RESUMO
Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.
Assuntos
Ataxina-2 , Proteínas de Drosophila , Drosophila melanogaster , RNA Mensageiro , Ribonucleoproteínas , Ataxina-2/genética , Ataxina-2/metabolismo , Animais , Humanos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/genética , Animais Geneticamente Modificados , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a DNARESUMO
Habituation, the process by which animals learn to ignore insignificant stimuli, facilitates engagement with salient features of the environment. However, neural mechanisms underlying habituation also allow responses to familiar stimuli to be reinstated when such stimuli become potentially significant. Thus, the habituated state must allow a mechanism for habituation override. The remarkably precise knowledge of cell identity, connectivity, and information coding in Drosophila sensory circuits, as well as the availability of tools to genetically target these cells, makes Drosophila a valuable and important organism for analysis of habituation and habituation-override mechanisms. Studies of olfactory and gustatory habituation in Drosophila suggest that potentiation of GABAergic neurons underlies certain timescales of habituation and have specified some elements of a gustatory habituation-override pathway. More detailed understanding of gustatory habituation and habituation-override mechanisms will benefit from access to robust behavioral assays for (a) the proboscis extension reflex (PER) elicited by a sweet stimulus, (b) exposure paradigms that result in PER habituation, and, most critically, (c) manipulations that result in PER-habituation override. Here, we describe simple protocols for persistent sucrose exposure of tarsal hairs that lead to habituation of proboscis extension and for presentation of a novel appetitive stimuli that reinstate robust PER to habituated flies. This detailed protocol of gustatory habituation provides (a) a simple method to induce habituation by continuous exposure of the flies to sucrose for 10 min without leading to ingestion and (b) a novel method to override habituation by presenting yeast to the proboscis. Key features ⢠A protocol for stimulation of Drosophila's taste (sugar) sensory neurons that induces gustatory habituation without satiation due to ingestion. ⢠A chemical (yeast) stimulation protocol that rapidly induces habituation override/dishabituation in sugar-habituated Drosophila.
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How compartment-specific local proteomes are generated and maintained is inadequately understood, particularly in neurons, which display extreme asymmetries. Here we show that local enrichment of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in axons of Drosophila mushroom body neurons is necessary for cellular plasticity and associative memory formation. Enrichment is achieved via enhanced axoplasmic translation of CaMKII mRNA, through a mechanism requiring the RNA-binding protein Mub and a 23-base Mub-recognition element in the CaMKII 3' UTR. Perturbation of either dramatically reduces axonal, but not somatic, CaMKII protein without altering the distribution or amount of mRNA in vivo, and both are necessary and sufficient to enhance axonal translation of reporter mRNA. Together, these data identify elevated levels of translation of an evenly distributed mRNA as a novel strategy for generating subcellular biochemical asymmetries. They further demonstrate the importance of distributional asymmetry in the computational and biological functions of neurons.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Neurônios , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Drosophila/genética , Corpos Pedunculados/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismoRESUMO
Habituated animals retain a latent capacity for robust engagement with familiar stimuli. In most instances, the ability to override habituation is best explained by postulating that habituation arises from the potentiation of inhibitory inputs onto stimulus-encoding assemblies and that habituation override occurs through disinhibition. Previous work has shown that inhibitory plasticity contributes to specific forms of olfactory and gustatory habituation in Drosophila Here, we analyze how exposure to a novel stimulus causes override of gustatory (proboscis extension reflex; PER) habituation. While brief sucrose contact with tarsal hairs causes naive Drosophila to extend their proboscis, persistent exposure reduces PER to subsequent sucrose stimuli. We show that in so habituated animals, either brief exposure of the proboscis to yeast or direct thermogenetic activation of sensory neurons restores PER response to tarsal sucrose stimulation. Similar override of PER habituation can also be induced by brief thermogenetic activation of a population of tyrosine hydroxylase (TH)-positive neurons, a subset of which send projections to the subesophageal zone (SEZ). Significantly, sensory-neuron induced habituation override requires transmitter release from these TH-positive cells. Treatments that cause override specifically influence the habituated state, with no effect on the naive sucrose response across a range of concentrations. Taken together with other findings, these observations in female flies are consistent with a model in which novel taste stimuli trigger activity in dopaminergic neurons which, directly or indirectly, inhibit GABAergic cells that drive PER habituation. The implications of these findings for general mechanisms of attentional and sensory override of habituation are discussed.SIGNIFICANCE STATEMENT Habituation can be overcome when a new context requires an enhanced response to a familiar stimulus. However, the underlying mechanisms remain incompletely understood. Previous studies have provided evidence that habituation of the sucrose-induced proboscis extension reflex (PER) in Drosophila occurs through potentiation of inhibition onto the PER pathway. This work defines controlled protocols for override of PER habituation and uses them to outline the underlying circuit mechanisms. The results presented support a model in which novel taste stimuli cause dishabituation by activating a subset of tyrosine hydroxylase (TH)-expressing neurons that inhibit GABAergic neurons whose potentiation underlies PER habituation. At a general level, these findings further highlight a central role for inhibition and disinhibition in the control of behavioral flexibility.
