InteractiveFly: GeneBrief

Tenascin accessory: Biological Overview | References


Gene name - Tenascin accessory

Synonyms -

Cytological map position - 11A7-11A8

Function - transmembrane receptor

Keywords - Teneurin, synapse assembly, neuromuscular junction, synaptic partner matching in olfactory system, fusion of central complex primordia, eye patterning, CNS

Symbol - Ten-a

FlyBase ID: FBgn0267001

Genetic map position - chrX:12089802-12226216

Classification - EGF domains protein & Rhs repeat-associated core

Cellular location - surface transmembrane



NCBI link: EntrezGene
Ten-a orthologs: Biolitmine
Recent literature
Osaka, J., Yasuda, H., Watanuki, Y., Kato, Y., Nitta, Y., Sugie, A., Sato, M. and Suzuki, T. (2023). Identification of genes regulating stimulus-dependent synaptic assembly in Drosophila using an automated synapse quantification system. Genes Genet Syst 97(6): 297-309. PubMed ID: 36878557
Summary:
Neural activity-dependent synaptic plasticity is an important physiological phenomenon underlying environmental adaptation, memory and learning. However, its molecular basis, especially in presynaptic neurons, is not well understood. Previous studies have shown that the number of presynaptic active zones in the Drosophila melanogaster photoreceptor R8 is reversibly changed in an activity-dependent manner. During reversible synaptic changes, both synaptic disassembly and assembly processes were observed. Although this study has established a paradigm for screening molecules involved in synaptic stability and several genes have been identified, genes involved in stimulus-dependent synaptic assembly are still elusive. Therefore, the aim of this study was to identify genes regulating stimulus-dependent synaptic assembly in Drosophila using an automated synapse quantification system. To this end, RNAi screening was performed against 300 memory-defective, synapse-related or transmembrane molecules in photoreceptor R8 neurons. Candidate genes were narrowed down to 27 genes in the first screen using presynaptic protein aggregation as a sign of synaptic disassembly. In the second screen, the decreasing synapse number was directly quantified using a GFP-tagged presynaptic protein marker. Custom-made image analysis software was used, which automatically locates synapses and counts their number along individual R8 axons, and identified cirl was used as a candidate gene responsible for synaptic assembly. Finally, a new model is presented of stimulus-dependent synaptic assembly through the interaction of cirl and its possible ligand, ten-a. This study demonstrates the feasibility of using the automated synapse quantification system to explore activity-dependent synaptic plasticity in Drosophila R8 photoreceptors in order to identify molecules involved in stimulus-dependent synaptic assembly.
BIOLOGICAL OVERVIEW

Synapse assembly requires trans-synaptic signals between the pre- and postsynapse, but 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. This study shows 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 alpha-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. This 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 (Mosca, 2012).

Vertebrate Teneurins are enriched in the developing brain, localized to synapses in culture and pattern visual connections. Both Drosophila Teneurins, Ten-m and Ten-a, function in olfactory synaptic partner matching and were further identified in neuromuscular junction (NMJ) defect screens, with Ten-m also affecting motor axon guidance (Zheng, 2011). This study examined their roles and underlying mechanisms in synapse development (Mosca, 2012).

Both Teneurins are enriched at the larval NMJ. Ten-a was detected at neuronal membranes: this staining was undetectable beyond background in ten-a null mutants and barely detectable following neuronal ten-a RNAi, indicating that Ten-a is predominantly presynaptic. Partial colocalization has been observed between Ten-a and the periactive zone marker Fasciclin II as well as the active zone marker Bruchpilot, suggesting a localization between these regions. Ten-m appeared strongly postsynaptic and surrounded each bouton. Muscle-specific ten-m RNAi eliminated the postsynaptic staining, but uncovered weak presynaptic staining that ubiquitous ten-m RNAi eliminated. Thus, the Ten-m signal was specific and, while partly presynaptic, enriched postsynaptically. Consistently, muscle Ten-m colocalized extensively with Dlg and completely with α-spectrin and is thus, likely coincident with all postsynaptic membranes (Mosca, 2012).

The localization of Ten-a and Ten-m suggested their transsynaptic interaction. To examine this, myc-tagged Ten-a was co-expressed in nerves using the Q system, and HA-tagged Ten-m was expressed in muscles using GAL4. Muscle Ten-m was able to co-immunoprecipitate nerve Ten-a from larval synaptosomes, suggesting that the Teneurins form a heterophilic transsynaptic receptor pair at the NMJ (Mosca, 2012).

To determine Teneurin function at the NMJ, the ten-a null allele and larvae with neuron or muscle RNAi of ten-a and/or ten-m were examined. Following such perturbations, bouton number and size were altered: the quantity was reduced by 55% and the incidence of large boutons markedly increased. 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. In the ten-a mutant, bouton morphogenesis was rescued by restoring Ten-a expression in neurons, but not muscles. Neuronal Ten-m overexpression could not substitute for the lack of Ten-a, revealing their nonequivalence. Neuronal knockdown of Ten-a or Ten-m both showed an impairment, indicating presynaptic function for both, though presynaptic Ten-a plays a more significant role. Moreover, knocking down postsynaptic Ten-m in the ten-a mutant did not enhance the phenotype. Thus, presynaptic Ten-a (and to a lesser extent, Ten-m) and postsynaptic Ten-m are required for synapse development (Mosca, 2012).

Perturbation of teneurins also caused defects in the apposition between presynaptic active zones (release sites) and postsynaptic glutamate receptor clusters: up to 15% of the active zones/receptor clusters lacked their partner compared to 1.8% in controls. Under electron microscopy, active zones are marked by electron dense membranes and single presynaptic specializations called T-bars, which enable synapse assembly, vesicle release and Ca2+ channel clustering. Teneurin disruption caused defects in T-bar ultrastructure, membrane organization and apposition to contractile tissue. Teneurin perturbation also impaired postsynaptic densities while increasing membrane ruffling, further indicating organizational impairment. These phenotypes resemble mutants with adhesion and T-bar biogenesis defects, 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. As these effects are not synonymous with active zone disruption, Teneurins are also required for synaptic vesicle organization (Mosca, 2012).

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. Spontaneous miniature EPSPs (mEPSPs) showed a 20% decrease in amplitude, a 46% decrease in frequency and an altered amplitude distribution compared with control). These defects resulted in a 20% reduction in quantal content, 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. The decay kinetics of responses were faster in ten-a mutants, suggesting additional postsynaptic effects on glutamate receptors and/or intrinsic membrane properties. Further, FM1-43 dye loading revealed markedly defective vesicle cycling in ten-a mutants. Consistent with physiological impairment, teneurin-perturbed larvae exhibited profound locomotor defects. In summary, Teneurins are required for multiple aspects of NMJ organization and function (Mosca, 2012).

As a potential mechanism for synaptic disorganization following teneurin perturbation, the pre- and postsynaptic cytoskeletons were examined. In the presynaptic terminal, organized microtubules contain Futsch (a microtubule-binding protein)-positive 'loops' while disorganized microtubules possess punctate, 'unbundled' Futsch. Each classification normally represented ~10% (often distal) of boutons. Following teneurin perturbation, many more boutons had unbundled Futsch while those with looped microtubules were decreased by 62%-95%. 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 (Mosca, 2012).

Removal of teneurins also severely disrupted the postsynaptic spectrin cytoskeleton, with which Ten-m colocalized. Postsynaptic α-spectrin normally surrounds the bouton. Perturbing neuronal or muscle Teneurins markedly reduced postsynaptic α-spectrin without affecting Dlg. Postsynaptic β-spectrin, Adducin and Wsp were similarly affected. In the muscle, α-spectrin is coincident with and essential for the integrity of the membranous subsynaptic reticulum (SSR). Consistent with this, teneurin disruption reduced SSR width up to 70% and increased the frequency of 'ghost' boutons, which are failures of postsynaptic membrane organization). Thus, Teneurins are involved in the organization of the pre- and postsynaptic cytoskeletons and postsynaptic membranes. Further, endogenous α-spectrin co-immunoprecipitates with muscle-expressed, FLAG-tagged Ten-m, suggesting that Ten-m physically links the synaptic membrane to the cytoskeleton (Mosca, 2012).

As the most severe defects following teneurin perturbation were cytoskeletal, it is proposed that Teneurins primarily organize the presynaptic microtubule and postsynaptic spectrin-based cytoskeletons. 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. Also, while active zone dynamics are affected by cytoskeletal perturbation, defects in apposition are not. Moreover, the T-bar structural defects more closely resemble synapse adhesion and active zone formation defects. Thus, Teneurins may regulate release site organization and synaptic adhesion independent of the cytoskeleton (Mosca, 2012).

These 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. Yet, in addition to the presynaptic phenotypes, many others were postsynaptic, including reduced muscle spectrin, SSR and membrane apposition. 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. 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 arevsimilarly severe and not enhanced by combination, they likely function in the same pathway. The finding that Ten-a and Ten-m co-immunoprecipitate from different cells in vitro (Hong, 2012) and across the NMJ in vivo 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. This suggests additional cell-autonomous roles unrelated to transsynaptic Teneurin signaling (Mosca, 2012).

