RESUMO
RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m6A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.
Assuntos
Proteínas de Drosophila/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , RNA Polimerase II/genética , Transcrição Gênica , Animais , Drosophila melanogaster/genética , Metiltransferases/genética , Regiões Promotoras Genéticas/genéticaRESUMO
The development of tissues and organs requires close interaction of cells. To achieve this, cells express adhesion proteins such as the neural cell adhesion molecule (NCAM) or its Drosophila ortholog Fasciclin 2 (Fas2). Both are members of the Ig-domain superfamily of proteins that mediate homophilic adhesion. These proteins are expressed as isoforms differing in their membrane anchorage and their cytoplasmic domains. To study the function of single isoforms, we have conducted a comprehensive genetic analysis of Fas2 We reveal the expression pattern of all major Fas2 isoforms, two of which are GPI anchored. The remaining five isoforms carry transmembrane domains with variable cytoplasmic tails. We generated Fas2 mutants expressing only single isoforms. In contrast to the null mutation, which causes embryonic lethality, these mutants are viable, indicating redundancy among the different isoforms. Cell type-specific rescue experiments showed that glial-secreted Fas2 can rescue the Fas2 mutant phenotype to viability. This demonstrates that cytoplasmic Fas2 domains have no apparent essential functions and indicate that Fas2 has function(s) other than homophilic adhesion. In conclusion, our data suggest novel mechanistic aspects of a long-studied adhesion protein.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Transdução de Sinais , Animais , Adesão Celular , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Movimento Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Glicosilfosfatidilinositóis/metabolismo , Mutação/genética , Neuroglia/metabolismo , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Traqueia/embriologia , Traqueia/metabolismoRESUMO
Visual motion cues provide animals with critical information about their environment and guide a diverse array of behaviors. The neural circuits that carry out motion estimation provide a well-constrained model system for studying the logic of neural computation. Through a confluence of behavioral, physiological, and anatomical experiments, taking advantage of the powerful genetic tools available in the fruit fly Drosophila melanogaster, an outline of the neural pathways that compute visual motion has emerged. Here we describe these pathways, the evidence supporting them, and the challenges that remain in understanding the circuits and computations that link sensory inputs to behavior. Studies in flies and vertebrates have revealed a number of functional similarities between motion-processing pathways in different animals, despite profound differences in circuit anatomy and structure. The fact that different circuit mechanisms are used to achieve convergent computational outcomes sheds light on the evolution of the nervous system.
Assuntos
Encéfalo/fisiologia , Drosophila melanogaster/fisiologia , Percepção de Movimento/fisiologia , Vias Visuais/fisiologia , Animais , Evolução Biológica , Drosophila melanogaster/anatomia & histologia , Modelos Animais , Vias Neurais/fisiologia , Lobo Óptico de Animais não Mamíferos/fisiologia , Vias Visuais/anatomia & histologiaRESUMO
The olfactory system translates chemical signals into neuronal signals that inform behavioral decisions of the animal. Odors are cues for source identity, but if monitored long enough, they can also be used to localize the source. Odor representations should therefore be robust to changing conditions and flexible in order to drive an appropriate behavior. In this review, we aim at discussing the main computations that allow robust and flexible encoding of odor information in the olfactory neural pathway.
