A genetic screen identifies that Axn, Slmb and Nkd regulate <i>br</i> expression.

<p>(A). Schematic diagram of genetic crosses for isolating mutations that derepress <i>br</i> expression in young larvae. <i>GAL4-PG12,UAS-mCD8GFP</i> (X chromosome) was used to monitor <i>br</i> expression. The lethal mutation or <i>P</i>-insertion on the 2^nd or 3^rd chromosome is represented by an asterisk (*). (B–E). GFP images show the expression of <i>GAL4-PG12,UASmCD8GFP</i> in 2^nd instar larvae. GFP was only expressed in the salivary gland of the wild type [B], but widely expressed in all tissues of <i>Axn^EY10228</i> [C], <i>slmb⁰⁰²⁹⁵</i> [D], and <i>nkd²</i> [E] mutant larvae. (B′–E′) White light images of the same organisms are shown in [B–E].</p

<i>GAL4-PG12</i> carries a <i>P</i>-element insertion in the first intron of <i>br</i> gene.

<p>(A) The flanking sequence of the <i>GAL4-PG12 P</i>-element insertion site identified by inverse PCR analysis. (B) The insertion site of <i>GAL4-PG12</i> was located within the first intron of the <i>br</i> gene by comparing the sequence with the <i>Drosophila</i> genome.</p

<i>GAL4-PG12</i> resembles endogenous <i>br</i> expression patterns.

<p>(A) Protein extracts isolated from wild type animals at different developmental stages were separated by SDS-PAGE. Br proteins were assessed by Western blotting using a Br-core antibody. Tubulin-β was used as a loading control. The Br proteins were only detected in the late 3^rd instar larval stage to pupal stage. All Br isoforms were expressed in the late 3^rd instar larvae and early pupae, but only Z1 and/or Z3 isoforms were expressed in the late pupae. (B–F) Expression of <i>GAL4-PG12</i> was marked by <i>GAL4-PG12,UAS-mCD8GFP</i>. Constitutive expression of <i>GAL4-PG12</i> in salivary glands (arrows) and auto-fluorescence of fly food in the midgut (arrowheads) are indicated. In tissues other than those from the salivary gland, <i>GAL4-PG12/UAS-mCD8GFP</i> was only expressed in late 3^rd instar larval and during early pupal stages (G and H). (B′–F′) White light images of the same organisms are shown in [B–F]. (G–I) <i>GAL4-PG12</i> expression was monitored by mCD8GFP (green) [G–I]. Endogenous Br proteins were recognized by a Br-core antibody (red) [G′–I′] and nuclei were marked with DAPI (blue) [G″–H″]. Neither endogenous Br nor <i>GAL4-PG12</i> were expressed in FB of the 2^nd instar or early 3^rd instar [G-G″ and H-H″], but both were expressed in FB of the late 3^rd instar [I-I″]. [I″] is a merged image of [I] and [I′].</p

Ectopic expression of JHE induces precocious <i>br</i> expression in the 2^nd instar larvae.

<p>Flies carrying two copies of <i>hs-jhe</i> transgenes (<i>GAL4-PG12, UAS-mCD8GFP/Fm7C; hs-jhe¹, hs-jhe²/+</i>) were reared on normal (−JHA) or 0.1 ppm pyriproxifen-containing (+JHA) food and were treated with (HS) or without (no-HS) heat shocking twice a day for 40 min at 37°C. <i>Br</i> expression was monitored by <i>GAL4-PG12,UAS-mCD8GFP</i> [A–D] and FB Br-core antibody staining in 2^nd instar larvae [E–H]. Precocious <i>br</i> expression occurred in 2^nd instar larvae that were reared on normal food and treated with heat shocking [B-B′ and F-F′]. However, this phenotype was blocked by JHA treatment [D-D′ and H-H′].</p

Precocious <i>br</i> expression occurs in <i>Axn</i> mutants in a tissue-specific manner.

<p>2^nd instar larvae of <i>Oregon</i> R and <i>Axn^EY10228</i> were dissected and stained with a Br-core antibody (red). Nuclei were labeled with DAPI (blue). Images show central nervous system (CNS) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026772#pone-0026772-g006" target="_blank">Fig. 6A and D</a>), fat body (FB) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026772#pone-0026772-g006" target="_blank">Fig. 6B and E</a>) and midgut (MG) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026772#pone-0026772-g006" target="_blank">Fig. 6C and F</a>).</p

Expression of <i>Met</i>, <i>gce</i> and <i>Kr-h1</i> is reduced in the <i>Axn</i>, <i>slmb</i> and <i>nkd</i> mutants.

<p>(A) Total RNAs were extracted from <i>Oregon</i> R, <i>Axn^EY10228</i>, <i>slmb⁰⁰²⁹⁵</i>, and <i>nkd²</i> 2^nd instar larvae. The mRNA levels of <i>Met</i>, <i>gce</i> and <i>Kr-h1</i> were assessed by quantitative real-time PCR and normalized to <i>rp49</i> mRNA. Values shown are the means of 4 independent experiments ± standard deviations. (B) The same total RNAs described in [A] were used as the templates for a 30-cycle reverse transcriptional PCR. The RT-PCR products were analyzed by DNA agarose gel electrophoresis.</p

Precocious <i>br</i> expression in <i>Axn</i>, <i>slmb</i> and <i>nkd</i> mutants is not prevented by JHA.

