Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice

Abstract

Synapse assembly requires trans-synaptic signals between the pre- and postsynapse, but our understanding of the essential organizational molecules involved in this process remains incomplete. Teneurin proteins are conserved, epidermal growth factor (EGF)-repeat-containing transmembrane proteins with large extracellular domains. Here we show that two Drosophila Teneurins, Ten-m and Ten-a, are required for neuromuscular synapse organization and target selection. Ten-a is presynaptic whereas Ten-m is mostly postsynaptic; neuronal Ten-a and muscle Ten-m form a complex in vivo. Pre- or postsynaptic Teneurin perturbations cause severe synapse loss and impair many facets of organization trans-synaptically and cell autonomously. These include defects in active zone apposition, release sites, membrane and vesicle organization, and synaptic transmission. Moreover, the presynaptic microtubule and postsynaptic spectrin cytoskeletons are severely disrupted, suggesting a mechanism whereby Teneurins organize the cytoskeleton, which in turn affects other aspects of synapse development. Supporting this, Ten-m physically interacts with α-Spectrin. Genetic analyses of teneurin and neuroligin reveal that they have differential roles that synergize to promote synapse assembly. Finally, at elevated endogenous levels, Ten-m regulates target selection between specific motor neurons and muscles. Our study identifies the Teneurins as a key bi-directional trans-synaptic signal involved in general synapse organization, and demonstrates that proteins such as these can also regulate target selection.

Figure 1. Teneurins are Enriched at and Interact across Drosophila Neuromuscular Synapses

a – e, Representative single confocal sections of synaptic boutons stained with antibodies against Ten-a (red) or Ten-m (green), HRP to mark neuronal membrane (blue) and a synaptic marker as indicated. Ten-a is associated with presynaptic membranes and Ten-m largely with the surrounding postsynapse (a). Ten-a has limited colocalization with a periactive zone marker Fasciclin II (b), and Bruchpilot (Brp), an active zone marker (c). Ten-m colocalizes with, and extends beyond, Dlg (d) and completely with muscle α-spectrin (e). f, Immunoblots (IB) of larval synaptosomes expressing neuronal FLAG-Myc-tagged Ten-a (N) and muscle FLAG-HA-tagged Ten-m (M) and immunoprecipitated (IP) using HA antibodies. Ten-a is detected in the pull-down, indicating that nerve Ten-a and muscle Ten-m interact across the NMJ. This is not seen in control lanes. Due to low expression, neither transgene product is detectable in input lysates, which are enriched in Brp. Scale = 5 μm.

Figure 2. Teneurins Affect Structure and Function of the Neuromuscular Synapse

a – f, Representative NMJs stained with antibodies to HRP. Muscle-specific ten-m RNAi (M-IR) and neuron-specific ten-a knockdown (N-IR) decreases bouton number. Neuronal (Ten-a N) but not muscle (Ten-a M) restoration of ten-a expression rescues this phenotype. These defects resemble dnlg1 mutants and are enhanced in ten-a dnlg1 double mutants. g, Quantification of bouton number. h – j, Representative NMJs stained with antibodies to Brp (green), the glutamate receptor DGluRIII (red) and HRP (blue). In control larvae (h), Brp and DGluRIII puncta properly appose. teneurin perturbations (i – j) disrupt this active zone (yellow arrowhead) and glutamate receptor apposition (white arrowhead). k, Quantification of unopposed active zone/glutamate receptor pairs. For each quantification, n≥8 larvae, 16 NMJs. l – q, Transmission electron microscopy of active zone T-bars (asterisks) in control larvae (l) and ten-a mutants (m–q) showing double T-bars (m), detached T-bars (n), misshapen T-bars (o), membrane ruffling (p, waved arrows) and T-bars facing contractile tissue (q). Some images show multiple defects. r, Distribution of T-bar defects as a percentage of the total T-bars. For each genotype, n≥3 larvae, 40 boutons. s – t, Representative evoked EPSP (s) and mEPSP (t) traces from control and ten-a mutant genotypes. u, Quantification of mean EPSP amplitude (black), mEPSP amplitude (red), mEPSP frequency (blue) and quantal content (QC, white) expressed as a percentage of the control average. For all genotypes, n≥7 larvae, 8 muscles. Error bars represent SEM. Scale = 10 μm (a – f), 5 μm (h – j), 100 nm (l – q). In this and all subsequent figures, error bars represent SEM; *** p<0.001, ** p<0.01, * p<0.05, ns = not significant. Statistical comparisons are with control unless noted.

Figure 3. Teneurin Perturbation Results in Marked Cytoskeletal Disorganization

a – c, Representative NMJs stained with antibodies to Futsch (green) and HRP (magenta). Arrowheads indicate looped organization. Arrows indicate unbundled Futsch. d, Quantification of the percent of total boutons with looped or unbundled microtubules. e – g, Representative NMJs stained with antibodies to α-spectrin (green), Dlg (red) and HRP (blue). Following teneurin perturbation, α-spectrin staining is largely lost. Axonal α-spectrin is unaffected by muscle teneurin RNAi (f). h, Quantification of α-spectrin (green) and Dlg (red) fluorescence. For all genotypes, n≥6 larvae, 12 NMJs. i, Immunoblots showing that α-spectrin is detected in the FLAG-immunoprecipitates of larvae expressing muscle FLAG-HA-tagged Ten-m but not in control larvae. Due to low expression, FLAG-HA-Ten-m is only detectable following IP enrichment. j, Model showing the roles of Teneurins, Neurexin and Neuroligin at the NMJ. Arrow size represents the relative contribution of each pathway to the cellular process as inferred from mutant phenotypic severity. Scale = 5 μm.

Figure 4. High-level Ten-m Expression Regulates Muscle Target Selection

a, Representative images of hemisegment A3 stained with antibodies to Dlg (blue), phalloidin (red) and expressing GFP via ten-m-GAL4 (green). High-level expression is observed in muscles 3 and 8 and basally in all muscles. b – c, Muscle 3 (b) and 4 (c) NMJs show differential Ten-m (red) but similar Syt I (green) expression (from a ten-m muscle knockdown animal). d, Quantification of presynaptic Ten-m (red) and Syt I (green) fluorescence at muscle 3 and 4 NMJs. e – f, NMJs at muscles 3 (e) and 4 (f) show differential Ten-m (red) but similar Syt I (green) expression in muscles (from a ten-m nerve knockdown). g, Quantification of postsynaptic Ten-m (red) and Syt I (green) fluorescence at muscle 3 and 4 NMJs. h – i, Representative images stained with phalloidin (blue) and antibodies to HRP (green) and Dlg (red) to visualize motoneurons and muscles in control (h) or ten-m-GAL4>ten-m RNAi larvae (i). j, Quantification of the hemisegment percentage with failed muscle 3 (red), 4 (black) or 2 (blue) innervation. k – l, Representative images of the muscle 6/7 NMJ labeled with antibodies to Dlg (green) and HRP (magenta). The characteristic wild-type arrangement of boutons (k) is shifted toward muscle 6 when Ten-m is overexpressed in that muscle and the innervating motoneurons (l). m, Quantification of the total bouton percentage on muscles 6 (blue) and 7 (red). All genotypes contain H94-GAL4, additional transgenes are indicated. The Ten-m-mediated shift is abolished by neuronal or muscle GAL80 transgenes. Scale = 100 μm (a), 5 μm (b – i), 10 μm (k – l). In all cases, n≥12 larvae.