Dynamic Switch of Negative Feedback Regulation in Drosophila Akt–TOR Signaling

Akt represents a nodal point between the Insulin receptor and TOR signaling, and its activation by phosphorylation controls cell proliferation, cell size, and metabolism. The activity of Akt must be carefully balanced, as increased Akt signaling is frequently associated with cancer and as insufficient Akt signaling is linked to metabolic disease and diabetes mellitus. Using a genome-wide RNAi screen in Drosophila cells in culture, and in vivo analyses in the third instar wing imaginal disc, we studied the regulatory circuitries that define dAkt activation. We provide evidence that negative feedback regulation of dAkt occurs during normal Drosophila development in vivo. Whereas in cell culture dAkt is regulated by S6 Kinase (S6K)–dependent negative feedback, this feedback inhibition only plays a minor role in vivo. In contrast, dAkt activation under wild-type conditions is defined by feedback inhibition that depends on TOR Complex 1 (TORC1), but is S6K–independent. This feedback inhibition is switched from TORC1 to S6K only in the context of enhanced TORC1 activity, as triggered by mutations in tsc2. These results illustrate how the Akt–TOR pathway dynamically adapts the routing of negative feedback in response to the activity load of its signaling circuit in vivo

A genetic strategy to measure insulin signaling regulation and physiology in Drosophila.

Insulin regulation is a hallmark of health, and impaired insulin signaling promotes metabolic diseases like diabetes mellitus. However, current assays for measuring insulin signaling in all animals remain semi-quantitative and lack the sensitivity, tissue-specificity or temporal resolution needed to quantify in vivo physiological signaling dynamics. Insulin signal transduction is remarkably conserved across metazoans, including insulin-dependent phosphorylation and regulation of Akt/Protein kinase B. Here, we generated transgenic fruit flies permitting tissue-specific expression of an immunoepitope-labelled Akt (AktHF). We developed enzyme-linked immunosorption assays (ELISA) to quantify picomolar levels of phosphorylated (pAktHF) and total AktHF in single flies, revealing dynamic tissue-specific physiological regulation of pAktHF in response to fasting and re-feeding, exogenous insulin, or targeted genetic suppression of established insulin signaling regulators. Genetic screening revealed Pp1-87B as an unrecognized regulator of Akt and insulin signaling. Tools and concepts here provide opportunities to discover tissue-specific regulators of in vivo insulin signaling responses

The PDGF/VEGF Receptor Controls Blood Cell Survival in Drosophila

The Drosophila PDGF/VEGF receptor (PVR) has known functions in the guidance of cell migration. We now demonstrate that during embryonic hematopoiesis, PVR has a role in the control of antiapoptotic cell survival. In Pvr mutants, a large fraction of the embryonic hemocyte population undergoes apoptosis, and the remaining blood cells cannibalistically phagocytose their dying peers. Consequently, total hemocyte numbers drop dramatically during embryogenesis, and large aggregates of engorged macrophages carrying multiple apoptotic corpses form. Hemocyte-specific expression of the pan-caspase inhibitor p35 in Pvr mutants eliminates hemocyte aggregates and restores blood cell counts and morphology. Additional rescue experiments suggest involvement of the Ras pathway in PVR-mediated blood cell survival. In cell culture, we demonstrate that PVR directly controls survival of a hemocyte cell line. This function of PVR shows striking conservation with mammalian hematopoiesis and establishes Drosophila as a model to study hematopoietic cell survival in development and disease. Copyright © 2004 Cell Press.</p

A Genetic Strategy to Measure Circulating <i>Drosophila</i> Insulin Reveals Genes Regulating Insulin Production and Secretion

<div><p>Insulin is a major regulator of metabolism in metazoans, including the fruit fly <i>Drosophila melanogaster</i>. Genome-wide association studies (GWAS) suggest a genetic basis for reductions of both insulin sensitivity and insulin secretion, phenotypes commonly observed in humans with type 2 diabetes mellitus (T2DM). To identify molecular functions of genes linked to T2DM risk, we developed a genetic tool to measure insulin-like peptide 2 (<i>Ilp2</i>) levels in <i>Drosophila</i>, a model organism with superb experimental genetics. Our system permitted sensitive quantification of circulating Ilp2, including measures of Ilp2 dynamics during fasting and re-feeding, and demonstration of adaptive Ilp2 secretion in response to insulin receptor haploinsufficiency. Tissue specific dissection of this reduced insulin signaling phenotype revealed a critical role for insulin signaling in specific peripheral tissues. Knockdown of the <i>Drosophila</i> orthologues of human T2DM risk genes, including <i>GLIS3</i> and <i>BCL11A</i>, revealed roles of these <i>Drosophila</i> genes in Ilp2 production or secretion. Discovery of <i>Drosophila</i> mechanisms and regulators controlling <i>in vivo</i> insulin dynamics should accelerate functional dissection of diabetes genetics.</p></div

IPC-specific regulators of circulating Ilp2HF.

