Selecting synaptic partners is a crucial step in neural circuit formation. Synapses are formed in vivo at specific locations, in part, under the control of target recognition molecules. Evidence shows that target selection is mediated not by a point-to-point system, but rather a combinatorial system. Localized attractants, repellants, and stabilization molecules act in combination to create precise neuromuscular contacts essential for muscle function.
Cell adhesion molecules (CAMs) are crucial for stabilizing synaptic connections. Changes in adhesive interactions can promote or restrict changes in synapse strength. Drosophila Fasciclin II (Fas II) is an immunoglobulin family CAM with sequence similarity to vertebrate Neural Cell Adhesion Molecule (NCAM; Chapter 6). Recent findings indicate that Fas II stabilizes the motor axon innervation pattern3. Fas II (If) mutants show reduced bouton number and size, while overexpression of Fas II causes synaptic overgrowth3. Fas II and the voltage-gated potassium channels bind to, and are clustered by, Discs Large (Dlg). Dlg is a membrane-associated guanylate kinase, a member of the Postsynaptic Density-95 (PSD-95) family10. The synaptic localization of Fas II by Dlg probably contributes to the stabilization of neuronal contacts and also reduces the probability of ectopic synapse formation.
Fasciclin III (Fas III), another homophilic CAM, is expressed on both the growth cone and the synaptic site on the muscle during NMJ development. Fas III acts as a sufficient, but not essential, synaptic target recognition molecule3. Ectopic expression of Fas III in muscle is sufficient to transform the muscle into an acceptable synaptic target. Target recognition is controlled by Fas III dosage and spatio-temporal expression.
Other CAMs shown to regulate target recognition include Sidestep, Connectin, and cadherins. Sidestep is an immunoglobulin CAM necessary for synaptic target recognition. Embryonic muscles express Sidestep when motor axons need to extend onto them. In sidestep mutants, axons fail to extend onto muscles, but instead they extend along the motor neurons28. Ectopic expression of Sidestep results in extensive and prolonged motor axon contact with inappropriate muscle targets. Connectin, a transmembrane glycoprotein that has homophilic cell adhesion properties, is expressed in a subset of muscles and the motor neurons that innervate them. Motor neurons inappropriately innervate neighboring nontarget muscle that ectopically express connectin3. Furthermore, the ectopic synapse formation is dependent on the endogenous connectin expression on the motor neurons. These results show that connectin can function as an attractive and homophilic target recognition molecule in vivo. Cadherins are implicated in target recognition or synaptic stabilization in various synapses. A role for cadherins at the NMJ is well characterized in vertebrates, but has not been shown in flies and worms. Most invertebrate studies have been conducted in the Drosophila retina. In N-cadherin mutant fly eyes, the characteristic spatial arrangement of axon terminals is disrupted as synaptogenesis proceeds. Although synapses form, underlying cytoplasmic structures are not fully specialized at both pre- and postsynaptic terminals and SVs abnormally accumulate29. Loss of the protocadherin, Flamingo, also disrupts the local pattern of synaptic terminals in the retina30. These studies suggest that the combinatorial effects of several cell adhesion molecules may be critical for target recognition.
Receptor protein tyrosine phosphatases (RPTP) regulate cell-cell adhesion at the synapse directly via homophilic binding or indirectly by association with other known CAMs. In flies, multiple RPTP genes, including dlar and dptp69d, participate in target selection both in the NMJ and in the retina31-33. C. elegans has a single Leukocyte common Antigen Related (LAR)-like RPTP gene, ptp-3 that produces at least two isoforms. The ptp-3b isoform affects axon outgrowth and targeting, while the ptp-3a isoform exhibits specific effects on synapse patterning34. In fly retina, loss of function in A-cadherin or in Dlar results in nearly identical target recognition defects35, suggesting Dlar and A-cadherin may act in the same pathway.
Heterophilic interactions between immunoglobulin CAMs also play an important role in the target selection of C. elegans HSNs. HSNs form NMJs on vulval muscles and stimulate egg laying. HSN synapse formation is guided by the epithelial cells of the developing vulva. SYG-1 (SYnaptoGenesis abnormal) and SYG-2 are members of the immunoglobulin superfamily15,36. SYG-1 functions cell autonomously in HSNs. SYG-2 is expressed in vulval epithelial cells. In syg-2 mutants, the SYG-1::GFP fusion protein fails to cluster at HSN synapses36. Thus, SYG-2 expressed in vulva epithelium acts as a ligand for neuronal SYG-1 to determine the site of the HSN synapse. The localized expression of SYG-2 may be regulated during vulva epithelium specification. These studies provide the first molecular mechanism for guidepost cells in synaptic target recognition. The homologs of SYG-1 are the vertebrate NEPH1 and Drosophila IrrecC and Kirre/DUF. The homologs of SYG-2 are vertebrate Nephrin and Drosophila Sticks and Stones (SNS) and Hibris (HIB). Both vertebrate homologs were identified for their roles in myo-blast fusion. The neuronal functions of the SYG-1/SYG-2 homologs remain to be determined.
The above-described CAMs and RPTPs all act as attractants and adhesion molecules in synaptic target recognition. However, it is poorly understood how these various molecules are regulated in time and cellular space.
5.1.2. Localized Inhibition and Synapse Destabilization
Localized inhibition and destabilization work in concert with localized attraction and stabilization to control synapse development. The transmembrane protein, Toll, is expressed in Drosophila embryo muscles and acts locally to inhibit synapse formation. In toll null mutants, the neuron innervates incorrect muscle cells, including those that normally express Toll37. Delayed expression of Toll leads to normal target recognition but delayed synaptic assembly. These studies show that both the temporal and spatial control of Toll expression is crucial for synapse formation.
Semaphorins are repulsive guidance molecules that signal through plexins to affect axon guidance and target recognition. Further evidence for repulsive signals in the body wall come from misexpression studies of Drosophila Semaphorin II (Sema II)38. Drosophila Sema II is transiently expressed in muscle during neurite outgrowth and synapse formation. Sema II (lf) mutants and ectopically or overexpressed Sema II reveal that it functions in vivo as a selective target-derived signal that inhibits synapse formation. A target recognition role for another axon guidance molecule, Netrin, has also been reported. Netrin B can also act as an inhibitor of target recognition, but does not require the Netrin receptor, Frazzled38.
These studies reveal that target recognition, like axon guidance, is based on measurement and response to a specific dynamic and malleable balance of repulsive and attractive forces. The targeting system uses combinatorial interactions between broadly and specifically expressed, temporal and spatial, repulsive and attractive forces. The specific combinations of these molecules create unique recognition signals. Future studies are needed to understand the precise regulation of the combinations of adhesive and repulsive molecules.
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