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Drosophila , Habituação Psicofisiológica , Animais , Drosophila/fisiologia , Feminino , Neurônios GABAérgicos/metabolismo , Habituação Psicofisiológica/fisiologia , Células Receptoras Sensoriais/metabolismo , Sacarose/farmacologia , Tirosina 3-Mono-OxigenaseRESUMO
Blood cells have a limited lifespan and are replenished by a small number of hematopoietic stem and progenitor cells (HSPCs). Adult vertebrate hematopoiesis occurs in the bone marrow, liver, and spleen, rendering a comprehensive analysis of the entire HSPC pool nearly impossible. The Drosophila blood system is well studied and has developmental, molecular, and functional parallels with that of vertebrates. Unlike vertebrates, post-embryonic hematopoiesis in Drosophila is essentially restricted to the larval lymph gland (LG), a multi-lobed organ that flanks the dorsal vessel. Because the anterior-most or primary lobes of the LG are easy to dissect out, their cellular and molecular characteristics have been studied in considerable detail. The 2-3 pairs of posterior lobes are more delicate and fragile and have largely been ignored. However, posterior lobes harbor a significant blood progenitor pool, and several hematopoietic mutants show differences in phenotype between the anterior and posterior lobes. Hence, a comprehensive analysis of the LG is important for a thorough understanding of Drosophila hematopoiesis. Most studies focus on isolating the primary lobes by methods that generally dislodge and damage other lobes. To obtain preparations of the whole LG, including intact posterior lobes, here we provide a detailed protocol for larval fillet dissection. This allows accessing and analyzing complete LG lobes, along with dorsal vessel and pericardial cells. We demonstrate that tissue architecture and integrity is maintained and provide methods for quantitative analysis. This protocol can be used to quickly and effectively isolate complete LGs from first instar larval to pupal stages and can be implemented with ease.
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Ataxin-2 (Atx2) is a translational control molecule mutated in spinocerebellar ataxia type II and amyotrophic lateral sclerosis. While intrinsically disordered domains (IDRs) of Atx2 facilitate mRNP condensation into granules, how IDRs work with structured domains to enable positive and negative regulation of target mRNAs remains unclear. Using the Targets of RNA-Binding Proteins Identified by Editing technology, we identified an extensive data set of Atx2-target mRNAs in the Drosophila brain and S2 cells. Atx2 interactions with AU-rich elements in 3'UTRs appear to modulate stability/turnover of a large fraction of these target mRNAs. Further genomic and cell biological analyses of Atx2 domain deletions demonstrate that Atx2 (1) interacts closely with target mRNAs within mRNP granules, (2) contains distinct protein domains that drive or oppose RNP-granule assembly, and (3) has additional essential roles outside of mRNP granules. These findings increase the understanding of neuronal translational control mechanisms and inform strategies for Atx2-based interventions under development for neurodegenerative disease.
