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Precise coordination of synaptic connections ensures proper information flow within circuits. The activity of presynaptic organizing molecules signaling to downstream pathways is essential for such coordination, though such entities remain incompletely known. We show that LRP4, a conserved transmembrane protein known for its postsynaptic roles, functions presynaptically as an organizing molecule. In the Drosophila brain, LRP4 localizes to the nerve terminals at or near active zones. Loss of presynaptic LRP4 reduces excitatory (not inhibitory) synapse number, impairs active zone architecture, and abolishes olfactory attraction - the latter of which can be suppressed by reducing presynaptic GABAB receptors. LRP4 overexpression increases synapse number in excitatory and inhibitory neurons, suggesting an instructive role and a common downstream synapse addition pathway. Mechanistically, LRP4 functions via the conserved kinase SRPK79D to ensure normal synapse number and behavior. This highlights a presynaptic function for LRP4, enabling deeper understanding of how synapse organization is coordinated. The connections between nerve cells, called synapses, often malfunction in disease, injury and during aging, and to understand how this happens we first need to know how they work normally. At a synapse, one nerve cell sends a signal to the other. The signal is a chemical substance, which binds to specialized proteins called receptors on the receiving nerve cell. At excitatory synapses, the chemical signal activates the receiver; at inhibitory synapses, it does the opposite. Communication at synapses typically only goes in one direction because the sender and receiver at a synapse are not interchangeable; they contain different molecules that support their distinct roles. To complicate matters, the same molecule may sometimes be present on both sides of a synapse with a different role in each. Moreover, not all synapses exist between two nerve cells; some synapses also form between nerve cells and muscle fibers to control the movement of the muscles. Mosca et al. set out to identify new players involved in forming synapses, and to identify differences in the formation of nerve cell-to-nerve cell versus nerve cell-to-muscle connections. Mosca et al. were interested in particular in a protein called LRP4. In mammals, LRP4 is largely present on the muscle side of nerve cell-to-muscle synapses, where it acts as a receptor for a chemical signal called Agrin. However, fruit flies — which lack Agrin – also possess the gene for LRP4, suggesting that it has other roles too. Mosca et al. now show that LRP4 is present in the nerve cell-to-nerve cell synapses found in the fruit fly’s brain. Further experiments reveal that fruit fly LRP4 plays an important role on the sender side of these synapses. Reducing the amount of LRP4 in the fruit fly brain reduces the number of excitatory, but not inhibitory, synapses. This suggests that fruit fly LRP4 may help regulate the formation of excitatory synapses. Understanding how synapses form, and the differences between excitatory and inhibitory connections, could provide new insights into disorders of impaired synapse formation such as schizophrenia. LRP4 has also been implicated in disorders, such as amyotrophic lateral sclerosis (ALS) and myasthenia gravis, in which impaired communication between nerves and muscles causes muscles to weaken. Improved understanding of how synapses work may lead to better drugs to treat these disorders.

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.

