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.

Fig. 1.

Scheme of 20E biosynthesis. The top portion represents a theoretical pathway in which plant sterols obtained in the diet are converted to the ketodiol (23). The bottom portion of the figure shows the final three steps in the Drosophila pathway that involve conversion of the ketotriol intermediate to 20-hydroxyecdysone by the activity of the P450 enzymes coded for by dib, sad, and shd (ref. and this work).

Fig. 2.

Phenotype of homozygous shd embryos and egg chambers. (a) Normal cuticle development in wild-type embryos. (b–d) Normal embryonic development of wild-type embryos. (e–h) Homozygous shd embryos. (e) Lack of cuticle differentiation in shd mutant embryos. (f) Normal embryonic development at stage 14. (g) Failure of head involution at embryonic stages 15–16 (see arrow). (h) Defect in dorsal closure and aberrant gut looping at embryonic stages 15–16 (see arrow). Embryos in b–d and f–h were stained with spectrin antibody. (i) Stage-15 wild-type embryo showing normal IMP-E1 expression. (j) Reduced IMP-E1 epidermal expression in stage 13 shd mutant embryos. The remaining gut staining is presumably under the influence of a nonecdysone response enhancer element (9, 16). (k) Wild-type egg chambers. (l) Egg chambers showing arrest and degeneration at stages 8–9 of oogenesis in rescued females of the genotype twist>gal4, shd^z329/UAS-shd, shd^z320.

Fig. 3.

In situ expression pattern of shd. (a) shd expression in the epidermis at the stage of germband extension (stage 10, 4:20–5:20 h). (b) shd expression in midgut (mg) and Malpighian tubules (mt) in third instar larval whole body but not in the brain-ring gland complex (brgc). (c) shd expression in third instar larval mg and mt. (d) Copper cells (cc) of the third instar larval midgut. (e) Third instar larval fat body. (f) Nurse cells of the adult ovary. (g) Centripetally migrating follicle cells. (h) RT-PCR study of shd tissue expression (Upper) and a control gene, rpL17A (Lower), in third instar larvae (Left) and adults (Right). RG, ring gland; Br, brain; SG, salivary glands; FB, fat body; Gu, gut; Ep, epidermis; He, head; Ov, ovary; Te, testis; Ca, carcass.

Fig. 4.

Immunolocalization of Halloween proteins. Confocal sections of S2 cells transfected with HA-tagged Sad (a–c), Dib (d–f), and Shd (g–i). HA immunoreactivity is shown in a, d, and g (green), MitoTracker Red signal is shown in b, e, and h (red), and the merge of HA and MitoTracker staining is shown in c, f, and i.

Fig. 5.

RP-HPLC/TLC/ESI-MS analysis of ecdysteroids after shd- or GFP-transfected S2 cell homogenate incubation (8 h) with [³H]E containing E (1 μg) and NADPH (30–100% methanol gradient). Radioactivity was measured after incubations with shd-transfected (red circles) or GFP-transfected (blue circles) cell homogenates with substrate. UV absorption was measured at 248 nm (solid line). (Upper Inset) TLC (chloroform/ethanol) of RP-HPLC-purified 20-hydroxyecdysone (20E) product (1/1,000th of total sample). (Lower Inset) RP-HPLC/ESI-MS on a TSQ Quantum (Thermo Finnigan, San Jose, CA) of the TLC-purified 20E product.