Signaling involving the transmembrane proteins Neurexin and Neuroligin also mediates synapse development (Craig, 2007). In Drosophila, Neurexin (dnrx) and Neuroligin1 (dnlg1) mutations cause phenotypes similar to teneurin perturbation: reduced boutons, active zone organization, transmission and SSR. dnlg1 and dnrx mutations do not enhance each other, suggesting their function in the same pathway. Consistently, this study found that dnrx and dnlg1 mutants exhibited largely similar phenotypes. To investigate the relationship between the teneurins and dnrx/dnlg1, focus was placed on the dnlg1 null mutant. Both Ten-m and DNlg1-eGFP occupy similar postsynaptic space. teneurin and dnlg1 loss-of-function also displayed similar bouton number reductions, vesicle disorganization and ghost bouton frequencies. Other phenotypes showed notable differences in severity. In dnlg1 mutants, there was a 29% failure of active zone/glutamate receptor apposition, compared to 15% for the strongest teneurin perturbation. For the cytoskeleton, dnlg1 mutants were mildly impaired compared to teneurin perturbations (Mosca, 2012).

To further examine their interplay, ten-a dnlg1 double mutants were analyzed. Both single mutants were viable, despite their synaptic defects. Double mutants, however, were larval lethal. Rare escapers were obtained that displayed a 72% reduction in boutons, compared to a 50%-55% decrease in single mutants. Active zone apposition in double mutants was enhanced synergistically over either single mutant. Cytoskeletal defects in the double mutant resembled the ten-a mutant. 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 (Mosca, 2012).

In the accompanying manuscript (Hong, 2012), it was shown 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, elevated ten-m expression was found at muscles 3 and 8 using the ten-m-GAL4 enhancer tra. This was confirmed for endogenous ten-m, and it was determined to be contributed by elevated Ten-m expression in both nerves and muscles. Indeed, ten-m-GAL4 is highly expressed in select motoneurons, including MN3-Ib, which innervates muscle 3. This elevated larval expression also varied along the anterior-posterior axis, and was specific for Ten-m as Ten-a expression did not differ within or between segments (Mosca, 2012).

To test whether elevated Ten-m expression in muscle 3 and MN3-Ib affects neuromuscular connectivity, ten-m RNAi was expressed using ten-m-GAL4. Wild-type muscle 3 was almost always innervated. However, following ten-m knockdown, muscle 3 innervation failed in 11% of hemisegments. 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. ten-a RNAi did not show this phenotype, 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. The low penetrance is likely due to redundant target selection mechanisms. Where innervation did occur, the terminal displayed similarly severe phenotypes to other NMJs. Thus, in addition to generally mediating synaptic organization, Ten-m also contributes to correct target selection at a specific NMJ (Mosca, 2012).

To determine whether Ten-m overexpression could alter connectivity, Ten-m was expressed 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. Ten-m overexpression caused a shift whereby 81% of boutons synapsed onto muscle 6 and only 19% onto muscle 7. This shift also required both neuronal and muscle Ten-m as neuronal or muscle GAL80 abrogated it. The effect was specific as Ten-a overexpression did not alter this synaptic balance, nor was it secondary to altered bouton number, which is unchanged. 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 vitro, supports a transsynaptic homophilic attraction model at the NMJ as in the olfactory system (Mosca, 2012).

In summary, this study has 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. Supporting this, the Teneurins can mediate homo-and heterophilic interactions in vitro and heterophilic interactions in vivo. 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 molecules to mediate precise neuromuscular connectivity. Teneurin-mediated target selection at the NMJ is analogous to its role in olfactory synaptic partner matching (Hong, 2012). As the Teneurins are expressed broadly throughout the antennal lobe, it remains an attractive possibility that they also regulate central synapse organization (Mosca, 2012).

Teneurins instruct synaptic partner matching in an olfactory map

Neurons are interconnected with extraordinary precision to assemble a functional nervous system. Compared to axon guidance, far less is understood about how individual pre- and postsynaptic partners are matched. To ensure the proper relay of olfactory information in the fruitfly Drosophila, axons of approximately 50 classes of olfactory receptor neurons (ORNs) form one-to-one connections with dendrites of approximately 50 classes of projection neurons (PNs). In this study, using genetic screens, two evolutionarily conserved, epidermal growth factor (EGF)-repeat containing transmembrane Teneurin proteins, Ten-m and Ten-a, were identified as synaptic-partner-matching molecules between PN dendrites and ORN axons. Ten-m and Ten-a are highly expressed in select PN-ORN matching pairs. Teneurin loss- and gain-of-function cause specific mismatching of select ORNs and PNs. Finally, Teneurins promote homophilic interactions in vitro, and Ten-m co-expression in non-partner PNs and ORNs promotes their ectopic connections in vivo. It is proposed that Teneurins instruct matching specificity between synaptic partners through homophilic attraction (Hong, 2012).

To identify potential PN-ORN matching molecules, select PN dendrites and ORN axons were simultaneously labeled in two colors, and two complementary genetic screens were performed. 410 candidate cell-surface molecules, comprising ~40% of the potential cell-recognition molecules in Drosophila were overexpressed. In the first screen, Mz19-GAL4 was used to label DA1, VA1d and DC3 PNs (referred to as Mz19 PNs), and Or47b-rCD2 was ised to label Or47b ORNs. Or47b ORN axons normally project to the VA1lm glomerulus and are adjacent to Mz19 PN dendrites without overlap. Candidate cell-surface molecules were overexpressed only in Mz19 PNs to identify those that promoted ectopic connections between Or47b axons and Mz19 dendrites. It was found that overexpression of ten-m produced ectopic connections (Hong, 2012).

In the second screen, Mz19 PNs were labelled as above and Or88a ORNs were labelled using Or88a-rCD2. Or88a ORN axons normally project to the VA1d glomerulus, intermingling extensively with VA1d PN dendrites. Candidate cell-surface molecules were overexpressed in Mz19 PNsm, and it was found that overexpression of ten-a partially disrupted the intermingling of Or88a axons and Mz19 dendrites (Hong, 2012).

In addition to impairing PN-ORN matching, ten-m and ten-a overexpression shifted Mz19 PN dendrite position. However, mismatching was not a secondary consequence of axon or dendrite mispositioning; mispositioning alone, caused by perturbation of other genes, does not alter PN-ORN matching. Furthermore, among 410 candidate molecules, only ten-m and ten-a overexpression exhibited mismatching defects, suggesting their specificity in PN-ORN matching (Hong, 2012).

Both ten-m and ten-a appear to encode type II transmembrane proteins. They possess highly similar domain compositions and amino acid sequences; each contains eight EGF-like and multiple YD (tyrosine-aspartate) repeats within its large C-terminal extracellular domain. Ten-m and Ten-a were discovered as Tenascin-like molecules, but vertebrate Teneurins were later identified as their true homologs based on sequence and domain similarity. Ten-m and Ten-a are referred to as Drosophila Teneurins. Teneurins are present in nematodes, flies and vertebrates. In human, Teneurin-1 and Teneurin-2 are located in chromosomal regions associated with mental retardation, and Teneurin-4 is linked to susceptibility to bipolar disorder (Hong, 2012).

Drosophila ten-m was originally identified as a pair-rule gene required for embryonic patterning, but was recently determined otherwise. Teneurins were implicated in synapse development at the neuromuscular junction, and Ten-m also regulates motor axon guidance. Neither the underlying mechanisms nor their potential roles in the central nervous system are known. Vertebrate Teneurins are widely expressed in the nervous system and interact homophilically in vitro, suggesting their potential role as homophilic cell adhesion molecules in patterning neuronal connectivity (Hong, 2012).

Both Drosophila Teneurins were endogenously expressed in the developing antennal lobe. At 48 hrs after puparium formation (APF), when individual glomeruli just become identifiable, elevated Teneurin expression was evident in select glomeruli. The subset of glomeruli expressing elevated Ten-m was distinct but partially overlapping with that expressing elevated Ten-a. Teneurins were also detected at a low level in all glomeruli. Both basal and elevated Teneurin expressions were eliminated by pan-neuronal RNAi targeting the corresponding gene, suggesting that Teneurins are produced predominantly by neurons. In a ten-a null mutant this study found, all Ten-a expression was eliminated, confirming antibody specificity (Hong, 2012).

The antennal lobe consists of ORN axons as well as PN and local interneuron dendrites. This study used intersectional analysis to determine the cellular source for elevated Teneurin expression. For ten-m, GAL4 enhancer traps near the ten-m gene were used, and NP6658 (hereafter as ten-m-GAL4) that recapitulated the glomerulus-specific Ten-m staining pattern, was identifed. A FLPout reporter UAS>stop>mCD8GFP or a PN-specific GH146-Flp were used to determine the intersection of ten-m-GAL4 and an ORN-specific ey-Flp. It was found that ten-m-GAL4 was selectively expressed in a subset of ORNs and PNs. Due to reagent availability, focused was placed on five glomeruli (DA1, VA1d, VA1lm, DC3, DA3), adjacently located on the lateral and anterior side of the antennal lobe. In these five glomeruli, Ten-m expression in PN and ORN classes matched: high levels in PNs corresponded to high levels in ORNs and vice versa (Hong, 2012).

To determine the cellular origin of elevated Ten-a expression, tissue-specific RNAi of endogenous Ten-a was performed, as no GAL4 enhancer trap is available near ten-a. To isolate Ten-a expression in ORNs, pan-neuronal ten-a RNAi was performed while specifically suppressing RNAi in ORNs using tubP>stop>GAL80 and ey-Flp. To restrict Ten-a expression to central neurons, ten-a RNAi was expressed in all ORNs. It was found that Ten-a was highly expressed in a subset of ORNs and central neurons, and also showed a matching expression in five glomeruli focused in this study. The glomerular-specific differential Ten-a expression in central neurons likely arises mainly from PNs as they target dendrites to specific glomeruli, and punctate Ten-a staining was observed in PN cell bodies. In summary, Ten-m and Ten-a are each highly expressed in a distinct, but partially overlapping, subset of matching ORNs and PNs (Hong, 2012).

