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1.
Dev Genes Evol ; 230(4): 279-294, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32623522

RESUMO

Genome studies have uncovered many examples of essential gene loss, raising the question of how ancient genes transition from essentiality to dispensability. We explored this process for the deeply conserved E3 ubiquitin ligase Murine double minute (Mdm), which is lacking in Drosophila despite the conservation of its main regulatory target, the cellular stress response gene p53. Conducting gene expression and knockdown experiments in the red flour beetle Tribolium castaneum, we found evidence that Mdm has remained essential in insects where it is present. Using bioinformatics approaches, we confirm the absence of the Mdm gene family in Drosophila, mapping its loss to the stem lineage of schizophoran Diptera and Pipunculidae (big-headed flies), about 95-85 million years ago. Intriguingly, this gene loss event was preceded by the de novo origin of the gene Companion of reaper (Corp), a novel p53 regulatory factor that is characterized by functional similarities to vertebrate Mdm2 despite lacking E3 ubiquitin ligase protein domains. Speaking against a 1:1 compensatory gene gain/loss scenario, however, we found that hoverflies (Syrphidae) and pointed-wing flies (Lonchopteridae) possess both Mdm and Corp. This implies that the two p53 regulators have been coexisting for ~ 150 million years in select dipteran clades and for at least 50 million years in the lineage to Schizophora and Pipunculidae. Given these extensive time spans of Mdm/Corp coexistence, we speculate that the loss of Mdm in the lineage to Drosophila involved further acquisitions of compensatory gene activities besides the emergence of Corp. Combined with the previously noted reduction of an ancestral P53 contact domain in the Mdm homologs of crustaceans and insects, we conclude that the loss of the ancient Mdm gene family in flies was the outcome of incremental functional regression over long macroevolutionary time scales.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Genes Essenciais/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Tribolium/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Evolução Molecular , Técnicas de Silenciamento de Genes , Genômica , Filogenia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Tribolium/embriologia , Proteína Supressora de Tumor p53/genética
2.
Proc Natl Acad Sci U S A ; 117(28): 16606-16615, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601210

RESUMO

Neural network function requires an appropriate balance of excitation and inhibition to be maintained by homeostatic plasticity. However, little is known about homeostatic mechanisms in the intact central brain in vivo. Here, we study homeostatic plasticity in the Drosophila mushroom body, where Kenyon cells receive feedforward excitation from olfactory projection neurons and feedback inhibition from the anterior paired lateral neuron (APL). We show that prolonged (4-d) artificial activation of the inhibitory APL causes increased Kenyon cell odor responses after the artificial inhibition is removed, suggesting that the mushroom body compensates for excess inhibition. In contrast, there is little compensation for lack of inhibition (blockade of APL). The compensation occurs through a combination of increased excitation of Kenyon cells and decreased activation of APL, with differing relative contributions for different Kenyon cell subtypes. Our findings establish the fly mushroom body as a model for homeostatic plasticity in vivo.


Assuntos
Drosophila/fisiologia , Corpos Pedunculados/fisiologia , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Homeostase , Neurônios/fisiologia , Odorantes/análise , Olfato
3.
Mol Cell ; 79(2): 268-279.e5, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32592682

RESUMO

Circular RNAs (circRNAs) are abundant and accumulate with age in neurons of diverse species. However, only few circRNAs have been functionally characterized, and their role during aging has not been addressed. Here, we use transcriptome profiling during aging and find that accumulation of circRNAs is slowed down in long-lived insulin mutant flies. Next, we characterize the in vivo function of a circRNA generated by the sulfateless gene (circSfl), which is consistently upregulated, particularly in the brain and muscle, of diverse long-lived insulin mutants. Strikingly, lifespan extension of insulin mutants is dependent on circSfl, and overexpression of circSfl alone is sufficient to extend the lifespan. Moreover, circSfl is translated into a protein that shares the N terminus and potentially some functions with the full-length Sfl protein encoded by the host gene. Our study demonstrates that insulin signaling affects global circRNA accumulation and reveals an important role of circSfl during aging in vivo.


