A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila

eLife, Год журнала: 2020, Номер 9

Опубликована: Фев. 13, 2020

Genetic screens are powerful tools for the functional annotation of genomes. In context multicellular organisms, interrogation gene function is greatly facilitated by methods that allow spatial and temporal control abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library efficient CRISPR-based disruption specific target genes in constitutive or conditional manner. The consists currently more than 2600 plasmids 1700 fly lines with focus on targeting kinases, phosphatases transcription factors, each expressing two sgRNAs under Gal4/UAS system. We show CRISPR mutagenesis robust across many can be efficiently employed various somatic tissues, as well germline. order to prevent artefacts commonly associated excessive amounts Cas9 protein, have developed series novel UAS-Cas9 transgenes, which fine tuning expression achieve high editing activity without detectable toxicity. Functional assays, direct sequencing genomic sgRNA sites, indicates vast majority mediate disruption. Furthermore, conducted so far largest fully screen any metazoan organism, further supported efficiency accuracy our revealed uncharacterized essential development.

Язык: Английский

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A transcriptomic taxonomy of Drosophila circadian neurons around the clock

eLife, Год журнала: 2021, Номер 10

Опубликована: Янв. 13, 2021

Many different functions are regulated by circadian rhythms, including those orchestrated discrete clock neurons within animal brains. To comprehensively characterize and assign cell identity to the 75 pairs of Drosophila neurons, we optimized a single-cell RNA sequencing method assayed neuron gene expression at times day. The data identify least 17 categories with striking spatial regulation expression. Transcription factor is prominent likely contributes robust oscillation many transcripts, that encode cell-surface proteins previously shown be important for recognition synapse formation during development. other clock-regulated genes also constitute an resource future mechanistic functional studies between and/or temporal signaling circuits elsewhere in fly brain.

Язык: Английский

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Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity

eLife, Год журнала: 2019, Номер 8

Опубликована: Окт. 15, 2019

Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian clocks continue to tick away without intercellular communication. To investigate issues Drosophila, we assayed behavior as well molecular within individual brain clock neurons while blocking communication the ca. 150 neuron network. We also generated CRISPR-mediated neuron-specific knockouts. The results point two key groups: loss of both regions but neither one alone has a strong behavioral phenotype darkness; between contributes period determination. Under dark conditions, region persists network famous PDF-expressing s-LNv however was strongly dependent on communication, likely because gene expression vulnerable sLNvs depends neuronal firing or light.

Язык: Английский

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Internal state configures olfactory behavior and early sensory processing in Drosophila larvae

Science Advances, Год журнала: 2021, Номер 7(1)

Опубликована: Янв. 1, 2021

The first olfactory processing center in the larval Drosophila brain uses information about feeding state to shape behavior.

Язык: Английский

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Drosophila clock cells use multiple mechanisms to transmit time-of-day signals in the brain

Proceedings of the National Academy of Sciences, Год журнала: 2021, Номер 118(10)

Опубликована: Март 3, 2021

Significance Despite our growing understanding of how the fly clock network maintains free-running rhythms behavior and physiology, little is known about information communicated from to rest brain regulate behavior. We identify glutamate acetylcholine as key neurotransmitters signaling neurons pars intercerebralis (PI), a output region regulating circadian sleep metabolism. report link between Drosophila evening PI, find that effect on neuron physiology varies, suggesting same cells use multiple mechanisms simultaneously drive cycling in neurons.

Язык: Английский

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Connectomic analysis of the Drosophila lateral neuron clock cells reveals the synaptic basis of functional pacemaker classes