To examine whether Teneurins are required for proper PN-ORN matching, tissue-specific RNAi was performed in all neurons using C155-GAL4, in PNs using GH146-GAL4, or in ORNs using peb-GAL4. To label specific subsets of PN dendrites independent of GAL4-UAS, the Q binary expression system was used, and Mz19-GAL4 was converted to Mz19-QF by BAC recombineering. It was thus possible to perform GAL4-based RNAi knockdown while labeling PN dendrites and ORN axons in two colors independent of GAL4. The analysis focused on Mz19 dendrites and Or47b axons, which innervate neighboring glomeruli but never intermingle in wild type (Hong, 2012).

Pan-neuronal RNAi of both teneurins shifted Or47b axons to a position between two adjacent Mz19 glomeruli, DA1 and VA1d. Moreover, Mz19 dendrites and Or47b axons intermingled without a clear border, reflecting a PN-ORN matching defect. This was confirmed using independent RNAi lines targeting different regions of the ten-m and ten-a transcripts. Further, knocking down teneurins only in PNs or ORNs also led to Mz19-Or47b intermingling, indicating that Teneurins are required in both PNs and ORNs to ensure proper matching (Hong, 2012).

Next, the contribution of each Teneurin was examined by individual RNAi knockdown in ORNs. Knocking down ten-m, and to a lesser extent, ten-a, caused mild mismatching. This was greatly enhanced by simultaneous teneurin knockdown, likely because Mz19-Or47b mismatching requires weakening connections with their respective endogenous partners. This synergy implies that multiple matching molecules can enhance partner matching robustness (Hong, 2012).

The functions of individual Teneurins in PNs was examined. It was found that the Mz19-Or47b mismatching was caused by PN-specific knockdown of ten-a, but not ten-m. As VA1d/DC3 and DA1 PNs arise from separate neuroblast lineages, MARCM neuroblast clones were generated to label and knock down ten-a in DA1 or VA1d/DC3 PNs. ten-a knockdown only in DA1 PNs (normally Ten-a high) caused dendrite mismatching with Or47b axons. By contrast, ten-a knockdown in VA1d/DC3 PNs (normally Ten-a low) did not cause mismatching. Similarly, MARCM loss-of-function of ten-a mutant in DA1 but not in VA1d/DC3 PNs resulted in mismatching with Or47b ORNs. Thus, removal of ten-a from Ten-a-high DA1 PNs caused dendrite mismatching with Ten-a-low Or47b ORNs. The differential requirements of Ten-m and Ten-a in ORNs or PNs in preventing Mz19-Or47b mismatching likely reflect differential expression of Ten-m and Ten-a in the mismatching partners (Hong, 2012).

The finding that loss of ten-a caused Ten-a-high PNs to mismatch with Ten-a-low ORNs, together with the matching expression of Teneurins in PNs and ORNs, raised the possibility that Teneurins instruct class-specific PN-ORN connections through homophilic attraction: PNs expressing high-level Ten-m or Ten-a connect to ORNs with high-level Ten-m or Ten-a, respectively (Hong, 2012).

This homophilic attraction hypothesis predicts that overexpression of a given Teneurin in PNs (1) should preferentially affect PNs normally expressing low levels of that Teneurin, causing their dendrites to lose endogenous connections with their cognate ORNs, and (2) should cause these PNs to make ectopic connections with ORNs expressing high levels of that Teneurin (Hong, 2012).

To test the first prediction,whether Teneurin overexpression in Mz19 PNs impaired their endogenous connections with cognate ORNs was examined. Consistently, Ten-m overexpression specifically disrupted the connections of DA1 PNs and Or67d ORNs, a PN-ORN pair expressing low-level Ten-m. Connections of the other two pairs were unaffected. Likewise, Ten-a overexpression specifically disrupted connections between VA1d PNs and Or88a ORNs, a PN-ORN pair expressing low-level Ten-a, but not between the other two PN-ORN pairs (Hong, 2012).

To test the second prediction, the specificity of ectopic connections made by Mz19 PNs overexpressing Teneurins were examined, and sampled with non-partner ORN classes that project axons nearby Mz19 dendrites. It was found that Ten-m overexpression in Mz19 PNs caused dendrite mismatching only with Or47b ORNs. To examine additional mismatching phenotypes that may occur within Mz19 glomeruli and to determine whether DA1 or VA1d/DC3 PNs contribute to the ectopic connections, MARCM was used to overexpress Ten-m in individual PN classes. It was found that Ten-m overexpression in DA1 PNs (Ten-m low) caused dendrite mismatching with Or47b and (to a lesser extent) Or88a ORNs, both endogenously expressing high-level Ten-m. By contrast, Ten-m overexpression in VA1d/DC3 PNs did not produce ectopic connections with any non-matching ORNs tested (Hong, 2012).

Likewise, Ten-a overexpression in Mz19 PNs caused dendrite mismatching only with Or23a ORNs among all non-matching ORN classes sampled outside the Mz19 region. Further, MARCM overexpression of Ten-a in VA1d/DC3 PNs (Ten-a low) caused dendrite mismatching specifically with Or23a and (to a lesser extent) Or67d ORNs, both endogenously expressing high-level Ten-a. By contrast, Ten-a overexpression in DA1 PNs (Ten-a high) did not produce ectopic connections with any non-matching ORNs tested. Thus, both Ten-m and Ten-a overexpression analyses support the homophilic attraction hypothesis (Hong, 2012).

The data also suggest that additional molecule(s) are required to completely determine the wiring specificity of the five PN-ORN pairs examined. For example, VA1d-Or88a and VAl1m-Or47b have indistinguishable Ten-m/Ten-a expression patterns, and may require additional molecules to distinguish target choice. Indeed, Ten-a knockdown or Ten-m overexpression caused DA1 PNs to mismatch preferentially with Or47b as opposed to Or88a axons. This suggests that the non-adjacent DA1 and VA1lm share a more similar Teneurin-independent cell-surface code than the adjacent VA1d and VA1lm. Likewise, Ten-a overexpression caused VA1d PNs to mismatch with the non-adjacent Or23a more so than the adjacent Or67d ORNs, even though both ORNs express high-level Ten-a. Finally, Ten-m overexpression in DC3 PNs, which express low-level Ten-m, did not change its matching specificity, suggesting that Teneurin-independent mechanisms are involved in matching DC3 PNs and Or83c ORNs (Hong, 2012).

In summary, this study has shown that Teneurin overexpression in Teneurin-low PNs caused their dendrites to lose endogenous connections with Teneurin-low ORNs and mismatch with Teneurin-high ORNs. However, Teneurin overexpression in Teneurin-high PNs did not disrupt their proper connections. These data strongly support that Teneurins instruct connection specificity likely through homophilic attraction, by matching Ten-m or Ten-a levels in PN and ORN partners (Hong, 2012).

To test whether Teneurins interact in vitro, two populations of Drosophila S2 cells were separately transfected with FLAG- and HA-tagged Teneurins, and co-immunoprecipitations were performed from lysates of these cells after mixing. Strong homophilic interactions were detected between FLAG- and HA-tagged Ten-m proteins, and to a lesser extent between FLAG- and HA-tagged Ten-a proteins. Ten-m and Ten-a also exhibited heterophilic interactions, which may account for their role in synapse organization (Hong, 2012).

Next, whether Teneurins can homophilically promote in vivo trans-cellular interactions between PN dendrites and ORN axons was tested. Ten-m was simultaneously overexpressed in Mz19 PNs using Mz19-QF, and Or67a and Or49a ORNs using AM29-GAL4. This enabled independently labeling and manipulation of Mz19 dendrites and AM29 axons with distinct markers and transgenes. AM29-GAL4 was chosed because of its early onset of expression, whereas other class-specific ORN drivers start to express only after PN-ORN connection is established. AM29 axons do not normally connect with Mz19 dendrites (Hong, 2012).

Simultaneous overexpression of Ten-m in both Mz19 PNs and AM29 ORNs produced ectopic connections between them, suggesting that Ten-m homophilically promotes PN-ORN attraction. By contrast, Ten-m overexpression only in PNs or ORNs did not produce any ectopic connections, despite causing dendrite or axon mistargeting, respectively. These data ruled out the involvement of heterophilic partners in Ten-m-mediated attraction. Simultaneous overexpression of Ten-a in Mz19 PNs and AM29 ORNs did not produce ectopic connections, possibly due to lower expression or weaker Ten-a homophilic interactions (Hong, 2012).

Finally, whether these ectopic connections lead to the formation of synaptic structures was examined. Indeed, the ectopic connections between Mz19 dendrites and AM29 axons were enriched in synaptotagmin-HA expressed from AM29 ORNs, suggesting that these connections can aggregate synaptic vesicles and could be functional. It is proposed that Teneurins promote attraction between PN-ORN synaptic partners through homophilic interactions, eventually leading to synaptic connections (Hong, 2012).

Compared to axon guidance, relatively little is known about synaptic target selection mechanisms. Among the notable examples, the graded expressions of vertebrate EphA and Ephrin-A instruct the topographic targeting of retinal ganglion cell axons. Chick DSCAMs and Sidekicks promote lamina-specific arborization of retinal neurons. Drosophila Capricious promotes target specificity of photoreceptor and motor axons. C. elegans SYG-1 and SYG-2 specify synapse location through interaction between pre-synaptic axons and intermediate guidepost cells>. However, it is unclear whether any of these molecules mediate direct, selective interactions between individual pre- and post-synaptic partners. Indeed, in complex neural circuits, it is not clear a priori whether molecular determinants mediate such interactions. For example, the final retinotopic map is thought to result from both Ephrin signaling and spontaneous activity. Mammalian ORN axon targeting involves extensive axon-axon interactions through activity-dependent and independent modes, with minimal participation of postsynaptic neurons identified thus far (Hong, 2012).

