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Transsynaptic Teneurin Signaling in Neuromuscular Synapse Organization and Target Choice

. Author manuscript; available in PMC: 2012 Oct 12.

Published in final edited form as: Nature. 2012 Mar 18;484(7393):237–241. doi: 10.1038/nature10923

Abstract

Synapse assembly requires transsynaptic signals between the pre- and postsynapse¹, but the understanding of essential organizational molecules remains incomplete². Teneurins are conserved, EGF-repeat containing transmembrane proteins with large extracellular domains³. 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 while 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 transsynaptically 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 their differential roles that synergize to promote synapse assembly. Finally, at elevated endogenous levels, Ten-m regulates specific motoneuron-muscle target selection. Our study identifies the Teneurins as a key bi-directional transsynaptic signal in general synapse organization, and demonstrates that such a molecule can also regulate target selection.

Vertebrate Teneurins are enriched in the developing brain^4,5, localized to synapses in culture⁶ and pattern visual connections⁷. Both Drosophila Teneurins, Ten-m and Ten-a, function in olfactory synaptic partner matching⁸ and were further identified in neuromuscular junction (NMJ) defect screens^9,10, with Ten-m also affecting motor axon guidance¹¹. We examine here their roles and underlying mechanisms in synapse development.

Both Teneurins were enriched at the larval NMJ (Fig. 1a, S1a). Ten-a was detected at neuronal membranes: this staining was undetectable beyond background in ten-a null mutants (Fig. S1b) and barely detectable following neuronal ten-a RNAi (Fig. S1c), indicating that Ten-a is predominantly presynaptic. Partial colocalization was observed between Ten-a and the periactive zone marker Fasciclin II¹² as well as the active zone marker Bruchpilot¹³ (Fig. 1b–c), suggesting a localization between these regions. Ten-m appeared strongly postsynaptic and surrounded each bouton (Fig. 1a, S1a, d). Muscle-specific ten-m RNAi eliminated the postsynaptic staining, but uncovered weak presynaptic staining (Fig. S1e) that ubiquitous ten-m RNAi eliminated (Fig. S1f). Thus, the Ten-m signal was specific and, while partly presynaptic, enriched postsynaptically. Consistently, muscle Ten-m colocalized extensively with Dlg (Fig. 1d) and completely with α-spectrin (Fig. 1e) and is thus, likely coincident with all postsynaptic membranes.

**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.

The localization of Ten-a and Ten-m suggested their transsynaptic interaction. To examine this, we co-expressed myc-tagged Ten-a in nerves using the Q system¹⁴ and HA-tagged Ten-m in muscles using GAL4. Muscle Ten-m was able to co-immunoprecipitate nerve Ten-a from larval synaptosomes (Fig. 1f), suggesting that the Teneurins form a heterophilic transsynaptic receptor pair at the NMJ.

To determine Teneurin function at the NMJ, we examined the ten-a null allele and larvae with neuron or muscle RNAi of ten-a and/or ten-m. Following such perturbations, bouton number and size were altered: the quantity was reduced by 55% (Fig 2a–c, g, S2) and the incidence of large boutons markedly increased (Fig. S2k). Both elements represent impaired synaptic morphogenesis. The reduction in bouton number was likely cumulative through development, as it was visible in first instar ten-a mutants and persisted (Fig. S2k). In the ten-a mutant, bouton morphogenesis was rescued by restoring Ten-a expression in neurons, but not muscles (Fig. 2d,g, S2). Neuronal Ten-m overexpression could not substitute for the lack of Ten-a, revealing their nonequivalence (Fig. S2e,l). Neuronal knockdown of Ten-a or Ten-m both showed an impairment (Fig. S2f–h,l), indicating presynaptic function for both, though presynaptic Ten-a plays a more significant role (Fig. S2l). Moreover, knocking down postsynaptic Ten-m in the ten-a mutant did not enhance the phenotype (Fig. 2g). Thus, presynaptic Ten-a (and to a lesser extent, Ten-m) and postsynaptic Ten-m are required for synapse development.

