Table of Contents

Volume 4, Issue 4

  October/November/December 2010

  Pages 267 - 364

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EDITOR'S PREVIEW

Open Access Article

In this issue of Fly...

Barbara P. Rattner
Pages 267 - 269
http://dx.doi.org/10.4161/fly.4.4.13341
PDF




EXTRA VIEWS

Open Access Article

Gene loss on Y chromosomes: a consequence of purifying selection?

Vera B. Kaiser
Pages 270 - 272
http://dx.doi.org/10.4161/fly.4.4.12669
Abstract | PDF

Recombination restriction between evolving sex chromosomes leads to the degeneration of the chromosome that is present only in the heterogametic sex (the Y chromosome in XY species). The evolutionary forces driving Y chromosome degeneration, however, are still under debate and include positive and negative selection models. In a recent study, we showed that the rate of accumulation of loss-of-function mutations on the neo-Y chromosome of Drosophila miranda is compatible with the process of Muller's ratchet, the stochastic loss of the best mutational class of individuals from a small asexual population. Purifying selection at amino acid sites can accelerate the ratchet, and the speed of degeneration depends on the number of genes still present on the evolving Y chromosome. Our study shows that Y chromosome degeneration does not require the action of selective sweeps at linked sites, and can take place under realistic parameters of purifying selection only.

Open Access Article

Establishing positional information through gradient dynamics: a lesson from the Hedgehog signaling pathway

Marcos Nahmad and Angela Stathopoulos
Pages 273 - 277
http://dx.doi.org/10.4161/fly.4.4.12677
Abstract | PDF

A longstanding question in developmental biology is how morphogen gradients establish positional information during development. Although the existence of gradients and their role in developmental patterning is no longer in doubt, the ability of cells to respond to different morphogen concentrations has been controversial. In the Drosophila wing disc, Hedgehog (Hh) forms a concentration gradient along the anterior-posterior axis and establishes at least three different gene expression patterns. In a recent study, we challenged the prevailing idea that Hh establishes positional information in a dose-dependent manner and proposed a model in which dynamics of the gradient, resulting from the Hh gene network architecture, determines pattern formation in the wing disc. In this Extra View, we discuss further the methodology used in this study, highlight differences between this and other models of developmental patterning, and also present some questions that remain to be answered in this system.

Open Access Article

Why Cyclin Y? A highly conserved cyclin with essential functions

Dongmei Liu, Stephen Guest and Russell L. Finley
Pages 278 - 282
http://dx.doi.org/10.4161/fly.4.4.12881
Abstract | PDF

Cyclin Y is one of the most highly conserved members of the cyclin superfamily of proteins, which are famous for their crucial roles in regulating the cell cycle and transcription. Despite this high degree of conservation, very little was known about Cyclin Y function prior to a handful of studies published in this past year. Cyclins typically function by activating cyclin dependent kinases (Cdks) and one insight has come from the identification of a Cdk that is activated by Cyclin Y. Yeast two-hybrid data first linked Cyclin Y with Cdk14, known as Eip63E in Drosophila or PFTAIRE1 in vertebrates. In Drosophila, both Cyclin Y and Eip63E are essential at many stages of development, from embryogenesis to metamorphosis and null mutants show a similar spectrum of developmental defects. In cultured cells, Cyclin Y and Eip63E were shown to phosphorylate the Wg/Wnt co-receptor Arrow/LRP6 in a ligand-independent manor. Eip63E is recruited to LRP6 at the plasma membrane by interacting with Cyclin Y, which is tethered to the membrane through an N-terminal myristoylation. Cyclin Y-dependent LRP6 phosphorylation appears to prime the receptor for subsequent ligand-dependent phosphorylation and activation of the canonical Wnt signaling pathway. Interestingly, Wnt receptor phosphorylation and signaling is maximal in G2/M when Cyclin Y is at its highest levels, suggesting that Cyclin Y may serve to entrain Wnt signaling to the cell cycle. Given the wide range of roles for Wnt signaling during development, these studies may help explain why Cyclin Y is required at several developmental stages and in turn why these proteins are so well conserved in metazoans.

