PLoS Biol. 9, e1001160 (2011)

Three minor ecdysone pulses in the third-instar (L3) larval stage of Drosophila melanogaster development are required to prepare the animal for metamorphosis. It has been difficult to track these pulses because intermediate steps in the ecdysone biosynthetic pathway are not fully understood. To better understand this process, Ou et al. demonstrated that decreased expression of the nuclear receptor Drosophila Hormone Receptor 4 (DHR4) resulted in precocious ecdysone pulses and elevated concentrations of ecdysteroids during the L3, indicating that DHR4 negatively regulates ecdysone pulses. The authors also noted that the localization of DHR4 oscillated between the nucleus and cytoplasm of ecdysone-producing cells and that nuclear localization of DHR4 was blocked by the activation of a neuropeptide hormone signaling pathway that controls the timing of major ecdysone peaks. Subsequent experiments revealed that the cytochrome P450 gene Cyt6t3 is a likely target of DHR4: Cyp6t3 transcript levels oscillated in the L3 larvae, and knockdown of Cyt6t3 by RNA interference resulted in phenotypes consistent with low ecdysone that could be rescued by feeding larvae 20-hydroxyecdysone (20E), ecdysone or 5′-ketodiol. Although Cyt6t3 overexpression alone was not sufficient to accelerate development of wild-type larvae, Cyt6t3 expression was necessary for the accelerated development of larvae with reduced DHR4. Taken together, these data indicate that Cyt6t3 encodes one of several enzymes that function in the intermediate steps of the 20E biosynthesis and that DHR4 directly represses Cyt6t3 expression to help terminate ecdysone pulses.