|Home | About | Journals | Submit | Contact Us | Français|
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 post-synaptic partners are matched. To ensure the proper relay of olfactory information in flies, axons of ~50 classes of olfactory receptor neurons (ORNs) form one-to-one connections with dendrites of ~50 classes of projection neurons (PNs). Using genetic screens, we identified two evolutionarily conserved EGF-repeat transmembrane Teneurins, Ten-m and Ten-a, 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. We propose that Teneurins instruct matching specificity between synaptic partners through homophilic attraction.
Sperry proposed the chemoaffinity hypothesis nearly 50 years ago to explain the exquisite target specificity of regenerating optic nerves: developing neurons “must carry individual identification tags, presumably cytochemical in nature, by which they are distinguished one from another almost, in many regions, to the level of the single neuron.”1 Many molecules are now known that guide axons to their target areas2,3, but few may mediate mutual selection and direct matching between individual pre- and post-synaptic partners. Here we show that the transmembrane Teneurins instruct the selection of specific synaptic partners in the Drosophila olfactory circuit (Fig. S1).
In Drosophila, individual classes of olfactory receptor neuron (ORN) axons make one-to-one connections with individual classes of second-order projection neuron (PN) dendrites within one of ~50 discrete glomeruli in the antennal lobe. We refer to this specific one-to-one connection as PN-ORN synaptic partner matching. Olfactory circuit assembly takes place in sequential steps before sensory activity begins4-6. PN dendrites first elaborate within and pattern the developing antennal lobe7-9, followed by ORN axons invasion10-14. Importantly, re-positioning PN dendrites redirects their partner ORN axons without disrupting the connections15, suggesting that proper PN-ORN connections likely involve direct recognition and matching between partners.
To identify potential PN-ORN matching molecules, we simultaneously labeled select PN dendrites and ORN axons in two colors and performed two complementary genetic screens (Fig. 1a,d). We overexpressed 410 candidate cell-surface molecules, comprising ~40% of the potential cell-recognition molecules in Drosophila16. In the first screen, we used Mz19-GAL4 to label DA1, VA1d and DC3 PNs (hereafter Mz19 PNs), and Or47b-rCD2 to label Or47b ORNs (Fig. 1a,b). Or47b ORN axons normally project to the VA1lm glomerulus and are adjacent to Mz19 PN dendrites without overlap. We overexpressed candidate cell-surface molecules only in Mz19 PNs to identify those that promoted ectopic connections between Or47b axons and Mz19 dendrites (Fig. 1a). We found that overexpression of ten-m (P[GS]9267, Fig. S2b) produced ectopic connections (Fig. 1c).
In the second screen, we labeled Mz19 PNs as above and Or88a ORNs using Or88a-rCD2 (Fig. 1d,e). Or88a ORN axons normally project to the VA1d glomerulus, intermingling extensively with VA1d PN dendrites (Fig. 1e). We overexpressed candidate cell-surface molecules in Mz19 PNs (Fig. 1d) as above and found that overexpression of ten-a (P[GE]1914, Fig. S2a) partially disrupted the intermingling of Or88a axons and Mz19 dendrites (Fig. 1f).
In addition to impairing PN-ORN matching, ten-m and ten-a overexpression shifted Mz19 PN dendrite position (Fig. 1c,f). 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 matching9,13,15. Furthermore, among 410 candidate molecules, only ten-m and ten-a overexpression exhibited mismatching defects, suggesting their specificity in PN-ORN matching.
Both ten-m and ten-a appear to encode type II transmembrane proteins17-19. 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 (Fig. 1g). Ten-m and Ten-a were discovered as Tenascin-like molecules20,21, but vertebrate Teneurins were later identified as their true homologs based on sequence and domain similarity (Fig. 1h). Thus, we refer to Ten-m and Ten-a 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 retardation17, and Teneurin-4 is linked to susceptibility to bipolar disorder22.
Drosophila ten-m was originally identified as a pair-rule gene required for embryonic patterning21,23, but was recently determined otherwise24. Teneurins were implicated in synapse development at the neuromuscular junction16,25 (see Ref. 26), and Ten-m also regulates motor axon guidance24. Neither the underlying mechanisms nor their potential roles in the central nervous system are known. Vertebrate Teneurins are widely expressed in the nervous system18,27 and interact homophilically in vitro28,29, suggesting their potential role as homophilic cell adhesion molecules in patterning neuronal connectivity.