This study has shown that Teneurins instruct PN-ORN matching through homophilic attraction. Although each glomerulus contains many synapses between cognate ORNs and PNs, these synapses transmit the same information and can be considered identical with regard to specificity. Thus, Teneurins represent a strong case in determining connection specificity directly between pre- and post-synaptic neurons. It was further demonstrated that molecular determinants can instruct connection specificity of a moderately complex circuit at the level of individual synapses (Hong, 2012).

This study reveals a requirement for PN-ORN attraction in the stepwise assembly of the olfactory circuit. PN dendrites and ORN axons first independently target to appropriate regions using global cues, dendrite-dendrite and axon-axon interactions. These initial, independent dendrite and axon targeting are eventually coordinated in their final one-to-one matching. Teneurins were identified as the first molecules to medicate this matching process, through direct PN-ORN attraction. These analyses have focused on a subset of ORN-PN pairs involving trichoid ORNs, including Or67d/Or88a/Or47b that are implicated in pheromone sensation. The partially overlapping expressions of Teneurins in other PN and ORN classes suggest a broader involvement of Teneurins. At the same time, additional cell-surface molecules are also needed to completely determine connection specificity of all 50 PN-ORN pairs (Hong, 2012).

Teneurins are present throughout Animalia. Different vertebrate Teneurins are broadly expressed in distinct and partially overlapping patterns in the nervous system. Teneurin-3 is expressed in the visual system and is required for ipsilateral retinogeniculate projections. This study suggests that differential Teneurin expression may play a general role in matching pre- and post-synaptic partners. Indeed, high-level Ten-m is involved in matching select motoneuron-muscle pairs. Furthermore, Teneurins also trans-synaptically mediate neuromuscular synapse organization. This suggests that the synapse partner matching function of Teneurins may have evolved from their basal role in synapse organization. Interestingly, synaptic partner matching only involves homophilic interactions, whereas synapse organization preferentially involves heterophilic interactions. This could not be fully accounted for by different strength of their homophilic and heterophilic interactions in vitro. Indeed, while heterophilic interactions occur in vitro, heterophilic overexpression of Ten-m and Ten-a in AM29 ORNs and Mz19 PNs did not produce ectopic connections. Thus it is speculated that these dual functions of Teneurins in vivo may engage signaling mechanisms that further distinguish homophilic versus heterophilic interactions (Hong, 2012).

Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins

Understanding information flow through neuronal circuits requires knowledge of their synaptic organization. This study utilized fluorescent pre- and postsynaptic markers to map synaptic organization in the Drosophila antennal lobe, the first olfactory processing center. Olfactory receptor neurons (ORNs) produce a constant synaptic density across different glomeruli. Each ORN within a class contributes nearly identical active zone number. Active zones from ORNs, projection neurons (PNs), and local interneurons have distinct subglomerular and subcellular distributions. The correct number of ORN active zones and PN acetylcholine receptor clusters requires the Teneurins, conserved transmembrane proteins involved in neuromuscular synapse organization and synaptic partner matching. Ten-a acts in ORNs to organize presynaptic active zones via the spectrin cytoskeleton. Ten-m acts in PNs autonomously to regulate acetylcholine receptor cluster number and transsynaptically to regulate ORN active zone number. These studies advanced the ability to assess synaptic architecture in complex CNS circuits and their underlying molecular mechanisms (Mosca, 2014).

A functional synapse consists of a presynaptic neurotransmitter release site and a postsynaptic neurotransmitter receptor cluster. Therefore, critical parameters of synaptic organization within a circuit not only include the location and number of presynaptic active zones, but also postsynaptic receptor clusters. Therefore, this study examined the organization of postsynapses. Given that ORNs are cholinergic, an ideal labeling strategy would image postsynaptic acetylcholine receptors (Mosca, 2014).

The Dα7 acetylcholine receptor subunit was chosen because it is endogenously expressed in the antennal lobe (Fayyazuddin, 2006) and it has been used to examine organization in the mushroom body, a higher olfactory center (Leiss, 2009a, 2009b; Kremer, 2010; Christiansen, 2011). A GFP-tagged Dα7 transgene under the control of the GAL4/UAS system was used to visualize postsynapses in vivo. Expression of Dα7-GFP in PNs revealed distinct puncta, possibly corresponding to acetylcholine receptor (AChR) clusters. These puncta were apposed to endogenous Brp puncta, as revealed by nc82 staining, consistent with these puncta representing bona fide synapses. To examine AChR clusters in PNs, Dα7-GFP was co-expressed with mtdT as a general neurite label. As such, the approach is analogous to a Brp-Short assay and yielded similar results, enabling a quantitative assessment of the number and density of AChR clusters (Mosca, 2014).

As the study was limited to genetically accessible PN subsets, focus was placed on identifying organizational parameters in the PNs that innervate the DA1 and VA1d glomeruli via the Mz19-GAL4 driver. As with ORN presynapses, the assay revealed that the number of AChR puncta scales with glomerular size. Further, known sex-specific differences in DA1, as seen in glomerular volume and in ORN synapses, were also observed. The differences between the Brp-Short and AChR assays for the DA1 and VA1d glomeruli may reflect the fact that the Brp-Short assay does not distinguish ORN synapses onto PNs and LNs, and the Dα7-GFP assay does not distinguish synapses from ORNs and LNs onto PNs. As these values are less than twofold different, this is consistent with the majority of synapses labeled being ORN to PN synapses. The similarity between the numbers of endogenous Brp and Brp-Short puncta suggests that Brp-Short is a more accurate estimator of absolute synapse number. The larger number of AChRs detected in each glomerulus may reflect an overestimation associated with full-length Dα7 overexpression or that these are postsynaptic not just to cholinergic ORNs, but also other excitatory neurons such as local interneurons or PN-PN chemical synapses (Mosca, 2014).

Calculation of AChR puncta density in PNs revealed subtle but significant differences across different glomeruli. In the VA1d glomerulus, the densities were identical between males and females. However, these were different from AChR puncta densities in the DA1 glomerulus. There was a modest but significant difference between both male and female AChR densities in DA1 and between both DA1 AChR densities and the shared VA1d AChR density. Unlike ORNs, where the Brp-Short density was identical across different classes of neurons, PNs can have different densities between distinct glomeruli and even between sexes for the same glomerulus. Thus, the parameters that govern presynaptic density may differ from those that govern postsynaptic density in the same glomerulus (Mosca, 2014).

To further examine if the Teneurins regulate postsynaptic acetylcholine receptor number and density, the Dα7-GFP assay was used to determine the effect of Teneurin perturbation on AChR puncta number. PNs were examined in DA1 and VA1d and the AChR puncta of both glomeruli were counted together as one measurement, as partner matching defects following Teneurin perturbation make it difficult to differentiate between the two glomeruli. In ten-a mutants, the number of AChR clusters in these glomeruli was decreased by 23%, compared to wild type. This is consistent with results from the Brp-Short assay. Moreover, PN neurite volume was unaffected, so AChR puncta density was similarly reduced. Thus, two independent assays, both pre- and postsynaptic, show the same phenotypes, demonstrating a clear effect of ten-a loss on synapse organization in olfactory neurons (Mosca, 2014).

At the NMJ, presynaptic Ten-a functions largely in a transsynaptic, heterophilic complex with postsynaptic Ten-m to regulate synapse organization. Ten-a functions presynaptically in ORNs to ensure proper synapse number. Thus, it was hypothesized that the loss of Ten-m in postsynaptic PNs should result in a similar phenotype. As the ten-m mutant is larval lethal, a previously validated transgenic RNAi line against ten-m was expressed in Mz19 PNs, and AChR puncta number was quantitated using the Dα7 assay. ten-m knockdown phenocopied the ten-a phenotype. As above, PN neurite volume was unaffected, leading to a concomitant decrease in AChR puncta density that also phenocopied the ten-a phenotype. Further, this reduction was not enhanced by knocking down ten-m in PNs of a ten-a null mutant, suggesting that the two function in the same genetic pathway (Mosca, 2014).

This study has identified parameters that govern synapse number, density, and subcellular organization using two fluorescently-tagged synaptic proteins expressed from single transgenes in combination with high-resolution confocal microscopy and image processing to visualize synapses in the Drosophila olfactory system in vivo. It was demonstrated that these methods are amenable to analysis in both individual neuronal classes and individual neurons. The study provides a synapse-level analysis of innervation of olfactory receptor neurons, projection neurons, and local interneurons in the antennal lobe, which has emerged as a model circuit for analyzing principles of information processing. Finally, using these synaptic tagging assays, it was shown that the Teneurins are required for the proper synapse number in ORNs and PNs as well as the structure of the active zones themselves. This reveals a critical role for these transmembrane proteins in organizing central synapses, likely by regulating the cytoskeleton. These approaches can be broadly used to study synaptic organization of neurons for which genetic access is available, and to investigate the functions of any other proteins in the organization and development of CNS synapses (Mosca, 2014).

