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Neverland is an evolutionally conserved Rieske-domain ... two distinct UAS-nvd-Dm-Inverted Repeat constructs, designated UAS-nvd-Dm-IR-1 and UAS-nvd-Dm-IR-2. UAS-nvd-Dm-IR-1 was a

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Text of Neverland is an evolutionally conserved Rieske-domain ... two distinct UAS-nvd-Dm-Inverted Repeat...

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    INTRODUCTION Steroid hormones are responsible for the coordination and regulation of many aspects of development, growth and differentiation of multicellular organisms. In insects and other arthropods, a strict regulation of titers of ecdysteroids, especially ecdysone (so called �-ecdysone) and 20-hydroxyecdysone (20E), plays central roles in development, especially in guiding transition from one developmental stage to the next via molting and metamorphosis (Thummel, 2001; Gilbert et al., 2002; Thummel and Chory, 2002). During larval and pupal development of insects, ecdysone is synthesized from dietary cholesterol or phytosterols via a series of hydroxylation and oxidation steps in the small endocrine organ called the prothoracic gland (PG) (Gilbert et al., 2002).

    Studies using Drosophila have identified several molecules that are involved in ecdysteroid biosynthesis in the PG. For example, ecdysone synthesis is regulated by itpr, which encodes inositol 1,4,5- trisphosphate receptor (Venkatesh and Hasan, 1997; Venkatesh et al., 2001); dare, which encodes adrenodoxin reductase (Freeman et al., 1999); ecdysoneless (ecd), which encodes an evolutionally conserved protein with no known motifs (Warren et al., 1996; Gaziova et al., 2004); and without children, which encodes a putative transcriptional regulator (Wismar et al., 2000; Warren et al., 2001). Ras-dependent signaling cascade and insulin-dependent PG cell growth are also essential for the ecdysone production and/or release (Caldwell et al., 2005; Mirth et al., 2005).

    Recently, five hydroxylase genes that are essential for ecdysteroid biosynthesis have been identified in Drosophila. All of the hydroxylase genes, Cyp306a1/phantom (phm), Cyp302a1/ disembodied (dib), Cyp315a1/shadow (sad), Cyp314a1/shade (shd) and Cyp307a1/spook (spo), are named the Halloween genes and encode cytochrome P450 mono-oxygenases (Chávez et al., 2000; Warren et al., 2002; Petryk et al., 2003; Niwa et al., 2004; Warren et al., 2004; Namiki et al., 2005). A combination of molecular and biochemical experiments have shown that Phm, Dib, Sad and Shd play pivotal roles in the final four steps of ecdysteroidogenesis, namely the conversion of 5�-ketodiol to 20E (Gilbert and Warren, 2005). Orthologs of the Halloween P450 genes have also been identified in the silkworm Bombyx mori and the tobacco hornmoth Manduca sexta. The expression patterns of these lepidopteran hydroxylase genes are spatially restricted to the PG and are temporally correlated with the ecdysteroids titer during larval development (Niwa et al., 2004; Warren et al., 2004; Namiki et al., 2005; Niwa et al., 2005; Rewitz et al., 2006). The identification of these genes provides the basis for investigating the regulation of insect hormone production in more detail. For example, it has been shown that the expression of phm and dib is regulated by the �FTZ- F1 transcription factor in Drosophila (Parvy et al., 2005). Similarly, molting defective, which encodes a putative transcription factor in Drosophila, influences the expression level of some Halloween P450 genes (Neubueser et al., 2005). In Bombyx, the expression of the dib ortholog is significantly induced by steroidogenic neuropeptide prothoracicotropic hormone in cultured PGs (Niwa et al., 2005).

    Although the enzymes involved in the final biochemical steps of ecdysteroid biosynthesis are relatively well characterized, little is known about the molecules involved in earlier steps (Gilbert and Warren, 2005). Dietary cholesterol (C) is first converted to 7- dehydrocholesterol (7dC) by 7,8-dehydrogenation in the endoplasmic reticulum. Conversion of the 7dC to the �4-diketol constitutes the so called ‘black box’. Subsequently, cytosolic 5�- reduction and microsomal 3�-reduction steps convert the �4-diketol

    Neverland is an evolutionally conserved Rieske-domain protein that is essential for ecdysone synthesis and insect growth Takuji Yoshiyama1, Toshiki Namiki1, Kazuei Mita2, Hiroshi Kataoka1,† and Ryusuke Niwa1,*,†

    Steroid hormones mediate a wide variety of developmental and physiological events in multicellular organisms. During larval and pupal stages of insects, the principal steroid hormone is ecdysone, which is synthesized in the prothoracic gland (PG) and plays a central role in the control of development. Although many studies have revealed the biochemical features of ecdysone synthesis in the PG, many aspects of this pathway have remained unclear at the molecular level. We describe the neverland (nvd) gene, which encodes an oxygenase-like protein with a Rieske electron carrier domain, from the silkworm Bombyx mori and the fruitfly Drosophila melanogaster. nvd is expressed specifically in tissues that synthesize ecdysone, such as the PG. We also show that loss of nvd function in the PG causes arrest of both molting and growth during Drosophila development. Furthermore, the phenotype is rescued by application of 20-hydroxyecdysone or the precursor 7-dehydrocholesterol. Given that the nvd family is evolutionally conserved, these results suggest that Nvd is an essential regulator of cholesterol metabolism or trafficking in steroid synthesis across animal phyla.

