Cell cycle genes regulate vestigial and scalloped to ensure normal proliferation in the wing disc of Drosophila melanogaster.

In Drosophila, the Vestigial-Scalloped (VG-SD) dimeric transcription factor is required for wing cell identity and proliferation. Previous results have shown that VG-SD controls expression of the cell cycle positive regulator dE2F1 during wing development. Since wing disc growth is a homeostatic process, we investigated the possibility that genes involved in cell cycle progression regulate vg and sd expression in feedback loops. We focused our experiments on two major regulators of cell cycle progression: dE2F1 and the antagonist dacapo (dap). Our results reinforce the idea that VG/SD stoichiometry is critical for correct development and that an excess in SD over VG disrupts wing growth. We reveal that transcriptional activity of VG-SD and the VG/SD ratio are both modulated upon down-expression of cell cycle genes. We also detected a dap-induced sd upregulation that disrupts wing growth. Moreover, we observed a rescue of a vg hypomorphic mutant phenotype by dE2F1 that is concomitant with vg and sd induction. This regulation of the VG-SD activity by dE2F1 is dependent on the vg genetic background. Our results support the hypothesis that cell cycle genes fine-tune wing growth and cell proliferation, in part, through control of the VG/SD stoichiometry and activity. This points to a homeostatic feedback regulation between proliferation regulators and the VG-SD wing selector

Integration of differentiation signals during indirect flight muscle formation by a novel enhancer of Drosophila vestigial gene

AbstractThe gene vestigial (vg) plays a key role in indirect flight muscle (IFM) development. We show here that vg is controlled by the Notch anti-myogenic signaling pathway in myoblasts and is regulated by a novel 822 bp enhancer during IFM differentiation. Interestingly, this muscle enhancer is activated in developing fibers and in a small number of myoblasts before the fusion of myoblasts with the developing muscle fibers. Moreover, we show that this enhancer is activated by Drosophila Myocyte enhancing factor 2 (MEF2), Scalloped (SD) and VG but repressed by Twist, demonstrating a sensitivity to differentiation in vivo. In vitro experiments reveal that SD can directly bind this enhancer and MEF2 can physically interact with both SD and TWI. Cumulatively, our data reveal the interplay between different myogenic factors responsible for the expression of an enhancer activated during muscle differentiation

in vivo analysis of Drosophila deoxyribonucleoside kinase function in cell cycle, cell survival and anti-cancer drugs resistance.

in vitro studies have shown that Drosophila melanogaster has a highly efficient single deoxyribonucleoside kinase (dNK) multisubstrate enzyme. dNK is related to the mammalian Thymidine Kinase 2 (TK2) group involved in the nucleotide synthesis salvage pathway. To study the dNK function in vivo, we constructed transgenic Drosophila strains and impaired the nucleotide de novo synthesis pathway, using antifolates such as aminopterin. Our results show that dNK overexpression rescues both cell death and cell cycle arrest triggered by this anti-cancer drug, and confers global resistance on the fly. Moreover, we show that fly viability and growth depend on the exquisite ratio between dNK expression and its substrate thymidine (dT) in the medium, and that increased dT concentrations trigger apoptosis and a decrease in body mass when dNK is mis-expressed. Finally, dNK expression, unlike that of TK2, is cell cycle dependent and under the control of CyclinE and the dE2F1 transcription factor involved in the G1/S transition. dNK is therefore functionally more closely related to mammalian TK1 than to TK2. This strongly suggest that dNK plays a role in cell proliferation in physiological conditions

The insulin receptor is required for the development of the Drosophila peripheral nervous system.

