Drosophila Gene Research Articles

Filtering of Data-Driven Gene Regulatory Networks Using Drosophila melanogaster as a Case Study.

Gene Regulatory Networks (GRNs) allow the study of regulation of gene expression of whole genomes. Among the most relevant advantages of using networks to depict this key process, there is the visual representation of large amounts of information and the application of graph theory to generate new knowledge. Nonetheless, despite the many uses of GRNs, it is still difficult and expensive to assign Transcription Factors (TFs) to the regulation of specific genes. ChIP-Seq allows the determination of TF Binding Sites (TFBSs) over whole genomes, but it is still an expensive technique that can only be applied one TF at a time and requires replicates to reduce its noise. Once TFBSs are determined, the assignment of each TF and its binding sites to the regulation of specific genes is not trivial, and it is often performed by carrying out site-specific experiments that are unfeasible to perform in all possible binding sites. Here, we addressed these relevant issues with a two-step methodology using Drosophila melanogaster as a case study. First, our protocol starts by gathering all transcription factor binding sites (TFBSs) determined with ChIP-Seq experiments available at ENCODE and FlyBase. Then each TFBS is used to assign TFs to the regulation of likely target genes based on the TFBS proximity to the transcription start site of all genes. In the final step, to try to select the most likely regulatory TF from those previously assigned to each gene, we employ GENIE3, a random forest-based method, and more than 9,000 RNA-seq experiments from D. melanogaster. Following, we employed known TF protein-protein interactions to estimate the feasibility of regulatory events in our filtered networks. Finally, we show how known interactions between co-regulatory TFs of each gene increase after the second step of our approach, and thus, the consistency of the TF-gene assignment. Also, we employed our methodology to create a network centered on the Drosophila melanogaster gene Hr96 to demonstrate the role of this transcription factor on mitochondrial gene regulation.

Sexual Antagonism, Temporally Fluctuating Selection, and Variable Dominance Affect a Regulatory Polymorphism in Drosophila melanogaster.

Understanding how genetic variation is maintained within species is a major goal of evolutionary genetics that can shed light on the preservation of biodiversity. Here, we examined the maintenance of a regulatory single-nucleotide polymorphism (SNP) of the X-linked Drosophila melanogaster gene fezzik. The derived variant at this site is at intermediate frequency in many worldwide populations but absent in populations from the ancestral species range in sub-Saharan Africa. We collected and genotyped wild-caught individuals from a single European population biannually over a period of 5 years, which revealed an overall difference in allele frequency between the sexes and a consistent change in allele frequency across seasons in females but not in males. Modeling based on the observed allele and genotype frequencies suggested that both sexually antagonistic and temporally fluctuating selection may help maintain variation at this site. The derived variant is predicted to be female-beneficial and mostly recessive; however, there was uncertainty surrounding our dominance estimates and long-term modeling projections suggest that it is more likely to be dominant. By examining gene expression phenotypes, we found that phenotypic dominance was variable and dependent upon developmental stage and genetic background, suggesting that dominance may be variable at this locus. We further determined that fezzik expression and genotype are associated with starvation resistance in a sex-dependent manner, suggesting a potential phenotypic target of selection. By characterizing the mechanisms of selection acting on this SNP, our results improve our understanding of how selection maintains genetic and phenotypic variation in natural populations.

Highly contiguous assemblies of 101 drosophilid genomes.

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

The Foraging Gene, a New Environmental Adaptation Player Involved in Xenobiotic Detoxification.

Foraging is vital for animals, especially for food. In Drosophila melanogaster, this behavior is controlled by the foraging gene (for) which encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG). In wild populations of Drosophila, rover individuals that exhibit long foraging trails and sitter individuals that exhibit short ones coexist and are characterized by high and low levels of PKG activity, respectively. We, therefore, postulated that rover flies are more exposed to environmental stresses, including xenobiotics contamination, than sitter flies. We then tested whether these flies differed in their ability to cope with xenobiotics by exposing them to insecticides from different chemical families. We performed toxicological tests and measured the activity and expression levels of different classes of detoxification enzymes. We have shown that a link exists between the for gene and certain cytochrome P450-dependent activities and that the expression of the insecticide-metabolizing cytochrome P450 Cyp6a2 is controlled by the for gene. An unsuspected regulatory pathway of P450s expression involving the for gene in Drosophila is revealed and we demonstrate its involvement in adaptation to chemicals in the environment. This work can serve as a basis for reconsidering adaptation to xenobiotics in light of the behavior of species, including humans.

