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1.
Sheng Wu Gong Cheng Xue Bao ; 36(10): 2171-2180, 2020 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-33169581

RESUMO

In most insects, polyunsaturated fatty acids (PUFAs) are mainly polyunsaturated fatty acids with a carbon-chain length less than 18 carbon atoms, hardly any long-chain polyunsaturated fatty acids such as C20 and C22 that are more valuable and bioactive. This study, by using Drosophila melanogaster (Fruit fly) as a model organism, optimized the Δ6-fatty acid elongase enzyme Elovl5 gene from mice and transferred it to fruit flies for expression. Vectors containing Elovl5 gene were successfully injected into drosophila embryo through the microscopic injection. There were enhanced green fluorescent proteins expressed in the whole developmental stage of Drosophila be means of fluorescence microscope. At the same time, expression of Elovl5 gene significantly contributed to the transformation of fruit flies C18-polyunsaturated fatty acids in the body towards the biosynthesis of longer-chain polyunsaturated fatty acids. The transgenic fruit fly model rich in long-chain polyunsaturated fatty acids such as C20 and C22 were obtained, providing a basis for further research on biosynthesis of polyunsaturated fatty acids in fruit flies.


Assuntos
Acetiltransferases , Drosophila melanogaster , Elongases de Ácidos Graxos , Ácidos Graxos , Acetiltransferases/genética , Animais , Drosophila melanogaster/genética , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , Técnicas de Transferência de Genes , Camundongos
2.
PLoS One ; 15(10): e0234223, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33057335

RESUMO

Matching habitat choice is gaining attention as a mechanism for maintaining biodiversity and driving speciation. It revolves around the idea that individuals select the habitat in which they perceive to obtain greater fitness based on a prior evaluation of their local performance across heterogeneous environments. This results in individuals with similar ecologically relevant traits converging to the same patches, and hence it could indirectly cause assortative mating when mating occurs in those patches. White-eyed mutants of Drosophila fruit flies have a series of disadvantages compared to wild type flies, including a poorer performance under bright light. It has been previously reported that, when given a choice, wild type Drosophila simulans preferred a brightly lit habitat while white-eyed mutants occupied a dimly lit one. This spatial segregation allowed the eye color polymorphism to be maintained for several generations, whereas normally it is quickly replaced by the wild type. Here we compare the habitat choice decisions of white-eyed and wild type flies in another species, D. melanogaster. We released groups of flies in a light gradient and recorded their departure and settlement behavior. Departure depended on sex and phenotype, but not on the light conditions of the release point. Settlement depended on sex, and on the interaction between phenotype and light conditions of the point of settlement. Nonetheless, simulations showed that this differential habitat use by the phenotypes would only cause a minimal degree of assortative mating in this species.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Drosophila simulans/fisiologia , Mutação , Animais , Comportamento Animal , Comportamento de Escolha , Drosophila melanogaster/classificação , Drosophila simulans/classificação , Ecossistema , Cor de Olho , Feminino , Luz , Masculino , Preferência de Acasalamento Animal , Fenótipo
3.
Nat Commun ; 11(1): 5451, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116142

RESUMO

The genetics of phenotypic responses to changing environments remains elusive. Using whole-genome quantitative gene expression as a model, here we study how the genetic architecture of regulatory variation in gene expression changed in a population of fully sequenced inbred Drosophila melanogaster strains when flies developed in different environments (25 °C and 18 °C). We find a substantial fraction of the transcriptome exhibited genotype by environment interaction, implicating environmentally plastic genetic architecture of gene expression. Genetic variance in expression increases at 18 °C relative to 25 °C for most genes that have a change in genetic variance. Although the majority of expression quantitative trait loci (eQTLs) for the gene expression traits in the two environments are shared and have similar effects, analysis of the environment-specific eQTLs reveals enrichment of binding sites for two transcription factors. Finally, although genotype by environment interaction in gene expression could potentially disrupt genetic networks, the co-expression networks are highly conserved across environments. Genes with higher network connectivity are under stronger stabilizing selection, suggesting that stabilizing selection on expression plays an important role in promoting network robustness.