Assuntos
Drosophila/fisiologia , Insulina/fisiologia , Longevidade/genética , RNA Circular/fisiologia , Envelhecimento , Animais , Animais Geneticamente Modificados , Drosophila/genética , Proteínas de Drosophila/genética , Feminino , Masculino , Mutação , Neurônios/fisiologia , Sulfotransferases/genética , Transcriptoma
4.
Nat Commun ; 11(1): 3095, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32555259

RESUMO

Genetically modified conditional lethal strains have been created to improve the control of insect pest populations damaging to human health and agriculture. However, understanding the potential for the genetic breakdown of lethality systems by rare spontaneous mutations, or selection for inherent suppressors, is critical since field release studies are in progress. This knowledge gap was addressed in a Drosophila tetracycline-suppressible embryonic lethality system by analyzing the frequency and structure of primary-site spontaneous mutations and second-site suppressors resulting in heritable survivors from 1.2 million zygotes. Here we report that F1 survivors due to primary-site deletions and indels occur at a 5.8 × 10-6 frequency, while survival due to second-site maternal-effect suppressors occur at a ~10-5 frequency. Survivors due to inherent lethal effector suppressors could result in a resistant field population, and we suggest that this risk may be mitigated by the use of dual redundant, albeit functionally unrelated, lethality systems.


Assuntos
Insetos/fisiologia , Controle da População/métodos , Animais , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Insetos/genética , Mutação/genética , Zigoto/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(21): 11573-11583, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404421

RESUMO

Animals interact with each other in species-specific reproducible patterns. These patterns of organization are captured by social network analysis, and social interaction networks (SINs) have been described for a wide variety of species including fish, insects, birds, and mammals. The aim of this study is to understand the evolution of social organization in Drosophila Using a comparative ecological, phylogenetic, and behavioral approach, the different properties of SINs formed by 20 drosophilids were compared. We investigate whether drosophilid network structures arise from common ancestry, a response to the species' past climate, other social behaviors, or a combination of these factors. This study shows that differences in past climate predicted the species' current SIN properties. The drosophilid phylogeny offered no value to predicting species' differences in SINs through phylogenetic signal tests. This suggests that group-level social behaviors in drosophilid species are shaped by divergent climates. However, we find that the social distance at which flies interact correlated with the drosophilid phylogeny, indicating that behavioral elements of SINs have remained largely unchanged in their evolutionary history. We find a significant correlation of leg length to social distance, outlining the interdependence of anatomy and complex social structures. Although SINs display a complex evolutionary relationship across drosophilids, this study suggests that the ecology, and not common ancestry, contributes to diversity in social structure in Drosophila.


Assuntos
Evolução Biológica , Drosophila , Meio Ambiente , Comportamento Social , Animais , Drosophila/classificação , Drosophila/genética , Drosophila/fisiologia , Feminino , Masculino , Filogenia
6.
Proc Natl Acad Sci U S A ; 117(24): 13757-13766, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467161

RESUMO

Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK-related K+ channels (TREK-1) and reversibly induce loss of consciousness. For 100 y, anesthetics were speculated to target cellular membranes, yet no plausible mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of phospholipase D2 (PLD2) localization to lipid rafts and subsequent production of signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK-1 currents in whole-cell patch-clamp recordings. Localization of PLD2 renders the TRAAK channel sensitive, a channel that is otherwise anesthetic insensitive. General anesthetics, such as chloroform, isoflurane, diethyl ether, xenon, and propofol, disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and PLDnull flies resist anesthesia. Our results establish a membrane-mediated target of inhaled anesthesia and suggest PA helps set thresholds of anesthetic sensitivity in vivo.


Assuntos
Anestésicos Inalatórios/administração & dosagem , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Clorofórmio/administração & dosagem , Drosophila/efeitos dos fármacos , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Isoflurano/administração & dosagem , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(24): 13541-13551, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467168

RESUMO

Within developing embryos, tissues flow and reorganize dramatically on timescales as short as minutes. This includes epithelial tissues, which often narrow and elongate in convergent extension movements due to anisotropies in external forces or in internal cell-generated forces. However, the mechanisms that allow or prevent tissue reorganization, especially in the presence of strongly anisotropic forces, remain unclear. We study this question in the converging and extending Drosophila germband epithelium, which displays planar-polarized myosin II and experiences anisotropic forces from neighboring tissues. We show that, in contrast to isotropic tissues, cell shape alone is not sufficient to predict the onset of rapid cell rearrangement. From theoretical considerations and vertex model simulations, we predict that in anisotropic tissues, two experimentally accessible metrics of cell patterns-the cell shape index and a cell alignment index-are required to determine whether an anisotropic tissue is in a solid-like or fluid-like state. We show that changes in cell shape and alignment over time in the Drosophila germband predict the onset of rapid cell rearrangement in both wild-type and snail twist mutant embryos, where our theoretical prediction is further improved when we also account for cell packing disorder. These findings suggest that convergent extension is associated with a transition to more fluid-like tissue behavior, which may help accommodate tissue-shape changes during rapid developmental events.