**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.

Perturbation of teneurins also caused defects in the apposition between presynaptic active zones (release sites) and postsynaptic glutamate receptor clusters¹⁵ (Fig. 2h, S3): up to 15% of the active zones/receptor clusters lacked their partner compared to 1.8% in controls (Fig. 2h–k). Under electron microscopy, active zones are marked by electron dense membranes and single presynaptic specializations called T-bars (Fig. 2l), which enable synapse assembly, vesicle release and Ca²⁺ channel clustering¹⁶. Teneurin disruption caused defects (Fig. 2m-r, S3) in T-bar ultrastructure (Fig. 2m–o), membrane organization and apposition to contractile tissue (Fig. 2p–q). Teneurin perturbation also impaired postsynaptic densities while increasing membrane ruffling (Table S1), further indicating organizational impairment. These phenotypes resemble mutants with adhesion and T-bar biogenesis defects^17,18, suggesting a role for Teneurins in synaptic adhesion and stability. Synaptic vesicle populations similarly required Teneurins for clustering at the bouton perimeter and proper density (Fig. S4). As these effects are not synonymous with active zone disruption¹⁹, Teneurins are also required for synaptic vesicle organization.

Synapses lacking teneurin were also functionally impaired. The mean amplitude of evoked excitatory postsynaptic potentials (EPSP) in larvae was decreased by 28% in the ten-a mutant (Fig. 2s, u). Spontaneous miniature EPSPs (mEPSPs) showed a 20% decrease in amplitude, a 46% decrease in frequency (Fig. 2t, u) and an altered amplitude distribution compared with control (Fig. S5a). These defects resulted in a 20% reduction in quantal content (Fig. 2u), which could be partly due to fewer boutons and release sites. However, release probability may also be reduced, as suggested by an increased paired pulse ratio in ten-a mutants (Fig. S5d,e). The decay kinetics of responses were faster in ten-a mutants, suggesting additional postsynaptic effects on glutamate receptors and/or intrinsic membrane properties (Fig. S5b–c). Further, FM1-43 dye loading revealed markedly defective vesicle cycling in ten-a mutants (Fig. S5f–h). Consistent with physiological impairment, teneurin-perturbed larvae exhibited profound locomotor defects (Fig. S5i). In summary, Teneurins are required for multiple aspects of NMJ organization and function.

As a potential mechanism for synaptic disorganization following teneurin perturbation, we examined the pre- and postsynaptic cytoskeletons. In the presynaptic terminal, organized microtubules contain Futsch (a microtubule-binding protein)-positive “loops” while disorganized microtubules possess punctate, “unbundled” Futsch²⁰. Each classification normally represented ~10% (often distal) of boutons (Fig. 3a, d, S6). Following teneurin perturbation, many more boutons had unbundled Futsch (Fig. 3b–c, S6) while those with looped microtubules were decreased by 62–95% (Fig. 3d). Therefore, proper microtubule organization requires pre- and postsynaptic Teneurins. In contrast to mild active zone/glutamate receptor apposition defects, most boutons displayed microtubule organizational defects.

**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.

Removal of teneurins also severely disrupted the postsynaptic spectrin cytoskeleton, with which Ten-m colocalized (Fig. 1e). Postsynaptic α-spectrin normally surrounds the bouton (Fig. 3e). Perturbing neuronal or muscle Teneurins markedly reduced postsynaptic α-spectrin without affecting Dlg (Fig. 3f–h, S7). Postsynaptic β-spectrin²¹, Adducin²² and Wsp were similarly affected (Fig. S8). In the muscle, α-spectrin is coincident with and essential for the integrity of the membranous subsynaptic reticulum (SSR)^21,23. Consistent with this, teneurin disruption reduced SSR width up to 70% (Fig. S9d–g) and increased the frequency of “ghost” boutons, which are failures of postsynaptic membrane organization²³ (Fig. S9a–d). Thus, Teneurins are involved in the organization of the pre- and postsynaptic cytoskeletons and postsynaptic membranes. Further, endogenous α-spectrin co-immunoprecipitated with muscle-expressed, FLAG-tagged Ten-m (Fig. 3i), suggesting that Ten-m physically links the synaptic membrane to the cytoskeleton.

