RESUMO
Social affiliation emerges from individual-level behavioural rules that are driven by conspecific signals1-5. Long-distance attraction and short-distance repulsion, for example, are rules that jointly set a preferred interanimal distance in swarms6-8. However, little is known about their perceptual mechanisms and executive neural circuits3. Here we trace the neuronal response to self-like biological motion9,10, a visual trigger for affiliation in developing zebrafish2,11. Unbiased activity mapping and targeted volumetric two-photon calcium imaging revealed 21 activity hotspots distributed throughout the brain as well as clustered biological-motion-tuned neurons in a multimodal, socially activated nucleus of the dorsal thalamus. Individual dorsal thalamus neurons encode local acceleration of visual stimuli mimicking typical fish kinetics but are insensitive to global or continuous motion. Electron microscopic reconstruction of dorsal thalamus neurons revealed synaptic input from the optic tectum and projections into hypothalamic areas with conserved social function12-14. Ablation of the optic tectum or dorsal thalamus selectively disrupted social attraction without affecting short-distance repulsion. This tectothalamic pathway thus serves visual recognition of conspecifics, and dissociates neuronal control of attraction from repulsion during social affiliation, revealing a circuit underpinning collective behaviour.
Assuntos
Aglomeração , Neurônios , Comportamento Social , Colículos Superiores , Tálamo , Vias Visuais , Peixe-Zebra , Animais , Mapeamento Encefálico , Cálcio/análise , Hipotálamo/citologia , Hipotálamo/fisiologia , Locomoção , Microscopia Eletrônica , Neurônios/citologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Reconhecimento Visual de Modelos , Estimulação Luminosa , Colículos Superiores/citologia , Colículos Superiores/fisiologia , Tálamo/citologia , Tálamo/fisiologia , Vias Visuais/citologia , Vias Visuais/fisiologia , Vias Visuais/ultraestrutura , Peixe-Zebra/fisiologiaRESUMO
Dense reconstruction of synaptic connectivity requires high-resolution electron microscopy images of entire brains and tools to efficiently trace neuronal wires across the volume. To generate such a resource, we sectioned and imaged a larval zebrafish brain by serial block-face electron microscopy at a voxel size of 14 × 14 × 25 nm3. We segmented the resulting dataset with the flood-filling network algorithm, automated the detection of chemical synapses and validated the results by comparisons to transmission electron microscopic images and light-microscopic reconstructions. Neurons and their connections are stored in the form of a queryable and expandable digital address book. We reconstructed a network of 208 neurons involved in visual motion processing, most of them located in the pretectum, which had been functionally characterized in the same specimen by two-photon calcium imaging. Moreover, we mapped all 407 presynaptic and postsynaptic partners of two superficial interneurons in the tectum. The resource developed here serves as a foundation for synaptic-resolution circuit analyses in the zebrafish nervous system.
Assuntos
Sinapses , Peixe-Zebra , Animais , Larva , Sinapses/ultraestrutura , Encéfalo/ultraestrutura , Microscopia EletrônicaRESUMO
Expansion microscopy (ExM) allows scalable imaging of preserved 3D biological specimens with nanoscale resolution on fast diffraction-limited microscopes. Here, we explore the utility of ExM in the larval and embryonic zebrafish, an important model organism for the study of neuroscience and development. Regarding neuroscience, we found that ExM enabled the tracing of fine processes of radial glia, which are not resolvable with diffraction-limited microscopy. ExM further resolved putative synaptic connections, as well as molecular differences between densely packed synapses. Finally, ExM could resolve subsynaptic protein organization, such as ring-like structures composed of glycine receptors. Regarding development, we used ExM to characterize the shapes of nuclear invaginations and channels, and to visualize cytoskeletal proteins nearby. We detected nuclear invagination channels at late prophase and telophase, potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic studies of how molecular components are configured in multiple contexts of interest to neuroscience and developmental biology.