<p>(A) Circulating hemolymph Ilp2HF levels in heterozygous <i>Ilp2¹ gd2HF</i> flies expressing control <i>tdTomato</i> or <i>Kir2.1</i> by <i>Ilp2-GeneSwitch (Ilp2GS)</i> for 2 days with or without Mifepristone feeding. (B) Quantification of IPC cell number in flies expressing control <i>tdTomato</i> or <i>Kir2.1</i> by <i>Ilp2-GeneSwitch (Ilp2GS)</i> for 2 days with Mifepristone feeding. IPCs were marked by <i>dilp215-1-HStinger</i>. (C) <i>Ilp2</i> mRNA levels in heterozygous <i>Ilp2¹ gd2HF</i> flies with IPC-specific RNAi knockdown of <i>CG9650</i>, <i>Glut1</i>, <i>lmd</i>, and <i>Ilp2</i> genes or control mCherry RNAi. (D) Total Ilp2HF protein content in heterozygous <i>Ilp2¹ gd2HF</i> flies with IPC-specific RNAi knockdown of <i>CG9650</i>, <i>Glut1</i>, <i>lmd</i>, and <i>Ilp2</i> genes or control mCherry RNAi. (E) Circulating Ilp2HF concentration (pM) or content per fly (pg) in heterozygous <i>Ilp2¹ gd2HF</i> flies with IPC-specific RNAi knockdown of <i>CG9650</i>, <i>Glut1</i>, <i>lmd</i>, and <i>Ilp2</i> genes or control mCherry RNAi. In all figures, center values are averages, error bars represent the standard deviation, and two-tailed <i>t</i>-tests were used to generate <i>p</i> values. * indicates <i>p</i><0.05, ** <i>p</i><0.01, and *** <i>p</i><0.001. N.S. indicates statistically not significant.</p

Enhanced insulin secretion from impaired peripheral insulin signaling in <i>Drosophila</i>.

<p>(A) Circulating trehalose levels in <i>InR</i> or <i>Akt1</i> heterozygous mutants and sibling wild type control flies. (B) Circulating Ilp2HF levels in <i>InR</i> or <i>Akt1</i> heterozygous mutants and sibling wild type control flies. (C) Total Ilp2HF amounts in <i>InR</i> heterozygous mutants and sibling wild type control flies. (D) Representative image of Ilp2 immunofluorescence in IPCs of <i>InR</i> heterozygous mutants and sibling wild type control flies, and average mean signal intensity quantified from summed z-projections (n = 9). Scale bar is 10 µm. (E) Circulating Ilp2HF levels in flies with tissue-specific RNAi knockdown of <i>InR</i>. <i>InR</i> was knocked down in IPCs (<i>Ilp2</i>>), muscle (<i>Mef</i>2>), or adult fat body (<i>Lk6></i> and <i>ppl</i>>) tissues. (F) Total Ilp2HF content in flies with adult fat body specific RNAi knockdown of <i>InR</i> using <i>ppl-GAL4</i>. In all figures, center values are averages, error bars represent the standard deviation, and two-tailed <i>t</i>-tests were used to generate <i>p</i> values. * indicates <i>p</i><0.05, ** <i>p</i><0.01, and *** <i>p</i><0.001. N.S. indicates statistically not significant.</p

Epitope-tagged Ilp2 rescues insulin deficiency phenotypes.

<p>(A) Prepro-Ilp2 peptide sequence and locations of HA (blue) and FLAG (purple) epitopes inserted in Ilp2HF sequence. The sequences in black represent the putative mature Ilp2. Gray bars indicate conserved cysteine bonds in insulin-like peptides. (B) 2.4 kilobase pair genomic fragment (black bar) containing <i>Ilp2</i> gene and its regulatory sequence. (C) Quantification of wing length in flies lacking <i>Ilp2</i>, <i>Ilp3</i> and <i>Ilp5</i> genes (<i>Ilp2–3, 5</i>) with or without <i>gd2</i>, <i>gd2HF</i>, and <i>gd2HF.C119Y</i> genomic rescue fragments, as indicated. (D) Measurement of hemolymph trehalose concentration in insulin deficient flies with or without <i>gd2</i> or <i>gd2HF</i>, and <i>gd2HF.C119Y</i>. (E) Expression of <i>Ilp2HF</i> in <i>Ilp2¹ gd2HF</i> adult insulin producing neurons (arrow) detected by anti-Ilp2, anti-FLAG, and anti-HA antibodies. In all figures, center values are averages, error bars represent the standard deviation, and two-tailed <i>t</i>-tests were used to generate <i>p</i> values. * indicates <i>p</i><0.05, ** <i>p</i><0.01, and *** <i>p</i><0.001. N.S. indicates statistically not significant.</p

A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research

Novel binary gene expression tools like the LexA-LexAop system could powerfully enhance studies of metabolism, development, and neurobiology in Drosophila. However, specific LexA drivers for neuroendocrine cells and many other developmentally relevant systems remain limited. In a unique high school biology course, we generated a LexA-based enhancer trap collection by transposon mobilization. The initial collection provides a source of novel LexA-based elements that permit targeted gene expression in the corpora cardiaca, cells central for metabolic homeostasis, and other neuroendocrine cell types. The collection further contains specific LexA drivers for stem cells and other enteric cells in the gut, and other developmentally relevant tissue types. We provide detailed analysis of nearly 100 new LexA lines, including molecular mapping of insertions, description of enhancer-driven reporter expression in larval tissues, and adult neuroendocrine cells, comparison with established enhancer trap collections and tissue specific RNAseq. Generation of this open-resource LexA collection facilitates neuroendocrine and developmental biology investigations, and shows how empowering secondary school science can achieve research and educational goals