In Drosophila, previous approaches to studying central synapses used tagged synaptic vesicle proteins to reveal putative synaptic sites. While consistent with synapses, they could also stain non-synaptic, trafficking vesicles. This study utilized a structural active zone component, Brp. By expressing a truncated Brp transgene, Brp-Short, using the GAL4/UAS system, this approach can label synapses in any neurons with genetic access. Brp-Short expression requires endogenous Brp for proper localization. Thus, it accurately reports endogenous active zones. Recently, an elegant technique, STaR, was developed that tags an additional BAC-sourced copy of Brp with an epitope tag whose expression is conditional upon FLP-recombinase-mediated excision of an intervening stop codon. An important advantage of STaR is that Brp expression is controlled by its endogenous promoter, thus guarding against mislocalization of Brp or perturbation of synaptic function due to overexpression. A caveat of Brp-Short is that it is controlled by GAL4 and thus the levels may be different from the endogenous level. While Brp-Short overexpression does not interfere with synaptic function, care must be taken not to overexpress it to a level that saturates the active zone localization machinery when utilized in new cell types. The advantages of Brp-Short over STaR are (a) that it does not require a cell type-specific FLP transgene, which is not as widely available as GAL4 lines, or a BAC-bearing copy of Brp, and (b) that it can be co-expressed with UAS-transgenes for rescue or RNAi for perturbation experiments, although STaR can also achieve this aspect by using a cell-type-specific GAL4 and an extra UAS-FLP transgene (Mosca, 2014).

To examine putative postsynaptic acetylcholine receptor clusters, a GFP-tagged subunit, Dα7, was used to study cholinergic synapses in the antennal lobe. This transgene has been used to examine synaptic organization. Though false positives can be associated with full-length protein overexpression, the current observation of endogenous Brp puncta apposed to these Dα7-GFP puncta suggests that these receptors are properly localized to endogenous synapses. This assay complements the Brp-Short presynaptic assay and can be adapted for other tagged postsynaptic receptor transgenes (Mosca, 2014).

Though considerable advances have been made in understanding of the wiring specificity and physiological properties of the Drosophila olfactory circuit in the antennal lobe, little is known about its synaptic organization. Since this system has emerged as a model neural network, a detailed mapping of synaptic organizational principles is integral towards advancing the study of circuit dynamics. Indeed, the juxtaposition of distinct types of synapses between multiple neuronal classes in the antennal lobe provides a model to study complex synaptic interactions compared to a neuromuscular synapse that features only two synaptic partners: the motoneuron and the muscle. This study utilized the Brp-Short and Dα7 assays to probe how synapses in ORNs, PNs, and LNs are organized in the antennal lobe with respect to their number, density, and location. This work offers the first comprehensive information on (1) the number of active zones made by each ORN within a glomerulus, (2) the stereotypy of synapse numbers between those individual ORNs, (3) the prominence of PN presynaptic inputs within a glomerulus, suggesting a robust feedback mechanism, and (4) the relatively small contribution of LN active zones to the antennal lobe circuit. These analyses suggest distinct rules that govern the synaptic organization of antennal lobe neurons. ORNs, the primary input neurons of the olfactory system, are diverse in their olfactory receptor expression, ligand specificity, and glomerular targeting specificity; this study now shows that they also differ in the absolute synapse number. However, despite such differences, all five classes examined (DA1, VA1d, VA1lm, DL4, and DM6 ORNs) have identical synaptic densities, suggesting that this represents a general rule for other ORN classes. This may further suggest that the primary job of the ORN is to convey information from the environment into the system as faithfully as possible, that all information is treated equally at this level, and that weighting computations occur downstream in the brain. Indeed, analyses indicate that each ORN makes an equivalent, discrete number of synapses within a given glomerulus with little variation, further supporting this hypothesis (Mosca, 2014).

Interestingly, the density of postsynaptic receptors differs between glomeruli and even within the same glomerulus between sexes. This variation can be due to technical caveats, such as Mz19-GAL4 does not label all PNs that innervate DA1 and VA1d, or that Dα7-GFP clusters do not reflect the absolute number of AChRs. As the relative numbers still show these differences, an interesting possibility suggested by these results is that postsynaptic PN AChRs already reflect a transformed olfactory representation compared to output synapses of ORNs. The difference in PN AChR density as compared to the constant density of ORN active zones suggests that different classes of PNs may modulate how information is received by regulating the number of acetylcholine receptors. This can thus contribute to the transformation of olfactory representation by antennal lobe neurons (Mosca, 2014).

Fine-scale analysis of synapse localization within projections of ORNs, LNs, and PNs suggested that these three types of neurons differ in their subglomerular organization. While occupying the vast majority of territory throughout the entire glomerulus, ORN processes and synapses leave distinct voids. A significant proportion of LNs form synapses in these voids, likely to other LNs or PNs. However, there is also overlap between LN and ORN processes and synapses, consistent with physiologically characterized ORN -> LN and LN -> ORN synapses. Within their respective neurites, ORNs and PNs display uneven distributions and synaptic clusters to varying degrees while active zones in LNs are more evenly distributed throughout their processes. These characterizations contribute to the growing repertoire of studies seeking to understand synaptic organization in Drosophila olfactory circuits, adding synapse-level imaging to physiological techniques. Finally, the existence of synaptic organization parameters detailing number and location suggests molecular mechanisms designed to enforce those rules, both at cellular and circuit levels. Indeed, such analysis represents an integral part of neuronal circuit analysis, as recent work on retinal neurons has shown that accounting for synapse position is a critical aspect of modeling connectivity (Mosca, 2014).

The data demonstrate that the Teneurins, a family of transsynaptic adhesion molecules, regulate one of these synaptic organizational paradigms: synapse number. Beyond molecules like RPTPs and Wnts , there is little known conservation between the organization mechanisms of central synapses and the NMJ. In vertebrates, previous studies have identified a number of synaptogenic signaling and cell adhesion molecules in the CNS, but in many cases, their roles at the NMJ are either minimal or unknown. Likewise, the roles of pathways including Rapsyn, Dok7, MuSK, and Tid1, which are well established at the NMJ, have no well-established roles at CNS synapses. Among the identified central synaptogenic molecules, no master controller of synapses, like Agrin, has been discovered. In the mammalian CNS, synaptic adhesion molecules like Neurexin and Neuroligin have demonstrated organizational roles but their role (if any) at the NMJ is largely unknown. In Drosophila, considerable work has been done at the NMJ to understand synapse formation and organization. For example, neuromuscular Neurexin and Neuroligin regulate synaptic development and assembly, but remain (as with many other identified molecules) largely untested in the CNS due to the absence of techniques for doing so (Mosca, 2014).

The approaches described in this study have now enabled such an examination, uncovering a strongly conserved synaptic organization function of the Teneurins from PNS to CNS. Recent work has shown that these evolutionarily conserved proteins are involved in synaptic partner matching between neurons in the Drosophila olfactory system and between muscles and motoneurons at the NMJ. As there are marked differences between central and peripheral synapses (like the NMJ), it is further unclear whether the mechanisms would be conserved or if they would be wholly different. This study found, as assayed by Brp-Short, ultrastructural, and Dα7 analyses, that Teneurins in olfactory neurons are required for normal synapse number. Teneurin perturbation also reduces synaptic density, a parameter that is highly invariable for ORNs under normal conditions. It was determined that presynaptic Ten-a in ORNs likely functions with postsynaptic Ten-m in PNs to regulate levels of the spectrin cytoskeleton. The evidence is consistent with spectrin and the Teneurins functioning in the same genetic pathway to regulate synapse organization and density. The perturbation of active zone number in ORNs by knocking down ten-m in PNs further suggests that Teneurins regulate synapse number and organization in the CNS via a transsynaptic mechanism. This highlights further conservation between central and peripheral synapse organization in the use of Teneurins. There are, however, some differences between the CNS and the PNS regarding the Teneurins. While presynaptic ten-m has a minor role in synaptic organization at the NMJ, the data suggests the lack of such a role in the CNS. Thus, these different systems may also use the Teneurins differently. Mammalian Teneurins organize the visual system and Ten-2 can serve as a ligand for Latrophilin and localize to synapses in cultured neurons. However, as proper synaptic function is impaired in many neuropsychiatric disorders and human Teneurin-4 is associated with increased susceptibility to bipolar disorder, understanding how the Teneurins regulate central synapses is a question with clinical relevance. Studies in vivo will be important to determine how mammalian Teneurins regulate synaptic organization and whether the different Teneurins can have specific roles at synapses (Mosca, 2014).

In summary, the results demonstrate a role for the Teneurins in regulating the number of central synapses and highlight mechanistic conservation between peripheral and central synapse formation. Moreover, the fact that ORNs can be mistargeted but still have the correct number of synapses suggests that target choice and synapse organization can be biologically separable, even when they employ the same molecules (Mosca, 2014).

Ten-a affects the fusion of central complex primordia in Drosophila

The central complex of Drosophila melanogaster plays important functions in various behaviors, such as visual and olfactory memory, visual orientation, sleep, and movement control. However little is known about the genes regulating the development of the central complex. This study reports that a mutant gene affecting central complex morphology, cbd (central brain defect), was mapped to ten-a, a type II trans-membrane protein coding gene. Down-regulation of ten-a in pan-neural cells contributed to abnormal morphology of central complex. Over-expression of ten-a by C767-Gal4 was able to partially restore the abnormal central complex morphology in the cbd mutant. Tracking the development of FB primordia revealed that the C767-Gal4 labeled interhemispheric junction, that separated fan-shaped body precursors at the larval stage, withdrew to allow the fusion of the precursors. While the C767-Gal4 labeled structure did not withdraw properly and detached from FB primordia, the two fan-shaped body precursors failed to fuse in the cbd mutant. It is proposed that the withdrawal of the C767-Gal4 labeled structure is related to the formation of the fan-shaped body. These result revealed the function of ten-a in central brain development, and possible cellular mechanism underlying Drosophila fan-shaped body formation (Cheng, 2013).

