Tenascin major


Transcriptional Regulation

Ten-m protein is under the control of fushi tarazu and even-skipped, but not of opa (Baumgartner, 1994).

Hormone-dependent control of developmental timing through regulation of chromatin accessibility

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. This study shows that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, it was demonstrated that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. A temporal cascade of transcription factors was uncovered following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, the ecdysone-induced transcription factor E93 was shown to control temporal identity by directly regulating chromatin accessibility across the genome. Notably, it was found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility (Uyehara, 2017).

The importance of master transcription factors in specifying spatial identity during development suggests that they may control where other transcription factors bind in the genome. One prediction of this model is that tissues whose identities are determined by different master transcription factors would exhibit different genome-wide DNA-binding profiles. However, it was recently found that the Drosophila appendages (wings, legs, and halteres), which use different transcription factors to determine their identities, share nearly identical open chromatin profiles. Moreover, these shared open chromatin profiles change coordinately over developmental time. There are two possible explanations for these findings. Either (1) different transcription factors produce the same open chromatin profiles in different appendages or (2) transcription factors shared by each appendage control open chromatin profiles instead of the master transcription factors of appendage identity. The second model is favored for several reasons. Since the appendage master transcription factors possess different DNA-binding domains with distinct DNA-binding specificities, it is unlikely for them to bind the same sites in the genome. Supporting this expectation, ChIP for Scalloped and Homothorax, two transcription factors important for appendage identity, shows clear tissue-specific binding in both the wing and eye–antennal imaginal discs. The second model is also preferred because it provides a relatively straightforward mechanism for the observed temporal changes in open chromatin: By changing the expression of the shared temporal transcription factor over time, the open chromatin profiles that it controls would change as well. In contrast, expression of appendage master transcription factors is relatively stable over time, making it unlikely for them to be sufficient for temporal changes in open chromatin (Uyehara, 2017).

It is proposed that control of chromatin accessibility in the appendages is mediated at least in part by transcription factors downstream from ecdysone signaling. According to this model, a systemic pulse of ecdysone initiates a temporal cascade of hormone-induced transcription factor expression in each of the appendages. These are referred to as 'temporal' transcription factors. Temporal transcription factors can directly regulate the accessibility of transcriptional enhancers by opening or closing them, thereby conferring temporal specificity to their activity and driving development forward in time. Master transcription factors then bind accessible enhancers depending on their DNA-binding preferences (or other means of binding DNA) and differentially regulate the activity of these enhancers to control spatial patterns of gene expression, thus shaping the unique identities of individual appendages (Uyehara, 2017).

The experiments with E93 provide direct support for this model. In wild-type wings, thousands of changes in open chromatin occur after the large pulse of ecdysone that triggers the end of larval development. In E93 mutants, ~40% of these open chromatin changes fail to occur. Importantly, nearly three-quarters of sites that depend on E93 for accessibility correspond to temporally dynamic sites in wild-type wings. Thus, chromatin accessibility is not grossly defective across the genome; instead, defects occur specifically in sites that change in accessibility over time. This finding, combined with the large fraction of temporally dynamic sites that depend on E93 for accessibility, indicates that E93 controls a genome-wide shift in the availability of temporal-specific transcriptional enhancers. Supporting this hypothesis, temporal-specific enhancers depend on E93 for both accessibility and activity. Since it is proposed that the response to ecdysone is shared across the appendages, it is predicted that similar defects occur in appendages besides the wing. It remains to be seen whether other ecdysone-induced transcription factors besides E93 control accessibility of enhancers at different developmental times. It also remains to be seen how the temporal transcription factors work with the appendage master transcription factors to control appendage-specific enhancer activity (Uyehara, 2017).

The findings suggest that E93 controls temporal-specific gene expression through three different modalities that potentially rely on three distinct biochemical activities. The enrichment of E93 motifs and binding of E93 to temporally dynamic sites indicate that it contributes to this regulation directly. It is proposed that these combined activities drive development forward in time by turning off early-acting enhancers and simultaneously turning on late-acting enhancers (Uyehara, 2017).

