Repulsions instruct synaptic partner matching in an olfactory circuit

Article Repulsions instruct synaptic partner matching in an olfactory circuit https://doi.org/10.1038/s41586-025-09768-4 Received: 24 February 2025 Zhuoran Li1,3, Cheng Lyu1,3, Chuanyun Xu1, Ying Hu1, David J. Luginbuhl1, Asaf B. Caspi-Lebovic2, Jessica M. Priest2, Engin Özkan2 & Liqun Luo1 ✉ Accepted: 16 October 2025 Published online: xx xx xxxx Open access Check for updates Neurons exhibit extraordinary precision in selecting synaptic partners. Although cell-surface proteins (CSPs) that mediate attractive interactions between developing axons and dendrites have been shown to instruct synaptic partner matching1,2, the degree to which repulsive interactions have a role is less clear. Here, using a genetic screen guided by single-cell transcriptomes3,4, we identified three CSP pairs, Toll2– Ptp10D, Fili–Kek1 and Hbs/Sns–Kirre, that mediate repulsive interactions between non-partner olfactory receptor neuron (ORN) axons and projection neuron (PN) dendrites in the developing Drosophila olfactory circuit. Each CSP pair exhibits inverse expression patterns in the select ORN–PN partners. Loss of each CSP in ORNs led to similar synaptic partner matching deficits as the loss of its partner CSP in PNs, and mistargeting phenotypes caused by overexpressing one CSP could be suppressed by loss of its partner CSP. All CSP pairs are also differentially expressed in other brain regions. Together, our data reveal that multiple repulsive CSP pairs work together to ensure precise synaptic partner matching during development by preventing neurons from forming connections with non-cognate partners. A fundamental question in neural development is how the vast number of neurons precisely select their synaptic partners to form functional circuits. Neural circuit wiring involves multiple coordinated developmental steps: axon guidance to target regions, dendrite patterning and synaptic partner matching followed by synaptogenesis5–7. Even though axon guidance and dendrite patterning can greatly reduce the number of potential partners a neuron encounters at a given time and region8, a developing axon must select specific partners among multiple nearby non-partners1,2. The mechanisms by which neural systems reduce multiple candidate synaptic partners to a specific one remain poorly understood. It is well established that axon guidance involves both attraction towards the target region and repulsion away from non-target regions9,10. Repulsion mediated by CSPs is also used in establishing topographic maps11, subregion target selection12, and dendritic and axonal self-avoidance2. However, most known CSPs that instruct the final steps of synaptic partner selection act through attraction. These include homophilic attraction of teneurins (Ten-m and Ten-a) in Drosophila olfactory and neuromuscular systems13,14, heterophilic attractions among members of the immunoglobulin superfamily of CSPs in multiple Drosophila circuits15–22, and homophilic attraction mediated by immunoglobulin23 or cadherin24,25 families of CSPs in the vertebrate retina. The few examples of repulsion include Drosophila motor axon target selection, controlled by Wnt4 from non-target muscles26, and olfactory neuron target selection by Fish-lips (Fili) from non-cognate partners27. How general repulsion is utilized as a guiding force in synaptic partner matching remains to be examined. In the Drosophila olfactory circuit, axons of about 50 types of ORNs form one-to-one precise synaptic connections with dendrites of 50 types of PNs in 50 glomeruli in the antennal lobe28. During development, PN dendrites coarsely pattern the antennal lobe first29,30. While extending across the antennal lobe prepatterned by PN dendrites, each ORN axon sends multiple transient branches along its trajectory. ORN axon branches that contact partner PN dendrites are stabilized and branch further, whereas the rest retract31,32. Since synaptic partner matching involves retraction of transient ORN axon branches in contact with non-partner PNs, we aimed to identify repulsive CSPs that might function to prevent the formation of misconnections between non-partner PNs and ORNs. Inverse expression of three CSP pairs VA1d and VA1v are neighbouring glomeruli that sense distinct pheromones33,34. Known homophilic attraction molecules that mediate matching between synaptic partners, Ten-m and Ten-a, cannot distinguish VA1d-PNs and VA1d-ORNs from VA1v-PNs and VA1v-ORNs, as they all express Ten-m at high levels and Ten-a at low levels13. We hypothesized that additional CSPs are differentially expressed and instruct synaptic partner matching in these adjacent glomeruli. To identify such CSPs, we performed a genetic screen focusing on PN–ORN matching in the VA1d and VA1v glomeruli (Fig. 1a). We first analysed the existing single-cell transcriptome data for developing PNs and ORNs3,4 at 24–30 h after puparium formation (APF), shortly before matching between ORN axons and PN dendrites occurs. We focused on CSPs (including both transmembrane and secreted proteins35) that are differentially expressed in VA1d-PNs and VA1v-PNs or in VA1d-ORNs and VA1v-ORNs. We identified 36 candidate genes with assistance from existing literature, including the list of top 100 CSPs enriched in 1 Department of Biology and Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA. 2Department of Biochemistry and Molecular Biology, The Neuroscience Institute and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA. 3These authors contributed equally: Zhuoran Li, Cheng Lyu. ✉e-mail: Nature | www.nature.com | 1 Article a e PN dendrites Antennal lobes VA1d VA1v 0.6 Preference index Adult Drosophila brain 9.1 × 10–3 0.011 –3 0.034 1.9 × 10 h ORN axons PN dendrites 0.4 0.2 VA1d 0 –0.2 –0.4 VA1v –0.6 ORN axons To ll2 Lateral Toll2 Ptp10D in O R To ll2 Ns Pt in p1 PN 0D s i Pt n O p1 R 0D Ns in PN s Dorsal HA Stop Myc FRT VA1v –0.4 0.6 k1 k1 in in O RN s Fili Kek1 Ke 3.9 × 10–4 1.5 × 10–4 5.8 × 10–4 0.22 0.4 j ORN axons PN dendrites VA1d 0.2 0 –0.2 VA1v –0.4 s rre in PN RN Hbs Sns Kirre Ki s Ki Sn bs H s –0.6 in * PN s RN s O Fi li in g O * –0.2 s VA1v MYC PN dendrites 0 PN VA1d Merge Toll2 in ORNs Ventral ORN axons –0.6 Preference index Ptp10D in PNs MYC Lateral d Toll2 in PNs Merge Ptp10D in ORNs c i VA1d 0.2 s Exons Exons 0.4 PN ORN-FLP or PN-FLP Myc FRT 0.99 1.1 × 10–4 0.26 0.6 in FRT f in Exons Exons Stop rre Genomic locus Stop Ke Endogenous conditional tagging Preference index b Fig. 1 | Inverse expression of three CSP pairs in the VA1d and VA1v glomeruli. a, Schematics of adult Drosophila brain and the antennal lobe. Axons of VA1dORNs and VA1v-ORNs (green) match with dendrites of VA1d-PNs and VA1v-PNs (magenta), respectively. b, Schematic of conditional tagging of CSPs to reveal their endogenous protein expression pattern (top) before—and in specific (...truncated)