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Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here, we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end, we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.
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Proteínas de Drosophila , Neurociencias , Animales , Drosophila/metabolismo , Neuronas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Sistema Nervioso Central/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Directed cellular movements are a universal feature of morphogenesis in multicellular organisms. Differential adhesion between the stationary and motile cells promotes these cellular movements to effect spatial patterning of cells. A prominent feature of Drosophila eye development is the 90 degrees rotational movement of the multicellular ommatidial precursors within a matrix of stationary cells. We demonstrate that the cell adhesion molecules Echinoid (Ed) and Friend of Echinoid (Fred) act throughout ommatidial rotation to modulate the degree of ommatidial precursor movement. We propose that differential levels of Ed and Fred between stationary and rotating cells at the initiation of rotation create a permissive environment for cell movement, and that uniform levels in these two populations later contribute to stopping the movement. Based on genetic data, we propose that ed and fred impart a second, independent, ;brake-like' contribution to this process via Egfr signaling. Ed and Fred are localized in largely distinct and dynamic patterns throughout rotation. However, ed and fred are required in only a subset of cells - photoreceptors R1, R7 and R6 - for normal rotation, cells that have only recently been linked to a role in planar cell polarity (PCP). This work also provides the first demonstration of a requirement for cone cells in the ommatidial rotation aspect of PCP. ed and fred also genetically interact with the PCP genes, but affect only the degree-of-rotation aspect of the PCP phenotype. Significantly, we demonstrate that at least one PCP protein, Stbm, is required in R7 to control the degree of ommatidial rotation.
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Moléculas de Adhesión Celular/fisiología , Proteínas de Drosophila/fisiología , Drosophila/crecimiento & desarrollo , Drosophila/fisiología , Ojo/citología , Ojo/crecimiento & desarrollo , Proteínas Represoras/fisiología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo , Adhesión Celular , Moléculas de Adhesión Celular/genética , Movimiento Celular , Polaridad Celular , Drosophila/genética , Proteínas de Drosophila/genética , Receptores ErbB/metabolismo , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Modelos Biológicos , Fenotipo , Proteínas Represoras/genética , Rotación , Transducción de SeñalRESUMEN
Flexible behaviors over long timescales are thought to engage recurrent neural networks in deep brain regions, which are experimentally challenging to study. In insects, recurrent circuit dynamics in a brain region called the central complex (CX) enable directed locomotion, sleep, and context- and experience-dependent spatial navigation. We describe the first complete electron microscopy-based connectome of the Drosophila CX, including all its neurons and circuits at synaptic resolution. We identified new CX neuron types, novel sensory and motor pathways, and network motifs that likely enable the CX to extract the fly's head direction, maintain it with attractor dynamics, and combine it with other sensorimotor information to perform vector-based navigational computations. We also identified numerous pathways that may facilitate the selection of CX-driven behavioral patterns by context and internal state. The CX connectome provides a comprehensive blueprint necessary for a detailed understanding of network dynamics underlying sleep, flexible navigation, and state-dependent action selection.
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Conectoma , Navegación Espacial , Animales , Encéfalo/fisiología , Drosophila/fisiología , Drosophila melanogaster/fisiología , Neuronas/fisiología , Navegación Espacial/fisiologíaRESUMEN
Cell shapes and contacts are dynamically regulated during organogenesis to enable contacts with relevant neighboring cells at appropriate times. During Drosophila larval eye development, an apical contact is established between one pair of non-neuronal cones cells, precluding contact between the opposing pair. Concurrent with changes in cell shape, these contacts reverse in early pupal life. The reversal in cone cell contacts occurs in a posterior to anterior gradient across the eye, following the developmental gradient established in the larval eye imaginal disc. Hibris (Hbs), an Immunoglobulin cell adhesion molecule homologous to vertebrate Nephrin, is required for cone cell morphogenesis. In hbs null mutants, a majority of cone cells fail to both establish wild-type contacts and achieve mature cone cell shapes. hbs acts cell autonomously in the cone cells to drive these changes. The work presented here indicates hbs contributes to the remodeling of cell contacts and cell shapes throughout development.