Assuntos
Microscopia/métodos , Peixe-Zebra/anatomia & histologia , Animais , Encéfalo/ultraestrutura , Núcleo Celular/ultraestrutura , Biologia do Desenvolvimento/métodos , Larva/anatomia & histologia , Neurociências/métodos , Sinapses/ultraestrutura , Peixe-Zebra/embriologiaRESUMO
All-optical methods enable the control and monitoring of neuronal activity with minimal perturbation of the system. Although imaging and optogenetic manipulations can be performed at cellular resolution, the morphology of single cells in a dense neuronal population has often remained unresolvable. Here we describe in detail two recently established optogenetic protocols for systematic description of function and morphology of single neurons in zebrafish. First, the Optobow toolbox allows unbiased mapping of excitatory functional connectivity. Second, the FuGIMA technique enables selective labeling and anatomical tracing of neurons that are responsive to a given sensory stimulus or correlated with a specific behavior. Both strategies can be genetically targeted to a neuronal population of choice using the Gal4/UAS system. As these in vivo approaches are non-invasive, we envision useful applications for the study of neuronal structure, function and connectivity during development and behavior.
Assuntos
Rede Nervosa/fisiologia , Neurônios/fisiologia , Optogenética/métodos , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Técnicas Biossensoriais/métodos , Encéfalo/citologia , Encéfalo/fisiologia , Cálcio/química , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/efeitos da radiação , Rede Nervosa/citologia , Estimulação Luminosa/métodos , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Occluding cell-cell junctions in epithelia form physical barriers that separate different membrane domains, restrict paracellular diffusion and prevent pathogens from spreading across tissues. In invertebrates, these functions are provided by septate junctions (SJs), the functional equivalent of vertebrate tight junctions. How the diverse functions of SJs are integrated and modulated in a multiprotein complex is not clear, and many SJ components are still unknown. Here we report the identification of Macroglobulin complement-related (Mcr), a member of the conserved α-2-macroglobulin (α2M) complement protein family, as a novel SJ-associated protein in Drosophila. Whereas α2M complement proteins are generally known as secreted factors that bind to surfaces of pathogens and target them for phagocytic uptake, Mcr represents an unusual α2M protein with a predicted transmembrane domain. We show that Mcr protein localizes to lateral membranes of epithelial cells, where its distribution overlaps with SJs. Several SJ components are required for the correct localization of Mcr. Conversely, Mcr is required in a cell-autonomous fashion for the correct membrane localization of SJ components, indicating that membrane-bound rather than secreted Mcr isoforms are involved in SJ formation. Finally, we show that loss of Mcr function leads to morphological, ultrastructural and epithelial barrier defects resembling mutants lacking SJ components. Our results, along with previous findings on the role of Mcr in phagocytosis, suggest that Mcr plays dual roles in epithelial barrier formation and innate immunity. Thus, Mcr represents a novel paradigm for investigating functional links between occluding junction formation and pathogen defense mechanisms.
Assuntos
Citocinas/fisiologia , Drosophila/genética , Células Epiteliais/fisiologia , Imunidade Inata/genética , Junções Intercelulares/fisiologia , Proteínas de Membrana/fisiologia , Serpinas/fisiologia , Animais , Drosophila/fisiologia , Proteínas de Drosophila , Imunofluorescência , Processamento de Imagem Assistida por Computador , Immunoblotting , Hibridização In Situ , Junções Intercelulares/genética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Oligonucleotídeos/genéticaRESUMO
Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but so far, tools for investigating the dynamics of membrane trafficking have been limited to cultured cells. Here, we present a system that enables acute manipulation and real-time visualization of membrane trafficking through the reversible retention of proteins in the endoplasmic reticulum (ER) in living multicellular organisms. By adapting the "retention using selective hooks" (RUSH) approach to Drosophila, we show that trafficking of GPI-linked, secreted, and transmembrane proteins can be controlled with high temporal precision in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and apical secretion and the spatiotemporal dynamics of tricellular junction assembly in epithelia of living embryos. Furthermore, we show that controllable ER retention enables tissue-specific depletion of secretory protein function. The system is broadly applicable to visualizing and manipulating membrane trafficking in diverse cell types in vivo.
