Teneurins instruct synaptic partner matching in an olfactory map

Abstract

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 ∼50 classes of olfactory receptor neurons (ORNs) form one-to-one connections with dendrites of ∼50 classes of projection neurons (PNs). Here, using genetic screens, we identified two evolutionarily conserved, epidermal growth factor (EGF)-repeat containing transmembrane Teneurin proteins, 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.

Figure 1. PN-ORN synaptic matching screens identify two Teneurins

a, d, Schematics showing two PN-ORN matching screens. PN dendrites are labeled by Mz19-GAL4 driving mCD8GFP and ORN axons by Or47b-rCD2 (a) or Or88a-rCD2 (d). Candidate cell-surface molecules are overexpressed only in Mz19 PNs. b-c, Or47b axons and Mz19 dendrites do not overlap in control (b), but form ectopic connections following Ten-m overexpression (c), as seen by axon-dendrite intermingling (arrowhead). e-f, Or88a axons and Mz19 dendrites connect at the VA1d glomerulus in control (e), but the connection is partially lost following Ten-a overexpression, as part of Or88a axons no longer intermingle with Mz19 dendrites (arrowhead). Target areas of Or47b (b-c) or Or88a (e-f) axons are outlined. Mismatching phenotypes are quantified in Fig. S9k and S10q. The first three columns in b,c,e,f show separate channels of the same section; the fourth shows higher magnification of the dashed squares (as in Fig. 3, 4, 5d-g.). Unless indicated, all images in this and subsequent figures are single confocal sections and all scale bars are 10 μm. g, Domain composition of Drosophila Ten-m and Ten-a. h, Phylogeny of the Drosophila Teneurins and related proteins in other species. Branch lengths represent units of substitutions per site of the sequence alignment. Teneurins are evolutionarily conserved in bilaterians and a unicellular choanoflagellate Monosiga brevicollis, but not in cnidarians.

Figure 2. Teneurins are differentially expressed in matching PN and ORN classes

a, Developing antennal lobes at 48 hrs APF stained by antibodies against Ten-m, Ten-a, and a neuropil marker, N-cadherin. Solid lines encircle the DA1 glomerulus (Ten-m low, Ten-a high). Dashed lines encircle the VA1d/VA1lm glomeruli (Ten-m high, Ten-a low). b-c, Ten-m and Ten-a proteins are undetectable following pan-neuronal RNAi of ten-m (b) and ten-a (c), respectively. d, A ten-a homozygous mutant eliminated the Ten-a antibody staining. e, Summary of elevated Ten-m and Ten-a expression in five select glomeruli. f-g, Expression of the Flp-out GFP reporter UAS>stop>mCD8GFP at the intersection of ten-m-GAL4 with ORN-specific ey-Flp (f) or with PN-specific GH146-Flp (g) in adult. h-i, Antibody staining of Ten-a in central neuron-specific RNAi (h) or in ORN-specific RNAi (i) at 48 hrs APF. Individual cell type-specific Teneurin expression patterns are schematically summarized at right (f-i). j, Combined expression patterns of Teneurins in PNs (left) and ORNs (right). Blue: Ten-m high; orange: Ten-a high.

Figure 3. Loss of Teneurins causes PN-ORN mismatching

a, Normally, Mz19 dendrites (green) innervate glomeruli adjacent to the VA1lm glomerulus, which is itself innervated by Or47b axons (red). The dashed line encircles Or47b axons. DC3 PNs are located posterior to DA1/VA1d PNs and Or47b ORNs, and are not visible in these sections. c, Mismatching phenotypes in ten-m and ten-a RNAi driven by the pan-neuronal driver C155-GAL4. Dashed lines encircle Or47b ORN axons, showing intermingling with Mz19 PN dendrites (arrowhead). e, Quantification of Mz19-Or47b mismatching phenotypes. For all genotypes, n≥15. f, In control, DA1 PNs do not intermingle with Or47b ORNs. h, MARCM expression of ten-a RNAi in DA1 PNs causes dendrite intermingling with Or47b axons (arrowhead). j, Quantification of mismatching phenotypes. For all genotypes, n≥6. Error bars represent S.E.M. ***, p<0.001 compared to control. b, d, g, i, Summary showing normal connectivity in control (a, f) and mismatching phenotypes following teneurin RNAi (c, h). Blue: Ten-m high; orange: Ten-a high. Green outlines: labeled PNs. Red outlines: labeled ORNs.

Figure 4. Teneurin overexpression in specific PN classes causes mismatching

Mismatching phenotypes following Ten-m (a-f) or Ten-a (g-l) overexpression in different PN classes. Specific PN classes are labeled by MARCM with Mz19-GAL4 and ORN axons using Or47b-rCD2 (a, d) or Or23a-mCD8GFP (g, j). In control, Mz19 PNs do not intermingle with Or47b ORNs (Fig. 1b). MARCM overexpression of Ten-m in DA1 PNs (a, arrowhead), but not VA1d/DC3 PNs (d), causes dendrite mismatching with Or47b axons. MARCM overexpression of Ten-a in VA1d/DC3 PNs (j, arrowhead), but not in DA1 PNs (g), causes their dendrites to mismatch with Or23a axons. (c, f, i, l) Quantification of mismatching phenotypes (n=9 for each). Error bars represent S.E.M. See Fig. S10 for details on some genotypes quantified here. (b, e, h, k) Schematic summarizing the mismatching phenotypes in Fig. 4, Fig. S9, and Fig. S10. Blue: Ten-m high; orange: Ten-a high.

Figure 5. Ten-m promotes homophilic interactions in vitro and in vivo

a, Co-immunoprecipitation of FLAG- and HA-tagged Teneurins from separately transfected S2 cells. Co-immunoprecipitated HA-tagged Teneurins are detected by anti-HA antibody and immunoprecipitated FLAG-tagged Teneurins by anti-FLAG antibody (upper two blots). The input lysates are immunoblotted for HA and FLAG (lower two blots). b, Schematic showing the relative positions of glomeruli targeted by Mz19 PN dendrites (green) and AM29 ORN axons (red). c, Quantification of mismatching between Mz19 PNs and AM29 ORNs (n=10 for each condition). d, In control, Mz19 dendrites do not connect with AM29 axons. e-f, Overexpression of Ten-m only in AM29 ORNs (e) or Mz19 PNs (f) does not produce mismatching between them. Following Ten-m overexpression, AM29 axons mistarget posteriorly to Mz19 dendrites and are therefore not visible in e. g, Simultaneous overexpression of Ten-m in both PNs and ORNs produces ectopic Mz19-AM29 connections (arrowhead). Schematics on the right show the Mz19-AM29 connectivity in different conditions. h, The synaptic vesicle marker Synaptotagmin is enriched at these Mz19-AM29 ectopic connections. AM29 axons are labeled by AM29-Gal4 with UAS-mtdT to visualize the entire axonal processes and UAS-synaptotagmin-HA to visualize synaptic vesicles in axon terminals. Mz19 dendrites are labeled by Mz19-QF driving QUAS-mCD8GFP.