Assuntos
Forma Celular , Drosophila/crescimento & desenvolvimento , Animais , Anisotropia , Drosophila/citologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epitélio/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo
8.
Nat Commun ; 11(1): 2073, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350270

RESUMO

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.


Assuntos
Doença/genética , Modelos Genéticos , Mutação de Sentido Incorreto/genética , PTEN Fosfo-Hidrolase/genética , Animais , Comportamento Animal , Caenorhabditis elegans/fisiologia , Células Cultivadas , Dendritos/fisiologia , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Ensaios Enzimáticos , Células HEK293 , Humanos , Neoplasias/genética , Sistema Nervoso/crescimento & desenvolvimento , Fosforilação , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Piramidais/metabolismo , Ratos Sprague-Dawley , Saccharomyces cerevisiae/metabolismo
9.
Nat Commun ; 11(1): 2084, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350282

RESUMO

Single-cell RNA sequencing (scRNA-seq) provides details for individual cells; however, crucial spatial information is often lost. We present SpaOTsc, a method relying on structured optimal transport to recover spatial properties of scRNA-seq data by utilizing spatial measurements of a relatively small number of genes. A spatial metric for individual cells in scRNA-seq data is first established based on a map connecting it with the spatial measurements. The cell-cell communications are then obtained by "optimally transporting" signal senders to target signal receivers in space. Using partial information decomposition, we next compute the intercellular gene-gene information flow to estimate the spatial regulations between genes across cells. Four datasets are employed for cross-validation of spatial gene expression prediction and comparison to known cell-cell communications. SpaOTsc has broader applications, both in integrating non-spatial single-cell measurements with spatial data, and directly in spatial single-cell transcriptomics data to reconstruct spatial cellular dynamics in tissues.


Assuntos
Transdução de Sinais/genética , Análise de Célula Única , Transcriptoma/genética , Animais , Comunicação Celular , Análise por Conglomerados , Bases de Dados Genéticas , Drosophila/embriologia , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Córtex Visual/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética
10.
PLoS Genet ; 16(5): e1008767, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32357156

RESUMO

Despite the importance of dendritic targeting in neural circuit assembly, the mechanisms by which it is controlled still remain incompletely understood. We previously showed that in the developing Drosophila antennal lobe, the Wnt5 protein forms a gradient that directs the ~45˚ rotation of a cluster of projection neuron (PN) dendrites, including the adjacent DA1 and VA1d dendrites. We report here that the Van Gogh (Vang) transmembrane planar cell polarity (PCP) protein is required for the rotation of the DA1/VA1d dendritic pair. Cell type-specific rescue and mosaic analyses showed that Vang functions in the olfactory receptor neurons (ORNs), suggesting a codependence of ORN axonal and PN dendritic targeting. Loss of Vang suppressed the repulsion of the VA1d dendrites by Wnt5, indicating that Wnt5 signals through Vang to direct the rotation of the DA1 and VA1d glomeruli. We observed that the Derailed (Drl)/Ryk atypical receptor tyrosine kinase is also required for the rotation of the DA1/VA1d dendritic pair. Antibody staining showed that Drl/Ryk is much more highly expressed by the DA1 dendrites than the adjacent VA1d dendrites. Mosaic and epistatic analyses showed that Drl/Ryk specifically functions in the DA1 dendrites in which it antagonizes the Wnt5-Vang repulsion and mediates the migration of the DA1 glomerulus towards Wnt5. Thus, the nascent DA1 and VA1d glomeruli appear to exhibit Drl/Ryk-dependent biphasic responses to Wnt5. Our work shows that the final patterning of the fly olfactory map is the result of an interplay between ORN axons and PN dendrites, wherein converging pre- and postsynaptic processes contribute key Wnt5 signaling components, allowing Wnt5 to orient the rotation of nascent synapses through a PCP mechanism.