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Ataxina-2/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , RNA Mensageiro/metabolismo , Animais , Ataxina-2/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Blood cells arise from diverse pools of stem and progenitor cells. Understanding progenitor heterogeneity is a major challenge. The Drosophila larval lymph gland is a well-studied model to understand blood progenitor maintenance and recapitulates several aspects of vertebrate hematopoiesis. However in-depth analysis has focused on the anterior lobe progenitors (AP), ignoring the posterior progenitors (PP) from the posterior lobes. Using in situ expression mapping and developmental and transcriptome analysis, we reveal PP heterogeneity and identify molecular-genetic tools to study this abundant progenitor population. Functional analysis shows that PP resist differentiation upon immune challenge, in a JAK-STAT-dependent manner. Upon wasp parasitism, AP downregulate JAK-STAT signaling and form lamellocytes. In contrast, we show that PP activate STAT92E and remain undifferentiated, promoting survival. Stat92E knockdown or genetically reducing JAK-STAT signaling permits PP lamellocyte differentiation. We discuss how heterogeneity and compartmentalization allow functional segregation in response to systemic cues and could be widely applicable.
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Drosophila melanogaster/imunologia , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Animais , Drosophila melanogaster/parasitologia , Hematopoese/fisiologia , Hemócitos/imunologia , Hemócitos/metabolismo , Janus Quinases/genética , Larva/imunologia , Larva/parasitologia , Linfonodos/fisiologia , Fatores de Transcrição STAT/genética , Transdução de Sinais , Células-Tronco , Vespas/fisiologiaRESUMO
BACKGROUND: Muscle myofibrils and sarcomeres present exceptional examples of highly ordered cytoskeletal filament arrays, whose distinct spatial organization is an essential aspect of muscle cell functionality. We utilized ultra-structural analysis to investigate the assembly of myofibrils and sarcomeres within developing myotubes of the indirect flight musculature of Drosophila. RESULTS: A temporal sequence composed of three major processes was identified: subdivision of the unorganized cytoplasm of nascent, multi-nucleated myotubes into distinct organelle-rich and filament-rich domains; initial organization of the filament-rich domains into myofibrils harboring nascent sarcomeric units; and finally, maturation of the highly-ordered pattern of sarcomeric thick (myosin-based) and thin (microfilament-based) filament arrays in parallel to myofibril radial growth. Significantly, organized microtubule arrays were present throughout these stages and exhibited dynamic changes in their spatial patterns consistent with instructive roles. Genetic manipulations confirm these notions, and imply specific and critical guidance activities of the microtubule-based cytoskeleton, as well as structural interdependence between the myosin- and actin-based filament arrays. CONCLUSIONS: Our observations highlight a surprisingly significant, behind-the-scenes role for microtubules in establishment of myofibril and sarcomere spatial patterns and size, and provide a detailed account of the interplay between major cytoskeletal elements in generating these essential contractile myogenic units.
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Citoesqueleto/metabolismo , Drosophila/crescimento & desenvolvimento , Desenvolvimento Muscular , Pupa/ultraestrutura , Sarcômeros/metabolismo , Animais , Drosophila/ultraestruturaRESUMO
Severe locomotor impairment is a common phenotype of neurodegenerative disorders such as Parkinson's disease (PD). Drosophila models of PD, studied for more than a decade, have helped in understanding the interaction between various genetic factors, such as parkin and PINK1, in this disease. To characterize locomotor behavioral phenotypes for these genes, fly climbing assays have been widely used. While these simple current assays for locomotor defects in Drosophila mutants measure some locomotor phenotypes well, it is possible that detection of subtle changes in behavior is important to understand the manifestation of locomotor disorders. We introduce a climbing behavior assay which provides such fine-scale behavioral data and tests this proposition for the Drosophila model. We use this inexpensive, fully automated assay to quantitatively characterize the climbing behavior at high parametric resolution in 3 contexts. First, we characterize wild-type flies and uncover a hitherto unknown sexual dimorphism in climbing behavior. Second, we study climbing behavior of heterozygous mutants of genes implicated in the fly PD model and reveal previously unreported prominent locomotor defects in some of these heterozygous fly lines. Finally, we study locomotor defects in a homozygous proprioceptory mutation (Trp-γ1 ) known to affect fine motor control in Drosophila Moreover, we identify aberrant geotactic behavior in Trp-γ1 mutants, thereby opening up a finer assay for geotaxis and its genetic basis. Our assay is therefore a cost-effective, general tool for measuring locomotor behaviors of wild-type and mutant flies in fine detail and can reveal subtle motor defects.
