My laboratory is interested in understanding the molecular mechanisms of neural circuit assembly at the level of synapse formation. The general wiring of the nervous system is achieved through a sequence of developmental events that include neuronal migration, axon guidance, axonal layer specificity, synaptic specificity, and activity-dependent modification of nascent synaptic circuits. Both anatomical and electrophysiological data suggest that certain synaptic connections form between specific synaptic partners at particular subcellular locations from the outset of synaptogenesis.
We are exploring the molecular mechanisms that specify synaptic connectivity in Caenorhabditis elegans. We are also interested in understanding how the presynaptic structures form: How are synaptic vesicle precursors trafficked specifically to axons? How do active zone proteins assemble into defined presynaptic apparatus? To study these questions, we have labeled the synapses of six different classes of C. elegans neurons with single-cell resolution in vivo and performed genetic analysis on them. This work has identified two Wnt family proteins that act as negative regulators of synapse formation: an E3 ubiquitin ligase complex that is required for selective synapse elimination; and a Netrin/UNC-6–DCC/UNC-40 pathway that coordinates axon guidance and synapse formation in two synaptic partners, resulting in the proper formation of a neural circuit.
We also have six other ongoing projects that study various aspects of synaptic assembly and specificity in C. elegans. Our data suggest that diverse mechanisms can modulate connectivity and generate specificity at the level of synapse formation. Synaptic connections can be specified by both positive and negative regulators of synaptogenesis, which can act either via contact-dependent mechanisms or as diffusible molecules. Furthermore, nonneuronal cells such as glial cells may play important roles in the assembly of neural circuits.
A Wnt/Fz Pathway as Inhibitor of Synapse Formation
Nervous system function is mediated by an elaborate and precisely patterned network of synaptic connections. We have shown that two immunoglobulin superfamily proteins, SYG-1 and SYG-2, are essential for the synaptic target decisions of a C. elegans neuron, HSNL. SYG-1 functions cell-autonomously in HSNL; SYG-2 functions in the transient guidepost cells. SYG-2 directly binds to SYG-1 and clusters it at the normal synaptic location to promote synaptogenesis. Thus, SYG-1 and SYG-2 act as contact-dependent positive regulators of synapse formation.
Although multiple other cell-adhesion and secreted molecules have been found to stimulate the assembly of synapses, the contribution of signals that negatively regulate synaptic development is not well understood. We examined synapse formation in the C. elegans cholinergic motor neuron DA9, whose en passant presynapses are restricted to a specific segment of its axon. We report that a signaling pathway composed of the Wnts lin-44 and egl-20, the Wnt receptor lin-17/Frizzled, and the cytoplasmic effector dsh-1/Dishevelled defines the subcellular location of DA9 presynapses by inhibiting their assembly in regions of the axon proximal to the sources of Wnt. LIN-44/Wnt is secreted from the tail hypodermis and localizes LIN-17/Frizzled to a subdomain of the DA9 axon that is devoid of presynaptic specializations (Figure 1). The dependence of receptor localization on its ligand might be a way to sharpen the cell's response to Wnts. When this signaling pathway is compromised, DA9 synapses develop ectopically in the proximal subdomain. Conversely, the ectopic overexpression of LIN-44 in cells adjacent to DA9 is sufficient to expand LIN-17 localization in the DA9 axon and concomitantly displace the assembly of presynaptic terminals. These results demonstrate a novel role for Wnt signaling in inhibiting synapse formation and suggest that morphogenetic signals can spatially regulate the patterning of synaptic connections by subdividing regions of the axon into discrete domains.
UNC-6/Netrin and Its Receptor UNC-5 Locally Exclude Presynaptic Components from Dendrites
Polarity is an essential feature of many cell types, including neurons that receive information from local inputs within their dendrites and propagate nerve impulses to distant targets through a single axon. It is generally believed that intrinsic structural differences between axons and dendrites dictate the polarized localization of axonal and dendritic proteins. However, whether extracellular cues also instruct this process in vivo has not been explored.
We have shown that the axon guidance cue UNC-6/netrin and its receptor UNC-5 act throughout development to exclude synaptic vesicle and active zone proteins from the dendrite of the C. elegans motor neuron DA9, which is proximal to a source of UNC-6/netrin. In unc-6/netrin and unc-5 loss-of-function mutants, presynaptic components mislocalize to the DA9 dendrite. In addition, ectopically expressed UNC-6/netrin, acting through UNC-5, is sufficient to exclude endogenous synapses from adjacent subcellular domains within the DA9 axon. Furthermore, this antisynaptogenic activity is interchangeable with that of LIN-44/Wnt, despite being transduced through different receptors, suggesting that extracellular cues such as Netrin and Wnts not only guide axon navigation but also regulate the polarized accumulation of presynaptic components through local exclusion.
SYG-1 Assembles Functional Synaptic Specializations through Key Scaffolding Molecules SYD-1 and SYD-2
Once the SYG-1–protected, synaptogenic zone is formed in HSNL, how are synaptic vesicles and active zone proteins recruited and assembled at the correct site? The rules for orderly recruitment of presynaptic components to synaptic sites are not well understood. By studying localization of synaptic proteins in various mutant backgrounds, we uncovered a three-layer hierarchical organization of presynaptic assembly. In the first layer, SYG-1 and SYG-2 define the location of the presynaptic apparatus and the selection of synaptic targets. In the second layer, two scaffolding proteins, SYD-1 and SYD-2/liprin, act downstream of SYG-1 to recruit synaptic vesicles and active zone proteins to the PSR.