Assuntos
Antenas de Artrópodes/crescimento & desenvolvimento , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Wnt/metabolismo , Animais , Antenas de Artrópodes/metabolismo , Axônios/metabolismo , Padronização Corporal , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Neurônios Receptores Olfatórios/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Proteínas Wnt/genética
11.
PLoS Genet ; 16(5): e1008815, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453722

RESUMO

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease.


Assuntos
Proteínas de Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Proteínas do Complexo SMN/genética , Animais , Regulação para Baixo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Olho/crescimento & desenvolvimento , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes Letais , Tamanho do Órgão , Proteínas de Ligação a RNA/metabolismo , Proteínas do Complexo SMN/metabolismo
12.
PLoS One ; 15(4): e0232046, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352996

RESUMO

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5' vs. 3' untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.


Assuntos
Engenharia Genética/métodos , RNA Catalítico/fisiologia , Biologia Sintética/métodos , Animais , Drosophila/genética , Eucariotos/genética , Eucariotos/metabolismo , Células Eucarióticas/metabolismo , Expressão Gênica/genética , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Conformação de Ácido Nucleico , Estudo de Prova de Conceito , RNA Catalítico/genética , RNA Mensageiro/metabolismo
13.
Nat Commun ; 11(1): 2228, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376880

RESUMO

Cell competition is an emerging principle that eliminates suboptimal or potentially dangerous cells. For 'unfit' cells to be detected, their competitive status needs to be compared to the collective fitness of cells within a tissue. Here we report that the NMDA receptor controls cell competition of epithelial cells and Myc supercompetitors in the Drosophila wing disc. While clonal depletion of the NMDA receptor subunit NR2 results in their rapid elimination via the TNF/Eiger>JNK signalling pathway, local over-expression of NR2 causes NR2 cells to acquire supercompetitor-like behaviour that enables them to overtake the tissue through clonal expansion that causes, but also relies on, the killing of surrounding cells. Consistently, NR2 is utilised by Myc clones to provide them with supercompetitor status. Mechanistically, we find that the JNK>PDK signalling axis in 'loser' cells reprograms their metabolism, driving them to produce and transfer lactate to winners. Preventing lactate transfer from losers to winners abrogates NMDAR-mediated cell competition. Our findings demonstrate a functional repurposing of NMDAR in the surveillance of tissue fitness.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Células Epiteliais/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Fatores de Transcrição/metabolismo , Asas de Animais/metabolismo , Animais , Apoptose/genética , Drosophila/genética , Proteínas de Drosophila/genética , Ácido Láctico/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Mitocôndrias/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Asas de Animais/crescimento & desenvolvimento
14.
J Vis Exp ; (157)2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32225149

RESUMO

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a cost-effective in vivo method to identify heterochromatin-promoting compounds by using Drosophila. The Drosophila's DX1 strain, having a variegated eye color phenotype that reflects the extents of heterochromatin formation, thereby providing a tool for a heterochromatin-promoting drug screen. In this screening method, eye variegation is quantified based on the surface area of red pigmentation occupying parts of the eye and is scored on a scale from 1 to 5. The screening method is straightforward and sensitive and allows for testing compounds in vivo. Drug screening using this method provides a fast and inexpensive way for identifying heterochromatin-promoting drugs that could have beneficial effects in cancer therapeutics. Identifying compounds that promote the formation of heterochromatin could also lead to the discovery of epigenetic mechanisms of cancer development.


Assuntos
Drosophila/genética , Desenho de Fármacos , Heterocromatina/metabolismo , Animais
15.
PLoS Comput Biol ; 16(4): e1007750, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251432

RESUMO

In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. In this work we investigate the robustness of the Dorsal/NF-κB signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and we found that an empirical description of the Dorsal gradient is highly sensitive to maternal dorsal dosage. In contrast, we found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, we analyzed a mechanistic model of the Dorsal signaling module and found that Cactus, a cytoplasmic inhibitor for Dorsal, must be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. These mechanisms synergistically contribute to changing the amplitude and shape of the active Dorsal gradient, which is required for robust gene expression. Our work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness.


Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese/genética , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(16): 9013-9021, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32245808

RESUMO

Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes, is needed for long-term success. Here, we show that resilience can be achieved through cycles of population modification with "Cleave and Rescue" (ClvR) selfish genetic elements. ClvR comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Cycles of modification can, in principle, be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvR n+1, carries a Rescue transgene from an earlier element, ClvR n ClvR n+1 should spread within a population of ClvR n, while also bringing about a decrease in its frequency. To test this hypothesis, we first show that multiple ClvRs, each targeting a different essential gene, function when located at a common chromosomal position in Drosophila We then show that when several of these also carry the Rescue from a different ClvR, they spread to transgene fixation in populations fixed for the latter and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control.