Open Access Article

Time to taste: Circadian clock function in the Drosophila gustatory system

Abhishek Chatterjee and Paul E. Hardin
Pages 283 - 287
http://dx.doi.org/10.4161/fly.4.4.13010
Abstract | PDF

Circadian clocks keep time in the digestive, circulatory, reproductive, excretory, and nervous systems even in absence of external cues. Central oscillators in the brain control locomotor activity of organisms ranging from fruit flies to man, but the functions of the clocks in peripheral nervous system are not well understood. The presence of autonomous peripheral oscillators in the major taste organ of Drosophila, the proboscis, prompted us to test whether gustatory responses are under control of the circadian clock. We find that synchronous rhythms in physiological and behavioral responses to attractive and aversive tastants are driven by oscillators in gustatory receptor neurons (GRNs); primary sensory neurons that carry taste information from the proboscis to the brain. During the middle of the night, high levels of G protein-coupled receptor kinase 2 (GPRK2) in the GRNs suppresses tastant-evoked responses. Flies with disrupted gustatory clocks are hyperphagic and hyperactive, recapitulating behaviors typically seen under the stress of starvation. Temporal plasticity in innate behaviors should offer adaptive advantages to flies. In this Extra View article we discuss how oscillators inside GRNs regulate responsiveness to tastants, and influence feeding, metabolism and general activity.

Open Access Article

Lgl/aPKC and Crb regulate the Salvador/Warts/Hippo pathway

Linda M Parsons, Nicola A Grzeschik, Melinda Allott and Helena Richardson
Pages 288 - 293
http://dx.doi.org/10.4161/fly.4.4.13116
Abstract | PDF

A key goal of developmental biology is to understand the mechanisms that coordinate organ growth. It has long been recognized that the genes that control apico-basal cell polarity also regulate tissue growth. How loss of cell polarity contributes to tissue overgrowth has been the subject of much speculation. Do loss-of-function mutations in cell polarity regulators result in secondary effects that globally deregulate cell proliferation, or do these genes specifically control growth pathways? Three recent papers have shown that the apico-basal polarity determinants Lgl/aPKC and Crb regulate tissue growth independently of their roles in cell polarity and coordinately regulate cell proliferation and cell death via the Salvador/Warts/Hippo (SWH) pathway. Lgl/aPKC are required for the correct localization of Hippo (Hpo)/Ras associated factor (RASSF), whilst Crb regulates the levels and localization of Expanded (Ex), indicating that cell polarity determinants modify SWH pathway activity by distinct mechanisms. Here, we review the key data that support these conclusions, highlight remaining questions and speculate on the underlying mechanisms by which the cell polarity complexes interact with the SWH pathway. Understanding the interactions between cell polarity regulators and the SWH pathway will improve our knowledge of how epithelial organization and tissue growth are coordinated during development and perturbed in disease states such as cancer.

Open Access Article

discs large in the Drosophila testis: an old player on a new task

Fani Papagiannouli and Bernard M. Mechler
Pages 294 - 298
http://dx.doi.org/10.4161/fly.4.4.13149
Abstract | PDF

Gamete development requires a coordinated soma-germ line interaction that ensures renewal and differentiation of germline and somatic stem cells. The physical contact between the germline and somatic cell populations is crucial because it allows the exchange of diffusible signals among them. The tumor suppressor gene discs large (dlg) encodes a septate junction protein with functions in epithelial cell polarity, asymmetric neuroblast division, and formation of neuromuscular junctions. Our recent work reveals a new role of dlg in the Drosophila testis, as mutations in dlg lead to testis defects and cell death. Dlg is required throughout spermatogenesis in the somatic lineage and its localization changes from a uniform distribution along the plasma membrane of somatic cells in the testis apex, to a restricted localization on the distally located somatic cell in growing cysts. The extensive defects in dlg testis underline the importance of the somatic cells in the establishment and maintenance of the male stem cell niche and somatic cell differentiation. Here, we discuss our latest findings on the role of dlg in the Drosophila testis, supporting the view that junction proteins are dynamic structures, which can provide guiding cues to recruit scaffold proteins or other signaling molecules.

