<i>Elo</i> genes control wing cell identity.

<p>(A): Wing from control <i>w¹¹¹⁸</i> fly (L1-L5: longitudinal veins; ACV and PCV: anterior and posterior cross-veins). (B, C): Wings from <i>+/EloB^EP3132</i> and <i>EloB^EP3132/Df(3R)BSC518</i> flies exhibit truncated L5. (D): Wings from <i>+/sd::Gal4</i> flies have a very faint ectopic vein phenotype and no margin phenotype. (E, F): Wings from flies over-expressing <i>EloA</i> exhibit ectopic vein and margin phenotypes. (G, H, I): <i>EloC^SH1520</i> and <i>EloC^SH1299</i> loss-of-function alleles diminish expressivity of the ectopic vein phenotype induced by the <i>bs^EY23316</i> loss-of-function allele. Strong phenotype: ectopic veins everywhere in the wing (shown in G). Mild phenotype: ectopic veins under the posterior cross-vein only (shown in H).</p

Down-regulation of <i>EloC</i> by RNA interference impairs both cell proliferation and cell differentiation in wing imaginal discs.

<p>(A): Clones expressing the <i>ValEloC</i> transgene (GFP⁺ cells, shown by white arrows) are located at the periphery of the disc and are very small compared to control clones. (B, C, D): Wings from pharates in which <i>ValEloC</i> is driven by <i>nub::Gal4</i> (C) or <i>rn::Gal4</i> (D), both expressed in the wing pouch <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077592#pone.0077592-StPierre1" target="_blank">[66]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077592#pone.0077592-Ng1" target="_blank">[67]</a> are small compared to wild-type pharate wings (B) and exhibit severe wing blade defects. By contrast, longitudinal veins (shown by asterisks) are formed in the proximal-most part of the wing blade where <i>nub::Gal4</i> and <i>rn::Gal4</i> are not expressed.</p

<i>corto</i> and several TrxG genes control wing cell identity.

<p>(A, B): Ectopic vein phenotypes induced by the <i>corto⁰⁷¹²⁸</i> loss-of-function allele (A) or by <i>corto⁴²⁰</i> loss-of-function clones (B). (C): <i>corto⁴²⁰</i> homozygous clones (GFP^- cells) in wing imaginal discs. (D, E, F): Ectopic vein phenotypes induced by <i>mor</i>, <i>kis</i> or <i>trx</i> loss-of-function alleles. In A, B, D, E, F, asterisks mark ectopic veins.</p

Decreasing <i>EloC</i> expression suppresses ectopic veins induced by <i>blistered</i> loss-of-function.

<p>The upper allele was brought by the mother. The number of <i>EloC</i>/<i>bs^EY23316</i> females with ectopic veins was compared to the number of <i>+/bs^EY23316</i> females with ectopic veins (z-test, ^a p<0.001). The mild ectopic vein phenotype corresponds to presence of ectopic veins distal to the posterior cross-vein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077592#pone-0077592-g005" target="_blank">Figure 5H</a>), whereas the strong ectopic vein phenotype corresponds to presence of ectopic veins everywhere in the wing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077592#pone-0077592-g005" target="_blank">Figure 5G</a>).</p

EloC and Corto bind <i>rho</i> in wing imaginal discs.

<p>(A, B): Wing phenotypes induced by <i>rho</i> loss-of-function (A) or over-expression (B). Asterisks mark truncated L5 (in A) or ectopic veins (in B). (C): Schematic structure of <i>rho</i> with exons represented by boxes and introns by lines. Black arrows show primer pairs used for ChIP experiments. (D): Binding of Corto chromodomain (FH-CortoCD) and EloC (FH-EloC) on <i>rho</i>. For each genotype, the mean of two independent experiments is shown. Error bars correspond to standard deviations.</p

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<p>The upper allele was brought by the mother. The number of females with ectopic veins among flies transheterozygous for <i>Elo</i> and <i>corto</i> mutations was compared to the number of females with ectopic veins among flies with a <i>corto</i> mutation only (z-test, ^a p<0.001).</p

Deregulation of <i>EloA, EloB</i> or <i>EloC</i> expression using <i>P</i>-element insertion lines.