Assuntos
Drosophila/genética , Tecnologia de Impulso Genético , Genes Essenciais/genética , Genética Populacional/métodos , Modelos Genéticos , Animais , Animais Geneticamente Modificados , Feminino , Heterozigoto , Masculino , Modelos Animais
17.
Arch Insect Biochem Physiol ; 104(2): e21675, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32285519

RESUMO

The Drosophila inner photoreceptors R7 and R8 are responsible for color vision and their differentiation starts at the third instar larval stage. Only a handful of genes with R7 or R8-cell-specific expression are known. We performed an enhancer-trap screen using a novel piggyBac transposable element, pBGay, carrying a Gal4 sequence under the control of the P promoter to identify novel genes expressed specifically in R7 or R8 cells. From this screen, three lines were analyzed in detail: piggyBacAC109 and piggyBacAC783 are expressed in R8 cells and piggyBacAC887 is expressed in R7 cells at the third instar larval stage and pupal stages. Molecular analysis showed that the piggyBac elements were inserted into the first intron of CG14160 and CG7985 genes and the second intron of unzipped. We show the expression pattern in the developing eye imaginal disc, pupal retina as well as the adult retina. The photoreceptor-specific expression of these genes is reported for the first time and we propose that these lines are useful tools for studying the development of the visual system.


Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Drosophila/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Fatores de Transcrição/metabolismo
18.
Mol Cell ; 78(4): 785-793.e8, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32229306

RESUMO

RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Fator de Transcrição TFIID/metabolismo , Transcrição Genética , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Células HCT116 , Humanos , Ligação Proteica , RNA Polimerase II/metabolismo , Fator de Transcrição TFIID/genética
19.
PLoS Comput Biol ; 16(3): e1007718, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226007

RESUMO

Mechanisms that control movements range from navigational mechanisms, in which the animal employs directional cues to reach a specific destination, to search movements during which there are little or no environmental cues. Even though most real-world movements result from an interplay between these mechanisms, an experimental system and theoretical framework for the study of interplay of these mechanisms is not available. Here, we rectify this deficit. We create a new method to stimulate the olfactory system in Drosophila or fruit flies. As flies explore a circular arena, their olfactory receptor neuron (ORNs) are optogenetically activated within a central region making this region attractive to the flies without emitting any clear directional signals outside this central region. In the absence of ORN activation, the fly's locomotion can be described by a random walk model where a fly's movement is described by its speed and turn-rate (or kinematics). Upon optogenetic stimulation, the fly's behavior changes dramatically in two respects. First, there are large kinematic changes. Second, there are more turns at the border between light-zone and no-light-zone and these turns have an inward bias. Surprisingly, there is no increase in turn-rate, rather a large decrease in speed that makes it appear that the flies are turning at the border. Similarly, the inward bias of the turns is a result of the increase in turn angle. These two mechanisms entirely account for the change in a fly's locomotion. No complex mechanisms such as path-integration or a careful evaluation of gradients are necessary.


Assuntos
Drosophila , Locomoção/genética , Odorantes , Neurônios Receptores Olfatórios/fisiologia , Animais , Comportamento Animal , Biologia Computacional , Drosophila/genética , Drosophila/fisiologia , Locomoção/fisiologia , Optogenética , Olfato/genética , Olfato/fisiologia
20.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118713, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32246948

RESUMO

WD40-Repeat Protein 62 (WDR62) is required to maintain neural and glial cell populations during embryonic brain growth. Although elevated expression of WDR62 is frequently associated with several tumour types, potential effects of excess WDR62 on proliferative growth remain undefined. Here, we demonstrate that glia specific overexpression of WDR62 in Drosophila larval brains resulted in increased cell size, over-proliferation and increased brain volume, without overt disruption of tissue organization. We further demonstrate WDR62 promoted over-proliferation and brain overgrowth by activating AURKA and pAKT signalling to increase MYC function in glial cells. Together these data suggest WDR62 normally functions in the glial lineage to activate oncogenic signalling networks, promoting proliferation and brain overgrowth.


Assuntos
Aurora Quinase A/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proliferação de Células/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Neurogênese/genética , Neuroglia/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Fuso Acromático/genética
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