International audienceThe Insulin Receptor (InR) in Drosophila presents features conserved in its mammalian counterparts. InR is required for growth; it is expressed in the central and embryonic nervous system and modulates the time of differentiation of the eye photoreceptor without altering cell fate. We show that the InR is required for the formation of the peripheral nervous system during larval development and more particularly for the formation of sensory organ precursors (SOPs) on the fly notum and scutellum. SOPs arise in the proneural cluster that expresses high levels of the proneural proteins Achaete (Ac) and Scute (Sc). The other cells will become epidermis due to lateral inhibition induced by the Notch (N) receptor signal that prevents its neighbors from adopting a neural fate. In addition, misexpression of the InR or of other components of the pathway (PTEN, Akt, FOXO) induces the development of an abnormal number of macrochaetes that are Drosophila mechanoreceptors. Our data suggest that InR regulates the neural genes ac, sc and sens. The FOXO transcription factor which is localized in the cytoplasm upon insulin uptake, displays strong genetic interaction with the InR and is involved in Ac regulation. The genetic interactions between the epidermal growth factor receptor (EGFR), Ras and InR/FOXO suggest that these proteins cooperate to induce neural gene expression. Moreover, InR/FOXO is probably involved in the lateral inhibition process, since genetic interactions with N are highly significant. These results show that the InR can alter cell fate, independently of its function in cell growth and proliferation

Induction by <i>InR</i> and <i>FOXO RNAi</i> of Ac and Sens.

<p>Confocal images of third instar wing discs stained with Ac (red) and Sens (green) antibodies. All disk are oriented A/P (antero/posterior) from left to right: <b>A</b>) <i>dpp>GFP</i> which serves as control, <b>B</b>) <i>dpp>GFP,InR</i> and <b>C</b>) <i>dpp>GFP,FOXO RNAi. </i><b>A' B' C'</b> are enlarged views of DC and SC clusters respectively. Note the ectopic expression of Ac (circle) and Sens (asterisk) cells along the A/P boundary in <b>B</b> and <b>C</b> identified by <i>dpp>GFP</i>.</p

Schematic outline of <i>Drosophila</i> InR/TOR signaling.

<p>Functional relationships between the InR (purple), TOR (blue), EGFR (green) and N (brown) pathways are indicated with black links. Arrows indicate activation, whereas bar-ended lines indicate inhibitory interactions. Broken lines indicate indirect interactions or interactions requiring further study.</p

Effects of InR on bristle formation.

<p><b>A</b>) Wild-type fly; anterior and posterior DC and SC macrochaetes, designated aDC, pDC and aSC, pSC are shown. <b>B and C</b>) <i>InR</i> null clones were generated crossing <i>y,w,Ubx-FLP;FRT82B</i> and <i>FRT82B, dInR^EX15</i>. Square area magnified showing lack of DC macrochaetes (arrow). In <b>D </b><i>sca>InR RNAi</i> notum. Some macrochaetes are missing from the scutellum. Overexpression of <i>Inr</i> with <i>sca-GAL4</i> (<b>E)</b>, <i>dpp-GAL4 </i><b>(F)</b> or <i>C253-GAL4</i> (<b>K</b>), led to extra macrochaetes and microchaetes. Note that most of the supplementary macrochaetes have a socket and shaft. In <b>I</b> overexpression of both <i>dilp2</i> and <i>InR</i> in <i>sca</i> strongly enhanced the <i>InR</i> overexpression phenotype. (<b>G</b> and <b>H</b>) <i>FOXO</i> and <i>InR</i> play opposite roles. In <b>G</b> lack of macrochaetes is observed on <i>sca>hFOXO^3a-TM</i> flies, and in <b>H</b>, it is a decrease of the <i>InR</i> overexpression phenotype (compared to <b>E)</b> in sca><i>Inr</i>,<i>hFOXO^3a-TM</i>. In <b>J </b><i>pnr>FOXO RNAi</i> fly, an increase in microchaetes and macrochaetes on the notum and scutellum are observed. Overexpression of <i>InR</i> (<b>K</b>), or <i>Ras^V12</i> (<b>L</b>), induces extra bristles, effect, stronger with both transgenes (<b>M</b>), supporting the hypothesis of genetic interaction between the two pathways. <i>sca>sc</i> generates extramacrochaetes <b>(N)</b>, a phenotype enhanced by overexpressing <i>InR </i><b>(O)</b>, and is decreased by <i>UAS-hFOXO^3a-TM</i><b>(P)</b>.</p

Relative quantification on Ac and Sens.