Genomic analyses of new genes and their phenotypic effects reveal rapid evolution of essential functions in Drosophila development.

It is a conventionally held dogma that the genetic basis underlying development is conserved in a long evolutionary time scale. Ample experiments based on mutational, biochemical, functional, and complementary knockdown/knockout approaches have revealed the unexpectedly important role of recently evolved new genes in the development of Drosophila. The recent progress in the genome-wide experimental testing of gene effects and improvements in the computational identification of new genes (< 40 million years ago, Mya) open the door to investigate the evolution of gene essentiality with a phylogenetically high resolution. These advancements also raised interesting issues in techniques and concepts related to phenotypic effect analyses of genes, particularly of those that recently originated. Here we reported our analyses of these issues, including reproducibility and efficiency of knockdown experiment and difference between RNAi libraries in the knockdown efficiency and testing of phenotypic effects. We further analyzed a large data from knockdowns of 11,354 genes (~75% of the Drosophila melanogaster total genes), including 702 new genes (~66% of the species total new genes that aged < 40 Mya), revealing a similarly high proportion (~32.2%) of essential genes that originated in various Sophophora subgenus lineages and distant ancestors beyond the Drosophila genus. The transcriptional compensation effect from CRISPR knockout were detected for highly similar duplicate copies. Knockout of a few young genes detected analogous essentiality in various functions in development. Taken together, our experimental and computational analyses provide valuable data for detection of phenotypic effects of genes in general and further strong evidence for the concept that new genes in Drosophila quickly evolved essential functions in viability during development.

Transcriptome analysis of chloride intracellular channel knockdown in Drosophila identifies oxidation-reduction function as possible mechanism of altered sensitivity to ethanol sedation.

Chloride intracellular channels (CLICs) are a unique family of evolutionarily conserved metamorphic proteins, switching between stable conformations based on redox conditions. CLICs have been implicated in a wide variety biological processes including ion channel activity, apoptosis, membrane trafficking, and enzymatic oxidoreductase activity. Understanding the molecular mechanisms by which CLICs engage in these activities is an area of active research. Here, the sole Drosophila melanogaster ortholog, Clic, was targeted for RNAi knockdown to identify genes and biological processes associated with Clic expression. Clic knockdown had a substantial impact on global transcription, altering expression of over 7% of transcribed Drosophila genes. Overrepresentation analysis of differentially expressed genes identified enrichment of Gene Ontology terms including Cytoplasmic Translation, Oxidation-Reduction Process, Heme Binding, Membrane, Cell Junction, and Nucleolus. The top term, Cytoplasmic Translation, was enriched almost exclusively with downregulated genes. Drosophila Clic and vertebrate ortholog Clic4 have previously been tied to ethanol sensitivity and ethanol-regulated expression. Clic knockdown-responsive genes from the present study were found to overlap significantly with gene sets from 4 independently published studies related to ethanol exposure and sensitivity in Drosophila. Bioinformatic analysis of genes shared between these studies revealed an enrichment of genes related to amino acid metabolism, protein processing, oxidation-reduction processes, and lipid particles among others. To determine whether the modulation of ethanol sensitivity by Clic may be related to co-regulated oxidation-reduction processes, we evaluated the effect of hyperoxia on ethanol sedation in Clic knockdown flies. Consistent with previous findings, Clic knockdown reduced acute ethanol sedation sensitivity in flies housed under normoxia. However, this effect was reversed by exposure to hyperoxia, suggesting a common set of molecular-genetic mechanism may modulate each of these processes. This study suggests that Drosophila Clic has a major influence on regulation of oxidative stress signaling and that this function overlaps with the molecular mechanisms of acute ethanol sensitivity in the fly.

Ergosta-7, 9 (11), 22-trien-3β-ol Interferes with LPS Docking to LBP, CD14, and TLR4/MD-2 Co-Receptors to Attenuate the NF-κB Inflammatory Pathway In Vitro and Drosophila.