As the most severe defects following teneurin perturbation were cytoskeletal, we propose that Teneurins primarily organize the presynaptic microtubule and postsynaptic spectrin-based cytoskeletons (Fig. 3j), which then organize additional synaptic aspects^20,21. However, such a solitary role cannot fully explain the observed phenotypes. The bouton number defects associated with cytoskeletal disruption are milder than those following teneurin disruption^20,21,24. Also, while active zone dynamics are affected by cytoskeletal perturbation²¹, defects in apposition are not^21,25. Moreover, the T-bar structural defects more closely resemble synapse adhesion and active zone formation defects^17,18. Thus, Teneurins may regulate release site organization and synaptic adhesion independent of the cytoskeleton (Fig. 3j).

Our data also indicate that Teneurins act bi-directionally across the synaptic cleft. Ten-a acts predominantly neuronally as evidenced by localization, phenotypes caused by neuronal (but not muscle) knockdown, and mutant rescue by neuronal (but not muscle) expression (Fig. 2–3, S2–4, S6–7, S9). Yet, in addition to the presynaptic phenotypes, many others were postsynaptic, including reduced muscle spectrin, SSR and membrane apposition (Fig. 3, S7–9). Similarly, although Ten-m is present both pre-and postsynaptically, muscle knockdown resulted in presynaptic defects, including microtubule and vesicle disorganization, reduced active zone apposition, and T-bar defects (Fig. 2–3, S3–4, S6–S7). Thus, Teneurins function in bi-directional transsynaptic signaling to organize neuromuscular synapses. This may involve downstream pathways or simply establish an organizational framework by the receptors themselves. Moreover, as the single disruptions of neuronal ten-a or muscle ten-m were similarly severe and not enhanced by combination (Fig. 2g, 3d, 3h, S2k), they likely function in the same pathway. Our finding that Ten-a and Ten-m co-immunoprecipitate from different cells in vitro⁸ and across the NMJ in vivo (Fig. 1f) further suggests a signal via trans-synaptic complex. Teneurin function, however, may not be solely transsynaptic. In some cases (vesicle density, SSR width), cell-autonomous knockdown showed stronger phenotypes than knocking down in synaptic partners (Fig. S3, S4, S9, Table S1). This suggests additional cell-autonomous roles unrelated to transsynaptic Teneurin signaling.

Signaling involving the transmembrane proteins Neurexin and Neuroligin also mediates synapse development²⁶. In Drosophila, Neurexin (dnrx) and Neuroligin1 (dnlg1) mutations cause phenotypes similar to teneurin perturbation: reduced boutons, active zone organization, transmission and SSR^27,28. dnlg1 and dnrx mutations do not enhance each other, suggesting their function in the same pathway²⁸. Consistently^27,28, we found that dnrx and dnlg1 mutants exhibited largely similar phenotypes (not shown). To investigate the relationship between the teneurins and dnrx/dnlg1, we focused on the dnlg1 null mutant. Both Ten-m and DNlg1-eGFP occupied similar postsynaptic space (Fig. S10a). teneurin and dnlg1 loss-of-function also displayed similar bouton number reductions (Fig. 2e, g), vesicle disorganization (Fig. S4) and ghost bouton frequencies (Fig. S9). Other phenotypes showed notable differences in severity. In dnlg1 mutants, there was a 29% failure of active zone/glutamate receptor apposition (Fig. 2k, S10d), compared to 15% for the strongest teneurin perturbation. For the cytoskeleton, dnlg1 mutants were mildly impaired compared to teneurin perturbations (Fig. 3d, 3h, S6, S7).

To further examine their interplay, we analyzed ten-a dnlg1 double mutants. Both single mutants were viable, despite their synaptic defects. Double mutants, however, were larval lethal. We obtained rare escapers, which displayed a 72% reduction in boutons, compared to a 50–55% decrease in single mutants (Fig. 2e). Active zone apposition in double mutants was enhanced synergistically over either single mutant (Fig. 2k, S10e). Cytoskeletal defects in the double mutant resembled the ten-a mutant (Fig. 3, S6–7). These data suggest that teneurins and dnrx/dnlg1 act in partially overlapping pathways, cooperating to properly organize synapses, with Teneurins contributing more to cytoskeletal organization and Neurexin/Neuroligin to active zone apposition (Fig. 3j).