Assuntos
Drosophila , Complexo de Golgi , Animais , Transporte Proteico/fisiologia , Complexo de Golgi/metabolismo , Transporte Biológico , ExocitoseRESUMO
Retinal axon projections form a map of the visual environment in the tectum. A zebrafish larva typically detects a prey object in its peripheral visual field. As it turns and swims towards the prey, the stimulus enters the central, binocular area, and seemingly expands in size. By volumetric calcium imaging, we show that posterior tectal neurons, which serve to detect prey at a distance, tend to respond to small objects and intrinsically compute their direction of movement. Neurons in anterior tectum, where the prey image is represented shortly before the capture strike, are tuned to larger object sizes and are frequently not direction-selective, indicating that mainly interocular comparisons serve to compute an object's movement at close range. The tectal feature map originates from a linear combination of diverse, functionally specialized, lamina-specific, and topographically ordered retinal ganglion cell synaptic inputs. We conclude that local cell-type composition and connectivity across the tectum are adapted to the processing of location-dependent, behaviorally relevant object features.
The retina is the thin layer of tissue in the eye that can receive light stimuli and convert them into electric signals to be transmitted to the brain. The cells that sense fine detail cluster at the center of the retina while the motion-sensing cells that keep track of movement lie at the periphery. When zebrafish larvae hunt, their motion-sensing cells are triggered as a prey crosses their peripheral field of view. They then turn and swim towards it. As they approach, the prey image moves to the detail-sensing part of the retina and appears larger, filling more of the field of view at close range. The signals are then processed in defined parts of the brain, in particular in a region called the optic tectum. How this area is organized in response to the organization of the eye and the requirements of the hunt is still unclear. Förster et al. set out to explore how the hunting routine of zebrafish larvae shapes the arrangement of neurons in the optic tectum. The larvae were exposed to different images representing the various aspects of the prey capture process: small moving dots represented passing prey at a distance, while large moving dots stood for prey just before capture. Measuring activity in the neurons of the optic tectum revealed that, like the eye, different areas specialize in different tasks. The back of the tectum was frequently activated by small dots and worked out which direction they were moving in during the first hunting steps. The front of the tectum responded best to large dots, often ignoring their direction, and helped the larvae to track their prey straight ahead. To test these findings, Förster et al. destroyed the large object-responsive cells with a laser and watched the larvae hunting real prey. Without the cells, the fish found it much harder to track and catch their targets. These results shed light on the link between behavior and how neurons are arranged in the brain. Future work could explore how the different neurons in the optic tectum are connected, and the behaviors they trigger in the fish. This could help to reveal general principles about how sensory information guides behavior and how evolution has shaped the layout of the brain.
Assuntos
Comportamento Predatório/fisiologia , Colículos Superiores/fisiologia , Percepção Visual , Peixe-Zebra/fisiologia , AnimaisRESUMO
Understanding brain-wide neuronal dynamics requires a detailed map of the underlying circuit architecture. We built an interactive cellular-resolution atlas of the zebrafish brain at 6 days post-fertilization (dpf) based on the reconstructions of over 2,000 individually GFP-labeled neurons. We clustered our dataset in "morphotypes," establishing a unique database of quantitatively described neuronal morphologies together with their spatial coordinates in vivo. Over 100 transgene expression patterns were imaged separately and co-registered with the single-neuron atlas. By annotating 72 non-overlapping brain regions, we generated from our dataset an inter-areal wiring diagram of the larval brain, which serves as ground truth for synapse-scale, electron microscopic reconstructions. Interrogating our atlas by "virtual tract tracing" has already revealed previously unknown wiring principles in the tectum and the cerebellum. In conclusion, we present here an evolving computational resource and visualization tool, which will be essential to map function to structure in a vertebrate brain. VIDEO ABSTRACT.