Assuntos
Drosophila melanogaster/genética , Interação Gene-Ambiente , Animais , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes de Insetos , Variação Genética , Genótipo , Masculino , Locos de Características Quantitativas , RNA-Seq , Temperatura , Transcriptoma
4.
PLoS One ; 15(10): e0237201, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33119606

RESUMO

Temperature varies on a daily and seasonal scale and thermal fluctuations are predicted to become even more pronounced under future climate changes. Studies suggest that plastic responses are crucial for species' ability to cope with thermal stress including variability in temperature, but most often laboratory studies on thermal adaptation in plant and ectotherm organisms are performed at constant temperatures and few species included. Recent studies using fluctuating thermal regimes find that thermal performance is affected by both temperature mean and fluctuations, and that plastic responses likely will differ between species according to life strategy and selective past. Here we investigate how acclimation to fluctuating or constant temperature regimes, but with the same mean temperature, impact on heat stress tolerance across a plant (Arabidopsis thaliana) and two arthropod species (Orchesella cincta and Drosophila melanogaster) inhabiting widely different thermal microhabitats and with varying capability for behavioral stress avoidance. Moreover, we investigate the underlying metabolic responses of acclimation using NMR metabolomics. We find increased heat tolerance for D. melanogaster and A. thaliana exposed to fluctuating acclimation temperatures, but not for O. cincta. The response was most pronounced for A. thaliana, which also showed a stronger metabolome response to thermal fluctuations than both arthropods. Generally, sugars were more abundant across A. thaliana and D. melanogaster when exposed to fluctuating compared to constant temperature, whereas amino acids were less abundant. This pattern was not evident for O. cincta, and generally we do not find much evidence for similar metabolomics responses to fluctuating temperature acclimation across species. Differences between the investigated species' ecology and different ability to behaviorally thermoregulate may have shaped their physiological responses to thermal fluctuations.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Artrópodes/crescimento & desenvolvimento , Regulação da Temperatura Corporal , Drosophila melanogaster/crescimento & desenvolvimento , Resposta ao Choque Térmico , Metaboloma , Animais , Arabidopsis/metabolismo , Artrópodes/metabolismo , Drosophila melanogaster/metabolismo , Masculino
5.
Nat Commun ; 11(1): 5350, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093442

RESUMO

Sociality is among the most important motivators of human behaviour. However, the neural mechanisms determining levels of sociality are largely unknown, primarily due to a lack of suitable animal models. Here, we report the presence of a surprising degree of general sociality in Drosophila. A newly-developed paradigm to study social approach behaviour in flies reveal that social cues perceive through both vision and olfaction converged in a central brain region, the γ lobe of the mushroom body, which exhibite activation in response to social experience. The activity of these γ neurons control the motivational drive for social interaction. At the molecular level, the serotonergic system is critical for social affinity. These results demonstrate that Drosophila are highly sociable, providing a suitable model system for elucidating the mechanisms underlying the motivation for sociality.


Assuntos
Comportamento Animal/fisiologia , Drosophila melanogaster/fisiologia , Comportamento Social , Animais , Animais Geneticamente Modificados , Encéfalo/fisiologia , Sinais (Psicologia) , Drosophila melanogaster/genética , Feminino , Humanos , Masculino , Motivação/fisiologia , Corpos Pedunculados/fisiologia , Rede Nervosa/fisiologia , Sensação/fisiologia , Neurônios Serotoninérgicos/fisiologia
6.
BMC Bioinformatics ; 21(1): 463, 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33076827

RESUMO

BACKGROUND: Repetitive sequences account for a large proportion of eukaryotes genomes. Identification of repetitive sequences plays a significant role in many applications, such as structural variation detection and genome assembly. Many existing de novo repeat identification pipelines or tools make use of assembly of the high-frequency k-mers to obtain repeats. However, a certain degree of sequence coverage is required for assemblers to get the desired assemblies. On the other hand, assemblers cut the reads into shorter k-mers for assembly, which may destroy the structure of the repetitive regions. For the above reasons, it is difficult to obtain complete and accurate repetitive regions in the genome by using existing tools. RESULTS: In this study, we present a new method called RepAHR for de novo repeat identification by assembly of the high-frequency reads. Firstly, RepAHR scans next-generation sequencing (NGS) reads to find the high-frequency k-mers. Secondly, RepAHR filters the high-frequency reads from whole NGS reads according to certain rules based on the high-frequency k-mer. Finally, the high-frequency reads are assembled to generate repeats by using SPAdes, which is considered as an outstanding genome assembler with NGS sequences. CONLUSIONS: We test RepAHR on five data sets, and the experimental results show that RepAHR outperforms RepARK and REPdenovo for detecting repeats in terms of N50, reference alignment ratio, coverage ratio of reference, mask ratio of Repbase and some other metrics.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequências Repetitivas de Ácido Nucleico/genética , Software , Animais , Sequência de Bases , Bases de Dados Genéticas , Drosophila melanogaster/genética , Biblioteca Gênica , Genoma Humano , Humanos , Camundongos , Padrões de Referência , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de DNA/métodos , Estatística como Assunto , Fatores de Tempo
7.
BMC Bioinformatics ; 21(1): 478, 2020 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-33099301