In the accompanying manuscript⁸, we showed that while the basal Teneurins are broadly expressed in the Drosophila antennal lobe, elevated expression in select glomeruli mediates olfactory neuron partner matching. At the NMJ, this basal level mediates synapse organization. Analogous to the antennal lobe, we found elevated ten-m expression at muscles 3 and 8 using the ten-m-GAL4 enhancer trap (Fig. 4a). We confirmed this for endogenous ten-m, and determined that it was contributed by elevated Ten-m expression in both nerves and muscles (Fig. 4b–g). Indeed, ten-m-GAL4 was highly expressed in select motoneurons, including MN3-Ib, which innervates muscle 3²⁹ (Fig. S11c). This elevated larval expression also varied along the anterior-posterior axis (Fig. S12), and was specific for Ten-m as Ten-a expression did not differ within or between segments (not shown).

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.

To test whether elevated Ten-m expression in muscle 3 and MN3-Ib affects neuromuscular connectivity, we expressed ten-m RNAi using ten-m-GAL4. Wild-type muscle 3 was almost always innervated (Fig. 4h). However, following ten-m knockdown, muscle 3 innervation failed in 11% of hemisegments (Fig. 4i–j). This required Ten-m on both sides of the synapse, as the targeting phenotype persisted following neuronal or muscle RNAi suppression using tissue-specific GAL80 transgenes (Fig. 4j). ten-a RNAi did not show this phenotype (Fig. 4j), consistent with homophilic target selection via Ten-m. The phenotype was specific to muscle 3, as innervation onto the immediately proximal or distal muscle was unchanged (Fig. 4j). The low penetrance is likely due to redundant target selection mechanisms³⁰. Where innervation did occur, the terminal displayed similarly severe phenotypes to other NMJs (not shown). Thus, in addition to generally mediating synaptic organization, Ten-m also contributes to correct target selection at a specific NMJ.

To determine whether Ten-m overexpression could alter connectivity, we expressed Ten-m in muscle 6 (but not the adjacent muscle 7), and the motoneurons innervating both muscles using H94-GAL4. Normally, 60% of the boutons at muscles 6/7 are present on muscle 6 with 40% on muscle 7 (Fig. 4k,m). Ten-m overexpression caused a shift whereby 81% of boutons synapsed onto muscle 6 and only 19% onto muscle 7 (Fig. 4l–m). This shift also required both neuronal and muscle Ten-m as neuronal or muscle GAL80 abrogated it (Fig. 4m). The effect was specific as Ten-a overexpression did not alter this synaptic balance (Fig. 4m), nor was it secondary to altered bouton number, which is unchanged (not shown). Therefore, elevated Ten-m on both sides of the NMJ can bias target choice. This, combined with evidence that Ten-m can engage homophilic interaction in vitro⁸, supports a transsynaptic homophilic attraction model at the NMJ as in the olfactory system.

In summary, we identified a two-tier mechanism for Teneurin function in synapse development at the Drosophila NMJ. At the basal level, Teneurins are expressed at all synapses and engage in hetero- and homophilic bi-directional transsynaptic signaling to properly organize synapses (Fig. 3j). Supporting this, the Teneurins can mediate homo-and heterophilic interactions in vitro⁸ and heterophilic interactions in vivo (Fig. 1f). At the synapse, Teneurins organize the cytoskeleton, interact with α-spectrin, and enable proper adhesion and release site formation. Further, elevated Ten-m expression regulates target selection in specific motoneurons and muscles via homophilic matching and functions with additional molecules³⁰ to mediate precise neuromuscular connectivity. Teneurin-mediated target selection at the NMJ is analogous to its role in olfactory synaptic partner matching⁸. As the Teneurins are expressed broadly throughout the antennal lobe, it remains an attractive possibility that they also regulate central synapse organization.

METHODS SUMMARY

Detailed methods on Drosophila stocks, immunostaining, electron microscopy, functional assays, construction of epitope-tagged Teneurin constructs, immunoprecipitation, imaging and statistical analysis can be found in Methods.