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Técnicas de Observação do Comportamento/métodos , Comportamento Animal/fisiologia , Locomoção/genética , Doença de Parkinson/genética , Propriocepção/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Doença de Parkinson/fisiopatologia , Proteínas Serina-Treonina Quinases/genética , Sensibilidade e Especificidade , Caracteres Sexuais , Canais de Potencial de Receptor Transitório/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Alary muscle syncytia in Drosophila larvae undergo a remarkable process of dedifferentiation into single cells that then fuse to become ventral longitudinal muscle in the adult. In this issue, Schaub et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201905048) identify the Hippo and JNK signaling pathways as key regulators of this process of developmental remodeling of cell fate.
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Drosophila melanogaster , Transdução de Sinais , Animais , Diferenciação Celular , Proteínas de Drosophila , Células Gigantes , Mioblastos , Proteínas Nucleares , Transativadores , Proteínas de Sinalização YAPRESUMO
Indirect flight muscles (IFMs) in adult Drosophila provide the key power stroke for wing beating. They also serve as a valuable model for studying muscle development. An age-dependent decline in Drosophila free flight has been documented, but its relation to gross muscle structure has not yet been explored satisfactorily. Such analyses are impeded by conventional histological preparations and imaging techniques that limit exact morphometry of flight muscles. In this study, we employ microCT scanning on a tissue preparation that retains muscle morphology under homeostatic conditions. Focusing on a subset of IFMs called the dorsal longitudinal muscles (DLMs), we find that DLM volumes increase with age, partially due to the increased separation between myofibrillar fascicles, in a sex-dependent manner. We have uncovered and quantified asymmetry in the size of these muscles on either side of the longitudinal midline. Measurements of this resolution and scale make substantive studies that test the connection between form and function possible. We also demonstrate the application of this method to other insect species making it a valuable tool for histological analysis of insect biodiversity.
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Drosophila/fisiologia , Músculo Esquelético/anatomia & histologia , Envelhecimento/fisiologia , Animais , Drosophila/anatomia & histologia , Feminino , Masculino , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/fisiologia , Asas de Animais/anatomia & histologia , Asas de Animais/diagnóstico por imagem , Asas de Animais/fisiologia , Microtomografia por Raio-XRESUMO
Inactivation of the tumor suppressor p53 is essential for unrestrained growth of cancers. However, only 11% of hematological malignancies have mutant p53. Mechanisms that cause wild-type p53 dysfunction and promote leukemia are inadequately deciphered. The stem cell protein Asrij/OCIAD1 is misexpressed in several human hematological malignancies and implicated in the p53 pathway and DNA damage response. However, Asrij function in vertebrate hematopoiesis remains unknown. We generated the first asrij null (knockout [KO]) mice and show that they are viable and fertile with no gross abnormalities. However, by 6 months, they exhibit increased peripheral blood cell counts, splenomegaly, and an expansion of bone marrow hematopoietic stem cells (HSCs) with higher myeloid output. HSCs lacking Asrij are less quiescent and more proliferative with higher repopulation potential as observed from serial transplantation studies. However, stressing KO mice with sublethal γ irradiation or multiple injections of 5-fluorouracil results in reduced survival and rapid depletion of hematopoietic stem/progenitor cells (HSPCs) by driving them into proliferative exhaustion. Molecular and biochemical analyses revealed increased polyubiquitinated protein levels, Akt/STAT5 activation and COP9 signalosome subunit 5 (CSN5)-mediated p53 ubiquitination, and degradation in KO HSPCs. Further, we show that Asrij sequesters CSN5 via its conserved OCIA domain, thereby preventing p53 degradation. In agreement, Nutlin-3 treatment of KO mice restored p53 levels and reduced high HSPC frequencies. Thus, we provide a new mouse model resembling myeloproliferative disease and identify a posttranslational regulator of wild-type p53 essential for maintaining HSC quiescence that could be a potential target for pharmacological intervention.