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Forma de la Célula/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Proteínas de la Membrana/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Animales , Cadherinas/metabolismo , Cadherinas/fisiología , Forma de la Célula/genética , Drosophila/citología , Drosophila/metabolismo , Proteínas de Drosophila/genética , Inmunohistoquímica , Proteínas de la Membrana/genética , Retina/citología , Retina/embriología , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/citologíaRESUMEN
Neural representations of head direction (HD) have been discovered in many species. Theoretical work has proposed that the dynamics associated with these representations are generated, maintained, and updated by recurrent network structures called ring attractors. We evaluated this theorized structure-function relationship by performing electron-microscopy-based circuit reconstruction and RNA profiling of identified cell types in the HD system of Drosophila melanogaster. We identified motifs that have been hypothesized to maintain the HD representation in darkness, update it when the animal turns, and tether it to visual cues. Functional studies provided support for the proposed roles of individual excitatory or inhibitory circuit elements in shaping activity. We also discovered recurrent connections between neuronal arbors with mixed pre- and postsynaptic specializations. Our results confirm that the Drosophila HD network contains the core components of a ring attractor while also revealing unpredicted structural features that might enhance the network's computational power.
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Encéfalo/ultraestructura , Movimientos de la Cabeza , Red Nerviosa/ultraestructura , Neuronas/ultraestructura , Navegación Espacial , Sinapsis/ultraestructura , Animales , Drosophila melanogaster , Microscopía Confocal , Microscopía Electrónica , Microscopía de Fluorescencia por Excitación Multifotónica , Vías Nerviosas , Vías VisualesRESUMEN
The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain.
Animal brains of all sizes, from the smallest to the largest, work in broadly similar ways. Studying the brain of any one animal in depth can thus reveal the general principles behind the workings of all brains. The fruit fly Drosophila is a popular choice for such research. With about 100,000 neurons compared to some 86 billion in humans the fly brain is small enough to study at the level of individual cells. But it nevertheless supports a range of complex behaviors, including navigation, courtship and learning. Thanks to decades of research, scientists now have a good understanding of which parts of the fruit fly brain support particular behaviors. But exactly how they do this is often unclear. This is because previous studies showing the connections between cells only covered small areas of the brain. This is like trying to understand a novel when all you can see is a few isolated paragraphs. To solve this problem, Scheffer, Xu, Januszewski, Lu, Takemura, Hayworth, Huang, Shinomiya et al. prepared the first complete map of the entire central region of the fruit fly brain. The central brain consists of approximately 25,000 neurons and around 20 million connections. To prepare the map or connectome the brain was cut into very thin 8nm slices and photographed with an electron microscope. A three-dimensional map of the neurons and connections in the brain was then reconstructed from these images using machine learning algorithms. Finally, Scheffer et al. used the new connectome to obtain further insights into the circuits that support specific fruit fly behaviors. The central brain connectome is freely available online for anyone to access. When used in combination with existing methods, the map will make it easier to understand how the fly brain works, and how and why it can fail to work correctly. Many of these findings will likely apply to larger brains, including our own. In the long run, studying the fly connectome may therefore lead to a better understanding of the human brain and its disorders. Performing a similar analysis on the brain of a small mammal, by scaling up the methods here, will be a likely next step along this path.
