Control of Synaptic Connectivity by a Network of Drosophila IgSF Cell Surface Proteins.

We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.

An extracellular interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks.

Extracellular domains of cell surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein "interactome" data sets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on a set of 202 proteins composed of the Drosophila melanogaster immunoglobulin superfamily (IgSF), fibronectin type III (FnIII), and leucine-rich repeat (LRR) families, which are known to be important in neuronal and developmental functions. Out of 20,503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We "deorphanized" the 20 member subfamily of defective-in-proboscis-response IgSF proteins, showing that they selectively interact with an 11 member subfamily of previously uncharacterized IgSF proteins. Both subfamilies interact with a single common "orphan" LRR protein. We also observed interactions between Hedgehog and EGFR pathway components. Several of these interactions could be visualized in live-dissected embryos, demonstrating that this approach can identify physiologically relevant receptor-ligand pairs.

Systematic expression profiling of Dpr and DIP genes reveals cell surface codes in Drosophila larval motor and sensory neurons.

In complex nervous systems, neurons must identify their correct partners to form synaptic connections. The prevailing model to ensure correct recognition posits that cell-surface proteins (CSPs) in individual neurons act as identification tags. Thus, knowing what cells express which CSPs would provide insights into neural development, synaptic connectivity, and nervous system evolution. Here, we investigated expression of Dpr and DIP genes, two CSP subfamilies belonging to the immunoglobulin superfamily, in Drosophila larval motor neurons (MNs), muscles, glia and sensory neurons (SNs) using a collection of GAL4 driver lines. We found that Dpr genes are more broadly expressed than DIP genes in MNs and SNs, and each examined neuron expresses a unique combination of Dpr and DIP genes. Interestingly, many Dpr and DIP genes are not robustly expressed, but are found instead in gradient and temporal expression patterns. In addition, the unique expression patterns of Dpr and DIP genes revealed three uncharacterized MNs. This study sets the stage for exploring the functions of Dpr and DIP genes in Drosophila MNs and SNs and provides genetic access to subsets of neurons.

Structural and Functional Synaptic Plasticity Induced by Convergent Synapse Loss in the Drosophila Neuromuscular Circuit.

Throughout the nervous system, the convergence of two or more presynaptic inputs on a target cell is commonly observed. The question we ask here is to what extent converging inputs influence each other's structural and functional synaptic plasticity. In complex circuits, isolating individual inputs is difficult because postsynaptic cells can receive thousands of inputs. An ideal model to address this question is the Drosophila larval neuromuscular junction (NMJ) where each postsynaptic muscle cell receives inputs from two glutamatergic types of motor neurons (MNs), known as 1b and 1s MNs. Notably, each muscle is unique and receives input from a different combination of 1b and 1s MNs; we surveyed multiple muscles for this reason. Here, we identified a cell-specific promoter that allows ablation of 1s MNs postinnervation and measured structural and functional responses of convergent 1b NMJs using microscopy and electrophysiology. For all muscles examined in both sexes, ablation of 1s MNs resulted in NMJ expansion and increased spontaneous neurotransmitter release at corresponding 1b NMJs. This demonstrates that 1b NMJs can compensate for the loss of convergent 1s MNs. However, only a subset of 1b NMJs showed compensatory evoked neurotransmission, suggesting target-specific plasticity. Silencing 1s MNs led to similar plasticity at 1b NMJs, suggesting that evoked neurotransmission from 1s MNs contributes to 1b synaptic plasticity. Finally, we genetically blocked 1s innervation in male larvae and robust 1b synaptic plasticity was eliminated, raising the possibility that 1s NMJ formation is required to set up a reference for subsequent synaptic perturbations.SIGNIFICANCE STATEMENT In complex neural circuits, multiple convergent inputs contribute to the activity of the target cell, but whether synaptic plasticity exists among these inputs has not been thoroughly explored. In this study, we examined synaptic plasticity in the structurally and functionally tractable Drosophila larval neuromuscular system. In this convergent circuit, each muscle is innervated by a unique pair of motor neurons. Removal of one neuron after innervation causes the adjacent neuron to increase neuromuscular junction outgrowth and functional output. However, this is not a general feature as each motor neuron differentially compensates. Further, robust compensation requires initial coinnervation by both neurons. Understanding how neurons respond to perturbations in adjacent neurons will provide insight into nervous system plasticity in both healthy and disease states.