    KEY WORDS: Bombyx mori, Cholesterol, Drosophila melanogaster, Ecdysone, Growth, Molting, Prothoracic gland, Rieske, Ring gland, Steroidogenesis

    Development 133, 2565-2574 (2006) doi:10.1242/dev.02428

    1Department of Integrated Biosciences, Rm201, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan. 2Laboratory of Insect Genome, National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8643, Japan.

    *Present address: Department of Molecular, Cellular and Developmental Biology, Yale University, KBT 938, P.O. Box 208103, 266 Whitney Ave., New Haven, CT 06520, USA †Authors for correspondence (e-mail: [email protected]; [email protected]

    Accepted 5 May 2006

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    to the 5�-ketodiol. Further identification and characterization of these ‘early’ ecdysteroidogenic genes are important for understanding the mechanisms by which ecdysteroidogenesis and developmental timing are precisely controlled in arthropods.

    To facilitate identification and characterization of components responsible for ecdysteroid biosynthesis, we have used Bombyx to identify genes predominantly expressed in the PG (Niwa et al., 2004; Namiki et al., 2005; Niwa et al., 2005; Yamanaka et al., 2005). Based on gene expression analysis using Bombyx cDNA microarrays that we have previously performed (Niwa et al., 2004), we now describe a novel gene named neverland (nvd), the expression of which is specifically enriched in ecdysone-synthesizing tissues, including the PG. We show that loss of nvd function in the PG causes growth arrest at the larval stages, and this phenotype is rescued by application of 20E or 7dC. Our results suggest that Nvd plays a pivotal role in the metabolism of cholesterol and steroid intermediates during ecdysteroidogenesis. Considering that the nvd gene family is evolutionally conserved, we propose that the nvd family of proteins is an essential regulator of steroid biosynthesis in various animal phyla.

    MATERIALS AND METHODS Animal strains and culture Culture and staging of silkworms, B. mori (KINSHU � SHOWA F1 hybrid), have been described previously (Niwa et al., 2004). All D. melanogaster flies were reared on a standard agar-cornmeal medium at 25°C under a 12-hour light/12-hour dark photoperiod unless otherwise specified. 2-286-GAL4 (Timmons et al., 1997; Andrews et al., 2002), AUG21-GAL4 (Siegmund and Korge, 2001) and AKH-GAL4 (Lee and Park, 2004) were kindly provided from C. S. Thummel, G. Korge and J. H. Park, respectively. wocrgl (Wismar et al., 2000) was generous gift from J. T. Warren and L. I. Gilbert. breathless- GAL4 (Shiga et al., 1996), elavc155-GAL4 (Luo et al., 1994) and teashirt- GAL4 (Shiga et al., 1996) were provided from Genetic Strains Research Center in National Institute of Genetics, Japan. scabrous-GAL4 (Klaes et al., 1994) and decapentaplegic-GAL4 (Staehling-Hampton et al., 1994) were obtained from the Bloomington stock center. Lsp2-GAL4 (Cherbas et al., 2003) and pGawB5015 were provided from the Drosophila Genetic Resource Center, Kyoto Institute of Technology. Although a previous study shows that a GAL4 in pGawB5015 is active in lymph gland and hematopoietic cells (Sinenko et al., 2004), the GAL4 was also expressed in the larval ring gland containing the PG cells (see Fig. S1 in the supplementary materials; J.-B. Peyre and T. Aigaki, personal communication). All flies were maintained in yw background. Cyo[y+] and TM3[y+] were used as balancers.

    Molecular cloning Because an EST clone prgv0382 from the Bombyx EST project (Mita et al., 2003) lacked the 5� region of full-length cDNA of Bombyx neverland (nvd- Bm), the 5� end of nvd-Bm cDNA was obtained by the 5� Rapid Amplification of cDNA ends (5� RACE) method using the GeneRacer Kit (Invitrogen). The first 5� RACE product was amplified with a gene-specific primer (5�-GGGCAGAAGTAAGGAGCGCCATCTCTGTG-3�) and GeneRacer 5� Primer. Then we performed the nested PCR with another gene-specific primer (5�-CCGCTGTAAAGAAGCCAATTAAGGTG- GCGC-3�) and GeneRacer 5� nested primer. The nucleotide sequence of Drosophila neverland (nvd-Dm) was identified from the Drosophila EST database (GenBank Accession Number BT021261). The cDNA containing the entire open reading frame (ORF) for nvd-Dm was amplified by PCR from wild-type Drosophila ring gland-derived cDNA using the following primers: forward, 5�-ATGACGAGCTACAGTTTATTTTGGATGTC-3�; reverse, 5�-CTACCAACCAATATTGGTTGCTTCAG-3�. The DNA sequen