RESUMO

BACKGROUND: Introns have been shown to be spliced in a defined order, and this order influences both alternative splicing regulation and splicing fidelity, but previous studies have only considered neighbouring introns. The detailed intron splicing order remains unknown. RESULTS: In this work, a method was developed that can calculate the intron splicing orders of all introns in each transcript. A simulation study showed that this method can accurately calculate intron splicing orders. I further applied this method to real S. pombe, fruit fly, Arabidopsis thaliana, and human sequencing datasets and found that intron splicing orders change from gene to gene and that humans contain more not in-order spliced transcripts than S. pombe, fruit fly and Arabidopsis thaliana. In addition, I reconfirmed that the first introns in humans are spliced slower than those in S. pombe, fruit fly, and Arabidopsis thaliana genome-widely. Both the calculated most likely orders and the method developed here are available on the web. CONCLUSIONS: A novel computational method was developed to calculate the intron splicing orders and applied the method to real sequencing datasets. I obtained intron splicing orders for hundreds or thousands of genes in four organisms. I found humans contain more number of not in-order spliced transcripts.


Assuntos
Arabidopsis/genética , Biologia Computacional/métodos , Drosophila melanogaster/genética , Íntrons/genética , Processamento de RNA/genética , Schizosaccharomyces/genética , Processamento Alternativo , Animais , Sequência de Bases , Humanos
8.
Zootaxa ; 4789(1): zootaxa.4789.1.1, 2020 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-33056443

RESUMO

The taxonomy of the subgenus Ashima of the genus Phortica is revised. A cladistic analysis of 66 morphological characters is conducted, covering 35 species (28 known and 7 new species: Phortica efragmentata sp. nov., P. andreagigoni sp. nov., P. watabei sp. nov., P. halimunensis sp. nov., P. akutsui sp. nov., P. kerinciensis sp. nov., and P. takehiroi sp. nov.) which correspond to 71.4% of 49 total spp. of Ashima. The resulting cladogram shows that the studied species are separated into several clades/subclades/cluster each highly supported with specific synapomorphies. Those clades/subclades/cluster are newly defined as the following species groups, subgroups or complex: the afoliolata, foliiseta, nigrifoliiseta and angulata species groups; the foliiseta, tanabei, nigrifoliiseta and glabra species subgroups; and the foliiseta species complex. The subgenus Ashima is peculiar in having the asymmetric male genitalia as the ground plan and showing the antisymmetry (i.e. intraspecific mirror-image variation) in some species but the directional asymmetry (i.e. side-fixed asymmetry) in others. The evolution of genital asymmetry in this subgenus is estimated by mapping the states (symmetry, directional asymmetry and antisymmetry) of bilateral structures of male genitalia on the cladogram. This ancestral state reconstruction estimates that the directional asymmetry of male genitalia has evolved at the ancestor of this subgenus and then changed to the antisymmetric state independently in two lineages, the angulata + nigrifoliiseta species groups and the foliiseta species complex. In this study, a standardized terminology recently proposed for the male terminalia of Drosophila melanogaster is extendedly adopted to describe the morphology of male terminalia in the subgenus Ashima, one group of the subfamily Steganinae.


Assuntos
Drosophilidae , Animais , Drosophila melanogaster , Genitália , Genitália Masculina , Masculino
9.
Science ; 370(6514)2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060329

RESUMO

Biological systems tailor their properties and behavior to their size throughout development and in numerous aspects of physiology. However, such size scaling remains poorly understood as it applies to cell mechanics and mechanosensing. By examining how the Drosophila pupal dorsal thorax epithelium responds to morphogenetic forces, we found that the number of apical stress fibers (aSFs) anchored to adherens junctions scales with cell apical area to limit larger cell elongation under mechanical stress. aSFs cluster Hippo pathway components, thereby scaling Hippo signaling and proliferation with area. This scaling is promoted by tricellular junctions mediating an increase in aSF nucleation rate and lifetime in larger cells. Development, homeostasis, and repair entail epithelial cell size changes driven by mechanical forces; our work highlights how, in turn, mechanosensitivity scales with cell size.