<p><i>Oregon</i> R, <i>Axn^EY10228</i>, <i>slmb⁰⁰²⁹⁵</i>, and <i>nkd²</i> mutants were reared on normal (−JHA) or 0.1 ppm pyriproxifen-containing (+JHA) food. Fat bodies of the 2^nd instar larvae were stained with a Br-core antibody (red). Nuclei were labeled with DAPI (blue).</p

A possible model showing the last two steps of biosynthesis and the molecular actions of the three sesquiterpenoids in <i>Drosophila</i>.

<p>In the CA, FA is the common precursors for JHB3, JH III, and MF biosynthesis. In the hemolymph and peripheral tissues, MF either directly acts through Met/Gce or is converted to JHB3. JHAMT only accounts for JHB3 biosynthesis; and other methyltransferases and P450 epoxidase with question marks have not been identified. Please see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005038#sec008" target="_blank">Discussion</a> for details on the model. Text and arrow sizes convey magnitude of treatment and response. The gray line separates CA from hemolymph and peripheral tissues.</p

Reduction of <i>hmgcr</i> expression in the CA of <i>jhamt</i> mutant results in complete lethality.

<p>(A and A’) The brain-RG complex in <i>jhamt-GAL4>UAS-GFP</i>. BR, brain; CA, corpus allatum. Observed under bright-field (A) or fluorescence (A’) using the same microscope. The CA cells expressing JHAMT were labeled with GFP. (B and B’) (B) Lethality of <i>jhamt-GAL4</i>><i>UAS-hmgcr dsRNA</i> during the embryonic, larval, and pupal stages. <i>jhamt-GAL4/+</i> and <i>UAS-hmgcr dsRNA/+</i> were used as the controls. (B’) Lethality of <i>jhamt</i>^<i>2</i>/<i>jhamt</i>^<i>2</i>; <i>jhamt-GAL4>UAS-hmgcr dsRNA</i> during the embryonic, larval, and pupal stages. <i>jhamt</i>^<i>2</i>/+; <i>jhamt-GAL4/+</i> and <i>jhamt</i>^<i>2</i><i>/+; UAS-hmgcr dsRNA/+</i> were used as the controls. (C) Images of various pupal lethal phenotypes of <i>jhamt</i>^<i>2</i><i>/jhamt</i>^<i>2</i>; <i>jhamt-GAL4>UAS-hmgcr dsRNA</i>. (1–6) the abdominal sides; (1’-6’) the dorsal sides. The black asterisks point to empty portions of the pupae; the white asterisks, eye defects showing no pigmentation; the red asterisks, wing defects showing a unilateral wing loss.</p

MF plays a dual role: as a JHB3 precursor and as a hormone.

<p>(A) Percentage of rescuing <i>Aug21-GAL4>UAS-Grim</i>, <i>jhamt</i>^<i>2</i>/<i>jhamt</i>^<i>2</i><i>; jhamt-GAL4>UAS-hmgcr dsRNA</i>, and <i>Met</i>^<i>27</i><i>gce</i>^{<i>2</i>.<i>5k</i>} to adults by topical application of methoprene, JHB3, JH III and MF (0.5×10^-2 μmol per larva) at 96h AEL. (B) Percentage of rescuing <i>jhamt</i>^<i>2</i>/<i>jhamt</i>^<i>2</i><i>; jhamt-GAL4>UAS-hmgcr dsRNA</i> to adults by topical application of a dose gradient of methoprene, JHB3, JH III, and MF (0.5×10^-9~-2 μmol per larva) at 96h AEL. (C) qPCR measurements of fold-changes of relative <i>Kr-h1</i> mRNA levels in Kc cells treated with methoprene, JHB3, JH III, and MF (1×10^-10~-6 M) for 30 min. (D) qPCR measurements of relative <i>Kr-h1</i> mRNA levels in fat body tissues isolated from <i>w</i>^<i>1118</i> and <i>Met</i>^<i>27</i><i>gce</i>^{<i>2</i>.<i>5k</i>} at 96h AEL after treatments with methoprene, JHB3, JH III, and MF (1×10^-6 M) for 30 min. (E) qPCR measurements of the relative <i>Kr-h1</i> mRNA levels in the fat body tissues isolated from <i>w</i>^<i>1118</i>, <i>jhamt</i>^<i>2</i>, <i>Met</i>^<i>27</i>, <i>gce</i>^{<i>2</i>.<i>5k</i>}, and <i>Met</i>^<i>27</i><i>gce</i>^{<i>2</i>.<i>5k</i>} at 3h AIW. (F) MF promotes interaction of Met and SRC in mouse embryonic fibroblast 3T3 cells. 3T3 cells were transiently transfected with GAL4:TcMet and TcSRC. And the transfected cells were cultured in the medium containing different concentrations of MF and JH III (DMSO as control). After 24 hours exposure to the ligands, cells were assayed for luciferase reporter activity. The luciferase activity was normalized based on the total protein concentration determined for cells in each well. (G-G”) Measurements whole body titers of JHB3 (G), JH III (G’), and MF (G”) in <i>jhamt</i>^<i>2</i>/<i>jhamt</i>^<i>2</i><i>; jhamt-GAL4>UAS-hmgcr dsRNA</i> at 3hAIW after topical application of MF (0.5×10^-2 μmol per larva; dissolved in acetone) at 96h AEL (about 24 hours after treatments).</p