<p><b>A.</b> Immunofluorescence labeling on wing imaginal discs performed with antibodies against Ac and Sens on <i>sca>GFP</i> (control), <i>sca>InR, sca>hFOXO^3A-TM</i>, <i>sca>InR; hFOXO^3A-TM</i> and <i>sca>FOXO RNAi</i>, showed higher staining of Sens when <i>InR</i> or <i>FOXO RNAi</i> were overexpressed. The Table presents the number of cells positive for Ac and Sens for the four genotypes for the DC and SC clusters. Data represent mean ± SEM. <b>B.</b> To evaluate the differences in the level of Ac and Sens expression in each cell in the DC and SC clusters between the genotypes, a relative quantification on confocal acquired images was performed after immunofluorescence labeling conducted simultaneously in control (<i>sca>GFP</i>), and <i>sca>InR</i> wing discs of the same age. The figure presents the number of cells in each cluster containing >10⁵; >10⁶; >10⁷ intensity units/cell. *<i>p</i><0.05.</p

Genetic interactions of <i>InR</i> on macrochaete formation.

<p>The number of bristles (n) counted on female heminota (except in Table C on male heminota), at aDC, pDC, aSC and pSC positions. The number of macrochaetes for each position was determined and 6 classes were established: n<1, n = 1, n = 2, n = 3, n = 4 or n≥ 5. Experiments were performed at 25°C, except when otherwise stated. A minimum of 24 hemi-thoraces were counted and the results are expressed in the medium percentage of the number of macrochaetes for a given position. The Fisher Exact Test with the “R” programming language was used to calculate p-values on the 6 classes to compare the phenotypes between two classes. Only significant results are indicated, using the following code: blue letters for 0.05</p

<i>InR</i> activates Ac and Sens expression and accelerates the time of sensory organ development.

<p>Confocal sections of the SC cluster expressing Ac (red) and Sens (green) in the control <b>(A–C)</b> and in <i>sca>InR </i><b>(D–F).</b> Note that when <i>InR</i> is overexpressed the number of cells labeled is higher (<b>F</b>) and the signal is stronger (<b>D</b>). Proliferation was tested in <b>G, H</b> by labeling with anti-PH3 antibodies. In <i>tub-GAL80^ts</i> and <i>sca>GFP</i>, 2 SOPs are labeled in the SC cluster (<b>G</b>). In <i>tub-GAL80^ts, sca>InR,GFP</i> (<b>H, I</b>) 7 SOPs have emerged, but no mitotic activity is detected. Inside the cluster, cells are blocked in G2 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071857#pone.0071857-Usui1" target="_blank">[29]</a>. In <b>J, K</b> (detailed notum) <i>sca>InR</i> early L3 wing disc shows strong Ac (red) labeling in most proneuronal clusters as usually observed in wild-type disks. No Sens (green) is detected in the DC or SC clusters when it is already visible in SOP at the DV boundary and hinge (arrow). Note that Sens does not appear earlier in SC and DC clusters on an L3 wing disc when <i>InR</i> was overexpressed. Wing imaginal disc from middle third instar larvae stained with anti-Sens and anti-Pro antibodies which specifically mark pIIb cells. In <b>L </b><i>sca>InR</i> and <b>N </b><i>sca>FOXO RNAi</i>, pIIb cells were detected (asterisk). On the contrary in <b>M </b><i>sca>hFOXO3A-TM</i>, only one pIIb dividing cell is seen at the hinge. Activation of the InR pathway accelerates the time of development after SOPs were formed.</p