29.degree. C. The mutant was used to prepare 3 samples of late 3rd instar larvae with different titers of ecdysone; the titer was low in 1 sample because of an earlier temperature shift, high in a 2nd sample because the larvae were subsequently transferred to ecdysone-supplemented food and also high in a 3rd sample that was kept at 20.degree. C, providing a control for normal development. The effect of the high titer of ecdysone on proteins of the larval fat bodies was examined by comparing 2-dimensional gel electrophoresis patterns of total proteins in stained gels. There were proteins at 5 positions in the gels for the high-ecdysone samples that were not detected at the corresponding positions in the gel for the low-ecdysone sample. The effect of ecdysone on these proteins was further studied by injecting [35S]methionine into the larvae at both early and late 3rd instar stages, to label proteins synthesized before and after the increase in ecdysone titer. Ecdysone induces 2 major responses in the fat bodies; certain proteins that were synthesized earlier in the fat bodies and secreted into the hemolymph are incorporated back into the fat bodies; other proteins are newly synthesized. Attempts to prematurely induce the synthesis of the new proteins by exposing early 3rd instar larvae to exogenous ecdysone were unsuccessful, suggesting that development must proceed further before the fat bodies can repond to ecdysone. By in vitro translation of RNA isolated from fat bodies of low-and high-ecdysone samples of larvae, ecdysone greatly increases the amount of translatable mRNA for one of the newly synthesized proteins. A clone of DNA complementary of the induced mRNA was isolated from a population of .lambda. bacteriophage carrying segments of the Drosophila genome. Using the cloned DNA to measure amounts of complementary poly(A)-RNA in the fat bodies by DNA.cntdot.RNA hybridization, .apprx. 50 times more complementary poly(A)-RNA was detected in the high-ecdysone sample of larvae than in the low-ecdysone sample. This finding provides direct evidence that ecdysone induces an increase in the amount of the mRNA. The ecdysone-induced appearance of a major mRNA in late 3rd instar larval fat bodies represents a developmental response to ecdysone that is apparently gene-specific, tissue-specific and stage-specific. It has exceptionally favorable features for further molecular studies of gene expression control by a steroid hormone.