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Conectoma/métodos , Drosophila melanogaster/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Encéfalo/fisiología , Femenino , MasculinoRESUMEN
Both dramatic and subtle morphogenetic movements are of paramount importance in molding cells and tissues into functional form. Cells move either independently or as populations and the distance traversed by cells varies greatly, but in all cases, the output is common: to organize cells into or within organs and epithelia. In the developing Drosophila eye, a highly specialized, 90 degrees rotational movement of subsets of cells imposes order by polarizing the retinal epithelium across its dorsoventral axis. This process was proposed to take place in two 45 degrees steps, with the second under control of the gene nemo (nmo), a serine/threonine kinase. While our analysis confirms that these subsets of cells, the ommatidial precursors, do stall at 45 degrees , we demonstrate that nmo is also required through most of the first 45 degrees of rotation to regulate the speed at which the ommatidial precursors move. In addition, although the precursors reach only the halfway point by the end of larval life, this work demonstrates that patterning events that occur during pupal life move the ommatidial units an additional 15 degrees . A re-analysis of nmo mosaic clones indicates that nmo is required in photoreceptors R1, R6 and R7 for normal orientation. This work also demonstrates that two major isoforms of nmo rescue the nmo(P1) phenotype. Finally, a dominant modifier screen of a nmo misexpression background identified genomic regions that potentially regulate rotation. The results presented here suggest a model in which a motor for rotation is established in a nemo-dependent fashion in a subset of cells.
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Proteínas de Drosophila/fisiología , Ojo/citología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Células Fotorreceptoras de Invertebrados/fisiología , Rotación , Alelos , Animales , Animales Modificados Genéticamente , Polaridad Celular , Clonación Molecular , Drosophila/citología , Drosophila/genética , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Ojo/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Metamorfosis Biológica/fisiología , Proteínas Quinasas Activadas por Mitógenos/genética , Modelos Biológicos , Mutación , Células Fotorreceptoras de Invertebrados/citología , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiología , Retina/citología , Retina/crecimiento & desarrollo , Retina/fisiología , TransgenesRESUMEN
The tissue polarity pathway is required for the establishment of epithelial polarity in a variety of vertebrate and invertebrate organs. Core tissue polarity proteins act in a dynamically regulated complex to direct the polarization of the Drosophila eye. We report the identification and characterization of bedraggled (bdg), a novel gene that regulates one output of the tissue polarity pathway--the establishment of the R3/R4 photoreceptor fates. bdg encodes a novel, putative transporter protein and interacts genetically with all of the core polarity genes to influence the specification of the R3 and R4 cell fates. Finally, bdg is required for both viability and the initial stages of imaginal disc development.
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Polaridad Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ojo/embriología , Animales , Animales Modificados Genéticamente , Northern Blotting , Diferenciación Celular , Linaje de la Célula , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Ojo/citología , Ojo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Masculino , Actividad Motora , Fenotipo , FilogeniaRESUMEN
The central complex, a set of neuropils in the center of the insect brain, plays a crucial role in spatial aspects of sensory integration and motor control. Stereotyped neurons interconnect these neuropils with one another and with accessory structures. We screened over 5,000 Drosophila melanogaster GAL4 lines for expression in two neuropils, the noduli (NO) of the central complex and the asymmetrical body (AB), and used multicolor stochastic labeling to analyze the morphology, polarity, and organization of individual cells in a subset of the GAL4 lines that showed expression in these neuropils. We identified nine NO and three AB cell types and describe them here. The morphology of the NO neurons suggests that they receive input primarily in the lateral accessory lobe and send output to each of the six paired noduli. We demonstrate that the AB is a bilateral structure which exhibits asymmetry in size between the left and right bodies. We show that the AB neurons directly connect the AB to the central complex and accessory neuropils, that they target both the left and right ABs, and that one cell type preferentially innervates the right AB. We propose that the AB be considered a central complex neuropil in Drosophila. Finally, we present highly restricted GAL4 lines for most identified protocerebral bridge, NO, and AB cell types. These lines, generated using the split-GAL4 method, will facilitate anatomical studies, behavioral assays, and physiological experiments.
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Encéfalo/anatomía & histología , Drosophila melanogaster/anatomía & histología , Neurópilo/citología , AnimalesRESUMEN
The coordinated polarization of cells within an epithelium is required for the development and function of some tissues. Recent work has shown that the EGF receptor signaling pathway plays a key role in establishing epithelial polarity in the compound eye of Drosophila.