METHODS

Drosophila Stocks

All Drosophila strains and controls were raised at 29°C to maximize GAL4 expression. All mutants and transgenes were maintained over GFP balancer chromosomes to enable larval selection. Mhc-GAL4 or DMef2-GAL4³¹ was used to drive expression in all somatic muscles. Nrv2-GAL4³² and elav-GAL4³³ were used to drive expression in all neurons. H94-GAL4 was used to drive expression in muscles 6, 13 and 4 and their corresponding motoneurons³⁴. Daughterless-GAL4 was used to drive expression ubiquitously³⁵. Synj-QF³⁶ was used to drive expression in all nerves. NP6658-GAL4 (ten-m-GAL4) was used to drive expression in the pattern of endogenous Ten-m expression⁸. The Df(X)ten-a deletion was used as a ten-a null mutant⁸. For dnlg1 mutants, the I960 and ex2.3 alleles were used in trans²⁸ and double mutant larvae with ten-a were obtained using optimized rearing conditions³⁷. Due to the early lethality of the ten-m mutant¹¹, and to independently assess ten-a, tissue-specific RNAi was utilized to assess teneurin perturbation using the following interfering RNA (IR) transgenic strains: for ten-m, UAS-ten-m^RNAi-V51173 and for ten-a⁸, UAS-ten-a^RNAi-V32482. The following transgenic strains were used: UAS-Dcr2³⁸, UAS-FasII³⁴, UAS-mCD8GFP³⁹, UAS-DNlg1-eGFP²⁸, UAS-Ten-a⁸, P[GS]9267 for ten-m overexpression⁸. In all cases, the efficacy of interfering RNA (IR, RNAi) transgenes, overexpression transgenes and the ten-a deletion mutant were assessed and verified by the alteration of antibody staining (loss, reduction or increase) using tissue-specific GAL4 drivers. For all cases, N-IR indicates neuronal RNAi, M-IR indicates muscle RNAi, U-IR indicates ubiquitous RNAi. For rescuing ten-a mutants, Ten-a N indicates neuronal overexpression with elav-GAL4 of UAS-ten-a and Ten-a M indicates muscle overexpression of UAS-ten-a with DMef2-GAL4.

Immunostaining

Wandering third instar larvae were processed as previously described²³. The following primary antibodies were used: mouse antibody to Ten-m (mAb20, 1:500)⁴⁰, guinea pig antibody to Ten-a (1:100)⁴¹, mouse antibody to Brp (mAbnc82, 1:250)⁴², rabbit antibody to Synaptotagmin I (1:4000)⁴³, mouse antibody to Cysteine String Protein (mAb6D6, 1:100)⁴⁴, mouse antibody to Dlg (mAb4F3, 1:500)⁴⁵, rabbit antibody to Dlg (1:40000)⁴⁶, mouse antibody to α-spectrin (mAb3A9, 1:50)⁴⁷, mouse antibody to Fasciclin II (mAb1D4, 1:20)⁴⁸, rabbit antibody to Fasciclin II (1:5000)⁴⁶, mouse antibody to Futsch (mAb22C10, 1:50)²⁰, rabbit antibody to DGluRIII (1:2500)¹⁵, rat antibody to Elav (mAb7E8A10, 1:25)⁴⁹, mouse antibody to Even-skipped (mAb3C10, 1:100)⁵⁰, rabbit antibody to β-spectrin (1:1000)⁵¹, mouse antibody to Hts (1:500)²², guinea pig antibody to Wsp (1:1000)⁵². Alexa488-, Alexa546-, or Alexa647-conjugated secondary antibodies were used at 1:250 (Invitrogen). Texas Red-conjugated Phalloidin was used at 1:300 FITC-, Cy3-, or Cy5-conjugated antibodies to horseradish peroxidase were used at 1:100 (Jackson ImmunoResearch).

Electron Microscopy

Wandering third instar larvae were processed and sectioned as described²³. Sections were imaged on a JEM-1400 (JEOL) transmission electron microscope at 3000X to 20000X magnification.