Both Drosophila Teneurins were endogenously expressed in the developing antennal lobe (Fig. 2a, S3). 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 (Fig. 2a,e). 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 (Fig. 2b,c), suggesting that Teneurins are produced predominantly by neurons. In a ten-a null mutant we generated (Fig. S2a), all Ten-a expression was eliminated, confirming antibody specificity (Fig. 2d).
The antennal lobe consists of ORN axons as well as PN and local interneuron dendrites. We used intersectional analysis to determine the cellular source for elevated Teneurin expression. For ten-m, we screened GAL4 enhancer traps near the ten-m gene, and identified NP6658 (hereafter as ten-m-GAL4; Fig. S2b) that recapitulated the glomerulus-specific Ten-m staining pattern (Fig. S4a-c). We used a FLPout reporter UAS>stop>mCD8GFP to determine the intersection of ten-m-GAL4 and an ORN-specific ey-Flp (Fig. 2f, S4d-f) or a PN-specific GH146-Flp (Fig. 2g, S4g-i). We found that ten-m-GAL4 was selectively expressed in a subset of ORNs and PNs. Due to reagent availability, we focused our analysis 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 (Fig. 2f-g).
To determine the cellular origin of elevated Ten-a expression, we performed tissue-specific RNAi of endogenous Ten-a, as no GAL4 enhancer trap is available near ten-a. To isolate Ten-a expression in ORNs, we drove pan-neuronal ten-a RNAi while specifically suppressing RNAi in ORNs using tubP>stop>GAL80 and ey-Flp (Fig. 2h). To restrict Ten-a expression to central neurons, we expressed ten-a RNAi in all ORNs (Fig. 2i). We 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 here (Fig. 2h-i). 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 (Fig. S5). In summary, Ten-m and Ten-a are each highly expressed in a distinct, but partially overlapping, subset of matching ORNs and PNs (Fig. 2j).
To examine whether Teneurins are required for proper PN-ORN matching, we performed tissue-specific RNAi (Fig. 3, S2c) 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, we used the Q binary expression system30, and converted Mz19-GAL4 to Mz19-QF by BAC recombineering (Fig. S2d). We could thus perform GAL4-based RNAi knockdown while labeling PN dendrites and ORN axons in two colors independent of GAL4. We focused our analysis on Mz19 dendrites and Or47b axons, which innervate neighboring glomeruli but never intermingle in wild type (Fig. 1b, 3a-b).
Pan-neuronal RNAi of both teneurins shifted Or47b axons to a position between two adjacent Mz19 glomeruli, DA1 and VA1d (Fig. 3c). Moreover, Mz19 dendrites and Or47b axons intermingled without a clear border (Fig. 3c, d), reflecting a PN-ORN matching defect. We confirmed this using independent RNAi lines targeting different regions of the ten-m and ten-a transcripts (Fig. S6). Further, knocking down teneurins only in PNs or ORNs also led to Mz19-Or47b intermingling (Fig. 3e, S7a,d), indicating that Teneurins are required in both PNs and ORNs to ensure proper matching.
Next, we examined the contribution of each Teneurin by individual RNAi knockdown in ORNs. Knocking down ten-m, and to a lesser extent, ten-a, caused mild mismatching (Fig. 3e, S7). This was greatly enhanced by simultaneous teneurin knockdown (Fig. 3e), likely because Mz19-Or47b mismatching requires weakening connections with their respective endogenous partners (Fig. S7g). This synergy implies that multiple matching molecules can enhance partner matching robustness.
We also tested the functions of individual Teneurins in PNs. We found that the Mz19-Or47b mismatching was caused by PN-specific knockdown of ten-a, but not ten-m (Fig. 3e, S7). As VA1d/DC3 and DA1 PNs arise from separate neuroblast lineages31, we generated MARCM neuroblast clones to label and knock down ten-a in DA1 or VA1d/DC3 PNs (Fig. 3f-j, see Methods). ten-a knockdown only in DA1 PNs (normally Ten-a high) caused dendrite mismatching with Or47b axons (Fig. 3h-j). By contrast, ten-a knockdown in VA1d/DC3 PNs (normally Ten-a low) did not cause mismatching (Fig. 3j, S8a,b). Similarly, MARCM loss-of-function of ten-a mutant in DA1 but not in VA1d/DC3 PNs resulted in mismatching with Or47b ORNs (Fig. 3j, S8c-d). Thus, removal of ten-a from Ten-a-high DA1 PNs caused dendrite mismatching with Ten-a-low Or47b ORNs (Fig. 3i). 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.