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Movimiento Celular/fisiología , Drosophila/embriología , Receptores ErbB/metabolismo , Células Fotorreceptoras de Invertebrados/embriología , Transducción de Señal/fisiología , Animales , Drosophila/fisiología , Epitelio/fisiología , Receptores ErbB/fisiología , Organogénesis/fisiologíaRESUMEN
The Drosophila eye is a polarized epithelium in which ommatidia of opposing chirality fall on opposite sides of the eye's midline, the equator. The equator is established in at least two steps: photoreceptors R3 and R4 adopt their fates, and then ommatidia rotate clockwise or counterclockwise in accordance with the identity of these photoreceptors. We report the role of two cadherins, Fat (Ft) and Dachsous (Ds), in conveying the polarizing signal from the D/V midline in the Drosophila eye. In eyes lacking Ft, the midline is abolished. In ft and ds mutant clones, wild-type tissue rescues genetically mutant tissue at the clonal borders, giving rise to ectopic equators. These ectopic equators distort a mosaic analysis of these genes and led to the possible misinterpretation that ft and ds are required to specify the R3 and R4 cell fates, respectively. Our interpretation of these data supports a significantly different model in which ft and ds are not necessarily required for fate determination. Rather, they are involved in long-range signaling during the formation of the equator, as defined by the presence of an organized arrangement of dorsal and ventral chiral ommatidial forms.
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Cadherinas/fisiología , Proteínas de Drosophila , Células Fotorreceptoras de Invertebrados/metabolismo , Transducción de Señal , Animales , Polaridad Celular , Drosophila , Células Fotorreceptoras de Invertebrados/citologíaRESUMEN
Many animals maintain an internal representation of their heading as they move through their surroundings. Such a compass representation was recently discovered in a neural population in the Drosophila melanogaster central complex, a brain region implicated in spatial navigation. Here, we use two-photon calcium imaging and electrophysiology in head-fixed walking flies to identify a different neural population that conjunctively encodes heading and angular velocity, and is excited selectively by turns in either the clockwise or counterclockwise direction. We show how these mirror-symmetric turn responses combine with the neurons' connectivity to the compass neurons to create an elegant mechanism for updating the fly's heading representation when the animal turns in darkness. This mechanism, which employs recurrent loops with an angular shift, bears a resemblance to those proposed in theoretical models for rodent head direction cells. Our results provide a striking example of structure matching function for a broadly relevant computation.
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Encéfalo/fisiología , Drosophila melanogaster/fisiología , Orientación Espacial , Animales , Calcio/análisis , Red Nerviosa/fisiología , Imagen Óptica , Técnicas de Placa-ClampRESUMEN
Insects exhibit an elaborate repertoire of behaviors in response to environmental stimuli. The central complex plays a key role in combining various modalities of sensory information with an insect's internal state and past experience to select appropriate responses. Progress has been made in understanding the broad spectrum of outputs from the central complex neuropils and circuits involved in numerous behaviors. Many resident neurons have also been identified. However, the specific roles of these intricate structures and the functional connections between them remain largely obscure. Significant gains rely on obtaining a comprehensive catalog of the neurons and associated GAL4 lines that arborize within these brain regions, and on mapping neuronal pathways connecting these structures. To this end, small populations of neurons in the Drosophila melanogaster central complex were stochastically labeled using the multicolor flip-out technique and a catalog was created of the neurons, their morphologies, trajectories, relative arrangements, and corresponding GAL4 lines. This report focuses on one structure of the central complex, the protocerebral bridge, and identifies just 17 morphologically distinct cell types that arborize in this structure. This work also provides new insights into the anatomical structure of the four components of the central complex and its accessory neuropils. Most strikingly, we found that the protocerebral bridge contains 18 glomeruli, not 16, as previously believed. Revised wiring diagrams that take into account this updated architectural design are presented. This updated map of the Drosophila central complex will facilitate a deeper behavioral and physiological dissection of this sophisticated set of structures.