Assuntos
Odorantes , Condutos Olfatórios/fisiologia , AnimaisRESUMO
The development of the nervous system requires tight control of cell division, fate specification and migration. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that affects different steps of cell cycle progression, as well as having postmitotic functions in nervous system development. It can therefore link different developmental stages in one tissue. The two adaptor proteins, Fizzy/Cdc20 and Fizzy-related/Cdh1, confer APC/C substrate specificity. Here, we show that two distinct modes of APC/C function act during Drosophila eye development. Fizzy/Cdc20 controls the early growth of the eye disc anlage and the concomitant entry of glial cells onto the disc. In contrast, fzr/cdh1 acts during neuronal patterning and photoreceptor axon growth, and subsequently affects neuron-glia interaction. To further address the postmitotic role of Fzr/Cdh1 in controlling neuron-glia interaction, we identified a series of novel APC/C candidate substrates. Four of our candidate genes are required for fzr/cdh1-dependent neuron-glia interaction, including the dynein light chain Dlc90F Taken together, our data show how different modes of APC/C activation can couple early growth and neuron-glia interaction during eye disc development.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/metabolismo , Proteínas Cdh1/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Olho/embriologia , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Comunicação Celular/fisiologia , Ciclo Celular/fisiologia , Dineínas do Citoplasma/metabolismo , Dineínas , Células Fotorreceptoras de Invertebrados/citologiaRESUMO
The computational organization of sensory systems depends on the diversification of individual cell types with distinct signal-processing capabilities. The Drosophila visual system, for instance, splits information into channels with different temporal properties directly downstream of photoreceptors in the first-order interneurons of the OFF pathway, L2 and L3. However, the biophysical mechanisms that determine this specialization are largely unknown. Here, we show that the voltage-gated Ka channels Shaker and Shal contribute to the response properties of the major OFF pathway input L2. L3 calcium response kinetics postsynaptic to photoreceptors resemble the sustained calcium signals of photoreceptors, whereas L2 neurons decay transiently. Based on a cell-type-specific RNA-seq data set and endogenous protein tagging, we identified Shaker and Shal as the primary candidates to shape L2 responses. Using in vivo two-photon imaging of L2 calcium signals in combination with pharmacological and genetic perturbations of these Ka channels, we show that the wild-type Shaker and Shal function is to enhance L2 responses and cell-autonomously sharpen L2 kinetics. Our results reveal a role for Ka channels in determining the signal-processing characteristics of a specific cell type in the visual system.
Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Interneurônios/metabolismo , Lobo Óptico de Animais não Mamíferos/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Canais de Potássio Shal/metabolismo , Visão Ocular , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Potenciais Evocados Visuais , Cinética , Lobo Óptico de Animais não Mamíferos/citologia , Estimulação Luminosa , Superfamília Shaker de Canais de Potássio/genética , Canais de Potássio Shal/genética , Vias Visuais/metabolismo , Percepção VisualRESUMO
Dopamine is synonymous with reward and motivation in mammals. However, only recently has dopamine been linked to motivated behaviour and rewarding reinforcement in fruitflies. Instead, octopamine has historically been considered to be the signal for reward in insects. Here we show, using temporal control of neural function in Drosophila, that only short-term appetitive memory is reinforced by octopamine. Moreover, octopamine-dependent memory formation requires signalling through dopamine neurons. Part of the octopamine signal requires the α-adrenergic-like OAMB receptor in an identified subset of mushroom-body-targeted dopamine neurons. Octopamine triggers an increase in intracellular calcium in these dopamine neurons, and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking octopamine. Analysis of the ß-adrenergic-like OCTß2R receptor reveals that octopamine-dependent reinforcement also requires an interaction with dopamine neurons that control appetitive motivation. These data indicate that sweet taste engages a distributed octopamine signal that reinforces memory through discrete subsets of mushroom-body-targeted dopamine neurons. In addition, they reconcile previous findings with octopamine and dopamine and suggest that reinforcement systems in flies are more similar to mammals than previously thought.
Assuntos
Dopamina/metabolismo , Drosophila melanogaster/metabolismo , Octopamina/metabolismo , Recompensa , Transdução de Sinais , Animais , Comportamento Apetitivo/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Dopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Feminino , Masculino , Memória de Curto Prazo/efeitos dos fármacos , Memória de Curto Prazo/fisiologia , Motivação/efeitos dos fármacos , Motivação/fisiologia , Corpos Pedunculados/citologia , Corpos Pedunculados/efeitos dos fármacos , Corpos Pedunculados/metabolismo , Octopamina/farmacologia , Receptores de Neurotransmissores/deficiência , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Paladar/efeitos dos fármacos , Paladar/fisiologiaRESUMO
Rhythmic motor behaviors such as feeding are driven by neural networks that can be modulated by external stimuli and internal states. In Drosophila, ingestion is accomplished by a pump that draws fluid into the esophagus. Here we examine how pumping is regulated and characterize motor neurons innervating the pump. Frequency of pumping is not affected by sucrose concentration or hunger but is altered by fluid viscosity. Inactivating motor neurons disrupts pumping and ingestion, whereas activating them elicits arrhythmic pumping. These motor neurons respond to taste stimuli and show prolonged activity to palatable substances. This work describes an important component of the neural circuit for feeding in Drosophila and is a step toward understanding the rhythmic activity producing ingestion.