eLife, Год журнала: 2022, Номер 11

Опубликована: Июнь 29, 2022

The circadian clock orchestrates daily changes in physiology and behavior to ensure internal temporal order optimal timing across the day. In animals, a central brain coordinates rhythms throughout body is characterized by remarkable robustness that depends on synaptic connections between constituent neurons. neuron network of Drosophila, which shares motifs with networks mammalian yet built many fewer neurons, offers powerful model for understanding properties timekeeping. Here, we report an assessment connectivity within network, focusing critical lateral (LN) classes Janelia hemibrain dataset. Our results reveal previously identified anatomical functional subclasses LNs represent distinct connectomic types. Moreover, identify small number non-clock cell subtypes representing highly synaptically coupled nodes network. This suggests neurons lacking molecular timekeeping likely play integral roles To our knowledge, this represents first comprehensive analysis neuronal network.Most organisms Earth possess system ensures bodily processes such as sleep, wakefulness or digestion take place at right time. These precise are kept check master brain. There, thousands – some carrying ‘molecular clock’ connect each other through structures known synapses. Exactly how resulting organised support remains unclear. explore question, Shafer, Gutierrez et al. focused fruit flies, recent efforts have systematically mapped every connection organism. Analysing available data from connectome project revealed most important were fact forming fewest synapses addition, without clocks mediated strong those did, suggesting ‘clockless’ cells still role With research, provide unexpected insights into organisation clock. Better underpin will help grasp why these disrupted obesity, depression Alzheimer’s disease.

Язык: Английский

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A split-GAL4 driver line resource forDrosophilaneuron types

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Янв. 10, 2024

Abstract Techniques that enable precise manipulations of subsets neurons in the fly central nervous system have greatly facilitated our understanding neural basis behavior. Split-GAL4 driver lines allow specific targeting cell types Drosophila melanogaster and other species. We describe here a collection 3060 range adult 1373 characterized third-instar larvae. These tools functional, transcriptomic, proteomic studies based on anatomical targeting. NeuronBridge search relate light microscopy images these split-GAL4 to connectomes reconstructed from electron images. The collections are result screening over 77,000 split hemidriver combinations. Previously published new included, all validated for expression curated optimal type specificity across diverse types. In addition stocks well-characterized lines, we make available 300,000 3D lines.

Язык: Английский

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Dopamine and GPCR-mediated modulation of DN1 clock neurons gates the circadian timing of sleep

Proceedings of the National Academy of Sciences, Год журнала: 2022, Номер 119(34)

Опубликована: Авг. 15, 2022

The metronome-like circadian regulation of sleep timing must still adapt to an uncertain environment. Recent studies in Drosophila indicate that neuromodulation not only plays a key role clock neuron synchronization but also affects interactions between the network and brain centers. We show here targets neuromodulators, G Protein Coupled Receptors (GPCRs), are highly enriched fly network. Single-cell sequencing indicates they differentially expressed contribute identity. generated comprehensive guide library mutagenize individual GPCRs specific neurons verified strategy by introducing targeted approach. Combined with behavioral screen, mutagenesis revealed dopamine identifying two receptors subpopulation gate sleep.

Язык: Английский

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Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms

Proceedings of the National Academy of Sciences, Год журнала: 2021, Номер 118(28)

Опубликована: Июль 7, 2021

Circadian clocks regulate ∼24-h oscillations in gene expression, behavior, and physiology. While the genetic molecular mechanisms of circadian rhythms are well characterized, what remains poorly understood intracellular dynamics clock components how they affect rhythms. Here, we elucidate spatiotemporal organization core proteins genes Drosophila neurons. Using high-resolution imaging DNA-fluorescence situ hybridization techniques, demonstrate that (PERIOD CLOCK) organized into a few discrete foci at nuclear envelope during repression phase play an important role subnuclear localization to control Specifically, show genes, period timeless, positioned close periphery by PERIOD protein specifically phase, suggesting might key their rhythmic expression. Finally, loss Lamin B receptor, protein, leads disruption PER per peripheral results rhythm defects. These hitherto unexpected reorganization rhythms, revealing function subcellular level. Our further suggest dynamic clustering spatial clock-regulated over behavior

Язык: Английский

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An extra-clock ultradian brain oscillator sustains circadian timekeeping

Science Advances, Год журнала: 2022, Номер 8(35)

Опубликована: Сен. 2, 2022

The master circadian clock generates 24-hour rhythms to orchestrate daily behavior, even running freely under constant conditions. Traditionally, the is considered self-sufficient in sustaining free-running timekeeping via its cell-autonomous molecular clocks and interneuronal communications within neural network. Here, we find a set of bona fide ultradian oscillators Drosophila brain that support timekeeping, despite being located outside circuit lacking gene expression. These extra-clock electrical (xCEOs) generate bursts, pacing widespread burst firing promoting rhythmic resting membrane potentials neurons parallel monosynaptic connections. Silencing xCEOs disrupts impairs cycling neuropeptide pigment dispersing factor, leading loss locomotor rhythms. Together, conclude not sustain behavior but requires additional endogenous inputs from oscillators.

Язык: Английский

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