Assuntos
Atlas como Assunto , Encéfalo/anatomia & histologia , Encéfalo/citologia , Peixe-Zebra/anatomia & histologia , Animais , Encéfalo/ultraestrutura , Mapeamento Encefálico , Cerebelo/anatomia & histologia , Conectoma , Expressão Gênica , Proteínas de Fluorescência Verde , Larva/anatomia & histologia , Larva/citologia , Neurônios/ultraestrutura , Transgenes , Vias Visuais/anatomia & histologiaRESUMO
Agouti-related protein (AgRP) is a hypothalamic regulator of food consumption in mammals. However, AgRP has also been detected in circulation, but a possible endocrine role has not been examined. Zebrafish possess two agrp genes: hypothalamically expressed agrp1, considered functionally equivalent to the single mammalian agrp, and agrp2, which is expressed in pre-optic neurons and uncharacterized pineal gland cells and whose function is not well understood. By ablation of AgRP1-expressing neurons and knockout of the agrp1 gene, we show that AgRP1 stimulates food consumption in the zebrafish larvae. Single-cell sequencing of pineal agrp2-expressing cells revealed molecular resemblance to retinal-pigment epithelium cells, and anatomic analysis shows that these cells secrete peptides, possibly into the cerebrospinal fluid. Additionally, based on AgRP2 peptide localization and gene knockout analysis, we demonstrate that pre-optic AgRP2 is a neuroendocrine regulator of the stress axis that reduces cortisol secretion. We therefore suggest that the ancestral role of AgRP was functionally partitioned in zebrafish by the two AgRPs, with AgRP1 centrally regulating food consumption and AgRP2 acting as a neuroendocrine factor regulating the stress axis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Estresse Fisiológico/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Técnicas de Inativação de Genes , Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glândula Pineal/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The nonsense-mediated messenger RNA (mRNA) decay (NMD) pathway is a cellular quality control and post-transcriptional gene regulatory mechanism and is essential for viability in most multicellular organisms . A complex of proteins has been identified to be required for NMD function to occur; however, there is an incomplete understanding of the individual contributions of each of these factors to the NMD process. Central to the NMD process are three proteins, Upf1 (SMG-2), Upf2 (SMG-3), and Upf3 (SMG-4), which are found in all eukaryotes, with Upf1 and Upf2 being absolutely required for NMD in all organisms in which their functions have been examined. The other known NMD factors, Smg1, Smg5, Smg6, and Smg7, are more variable in their presence in different orders of organisms and are thought to have a more regulatory role. Here we present the first genetic analysis of the NMD factor Smg5 in Drosophila Surprisingly, we find that unlike the other analyzed Smg genes in this organism, Smg5 is essential for NMD activity. We found this is due in part to a requirement for Smg5 in both the activity of Smg6-dependent endonucleolytic cleavage, as well as an additional Smg6-independent mechanism. Redundancy between these degradation pathways explains why some Drosophila NMD genes are not required for all NMD-pathway activity. We also found that while the NMD component Smg1 has only a minimal role in Drosophila NMD during normal conditions, it becomes essential when NMD activity is compromised by partial loss of Smg5 function. Our findings suggest that not all NMD complex components are required for NMD function at all times, but instead are utilized in a context-dependent manner in vivo.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Animais , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Endorribonucleases/metabolismo , Regulação da Expressão Gênica , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Optical imaging approaches have revolutionized our ability to monitor neural network dynamics, but by themselves are unable to link a neuron's activity to its functional connectivity. We present a versatile genetic toolbox, termed 'Optobow', for all-optical discovery of excitatory connections in vivo. By combining the Gal4-UAS system with Cre/lox recombination, we target the optogenetic actuator ChrimsonR and the sensor GCaMP6 to stochastically labeled, nonoverlapping and sparse subsets of neurons. Photostimulation of single cells using two-photon computer-generated holography evokes calcium responses in downstream neurons. Morphological reconstruction of neurite arbors, response latencies and localization of presynaptic markers suggest that some neuron pairs recorded here are directly connected, while others are two or more synapses apart from each other. With this toolbox, we discover wiring principles between specific cell types in the larval zebrafish tectum. Optobow should be useful for identification and manipulation of networks of interconnected neurons, even in dense neural tissues.Mechanisms of neural processing can only be understood by revealing patterns of connectivity among the cellular components of the circuit. Here the authors report a new genetic toolbox, 'Optobow', which enables simultaneous optogenetic activation of single neurons in zebrafish and measuring the activity of downstream neurons in the network.