Assuntos
Epitélio/fisiologia , Mecanotransdução Celular , Fibras de Estresse/fisiologia , Estresse Mecânico , Animais , Caderinas/metabolismo , Tamanho Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células Epiteliais/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miosina Tipo II/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/metabolismo
10.
Science ; 370(6514): 321-327, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060356

RESUMO

Morphogen gradients provide positional information during development. To uncover the minimal requirements for morphogen gradient formation, we have engineered a synthetic morphogen in Drosophila wing primordia. We show that an inert protein, green fluorescent protein (GFP), can form a detectable diffusion-based gradient in the presence of surface-associated anti-GFP nanobodies, which modulate the gradient by trapping the ligand and limiting leakage from the tissue. We next fused anti-GFP nanobodies to the receptors of Dpp, a natural morphogen, to render them responsive to extracellular GFP. In the presence of these engineered receptors, GFP could replace Dpp to organize patterning and growth in vivo. Concomitant expression of glycosylphosphatidylinositol (GPI)-anchored nonsignaling receptors further improved patterning, to near-wild-type quality. Theoretical arguments suggest that GPI anchorage could be important for these receptors to expand the gradient length scale while at the same time reducing leakage.


Assuntos
Padronização Corporal , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Fluorescência Verde/genética , Discos Imaginais/crescimento & desenvolvimento , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Asas de Animais/crescimento & desenvolvimento
11.
Science ; 370(6514): 327-331, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060357

RESUMO

In metazoan tissues, cells decide their fates by sensing positional information provided by specialized morphogen proteins. To explore what features are sufficient for positional encoding, we asked whether arbitrary molecules (e.g., green fluorescent protein or mCherry) could be converted into synthetic morphogens. Synthetic morphogens expressed from a localized source formed a gradient when trapped by surface-anchoring proteins, and they could be sensed by synthetic receptors. Despite their simplicity, these morphogen systems yielded patterns reminiscent of those observed in vivo. Gradients could be reshaped by altering anchor density or by providing a source of competing inhibitor. Gradient interpretation could be altered by adding feedback loops or morphogen cascades to receiver cell response circuits. Orthogonal cell-cell communication systems provide insight into morphogen evolution and a platform for engineering tissues.


Assuntos
Padronização Corporal , Proteínas de Fluorescência Verde/metabolismo , Engenharia Tecidual/métodos , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Fibroblastos , Proteínas de Fluorescência Verde/genética , Engenharia de Proteínas , Receptores Notch/genética , Receptores Notch/metabolismo
12.
Mol Cell ; 80(1): 156-163.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007255

RESUMO

The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Éxons/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Masculino , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
13.
Mol Cell ; 80(1): 140-155.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007254

RESUMO

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.


Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Neurônios/metabolismo , Processamento Alternativo/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas ELAV/química , Larva/metabolismo , Mutação/genética , Poli A/metabolismo , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Mol Cell ; 80(2): 246-262.e4, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32949493

RESUMO

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.


Assuntos
Deleção de Genes , Tecnologia de Impulso Genético , Animais , Proteína 9 Associada à CRISPR/metabolismo , Cromossomos/genética , Drosophila melanogaster/genética , Feminino , Proteínas de Fluorescência Verde/metabolismo , Padrões de Herança/genética , Mutagênese/genética , RNA Guia/genética , Transgenes
15.
Neuron ; 107(6): 1071-1079.e2, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32931755

RESUMO

Drosophila melanogaster is an established model for neuroscience research with relevance in biology and medicine. Until recently, research on the Drosophila brain was hindered by the lack of a complete and uniform nomenclature. Recognizing this, Ito et al. (2014) produced an authoritative nomenclature for the adult insect brain, using Drosophila as the reference. Here, we extend this nomenclature to the adult thoracic and abdominal neuromeres, the ventral nerve cord (VNC), to provide an anatomical description of this major component of the Drosophila nervous system. The VNC is the locus for the reception and integration of sensory information and involved in generating most of the locomotor actions that underlie fly behaviors. The aim is to create a nomenclature, definitions, and spatial boundaries for the Drosophila VNC that are consistent with other insects. The work establishes an anatomical framework that provides a powerful tool for analyzing the functional organization of the VNC.