First, as in the case of the tenectin tncblade enhancer, active most strongly in the interveins between the first and second and between the fourth and fifth longitudinal veins and in cells near the proximal posterior margin, E93 appears to function as a conventional activator. In the absence of E93, tncblade fails to express at high levels, but the accessibility of the enhancer does not measurably change. This suggests that binding of E93 to tncblade is required to recruit an essential coactivator. Importantly, this finding demonstrates that E93 is not solely a regulator of chromatin accessibility. E93 binds many open chromatin sites in the genome without regulating their accessibility and thus may regulate the temporal-specific activity of many other enhancers. In addition, since the tncblade enhancer opens between L3 and 24 h even in the absence of E93, there must be other factors that control its accessibility, perhaps, for example, transcription factors induced by ecdysone earlier in the temporal cascade (Uyehara, 2017).

Second, as in the case of the nubvein enhancer, E93 is required to promote chromatin accessibility. In this capacity, E93 may function as a pioneer transcription factor to open previously inaccessible chromatin. Alternatively, E93 may combine with other transcription factors, such as the wing master transcription factors, to compete nucleosomes off DNA. Testing the ability of E93 to bind nucleosomal DNA will help to discriminate between these two alternatives. In either case, it is proposed that this function of E93 is necessary to activate late-acting enhancers across the genome. Since only half of E93-dependent enhancers are directly bound by E93 at 24 h, it is also possible that E93 regulates the expression of other transcription factors that control chromatin accessibility. Alternatively, if E93 uses a “hit and run” mechanism to open these enhancers, the ChIP time point may have been too late to capture E93 binding at these sites (Uyehara, 2017).

Finally, as in the case of the broad brdisc enhancer, E93 is required to decrease chromatin accessibility. It is proposed that this function of E93 is necessary to inactivate early-acting enhancers across the genome. Current models of gene regulation do not adequately explain how sites of open chromatin are rendered inaccessible, but the ability to turn off early-acting enhancers is clearly an important requirement in developmental gene regulation. It may also be an important contributor to diseases such as cancer, which exhibits widespread changes in chromatin accessibility relative to matched normal cells. Thus, this role of E93 may represent a new functional class of transcription factor (“reverse pioneer”) or conventional transcriptional repressor activity. Additional work is required to decipher the underlying mechanisms. Notably, recent work on the temporal dynamics of iPS cell reprogramming suggest a similar role for Oct4, Sox2, and Klf4 in closing open chromatin to inactivate somatic enhancers (Chronis, 2017; Uyehara, 2017 and references therein).

Targets of Activity

At least two pair-rule genes, paired and sloppy paired, and all segment-polarity genes analysed to date are under the control of Ten-m. Ten-m initiates a signal transduction cascade which acts either in concert with or via odd-paired, on downstream targets such as prd, slp and gooseberry, and through them, engrailed and wingless, leading to an opa-like phenotype (Baumgartner, 1994).

Protein Interactions

Ten-m is phosphorylated on tyrosine, resulting in an altered electrophoretic mobility. There are five putative tyrosine phosphorylation sites (Levine, 1994).

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

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).



The Ten-m protein is found in seven stripes during the blastoderm stage, each stripe overlapping the Even-skipped stripes (Baumgartner, 1994). Additional protein is seen in the lymph gland, cardiac cells, posterior spiracles and trachea. Expression is seen in the ventral nerve cord (CNS), the supraesophageal ganglia, and in hemocytes (Baumgartner, 1994).

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).

Effects of Mutation or Deletion

Ten-m mutants show a phenotype resembling that of odd-paired, a member of the pair-rule class of segmentation genes (Baumgartner, 1994).


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Tenascin major: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 23 August 2017

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