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In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.
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Mosquitoes are strongly attracted to humans, and their bites not only cause intense itch but can beget severe diseases. In this issue of Cell, Herre et al. reveal that non-canonical olfactory circuit organization and coding likely endow mosquitoes with a robust ability to locate human hosts.
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Aedes , Anopheles , Animales , Humanos , Odorantes , FeromonasRESUMEN
Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.
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Vías Olfatorias/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Proteómica/métodos , Animales , Axones/metabolismo , Encéfalo/metabolismo , Dendritas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Neurogénesis/fisiología , Nervio Olfatorio/metabolismo , Vías Olfatorias/citología , Vías Olfatorias/fisiología , Receptores de Lipoproteína/metabolismo , Olfato/fisiologíaRESUMEN
Elucidating signal transduction mechanisms of innate immune pathways is essential to defining how they elicit distinct cellular responses. Toll-like receptors (TLR) signal through their cytoplasmic TIR domains which bind other TIR domain-containing adaptors. dSARM/SARM1 is one such TIR domain adaptor best known for its role as the central axon degeneration trigger after injury. In degeneration, SARM1's domains have been assigned unique functions: the ARM domain is auto-inhibitory, SAM-SAM domain interactions mediate multimerization, and the TIR domain has intrinsic NAD+ hydrolase activity that precipitates axonal demise. Whether and how these distinct functions contribute to TLR signaling is unknown. Here we show divergent signaling requirements for dSARM in injury-induced axon degeneration and TLR-mediated developmental glial phagocytosis through analysis of new knock-in domain and point mutations. We demonstrate intragenic complementation between reciprocal pairs of domain mutants during development, providing evidence for separability of dSARM functional domains in TLR signaling. Surprisingly, dSARM's NAD+ hydrolase activity is strictly required for both degenerative and developmental signaling, demonstrating that TLR signal transduction requires dSARM's enzymatic activity. In contrast, while SAM domain-mediated dSARM multimerization is important for axon degeneration, it is dispensable for TLR signaling. Finally, dSARM functions in a linear genetic pathway with the MAP3K Ask1 during development but not in degenerating axons. Thus, we propose that dSARM exists in distinct signaling states in developmental and pathological contexts.
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Proteínas del Dominio Armadillo , NAD , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Proteínas del Citoesqueleto/genética , Hidrolasas/metabolismo , Fagocitosis/genética , Transducción de Señal/genéticaRESUMEN
Any adult who has tried to take up the piano or learn a new language is faced with the sobering realization that acquiring such skills is more challenging as an adult than as a child. Neuronal plasticity, or the malleability of brain circuits, declines with age. Young neurons tend to be more adaptable and can alter the size and strength of their connections more readily than can old neurons. Myriad circuit- and synapse-level mechanisms that shape plasticity have been identified. Yet, molecular mechanisms setting the overall competence of young neurons for distinct forms of plasticity remain largely obscure. Recent studies indicate evolutionarily conserved roles for FoxO proteins in establishing the capacity for cell-fate, morphological, and synaptic plasticity in neurons.
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Factores de Transcripción Forkhead/genética , Células-Madre Neurales/fisiología , Plasticidad Neuronal/fisiología , Animales , Proteínas de Caenorhabditis elegans/genética , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Transcripción Forkhead/metabolismo , Humanos , Neuronas/citología , Neuronas/fisiología , Sinapsis/fisiologíaRESUMEN
Many insect cells are encapsulated within the exoskeleton and cannot be dissociated intact, making them inaccessible to single-cell transcriptomic profiling. We have used single-nucleus RNA sequencing to extract transcriptomic information from multiple Drosophila tissues. Here, we describe procedures for the (1) dissociation of single nuclei, (2) isolation of single nuclei using two popular cell sorters, and (3) preparation of libraries for Smart-seq2 and 10× Genomics. This protocol enables generation of high-quality transcriptomes from single nuclei and can be applied to other species. For complete details on the use and execution of this protocol, please refer to McLaughlin et al. (2021) and Li et al. (2022).
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Drosophila , Perfilación de la Expresión Génica , Animales , Secuencia de Bases , Drosophila/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Secuenciación del ExomaRESUMEN
For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.
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Drosophila melanogaster/citología , Drosophila melanogaster/genética , Transcriptoma , Animales , Núcleo Celular/metabolismo , Bases de Datos Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Femenino , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genes de Insecto , Masculino , RNA-Seq , Caracteres Sexuales , Análisis de la Célula Individual , Factores de Transcripción/genéticaRESUMEN
Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes of Drosophila olfactory- (ORNs), thermo-, and hygro-sensory neurons at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuron types across multiple developmental stages. We found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. We uncovered stage-specific genes that could regulate the wiring and sensory responses of distinct ORN types. Collectively, our data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.
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Drosophila melanogaster/citología , Drosophila melanogaster/genética , Neuronas Receptoras Olfatorias/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/fisiología , Femenino , Masculino , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Olfato , TranscriptomaRESUMEN
Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage-neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.
