Transcriptional Control of Steroid Biosynthesis Genes in the Drosophila Prothoracic Gland by Ventral Veins Lacking and Knirps.

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development

Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects

Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. We also show that prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH

Vvl and Kni have binding sites in the promoters and enhancers of the ecdysone biosynthetic genes and are expressed in the PG.

<p>(A) An illustration showing binding sites in the PG specific cis-regulatory elements of <i>spok</i> and <i>phm</i> and <i>dib</i>. Binding sites are indicated by squares (Vvl) and pentagons (Kni) with shades indicating the conservation of the site between <i>Drosophila</i> species. Conservation tracks were obtained from the UCSC genome browser. (B) <i>In situ</i> hybridization of embryos and third instar larval brains and ring glands with antisense probes for <i>vvl</i> (<i>a</i>′–<i>d</i>′) or <i>kni</i> (<i>e</i>′–<i>g</i>′). (<i>a</i>′) Stage 11, shows <i>vvl</i> expression in the primordial cells of the trachea, while (<i>b</i>′) stage 13, (<i>c</i>′) stage 16 and (<i>d</i>′) L3 show strong <i>vvl</i> expression in the PG cells of the ring gland. (<i>e</i>′<i>)</i> stage 4, (<i>f</i>′<i>)</i> stage 16 and (<i>g</i>′) L3 show <i>kni</i> expression in the PG of L3 larvae, but not clearly in embryos. (C) Immunostaining of the PG from L3 larvae with antibodies against Kni and Vvl (magenta) and Phm (green). Co-localization with nuclei staining (DAPI: gray) indicates that Vvl and Kni are expressed in the nucleus of the PG cells. (D) Expression of <i>phm</i>, <i>vvl</i>, <i>kni</i> and <i>mld</i> measured by qPCR in tissue from whole body L3 larvae or dissected ring glands containing the PG of L3 larvae (n = 4). <i>vvl</i> is highly expressed in the ring gland compared to whole body, like <i>phm</i>, while the expression of <i>kni</i> and <i>mld</i> show a minor enrichment in the gland. Error bars indicate s.e.m. **<i>P</i><0.01, versus whole body.</p

<i>vvl</i>, <i>kni</i> and <i>mld</i> are required for the expression of genes in the ecdysone biosynthetic pathway.

<p>(A) Knock down of <i>vvl</i>, <i>kni</i> and <i>mld</i> in the PG reduces expression of genes in the steroidogenic pathway. <i>vvl</i> knock down results in a down-regulation of <i>spok</i> and <i>sro</i>, catalyzing early steps in the pathway, as well as a reduction of <i>phm</i>, <i>dib</i> and <i>sad</i> mediating the last three steps in the biosynthetic pathway. Knock down of <i>kni</i> results in down-regulation of <i>phm</i>, <i>dib</i> and <i>sad</i>, while knock down of <i>mld</i> causes a specific down-regulation of <i>spok</i> and a moderate reduction of <i>sro</i>. Expression was measured in mid-first instar larvae 36 hours AEL. Error bars indicate s.e.m. (n = 4). *<i>P</i><0.05, **<i>P</i><0.01, versus the <i>phm</i>>+ control. (B, C) Direct binding of Vvl or Kni to the regulatory sites in <i>phm</i> promoter indicated by electrophoretic mobility shift assay (EMSA). Nuclear extract was incubated with [γ32]ATP-labeled oligonucleotide sequences of <i>phm</i> promoter containing the <i>vvl</i> (B) or the <i>kni</i> sites (C) and resulted in shifted DNA-protein bands (lane 1). Competition assays were performed with unlabeled non-specific random oligonucleotide sequences (lane 2), the <i>phm</i> promoter containing the <i>vvl</i> or <i>kni</i> sites (lane 3), the <i>phm</i> promoter with mutated <i>vvl</i> or <i>kni</i> sites (lane 4), an oligonucleotide sequence with <i>vvl</i> or <i>kni</i> consensus motif sequence (lane 5), or with the consensus motif mutated (lane 6).</p

Ecdysone and 20E efficiently rescue loss of <i>vvl</i> and <i>kni</i> in the PG.

<p>(A) Ecdysone biosynthetic scheme showing steps in the conversion of cholesterol to 20-hydroxyecdysone (20E). Note that ecdysone produced and released from the PG is converted to its active form 20E in peripheral tissues. (B) Percentage of larvae developing to the indicated stage. L1; first instar larvae, L2; second instar larvae, L3; third instar larvae. Resupplying precursors later in the pathway is gradually more efficient in rescuing arrest of larvae with reduced expression of <i>vvl</i> and <i>kni</i> in the PG. In contrast, only precursors downstream of the black box efficiently rescue <i>mld-RNAi</i> larvae, indicating that Mld regulates a gene product(s) involved in the reactions upstream of the 5β-ketodiol. C; cholesterol, 7dC; 7-dehydrocholesterol, KD; 5β-ketodiol, KT; 5β-ketotriol, 2dE; 2-deoxyecdysone, E; ecdysone, 20E; 20-hydroxyecdysone.</p

Knock down of <i>vvl</i>, <i>kni</i> and <i>mld</i> in the PG results in developmental arrest and reduces ecdysteroid levels.

