Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the ‘Black Box’ of the ecdysteroid biosynthesis pathway

Ryusuke Niwa,Satoru Kobayashi,Yuko Shimada-Niwa,Yutaka Banno,Toru Shimada,Katsuhiko Ito,Shuji Shigenobu,Toshiki Namiki,Takumi Kayukawa,Susumu Katsuma,Shinpei Kawaoka,Makoto Kiuchi,Yoshinori Fujimoto,Tetsuro Shinoda

doi:10.1242/dev.045641

Abstract

In insects, the precise timing of molting and metamorphosis is strictly guided by a principal steroid hormone, ecdysone. Among the multiple conversion steps for synthesizing ecdysone from dietary cholesterol, the conversion of 7-dehydrocholesterol to 5beta-ketodiol, the so-called 'Black Box', is thought to be the important rate-limiting step. Although a number of genes essential for ecdysone synthesis have recently been revealed, much less is known about the genes that are crucial for functioning in the Black Box. Here we report on a novel ecdysteroidgenic gene, non-molting glossy (nm-g)/shroud (sro), which encodes a short-chain dehydrogenase/reductase. This gene was first isolated by positional cloning of the nm-g mutant of the silkworm Bombyx mori, which exhibits a low ecdysteroid titer and consequently causes a larval arrest phenotype. In the fruit fly, Drosophila melanogaster, the closest gene to nm-g is encoded by the sro locus, one of the Halloween mutant members that are characterized by embryonic ecdysone deficiency. The lethality of the sro mutant is rescued by the overexpression of either sro or nm-g genes, indicating that these two genes are orthologous. Both the nm-g and the sro genes are predominantly expressed in tissues producing ecdysone, such as the prothoracic glands and the ovaries. Furthermore, the phenotypes caused by the loss of function of these genes are restored by the application of ecdysteroids and their precursor 5beta-ketodiol, but not by cholesterol or 7-dehydrocholesterol. Altogether, we conclude that the Nm-g/Sro family protein is an essential enzyme for ecdysteroidogenesis working in the Black Box.

Highlights

In insects and other arthropods, steroid hormones designated as ecdysteroids, such as ecdysone and its derivative, 20hydroxyecdysone (20E), control the precise temporal progression of development (Thummel, 2001; Gilbert et al, 2002; Mirth and Riddiford, 2007; Spindler et al, 2009)
To uncover an unidentified gene responsible for ecdysteroid biosynthesis, we focused on a genetic mutant designated nonmolting glossy of the silkworm Bombyx mori, another classical model insect utilized for the study of endocrinology
Because the insertion disrupted the entire BGIBMGA007047 open reading frame (ORF), we concluded that BGIBMGA007047 was the gene responsible for nm-g mutants (+nm-g)

Summary

Introduction

In insects and other arthropods, steroid hormones designated as ecdysteroids, such as ecdysone and its derivative, 20hydroxyecdysone (20E), control the precise temporal progression of development (Thummel, 2001; Gilbert et al, 2002; Mirth and Riddiford, 2007; Spindler et al, 2009). The terminal hydroxylation steps in ecdysteroid biosynthesis in the PG, namely 5 -ketodiol to ecdysone, are catalyzed by three cytochrome P450 monooxygenases: Phantom (Phm; CYP306A1), Disembodied (Dib; CYP302A1) and Shadow (Sad; CYP315A1) (Chávez et al, 2000; Warren et al, 2002; Niwa et al, 2004; Warren et al, 2004; Niwa et al, 2005) (Fig. 1). The conversion of ecdysone to 20E is mediated by a P450 mono-oxygenase, Shade (Shd; CYP314A1), in the peripheral tissues (Petryk et al, 2003). All of these genes encoding P450 enzymes have been identified from embryonic lethal mutants called Halloween mutants that exhibit ecdysone deficiency in embryos (Chávez et al, 2000; Gilbert and Warren, 2005)

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Conclusion