How does wiring specificity of neural maps emerge during development? Formation of the adult Drosophila olfactory glomerular map begins with the patterning of projection neuron (PN) dendrites at the early pupal stage. To better understand the origin of wiring specificity of this map, we created genetic tools to systematically characterize dendrite patterning across development at PN type–specific resolution. We find that PNs use lineage and birth order combinatorially to build the initial dendritic map. Specifically, birth order directs dendrite targeting in rotating and binary manners for PNs of the anterodorsal and lateral lineages, respectively. Two-photon– and adaptive optical lattice light-sheet microscope–based time-lapse imaging reveals that PN dendrites initiate active targeting with direction-dependent branch stabilization on the timescale of seconds. Moreover, PNs that are used in both the larval and adult olfactory circuits prune their larval-specific dendrites and re-extend new dendrites simultaneously to facilitate timely olfactory map organization. Our work highlights the power and necessity of type-specific neuronal access and time-lapse imaging in identifying wiring mechanisms that underlie complex patterns of functional neural maps. The brain’s ability to sense, act and remember relies on the intricate network of connections between neurons. Organization of these connections into neural maps is critical for processing sensory information. For instance, different odors are represented by specific neurons in a part of the brain known as the olfactory bulb, allowing animals to distinguish between smells. Projection neurons in the olfactory bulb have extensions known as dendrites that receive signals from sensory neurons. Scientists have extensively used the olfactory map in adult fruit flies to study brain wiring because of the specific connections between their sensory and projection neurons. This has led to the discovery of similar wiring strategies in mammals. But how the olfactory map is formed during development is not fully understood. To investigate, Wong et al. built genetic tools to label specific types of olfactory projection neurons during the pupal stage of fruit fly development. This showed that a group of projection neurons directed their dendrites in a clockwise rotation pattern depending on the order in which they were born: the first-born neuron sent dendrites towards the top right of the antennal lobe (the fruit fly equivalent of the olfactory bulb), while the last-born sent dendrites towards the top left. Wong et al. also carried out high-resolution time-lapse imaging of live brains grown in the laboratory to determine how dendrites make wiring decisions. This revealed that projection neurons send dendrites in all directions, but preferentially stabilize those that extend in the direction which the neurons eventually target. Also, live imaging showed neurons could remove old dendrites (used in the larvae) and build new ones (to be used in the adult) simultaneously, allowing them to quickly create new circuits. These experiments demonstrate the value of imaging specific types of neurons to understand the mechanisms that assemble neural maps in the developing brain. Further work could use the genetic tools created by Wong et al. to study how wiring decisions are determined in this and other neural maps by specific genes, potentially yielding insights into neurological disorders associated with wiring defects.

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 Drosophila 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 Drosophila. 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 Drosophila olfactory circuit through temporally-regulated expression patterns and expression level-dependent effects.

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 Drosophila 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 Drosophila 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.

Neurons exhibit extraordinary precision in selecting synaptic partners. Although cell-surface proteins (CSPs) mediating attractive interactions between developing axons and dendrites have been shown to instruct synaptic partner matching , the degree to which repulsive interactions play a role is less clear. Here, using a genetic screen guided by single-cell transcriptomes , we identified three CSP pairs-Toll2-Ptp10D, Fili-Kek1, and Hbs/Sns-Kirre-in mediating repulsive interactions between non-partner olfactory receptor neuron (ORN) axons and projection neuron (PN) dendrites in the developing olfactory circuit. Each CSP pair exhibits inverse expression patterns in the select ORN-PN partners. Loss of each CSP in ORNs led to similar synaptic partner matching deficits as the loss of its partner CSP in PNs, and mistargeting phenotypes caused by overexpressing one CSP could be suppressed by loss of its partner CSP. All CSP pairs are also differentially expressed in other brain regions. Together, our data reveal that multiple repulsive CSP pairs work together to ensure precise synaptic partner matching during development by preventing neurons from forming connections with non-cognate partners.

The distribution of postsynaptic partners in three-dimensional (3D) space presents complex choices for a navigating axon. Here, we discovered a dimensionality reduction principle in establishing the 3D glomerular map in the fly antennal lobe. Olfactory receptor neuron (ORN) axons first contact partner projection neuron (PN) dendrites at the 2D spherical surface of the antennal lobe during development, regardless of whether the adult glomeruli are at the surface or interior of the antennal lobe. Along the antennal lobe surface, axons of each ORN type take a specific 1D arc-shaped trajectory that precisely intersects with their partner PN dendrites. Altering axon trajectories compromises synaptic partner matching. Thus, a 3D search problem is reduced to 1D, which simplifies synaptic partner matching and may generalize to the wiring process of more complex brains.

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H O . Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O instead of toxic H O , and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non- partner cells. Evolutionally conserved teneurins were the first identified transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurin's functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of 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.Competing Interest StatementThe authors have declared no competing interest.