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

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

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