The central complex is an interconnected neuropil structure across and along the sagittal mid-section of the fly brain and includes the protocerebral bridge (PB), the fan-shaped body (FB), the paired nodule (NO), and the ellipsoid body (EB). It is involved in multi-modal behavioral control, such as locomotion, visual pattern memory and spatial orientation. The development of the central complex can be traced back to the larval stage. Lineage analysis has revealed the neurons that contribute to the central complex, but the molecular and cellular mechanism of central complex formation is not fully understood (Cheng, 2013).

In the 1980s, Martin Heisenberg and coworkers generated a series of structural mutants, in which the morphology of adult central brain structures like mushroom bodies and the central complex were destroyed. Among these mutants, mbm (mushroom body miniature), ceb (central brain deranged) and nob (no-bridge) have been identified. mbm was found to be a transcription factor, a nucleic acid-binding zinc finger protein, while ceb was reported to encode Neuroglian, a cell adhesion molecule that is crucial for axonal development, synapse formation and female receptivity. As to nob, it interacted with drl at the interhemispheric junction to affect the formation of protocerebral bridge. Another mutant type is central body defect (cbd), of which the most typical phenotype is that the fan-shaped body and the ellipsoid body are fragmented in the middle, or some fusion of the fan-shaped body and the ellipsoid body. So far, the molecular basis of most structural mutants is unclear (Cheng, 2013).

Ten-a belongs to a large protein family, Teneurin, which contains an N-terminal intracellular domain, a single transmembrane domain, eight EGF-like domains, a 6-blade β-propeller TolB-like domain, and 26 YD repeats. From invertebrates to vertebrates, Teneurins function as signaling molecules at the cell surface as type II transmembrane receptors, while the intracellular domain cleaved from membrane works as a transcription regulator and carboxyl terminus functions as a bioactive peptide. The Teneurin family members are thought to be important for establishment and maintenance of neuronal connections, neurite outgrowth and axon guidance. Recent reports showed that two Drosophila Teneurin members, Ten-a and Ten-m, are crucial for proper synaptic matching and the maintenance of neuromuscular junction. Although Teneurin may play a role in mammalian brain functio, detailed study is still largely lacking (Cheng, 2013).

This study reports that the Drosophila structural mutant gene cbd, the most typical phenotype of which is the fragmented fan-shaped body and ellipsoid body, is ten-a. The cbd mutation disrupts the formation of the FB, by preventing the merging of the two FB parts. This defect was rescued by over-expression of ten-a in a C767-Gal4 labeled structure which separated the FB parts but later disappeared to allow the merging of the two FB primordia. These results might reveal the molecular and cellular mechanism of Drosophila central complex development (Cheng, 2013).

This study found that the Drosophila central brain morphological mutant cbd is actually ten-a, a member of the teneurin family. Ten-a is required for fusion of the fan-shaped body precursors, before the formation of the complete normal FB. Mutation in ten-a leads to the failure of the two FB precursors to merge and consequently to the deranged fan-shaped body in adult flies (Cheng, 2013).

Aside from the FB morphological defect itself, ten-a mutation might cause other abnormalities that contribute to the morphological defect. For example, Ten-a might affect the projections and contra-lateral crossing of FB neurons resulting from lineages of FBP1 and FBP2, which contribute to two staves of the fan-shaped body, consequently led to a cleaved fan-shaped body. Nevertheless, the generation and projection of large field ExFl neurons labeled by NP6510-Gal4 or C205-Gal4 are not affected when ten-a mutated, which suggested that ten-a mainly produce the morphological defect by exerting its effect on FBP1 and FBP2 neuron arborizations. Actually, based on the morphological observation in cbd KS171 flies and the rescue results, it was postulated that the interhemispheric structure C767-Gal4 labeled was related to FB primordial fusion. But it seemed to have no effect on axonal projections and terminal arborization of F1 and F5 neurons (Cheng, 2013).

ten-a knockdown results showed that the neuronal ten-a was required for the central complex formation. Further rescue experiments suggested that neither neurons nor glial cells alone were sufficient for normal central complex formation. After screening, one Gal4 line was found finally. C767-Gal4 could be used to rescue the cbd mutant phenotype significantly. To identify the cell types labeled by C767-Gal4, neuron specific marker ELAV or glial cell specific marker REPO were used to co-stain with C767-Gal4 labeling cells. The results showed that nlsGFP driven by C767-Gal4 was co-localized with both neuronal and glial markers from larval to early pupal stages. Since previous studies showed some adhesion molecules were expressed both in neurons and glia for mediating the fasciculation of axon bundles, axon guidance or targeting, it is suggested that the rescue results by C767-Gal4 might just attribute to that the Gal4 expressed both in neurons and glial cells. That is to say, only when ten-a functions in certain neurons and glial cells together, the FB precursors could merge normally. However, which neurons and glial cells were required for the partial rescue could not be identified from current results. To solve this problem, more Gal4 lines which can rescue the cbd mutant phenotype are needed. Then, dependent on the expression patterns of these Gal4 lines, the neurons and glial cells which ten-a functions in may be identified (Cheng, 2013).

If Ten-a functions in C767-Gal4 labeled cells to influence the merging of FB primordia, what is its working partner for the arborization of FB neurons? As a Drosophila homolog of vertebrate Teneurin, Ten-a has been reported to be involved in embryo development, especially in the central nervous system. Ten-a, as well as its homologue Ten-m, was recently found to be required for synaptic matching between olfactory receptor neurons and corresponding projection neurons. Ten-a and Ten-m were also important for establishing the correct connection in the larval neuromuscular junction. In the current work, lack of normal Ten-a function led to failure in merging of FB precursors. It is possible to assume that Ten-a itself mediates homophilic interaction between neurons and glial cells to regulate the fusion of the central complex, such as Nrg, which is expressed on both neurons and glial cells and interacts to control axonal sprouting and dendrite branching. Meanwhile, Ten-a may interact with other molecules such as Ten-m, or other membrane proteins that function in heterophilic way at the cell surface. Further molecular and cellular experiments are needed to elaborate this important issue (Cheng, 2013).

Vertebrate Teneurins have been suggested to be related to mental diseases, and the discovery of Ten-a function in Drosophila brain development seems to support the hypothesis. Neuroglian (Nrg), whose vertebrate homologue L1-CAM has been implicated in neurological disorders, is also required for development of normal brain morphology in Drosophila. Considering that both Nrg and Ten-a are type-II transmembrane proteins with extracellular EGF repeats and also function in glial cells for brain development, it is possible that Teneurins in vertebrates also affect brain development, and probably synapse formation, as vertebrate Nrg does (Cheng, 2013).

In summary, this work elucidates the function of ten-a in development of the Drosophila central brain, and the cellular mechanism underlying FB formation (Cheng, 2013).

Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: regulation by alternative splicing

Latrophilin-1, -2, and -3 are adhesion-type G protein-coupled receptors that are auxiliary alpha-latrotoxin receptors, suggesting that they may have a synaptic function. Using pulldowns, this study identified teneurins, type II transmembrane proteins that are also candidate synaptic cell-adhesion molecules, as interactors for the lectin-like domain of latrophilins. Teneurin are shown to bind to latrophilins with nanomolar affinity, and this binding mediates cell adhesion, consistent with a role of teneurin binding to latrophilins in trans-synaptic interactions. All latrophilins are subject to alternative splicing at an N-terminal site; in latrophilin-1, this alternative splicing modulates teneurin binding but has no effect on binding of latrophilin-1 to another ligand, FLRT3. Addition to cultured neurons of soluble teneurin-binding fragments of latrophilin-1 decreased synapse density, suggesting that latrophilin binding to teneurin may directly or indirectly influence synapse formation and/or maintenance. These observations are potentially intriguing in view of the proposed role for Drosophila teneurins in determining synapse specificity. However, teneurins in Drosophila were suggested to act as homophilic cell-adhesion molecules, whereas the current findings suggest a heterophilic interaction mechanism. Thus, whether mammalian teneurins also are homophilic cell-adhesion molecules was tested, in addition to binding to latrophilins as heterophilic cell-adhesion molecules. Strikingly, it was found that although teneurins bind to each other in solution, homophilic teneurin-teneurin binding is unable to support stable cell adhesion, different from heterophilic teneurin-latrophilin binding. Thus, mammalian teneurins act as heterophilic cell-adhesion molecules that may be involved in trans-neuronal interaction processes such as synapse formation or maintenance (Boucard, 2014).

Drosophila Ten-a is a maternal pair-rule and patterning gene

The Ten-a gene of Drosophila encodes several alternative variants of a full length member of the Odz/Tenm protein family. A number of Ten-a mutants created by inexact excisions of a resident P-element insertion are embryonic lethal, but show no pair-rule phenotype. In contrast, these mutants, and deficiencies removing Ten-a, do enhance the segmentation phenotype of a weak allele of the paralog gene odz (or Ten-m) to the odz amorphic phenotype. Germ line clone derived Ten-a embryos display a pair-rule phenotype which phenocopies that of odz. Post segmentation eye patterning phenotypes of Ten-a mutants establish it as a pleiotropic patterning co-partner of odz (Rakovitsky, 2007).

A recently produced deletion mutation of the Drosophila melanogaster gene Ten-a removes the entire gene, yet does not lead to lethality (Zheng, 2011). Thus the lethality and the pair-rule gene roles reported for Ten-a, cannot be attributed to this gene, but to second site loci in mutants studied. Given the centrality of the pair-rule claim for this paper, it has been retracted. The other findings of the paper remain as reported, particularly molecular and biochemical characterizations of the Ten-a gene, its alleles and its transcripts (Rakovitsky, 2011).

