Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera

Abstract

Ecdysteroids regulate many key developmental events in arthropods including molting and metamorphosis. Recently, members of the Drosophila Halloween group of genes, that are required for embryonic viability and cuticle deposition, have been shown to code for several cytochrome P450 enzymes that catalyze the terminal hydroxylation steps in the conversion of cholesterol to the molting hormone 20-hydroxyecdysone. These P450s are conserved in other insects and each is thought to function throughout development as the sole mediator of a particular biosynthetic step since, where analyzed, each is expressed at all stages of development and shows no closely related homolog in their respective genomes. In contrast, we show here that several dipteran genomes encode two novel, highly related, microsomal P450 enzymes, Cyp307A1 and Cyp307A2, that likely participate as stage-specific components of the ecdysone biosynthetic machinery. This hypothesis comes from the observation that Cyp307A1 is encoded by the Halloween gene spook (spo), but unlike other Halloween class genes, Dmspo is not expressed during the larval stages. In contrast, Cyp307a2, dubbed spookier (spok), is expressed primarily during larval stages within the prothoracic gland cells of the ring gland. RNAi mediated reduction in the expression of this heterochromatin localized gene leads to arrest at the first instar stage which can be rescued by feeding the larva 20E, E or ketodiol but not 7dC. In addition, spok expression is eliminated in larvae carrying mutations in molting defective (mld), a gene encoding a nuclear zinc finger protein that is required for production of ecdysone during Drosophila larval development. Intriguingly, mld is not present in the Bombyx mori genome, and we have identified only one spook homolog in both Bombyx and Manduca that is expressed in both embryos and larva. These studies suggest an evolutionary split between Diptera and Lepidoptera in how the ecdysone biosynthetic pathway is regulated during development.

Characterization of Drosophila melanogaster cytochrome P450 genes

“…Cyp302a1, Cyp306a1, Cyp307a2, and Cyp315a1 were detected in the prothoracic cells of the ring gland in embryos and larvae (Fig. 3A), as per previous studies (18,19,21,57). Five P450s were detected in the hindgut (Cyp6a17, Cyp301a1, Cyp4c3, Cyp49a1, and Cyp4aa1) (Fig.…”

“…One exception is the involvement of P450s in the biosynthesis of the major insect hormone 20-hydroxyecdysone (20H) from plant sterols, where in Drosophila melanogaster at least six P450s are involved (17). Cyp302a1, Cyp306a1, Cyp315a1, and Cyp307a2 are all expressed in the prothoracic glands of late embryos and larvae and catalyze steps in the ecdysone biosynthetic pathway (18)(19)(20)(21), while Cyp314a1 (shade), responsible for catalyzing the conversion from ecdysone to 20H, is expressed in the midgut, Malpighian tubules, and the fat body (22). Cyp307a1 (spook) has also been suggested to play a role in the synthesis of ecdysone, but it is not expressed in larvae (21).…”

“…Cyp302a1, Cyp306a1, Cyp315a1, and Cyp307a2 are all expressed in the prothoracic glands of late embryos and larvae and catalyze steps in the ecdysone biosynthetic pathway (18)(19)(20)(21), while Cyp314a1 (shade), responsible for catalyzing the conversion from ecdysone to 20H, is expressed in the midgut, Malpighian tubules, and the fat body (22). Cyp307a1 (spook) has also been suggested to play a role in the synthesis of ecdysone, but it is not expressed in larvae (21). P450s involved in other important endogenous processes in D. melanogaster have also been identified, including Cyp303a1, required for the development and structure of sensory bristles (23), and Cyp4g1, an oenocytespecific P450 required for correct triacylglycerol composition (24).…”

The genome of the model beetle and pest Tribolium castaneum

“…Most insect cytochrome P450 proteins (CYPs) are thought to be involved in metabolic detoxification of host plant allelochemicals and toxicants, and several are insecticide resistance genes 36 . Other CYPs act in the synthesis and degradation of lipid signalling molecules, such as ecdysteroids 37 . Similarly to mosquitoes, especially Aedes, Tribolium has an independently expanded CYP gene family, particularly those involved in environmental response (Supplementary Table 16).…”

Steroid signaling in mature follicles is important for Drosophila ovulation

“…Many enzymes involved in the ecdysone biosynthesis pathway have been identified, including Phantom (Phm), Disembodied (Dib), Shadow (Sad), and Shade (Shd), which carry out the final four steps of 20E synthesis (17)(18)(19)(20). In addition, enzymes encoded by spook (spo) and shroud (sro) act more upstream for chemical reactions designated as the black box (21,22). Ecdysteroids signal through a heterodimeric nuclear receptor complex comprised of Ultraspiracle (Usp) and Ecdysone receptor (EcR) (23,24).…”