Electrophysiology

Larvae were dissected in HL3 saline⁵³ containing 0 mM Ca²⁺ and 4 mM Mg²⁺. They were then transferred to saline containing 0.6 mM Ca²⁺ and recordings conducted by impaling larval muscle 6 in body wall segments A3 and A4 using sharp intracellular electrodes (10–20 MΩ), fabricated from borosilicate glass capillaries and filled with 3 M KCl solution. For evoked EPSPs, severed nerve bundles were stimulated using a suction electrode connected to a linear stimulus isolated (A395, World Precision Instruments). Data, acquired using Multiclamp 700B amplifiers (Molecular Devices), was low-pass filtered at 3 kHz and digitized at 10 kHz. Recordings were acquired and analyzed using Igor Pro software (Wavemetrics) and custom-written programs. All recordings in which the resting membrane potential was > −60 mV and/or whose resting potential, input resistance or access resistance changed by more than 20% during the duration of data acquisition were excluded from analysis. All recordings and data analyses were performed blind to the genotype. Quantal content was corrected for nonlinear summation⁵⁴.

Larval Locomotion

Crawling assays were conducted as described⁵⁵.

FM1-43 Dye Loading

FM1-43 (Invitrogen) dye loading was conducted as described⁵⁶ with the following modifications: loading was conducted in 1.5 mM Ca²⁺, 90 mM K⁺ saline for 1 minute followed by six 2-minute washes in 0 mM Ca²⁺ saline. Imaging was conducted on a Zeiss LSM 510 Meta Confocal (Carl Zeiss) with a 40X, PlanApo NA 1.0 water immersion lens (Carl Zeiss).

Construction of Epitope-tagged Teneurin Transgenes

The ten-m and ten-a coding sequences lacking the stop codon were cloned into pENTR-D/TOPO (Invitrogen) from pENTR-ten-m and pENTR-ten-a⁸. pENTR-ten-m(-stop) and pENTR-ten-a(-stop) were recombined into the destination vectors pUASTattB-gtw-tFHAH and pQUASTattB-gtw-tFMH, respectively, using LR Clonase II (Invitrogen). pUASTattB-gtw-tFHAH is a pUASt-Gateway-attB based vector with a C-terminal TEV recognition site and 3xFLAG, 3xHA and 10xHis tags. pQUASTattB-gtw-tFMH is a pQUASt-Gateway-attB⁸ based vector with a C-terminal TEV recognition site and 3xFLAG, 6xMyc and 10xHis tags. The resulting constructs were verified by restriction digest and sequencing and integrated into the attP24 or 86Fb landing sites on the second and third chromosomes⁵⁷. Transgenic flies were verified by IP on Western blot and overexpression experiments.

Immunoprecipitation, Western blots and SDS-PAGE Analysis

For Ten-m and Ten-a, QUAS-Ten-a-FLAG-Myc was expressed in nerves using Synj-QF and UAS-Ten-m-FLAG-HA in muscles using Mhc-GAL4. Larval synaptosomes were prepared from larval body wall fillets as described⁵⁸. For Ten-m and α-spectrin, control larvae consisted of DMef2-GAL4 without UAS-Ten-m-FLAG-HA while experimental flies combined the two. Immunoprecipitation was conducted as described using M2-anti-FLAG-conjugated agarose (Sigma) or Affi-Prep Protein A beads (Bio-Rad) and rat antibodies to HA (Roche)²³. Proteins were separated on NuPAGE 3 – 8% Tris-Acetate Gels (Invitrogen) and transferred to nitrocellulose. Primary antibodies were applied overnight at 4°C and secondary antibodies at 21°C for 1 hour. The following primary antibodies were used: mouse antibody to α-spectrin (mAb3A9, 1:2000), mouse antibody to Brp (mAbnc82, 1:100), mouse antibody to FLAG (M2, 1:5000, Sigma-Aldrich), mouse antibody to Myc (3E10, 1:1500, Santa Cruz Biotechnology), rat antibody to HA (3F10, 1:1500, Roche). HRP-conjugated secondary antibodies (Jackson ImmunoResearch) were used at 1:10000. Blots were developed using the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoScientific).