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Complexo do Signalossomo COP9/metabolismo , Divisão Celular , Proteínas F-Box/metabolismo , Hematopoese , Células-Tronco Hematopoéticas , Transtornos Mieloproliferativos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Complexo do Signalossomo COP9/genética , Diferenciação Celular , Modelos Animais de Doenças , Proteínas F-Box/genética , Camundongos , Camundongos Knockout , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Peptídeo Hidrolases/genética , Proteólise , Proteína Supressora de Tumor p53/genéticaRESUMO
Human Ataxin-2 is implicated in the cause and progression of amyotrophic lateral sclerosis (ALS) and type 2 spinocerebellar ataxia (SCA-2). In Drosophila, a conserved atx2 gene is essential for animal survival as well as for normal RNP-granule assembly, translational control, and long-term habituation. Like its human homolog, Drosophila Ataxin-2 (Atx2) contains polyQ repeats and additional intrinsically disordered regions (IDRs). We demonstrate that Atx2 IDRs, which are capable of mediating liquid-liquid phase transitions in vitro, are essential for efficient formation of neuronal mRNP assemblies in vivo. Remarkably, ΔIDR mutants that lack neuronal RNP granules show normal animal development, survival, and fertility. However, they show defects in long-term memory formation/consolidation as well as in C9ORF72 dipeptide repeat or FUS-induced neurodegeneration. Together, our findings demonstrate (1) that higher-order mRNP assemblies contribute to long-term neuronal plasticity and memory, and (2) that a targeted reduction in RNP-granule formation efficiency can alleviate specific forms of neurodegeneration.
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Ataxina-2/genética , Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/genética , Proteínas Intrinsicamente Desordenadas/genética , Memória de Longo Prazo , Doenças Neurodegenerativas/genética , Ribonucleoproteínas/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Ataxina-2/metabolismo , Proteína C9orf72 , Drosophila , Proteínas de Drosophila/metabolismo , Fertilidade , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H , Proteínas Intrinsicamente Desordenadas/metabolismo , Olfato , Ataxias Espinocerebelares/genética , SobrevidaRESUMO
Rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) is a cytoskeletal protein that promotes directional cell migration and angiogenesis in vitro and is implicated in human carcinomas and coronary artery disease. To study the role of Rudhira during development in vivo, we generated the first knockout mouse for rudhira and show that Rudhira is essential for mouse development. Rudhira null embryos die at embryonic day (E) 9.5 accompanied by severe vascular patterning defects in embryonic and extra-embryonic tissues. To identify the molecular processes downstream of rudhira, we analyzed the transcriptome of intact knockout yolk sacs. Genome-wide transcriptome analysis showed that Rudhira functions in angiogenesis and its related processes such as cell adhesion, extracellular matrix organization, peptidase activity and TGFß signaling. Since Rudhira is also expressed in endothelial cells (ECs), we further generated Tie2Cre-mediated endothelial knockout (CKO) of rudhira. CKO embryos survive to E11.5 and similar to the global knockout, display gross vascular patterning defects, showing that endothelial Rudhira is vital for development. Further, Rudhira knockdown ECs in culture fail to sprout in a spheroid-sprouting assay, strongly supporting its role in vascular patterning. Our study identifies an essential role for Rudhira in blood vessel remodeling and provides a mouse model for cardiovascular development.
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Sistema Cardiovascular/crescimento & desenvolvimento , Embrião de Mamíferos/citologia , Endotélio Vascular/citologia , Redes Reguladoras de Genes , Neovascularização Fisiológica , Proteínas/fisiologia , Animais , Adesão Celular , Movimento Celular , Células Cultivadas , Embrião de Mamíferos/metabolismo , Endotélio Vascular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Drosophila has become an excellent model system for investigating the organization and function of the gustatory system due to the relatively simple neuroanatomical organization of its brain and the availability of powerful genetic and transgenic technology. Thus, at the molecular and cellular levels, a great deal of insight into the peripheral detection and coding of gustatory information has already been attained. In contrast, much less is known about the central neural circuits that process this information and induce behaviorally appropriate motor output. Here, we combine functional behavioral tests with targeted transgene expression through specific driver lines to identify a single bilaterally homologous pair of bitter-sensitive interneurons that are located in the subesophageal zone of the brain. Anatomical and functional data indicate that these interneurons receive specific synaptic input from bitter-sensitive gustatory receptor neurons. Targeted transgenic activation and inactivation experiments show that these bitter-sensitive interneurons can largely suppress the proboscis extension reflex to appetitive stimuli, such as sugar and water. These functional experiments, together with calcium-imaging studies and calcium-modulated photoactivatable ratiometric integrator (CaMPARI) labeling, indicate that these first-order local interneurons play an important role in the inhibition of the proboscis extension reflex that occurs in response to bitter tastants. Taken together, our studies present a cellular identification and functional characterization of a key gustatory interneuron in the bitter-sensitive gustatory circuitry of the adult fly.