Ergosta-7, 9 (11), 22-trien-3β-ol (EK100) was isolated from Cordyceps militaris, which has been used as a traditional anti-inflammatory medicine. EK100 has been reported to attenuate inflammatory diseases, but its anti-inflammatory mechanism is still unclear. We were the first to investigate the effect of EK100 on the Toll-like receptor 4 (TLR4)/nuclear factor of the κ light chain enhancer of B cells (NF-κB) signaling in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells and the green fluorescent protein (GFP)-labeled NF-κB reporter gene of Drosophila. EK100 suppressed the release of the cytokine and attenuated the mRNA and protein expression of pro-inflammatory mediators. EK100 inhibited the inhibitor kappa B (IκB)/NF-κB signaling pathway. EK100 also inhibited phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (Akt) signal transduction. Moreover, EK100 interfered with LPS docking to the LPS-binding protein (LBP), transferred to the cluster of differentiation 14 (CD14), and bonded to TLR4/myeloid differentiation-2 (MD-2) co-receptors. Compared with the TLR4 antagonist, resatorvid (CLI-095), and dexamethasone (Dexa), EK100 suppressed the TLR4/AKT signaling pathway. In addition, we also confirmed that EK100 attenuated the GFP-labeled NF-κB reporter gene expression in Drosophila. In summary, EK100 might alter LPS docking to LBP, CD14, and TLR4/MD-2 co-receptors, and then it suppresses the TLR4/NF-κB inflammatory pathway in LPS-stimulated RAW264.7 cells and Drosophila.

Adaptation of Drosophila subobscura chromosomal inversions to climatic variables: the Balkan natural population of Avala.

The adaptive value of chromosomal inversions continues raising relevant questions in evolutionary biology. In many species of the Drosophila genus, different inversions have been recognized to be related to thermal adaptation, but it is necessary to determine to which specific climatic variables the inversions are adaptive. With this aim, the behavior of thermal adapted inversions of Drosophila subobscura regarding climatic variables was studied in the natural population of Avala (Serbia) during the 2014-2017 period. The results obtained were compared with those previously reported in the Font Groga (Barcelona, Spain) population, which presents different climatic and environmental conditions. In both populations, it was observed that most thermal adapted inversions were significantly associated with the first, second or both principal components, which were related with maximum, minimum and mean temperatures. Moreover, a significant increase over years (2004-2017) for the minimum temperature was detected. In parallel, a significant variation over time in Avala was only observed for the frequencies of 'warm' and 'non-thermal' adapted inversions of the U chromosome. However, stability in the chromosomal inversion polymorphism was observed for the 2014-2017 period which might result from the temporal span of the study and/or selective process acting on the population.

A cross-species approach for the identification of Drosophila male sterility genes.

Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing our understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse, and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. We focused on one such gene, Rack 1, and found that it plays an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful.

The Adenine/Thymine Deleterious Selection Model for GC Content Evolution at the Third Codon Position of the Histone Genes in Drosophila.

The evolution of the GC (guanine cytosine) content at the third codon position of the histone genes (H1, H2A, H2B, H3, H4, H2AvD, H3.3A, H3.3B, and H4r) in 12 or more Drosophila species is reviewed. For explaining the evolution of the GC content at the third codon position of the genes, a model assuming selection with a deleterious effect for adenine/thymine and a size effect is presented. The applicability of the model to whole-genome genes is also discussed.

The RNA-Binding Protein SBR (Dm NXF1) Is Required for the Constitution of Medulla Boundaries in Drosophila melanogaster Optic Lobes.

Drosophila melanogaster sbr (small bristles) is an orthologue of the Nxf1 (nuclear export factor 1) genes in different Opisthokonta. The known function of Nxf1 genes is the export of various mRNAs from the nucleus to the cytoplasm. The cytoplasmic localization of the SBR protein indicates that the nuclear export function is not the only function of this gene in Drosophila. RNA-binding protein SBR enriches the nucleus and cytoplasm of specific neurons and glial cells. In sbr12 mutant males, the disturbance of medulla boundaries correlates with the defects of photoreceptor axons pathfinding, axon bundle individualization, and developmental neurodegeneration. RNA-binding protein SBR participates in processes allowing axons to reach and identify their targets.

Drosophilid species (Insecta, Diptera) in the Brazilian savanna are more symmetric in the dry season

The development of an organism is a controlled process, which can be disrupted by genetic or environmental stress. Although fluctuating asymmetry is widely used as an indicator of developmental instability, its effectiveness has been questioned due to the contradictory results produced by this technique which, at least in part, probably reflects methodological inappropriateness. Here, we investigated if wing asymmetry of drosophilids increases when they develop during the dry season in the Brazilian savanna, considered a stressful season for these insects. Using protocols designed to avoid methodological problems, we analysed the wings of Zaprionus indianus and three species of the genus Drosophila (D. mercatorum, D. simulans, and D. sturtevanti). Surprisingly, the flies were more asymmetrical in the rainy season than in the dry season, although this difference was not always significant. It is suggested that the wings of these insects are submitted to strong natural selection throughout the dry season, when the asymmetric individuals would have less chance of surviving. During the wet season, on the other hand, selective pressures would be more relaxed. Therefore, the results of this study suggest that asymmetry of the wings is not a good indicator of natural environmental stress in drosophilids. Future research should focus on selectively neutral characters.