The translational regulator Cup controls NMJ presynaptic terminal morphology.

During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. We and others have previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. Here we examine the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. In this paper, we show that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons. They also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and we show that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with two genes, EndoA and Dap160, that encode proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. Our results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal.

Immunohistochemistry and Morphometric Analysis of Drosophila Larval Body Wall Neuromuscular Junction Preparations.

The Drosophila neuromuscular junction (NMJ) is an excellent model for studying vertebrate glutamatergic synapses. Researchers have uncovered fundamental mechanisms at the fly NMJ that are conserved in higher-order organisms. To gain molecular and structural insight into these and other structures, immunolabeling is invaluable. In this protocol, we describe how to use immunolabeling to visualize embryonic/larval presynaptic and postsynaptic structures at the NMJ. We also include details about amplification of weak immunohistochemistry signals and how to use these signals to quantify synaptic growth via bouton counting. Boutons are bead-like structures at motor axon terminals that house synapses, and the number of boutons reflects the size of the NMJ. We also describe how to identify the different bouton types.

Using the Proximity Ligation Assay to Visualize Colocalization of Proteins at the Drosophila Larval Neuromuscular Junction.

In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized. Early work relied on fluorescent signals to determine whether proteins localized to the pre- and postsynaptic regions. As more synaptic molecules were identified, determining the localization of these proteins relative to each other became important. Conventional microscopy lacks the resolving power to assess whether two proteins are within an appropriate distance to bind directly or be part of a larger complex. Super-resolution and immunoelectron microscopies can improve spatial resolution, but these techniques can be difficult to execute and troubleshoot, and access to these instruments is limiting. However, another approach, proximity labeling, overcomes many of these limitations by using a DNA secondary label that can only be amplified if the two proteins of interest are within 40 nm of each other, which is ∼5× greater than the resolving power of conventional microscopy. In this protocol, we describe the use of the proximity ligation assay, which combines immunohistochemistry with DNA amplification, to reveal protein colocalization in the Drosophila NMJ.

Labeling of Cell Surface Proteins at the Drosophila Larval Neuromuscular Junction Using Binding Partner Peptides.

Determining the precise localization of interacting proteins provides fundamental insight into their putative function. Classically, immunolabeling of endogenous proteins or generating tagged versions of proteins has been used to localize interacting proteins. However, in many cases, the interacting partner of a protein of interest is unknown. For cell surface proteins, it is possible to determine the localization of interacting proteins if one of the binding partners is known. This approach is based on generating purified, recombinant, tagged extracellular domains (ECDs) of a protein of interest, and incubating tissue to allow the recombinant protein to bind to its interacting partner(s). In this protocol, we detail the cloning of secreted, tagged ECDs from cell surface proteins, transfection of cloned plasmids into S2 cells, collection of secreted domains, concentration of the cell culture medium to enrich for the ECDs, and labeling of tissue with these ECDs.

Cell Ablation Techniques for the Larval Drosophila Neuromuscular System.

Tissue development requires local and long-distance communication between cells. Cell ablation experiments have provided critical insights into the functions of specific cell types and the tissue surrounding the dead cells. In the Drosophila neuromuscular system, ablation of motor neurons and muscles has revealed the roles of the ablated cells in axon pathfinding and circuit wiring. For example, when muscles are denervated due to laser ablation of their motor neuron inputs, they receive ectopic innervation from neighboring motor neurons. Here, we describe two methods of specific cell ablation. The first is a genetic ablation approach that uses GAL4 (ideally expressed in a small subset of cells) to drive expression of cell death genes reaper and head involution defective The second method relies on reactive oxygen species produced by light activation of the Arabidopsis-derived Singlet Oxygen Generator, miniSOG2, expressed in a subset of cells. For the latter, the precision stems from both the GAL4 and the restricting of the blue-light stimulation area.