<p>(A) RNAi mediated knock down of <i>vvl</i>, <i>kni</i> or <i>mld</i> in the PG using a PG specific driver (<i>phm</i>>) results in developmental L1 arrest for <i>phm>vvl-RNAi</i> and <i>phm>mld-RNAi</i> and L1 and L2 arrest for <i>phm>kni-RNAi</i> larvae. The morphology of the cells in the PG (GFP; green in the top left corner) is normal in <i>phm</i>><i>GFP</i>,<i>vvl-RNAi</i>, <i>phm</i>><i>GFP</i>,<i>kni-RNAi</i> and <i>phm</i>><i>GFP</i>,<i>mld-RNAi</i> animals 36 hours AEL (scale bars, 20 µm). Supplying <i>phm</i>><i>vvl-RNAi</i>, <i>phm</i>><i>kni-RNAi</i> and <i>phm</i>><i>mld-RNAi</i> larvae with 20-hydroxyecdysone (20E) rescues the developmental arrest. (B) Ecdysone levels, as measured by the ecdysone inducible gene <i>E75B</i>, is reduced in the mid-first instar (36 hours AEL) by knock down of <i>vvl</i>, <i>kni</i> or <i>mld</i> in the PG. (C) Ecdysteroid levels measured by ELISA confirm that L1 larvae with reduced expression of <i>vvl</i>, <i>kni</i> or <i>mld</i> in the PG have low levels of ecdysteroids 36 hours AEL compared to the control. Error bars indicate s.e.m. (n = 4). *<i>P</i><0.05, ***<i>P</i><0.001, versus the <i>phm</i>>+ control.</p

<i>vvl</i>, <i>kni</i> and <i>mld</i> have a specific role in regulating ecdysone biosynthesis in the L3 stage.

<p>(A) Expression of steroidogenic genes in ring glands from wild type (<i>phm-GFP</i>) larvae increases little from early (72 hours AEL) to mid (96 hours AEL) third instar, but rises dramatically in the late (120 hours AEL) third instar. <i>vvl</i> expression exhibits a minor increase in the late third instar, while <i>mld</i>, <i>kni</i> and especially <i>Br-Z4</i> show a strong increase (n = 4). (B) Expression in the ring gland from larvae with knock down of <i>vvl</i>, <i>kni</i> or <i>mld</i> during the L3 stage two days after temperature induced activation of the RNAi with the <i>Gal80^ts</i>;<i>phm</i>> driver 96 hours AEL. Expression of all the steroidogenic genes were significantly reduced in animals with reduces <i>vvl</i> or <i>kni</i> expression. Knock down of <i>mld</i> results in a dramatic reduction in expression of <i>nvd</i> and <i>spok</i> that mediate two early steps in the biosynthesis of ecdysone (n = 4). (C) Quantified level of Phm protein in brain-ring gland complexes (BRGCs) from L3 larvae two days after temperature induced RNAi (96 hours AEL) normalized to Tubulin levels determined by immunoblotting (top panel) (n = 3). (D) Ecdysteroid levels determined by ELISA in L3 larvae with reduced PG expression of <i>vvl</i>, <i>kni</i> or <i>mld</i> two days after temperature induced activation of the RNAi effect (96 hours AEL) (n = 4). Error bars indicate s.e.m. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, versus the <i>Gal80^ts;phm>+</i> control.</p

A model for sex-specific synthesis and action of ecdysteroids in adults.

<p>The ovaries of female <i>Tribolium</i> express <i>spo</i> and the genes for the terminal hydroxylases, including <i>shd</i>, required for synthesis of 20E. Male accessory glands also express a <i>spo</i>-like gene, <i>spot</i>, and the genes for the terminal hydroxylases required for synthesis of E, but not <i>shd</i>. However, s<i>hd</i> expression was detected in the carcass without the reproductive system indicating that E synthesized by the accessory gland might be converted to 20E in peripheral tissues, like during the larval stages. Alternatively, E produced by the accessory gland may be involved in male-specific hormone signaling or be transferred to females during mating. Multiple arrows indicate several steps in the biosynthetic pathway.</p

Halloween gene expression during development and in adult <i>Tribolium</i>.

<p>The relative mRNA distribution of the Halloween genes during development and in adults was measured by qPCR. Expression was normalized to <i>rpS3</i> mRNA levels. The step in the ecdysteroid biosynthetic pathway catalyzed by each enzyme is shown on the right. Numbers on the x-axis indicate the day of each stage. E; embryo, F; females, O; ovaries, M; males, R; reproductive systems of males, 7dC; 7-dehydrocholesterol, 2,22,25-dE; 2,22,25-trideoxyecdysone (ketodiol), 2,22-dE; 2,22-dideoxyecdysone (ketotriol), 2-dE; 2-deoxyecdysone, 20E; 20-hydroxyecdysone. Note that the Black Box is believed to include multiple uncharacterized reactions converting 7dC to the ketodiol.</p