Open Access Article

Positive regulation of euchromatic gene expression by HP1a

Lucia Piacentini and Pimpinelli Sergio
Pages 299 - 301
http://dx.doi.org/10.4161/fly.4.4.13261
Abstract | PDF

HP1 is a conserved prototype protein that plays an essential role in heterochromatin formation and epigenetic gene silencing through its interaction with histone methyltransferase enzymes (HMTases) and the histone H3 at lysine 9 (H3-MeK9). HP1 is also involved in telomere capping and, more surprisingly, in positive regulation of gene expression. Recently, a wide expression analysis, using a RIP-chip assays (RNA-immunoprecipitation on microarrays), has shown that HP1 associates with the transcripts of more than one hundred euchromatic genes and interacts with the heterogeneous nuclear ribonucleoproteins (hnRNPs) that are known to be involved in RNA processing. By these results, HP1 seems to be part of a complex that stabilizes RNA transcripts. Though previously unsuspected, it was also found that HP1-interacting hnRNPs have a functional role in heterochromatin formation. These proteins bind heterochromatin and are dominant suppressors of position effect variegation. Taken together, the results in the paper by Piacentini et al., open a window on a possible new conceptual landscape in which similar epigenetic mechanisms could have a significant role, both in the metabolism of RNA transcripts and in heterochromatin formation, producing opposite functional effects. These data seem to establish a functional link between euchromatin and heterochromatin.

Open Access Article

Vesicular neurotransmitter transporter trafficking in vivo: Moving from cells to flies

Anna Grygoruk, Hao Fei, Richard W. Daniels, Brian C. Miller, Audrey Chen, Aaron DiAntonio and David E. Krantz
Pages 302 - 305
http://dx.doi.org/10.4161/fly.4.4.13305
Abstract | PDF

During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific vesicular transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study vesicular transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT may undergo different modes of trafficking at the synapse. Further in vivo studies of DVMAT trafficking mutants will allow us to determine how changes in the localization of vesicular transporters affect the nervous system as a whole and complex behaviors mediated by aminergic circuits.




RESEARCH PAPERS

Open Access Article

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

Alina Garbuzov and Marc Tatar
Pages 306 - 311
http://dx.doi.org/10.4161/fly.4.4.13008
Abstract | PDF

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode Caenorhabditis elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila melanogaster, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. Here we used Drosophila S2 cells to analyze the effects of 20-HE on D. melanogaster microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNAi knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNA, we show that nanomolar concentrations of 20-HE primes cells to subsequently express microRNA when exposed to micromolar levels of 20-HE. We then explore the role microRNA plays in the established relationship between 20-HE and the induction of innate immunity. We show that the 3'UTR of the antimicrobial peptide diptericin has a let-7 binding site and that let-7 represses translation from this site. We conclude that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNA control of antimicrobial peptide translation.

Open Access Article

Effects of dietary folic acid level and symbiotic folate production on fitness and development in the fruit fly Drosophila melanogaster

Sydella Blatch, Kyle W. Meyer and Jon F. Harrison
Pages 312 - 319
http://dx.doi.org/10.4161/fly.4.4.13258
Abstract | PDF