Drosophila Late Embryonic through Late Larval Stage Body Wall Dissection: Dissection Tools and Techniques.

One of the challenges of studying synaptic structure and function is accessibility. Some of the earliest readily identifiable and accessible synapses were from the frog and various arthropods. To address questions regarding mechanisms that underlie synaptic development and function, genetically tractable systems were required, and researchers turned to the Drosophila melanogaster embryonic/larval neuromuscular preparation. Drosophila embryos are transparent and can be labeled with antibodies or probes and imaged in whole-mount preparation for structural analysis. Embryos can also be dissected to visualize the entire body wall musculature as well as finer details including live protein trafficking and protein-protein interactions. Whereas younger dissected embryos can be mounted directly onto charged slides, more mature embryos and larvae develop a cuticle that impedes this adherence, so different techniques must be applied. In this protocol, we detail how to manufacture dissection tools and collect embryos, and discuss the individual steps of dissecting late-stage embryos, early first-instar larvae, and late-stage third-instar larvae.

The Drosophila Larval Neuromuscular Junction: Developmental Overview.

For decades, the Drosophila larval neuromuscular junction (NMJ) has been a go-to model for synaptic development. This simple, accessible system is composed of a repeating pattern of 33 distinct neurons that stereotypically innervate 30 muscles. Fundamental mechanisms that underlie diverse aspects of axon pathfinding, synaptic form, and function have been uncovered at the NMJ, and new pathways continue to be uncovered. These discoveries are fueled by the ease of dissections and an extensive array of techniques. Chief among these techniques are various microscopy approaches, including super-resolution and electron microscopy. Functionally, the Drosophila NMJ is glutamatergic, similar to the vertebrate central synapses, making it a great model to study normal development and neurological diseases. Here we provide a brief overview of the larval neuromuscular system, highlighting the connectivity patterns, development, and some of the mechanisms underlying these processes.

Neuronal Wiring Receptors Dprs and DIPs Are GPI Anchored and This Modification Contributes to Their Cell Surface Organization.

The Drosophila Dpr and DIP proteins belong to the immunoglobulin superfamily of cell surface proteins (CSPs). Their hetero- and homophilic interactions have been implicated in a variety of neuronal functions, including synaptic connectivity, cell survival, and axon fasciculation. However, the signaling pathways underlying these diverse functions are unknown. To gain insight into Dpr-DIP signaling, we sought to examine how these CSPs are associated with the membrane. Specifically, we asked whether Dprs and DIPs are integral membrane proteins or membrane anchored through the addition of glycosylphosphatidylinositol (GPI) linkage. We demonstrate that most Dprs and DIPs are GPI anchored to the membrane of insect cells and validate these findings for some family members in vivo using Drosophila larvae, where GPI anchor cleavage results in loss of surface labeling. Additionally, we show that GPI cleavage abrogates aggregation of insect cells expressing cognate Dpr-DIP partners. To test if the GPI anchor affects Dpr and DIP localization, we replaced it with a transmembrane domain and observed perturbation of subcellular localization on motor neurons and muscles. These data suggest that membrane anchoring of Dprs and DIPs through GPI linkage is required for localization and that Dpr-DIP intracellular signaling likely requires transmembrane coreceptors.

hkb is required for DIP-α expression and target recognition in the Drosophila neuromuscular circuit.