Our finding that loss of ten-a caused Ten-a-high PNs to mismatch with Ten-a-low ORNs (Fig. 3i,j), 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.
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.
To test the first prediction, we examined whether Teneurin overexpression in Mz19 PNs impaired their endogenous connections with cognate ORNs. Consistently, Ten-m overexpression specifically disrupted the connections of DA1 PNs and Or67d ORNs, a PN-ORN pair expressing low-level Ten-m (Fig. S9b,e). Connections of the other two pairs were unaffected (Fig. S9a,c,d,f). Likewise, Ten-a overexpression specifically disrupted connections between VA1d PNs and Or88a ORNs, a PN-ORN pair expressing low-level Ten-a (Fig. S9g), but not between the other two PN-ORN pairs (Fig. S9h-i).
To test the second prediction, we examined the specificity of ectopic connections made by Mz19 PNs overexpressing Teneurins, and sampled with non-partner ORN classes that project axons nearby Mz19 dendrites (Fig. S10). We found that Ten-m overexpression in Mz19 PNs caused dendrite mismatching only with Or47b ORNs (Fig. S10f). 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, we used MARCM to overexpress Ten-m in individual PN classes. We found that Ten-m overexpression in DA1 PNs (Ten-m low) caused dendrite mismatching with Or47b (Fig. 4a-b) and (to a lesser extent) Or88a ORNs (Fig. 4b-c), 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 (Fig. 4d-f).
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 (Fig. S10l). Further, MARCM overexpression of Ten-a in VA1d/DC3 PNs (Ten-a low) caused dendrite mismatching specifically with Or23a (Fig. 4j-k) and (to a lesser extent) Or67d ORNs (Fig. 4k-l), both endogenously expressing high-level Ten-a (Fig. 4l). By contrast, Ten-a overexpression in DA1 PNs (Ten-a high) did not produce ectopic connections with any non-matching ORNs tested (Fig. 4g-i). Thus, both Ten-m and Ten-a overexpression analyses support the homophilic attraction hypothesis.
Our 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 (Fig. 2j), and may require additional molecules to distinguish target choice. Indeed, Ten-a knockdown (Fig. 3h-j, S8e-f) or Ten-m overexpression (Fig. 4b,c) 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 (Fig. 4k,l). Finally, Ten-m overexpression in DC3 PNs, which express low-level Ten-m, did not change its matching specificity (Fig. 4f, S9f), suggesting that Teneurin-independent mechanisms are involved in matching DC3 PNs and Or83c ORNs.
In summary, we showed that Teneurin overexpression in Teneurin-low PNs caused their dendrites to lose endogenous connections with Teneurin-low ORNs and mismatch with Teneurin-high ORNs (Fig. 4b,k). However, Teneurin overexpression in Teneurin-high PNs did not disrupt their proper connections (Fig. 4e,h). 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.
To test whether Teneurins interact in vitro, we separately transfected two populations of Drosophila S2 cells with FLAG- and HA-tagged Teneurins, and performed co-immunoprecipitations from lysates of these cells after mixing. We detected strong homophilic interactions between FLAG- and HA-tagged Ten-m proteins, and to a lesser extent between FLAG- and HA-tagged Ten-a proteins (Fig. 5a). Ten-m and Ten-a alsoexhibited heterophilic interactions (Fig. 5a), which may account for their role in synapse organization26.
Next, we tested if Teneurins can homophilically promote in vivo trans-cellular interactions between PN dendrites and ORN axons. We simultaneously overexpressed Ten-m in Mz19 PNs using Mz19-QF, and Or67a and Or49a ORNs using AM29-GAL432 (Fig. 5b). This enabled us to independently label and manipulate Mz19 dendrites and AM29 axons with distinct markers and transgenes. We chose AM29-GAL4 because of its early onset of expression, whereas other class-specific ORN drivers start to express only after PN-ORN connection is established5,6. AM29 axons do not normally connect with Mz19 dendrites (Fig. 5c-d).
