Our understanding of the mechanisms of neural circuit assembly is far from complete. Identification of wiring molecules with novel mechanisms of action will provide insights into how complex and heterogeneous neural circuits assemble during development. In the olfactory system, 50 classes of olfactory receptor neurons (ORNs) make precise synaptic connections with 50 classes of partner projection neurons (PNs). Here, we performed an RNA interference screen for cell surface molecules and identified the leucine-rich repeat-containing transmembrane protein known as Fish-lips (Fili) as a novel wiring molecule in the assembly of the olfactory circuit. Fili contributes to the precise axon and dendrite targeting of a small subset of ORN and PN classes, respectively. Cell-type-specific expression and genetic analyses suggest that Fili sends a transsynaptic repulsive signal to neurites of nonpartner classes that prevents their targeting to inappropriate glomeruli in the antennal lobe.

Plexins exhibit multitudinous, evolutionarily conserved functions in neural development. How Plexins employ their diverse structural motifs in vivo to perform distinct roles is unclear. We previously reported that Plexin B (PlexB) controls multiple steps during the assembly of the olfactory circuit (Li et al., 2018b). Here, we systematically mutagenized structural motifs of PlexB and examined the function of these variants in these multiple steps: axon fasciculation, trajectory choice, and synaptic partner selection. We found that the extracellular Sema domain is essential for all three steps, the catalytic site of the intracellular RapGAP is engaged in none, and the intracellular GTPase-binding motifs are essential for trajectory choice and synaptic partner selection, but are dispensable for fasciculation. Moreover, extracellular PlexB cleavage serves as a regulatory mechanism of PlexB signaling. Thus, the divergent roles of PlexB motifs in distinct steps of neural development contribute to its functional versatility in neural circuit assembly.

The precise assembly of a neural circuit involves many consecutive steps. The conflict between a limited number of wiring molecules and the complexity of the neural network impels each molecule to execute multiple functions at different steps. Here, we examined the cell-type specific distribution of endogenous levels of axon guidance receptor Plexin B (PlexB) in the developing antennal lobe, the first olfactory processing center in . We found that different classes of olfactory receptor neurons (ORNs) express PlexB at different levels in two wiring steps - axonal trajectory choice and subsequent target selection. In line with its temporally distinct patterns, the proper levels of PlexB control both steps in succession. Genetic interactions further revealed that the effect of high-level PlexB is antagonized by its canonical partner Sema2b. Thus, PlexB plays a multifaceted role in instructing the assembly of the olfactory circuit through temporally-regulated expression patterns and expression level-dependent effects.

Understanding signaling pathways in neuroscience requires high-resolution maps of the underlying protein networks. Proximity-dependent biotinylation with engineered enzymes, in combination with mass spectrometry-based quantitative proteomics, has emerged as a powerful method to dissect molecular interactions and the localizations of endogenous proteins. Recent applications to neuroscience have provided insights into the composition of sub-synaptic structures, including the synaptic cleft and inhibitory post-synaptic density. Here we compare the different enzymes and small-molecule probes for proximity labeling in the context of cultured neurons and tissue, review existing studies, and provide technical suggestions for the in vivo application of proximity labeling.

Brain function requires precise neural circuit assembly during development. Establishing a functional circuit involves multiple coordinated steps ranging from neural cell fate specification to proper matching between pre- and post-synaptic partners. How neuronal lineage and birth timing influence wiring specificity remains an open question. Recent findings suggest that the relationships between lineage, birth timing, and wiring specificity vary in different neuronal circuits. In this review, we summarize our current understanding of the cellular, molecular, and developmental mechanisms linking neuronal lineage and birth timing to wiring specificity in a few specific systems in Drosophila and mice, and review different methods employed to explore these mechanisms.

The definition of neuronal type and how it relates to the transcriptome are open questions. Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: different PN classes target dendrites to distinct olfactory glomeruli, while PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomes of most PN classes and unequivocally mapped transcriptomes to specific olfactory function for six classes. Transcriptomes of closely related PN classes exhibit the largest differences during circuit assembly but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Transcription factors and cell-surface molecules are the most differentially expressed genes between classes and are highly informative in encoding cell identity, enabling us to identify a new lineage-specific transcription factor that instructs PN dendrite targeting. These findings establish that neuronal transcriptomic identity corresponds with anatomical and physiological identity defined by connectivity and function.

No abstract available.