The third layer includes synaptic vesicles, active zone proteins, and other presynaptic proteins, including UNC-57/endophilin, SNN-1/synapsin, ELKS-1/ERC/CAST, GIT, and SAD-1. Their localization is dependent on SYD-1 and SYD-2 but largely independent of each other (Figure 2). This suggests that SYD-1 and SYD-2 are key scaffolding proteins that act as master regulators of synaptic assembly. SYD-1 likely functions upstream of SYD-2 and regulates its activity.
To understand the biochemical process that leads to activation of SYD-2, we are addressing two related issues. First, we are identifying molecules that are important for localizing SYD-1 and SYD-2 to synapses, since we did not find strong direct binding between SYG-1 and SYD-1/SYD-2. Using the candidate approach, we have identified a gene that is likely to bridge SYG-1 and the SYD proteins. Unlike all other presynaptic active zone proteins, the presynaptic localization of this protein is independent of SYD-1 or SYD-2, consistent with a role upstream of SYD-1/SYD-2 function. Furthermore, this gene showed genetic interaction with syd-1 and syd-2. We are characterizing the potential direct binding between this protein and SYD-1, SYD-2, and SYG-1.
Second, we are studying how SYD-1 activates the assembly activity of SYD-2. We have performed a suppressor screen in the syd-1 mutant background and have isolated two suppressors. Molecular cloning of one of the suppressors led to the recent discovery of a novel gene, rsy-1 (regulator of synaptogenesis), which acts as an inhibitor of SYD-1. In rsy-1 single mutants, more synapses form than in the wild-type animals. RSY-1 is a coiled-coil protein that colocalizes with SYD-1 at the HSNL presynapse. Its localization depends on SYD-1, suggesting that RSY-1 is recruited to synapses by SYD-1. Consistent with this hypothesis, SYD-1 and RSY-1 directly bind to each other when expressed in COS cells. These data suggest that SYD-1 may bring its own negative regulator to synaptic assembly sites.
Our experiments studying HSNL synapse formation revealed that presynaptic assembly is a highly regulated, hierarchical process, controlled both by positive regulators (such as SYD-1) and negative regulators (such as RSY-1). An understanding of the interplay between such regulators will be crucial to elucidate how the appropriate size and number of synapses are determined during development.
A Novel Split-GFP–Based Intersynaptic Marker to Study Synaptic Connectivity In Vivo
Understanding the molecular mechanisms of synaptic target selection requires labeling synapses with high resolution in vivo, which has proved to be difficult due to the complexity of innervation. We have made progress on this problem by labeling synapses made by single neurons in C. elegans and using the subcellular distribution of synapses as readout for synaptic target choice. However, such an approach has many limitations.
First, this approach can only be used to study neurons with simple connectivity patterns. For example, if neuron A only synapses onto neuron B, one would expect that when the A-B target selection is disrupted, then all the synapses from A would be affected. This is likely to lead to a dramatic change in the distribution of the synapses from A. However, if A synapses onto B and three other cells and their synapses intermingle with each other, then the A-B synaptic recognition defect would likely only affect a small fraction of the A synapses, which will be difficult to detect with the conventional methods of labeling. Most of the neurons in C. elegans or other organisms have complex connectivity patterns, making them difficult to analyze by conventional labeling methods.
To overcome the caveats of conventional synaptic labeling and to study the molecular mechanisms of synaptic connectivity, we have developed an intersynaptic marker that allows precise characterization of synaptic target choices. We constructed the intersynaptic marker by fusing complementary GFP (green fluorescent protein) halves, or split GFPs, to extracellular domains of pre- and postsynaptically localized proteins. The two GFP fragments fluoresce only when they associate across the synaptic cleft. The advantage of this labeling scheme is that it can mark a subset of synapses from any given neuron that synapses onto particular postsynaptic targets. By expressing these markers in characterized pre- and postsynaptic neuron pairs, we have visualized GFP puncta in the discrete region where synaptic connections between the neurons are formed. Using existing synaptic-specificity mutants syg-1 and syg-2, we can detect changes in synaptic connectivity in the HSNL synaptic circuit.
We have used this intersynaptic labeling method to tag synapses between four different groups of defined pre- and postsynaptic neurons. We are in the process of establishing strains that will be used for forward genetic screens, aiming to isolate mutants with specific defects in synaptic target selection. We anticipate that target selection mutants will exhibit a lack of intersynaptic labeling, therefore showing a loss of fluorescence. It is far easier to identify this phenotype than to identify subtle changes in synaptic distribution. The screen can easily be performed with worms colabeled with conventional synaptic markers in the red fluorescence channel to rule out the cell fate and axon guidance mutants. We hope to use this marker to study synaptic specificity between neurons in a complex environment in which changes in synaptic connectivity would be nearly impossible to detect with conventional markers.