Folic acid is a vitamin for probably all animals. When converted to folate forms, it is used in DNA synthesis and amino acid metabolism. Literature suggests insects must consume folates, folates do not affect others, is a toxin for some, and that a few insects synthesize it. It has been reported that Drosophila melanogaster does not consistently need dietary folate because it can synthesize it. This seems unlikely since animals generally lack this ability. More likely, folates thought to have been made by the fly came from microbial symbionts. We aimed to clarify how dietary folic acid affects fitness and development in fruit flies and whether flies may receive folates from microbial symbionts. We found larvae were more viable and developed faster with increasing dietary folic acid, with the surprising exception that larvae fed nearly-zero folic acid developed faster. Their body folate levels did not significantly differ from those that consumed up to 600 times more folic acid. However, these flies fed little folate only achieved normal body folate levels and development times when antibiotics were excluded from the diet. When flies consumed near-zero folates with antibiotics, their body folate levels decreased and development was prolonged. An assay for the endosymbiont Wolbachia in flies used to generate the experimental flies did not show presence of these bacteria. Our data suggest D. melanogaster can harbor unknown bacterial symbiont(s) that provide essential folates to their host when it is scarce in the diet, allowing the fruit fly to maintain growth and development.




BRIEF COMMUNICATIONS

Open Access Article

Alteration of wing size through over-expression of scribbler isoforms

Dennis R. LaJeuensse
Pages 320 - 326
http://dx.doi.org/10.4161/fly.4.4.13148
Abstract | PDF

scribbler was identified as a genetic modifier of the Drosophila tumor suppressor gene Merlin. Loss of Merlin results in hyperplasia in a number of different epithelial tissues including the wing and eye imaginal discs, however loss of scribbler does not. The lack of an overt proliferation sbb phenotype has complicated the mechanistic link between sbb and Merlin. Scribbler encodes two novel transcriptional repressors which function in numerous processes including axon guidance and pattern formation within the wing. While the two sbb isoforms have some redundant functions over-expression of two sbb isoforms within the wing show distinct and opposite effects. Over-expression of the smaller SbbA isoform results in a larger wing, while over-expression of larger SbbB isoform results in a smaller wing with defects in venation. Co-expression of sbb isoforms ameliorates the effects of expression of either isoform alone, suggesting that a balance between the express of each scribbler isoform is required to ensure proper size of the wing.

Open Access Article

Large standard deviations and logarithmic-normality: the truth about hemocyte counts in Drosophila

Richard P. Sorrentino
Pages 327 - 332
http://dx.doi.org/10.4161/fly.4.4.13260
Abstract | PDF

While many quantifiable biological phenomena can be described by making use of an assumption of normality in the distribution of individual values, many biological phenomena are not accurately described by the normal distribution. An unquestioned assumption of normality of distribution of possible outcomes can lead to misinterpretation of data, which could have serious consequences. Thus it is extremely important to test the validity of an assumption of normality of possible outcomes. As it turns out, the logarithmic-normal (log-normal) distribution pattern is often far more accurate in describing statistical biological phenomena. Herein I examine large samples of values for circulating blood cell (hemocyte) concentration (CHC) among both wild-type and mutant Drosophila larvae, and demonstrate in both cases that the distribution of individual values does not conform to normality, but does conform to log-normality.




EXPRESSIONS AND PHENOTYPES

Open Access Article

A GFP-tagged Muscleblind C protein isoform reporter construct

Maya Pascual, Lidon Monferrer, Juan Miguel Fernandez-Costa, Ariadna Bargiela, Ruben Artero and Beatriz Llamusi
Pages 333 - 337
http://dx.doi.org/10.4161/fly.4.4.13259
Abstract | PDF

Drosophila muscleblind (mbl), the ortholog of human Muscleblind-like 1 (MBNL1) gene involved in Myotonic Dystrophy (DM), gives raise to protein isoforms MblA to G. The specific functions and subcellular distribution of isoforms are still largely unknown. To overcome the lack of isoform-specific antibodies we generated transgenic flies that express a GFP:MblC fusion protein under the control of the Gal4/UAS system. The reporter fusion protein was able to functionally complement mbl loss of function mutations, demonstrating activity, and accumulated predominantly in adult muscle nuclei. The fluorescent nature of the reporter makes it appropriate for live imaging detection of MblC protein isoform.




