Ectopic expression in commonly used transgenic Drosophila GAL4 driver lines.

Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previously discovered unexpected expression of the commonly used dilp2-GAL4 line in tracheal tissue which significantly impacted growth phenotypes. We realized that few GAL4 lines have been thoroughly characterized, particularly when considering transient activity that may have significant impact on phenotypic readouts. Here, we characterized a further subset of 12 reportedly tissue-specific GAL4 lines commonly used in genetic studies of development, growth, endocrine regulation, and metabolism. Ten out of 12 GAL4 lines exhibited ectopic activity in other larval tissues, with seven being active in the larval trachea. Since this ectopic activity may result in phenotypes that do not depend on the manipulation in the intended target tissue, it is recommended to carefully analyze the outcome while taking this aspect into consideration.

Genome-wide analysis identifies Homothorax and Extradenticle as regulators of insulin in Drosophila Insulin-Producing cells.

Drosophila Insulin-Producing Cells (IPCs) are the main production site of the Drosophila Insulin-like peptides or dilps which have key roles in regulating growth, development, reproduction, lifespan and metabolism. To better understand the signalling pathways and transcriptional networks that are active in the IPCs we queried publicly available transcriptome data of over 180 highly inbred fly lines for dilp expression and used dilp expression as the input for a Genome-wide association study (GWAS). This resulted in the identification of variants in 125 genes that were associated with variation in dilp expression. The function of 57 of these genes in the IPCs was tested using an RNAi-based approach. We found that IPC-specific depletion of most genes resulted in differences in expression of one or more of the dilps. We then elaborated further on one of the candidate genes with the strongest effect on dilp expression, Homothorax, a transcription factor known for its role in eye development. We found that Homothorax and its binding partner Extradenticle are involved in regulating dilp2, -3 and -5 expression and that genetic depletion of both TFs shows phenotypes associated with reduced insulin signalling. Furthermore, we provide evidence that other transcription factors involved in eye development are also functional in the IPCs. In conclusion, we showed that this expression level-based GWAS approach identified genetic regulators implicated in IPC function and dilp expression.

Glial and Neuronal Neuroglian, Semaphorin-1a and Plexin A Regulate Morphological and Functional Differentiation of Drosophila Insulin-Producing Cells.

The insulin-producing cells (IPCs), a group of 14 neurons in the Drosophila brain, regulate numerous processes, including energy homeostasis, lifespan, stress response, fecundity, and various behaviors, such as foraging and sleep. Despite their importance, little is known about the development and the factors that regulate morphological and functional differentiation of IPCs. In this study, we describe the use of a new transgenic reporter to characterize the role of the Drosophila L1-CAM homolog Neuroglian (Nrg), and the transmembrane Semaphorin-1a (Sema-1a) and its receptor Plexin A (PlexA) in the differentiation of the insulin-producing neurons. Loss of Nrg results in defasciculation and abnormal neurite branching, including ectopic neurites in the IPC neurons. Cell-type specific RNAi knockdown experiments reveal that Nrg, Sema-1a and PlexA are required in IPCs and glia to control normal morphological differentiation of IPCs albeit with a stronger contribution of Nrg and Sema-1a in glia and of PlexA in the IPCs. These observations provide new insights into the development of the IPC neurons and identify a novel role for Sema-1a in glia. In addition, we show that Nrg, Sema-1a and PlexA in glia and IPCs not only regulate morphological but also functional differentiation of the IPCs and that the functional deficits are likely independent of the morphological phenotypes. The requirements of nrg, Sema-1a, and PlexA in IPC development and the expression of their vertebrate counterparts in the hypothalamic-pituitary axis, suggest that these functions may be evolutionarily conserved in the establishment of vertebrate endocrine systems.

Growth control through regulation of insulin signalling by nutrition-activated steroid hormone in Drosophila.

Growth and maturation are coordinated processes in all animals. Integration of internal cues, such as signalling pathways, with external cues, such as nutritional status, is paramount for an orderly progression of development and growth. In Drosophila, this involves insulin and steroid signalling, but the underlying mechanisms and their coordination are incompletely understood. We show that bioactive 20-hydroxyecdysone production by the enzyme Shade in the fat body is a nutrient-dependent process. We demonstrate that under fed conditions, Shade plays a role in growth control. We identify the trachea and the insulin-producing cells in the brain as direct targets through which 20-hydroxyecdysone regulates insulin signalling. The identification of trachea-dependent regulation of insulin signalling exposes an important variable that may have been overlooked in other studies focusing on insulin signalling in Drosophila Our findings provide a potentially conserved, novel mechanism by which nutrition can modulate steroid hormone bioactivation, reveal an important caveat of a commonly used transgenic tool to study insulin-producing cell function, and yield further insights into how steroid and insulin signalling are coordinated during development to regulate growth and developmental timing.

Bacteriocyte cell death in the pea aphid/Buchnera symbiotic system.

Symbiotic associations play a pivotal role in multicellular life by facilitating acquisition of new traits and expanding the ecological capabilities of organisms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells (bacteriocytes) or organs (bacteriomes) have evolved for harboring beneficial microbial partners. The processes regulating the cellular life cycle of these endosymbiont-bearing cells, such as the cell-death mechanisms controlling their fate and elimination in response to host physiology, are fundamental questions in the biology of symbiosis. Here we report the discovery of a cell-death process involved in the degeneration of bacteriocytes in the hemipteran insect Acyrthosiphon pisum This process is activated progressively throughout aphid adulthood and exhibits morphological features distinct from known cell-death pathways. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of nonautophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, activation of reactive oxygen species, and Buchnera endosymbiont degradation by the lysosomal system. We showed that this multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization spread throughout the entire cytoplasm and surrounding Buchnera aphidicola endosymbionts. Our findings provide insights into the cellular and molecular processes that coordinate eukaryotic host and endosymbiont homeostasis and death in a symbiotic system and shed light on previously unknown aspects of bacteriocyte biological functioning.