Assuntos
Drosophila melanogaster/fisiologia , Ingestão de Alimentos/fisiologia , Comportamento Alimentar/fisiologia , Neurônios Motores/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Canais Iônicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Neurônios Motores/metabolismo , Contração Muscular/fisiologia , Sacarose/administração & dosagem , Sacarose/farmacologia , Canal de Cátion TRPA1 , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Paladar/fisiologia , TemperaturaRESUMO
Tissue-specific gene expression using the upstream activating sequence (UAS)GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we developed a convertible genetic platform, the integrase swappable in vivo targeting element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient in vivo manipulations, making it possible to generate many swaps in parallel. This system is modular, allowing future genetic tools to be easily incorporated into the existing framework.
Assuntos
Drosophila melanogaster/genética , Perfilação da Expressão Gênica/métodos , Expressão Gênica , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Dados de Sequência Molecular , Recombinação Genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina Endopeptidases/genética , Fatores de Transcrição/genéticaRESUMO
Visual systems are homogeneous structures, where repeating columnar units retinotopically cover the visual field. Each of these columns contain many of the same neuron types that are distinguished by anatomic, genetic and - generally - by functional properties. However, there are exceptions to this rule. In the 800 columns of the Drosophila eye, there is an anatomically and genetically identifiable cell type with variable functional properties, Tm9. Since anatomical connectivity shapes functional neuronal properties, we identified the presynaptic inputs of several hundred Tm9s across both optic lobes using the full adult female fly brain (FAFB) electron microscopic dataset and FlyWire connectome. Our work shows that Tm9 has three major and many sparsely distributed inputs. This differs from the presynaptic connectivity of other Tm neurons, which have only one major, and more stereotypic inputs than Tm9. Genetic synapse labeling showed that the heterogeneous wiring exists across individuals. Together, our data argue that the visual system uses heterogeneous, distributed circuit properties to achieve robust visual processing.
Assuntos
Artrópodes , Neurônios , Humanos , Animais , Feminino , Neurônios/fisiologia , Drosophila/fisiologia , Sinapses/fisiologia , Percepção Visual , Encéfalo , Vias Visuais/fisiologiaRESUMO
Animals navigating in natural environments must handle vast changes in their sensory input. Visual systems, for example, handle changes in luminance at many timescales, from slow changes across the day to rapid changes during active behavior. To maintain luminance-invariant perception, visual systems must adapt their sensitivity to changing luminance at different timescales. We demonstrate that luminance gain control in photoreceptors alone is insufficient to explain luminance invariance at both fast and slow timescales and reveal the algorithms that adjust gain past photoreceptors in the fly eye. We combined imaging and behavioral experiments with computational modeling to show that downstream of photoreceptors, circuitry taking input from the single luminance-sensitive neuron type L3 implements gain control at fast and slow timescales. This computation is bidirectional in that it prevents the underestimation of contrasts in low luminance and overestimation in high luminance. An algorithmic model disentangles these multifaceted contributions and shows that the bidirectional gain control occurs at both timescales. The model implements a nonlinear interaction of luminance and contrast to achieve gain correction at fast timescales and a dark-sensitive channel to improve the detection of dim stimuli at slow timescales. Together, our work demonstrates how a single neuronal channel performs diverse computations to implement gain control at multiple timescales that are together important for navigation in natural environments.
