Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone

Abstract

The steroid 20-hydroxyecdysone (20E) is the primary regulatory hormone that mediates developmental transitions in insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any insect. We show here that the Drosophila E-20-monooxygenase (E20MO) is the product of the shade (shd) locus (cytochrome p450, CYP314a1). When shd is transfected into Drosophila S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous shd embryos, no E20MO activity is apparent either in vivo or in vitro. Mutations in shd lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in disembodied (dib) and shadow (sad) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike dib and sad, shd is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of insects. However, the tissue in which shd is expressed does not appear to be important for developmental function because misexpression of shd in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which shd is expressed, rescues embryonic lethality.

Discrete pulses of molting hormone, 20‐hydroxyecdysone, during late larval development of Drosophila melanogaster: Correlations with changes in gene activity

“…Overall, these data are consistent with prior semi-quantitative RT-PCR and immunohistochemical data, indicating relatively constant phm and dib expression during the early third instar, although the expression of both phm and dib does appear later in the instar to be under the control of the nuclear receptor ␤Ftz-F1 in the ring gland (Parvy et al, 2005). Only the analysis of specific Phm and Dib enzymatic activities during this period, along with that of the 2-hydroxylase, Shadow (Warren et al, 2002) and the 20-hydroxylase, Shade (Petryk et al, 2003), will clarify the importance of CYP enzyme regulation to the changing titers of active molting hormone during Drosophila ecdysteroidogenesis (Fig. 4).…”

“…However, in contrast to the findings of Berreur et al (1984), little if any free E was detected in any of these early third instar extracts. Yet these repetitive, discrete elevations of 20E and 26E are the likely result of the increased secretion of E by the prothoracic gland cells of the ring gland, followed by its very rapid and efficient conversion to 20E by peripheral 20-hydroxylase (ecdysone 20-monooxygenase) activity (E20MO) (see Petryk et al, 2003) and/or to 26E by tissue 26-hydroxylase activity (Kayser et al, 1997;Williams et al, 2000) (Fig. 4).…”

“…This relationship remains true even in transgenic animals expressing the 20-hydroxylase. That is, when the specific P450 enzyme catalyzing this ecdysteroid activation reaction (CYP315A1, Shade) is expressed ectopically in embryonic, larval, and pupal tissues that normally do not exhibit this activity, normal development to the adult is observed (Petryk et al, 2003). However, ovarian development stops at stage 8 -9 and these animals are sterile unless Shade is expressed within the ovary, the site of adult ecdysteroid biosynthesis (Warren et al, 1996).…”

“…4) (Warren et al, 2002(Warren et al, , 2004Petryk et al, 2003;Niwa et al, 2004). Quantitative Western blot analysis, performed on the insoluble protein remaining after ecdysteroid extraction (see Experimental Procedures section), reveals significant expression throughout the third instar of both Phantom (Phm, the terminal 25-hydroxylase) and Disembodied (Dib, the subsequent 22-hydroxylase) (Fig.…”

Queen mandibular pheromone modulates hemolymph ecdysteroid titers in adult Apis mellifera workers

“…The site of steroid synthesis in females then shifts to ovarian follicle cells (Lafont et al 2005;Brown et al 2009). The primary steroid produced by insect ovaries is ecdysone (C 27 H 44 O 6 ), which is converted to 20-hydroxyecdysone (20E) by peripheral tissues such as fat body (Petryk et al 2003). An important action of 20E in adult female insects is regulation of vitellogenin expression by fat body cells (Raikhel et al 2002).…”