GFP expression driven by the fourth <i>Ndae1</i> intron.

<p>All embryos carry the pMC035 reporter construct and are stained by immunofluorescence with anti-GFP antibodies (green). (A) Magnification of a dorsal view of a stage 14 embryo double-stained with anti-Mef2 antibodies (red), labeling muscle cells including the cardiac tube. GFP is expressed in amnioserosa cells. (B) Lateral view of a stage 16 embryo double labeled with anti Mef-2 antibodies (red). (C) Magnification of a boxed portion of embryo in (B). The GFP-positive cells do not overlap the Mef-2-positive cells. (D) Lateral view of a stage 16 embryo double labeled with 22C10 antibodies (red). (E) Magnification of a boxed portion of embryo in (D). The GFP-positive cells lay below the 22C10-positive neurons.</p

<i>Ndae1</i> expression in early embryogenesis.

<p><i>Ndae1</i> expression patterns in embryos detected by <i>in situ</i> hybridization with TSA amplification followed by histochemistry (same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092956#pone-0092956-g002" target="_blank">figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092956#pone-0092956-g003" target="_blank">3</a>). (A) Ventral expression in a ventral view of a stage 5 embryo. (B) Ventral expression in a lateral view of a stage 5 embryo. (C) Lateral view of the posterior end of a stage 5 embryo. <i>Ndae1</i> is not expressed in pole cells (arrow). (D) Lateral view of the posterior end of a stage 6 embryo. No <i>Ndae1</i> transcripts are observed in pole cells (arrow). (E) Lateral view of a gastrulating stage 6 embryo showing strong expression in the head and weak in the tail. (F) Lateral view of a stage 10 embryo. <i>Ndae1</i> is likely expressed in yolk cells. (G) Stage 12 embryo with amnioserosa expression (arrows) and the beginning of anal pad expression (block arrow). (H) Lateral view of a stage 14 embryo showing expression in lateral cells (arrowhead) and in the anal pads (block arrow).</p

<i>Cis-</i>regulatory sequence analysis of <i>Ndae1</i>.

<p>(A) Genomic region of <i>Ndae1</i> (2L:7223328..7249343 in the R5.50 <i>Drosophila</i> genome sequence). Two of the nine splicing variants of <i>Ndae1</i> transcripts are represented. Genomic fragments cloned in reporter constructs are indicated by black or green lines corresponding to <i>lacZ</i> or <i>GFP</i> reporter constructs, respectively. Below each fragment there are representative stained embryos. Crossed lines indicate those fragments that do not drive any expression. Reporter constructs expression patterns are detected by immunohistochemistry. (B, C) β-Galactosidase (B) and lacZ mRNA (C) expression directed by pMC024 in rapidly changing cells from stage 10 until stage 15, likely being hemocytes. pMC028 (D) and pMC035 (E) drive expression in the CNS, in lateral cells (arrowheads) and in a dorsal line of amnioserosa cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092956#pone-0092956-g005" target="_blank">figure 5</a>). Fragments pMC025 (F), pMC027 (G) and pMC029 (H) drive the same expression pattern in the anal pads.</p

<i>Ndae1</i> expression in stage 15–16 embryos.

<p>(A) Lateral view of a stage 15 embryo showing expression in the CNS, in lateral cells and in the anal pads. (B) Ventral view of a stage 16 embryo expressing <i>Ndae1</i> in the CNS, in lateral cells and in the anal pads. (C) Magnification of a ventral view of a stage 16 embryo expressing <i>Ndae1</i> in the CNS and in lateral cells. (D) Magnification of the posterior end of a stage 16 embryo showing strong expression in the anal pads. Arrowheads: lateral cells. Block arrows: anal pads.</p

<i>Ndae1</i> expression in mutants of the DV pathway.

<p>(A) <i>dl¹/dl⁴</i> embryo. (B) <i>Tl^10B</i> embryo. (C) <i>twi¹</i> embryo. (D) <i>sna¹⁸</i> embryo. <i>Ndae1</i> is not expressed in <i>dl</i>, <i>twi</i> and <i>sna</i> mutants, while it is expressed ectopically in <i>Tl</i> mutants. (E) Overstained <i>dl¹/dl⁴</i> embryo in which residual anterior expression is observed. All embryos are at cellular blastoderm (stage 5).</p

Genetic analysis of <i>ACC</i> by tissue-targeted knockdown.

