Table of Contents

Volume 6, Issue 3

  July/August/September 2012

  Pages 133 - 210

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EDITOR'S PREVIEW

Open Access Article

In this issue of Fly

Barbara Rattner
Pages 133 - 134
http://dx.doi.org/10.4161/fly.21538
Full Text | PDF




EXTRA VIEWS

The complexities of G-protein-coupled receptor kinase function in Hedgehog signaling

Dominic Maier, Shuofei Cheng and David R. Hipfner
Pages 135 - 141
http://dx.doi.org/10.4161/fly.20245
Abstract | Full Text | PDF

Hedgehog (Hh) signaling is essential for proper tissue patterning and maintenance and has a substantial impact on human disease. While many of the main components and mechanisms involved in transduction of the Hh signal have been identified, the details of how the pathway functions are continually being refined. One aspect that has attracted much attention recently is the involvement of G-protein-coupled receptor kinases (GRKs) in the pathway. These regulators of G-protein-coupled receptor (GPCR) signaling have an evolutionarily-conserved function in promoting high-threshold Hh target gene expression through regulation of Smoothened (Smo), a GPCR family member that activates intracellular Hh signaling. Several models of how GRKs impact on Smo to increase downstream signaling have been proposed. Recently, we demonstrated that these kinases have surprisingly complex and conflicting roles, acting to limit signaling through the pathway while also promoting Smo activity. In addition to the previously described direct effects of Gprk2 on Smo activation, Gprk2 also indirectly affects Hh signaling by controlling production of the second messenger cyclic AMP to influence Protein kinase A activity.

Coordinated control of lumen size and collective migration in the salivary gland

Na Xu and Monn Monn Myat
Pages 142 - 146
http://dx.doi.org/10.4161/fly.20246
Abstract | Full Text | PDF

The Drosophila embryonic salivary gland is a powerful system to analyze the cellular and molecular mechanisms of tubulogenesis in vivo. The secretory portion of the salivary gland (referred to here as the salivary gland), is a pair of elongated epithelial tubes with a single central lumen, that is formed by the invagination and collective migration of gland cells from the ventral surface of the embryo.^1,2 As the salivary gland elongates during migration, the central lumen elongates and narrows at the proximal end.³ A number of genes are known to regulate salivary gland migration and/or lumen size (Table 1); however, it is only recently that we have begun to analyze how control of salivary gland migration is coordinated with control of lumen size. To understand coordinated control of salivary gland migration and lumen size, we analyzed the role of the single Drosophila Rho GTPase, Rho1, in salivary gland tubulogenesis. In addition to the requirement of Rho1 in salivary gland invagination and migration (Xu et al., 2008), our recent studies show that Rho1 controls salivary gland lumen width and length during the migration process (Fig. 1). These studies reveal that Rho1-mediated processes at the proximal end of the migrating salivary gland, such as cell shape change, cell rearrangement and apical domain elongation, contribute to collective migration, and narrowing and elongation of the lumen.⁴

Open Access Article

Plastic flies: The regulation and evolution of trait variability in Drosophila

Alexander W. Shingleton and Hui Yuan Tang
Pages 147 - 152
http://dx.doi.org/10.4161/fly.20323
Abstract | Full Text | PDF

Individuals within species and populations vary. Such variation arises through environmental and genetic factors and ensures that no two individuals are identical. However, it is clear that not all traits show the same degree of intraspecific variation. Some traits, in particular secondary sexual characteristics used by males to compete for and attract females, are extremely variable among individuals in a population. Other traits, for example brain size in mammals, are not. Recent research has begun to explore the possibility that the extent of phenotypic variation (here referred to as “variability”) may be a character itself and subject to natural selection. While these studies support the concept of variability as an evolvable trait, controversy remains over what precisely the trait is. At the heart of this controversy is the fact that there are very few examples of developmental mechanisms that regulate trait variability in response to any source of variation, be it environmental or genetic. Here, we describe a recent study from our laboratory that identifies such a mechanism. We then place the study in the context of current research on the regulation of trait variability, and discuss the implications for our understanding of the developmental regulation and evolution of phenotypic variation.

Homeostatic plasticity in Drosophila central neurons, and implications in human diseases

Yong Ping and Susan Tsunoda
Pages 153 - 157
http://dx.doi.org/10.4161/fly.20775
Abstract | Full Text | PDF

Synaptic homeostasis is a form of neuronal plasticity that stabilizes activity of neural networks. Both presynaptic and postsynaptic effects are well documented in response to activity changes. The electrical signaling machinery of individual neurons, or intrinsic properties, have also been implicated in this plasticity. How synaptic and intrinsic changes are coordinated, however, is still a puzzle. A recent study by Ping and Tsunoda shows both synaptic and intrinsic changes in Drosophila central neurons in response to prolonged inactivity.¹ Changes include the upregulation of Dα7 nicotinic acetylcholine receptors (nAChRs) and Shal (K_v4) potassium channels. This work has two noteworthy findings. First, although mediated by different receptors, synaptic homeostasis in the central nervous system (CNS) is conserved across species. This is perhaps the most direct demonstration that nAChRs mediate synaptic homeostasis. Changes in the expression of nAChRs have long been noted during development, as well as during pathological conditions, such as nicotine addiction² and Alzheimer disease.³ The second interesting finding is the relationship between synaptic and intrinsic plasticity: nAChRs are upregulated immediately, subsequently triggering a rapid increase in Shal K⁺ channels. This novel mechanism regulates synaptic homeostasis to stabilize synaptic potentials. This study sets the stage for Drosophila central neurons as a model for cholinergic synaptic homeostasis, its regulation and role in disease.

Recent progress on the identity and characterization of factors that shape gene organization during eukaryotic evolution

Carlos Díaz-Castillo and José M. Ranz
Pages 158 - 161
http://dx.doi.org/10.4161/fly.20861
Abstract | Full Text | PDF

Comparative genomics has identified regions of chromosomes susceptible to participate in rearrangements that modify gene order and genome architecture. Additionally, despite the high levels of genome rearrangement, unusually large regions that remain unaffected have also been uncovered. Functional constraints, such as long-range enhancers or local coregulation of neighboring genes, are thought to explain the maintenance of gene order (i.e., collinearity conservation) among distantly related species since the disruption of these protected regions would cause detrimental misregulation of gene expression. Local enrichment of certain genetic elements in regions of conserved collinearity has been used to support the existence of regulatory-based constraints, although the evidence is largely circumstantial. Indeed, a mechanism of chromosome evolution based only on the existence of fragile regions (i.e., those more susceptible to breaks) can also give rise to extended collinearity conservation, making it difficult to determine whether conserved gene organization is actually caused by functional constraints. Chromosome engineering coupled with genome wide expression profiling and phenotypic assays can provide unambiguous evidence for the presence of functional constraints acting on particular genomic regions. We have recently used this integrated approach to evaluate the presence and nature of putative constraints acting on one of the largest chromosomal regions conserved across nine species of Drosophila. We propose that regulatory-based constraints might not suffice to explain the maintenance of gene organization of some chromosome domains over evolutionary time.

Histone H2A monoubiquitination and Polycomb repression: The missing pieces of the puzzle

Johanna C. Scheuermann, Luis Gutiérrez and Jürg Müller
Pages 162 - 168
http://dx.doi.org/10.4161/fly.20986
Abstract | Full Text | PDF

Polycomb group (PcG) proteins were originally identified as negative regulators of HOX genes in Drosophila but have since emerged as a widely used transcriptional repression system that controls a variety of developmental processes in animals and plants. PcG proteins exist in multi-protein complexes that comprise specific chromatin-modifying enzymatic activities. Genome-wide binding studies in Drosophila and in mammalian cells revealed that these complexes co-localize at a large set of genes encoding developmental regulators. Recent analyses in Drosophila have begun to explore how the different chromatin-modifying activities of PcG protein complexes contribute to the repression of individual target genes. These studies suggest that monoubiquitination of histone H2A (H2Aub) by the PcG protein Sce is only essential for repression of a subset of PcG target genes but is not required for the Polycomb-mediated repression of other targets. Calypso/dBap1, a major deubiquitinase for H2Aub is also critically needed for repression of a subset of PcG target genes. Here, we review our current understanding of the role of H2A monoubiquitination and deubiquitination in Polycomb repression in Drosophila. We discuss unresolved issues concerning the immunological detection of H2Aub and critically evaluate experiments that used Sce and Ring1B point mutants with impaired H2A ubiquitinase activity to study H2Aub-dependent and -independent functions of these proteins in transcriptional repression.

Drosophila innate immune response pathways moonlight in neurodegeneration

Andrew J. Petersen and David A. Wassarman
Pages 169 - 172
http://dx.doi.org/10.4161/fly.20999
Abstract | Full Text | PDF

In this Extra View, we highlight recent Drosophila research that has uncovered a new role for the innate immune response. The research indicates that, in addition to combating infection, the innate immune response promotes neurodegeneration. Our publication (Petersen et al., 2012) reveals a correlative relationship between the innate immune response and neurodegeneration in a model of the human disease Ataxia-telangiectasia (A-T). We also found that glial cells are responsible for the innate immune response in the A-T model, and work by others implicates glial cells in neurodegeneration. Additionally, publications by Chinchore et al. (2012) and Tan et al. (2008) reveal a causative role for the innate immune response in models of human retinal degenerative disorders and Alzheimer disease, respectively. Collectively, these findings suggest that activation of the innate immune response is a shared cause of neurodegeneration in different human diseases.




RESEARCH PAPER

Characterization of null and hypomorphic alleles of the Drosophila l(2)dtl/cdt2 gene: Larval lethality and male fertility

Roketa S. Sloan, Christina I. Swanson, Lily Gavilano, Kristen N. Smith, Pamela Y. Malek, Mayronne Snow-Smith, Robert J. Duronio and S. Catherine Silver Key
Pages 173 - 183
http://dx.doi.org/10.4161/fly.20247
Abstract | Full Text | PDF

The Drosophila lethal(2)denticleless (l(2)dtl) gene was originally reported as essential for embryogenesis and formation of the rows of tiny hairs on the larval ventral cuticle known as denticle belts. It is now well-established that l(2)dtl (also called cdt2) encodes a subunit of a Cullin 4-based E3 ubiquitin ligase complex that targets a number of key cell cycle regulatory proteins, including p21, Cdt1, E2F1 and Set8, to prevent replication defects and maintain cell cycle control. To investigate the role of l(2)dtl/cdt2 during development, we characterized existing l(2)dtl/cdt2 mutants and generated new deletion alleles, using P-element excision mutagenesis. Surprisingly, homozygous l(2)dtl/cdt2 mutant embryos developed beyond embryogenesis, had intact denticle belts, and lacked an observable embryonic replication defect. These mutants died during larval stages, affirming that loss of l(2)dtl/cdt2 function is lethal. Our data show that L(2)dtl/Cdt2 is maternally deposited, remains nuclear throughout the cell cycle, and has a previously unreported, elevated expression in the developing gonads. We also find that E2f1 regulates l(2)dtl/cdt2 expression during embryogenesis, possibly via several highly conserved putative E2f1 binding sites near the l(2)dtl/cdt2 promoter. Finally, hypomorphic allele combinations of the l(2)dtl/cdt2 gene result in a novel phenotype: viable, low-fertility males. We conclude that “denticleless” is a misnomer, but that l(2)dtl/cdt2 is an essential gene for Drosophila development.




METHODS AND TECHNICAL ADVANCES

Rapid phenotypic analysis of uncoated Drosophila samples with low-vacuum scanning electron microscopy

Nicholas J. Tardi, Martha E. Cook and Kevin A. Edwards
Pages 184 - 192
http://dx.doi.org/10.4161/fly.20525
Abstract | Full Text | PDF

Research projects featuring repetitive phenotypic analysis of insects, such as taxonomic studies, quantitative genetics, and mutant screens, could be greatly facilitated by a simpler approach to scanning electron microscopy (SEM). Here, we have applied low-vacuum SEM to wild type and mutant Drosophila and demonstrate that high quality ultrastructure data can be obtained quickly using minimal preparation. Adult flies, frozen live for storage, were mounted on aluminum stubs with carbon cement and directly imaged, with no chemical treatment or sputter coating. The key imaging parameters were identified and optimized, including chamber pressure, beam size, accelerating voltage, working distance and beam exposure. Different optimal conditions were found for eyes, wings, and bristles; in particular, surface features of bristles were obscured at higher accelerating voltages. The chief difficulties were charging, beam damage, and sample movement. We conclude that our optimized protocol is well suited to large-scale ultrastructural phenotypic analysis in insects.

Fly culture collapse disorder: detection, prophylaxis and eradication of the microsporidian parasite Tubulinosema ratisbonensis infecting Drosophila melanogaster

Sebastian Niehus, Philippe Giammarinaro, Samuel Liégeois, Jessica Quintin and Dominique Ferrandon
Pages 193 - 204
http://dx.doi.org/10.4161/fly.20896
Abstract | Full Text | PDF

Drosophila melanogaster is a robust model to investigate many biological problems. It is however prone to some infections, which may endanger fly stocks if left unchecked for. One such infection is caused by an obligate fungal intracellular parasite, Tubulinosema ratisbonensis, which can be found in laboratory stocks. Here, we identify and briefly characterize a T. ratisbonensis strain that was infesting our Drosophila cultures and that required intensive measures to contain and eradicate the infection. We describe the phenotypes of infested stocks. We also report PCR-based techniques that allow the detection of infested stocks with a high sensitivity. We have developed a high-throughput qPCR assay that allows the efficient parallel screening of a large number of potentially-infested stocks. We also have investigated several prophylactic measures to prevent the further contamination of stocks, namely UV-exposure, ethanol treatment, bleaching, and desiccation. Bleaching was found to kill all spores. Other treatments were less effective but were found to be sufficient to prevent further contamination of noninfested stocks. Two treatments were efficacious in curing infested stocks (1) bleaching of eggs and subsequent raising of the larvae in clean vials; (2) fumagillin treatment. These cures only work on stocks that have not become too weak to withstand the procedures.

Estimating the speed of Drosophila locomotion using an automated behavior detection and analysis system

Kimberly L. Chan, Omer Inan, Sharmila Bhattacharya and Oana Marcu
Pages 205 - 210
http://dx.doi.org/10.4161/fly.20987
Abstract | Full Text | PDF

A fundamental phenotypic trait in Drosophila melanogaster is the speed of movement. Its quantification in response to environmental and experimental factors is highly useful for behavioral and neurological studies. Quantifying this behavioral characteristic in freely moving flies is difficult, and many current systems are limited to evaluating the speed of movement of one fly at a time or rely on expensive, time-consuming methods. Here, we present a novel signal processing method of quantifying the speed of multiple flies using a system with automatic behavior detection and analysis that we previously developed to quantify general activity. By evaluating the shape of the signal wave from recordings of a live and simulated single fly, a metric for speed of movement was found. The feasibility of using this metric to estimate the speed of movement in a population of flies was then confirmed by evaluating recordings taken from populations of flies maintained at two different temperatures. The results were consistent with those reported in the literature. This method provides an automated way of measuring speed of locomotion in a fly population, which will further quantify fly behavioral responses to the environment.