False and true positives in arthropod thermal adaptation candidate gene lists

Genome-wide studies are prone to false positives due to inherently low priors and statistical power. One approach to ameliorate this problem is to seek validation of reported candidate genes across independent studies: genes with repeatedly discovered effects are less likely to be false positives. Inversely, genes reported only as many times as expected by chance alone, while possibly representing novel discoveries, are also more likely to be false positives. We show that, across over 30 genome-wide studies that reported Drosophila and Daphnia genes with possible roles in thermal adaptation, the combined lists of candidate genes and orthologous groups are rapidly approaching the total number of genes and orthologous groups in the respectivegenomes. This isconsistent with the expectation of high frequency of false positives. The majority of these spurious candidates have been identified by one or a few studies, as expected by chance alone. In contrast, a noticeable minority of genes have been identified by numerous studies with the probabilities of such discoveries occurring by chance alone being exceedingly small. For this subset of genes, different studies are in agreement with each other despite differences in the ecological settings, genomic tools and methodology, and reporting thresholds. We provide a reference set of presumed true positives among Drosophila candidate genes and orthologous groups involved in response to changes in temperature, suitable for cross-validation purposes. Despite this approach being prone to false negatives, this list of presumed true positives includes several hundred genes, consistent with the "omnigenic" concept of genetic architecture of complex traits.

The spontaneous mutation rate of Drosophila pseudoobscura.

The spontaneous mutation rate is a very variable trait that is subject to drift, selection and is sometimes highly plastic. Consequently, its variation between close species, or even between populations from the same species, can be very large. Here, I estimated the spontaneous mutation rate of Drosophila pseudoobscura and Drosophila persimilis crosses to explore the mutation rate variation within the Drosophila genus. All mutation rate estimations in Drosophila varied fourfold, probably explained by the sensitivity of the mutation rate to environmental and experimental conditions. Moreover, I found a very high mutation rate in the hybrid cross between D. pseudoobscura and D. persimilis, in agreement with known elevated mutation rate in hybrids. This mutation rate increase can be explained by heterozygosity and fitness decrease effects in hybrids.

Karyotype Description of Two Andean Species of the guarani Group of Drosophila (Díptera: Drosophilidae) and Cytological Notes.

The guarani group of Drosophila genus (Diptera: Drosophilidae) is formed by 24 species however the relationship of these species is not clear. In the present study are described the karyotypes of Drosophila sachapuyu Peñafiel and Rafael, 2018 and Drosophila zamorana Peñafiel and Rafael, 2018, two Andean species members of the guarani group. Mitotic chromosomes from cerebral ganglia of third stand larval were obtained by thermal shock and cell suspension techniques. The karyotype of D. sachapuyu, presents 2n = 10 (4R, 1V; X = R, Y = R) while D. zamorana exhibits karyotype 2n = 12 (5R, 1V; X = V, Y = R).

Sight of parasitoid wasps accelerates sexual behavior and upregulates a micropeptide gene in Drosophila

Parasitoid wasps inflict widespread death upon the insect world. Hundreds of thousands of parasitoid wasp species kill a vast range of insect species. Insects have evolved defensive responses to the threat of wasps, some cellular and some behavioral. Here we find an unexpected response of adult Drosophila to the presence of certain parasitoid wasps: accelerated mating behavior. Flies exposed to certain wasp species begin mating more quickly. The effect is mediated via changes in the behavior of the female fly and depends on visual perception. The sight of wasps induces the dramatic upregulation in the fly nervous system of a gene that encodes a 41-amino acid micropeptide. Mutational analysis reveals that the gene is essential to the behavioral response of the fly. Our work provides a foundation for further exploration of how the activation of visual circuits by the sight of a wasp alters both sexual behavior and gene expression.

Interrogating the 5'UTR tandem repeats of retrotransposon roo of Drosophila about horizontal transfer.