Assuntos
Sensibilidades de Contraste , Drosophila , Animais , Visão Ocular , Células Fotorreceptoras , Neurônios/fisiologia , Estimulação Luminosa/métodosRESUMO
Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.
Assuntos
Antioxidantes , Corpo Estriado , Estresse Oxidativo , Animais , Camundongos , Antioxidantes/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Olho/metabolismo , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Corpo Estriado/fisiologiaRESUMO
Self-motion generates visual patterns on the eye that are important for navigation. These optic flow patterns are encoded by the population of local directionselective cells in the mouse retina, whereas in flies, local directionselective T4/T5 cells are thought to be uniformly tuned. How complex global motion patterns can be computed downstream is unclear. We show that the population of T4/T5 cells in Drosophila encodes global motion patterns. Whereas the mouse retina encodes four types of optic flow, the fly visual system encodes six. This matches the larger number of degrees of freedom and the increased complexity of translational and rotational motion patterns during flight. The four uniformly tuned T4/T5 subtypes described previously represent a local subset of the population. Thus, a population code for global motion patterns appears to be a general coding principle of visual systems that matches local motion responses to modes of the animal's movement.
RESUMO
The accurate processing of contrast is the basis for all visually guided behaviors. Visual scenes with rapidly changing illumination challenge contrast computation because photoreceptor adaptation is not fast enough to compensate for such changes. Yet, human perception of contrast is stable even when the visual environment is quickly changing, suggesting rapid post receptor luminance gain control. Similarly, in the fruit fly Drosophila, such gain control leads to luminance invariant behavior for moving OFF stimuli. Here, we show that behavioral responses to moving ON stimuli also utilize a luminance gain, and that ON-motion guided behavior depends on inputs from three first-order interneurons L1, L2, and L3. Each of these neurons encodes contrast and luminance differently and distributes information asymmetrically across both ON and OFF contrast-selective pathways. Behavioral responses to both ON and OFF stimuli rely on a luminance-based correction provided by L1 and L3, wherein L1 supports contrast computation linearly, and L3 non-linearly amplifies dim stimuli. Therefore, L1, L2, and L3 are not specific inputs to ON and OFF pathways but the lamina serves as a separate processing layer that distributes distinct luminance and contrast information across ON and OFF pathways to support behavior in varying conditions.
Assuntos
Percepção de Movimento , Visão Ocular , Animais , Sensibilidades de Contraste , Drosophila , Interneurônios/fisiologia , Movimento (Física) , Percepção de Movimento/fisiologia , Estimulação Luminosa , Vias Visuais/fisiologiaRESUMO
Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.
Assuntos
Actinas , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Humanos , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Complexo Piruvato Desidrogenase/metabolismoRESUMO
In Drosophila, direction-selective neurons implement a mechanism of motion computation similar to cortical neurons, using contrast-opponent receptive fields with ON and OFF subfields. It is not clear how the presynaptic circuitry of direction-selective neurons in the OFF pathway supports this computation if all major inputs are OFF-rectified neurons. Here, we reveal the biological substrate for motion computation in the OFF pathway. Three interneurons, Tm2, Tm9 and CT1, provide information about ON stimuli to the OFF direction-selective neuron T5 across its receptive field, supporting a contrast-opponent receptive field organization. Consistent with its prominent role in motion detection, variability in Tm9 receptive field properties transfers to T5, and calcium decrements in Tm9 in response to ON stimuli persist across behavioral states, while spatial tuning is sharpened by active behavior. Together, our work shows how a key neuronal computation is implemented by its constituent neuronal circuit elements to ensure direction selectivity.