Our nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins. In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth, and cell survival. However, the upstream regulatory mechanisms of Dprs and DIPs are not clear. On the other hand, while transcription factors have been implicated in target recognition, their downstream cell surface proteins remain mostly unknown. We conduct an F1 dominant modifier genetic screen to identify regulators of Dprs and DIPs. We identify huckebein (hkb), a transcription factor previously implicated in target recognition of the dorsal Is motor neuron. We show that hkb genetically interacts with DIP-α and loss of hkb leads to complete removal of DIP-α expression specifically in dorsal Is motor neurons. We then confirm that this specificity is through the dorsal Is motor neuron specific transcription factor, even-skipped (eve), which acts downstream of hkb. Analysis of the genetic interaction between hkb and eve reveals that they act in the same pathway to regulate dorsal Is motor neuron connectivity. Our study provides insight into the transcriptional regulation of DIP-α and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons.

Glial Draper signaling triggers cross-neuron plasticity in bystander neurons after neuronal cell death in Drosophila.

Neuronal cell death and subsequent brain dysfunction are hallmarks of aging and neurodegeneration, but how the nearby healthy neurons (bystanders) respond to the death of their neighbors is not fully understood. In the Drosophila larval neuromuscular system, bystander motor neurons can structurally and functionally compensate for the loss of their neighbors by increasing their terminal bouton number and activity. We term this compensation as cross-neuron plasticity, and in this study, we demonstrate that the Drosophila engulfment receptor, Draper, and the associated kinase, Shark, are required for cross-neuron plasticity. Overexpression of the Draper-I isoform boosts cross-neuron plasticity, implying that the strength of plasticity correlates with Draper signaling. In addition, we find that functional cross-neuron plasticity can be induced at different developmental stages. Our work uncovers a role for Draper signaling in cross-neuron plasticity and provides insights into how healthy bystander neurons respond to the loss of their neighboring neurons.

Glial Draper signaling triggers cross-neuron plasticity in bystander neurons after neuronal cell death.

Neuronal cell death and subsequent brain dysfunction are hallmarks of aging and neurodegeneration, but how the nearby healthy neurons (bystanders) respond to the cell death of their neighbors is not fully understood. In the Drosophila larval neuromuscular system, bystander motor neurons can structurally and functionally compensate for the loss of their neighbors by increasing their axon terminal size and activity. We termed this compensation as cross-neuron plasticity, and in this study, we demonstrated that the Drosophila engulfment receptor, Draper, and the associated kinase, Shark, are required in glial cells. Surprisingly, overexpression of the Draper-I isoform boosts cross-neuron plasticity, implying that the strength of plasticity correlates with Draper signaling. Synaptic plasticity normally declines as animals age, but in our system, functional cross-neuron plasticity can be induced at different time points, whereas structural cross-neuron plasticity can only be induced at early stages. Our work uncovers a novel role for glial Draper signaling in cross-neuron plasticity that may enhance nervous system function during neurodegeneration and provides insights into how healthy bystander neurons respond to the loss of their neighboring neurons.

hkb is required for DIP-α expression and target recognition in the Drosophila neuromuscular circuit.

Our nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins (CSPs). In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth and cell survival. However, the upstream regulation and downstream signaling mechanisms of Dprs and DIPs are not clear. In the Drosophila larval neuromuscular system, DIP-α is expressed in the dorsal and ventral type-Is motor neurons (MNs). We conducted an F1 dominant modifier genetic screen to identify regulators of Dprs and DIPs. We found that the transcription factor, huckebein (hkb), genetically interacts with DIP-α and is important for target recognition specifically in the dorsal Is MN, but not the ventral Is MN. Loss of hkb led to complete removal of DIP-α expression. We then confirmed that this specificity is through the dorsal Is MN specific transcription factor, even-skipped (eve), which acts downstream of hkb. Genetic interaction between hkb and eve revealed that they act in the same pathway to regulate dorsal Is MN connectivity. Our study provides insight into the transcriptional regulation of DIP-α and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons.

Dpr10 and Nocte are required for Drosophila motor axon pathfinding.

