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

The <i>Drosophila</i> Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of <i>spookier</i>

<div><p>Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C₂H₂-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly <i>Drosophila melanogaster</i>. Expression of <i>ouib</i> is predominantly limited to the PG, and genetic null mutants of <i>ouib</i> result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, <i>ouib</i> mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, <i>spookier</i>. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of <i>spok</i> by binding to a specific ~15 bp response element in the <i>spok</i> PG enhancer element. Most remarkable, the developmental arrest phenotype of <i>ouib</i> mutants is rescued by over-expression of a functionally-equivalent paralog of <i>spookier</i>. These observations imply that the main biological function of Ouib is to specifically regulate <i>spookier</i> transcription during <i>Drosophila</i> development.</p></div

Expression analysis of <i>ouib</i> in <i>Drosophila</i> larva and embryo.

<p>(A, B) RNA <i>in situ</i> hybridization of stage 14 (A) and stage 16 (B) embryos with the <i>ouib</i> antisense RNA probe. Dorsal views are shown. <i>ouib</i> signal was detected in the primordia of PG cells (arrows). An image with sense RNA probe is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005712#pgen.1005712.s004" target="_blank">S1 Fig</a> (C, D) <i>in situ</i> hybridization of third instar larval brain-ring gland complexes with the <i>ouib</i> antisense (C) and sense (D) RNA probes. <i>ouib</i> signal is detected in the ring gland including the PG cells (arrow). (E) The expression levels of <i>ouib</i> in several tissues quantified by qRT-PCR (N = 3). Total RNA was prepared from wandering third instar larvae. BR, brain; RG, ring gland; ID, imaginal disc; IN, intestine; FB, fat body; SG, salivary gland. Error bars indicate the s.e.m.</p

DNA-Binding analysis of Ouib for the upstream element of <i>spok</i>.

<p>(A) ABCD assay with nuclear extracts from S2 cells and avidin-biotin-conjugated double-stranded DNA probes. After pull-down, proteins were detected by western blotting using anti-FLAG antibody. (B) EMSA using recombinant proteins of GST alone or GST-fusion zinc finger domains of Ouib (amino acids 150–313) with ³²P-labeled double-stranded oligonucleotide probes containing the wild type 15 bp Ouib-response element (ORE). The x100 and x200 amounts of the non-labeled probes of the wild type ORE (ORE WT), the mutated ORE (ORE Mut) and the wild type M1BP-response element (MRE WT) were used as cold competitors. (C) Densitometric analysis of the EMSA band radioactive intensities in the same experimental condition as B with 3 independent replicates. Average radioactivity of the ORE/GST-Ouib-Zf complex is set as 100%. Note that the complexes with the ORE were outcompeted by the unlabeled ORE WT probe, but not by the unlabeled MRE WT probe. *<i>P</i><0.05 and **<i>P</i><0.01 by Tukey's multiple comparisons test. n.s., not significant. (D) EMSA using recombinant proteins of GST-fusion zinc finger domains of Ouib (amino acids 150–313) with ³²P-labeled double-stranded oligonucleotide probes of ORE WT, ORE Mut and MRE WT.</p

Larval lethality and developmental arrest phenotype of <i>ouib</i> mutant larvae.

<p>(A) The survival rate and developmental progression of control and <i>ouib</i> mutant animals (N = 50~100). (B) Comparison of body size and developmental stage between control (right and middle) and <i>ouib</i> mutant (left) at 108 hours AEL. Control animals became third instar larvae, while <i>ouib</i> mutant animals were first instar larvae. Scale bar: 1 mm. (C) Ecdysteroid levels in control and <i>ouib</i> mutant first instar larvae at 12 hours AH measured by ELISA (N = 4). (D) Ecdysteroid levels, as measured by the ecdysone inducible gene <i>E75A</i>, in control and <i>ouib</i> mutant first instar larvae at 36 hours AEL measured by qRT-PCR (N = 3). Error bars indicate s.e.m. **; <i>P</i><0.01 with Student’s <i>t</i>-test.</p

Expression analysis of ecdysteroidogenic genes and feeding rescue experiment in <i>ouib</i> mutant larvae.

<p>(A) The expression levels of ecdysteroidogenic genes in control and <i>ouib</i> mutant first instar larvae at 38 hours AEL measured by qRT-PCR (N = 3). (B) Immunostaining of the PG cells from control and <i>ouib</i> mutant first instar larvae at 36 hours AEL with antibodies against Spok (green) and Sro (magenta). Scale bar: 25 μm. (C) The transcriptional expression profiles of <i>ouib</i> and <i>spok</i> in <i>w</i>^<i>1118</i> during larval development measured by qRT-PCR (N = 3). The solid line and dashed lines indicate the expression levels of <i>ouib</i> and <i>spok</i>, respectively. (D) Feeding rescue experiments for <i>ouib</i> mutant (<i>ouib</i>^<i>29</i><i>/ouib</i>^<i>74</i>) larvae. Mutant animals fed 5β-ketodiol (5βkd) and 20-hydroxyecdysone (20E) became third instar larvae, while animals fed cholesterol (C), 7-dehydrocholesterol (7dC) and none remained first instar larvae. The lethal stages in each experimental condition were scored and shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005712#pgen.1005712.t002" target="_blank">Table 2</a>. Scale bar: 1 mm. Error bars indicate s.e.m. *; <i>P</i><0.05, **; <i>P</i><0.01 with Student’s <i>t</i>-test.</p

Rescue of <i>ouib</i> null mutant animals by oral administration of sterols and ecdysteroids.

<p>The first instar larvae of <i>ouib</i>^<i>29</i>/<i>ouib</i>^<i>74</i> or <i>ouib</i>^<i>74</i><i>/Df</i> animals were collected 36 hours AEL, and then fed yeast pastes containing 0.5% (w/w) each steroid. The number of dead animals at each stage was counted. C, cholesterol; 7dC, 7-dehydrocholesterol; 5βkd, 5β-ketodiol; 20E, 20-hydroxyecdysone; -, not determined.</p><p>Rescue of <i>ouib</i> null mutant animals by oral administration of sterols and ecdysteroids.</p