Assuntos
Rede Nervosa/metabolismo , Neurônios/metabolismo , Optogenética/métodos , Sinapses/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Neurológicos , Neurônios/citologia , Colículos Superiores/citologia , Colículos Superiores/metabolismo , Peixe-ZebraRESUMO
Genetic access to small, reproducible sets of neurons is key to an understanding of the functional wiring of the brain. Here we report the generation of a new Gal4- and Cre-driver resource for zebrafish neurobiology. Candidate genes, including cell type-specific transcription factors, neurotransmitter-synthesizing enzymes and neuropeptides, were selected according to their expression patterns in small and unique subsets of neurons from diverse brain regions. BAC recombineering, followed by Tol2 transgenesis, was used to generate driver lines that label neuronal populations in patterns that, to a large but variable extent, recapitulate the endogenous gene expression. We used image registration to characterize, compare, and digitally superimpose the labeling patterns from our newly generated transgenic lines. This analysis revealed highly restricted and mutually exclusive tissue distributions, with striking resolution of layered brain regions such as the tectum or the rhombencephalon. We further show that a combination of Gal4 and Cre transgenes allows intersectional expression of a fluorescent reporter in regions where the expression of the two drivers overlaps. Taken together, our study offers new tools for functional studies of specific neural circuits in zebrafish.
Assuntos
Encéfalo/fisiologia , Cromossomos Artificiais Bacterianos , Marcação de Genes , Neurônios/fisiologia , Transgenes , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismoRESUMO
Although many organ functions rely on epithelial tubes with correct dimensions, mechanisms underlying tube size control are poorly understood. We analyse the cellular mechanism of tracheal tube elongation in Drosophila, and describe an essential role of the conserved tyrosine kinase Src42A in this process. We show that Src42A is required for polarized cell shape changes and cell rearrangements that mediate tube elongation. In contrast, diametric expansion is controlled by apical secretion independently of Src42A. Constitutive activation of Src42A induces axial cell stretching and tracheal overelongation, indicating that Src42A acts instructively in this process. We propose that Src42A-dependent recycling of E-Cadherin at adherens junctions is limiting for cell shape changes and rearrangements in the axial dimension of the tube. Thus, we define distinct cellular processes that independently control axial and diametric expansion of a cylindrical epithelium in a developing organ. Whereas exocytosis-dependent membrane growth drives circumferential tube expansion, Src42A is required to orient membrane growth in the axial dimension of the tube.
Assuntos
Polaridade Celular , Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Animais , Caderinas/metabolismoRESUMO
Epithelial tubes in developing organs, such as mammalian lungs and insect tracheae, need to expand their initially narrow lumina to attain their final, functional dimensions. Despite its critical role for organ function, the cellular mechanism of tube expansion remains unclear. Tracheal tube expansion in Drosophila involves apical secretion and deposition of a luminal matrix, but the mechanistic role of secretion and the nature of forces involved in the process were not previously clear. Here we address the roles of cell-intrinsic and extrinsic processes in tracheal tube expansion. We identify mutations in the sec24 gene stenosis, encoding a cargo-binding subunit of the COPII complex. Via genetic-mosaic analyses, we show that stenosis-dependent secretion drives tube expansion in a cell-autonomous fashion. Strikingly, single cells autonomously adjust both tube diameter and length by implementing a sequence of events including apical membrane growth, cell flattening, and taenidial cuticle formation. Known luminal components are not required for this process. Thus, a cell-intrinsic program, rather than nonautonomous extrinsic cues, controls the dimensions of tracheal tubes. These results indicate a critical role of membrane-associated proteins in the process and imply a mechanism that coordinates autonomous behaviors of individual cells within epithelial structures.