Simultaneous overexpression of Ten-m in both Mz19 PNs and AM29 ORNs produced ectopic connections between them (Fig. 5c,g), 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 (Fig. 5c,e,f). 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 (not shown), possibly due to lower expression or weaker Ten-a homophilic interactions (Fig. 5a).
Finally, we examined whether these ectopic connections lead to the formation of synaptic structures. Indeed, the ectopic connections between Mz19 dendrites and AM29 axons were enriched in synaptotagmin-HA expressed from AM29 ORNs (Fig. 5h), suggesting that these connections can aggregate synaptic vesicles and could be functional. We propose that Teneurins promote attraction between PN-ORN synaptic partners through homophilic interactions, eventually leading to synaptic connections.
Compared to axon guidance, relatively little is known about synaptic target selection mechanisms2-4. Among the notable examples, the graded expressions of vertebrate EphA and Ephrin-A instruct the topographic targeting of retinal ganglion cell axons4,33-35. Chick DSCAMs and Sidekicks promote lamina-specific arborization of retinal neurons36. Drosophila Capricious promotes target specificity of photoreceptor and motor axons16,37-39. C. elegans SYG-1 and SYG-2 specify synapse location through interaction between pre-synaptic axons and intermediate guidepost cells40. 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 activity41,42. Mammalian ORN axon targeting involves extensive axon-axon interactions through activity-dependent and independent modes43,44, with minimal participation of postsynaptic neurons identified thus far.
Here, we show 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. We further demonstrate that molecular determinants can instruct connection specificity of a moderately complex circuit at the level of individual synapses.
Our 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 interactions8,9,12-14. These initial, independent dendrite and axon targeting are eventually coordinated in their final one-to-one matching. We identified Teneurins as the first molecules to medicate this matching process, through direct PN-ORN attraction. Our analyses have focused on a subset of ORN-PN pairs involving trichoid ORNs45, including Or67d/Or88a/Or47b that are implicated in pheromone sensation46. The partially overlapping expressions of Teneurins in other PN and ORN classes (Fig. 2, S4) 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.
Teneurins are present throughout Animalia (Fig. 1h). Different vertebrate Teneurins are broadly expressed in distinct and partially overlapping patterns in the nervous system18. Teneurin-3 is expressed in the visual system and is required for ipsilateral retinogeniculate projections47. Our 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 pairs26. Furthermore, Teneurins also trans-synaptically mediate neuromuscular synapse organization26. 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 (this study and ref. 26), whereas synapse organization preferentially involves heterophilic interactions26. This could not be fully accounted for by different strength of their homophilic and heterophilic interactions in vitro (Fig. 5a). Indeed, while heterophilic interactions occur in vitro (Fig. 5a), heterophilic overexpression of Ten-m and Ten-a in AM29 ORNs and Mz19 PNs did not produce ectopic connections (not shown). Thus we speculate that these dual functions of Teneurins in vivo may engage signaling mechanisms that further distinguish homophilic versus heterophilic interactions.
Detailed methods on fly stocks, generation of the ten-a allele, construction of transgenic flies, clonal analysis, histology, imaging, quantification and statistical analysis, epitope-tagged constructs, and co-immunoprecipitation can be found in Methods.
We thank V. Favaloro for advice on biochemistry and D. Luginbuhl for technical assistance; K. Zinn for the EP collection; R. Wides and S. Baumgartner for teneurin reagents; B. Zhang, Bloomington, Kyoto, and Harvard and Vienna Stock Centers for fly stocks; BestGene Inc. for injection service; and K. Shen, T. Clandinin, D. Berns, V. Favaloro, X. Gao, S. Hippenmeyer, C. Liu, K. Miyamichi, and X. Yu for critiques. Supported by an NIH grant (R01 DC-005982 to L.L.), and Epilepsy, Neonatology and Developmental Biology Training Grants (NIH 5T32 NS007280 and HD007249 to T.J.M.). L.L. is an investigator of the Howard Hughes Medical Institute.
Author Contributions W.H. designed and performed all experiments. T.J.M. assisted in some experiments. L.L. supervised the project. W.H. and L.L. wrote the manuscript with feedback from T.J.M.
Author Information The authors declare no competing financial interests.