METHODS AND TECHNICAL ADVANCES

Open Access Article

Specific cell ablation in Drosophila using the toxic viral protein M2(H37A)

Victoria K. Lam, Tsuyoshi Tokusumi, Donna Cerabona and Robert A. Schulz
Pages 338 - 343
http://dx.doi.org/10.4161/fly.4.4.13114
Abstract | PDF

The expression of toxic viral proteins for the purpose of eliminating distinct populations of cells, while leaving the rest of an organism unaffected, is a valuable method for analyzing development. Using the Gal4-UAS system, we employed the M2(H37A) toxic ion channel of the influenza-A virus to selectively ablate the Drosophila eye-antennal imaginal discs, hemocytes, dorsal vessel and nervous tissue, and comparatively monitored the effects of expressing the apoptosis-promoting protein Reaper in identical cell populations. In this report, we demonstrate the effectiveness of M2(H37A)-mediated ablation as a new means to selectively eliminate cells of interest during Drosophila development.

Open Access Article

The FLIGHT Drosophila RNAi database: 2010 Update

David Sims, Borisas Bursteinas, Ekta Jain, Qiong Gao, Buzz Baum and Marketa Zvelebil
Pages 344 - 348
http://dx.doi.org/10.4161/fly.4.4.13303
Abstract | PDF

FLIGHT (http://flight.icr.ac.uk/) is an online resource compiling data from high-throughput Drosophila in vivo and in vitro RNAi screens. FLIGHT includes details of RNAi reagents and their predicted off-target effects, alongside RNAi screen hits, scores and phenotypes, including images from high-content screens. The latest release of FLIGHT is designed to enable users to upload, analyze, integrate and share their own RNAi screens. Users can perform multiple normalizations, view quality control plots, detect and assign screen hits and compare hits from multiple screens using a variety of methods including hierarchical clustering. FLIGHT integrates RNAi screen data with microarray gene expression as well as genomic annotations and genetic/physical interaction datasets to provide a single interface for RNAi screen analysis and data-mining in Drosophila.

Open Access Article

Transgenic tools for members of the genus Drosophila with sequenced genomes

Stacy Holtzman, David Miller, Robert C. Eisman, Hisashi Kuwayama, Teruyuki Niimi and Thomas C. Kaufman
Pages 349 - 362
http://dx.doi.org/10.4161/fly.4.4.13304
Abstract | PDF

The sequencing of the genomes of 12 Drosophila species has created an opportunity for much in the way of comparative molecular analyses amongst these species. To aid that endeavor, we have made several transformation vectors based on the piggyBac transposon with 3xP3-EGFP and -ECFP transgenic markers that should be useful for mutagenesis and establishing the GAL4/UAS system in these species. We have tested the ability of mini-white to be used as a marker for insertional mutagenesis, and have observed mini-white derived pigmentation of the testes sheath in a subset of lines from D. pseudoobscura and D. virilis. We have incorporated a source of piggyBac transposase into nine Drosophila species, and have demonstrated the functionality of these transposase lines for mobilization of marked inserts in vivo. Additionally, we tested the ability of a D. melanogaster nanos enhancer element to drive expression of GAL4 in D. melanogaster, D. simulans, D. erecta, D. yakuba, D. pseudoobscura, and D. virilis. The efficacy of the nos-Gal4 transgene was determined by measuring the response of UAS-EGFPtub in all six species. Our results show that D. melanogaster nos-Gal4 drives expression in other species, to varying degrees, in similar spatiotemporal domains in the ovaries, testes, and embryos as seen in D. melanogaster. However, expression levels are variable, demonstrating the possible need to use species-specific promoters in some cases. In summary, we hope to provide a set of guidelines and basic tools, based upon this work, for both insertional mutagenesis and GAL4/UAS system-based experiments in multiple species of Drosophila.




LANDES HIGHLIGHTS

Open Access Article

Landes Highlights

Barbara P. Rattner
Pages 363 - 364
http://dx.doi.org/10.4161/fly.4.4.13819
PDF