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Encéfalo/anatomía & histología , Proteínas de Drosophila/metabolismo , Red Nerviosa/anatomía & histología , Neuroanatomía , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Drosophila/anatomía & histología , Red Nerviosa/metabolismo , Neuronas/citología , Neurópilo/metabolismoRESUMEN
This protocol is used for light microscopic analysis of the adult Drosophila eye. It provides nice preservation of pigment granules and photoreceptor rhabdomeres. Therefore, it is used primarily for phenotypic and mosaic analysis of adult eyes. Because this fixation method does not preserve either cell membranes or subcellular structures, transmission electron microscopy (TEM) is recommended for visualizing these structures.
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Drosophila/citología , Microtomía/métodos , Animales , Microscopía Electrónica de Transmisión/métodos , Retina/citologíaRESUMEN
Scanning electron microscopy (SEM) allows for high-magnification analysis of the external structures of the Drosophila eye. In this article, three methods are provided: one for eyes that are to be critical-point-dried (CPD) and two alternatives if a critical point drier is not available. The major difference between the first method and the other two is a preliminary fixation step. Drosophila eyes have a tendency to collapse when CPD, so fixing the animals before critical point drying improves the yield of good specimens.
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Drosophila/ultraestructura , Entomología/métodos , Microscopía Electrónica de Rastreo/métodos , Animales , Ojo/ultraestructuraRESUMEN
Transmission electron microscopy (TEM) allows for high-magnification ultrastructural analysis of the Drosophila eye. The fixation protocol described here works well for Drosophila eye tissue, including larval imaginal discs and pupal and adult retinas.
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Drosophila/ultraestructura , Entomología/métodos , Microscopía Electrónica de Transmisión/métodos , Animales , Ojo/ultraestructura , Larva/ultraestructura , Pupa/ultraestructura , Fijación del Tejido/métodosRESUMEN
This protocol provides a method for identification of specific cell types in frozen adult Drosophila retina using beta-galactosidase staining.
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Técnicas Citológicas , Drosophila/citología , Coloración y Etiquetado/métodos , beta-Galactosidasa/análisis , Animales , Retina/citología , Retina/enzimologíaRESUMEN
INTRODUCTIONThe Drosophila eye has been used to study a broad range of topics, such as signal transduction, cell-fate specification, morphogenesis, and cell death. The eye has a relatively simple cellular architecture: the ommatidia (unit eyes of the compound eye) are composed of a small number of cells, and these cells can be readily identified on the basis of their shape and location within an ommatidium. Also, because the eye is a monolayer epithelium, all cells can be viewed in whole-mount preparations in both the larval imaginal disc and the pupal eye. Mosaic analysis is a powerful genetic tool that is routinely used in the eye to determine a given cell's requirement for a gene of interest. Such analyses are possible because cell fates are determined on the basis of positional cues. Furthermore, pattern formation proceeds as a wave of morphogenesis sweeps across the eye disc. Thus, all early patterning events are laid out in a single imaginal disc preparation. A final attractive feature of the eye is that it is dispensable for viability and development, and therefore a number of tools have been developed for its manipulation.
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INTRODUCTIONStaining Drosophila tissues with antibodies is generally straightforward. Ideal concentrations must be determined for each antibody: generally, dilute monoclonal supernatants 1:1 and ascites or sera 1:250 to 1:5000. If double-labeling, confirm that the secondary antibodies used will not recognize each other. Cross-reactivity can be avoided if secondary antibodies are raised in a single species. Fluorescent double-labeling generally does not work if both primaries are raised in the same species unless the primaries are directly conjugated to fluorochromes. If using primary antibodies raised in the same species, the use of HRP-conjugated antibodies is recommended. Double-labeling using DAB requires that the reaction product for one of the antibodies be intensified. This process is sensitive and should be used to detect the less abundant antigen. Similarly, if one of the primary antibodies gives a high background or is widely expressed throughout the tissue, this signal should not be enhanced. If one antigen is nuclear and the second is either cytoplasmic or on the cell surface, it is better to intensify the nuclear signal; the enhanced product can obscure details recognized by the cytoplasmic/cell surface antibody. If one antigen is less stable (e.g., extracted by detergent), this antigen should be detected first.