Assuntos
Drosophila/metabolismo , Percepção de Movimento/fisiologia , Movimento (Física) , Neurônios/metabolismo , Animais , Cálcio/metabolismo , Clorfenamidina , Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Interneurônios/metabolismoRESUMO
Visual perception scales with changes in the visual stimulus, or contrast, irrespective of background illumination. However, visual perception is challenged when adaptation is not fast enough to deal with sudden declines in overall illumination, for example, when gaze follows a moving object from bright sunlight into a shaded area. Here, we show that the visual system of the fly employs a solution by propagating a corrective luminance-sensitive signal. We use in vivo 2-photon imaging and behavioral analyses to demonstrate that distinct OFF-pathway inputs encode contrast and luminance. Predictions of contrast-sensitive neuronal responses show that contrast information alone cannot explain behavioral responses in sudden dim light. The luminance-sensitive pathway via the L3 neuron is required for visual processing in such rapidly changing light conditions, ensuring contrast constancy when pure contrast sensitivity underestimates a stimulus. Thus, retaining a peripheral feature, luminance, in visual processing is required for robust behavioral responses.
Assuntos
Drosophila melanogaster/fisiologia , Percepção Visual/fisiologia , Animais , Sensibilidades de Contraste/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Estimulação LuminosaRESUMO
The function of a complex nervous system depends on an intricate interplay between neuronal and glial cell types. One of the many functions of glial cells is to provide an efficient insulation of the nervous system and thereby allowing a fine tuned homeostasis of ions and other small molecules. Here, we present a detailed cellular analysis of the glial cell complement constituting the blood-brain barrier in Drosophila. Using electron microscopic analysis and single cell-labeling experiments, we characterize different glial cell layers at the surface of the nervous system, the perineurial glial layer, the subperineurial glial layer, the wrapping glial cell layer, and a thick layer of extracellular matrix, the neural lamella. To test the functional roles of these sheaths we performed a series of dye penetration experiments in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes in different mutants allowed to conclude that most of the barrier function is mediated by the septate junctions formed by the subperineurial cells, whereas the perineurial glial cell layer and the neural lamella contribute to barrier selectivity against much larger particles (i.e., the size of proteins). We further compare the requirements of different septate junction components for the integrity of the blood-brain barrier and provide evidence that two of the six Claudin-like proteins found in Drosophila are needed for normal blood-brain barrier function.
Assuntos
Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/fisiologia , Drosophila/anatomia & histologia , Drosophila/fisiologia , Neuroglia/fisiologia , Animais , Animais Geneticamente Modificados , Barreira Hematoencefálica/embriologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Mutação , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Neuroglia/ultraestruturaRESUMO
Chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) is a powerful technique to study transcriptional regulation. However, the requirement of millions of cells to generate results with high signal-to-noise ratio precludes it in the study of small cell populations. Here, we present a tagmentation-assisted fragmentation ChIP (TAF-ChIP) and sequencing method to generate high-quality histone profiles from low cell numbers. The data obtained from the TAF-ChIP approach are amenable to standard tools for ChIP-Seq analysis, owing to its high signal-to-noise ratio. The epigenetic profiles from TAF-ChIP approach showed high agreement with conventional ChIP-Seq datasets, thereby underlining the utility of this approach.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Drosophila/genética , Histonas/metabolismo , Animais , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células K562 , Razão Sinal-Ruído , Software , Sequenciamento Completo do GenomaRESUMO
Sensory systems sequentially extract increasingly complex features. ON and OFF pathways, for example, encode increases or decreases of a stimulus from a common input. This ON/OFF pathway split is thought to occur at individual synaptic connections through a sign-inverting synapse in one of the pathways. Here, we show that ON selectivity is a multisynaptic process in the Drosophila visual system. A pharmacogenetics approach demonstrates that both glutamatergic inhibition through GluClα and GABAergic inhibition through Rdl mediate ON responses. Although neurons postsynaptic to the glutamatergic ON pathway input L1 lose all responses in GluClα mutants, they are resistant to a cell-type-specific loss of GluClα. This shows that ON selectivity is distributed across multiple synapses, and raises the possibility that cell-type-specific manipulations might reveal similar strategies in other sensory systems. Thus, sensory coding is more distributed than predicted by simple circuit motifs, allowing for robust neural processing.