Paradigms and Our Shrinking Bioethics

Symposium: Emerging Paradigms in Bioethihc

Cryptocephal, the Drosophila melanogaster ATF4, Is a Specific Coactivator for Ecdysone Receptor Isoform B2

We thank Yoonseung Park (Kansas State University) and Michael Adams (UC Riverside) for the ETH-GeneSwitch line, and David Durica, Lauren Evans, and Dahong Chen (University of Oklahoma) and Nancy Thompson (Indiana University) for technical assistance.Author Summary Nuclear receptors are proteins that regulate gene expression in response to steroid and thyroid hormones and other small lipid-soluble signaling molecules. In many cases, nuclear receptor genes encode multiple variants (isoforms) that direct tissue- and stage-specific hormonal responses. The sequence differences among isoforms are often found at the protein N-terminus, which mediates hormone-independent interactions with unknown regulatory partners to control target gene expression. Here, we show that the fruit fly Cryptocephal (CRC) protein is a specific coactivator for one of three isoforms of the receptor for the insect molting steroid, ecdysone. Our findings reveal a mechanism for differential activation of gene expression in response to ecdysone during insect molting and metamorphosis, and contribute to our understanding of isoform-specific functions of nuclear hormone receptors.Yeshttp://www.plosgenetics.org/static/editorial#pee

Spatial control of Draper receptor signaling initiates apoptotic cell engulfment.

The engulfment of apoptotic cells is essential for tissue homeostasis and recovering from damage. Engulfment is mediated by receptors that recognize ligands exposed on apoptotic cells such as phosphatidylserine (PS). In this study, we convert Drosophila melanogaster S2 cells into proficient phagocytes by transfecting the Draper engulfment receptor and replacing apoptotic cells with PS-coated beads. Similar to the T cell receptor (TCR), PS-ligated Draper forms dynamic microclusters that recruit cytosolic effector proteins and exclude a bulky transmembrane phosphatase, consistent with a kinetic segregation-based triggering mechanism. However, in contrast with the TCR, localized signaling at Draper microclusters results in time-dependent depletion of actin filaments, which facilitates engulfment. The Draper-PS extracellular module can be replaced with FRB and FKBP, respectively, resulting in a rapamycin-inducible engulfment system that can be programmed toward defined targets. Collectively, our results reveal mechanistic similarities and differences between the receptors involved in apoptotic corpse clearance and mammalian immunity and demonstrate that engulfment can be reprogrammed toward nonnative targets

Genome-wide examination of the transcriptional response to ecdysteroids 20-hydroxyecdysone and ponasterone A in Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>The 20-hydroxyecdysone (20E) hierarchy of gene activation serves as an attractive model system for studying the mode of steroid hormone regulated gene expression and development. Many structural analogs of 20E exist in nature and among them the plant-derived ponasterone A (PoA) is the most potent. PoA has a higher affinity for the 20E nuclear receptor, composed of the ecysone receptor (EcR) and Ultraspiracle proteins, than 20E and a comparison of the genes regulated by these hormones has not been performed. Furthermore, in <it>Drosophila </it>different cell types elicit different morphological responses to 20E yet the cell type specificity of the 20E transcriptional response has not been examined on a genome-wide scale. We aim to characterize the transcriptional response to 20E and PoA in <it>Drosophila </it>Kc cells and to 20E in salivary glands and provide a robust comparison of genes involved in each response.</p> <p>Results</p> <p>Our genome-wide microarray analysis of Kc167 cells treated with 20E or PoA revealed that far more genes are regulated by PoA than by 20E (256 vs 148 respectively) and that there is very little overlap between the transcriptional responses to each hormone. Interestingly, genes induced by 20E relative to PoA are enriched in functions related to development. We also find that many genes regulated by 20E in Kc167 cells are not regulated by 20E in salivary glands of wandering 3^rdinstar larvae and we show that 20E-induced levels of <it>EcR </it>isoforms <it>EcR-RA, ER-RC</it>, and <it>EcR-RD/E </it>differ between Kc cells and salivary glands suggesting a possible cause for the observed differences in 20E-regulated gene transcription between the two cell types.</p> <p>Conclusions</p> <p>We report significant differences in the transcriptional responses of 20E and PoA, two steroid hormones that differ by only a single hydroxyl group. We also provide evidence that suggests that PoA induced death of non-adapted insects may be related to PoA regulating different set of genes when compared to 20E. In addition, we reveal large differences between Kc cells and salivary glands with regard to their genome-wide transcriptional response to 20E and show that the level of induction of certain EcR isoforms differ between Kc cells and salivary glands. We hypothesize that the differences in the transcriptional response may in part be due to differences in the EcR isoforms present in different cell types.</p

Generation of stable Drosophila cell lines using multicistronic vectors

Insect cell culture is becoming increasingly important for applications including recombinant protein production and cell-based screening with chemical or RNAi libraries. While stable mammalian cell lines expressing a protein of interest can be efficiently prepared using IRES-based vectors or viral-based approaches, options for stable insect cell lines are more limited. Here, we describe pAc5-STABLEs, new vectors for use in Drosophila cell culture to facilitate stable transformation. We show that viral-derived 2A-like (or "CHYSEL") peptides function in Drosophila cells and can mediate the multicistronic expression of two or three proteins of interest under control of the Actin5C constitutive promoter. The current vectors allow mCherry and/or GFP fusions to be generated for positive selection by G418 resistance in cells and should serve as a flexible platform for future applications

Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods

In Drosophila melanogaster, the iab-4/iab-8 locus encodes bi-directionally transcribed microRNAs that regulate the function of flanking Hox transcription factors. We show that bi-directional transcription, temporal and spatial expression patterns and Hox regulatory function of the iab-4/iab-8 locus are conserved between fly and the beetle Tribolium castaneum. Computational predictions suggest iab-4 and iab-8 microRNAs can target common sites, and cell-culture assays confirm that iab-4 and iab-8 function overlaps on Hox target sites in both fly and beetle. However, we observe key differences in the way Hox genes are targeted. For instance, abd-A transcripts are targeted only by iab-8 in Drosophila, whereas both iab-4 and iab-8 bind to Tribolium abd-A. Our evolutionary and functional characterization of a bi-directionally transcribed microRNA establishes the iab-4/iab-8 system as a model for understanding how multiple products from sense and antisense microRNAs target common sites

Fat Body Cells Are Motile and Actively Migrate to Wounds to Drive Repair and Prevent Infection

Adipocytes have many functions in various tissues beyond energy storage, including regulating metabolism, growth, and immunity. However, little is known about their role in wound healing. Here we use live imaging of fat body cells, the equivalent of vertebrate adipocytes in Drosophila, to investigate their potential behaviors and functions following skin wounding. We find that pupal fat body cells are not immotile, as previously presumed, but actively migrate to wounds using an unusual adhesion-independent, actomyosin-driven, peristaltic mode of motility. Once at the wound, fat body cells collaborate with hemocytes, Drosophila macrophages, to clear the wound of cell debris; they also tightly seal the epithelial wound gap and locally release antimicrobial peptides to fight wound infection. Thus, fat body cells are motile cells, enabling them to migrate to wounds to undertake several local functions needed to drive wound repair and prevent infections

A Role for Adenosine Deaminase in Drosophila Larval Development

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals