P-element Insertion Research Articles

Adhesive pad differentiation in Drosophila melanogaster depends on the Polycomb group gene Su(z)2.

The ability of many insects to walk on vertical smooth surfaces such as glass or even on the ceiling has fascinated biologists for a long time, and has led to the discovery of highly specialized adhesive organs located at the distal end of the animals' legs. So far, research has primarily focused on structural and ultrastructural investigations leading to a deeper understanding of adhesive organ functionality and to the development of new bioinspired materials. Genetic approaches, e.g. the analysis of mutants, to achieve a better understanding of adhesive organ differentiation have not been used so far. Here, we describe the first Drosophila melanogaster mutant that develops malformed adhesive organs, resulting in a complete loss of climbing ability on vertical smooth surfaces. Interestingly, these mutants fail to make close contact between the setal tips and the smooth surface, a crucial condition for wet adhesion mediated by capillary forces. Instead, these flies walk solely on their claws. Moreover, we were able to show that the mutation is caused by a P-element insertion into the Su(z)2 gene locus. Remobilization of the P-element restores climbing ability. Furthermore, we provide evidence that the P-element insertion results in an artificial Su(z)2 transcript, which most likely causes a gain-of-function mutation. We presume that this transcript causes deregulation of yet unknown target genes involved in pulvilli differentiation. Our results nicely demonstrate that the genetically treatable model organism Drosophila is highly suitable for future investigations on adhesive organ differentiation.

Fine-mapping and selective sweep analysis of QTL for cold tolerance in Drosophila melanogaster.

There is a growing interest in investigating the relationship between genes with signatures of natural selection and genes identified in QTL mapping studies using combined population and quantitative genetics approaches. We dissected an X-linked interval of 6.2 Mb, which contains two QTL underlying variation in chill coma recovery time (CCRT) in Drosophila melanogaster from temperate (European) and tropical (African) regions. This resulted in two relatively small regions of 131 kb and 124 kb. The latter one co-localizes with a very strong selective sweep in the European population. We examined the genes within and near the sweep region individually using gene expression analysis and P-element insertion lines. Of the genes overlapping with the sweep, none appears to be related to CCRT. However, we have identified a new candidate gene of CCRT, brinker, which is located just outside the sweep region and is inducible by cold stress. We discuss these results in light of recent population genetics theories on quantitative traits.

Do candidate genes mediating conspecific sperm precedence affect sperm competitive ability within species? A test case in Drosophila.

When females mate to multiple males, the last male to mate fathers the majority of progeny. When males of different species inseminate a female, the sperm of the male conspecific to the female is favored in fertilization in a process known as conspecific sperm precedence (CSP). A large number of studies in Drosophila have assayed the genetic basis of sperm competition, with a main focus on D. melanogaster and accessory gland protein genes. Only a few studies have attempted to disentangle the genetic basis of CSP between related species of Drosophila. Although there is no a priori reason to believe that genes influencing intraspecific sperm competitive ability might also mediate conspecific sperm precedence, no study has addressed the question. Here, we test a group of candidate CSP genes between D. simulans and D. mauritiana for their effect on sperm competition in D. melanogaster. The use of P-element insertion lines identified CG14891 gene disruption as the only one causing a significant decrease in second male paternity success relative to wild-type and ebony tester males. The gene disruption affected both sperm displacement and the sperm fertilizing ability. Out of five genes tested using RNA interference, only gene knockdown of CG6864 (Mst89B) significantly reduced the male’s ability to father progeny when second to mate. Our results suggest that CG14891 and CG6864 might have been co-opted from an intraspecies gene function (i.e., sperm competition) into an interspecies avoidance phenotype (i.e., CSP). Alternatively, the dual role of these genes could be a consequence of their pleiotropic roles.

Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster

The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster.

Disruption of the protein kinase N gene of drosophila melanogaster results in the recessive delorean allele (pkndln) with a negative impact on wing morphogenesis.

We describe the delorean mutation of the Drosophila melanogaster protein kinase N gene (pkndln) with defects in wing morphology. Flies homozygous for the recessive pkndln allele have a composite wing phenotype that exhibits changes in relative position and shape of the wing blade as well as loss of specific vein and bristle structures. The pkndln allele is the result of a P-element insertion in the first intron of the pkn locus, and the delorean wing phenotype is contingent upon the interaction of insertion-bearing alleles in trans. The presence of the insertion results in production of a novel transcript that initiates from within the 3′ end of the P-element. The delorean-specific transcript is predicted to produce a wild-type PKN protein. The delorean phenotype is not the result of a reduction in pkn expression, as it could not be recreated using a variety of wing-specific drivers of pkn-RNAi expression. Rather, it is the presence of the delorean-specific transcript that correlates with the mutant phenotype. We consider the delorean wing phenotype to be due to a pairing-dependent, recessive mutation that behaves as a dosage-sensitive, gain of function. Our analysis of genetic interactions with basket and nemo reflects an involvement of pkn and Jun-terminal kinase signaling in common processes during wing differentiation and places PKN as a potential effector of Rho1’s involvement in the Jun-terminal kinase pathway. The delorean phenotype, with its associated defects in wing morphology, provides evidence of a role for PKN in adult morphogenetic processes.

Increased Actin Polymerization and Stabilization Interferes with Neuronal Function and Survival in the AMPKγ Mutant Loechrig

loechrig (loe) mutant flies are characterized by progressive neuronal degeneration, behavioral deficits, and early death. The mutation is due to a P-element insertion in the gene for the γ-subunit of the trimeric AMP-activated protein kinase (AMPK) complex, whereby the insertion affects only one of several alternative transcripts encoding a unique neuronal isoform. AMPK is a cellular energy sensor that regulates a plethora of signaling pathways, including cholesterol and isoprenoid synthesis via its downstream target hydroxy-methylglutaryl (HMG)-CoA reductase. We recently showed that loe interferes with isoprenoid synthesis and increases the prenylation and thereby activation of RhoA. During development, RhoA plays an important role in neuronal outgrowth by activating a signaling cascade that regulates actin dynamics. Here we show that the effect of loe/AMPKγ on RhoA prenylation leads to a hyperactivation of this signaling pathway, causing increased phosphorylation of the actin depolymerizating factor cofilin and accumulation of filamentous actin. Furthermore, our results show that the resulting cytoskeletal changes in loe interfere with neuronal growth and disrupt axonal integrity. Surprisingly, these phenotypes were enhanced by expressing the Slingshot (SSH) phosphatase, which during development promotes actin depolymerization by dephosphorylating cofilin. However, our studies suggest that in the adult SSH promotes actin polymerization, supporting in vitro studies using human SSH1 that suggested that SSH can also stabilize and bundle filamentous actin. Together with the observed increase in SSH levels in the loe mutant, our experiments suggest that in mature neurons SSH may function as a stabilization factor for filamentous actin instead of promoting actin depolymerization.

Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Ab42-induced neurodegeneration.

The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, we identified the Drosophila homolog, CG2789/dTSPO, and studied the effects of its inactivation by P-element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5-4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to γ-irradiation or H2O2 exposure, as well as extended male fly lifespan and inhibited Aβ42-induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target.

Comparison of Ultra-Conserved Elements in Drosophilids and Vertebrates

Metazoan genomes contain many ultra-conserved elements (UCEs), long sequences identical between distant species. In this study we identified UCEs in drosophilid and vertebrate species with a similar level of phylogenetic divergence measured at protein-coding regions, and demonstrated that both the length and number of UCEs are larger in vertebrates. The proportion of non-exonic UCEs declines in distant drosophilids whilst an opposite trend was observed in vertebrates. We generated a set of 2,126 Sophophora UCEs by merging elements identified in several drosophila species and compared these to the eutherian UCEs identified in placental mammals. In contrast to vertebrates, the Sophophora UCEs are depleted around transcription start sites. Analysis of 52,954 P-element, piggyBac and Minos insertions in the D. melanogaster genome revealed depletion of the P-element and piggyBac insertions in and around the Sophophora UCEs. We examined eleven fly strains with transposon insertions into the intergenic UCEs and identified associated phenotypes in five strains. Four insertions behave as recessive lethals, and in one case we observed a suppression of the marker gene within the transgene, presumably by silenced chromatin around the integration site. To confirm the lethality is caused by integration of transposons we performed a phenotype rescue experiment for two stocks and demonstrated that the excision of the transposons from the intergenic UCEs restores viability. Sequencing of DNA after the transposon excision in one fly strain with the restored viability revealed a 47 bp insertion at the original transposon integration site suggesting that the nature of the mutation is important for the appearance of the phenotype. Our results suggest that the UCEs in flies and vertebrates have both common and distinct features, and demonstrate that a significant proportion of intergenic drosophila UCEs are sensitive to disruption.

D. melanogaster imaginal disk mitotic abnormalities induced by a tumor-suppressor Dlg-silencing construction

Mitosis, cytokinesis and nuclear texture of wing imaginal disk cells silenced by UAS-RNAi-dlg construct activated by a 1096-Gal4 driver were studied. The construct is a P-element genomic insertion with a UAS-promoter driving the silencing element. The latter contains a coding region of the dlg gene and complementary region. They produce an RNA hairpin processed by the endogenous protein Dicer (Dietzl et al., 2007). The resulting RNA fragments silence dlg mRNA. The complex is cleaved with the genomic Dicer, and dlg mRNA disintegrates. The tumor suppressor gene dlg encodes 21 transcripts. The construct UAS-RNAi-dlg inactivates 14 transcripts (RE, RH, RQ, RS, RG, RD, RL, RB, RK, RR, RT, RN, RA, RP) and does not silence 7 others (RO, RF, RI, RU, RJ, RC, RM). This permits one to study the function of proteins containing guanilate-kinase domain IPR008145 at the protein C-end. The most important consequences of the silencing are the abnormal mitoses exit and binuclear cells formation. Quantitative fluorescence measurements of anti-H3-p histone and DAPI signals showed phase-specific changes in nuclear texture. The most probable dlg target is inactivation of cellular cortex polarization in mitosis.

Gene-by-temperature interactions and candidate plasticity genes for morphological traits in Drosophila melanogaster.

Understanding the genetic architecture of any quantitative trait requires identifying the genes involved in its expression in different environmental conditions. This goal can be achieved by mutagenesis screens in genetically tractable model organisms such as Drosophila melanogaster. Temperature during ontogenesis is an important environmental factor affecting development and phenotypic variation in holometabolous insects. In spite of the importance of phenotypic plasticity and genotype by environment interaction (GEI) for fitness related traits, its genetic basis has remained elusive. In this context, we analyzed five different adult morphological traits (face width, head width, thorax length, wing size and wing shape) in 42 co-isogenic single P-element insertional lines of Drosophila melanogaster raised at 17°C and 25°C. Our analyses showed that all lines differed from the control for at least one trait in males or females at either temperature. However, no line showed those differences for all traits in both sexes and temperatures simultaneously. In this sense, the most pleiotropic candidate genes were CG34460, Lsd-2 and Spn. Our analyses also revealed extensive genetic variation for all the characters mostly indicated by strong GEIs. Further, our results indicate that GEIs were predominantly explained by changes in ranking order in all cases suggesting that a moderate number of genes are involved in the expression of each character at both temperatures. Most lines displayed a plastic response for at least one trait in either sex. In this regard, P-element insertions affecting plasticity of a large number of traits were associated to the candidate genes Btk29A, CG43340, Drak and jim. Further studies will help to elucidate the relevance of these genes on the morphogenesis of different body structures in natural populations of D. melanogaster.

Integrin αPS3/βν-mediated Phagocytosis of Apoptotic Cells and Bacteria in Drosophila

Integrins exert a variety of cellular functions as heterodimers of two transmembrane subunits named α and β. Integrin βν, a β-subunit of Drosophila integrin, is involved in the phagocytosis of apoptotic cells and bacteria. Here, we searched for an α-subunit that forms a complex and cooperates with βν. Examinations of RNAi-treated animals suggested that αPS3, but not any of four other α-subunits, is required for the effective phagocytosis of apoptotic cells in Drosophila embryos. The mutation of αPS3-encoding scb, deficiency, insertion of P-element, or alteration of nucleotide sequences, brought about a reduction in the level of phagocytosis. The defect in phagocytosis by deficiency was reverted by the forced expression of scb. Furthermore, flies in which the expression of both αPS3 and βν was inhibited by RNAi showed a level of phagocytosis almost equal to that observed in flies with RNAi for either subunit alone. A loss of αPS3 also decreased the activity of larval hemocytes in the phagocytosis of Staphylococcus aureus. Finally, a co-immunoprecipitation analysis using a Drosophila cell line treated with a chemical cross-linker suggested a physical association between αPS3 and βν. These results collectively indicated that integrin αPS3/βν serves as a receptor in the phagocytosis of apoptotic cells and bacteria by Drosophila phagocytes.

The M-Line Protein Obscurin in the early Development of Drosophila Flight Muscle

Obscurin is a 475 kDa protein in the M-line of Drosophila muscles. The protein is made up of Ig and Fn3 domains, a Rho-GEF domain near the N-terminus and two kinase domains near the C-terminus. The expression of obscurin was reduced by a P-element insertion, or by RNAi. Knockdown of obscurin by RNAi was targeted to all muscles, or specifically to the flight muscle (IFM). In P-element mutants and RNAi lines, embryo, larva and pupa developed normally; adults could walk and jump, but were flightless. In the wild-type pupa, obscurin in the IFM appeared in striations at 30 hours after puparium formation, when kettin (a Z-disc protein) and myosin were in amorphous strands. Thus, obscurin in the M-line precedes the regular assembly of the Z-disc and A-band. In the IFM of flies with reduced obscurin, the sarcomere length was normal but the M-line was missing and H-zone irregular. Isolated thick filaments were asymmetrical with the bare zone shifted from the middle of the filaments. In the sarcomere, the length and polarity of thin filaments depended on the position of the bare zone in adjacent thick filaments. Thus, the early expression of obscurin nucleates the assembly of a symmetrical thick filament, which leads to thin filaments of uniform length. Ligands of the kinase domains are: ball (another kinase) binding to kinase 1 and MASK (an ankyrin-repeat protein) binding to kinase 2. Confirmation of these interactions by injecting embryos with kinase constructs will be described. The abnormalities in the IFM sarcomere in RNAi lines with reduced ball or MASK were similar to those in obscurin knockdown flies. The expression of obscurin was normal in RNAi lines of both ligands, demonstrating the importance of obscurin, ball and MASK to sarcomere assembly.

Gene knockout by inducing P-element transposition in Drosophila

The P-element has been successfully used in germline transformation to create transgenic flies and as an insertion mutagenesis agent to disrupt genes. Moreover, P-element can also be used to knockout genes that are near the insertion sites by inducing its imprecise transposition. In this article, P-element insertion lines were crossed with transposase expression fly to recover the transposon, and the deletion of flanking sequence, which was created by imprecise excision, was detected by PCR. Through this method, null alleles of seven genes that spread over three major chromosomes (X, second, third) were generated in Drosophila melanogaster. Results show that the frequency of flanking deletions is expected to be 1%, and it is enough to pick up at least one ideal deletion line from 200 independent recovery lines in general. These data suggest that although the frequency of disrupted gene varied greatly, from 0.13 to 2.34%, gene knockout by inducing P-element transposition appears to be a feasible and effective strategy, compared to the complicated process of gene targeting based on homologous recombination.

Identification of Genes Underlying Hypoxia Tolerance inDrosophilaby a P-element Screen

Hypoxia occurs in physiologic conditions (e.g. high altitude) or during pathologic states (e.g. ischemia). Our research is focused on understanding the molecular mechanisms that lead to adaptation and survival or injury to hypoxic stress using Drosophila as a model system. To identify genes involved in hypoxia tolerance, we screened the P-SUP P-element insertion lines available for all the chromosomes of Drosophila. We screened for the eclosion rates of embryos developing under 5% O2 condition and the number of adult flies surviving one week after eclosion in the same hypoxic environment. Out of 2187 lines (covering ∼1870 genes) screened, 44 P-element lines representing 44 individual genes had significantly higher eclosion rates (i.e. >70%) than those of the controls (i.e. ∼7–8%) under hypoxia. The molecular function of these candidate genes ranged from cell cycle regulation, DNA or protein binding, GTP binding activity, and transcriptional regulators. In addition, based on pathway analysis, we found these genes are involved in multiple pathways, such as Notch, Wnt, Jnk, and Hedgehog. Particularly, we found that 20 out of the 44 candidate genes are linked to Notch signaling pathway, strongly suggesting that this pathway is essential for hypoxia tolerance in flies. By employing the UAS/RNAi-Gal4 system, we discovered that genes such as osa (linked to Wnt and Notch pathways) and lqf (Notch regulator) play an important role in survival and development under hypoxia in Drosophila. Based on these results and our previous studies, we conclude that hypoxia tolerance is a polygenic trait including the Notch pathway.

The Drosophila Homologue of Tubulin-Specific Chaperone E–Like Protein Is Required for Synchronous Sperm Individualization and Normal Male Fertility

The tubulin-specific chaperone E–like protein (TBCEL or E-like) of vertebrates shows sequence homology to TBCE, a component of the multimolecular complex required for tubulin heterodimer formation in all eukaryotic cells. TBCEL apparently serves more specific functions, as it is found only in animals. At the cellular level, TBCEL plays a role as a regulator of tubulin stability. It is strongly expressed in human testes, but its systemic function is not known. The gene CG12214 codes for the Drosophila homologue of the vertebrate TBCEL protein. Here we show that disruption of the Drosophila Tbcel gene causes defects in spermatid individualixation, which leads to dispersed migration of F-actin–rich investment cones. Mutations affecting the Tbcel gene cause strong reduction in male, but not female, fertility. However, mature sperm function apparently is not impaired. We generated polyclonal antisera against TBCEL to study its localization and distribution in Drosophila tissues. Immunostainings of wild-type and null mutant testes demonstrated that TBCEL is localized in testes, presumably associated with axoneme bundles prior to spermatid individualization. Molecular analysis of the transposon insertion site in the mutant mulet (mlt), for which male sterility and sperm individualization defects have previously been described, demonstrates that the mlt P-element insertion resides in the Tbcel gene. Our results show that loss of TBCEL in Drosophila is compatible with viability and normal female fertility but causes reduced male fertility. We conclude that Drosophila TBCEL is strongly expressed in testes and plays an important role in sperm individualization during spermatogenesis. The high level of Tbcel mRNA in human testes suggests a general role of TBCEL in animal spermatogenesis. However, Western blots and courtship analysis suggest that TBCEL may have additional functions in the nervous system of Drosophila that could contribute to the observed reduced male fertility. These functions now have to be investigated.

Genetic knockdown of a single organic anion transporter alters the expression of functionally related genes in Malpighian tubules ofDrosophila melanogaster

Insects excrete a wide variety of toxins via the Malpighian (renal) tubules. Previous studies have implicated three transporters in the secretion of the organic anion (OA) methotrexate (MTX) by the Drosophila Malpighian tubule: Drosophila multidrug resistance-associated protein (dMRP, CG6214), a multidrug efflux transporter (MET, CG30344), and an organic anion transporting polypeptide 58Dc (OATP58Dc, CG3380). RNA interference (RNAi) knockdown and P-element insertion mutation of single OA transporter genes were used to evaluate the importance of these three putative transporters in the secretion of MTX by the Malpighian tubules of Drosophila melanogaster. A major finding is that genetic knockdown of a single OA transporter gene leads to reductions in the expression of at least one other OA transporter gene and in secretion of MTX by Malpighian tubules isolated from flies reared on a standard diet. The pattern of changes indicates that decreases in MTX secretion do not correspond to decreases in dMRP expression in all of the RNAi lines. Genetic knockdown of a single OA transporter gene also alters the extent of upregulation of multiple OA transporter genes in the tubules in response to dietary MTX. Knockdown of dMRP is associated with a decrease in MET expression but an increase in OATP expression when flies are reared on MTX-enriched diet. Our results indicate that dMRP and MET are not the dominant MTX transporters in the tubules when flies are reared on MTX-enriched diets. At least one additional transporter, and possibly OATP, are required for MTX secretion. The implications of our results for studies using genetic knockdown techniques to identify OA transporters in whole tissues such as Malpighian tubules are discussed.

Mechanism of Evenness Interrupted (Evi)-Exosome Release at Synaptic Boutons

Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.

Fhos encodes a Drosophila Formin‐Like Protein participating in autophagic programmed cell death

Larval tissues undergo programmed cell death (PCD) during Drosophila metamorphosis. PCD is triggered in a stage and tissue-specific fashion in response to ecdysone pulses. The understanding of how ecdysone induces the stage and tissue-specificity of cell death remains obscure. Several steroid-regulated primary response genes have been shown to act as key regulators of cellular responses to ecdysone by inducing a cascade of transcriptional regulation of late responsive genes. In this article, the authors identify Fhos as a gene that is required for Drosophila larval salivary gland destruction. Animals with a P-element mutation in Fhos possess persistent larval salivary glands, and precise excisions of this P-element insertion resulted in reversion of this salivary gland mutant phenotype. Fhos encodes the Drosophila homolog of mammalian Formin Fhos. Fhos is differentially transcribed during development and responds to ecdysone in a method that is similar to other cell death genes. Similarly to what has been shown for its mammalian counterpart, FHOS protein is translocated to the nucleus at later stages of cell death. Fhos mutants posses disrupted actin cytoskeleton dynamics in persistent salivary glands. Together, our data indicate that Fhos is a new ecdysone-regulated gene that is crucial for changes in the actin cytoskeleton during salivary gland elimination in Drosophila.

Genome‐Wide P‐element Screen for Hypoxia Tolerance Genes in Drosophila melanogaster

Hypoxia occurs during normal conditions (high altitude) or during pathologic states (sickle cell anemia). Our research is focused on understanding the molecular mechanisms that lead to injury or adaptation to hypoxic stress using Drosophila as a model system. To identify the genes involved in hypoxia tolerance, we screened P‐element insertion lines generated by Gene Disruption Project. For hypoxia tolerance, we screened for a) eclosion rates after following the development of embryos placed in 5% O2 to eclosion and adulthood, b) number of adult flies surviving after eclosion. Out of 2367 lines screened, 44 P‐element lines had eclosion rates significantly higher (>70% eclosion) than the controls (eclosion rate ~7–8%) under hypoxia. The molecular function of these lines ranged from cell cycle regulation, DNA or protein binding, ATPase activity, and transcriptional co‐regulators. In this screen, we found certain interesting candidate genes such as sec8, cyclin E, osa, l(3)mbn, lqf which show tremendous hypoxia tolerance during all stages of the developmental cycle. Furthermore, we employed the UAS–Gal4 system to further dissect the role of these genes ubiquitously or in specific tissues in vivo. Interestingly, we found that regulation by single genes, such as osa can play an important role in survival and development during hypoxia.This work has been supported by NIH grant (RO1 NS037756) to Dr. Gabriel Haddad.

Extensive epistasis for olfactory behaviour, sleep and waking activity in Drosophila melanogaster.

SummaryEpistasis is an important feature of the genetic architecture of quantitative traits, but the dynamics of epistatic interactions in natural populations and the relationship between epistasis and pleiotropy remain poorly understood. Here, we studied the effects of epistatic modifiers that segregate in a wild-derived Drosophila melanogaster population on the mutational effects of P-element insertions in Semaphorin-5C (Sema-5c) and Calreticulin (Crc), pleiotropic genes that affect olfactory behaviour and startle behaviour and, in the case of Crc, sleep phenotypes. We introduced Canton-S B (CSB) third chromosomes with or without a P-element insertion at the Crc or Sema-5c locus in multiple wild-derived inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and assessed the effects of epistasis on the olfactory response to benzaldehyde and, for Crc, also on sleep. In each case, we found substantial epistasis and significant variation in the magnitude of epistasis. The predominant direction of epistatic effects was to suppress the mutant phenotype. These observations support a previous study on startle behaviour using the same D. melanogaster chromosome substitution lines, which concluded that suppressing epistasis may buffer the effects of new mutations. However, epistatic effects are not correlated among the different phenotypes. Thus, suppressing epistasis appears to be a pervasive general feature of natural populations to protect against the effects of new mutations, but different epistatic interactions modulate different phenotypes affected by mutations at the same pleiotropic gene.