Unlike other pair-rule (P-R) genes, the Drosophila zygotic P-R gene odd Oz (odz or Ten-m) encodes a membrane anchored cell surface protein, and not an obvious transcription factor, with many indications that it is involved in patterning. These appear to be receptor like proteins, with discordant yet convergent evidence that discrete domains of the proteins might be processed into elements involved in transcription. The first phenotypes for non-Drosophilan Odz/Tenm mutations were recently described, further supporting this family's requirement for proper patterning and development. Phenotypes of mouse Odz4 mutations include failure in gastrulation and somite formation, and a small deletion of the C. elegans ortholog, Ten1, leads to early embryo arrest and gonadal defects (Lossie, 2005; Drabikowski, 2005; Rakovitsky, 2007 and references therein).

Ten-a was initially identified as a gene encoding a relatively short protein with EGF-like repeats at its C-terminus. Genomic sequence made it clear that the D. melanogaster genome project Ten-a gene model, once 'merged' with gene model annotations lying adjacent and 'downstream' to Ten-a on the X chromosome (gene annotations CG2590 and CG2578), constitutes a full length gene paralog of Ten-m/odz. An examination of cDNAs verified that a Ten-a transcript exists that encodes a full length 300 kDa protein of the Odz/Tenm family, with 48% amino acid similarity to Drosophila odz. A number of the expression sites of Ten-a in embryos suggest extensive overlap with odz expression (Rakovitsky, 2007).

This study has described lethal and semi-lethal mutations caused by small deletions that remove a region containing an exon of an alternative splice form of Ten-a transcripts. It is likely that it will be necessary to examine mutations that disrupt exons shared between all of the splice forms in order to see truly amorphic phenotypic consequences. Nonetheless, the levels of all tested Ten-a transcripts are reduced significantly in the small lethal deletions. This is documented by RT-PCRs performed on RNA from post-gastrulation male embryos, a stage when any maternal stores should be exhausted. A lack of transcripts was shown using a primer pair bridging the fourth to fifth exons, and a significant reduction was shown using a primer pair bridging the eighth to ninth exons, in coding regions shared by essentially all protein forms of Ten-a. The loss of transcripts, and the phenotypes affecting viability, are not likely due solely to the consequences of the loss of exon 1d. Rather, it is assumed that some crucial regulatory region or elements must be impacted by these small deletions (Rakovitsky, 2007).

There are predicted full length Ten-a proteins that span the original CG15733, CG12720, and Ten-a annotations, as well as full length Ten-a proteins that are derived from only the original Ten-a annotation's exons. Yet other transcripts encode both a full length Ten-a protein as well as exons in the transcript's 5' UTR with capacity to direct attenuated translation of smaller upstream gene products. This implies the possible alternative translation of cryptic regulatory short polypeptides from the transcript versus the full Ten-a protein reading frame, through regulation of the utilization of IRES sequences. Perhaps most intriguing is the existence of protein domains (domains of the original CG12720 annotation's exons) that appear alternatively in the attenuated polypeptide products from some transcripts, and at other times as the N-terminus of full length Ten-a protein from other transcripts. It should be noted that these N-terminal regions are among the most poorly conserved among proteins in the family, so that inferences for other homologs can not immediately be drawn. What is shared among all homologs are proportionately very large 5' introns among all sequenced species (except for C. elegans, which employs trans-splicing). This implies further extensive regulation in the 5' region of the genes, as do IRES sequences resident in the most upstream exons (Rakovitsky, 2007).

Among the alternatively spliced messages uncovered, there are encoded proteins lacking the hydrophobic domain close to the N-terminus present in all family homologs, and others with only a subset of the EGF-like repeats. These many variations are reminiscent of reports of Odz/Tenm protein variants in other metazoans. The existence of varied validated Mouse Odz4 proteins, from different regions of the gene and of different lengths, emphasizes the complexity of the gene products deriving from Odz/Tenm family loci (Lossie, 2005). Ultimately, the many optional messages support new predictions for membrane-bound and other deployments for different variants of these proteins. Perhaps these variants and isoforms can explain the differing and sometimes mutually exclusive results that have been reported for proteins of this family in the past. The multiple splice variants and resulting protein forms can explain the different biochemical, immunocytochemical, and membrane deployment observations that have been made for this family's homolog gene products (Rakovitsky, 2007).

Ten-a impacts proper segmentation through both zygotic and maternal contributions. It zygotically enhances a weak odz cuticle phenotype with high penetrance. In germ line clones it leads to a very strong, if not canonical, pair-rule phenotype likely caused by maternal and zygotic contributions. This occurs in a minority of the embryos, in a population that includes those with weak and no segmentation phenotypes. Both maternal and zygotic activities appear necessary, but a clearcut 'ratio' of weights of these two contributions will likely only be clear when null Ten-a mutations will be examined (Rakovitsky, 2007).

The germ line mosaic derived embryos deprived of Ten-a contribution provide evidence that activity from the Ten-a gene locus is necessary for segmentation at the pair-rule stage of segmentation. These embryos display a full-blown pair-rule phenotype in the same register as odz mutants. This case of a gene displaying a maternally dependent P-R phenotype is rare, but has been seen previously in kismet. It is likely that Ten-a acts in a direct manner, and not through downstream events, such as is the case for kismet. This is most evident from the disruption of slp expression in the odd parasegments of embryos mutant for odz and Ten-a. Striped slp RNA expression is not initiated, most likely due to the involvement of these paralogous gene products in transcription (Rakovitsky, 2007).

Ten-a mutants and deficiency chromosomes zygotically enhance weak odz segmentation phenotypes in a highly specific manner. There are indications that Odz and Ten-a proteins form heterodimers, as has been well established for the four vertebrate homologs (Feng, 2002). The segmentation phenotypes of each gene's mutations are in the same segmental register, and essentially are identical. Therefore, whether or not they prove to act as dimer proteins in this context, they are at least involved in the same process at the same time. It is thought that both of the two gene products are necessary for a concerted, coordinated activity that contributes to proper segmentation at the P-R stage. While each alone can cause the 'odd-fused' phenotype in the proper zygotic or maternal mutant context, it is assumed that mutants of each deplete the same active complex. Given the Ten-a maternal contribution, it is possible that the Ten-a protein is ubiquitous throughout the early embryo. It is therefore envisioned that the region of activity of both family-paralogs as spatially delimited to the striped domain of deployment of the Odz protein in the context of segmentation (Rakovitsky, 2007).

slp expression in odd parasegments requires Runt and Odd paired (Opa) activity, as opposed to slp expression's dependence on Hairy and Ftz in even parasegments. This study showns that the initiation of the odd parasegment expression is also dependent on Odz and Ten-a. The Odz striped domain covers odd parasegments, whereas Runt is expressed in the posterior half of the odd parasegment and anterior half of the even parasegment, and whereas Opa and Ten-a are ubiquitous. The domain of Odz and Runt expression overlap is therefore in the posterior half of the odd parasegment. This overlap corresponds to the unique domain of slp odd parasegment transcriptional activation. A complex, or confluence of activities, of: the two Odz/Tenm family homologs; Runt; and Opa can be envisioned turning on slp expression and provide an excellent context to be followed in order to establish the nature of downstream outcomes of odz homologs' activities. Whether the two Tenm/Odz paralogs' proteins interact directly with Runt and Opa in transcription, or whether they initiate a chain of interactions that results in these transcription factors' alterations, must still be clarified. It is also an alternative possibility that Odz and Ten-a influence the levels of odd parasegment slp RNA post-transcriptionally, for instance through an effect on slp transcript stability (Rakovitsky, 2007).

The atypical nature of odz and Ten-a as P-R genes extends to more than their proteins' structures and subcellular deployment. These are extremely large genes, transcribing larger than 120 kb nascent messages. This is essentially unheard of for early segmentation genes in Drosophila, where their nascent transcript sizes cluster around 2 kb. At a transcription rate of 1.4 kb per minute, interphase lengths as short as 3.5 min during syncitial blastoderm, and evident transcription abortion at each mitosis, transcription units are thought to be limited to 5 kb in order to function at early embryonic stages. This size restriction was shown to be the causal difference between knirps acting as a segmentation gap-gene, versus knirps-related only functioning at later stages, due to its larger transcript size (Rakovitsky, 2007).

This renders Ten-a and odz zygotic gene function complex to invoke, given the 1.5 h needed to transcribe them. They are proposed to act first at the cellular blastoderm, given the first appearance of Odz protein, given the timing of appearance of genes proven downstream to them, and given their cell-transmembrane deployment. Yet even the cycle 14 interphase is not clearly adequate to allow for synthesis of their full transcription unit. Instead it is most likely that the key regulatory step to produce these proteins, when needed just post-cellularization, is translation of partial messages or pre-existing messages. For odz, RNA has never been observed in a stripewise manner in the cellular blastoderm, unlike the seven observed stripes of protein. This implies possible regulated translation, which can be consistent with the presence of IRES sequences in Odz transcripts. This study showns that Ten-a has a maternally provided component, but have not examined whether or not odz does. In both cases, the zygotic contribution is unquestionably critical, raising the possibility that some length transcript form is adequately synthesized at cellular blastoderm. How this compliments gene activity afforded by the maternal contribution will need to be clarified in future work (Rakovitsky, 2007).

Phenotypes of Ten-a mutant eyes, and of Ten-a plus odz transhetereozygous combinations, attest to the importance of Ten-a in this second tissue system. Given the nature of the phenotypes, and within the context of what is known about Odz function, the importance of Ten-a in these cases is likely to center on its patterning roles. The strong phenotypes of transheterozygous combinations of odz and Ten-a alleles, that each alone display no eye phenotypes, suggest that they cooperate closely in eye patterning. Thus Ten-a is a maternally required pair-rule gene with likely far reaching patterning dimensions in many contexts. Understanding of Odz/Tenm family contributions to metazoan patterning can now be furthered in a system in which all (both) members can now be coordinately manipulated and studied (Rakovitsky, 2007).

Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities

Latrophilin 1 (LPH1), a neuronal receptor of alpha-latrotoxin, is implicated in neurotransmitter release and control of presynaptic Ca(2+). As an 'adhesion G-protein-coupled receptor,' LPH1 can convert cell surface interactions into intracellular signaling. To examine the physiological functions of LPH1, wLPH1's extracellular domain was used to purify its endogenous ligand. A single protein of approximately 275 kDa was isolated from rat brain and termed Lasso. Peptide sequencing and molecular cloning have shown that Lasso is a splice variant of teneurin-2, a brain-specific orphan cell surface receptor with a function in neuronal pathfinding and synaptogenesis. This study shows that LPH1 and Lasso interact strongly and specifically. They are always copurified from rat brain extracts. Coculturing cells expressing LPH1 with cells expressing Lasso leads to their mutual attraction and formation of multiple junctions to which both proteins are recruited. Cells expressing LPH1 form chimerical synapses with hippocampal neurons in cocultures; LPH1 and postsynaptic neuronal protein PSD-95 accumulate on opposite sides of these structures. Immunoblotting and immunoelectron microscopy of purified synapses and immunostaining of cultured hippocampal neurons show that LPH1 and Lasso are enriched in synapses; in both systems, LPH1 is presynaptic, whereas Lasso is postsynaptic. A C-terminal fragment of Lasso interacts with LPH1 and induces Ca(2+) signals in presynaptic boutons of hippocampal neurons and in neuroblastoma cells expressing LPH1. Thus, LPH1 and Lasso can form transsynaptic complexes capable of inducing presynaptic Ca(2+) signals, which might affect synaptic functions (Silva, 2011).

LPH1 has been implicated in multiple phenomena, including binding of α-LTX, release of neurotransmitters, intracellular signaling, neuronal morphogenesis, and mental conditions. However, further studies of LPH1 require the identification of its endogenous ligand. Using LPH1-affinity chromatography, this study has now isolated such a ligand. Lasso is a splice variant of teneurin-2. It interacts with LPH1 specifically and strongly, but binds very weakly to LPH2 and does not bind to LPH3. Reciprocally, sequencing results and LPH1 binding suggest that LPH1 interacts with Lasso/teneurin-2 only, and not with teneurin-1, -3, or -4 (Silva, 2011).

Both LPH1 and Lasso/teneurin-2 are highly abundant in the brain. Can they mediate neuronal cell interaction? Teneurin is proteolyzed between the TMR and EGF repeats (see Kenzelmann, 2008), and this could preclude its receptor activity. However, only a proportion of teneurin is cleaved, resulting in two bands on reducing SDS gels. The fragment remains anchored on the cell surface, probably as part of the homodimer. This makes Lasso a bona fide cell-surface receptor (Silva, 2011).

Accordingly, Lasso and LPH1 mediate heterophilic cell-cell contacts between expressing cells in cocultures and formation of artificial synapses between fibroblasts and neurons. Moreover, the data indicate that in cultured neurons and mature brain, LPH1 is localized in the presynaptic membranes, whereas Lasso is mostly postsynaptic. Given the size of LPH1 (14 nm) and Lasso (~30 nm), their complex can span the synaptic cleft (20-30 nm), allowing this receptor pair to connect neurons at synapses (Silva, 2011).

The LPH1-Lasso interaction is not purely structural. LPH1 mediates signaling induced by LTXN4C both in model cells expressing exogenous LPH1 and in organotypic hippocampal cultures. This signaling requires the CTF and involves the activation of phospholipase C, production of inositol-trisphosphate, and release of stored Ca2+. This study demonstrates that a soluble C-terminal fragment of Lasso also induces an increase in cytosolic Ca2+ in NB2a cells expressing LPH1 and in hippocampal neurons. Such a rise in cytosolic Ca2+ in neurons could modulate neurotransmitter release (Silva, 2011).

Interestingly, teneurin contains a sequence termed teneurin C-terminal-associated peptide (TCAP) that resembles corticotropin-releasing factor. It is hypothesized that TCAP is cleaved from teneurin and acts as a soluble ligand of unknown receptors (Wang, 2005). Synthetic TCAP regulates cAMP in immortalized neurons and, when injected cerebrally, affects behaviors related to stress and anxiety. However, there is no evidence that TCAP is released in vivo. The current work suggests an alternative possibility: TCAP, being part of Lasso, affects animal behavior by stimulating LPH1. This is supported by LPH being implicated in schizophrenia, anxiety (offspring killing by LPH1−/− mice), and attention deficit/hyperactivity disorder (Silva, 2011).

The current findings raise several interesting questions. Does Lasso send an intracellular signal in response to LPH1 binding? What are the relationships among the four teneurin homologs and the three LPH proteins found in most vertebrates? Can the LPH1–Lasso interaction affect synaptogenesis, neurotransmitter release, or synaptic plasticity? Answering these questions will provide important insights into the physiological functions of these two families of neuronal cell surface receptors (Silva, 2011).


REFERENCES

Search PubMed for articles about Drosophila Ten-a

Boucard, A. A., Maxeiner, S. and Sudhof, T. C. (2014). Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: regulation by alternative splicing. J Biol Chem 289: 387-402. PubMed ID: 24273166

Cheng, X., Jiang, H., Li, W., Lv, H., Gong, Z. and Liu, L. (2013). Ten-a affects the fusion of central complex primordia in Drosophila. PLoS One 8: e57129. PubMed ID: 23437330

Christiansen, F., Zube, C., Andlauer, T. F., Wichmann, C., Fouquet, W., Owald, D., Mertel, S., Leiss, F., Tavosanis, G., Luna, A. J., Fiala, A. and Sigrist, S. J. (2011). Presynapses in Kenyon cell dendrites in the mushroom body calyx of Drosophila. J Neurosci 31: 9696-9707. PubMed ID: 21715635

Craig, A. M. and Kang, Y. (2007). Neurexin-neuroligin signaling in synapse development. Curr Opin Neurobiol 17: 43-52. PubMed ID: 17275284

Fayyazuddin, A., Zaheer, M. A., Hiesinger, P. R. and Bellen, H. J. (2006). The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila. PLoS Biol 4: e63. PubMed ID: 16494528

Feng, K., Zhou, X. H., Oohashi, T., Morgelin, M., Lustig, A., Hirakawa, S., Ninomiya, Y., Engel, J., Rauch, U. and Fassler, R. (2002). All four members of the Ten-m/Odz family of transmembrane proteins form dimers. J Biol Chem 277: 26128-26135. PubMed ID: 12000766

Hong, W., Mosca, T. J. and Luo, L. (2012). Teneurins instruct synaptic partner matching in an olfactory map. Nature 484: 201-207. PubMed ID: 22425994

Kenzelmann, D., Chiquet-Ehrismann, R., Leachman, N. T. and Tucker, R. P. (2008). Teneurin-1 is expressed in interconnected regions of the developing brain and is processed in vivo. BMC Dev Biol 8: 30. PubMed ID: 18366734

Kremer, M. C., Christiansen, F., Leiss, F., Paehler, M., Knapek, S., Andlauer, T. F., Forstner, F., Kloppenburg, P., Sigrist, S. J. and Tavosanis, G. (2010). Structural long-term changes at mushroom body input synapses. Curr Biol 20: 1938-1944. PubMed ID: 20951043

Leiss, F., Koper, E., Hein, I., Fouquet, W., Lindner, J., Sigrist, S. and Tavosanis, G. (2009a). Characterization of dendritic spines in the Drosophila central nervous system. Dev Neurobiol 69: 221-234. PubMed ID: 19160442

Leiss, F., Groh, C., Butcher, N. J., Meinertzhagen, I. A. and Tavosanis, G. (2009b). Synaptic organization in the adult Drosophila mushroom body calyx. J Comp Neurol 517: 808-824. PubMed ID: 19844895

Mosca, T. J., Hong, W., Dani, V. S., Favaloro, V. and Luo, L. (2012). Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice. Nature 484: 237-241. Pubmed: 22426000

Mosca, T. J. and Luo, L. (2014). Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins. Elife 3:e03726. PubMed ID: 25310239

Rakovitsky, N., et al. (2007). Drosophila Ten-a is a maternal pair-rule and patterning gene. Mech. Dev. 124(11-12): 911-24. PubMed citation: 17890064

Silva, J. P., Lelianova, V. G., Ermolyuk, Y. S., Vysokov, N., Hitchen, P. G., Berninghausen, O., Rahman, M. A., Zangrandi, A., Fidalgo, S., Tonevitsky, A. G., Dell, A., Volynski, K. E. and Ushkaryov, Y. A. (2011). Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities. Proc Natl Acad Sci U S A 108: 12113-12118. PubMed ID: 21724987

Wang, L., Rotzinger, S., Al Chawaf, A., Elias, C. F., Barsyte-Lovejoy, D., Qian, X., Wang, N. C., De Cristofaro, A., Belsham, D., Bittencourt, J. C., Vaccarino, F. and Lovejoy, D. A. (2005). Teneurin proteins possess a carboxy terminal sequence with neuromodulatory activity. Brain Res Mol Brain Res 133: 253-265. PubMed ID: 15710242

Zheng, L. et al, (2011). Drosophila Ten-m and filamin affect motor neuron growth cone guidance. PLoS One 6(8): e22956. PubMed Citation: 21857973


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date revised: 25 September 2023

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