The formation of complex but highly organized neural circuits requires interactions between neurons and glia. During the assembly of the olfactory circuit, 50 olfactory receptor neuron (ORN) classes and 50 projection neuron (PN) classes form synaptic connections in 50 glomerular compartments in the antennal lobe, each of which represents a discrete olfactory information-processing channel. Each compartment is separated from the adjacent compartments by membranous processes from ensheathing glia. Here we show that Thisbe, an FGF released from olfactory neurons, particularly from local interneurons, instructs ensheathing glia to wrap each glomerulus. The Heartless FGF receptor acts cell-autonomously in ensheathing glia to regulate process extension so as to insulate each neuropil compartment. Overexpressing Thisbe in ORNs or PNs causes overwrapping of the glomeruli their axons or dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon targeting and discrete glomerular formation.

strain WP3 belongs to the group 1 branch of the genus and is a piezotolerant and psychrotolerant species isolated from the deep sea. In this study, a genome-scale model was constructed for WP3 using a combination of genome annotation, ortholog mapping, and physiological verification. The metabolic reconstruction contained 806 genes, 653 metabolites, and 922 reactions, including central metabolic functions that represented nonhomologous replacements between the group 1 and group 2 species. Metabolic simulations with the WP3 model demonstrated consistency with existing knowledge about the physiology of the organism. A comparison of model simulations with experimental measurements verified the predicted growth profiles under increasing concentrations of carbon sources. The WP3 model was applied to study mechanisms of anaerobic respiration through investigating energy conservation, redox balancing, and the generation of proton motive force. Despite being an obligate respiratory organism, WP3 was predicted to use substrate-level phosphorylation as the primary source of energy conservation under anaerobic conditions, a trait previously identified in other species. Further investigation of the ATP synthase activity revealed a positive correlation between the availability of reducing equivalents in the cell and the directionality of the ATP synthase reaction flux. Comparison of the WP3 model with an existing model of a group 2 species, MR-1, revealed that the WP3 model demonstrated greater flexibility in ATP production under the anaerobic conditions. Such flexibility could be advantageous to WP3 for its adaptation to fluctuating availability of organic carbon sources in the deep sea. The well-studied nature of the metabolic diversity of bacteria makes species from this genus a promising platform for investigating the evolution of carbon metabolism and energy conservation. The phylogeny is diverged into two major branches, referred to as group 1 and group 2. While the genotype-phenotype connections of group 2 species have been extensively studied with metabolic modeling, a genome-scale model has been missing for the group 1 species. The metabolic reconstruction of strain WP3 represented the first model for group 1 and the first model among piezotolerant and psychrotolerant deep-sea bacteria. The model brought insights into the mechanisms of energy conservation in WP3 under anaerobic conditions and highlighted its metabolic flexibility in using diverse carbon sources. Overall, the model opens up new opportunities for investigating energy conservation and metabolic adaptation, and it provides a prototype for systems-level modeling of other deep-sea microorganisms.

Mechanosensation, one of the fastest sensory modalities, mediates diverse behaviors including those pertinent for survival. It is important to understand how mechanical stimuli trigger defensive behaviors. Here, we report that Drosophila melanogaster adult flies exhibit a kicking response against invading parasitic mites over their wing margin with ultrafast speed and high spatial precision. Mechanical stimuli that mimic the mites' movement evoke a similar kicking behavior. Further, we identified a TRPV channel, Nanchung, and a specific Nanchung-expressing neuron under each recurved bristle that forms an array along the wing margin as being essential sensory components for this behavior. Our electrophysiological recordings demonstrated that the mechanosensitivity of recurved bristles requires Nanchung and Nanchung-expressing neurons. Together, our results reveal a novel neural mechanism for innate defensive behavior through mechanosensation.

SIGNIFICANCE STATEMENT: We discovered a previously unknown function for recurved bristles on the Drosophila melanogaster wing. We found that when a mite (a parasitic pest for Drosophila) touches the wing margin, the fly initiates a swift and accurate kick to remove the mite. The fly head is dispensable for this behavior. Furthermore, we found that a TRPV channel, Nanchung, and a specific Nanchung-expressing neuron under each recurved bristle are essential for its mechanosensitivity and the kicking behavior. In addition, touching different regions of the wing margin elicits kicking directed precisely at the stimulated region. Our experiments suggest that recurved bristles allow the fly to sense the presence of objects by touch to initiate a defensive behavior (perhaps analogous to touch-evoked scratching; Akiyama et al., 2012).