<p>Column 1 (Line) lists the tests referred to in the text. Column 2 (Genotype) summarizes the mutant (<i>ACC^B131</i>) or transgenic combinations used. Column 3 (Specificity) indicates the organ targeted by the <i>Gal4</i>-driver, or the use of the <i>ACC^B131</i> insertion mutant. Column 4 (Phenotype) summarizes the resulting viability (viable), lethality (†) or semi-lethality (½†). The developmental stage at which death occurs is indicated; L2/L3 refers to animals at the second/third larval stage transition. @4–5d indicates the day of lethality after egg deposit.</p

A VLCFA–dependent remote signal from the oenocytes controls lipid transfer within the spiracles.

<p>The default of VLCFA synthesis within the oenocytes provokes the failure to transfer lipids through the spiracular ducts from the spiracular gland to the spiracular opening. The gene products identified to be involved in this metabolic pathway within the oenocytes are indicated (oval forms surrounded in black). The signal running from the oenocytes to the spiracles is yet unidentified.</p

Oenocytes ACC signals to the tracheal system.

<p>(A) Dorsal view of a wild-type larva showing the two dorsal main trunks of the tracheal system, which extend from the anterior to the posterior spiracles (arrowheads). Air-filling phenotypes in the tracheal systems of <i>BO>grim</i> (B), <i>BO>ACC-RNAi</i> (C), <i>ACC^oeTD</i> mutant (D), <i>ACC^B131</i> mutant rescued by ubiquitous <i>UAS-ACC</i> (E) and <i>BO>KAR-RNAi</i> (F) animals. The portions of the dorsal main trunks filled with an aqueous solution are difficult to distinguish (arrows). The larvae have been selected early after the L2/L3 molting transition (prior to death in B,C,D,F). Larvae oriented anterior to the top. (G) Higher magnification of tracheal branches in early L3 <i>ACC^oeTD</i> larvae; a branch that originates from the air-filled part of the main trunk is entirely filled with air (arrow) whereas a branch that originates from the liquid-filled part of the main trunk is filled with liquid in its proximal end but not in the distal sub-branches (arrowhead). Scale bars: 100 µm. (H) Transcriptional expression of the <i>charybdis</i> and <i>scylla</i> hypoxic-responsive genes in control, cell-ablated, ACC or KAR deficient animals. Symbols used: (<i>WT</i>) control; (<i>grim</i>) <i>BO>grim</i>; (<i>ACC-Ri</i>) <i>BO>ACC-RNAi</i>; (<i>oeTD</i>) <i>ACC^oeTD</i>; (<i>Resc</i>) <i>ACC^B131;da-Gal4>UAS-ACC</i>; (<i>KAR-Ri</i>) <i>BO-Gal4>KAR-RNAi</i>. Genotypes behaved differently in their hypoxic response (ANOVA: genotype: F5,32 = 36.19 P<10⁻³; gene F1,32 = 1.24 P = 0.27; genotype×gene: F5,32 = 0.47 P = 0.795). Dunnet T test: *: P<0.05; **: P<0.01; ***: P<0.001.</p

Immunodetection of ACC.

<p>Direct GFP fluorescence (A,C,E,G), and staining to ACC (B,D,F,H,J) and nuclei (B,D). ACC expression was very high in the FB (B) and in the oenocytes (D) of L3 larvae, and thus detection was performed with very low laser intensity. The ACC signal was lower in the gut (F) and in the imaginal discs (H) of L3 larvae, and thus detection was performed with high laser intensity. Specificity of the ACC signal was monitored by generating homozygote <i>FRT</i>-<i>ACC^B131</i> clones (identified by the lack of GFP staining; arrowheads in A,C,E,G), which do not express the ACC protein in the corresponding pictures (arrowheads in B,D,F,H respectively). In the embryo, the strongest ACC staining was observed in the oenocytes (J) co-labeled to GFP driven by <i>BO-Gal4</i> (I). Scale bars: 20 µm.</p

Metabolic defects due to ACC disruption.

<p>(A, B) LD contents labeled by Nile red staining (B) in the FB of well fed animals. Note the drop of LD accumulation (B) in homozygote <i>FRT-ACC^B131</i> mutant clones marked by the absence of GFP (A); the dotted yellow line surrounds the GFP-negative clone (Scale bar: 20 µm). (C) Mean weight (mg) of 0–4 h prepupal female. (D–H) Concentration of TGs (D), proteins (E), trehalose (F), glucose (G) and glycogen (H) levels in prepupae. The values represent the concentration of each metabolite in µg per mg of 0–4 h prepupae. (I–N) Relative concentration (arbitrary units) of tetradecanoic (I), palmitic (J) palmitoleic (K), stearic (L), oleic (M) and linoleic (N) acid in 100 mg of 0–4 h prepupae. Color symbols (C–N): control (white bar) or expressing an <i>ACC-RNAi</i> in the FB (grey bar). T test: *: P<0.05; **: P<0.01; ***: P<0.001.</p