Horizontal transfer in Drosophila has been inferred for several families of transposable elements. Specifically, the retroelement roo has been suggested to have been horizontally transferred between the species D. melanogaster, D. simulans, D. sechellia and D. yakuba. The inferences were based on the observation that divergence between transposable elements in different species was lower than the divergence found in typical nuclear genes and in the incongruence of phylogenies of the species and their TEs. Here, we address the question of the possible horizontal transfer of roo between species of the Drosophila genus by studying the presence absence of a duplication of 99bp in the 5'UTR of the transposon, as well as comparing the sequences of the paralogous and orthologous duplicated repeats within and between species. First, the repeats were only found in five species of the melanogaster subgroup. Second, the date of occurrence of the duplication event originating the repeats was posterior to the split of the subgroup. The duplication date suggests an origin previous to the split of D. simulans and D. sechellia and close to the divergence of D. melanogaster from the D. simulans complex. These data point to horizontal transfer to the afrotropical species D. yakuba and D. erecta from one of the cosmopolitan species D. melanogaster or D. simulans. We propose that the parasitoid wasp Leptopilina could have been the vector of horizontal transfer after the observation that a sequence of 845bp with high homology to a fragment of roo was isolated from this wasp.

Functional assessment of the "two-hit" model for neurodevelopmental defects in Drosophila and X. laevis.

We previously identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. We hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while "second-hits" in the genetic background modulate the phenotypic trajectory. To test this model, we examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of "second-hit" genes in Drosophila melanogaster and Xenopus laevis. We observed that knockdown of 16p12.1 homologs affect multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. Compared to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs were less likely to interact with each other in Drosophila models or a human brain-specific interaction network, suggesting that interactions with "second-hit" genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 pairwise interactions in Drosophila between 16p12.1 homologs and 76 homologs of patient-specific "second-hit" genes (such as ARID1B and CACNA1A), genes within neurodevelopmental pathways (such as PTEN and UBE3A), and transcriptomic targets (such as DSCAM and TRRAP) identified genetic interactions in 63% of the tested pairs. In 11 out of 15 families, patient-specific "second-hits" enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs in 32/96 pairwise combinations tested. In fact, homologs of SETD5 synergistically interacted with homologs of MOSMO in both Drosophila and X. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. Our results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with "second-hit" genes determine the ultimate phenotypic manifestation.

Importin-9 regulates chromosome segregation and packaging in Drosophila germ cells.

Germ cells undergo distinct nuclear processes as they differentiate into gametes. Although these events must be coordinated to ensure proper maturation, the stage-specific transport of proteins in and out of germ cell nuclei remains incompletely understood. Our efforts to genetically characterize Drosophila genes that exhibit enriched expression in germ cells led to the finding that loss of the highly conserved Importin β/karyopherin family member Importin-9 (Ipo9, herein referring to Ranbp9) results in female and male sterility. Immunofluorescence and fluorescent in situ hybridization revealed that Ipo9KO mutants display chromosome condensation and segregation defects during meiosis. In addition, Ipo9KO mutant males form abnormally structured sperm and fail to properly exchange histones for protamines. Ipo9 physically interacts with proteasome proteins, and Ipo9 mutant males exhibit disruption of the nuclear localization of several proteasome components. Thus, Ipo9 coordinates the nuclear import of functionally related factors necessary for the completion of gametogenesis. This article has an associated First Person interview with the first author of the paper.

Functional analysis of SARS-CoV-2 proteins in Drosophila identifies Orf6-induced pathogenic effects with Selinexor as an effective treatment

BackgroundSARS-CoV-2 causes COVID-19 with a widely diverse disease profile that affects many different tissues. The mechanisms underlying its pathogenicity in host organisms remain unclear. Animal models for studying the pathogenicity of SARS-CoV-2 proteins are lacking.MethodsUsing bioinformatic analysis, we found that 90% of the virus-host interactions involve human proteins conserved in Drosophila. Therefore, we generated a series of transgenic fly lines for individual SARS-CoV-2 genes, and used the Gal4-UAS system to express these viral genes in Drosophila to study their pathogenicity.ResultsWe found that the ubiquitous expression of Orf6, Nsp6 or Orf7a in Drosophila led to reduced viability and tissue defects, including reduced trachea branching as well as muscle deficits resulting in a “held-up” wing phenotype and poor climbing ability. Furthermore, muscles in these flies showed dramatically reduced mitochondria. Since Orf6 was found to interact with nucleopore proteins XPO1, we tested Selinexor, a drug that inhibits XPO1, and found that it could attenuate the Orf6-induced lethality and tissue-specific phenotypes observed in flies.ConclusionsOur study established Drosophila as a model for studying the function of SARS-CoV2 genes, identified Orf6 as a highly pathogenic protein in various tissues, and demonstrated the potential of Selinexor for inhibiting Orf6 toxicity using an in vivo animal model system.