Imaging Analysis

Larvae were imaged with a Zeiss LSM 510 Meta laser-scanning confocal microscope (Carl Zeiss) using either a 63X 1.4 NA or a 40X 1.0 NA objective. NMJ images were taken as confocal z-stacks with the upper and lower bounds defined by HRP staining unless otherwise noted. For all metrics, boutons were assessed in segment A3 at muscle 6/7 and muscle 4 on both the left and right sides. Fluorescence intensity measurements were taken from terminals on muscle 4. All phenotypes, however, were observed at all synapses regardless of muscle fiber or segment. For membrane organization and Teneurin colocalization, NMJ images were taken as single optical sections at the precise center of the bouton as determined by HRP staining. Images were processed with the LSM software and Adobe Photoshop CS4. Bouton number, active zone / glutamate receptor apposition, fluorescent intensity and microtubule organization were quantified as previously described²³. Targeting errors for each larva were quantified as the percentage of hemisegments from A1 to A7 in a single animal with a failure of target innervation. There was no difference in targeting errors based on body wall segment. Experiments using H94-GAL4 were conducted as described³⁴, and their effects confirmed using Fasciclin II overexpression (Control = 58.1% of boutons on muscle 6, 41.9% on muscle 7; Fas II overexpression = 73.0% on muscle 6, 27.0% on muscle 7; n = 8 animals for each, p < 0.0001)³⁴.

In electron micrographs, parameters were quantified as previously described using ImageJ (National Institutes of Health)²³. T-bar defects were classified into one of five categories: Normal (no discernible defect), Double (two T-bars were observed in the same, continuous active zone), Detached (where the T-bar was clearly visible but was not explicitly connected to the membrane associated with the nearest PSD), Apposite Contractile Tissue (where the T-bar was not apposed to the SSR, but rather, the contractile tissue of the muscle), Misshapen (where an electron-dense T-bar was visible but did not conform to the “T” shape. Often, the T-bars were “X” shaped). For Fig. 2r, each defect is expressed as a percentage of the total number of T-bars observed in a particular genotype.

For Ten-m gradient calculation, single optical sections were taken through the center of the NMJ on muscle 3 or muscle 4, as determined by HRP immunoreactivity. The GFP signal (ten-m-GAL4) or antibody signal was then measured using ImageJ (National Institutes of Health). For each larva, measurements were taken on the right and left sides of each indicated segment. The fluorescence for each segment was expressed as a percentage of the fluorescence from segment A1 in the same animal, on the same side of the larvae. For all larvae, segment A1 represented the maximal fluorescence.

Statistical Analysis

Statistical analysis used GraphPad Prism 5 (Graphpad Software). In all cases involving more than two samples, significance was calculated using ANOVA followed by a Dunnett post-hoc test to the control sample and a Bonferroni post-hoc test among all samples. For two-sample cases, an unpaired student’s t-test was used to assess significance, unless otherwise indicated. In all cases, both methods provided similar significance measurements. In all figures, significance is with respect to control genotypes unless otherwise noted.

Supplementary Material

Acknowledgments

We thank H. Aberle, V. Budnik, A. DiAntonio, R. Dubreuil, D. Featherstone, N. Reist, T. Schwarz, S. Stowers, D. Van Vactor, R. Wides, the Bloomington Stock Center and the Developmental Studies Hybridoma Bank for fly stocks, antibodies and reagents; J. Perrino and D. Luginbuhl for technical assistance; K. Shen, D. Banovic, D. Berns, Y. Chou, X. Gao, S. Hippenmeyer, K. Miyamichi, B. Tasic, X. Yu and S. Zosimus for critiques. Supported by an NIH grant (R01 DC-005982 to L.L.), and Epilepsy, Neonatology and Developmental Biology Training Grants (NIH 5T32 NS007280 and HD007249 to T.J.M.). L.L. is an investigator of the Howard Hughes Medical Institute.

Footnotes

Author Contributions T.J.M. designed and performed all experiments (save electrophysiology). W.H. characterized and provided new reagents, and assisted in some experiments. V.S.D. and T.J.M. designed and V.S.D. performed electrophysiology experiments with assistance from T.J.M. V.F. provided new reagents. L.L. supervised the project. T.J.M. wrote the manuscript with feedback from all authors.

The authors declare no competing financial interests.

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