The paths axons travel to reach their targets and the subsequent synaptic connections they form are highly stereotyped. How cell surface proteins (CSPs) mediate these processes is not completely understood. The Drosophila neuromuscular junction (NMJ) is an ideal system to study how pathfinding and target specificity are accomplished, as the axon trajectories and innervation patterns are known and easily visualized. Dpr10 is a CSP required for synaptic partner choice in the neuromuscular and visual circuits and for axon pathfinding in olfactory neuron organization. In this study, we show that Dpr10 is also required for motor axon pathfinding. To uncover how Dpr10 mediates this process, we used immunoprecipitation followed by mass spectrometry to identify Dpr10 associated proteins. One of these, Nocte, is an unstructured, intracellular protein implicated in circadian rhythm entrainment. We mapped nocte expression in larvae and found it widely expressed in neurons, muscles, and glia. Cell-specific knockdown suggests nocte is required presynaptically to mediate motor axon pathfinding. Additionally, we found that nocte and dpr10 genetically interact to control NMJ assembly, suggesting that they function in the same molecular pathway. Overall, these data reveal novel roles for Dpr10 and its newly identified interactor, Nocte, in motor axon pathfinding and provide insight into how CSPs regulate circuit assembly.

Temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships.

How circuit wiring is specified is a key question in developmental neurobiology. Previously, using the Drosophila motor system as a model, we found the classic temporal transcription factor Hunchback acts in NB7-1 neuronal stem cells to control the number of NB7-1 neuronal progeny form functional synapses on dorsal muscles (Meng et al., 2019). However, it is unknown to what extent control of motor neuron-to-muscle synaptic partnerships is a general feature of temporal transcription factors. Here, we perform additional temporal transcription factor manipulations-prolonging expression of Hunchback in NB3-1, as well as precociously expressing Pdm and Castor in NB7-1. We use confocal microscopy, calcium imaging, and electrophysiology to show that in every manipulation there are permanent alterations in neuromuscular synaptic partnerships. Our data show temporal transcription factors, as a group of molecules, are potent determinants of synaptic partner choice and therefore ultimately control circuit membership.

Molecular basis of synaptic specificity by immunoglobulin superfamily receptors in Drosophila.

In stereotyped neuronal networks, synaptic connectivity is dictated by cell surface proteins, which assign unique identities to neurons, and physically mediate axon guidance and synapse targeting. We recently identified two groups of immunoglobulin superfamily proteins in Drosophila, Dprs and DIPs, as strong candidates for synapse targeting functions. Here, we uncover the molecular basis of specificity in Dpr-DIP mediated cellular adhesions and neuronal connectivity. First, we present five crystal structures of Dpr-DIP and DIP-DIP complexes, highlighting the evolutionary and structural origins of diversification in Dpr and DIP proteins and their interactions. We further show that structures can be used to rationally engineer receptors with novel specificities or modified affinities, which can be used to study specific circuits that require Dpr-DIP interactions to help establish connectivity. We investigate one pair, engineered Dpr10 and DIP-α, for function in the neuromuscular circuit in flies, and reveal roles for homophilic and heterophilic binding in wiring.

Transsynaptic interactions between IgSF proteins DIP-α and Dpr10 are required for motor neuron targeting specificity.

The Drosophila larval neuromuscular system provides an ideal context in which to study synaptic partner choice, because it contains a small number of pre- and postsynaptic cells connected in an invariant pattern. The discovery of interactions between two subfamilies of IgSF cell surface proteins, the Dprs and the DIPs, provided new candidates for cellular labels controlling synaptic specificity. Here we show that DIP-α is expressed by two identified motor neurons, while its binding partner Dpr10 is expressed by postsynaptic muscle targets. Removal of either DIP-α or Dpr10 results in loss of specific axonal branches and NMJs formed by one motor neuron, MNISN-1s, while other branches of the MNISN-1s axon develop normally. The temporal and spatial expression pattern of dpr10 correlates with muscle innervation by MNISN-1s during embryonic development. We propose a model whereby DIP-α and Dpr10 on opposing synaptic partners interact with each other to generate proper motor neuron connectivity.