<p>(A): Structure of <i>EloA</i>, <i>EloB</i> and <i>EloC</i> genes showing localization of the <i>P</i>-elements used in this study. Exons are represented by boxes, and introns by lines. Black arrowheads show positions of primer pairs used to quantify <i>Elo</i> gene expression. (B): Quantification of <i>Elo</i> gene expression in <i>EloA^G4930</i>, <i>EloB^EP3132</i>, <i>EloC^SH1520</i> or <i>EloC^SH1299</i> homozygous or heterozygous larvae. (C): Quantification of <i>Elo</i> gene expression in <i>da::Gal4>>EloA^G4930</i> or <i>da::Gal4>>EloB^EP3132</i> larvae. (D): Quantification of <i>EloC</i> expression in <i>da::Gal4</i>>><i>ValEloC</i> embryos. Relative <i>Elo</i> expression levels were obtained by normalization to <i>Rp49</i> (black bars, B to D), <i>RpL12</i> (grey bars, B, C) or <i>eIF-2α</i> (grey bars, D).</p

Functional subnetwork identified in wing imaginal discs expressing <i>CycG</i>^<i>ΔP</i>.

<p>Schematic representation of a sub-network of 222 genes centred on Cyclin G (CycG_subnetwork.xmml) and identified using JactiveModules (Z score 48.53). In this sub-network, 65 genes were up-regulated in <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i> <i>vs da-Gal4/+</i> wing imaginal discs (green gradient), 124 genes were down-regulated (red gradient), and 33 genes were not significantly deregulated (grey). Genes bound by Cyclin G are circled in blue. Transcription factor genes are represented by squares. Genes were clustered depending on their function. Black edges correspond to interactions discovered in the present study. Grey edges correspond to interactions described in the literature and imported into the WID network using DroID.</p

The ETP interacting domain limits <i>CycG</i>-induced FA.

<p><b>A–</b>Map of the 566 amino-acid Cyclin G protein showing the ETP interacting and PEST domains. <b>B–</b>Wing centroid size FA (FA10) of females <i>da-Gal4</i>/+ (+), <i>+/UAS-CycG</i>^<i>FL</i><i>; da-Gal4/+</i>, (<i>CycG</i>^<i>FL</i>) and <i>+/UAS-CycG</i>^<i>ΔE</i>; <i>da-Gal4</i>, (<i>CycG</i>^<i>ΔE</i>). <b>C–</b>Wing centroid size FA (FA10) of females <i>da-Gal4</i>/+ (+), <i>+/ UAS-CycG</i>^<i>ΔP</i><i>; da-Gal4/+</i> (<i>CycG</i>^<i>ΔP</i>) and <i>+/UAS-CycG</i>^{<i>ΔEΔP</i>}<i>; da-Gal4/+</i> (<i>CycG</i>^{<i>ΔEΔP</i>}). (F-tests, *** p-value<0.001, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s005" target="_blank">S3 Table</a>). Source data are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s006" target="_blank">S4 Table</a>.</p

Genes deregulated in wing imaginal discs expressing <i>CycG</i>^<i>ΔP</i>.

<p><b>A–</b>RT-qPCR analysis of endogenous <i>CycG</i> expression in <i>da-Gal4</i>,<i>UAS-CycG</i>^<i>ΔP</i><i>/+</i> and <i>da-Gal4/+</i> wing imaginal discs. Expression of <i>CycG</i> was normalized on the geometric mean of <i>Lam</i> and <i>rin</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s010" target="_blank">S8 Table</a>). t-tests, ** p-value<0.01. Error bars correspond to standard deviations. <b>B–</b>Ontology of up-regulated and down-regulated genes in <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i><i>/+ vs da-Gal4/+</i> wing imaginal discs. Gene ontology analysis was performed with DAVID (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s011" target="_blank">S9 Table</a>). <b>C–</b>RT-qPCR analysis of <i>RPL15</i>, <i>RPL7</i> and <i>Rack1</i> expression in <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i><i>/+</i> and <i>da-Gal4/+</i> wing imaginal discs. Expression of <i>RPL15</i>, <i>RPL7</i> and <i>Rack1</i> were normalized on the geometric mean of <i>Lam</i> and <i>rin</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s012" target="_blank">S10 Table</a>). t-tests, ** p-value<0.01. Error bars correspond to standard deviations. t-tests, ** p-value<0.01; *** p-value<0.001.</p