Assuntos
Interneurônios/fisiologia , Percepção Gustatória/fisiologia , Animais , Animais Geneticamente Modificados , Encéfalo/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Interneurônios/metabolismo , Fenômenos Fisiológicos do Sistema Nervoso , Receptores de Superfície Celular/metabolismo , Células Receptoras Sensoriais/fisiologia , Paladar/fisiologia , Transgenes/genéticaRESUMO
Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. Here we take advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, we have developed an algorithm for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, we use genetic reagents for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila Our findings indicate that targeted down-regulation of the GABAA receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila.
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Drosophila/fisiologia , Locomoção/fisiologia , Neurônios Motores/fisiologia , Caminhada/fisiologia , Algoritmos , Animais , Animais Geneticamente Modificados , Eletromiografia , Processamento Eletrônico de Dados , Extremidades/fisiologia , Retroalimentação Sensorial , Imuno-Histoquímica , Interneurônios/fisiologia , Íntrons , Masculino , Microscopia Confocal , Neurotransmissores/fisiologia , Periodicidade , Fenótipo , Interferência de RNA , Processamento de Sinais Assistido por Computador , Gravação em VídeoRESUMO
Inducing an injury specifically to Drosophila flight muscles is a difficult task, owing to the small size of the muscles and the presence of the cuticle. The protocol described below provides an easy and reproducible method to induce injury in the Drosophila flight muscles.
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[This corrects the article DOI: 10.1371/journal.pgen.1003452.].
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Myogenesis is a highly orchestrated, complex developmental process by which cell lineages that are mesodermal in origin generate differentiated multinucleate muscle cells as a final product. Considerable insight into the process of myogenesis has been obtained for the embryonic development of the larval muscles of Drosophila. More recently, the postembryonic development of the muscles of the adult fly has become a focus of experimental investigation of myogenesis since specific flight muscles of the fly manifest remarkable similarities to vertebrate muscles in their development and organization. In this review, we catalog some of the milestones in the study of myogenesis in the large adult-specific flight muscles of Drosophila. The identification of mesoderm-derived muscle stem cell lineages, the characterization of the symmetric and asymmetric divisions through which they produce adult-specific myoblasts, the multifaceted processes of myoblast fusion, and the unexpected discovery of quiescent satellite cells that can be activated by injury are discussed. Moreover, the finding that all of these processes incorporate a plethora of signaling interactions with other myogenic cells and with niche-like neighboring tissue is considered. Finally, we briefly point out possible future developments in the area of Drosophila myogenesis that may lead to of new avenues of genetic research into the roles of muscle stem cells in development, disease and aging.
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Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/genética , Músculos/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Modelos Genéticos , Morfogênese/genética , Fibras Musculares Esqueléticas/metabolismo , Músculos/fisiologia , Mioblastos/metabolismo , Regeneração/genéticaRESUMO
Work on genetic model systems such as Drosophila and mouse has shown that the fundamental mechanisms of myogenesis are remarkably similar in vertebrates and invertebrates. Strikingly, however, satellite cells, the adult muscle stem cells that are essential for the regeneration of damaged muscles in vertebrates, have not been reported in invertebrates. In this study, we show that lineal descendants of muscle stem cells are present in adult muscle of Drosophila as small, unfused cells observed at the surface and in close proximity to the mature muscle fibers. Normally quiescent, following muscle fiber injury, we show that these cells express Zfh1 and engage in Notch-Delta-dependent proliferative activity and generate lineal descendant populations, which fuse with the injured muscle fiber. In view of strikingly similar morphological and functional features, we consider these novel cells to be the Drosophila equivalent of vertebrate muscle satellite cells.