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Drosophila melanogaster/metabolismo , Neuritas/metabolismo , Nervio Olfatorio/metabolismo , Transcriptoma , Animales , Análisis de la Célula Individual , Factores de TiempoRESUMEN
The regulatory mechanisms by which neurons coordinate their physiology and connectivity are not well understood. The Drosophila olfactory receptor neurons (ORNs) provide an excellent system to investigate this question. Each ORN type expresses a unique olfactory receptor, or a combination thereof, and sends their axons to a stereotyped glomerulus. Using single-cell RNA sequencing, we identified 33 transcriptomic clusters for ORNs and mapped 20 to their glomerular types, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN types. Each ORN type expresses hundreds of transcription factors. Transcriptome-instructed genetic analyses revealed that (1) one broadly expressed transcription factor (Acj6) only regulates olfactory receptor expression in one ORN type and only wiring specificity in another type, (2) one type-restricted transcription factor (Forkhead) only regulates receptor expression, and (3) another type-restricted transcription factor (Unplugged) regulates both events. Thus, ORNs utilize diverse strategies and complex regulatory networks to coordinate their physiology and connectivity.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Factores de Transcripción Forkhead/genética , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Neuronas Receptoras Olfatorias/fisiología , Factores del Dominio POU/genética , Receptores Odorantes/genética , Transcriptoma , Animales , Axones/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores del Dominio POU/metabolismo , Receptores Odorantes/metabolismo , Análisis de la Célula Individual , Olfato/fisiologíaRESUMEN
Glia continuously survey neuronal health during development, providing trophic support to healthy neurons while rapidly engulfing dying ones. These diametrically opposed functions necessitate a foolproof mechanism enabling glia to unambiguously identify those neurons to support versus those to engulf. To ensure specificity, glia are proposed to interact with dying neurons via a series of carefully choreographed steps. However, these crucial interactions are largely obscure. Here we show that dying neurons and glia communicate via Toll-receptor-regulated innate immune signaling. Neuronal apoptosis drives processing and activation of the Toll-6 ligand, Spätzle5. This cue activates a dSARM-mediated Toll-6 transcriptional pathway in glia, which controls the expression of the Draper engulfment receptor. Pathway loss drives early-onset neurodegeneration, underscoring its functional importance. Our results identify an upstream priming signal that prepares glia for phagocytosis. Thus, a core innate immune pathway plays an unprecedented role setting the valence of neuron-glia interactions during development.
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Encéfalo/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Fagocitosis/fisiología , Animales , Animales Modificados Genéticamente , Apoptosis/fisiología , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Humanos , Proteínas de la Membrana/metabolismoRESUMEN
Synaptic plasticity occurs in response to intrinsic and extrinsic cues and is a key step in the formation of mature neuronal circuits. In this issue of Developmental Cell, Meng et al. (2017) find that two conserved Myrf transcription factors coexist in the same complex to promote developmental circuit remodeling.
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Proteínas de la Membrana/metabolismo , Neurogénesis/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Factores de Transcripción/metabolismo , Animales , Humanos , Red Nerviosa/fisiologíaRESUMEN
FoxO proteins are evolutionarily conserved regulators of neuronal structure and function, yet the neuron-specific pathways within which they act are poorly understood. To elucidate neuronal FoxO function in Drosophila melanogaster, we first screened for FoxO's upstream regulators and downstream effectors. On the upstream side, we present genetic and molecular pathway analyses indicating that the Toll-6 receptor, the Toll/interleukin-1 receptor domain adaptor dSARM, and FoxO function in a linear pathway. On the downstream side, we find that Toll-6-FoxO signaling represses the mitotic kinesin Pavarotti/MKLP1 (Pav-KLP), which itself attenuates microtubule (MT) dynamics. We next probed in vivo functions for this novel pathway and found that it is essential for axon transport and structural plasticity in motoneurons. We demonstrate that elevated expression of Pav-KLP underlies transport and plasticity phenotypes in pathway mutants, indicating that Toll-6-FoxO signaling promotes MT dynamics by limiting Pav-KLP expression. In addition to uncovering a novel molecular pathway, our work reveals an unexpected function for dynamic MTs in enabling rapid activity-dependent structural plasticity.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas Motoras/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismo , Animales , Proteínas del Dominio Armadillo/metabolismo , Axones/metabolismo , Núcleo Celular/metabolismo , Sistema Nervioso Central/metabolismo , Proteínas del Citoesqueleto/metabolismo , Modelos Biológicos , Mutación/genética , Unión Neuromuscular/metabolismo , Plasticidad Neuronal , Transporte de Proteínas , Sinapsis/metabolismoRESUMEN
Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal.
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Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Drosophila melanogaster/genética , Neuronas Motoras/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The ventral medial prefrontal cortex (vmPFC) controls vulnerability to the negative effects of chronic or uncontrollable stress. Dominance status alters responses to social defeat in the conditioned defeat model, which is a model characterized by loss of territorial aggression and increased submissive and defensive behavior following an acute social defeat. We have previously shown that dominant individuals show a reduced conditioned defeat response and increased defeat-induced neural activation in the vmPFC compared to subordinates. Here, we tested the hypothesis that defeat-induced activation of the vmPFC is necessary to confer resistance to conditioned defeat in dominants. We paired weight-matched male Syrian hamsters (Mesocricetus auratus) in daily 5-min aggressive encounters for 2 weeks and identified dominants and subordinates. Twenty-four hours after the final pairing, animals were bilaterally injected with 200 nl of the GABAA receptor agonist muscimol (1.1 nmol) or 200 nl of saline vehicle 5 min prior to social defeat. Defeat consisted of 3, 5-min encounters with resident aggressor hamsters at 10-min intervals. Twenty-four hours following social defeat, animals received conditioned defeat testing which involved a 5-min social interaction test with a non-aggressive intruder. Muscimol injection prior to social defeat prevented the reduced conditioned defeat response observed in vehicle-treated dominants. Further, there was no effect of muscimol injection on the conditioned defeat response in subordinates or controls. These data support the conclusion that activation of the vmPFC during social defeat is necessary for the protective effects of dominant social status on the acquisition of conditioned defeat.