Assuntos
Drosophila melanogaster/citologia , Gânglios dos Invertebrados/citologia , Rede Nervosa/citologia , Neurônios/classificação , Terminologia como Assunto , Animais , Linhagem da Célula , Drosophila melanogaster/fisiologia , Gânglios dos Invertebrados/fisiologia , Rede Nervosa/fisiologia , Neurônios/citologia , Neurônios/fisiologia
16.
J Vis Exp ; (163)2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986033

RESUMO

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.


Assuntos
Drosophila melanogaster/embriologia , Embrião não Mamífero/diagnóstico por imagem , Imageamento Tridimensional , Microscopia de Fluorescência/métodos , Tribolium/embriologia , Animais , Animais Geneticamente Modificados , Calibragem , Análise de Dados , Desenvolvimento Embrionário , Hipoclorito de Sódio
17.
Proc Natl Acad Sci U S A ; 117(38): 23960-23969, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900926

RESUMO

Many organisms enter a dormant state in their life cycle to deal with predictable changes in environments over the course of a year. The timing of dormancy is therefore a key seasonal adaptation, and it evolves rapidly with changing environments. We tested the hypothesis that differences in the timing of seasonal activity are driven by differences in the rate of development during diapause in Rhagoletis pomonella, a fly specialized to feed on fruits of seasonally limited host plants. Transcriptomes from the central nervous system across a time series during diapause show consistent and progressive changes in transcripts participating in diverse developmental processes, despite a lack of gross morphological change. Moreover, population genomic analyses suggested that many genes of small effect enriched in developmental functional categories underlie variation in dormancy timing and overlap with gene sets associated with development rate in Drosophila melanogaster Our transcriptional data also suggested that a recent evolutionary shift from a seasonally late to a seasonally early host plant drove more rapid development during diapause in the early fly population. Moreover, genetic variants that diverged during the evolutionary shift were also enriched in putative cis regulatory regions of genes differentially expressed during diapause development. Overall, our data suggest polygenic variation in the rate of developmental progression during diapause contributes to the evolution of seasonality in R. pomonella We further discuss patterns that suggest hourglass-like developmental divergence early and late in diapause development and an important role for hub genes in the evolution of transcriptional divergence.


Assuntos
Adaptação Fisiológica/genética , Diapausa/genética , Tephritidae , Transcriptoma/genética , Animais , Drosophila melanogaster/genética , Estudo de Associação Genômica Ampla , Estações do Ano , Tephritidae/genética , Tephritidae/crescimento & desenvolvimento
18.
Mol Cell ; 80(2): 345-358.e9, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32966759

RESUMO

Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here we report that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases.


Assuntos
Proteínas de Drosophila/metabolismo , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/metabolismo , Terminação da Transcrição Genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Conservada , Proteínas de Drosophila/química , Drosophila melanogaster , Regulação da Expressão Gênica , Loci Gênicos , Humanos , Fosforilação , Regiões Promotoras Genéticas , Subunidades Proteicas/química , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Transdução de Sinais , Especificidade por Substrato
19.
PLoS Genet ; 16(9): e1009011, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986715

RESUMO

Neuronal precursor cells undergo self-renewing and non-self-renewing asymmetric divisions to generate a large number of neurons of distinct identities. In Drosophila, primary precursor neuroblasts undergo a varying number of self-renewing asymmetric divisions, with one known exception, the MP2 lineage, which undergoes just one terminal asymmetric division similar to the secondary precursor cells. The mechanism and the genes that regulate the transition from self-renewing to non-self-renewing asymmetric division or the number of times a precursor divides is unknown. Here, we show that the T-box transcription factor, Midline (Mid), couples these events. We find that in mid loss of function mutants, MP2 undergoes additional self-renewing asymmetric divisions, the identity of progeny neurons generated dependent upon Numb localization in the parent MP2. MP2 expresses Mid transiently and an over-expression of mid in MP2 can block its division. The mechanism which directs the self-renewing asymmetric division of MP2 in mid involves an upregulation of Cyclin E. Our results indicate that Mid inhibits cyclin E gene expression by binding to a variant Mid-binding site in the cyclin E promoter and represses its expression without entirely abolishing it. Consistent with this, over-expression of cyclin E in MP2 causes its multiple self-renewing asymmetric division. These results reveal a Mid-regulated pathway that restricts the self-renewing asymmetric division potential of cells via inhibiting cyclin E and facilitating their exit from cell cycle.


Assuntos
Divisão Celular/genética , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Ciclo Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Hormônios Juvenis/genética , Hormônios Juvenis/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas com Domínio T/genética , Fatores de Transcrição/genética
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