The Olfactomedin domain of LPHN3 is required for development of normal synaptic output density. (A) Example images of L2/3 axons in L5 from P14 mice electroporated with Syp-GFP shLPHN3 plasmids and wild-type (WT) or mutant LPHN3 plasmids. Scale bar = 5 μm. (B) Deleting domains of LPHN3 differentially affects the capability of the protein to rescue axonal synapse density (One-way ANOVA, F(4,299) = 10.07, P <0.0001: WT Rescue, 1.00 ± 0.06, n = 63; shLPHN3, 0.72 ± 0.06, n = 55; ΔLec 0.95 ± 0.05, n = 66; ΔOlf 0.76 ± 0.04, n = 63; ΔHRD 1.13 ± 0.05, n = 57). Post-hoc Dunnett tests show that the shLPHN3 and ΔOlf conditions differ from the WT Rescue (P <0.01). (C) Schematic of the domain organization of LPHN3 and summary of structure-function results. Conserved protein-protein interaction domains are labeled in the schematic, with the functions they subserve indicated by the bars above them. The extracellular domain of LPHN3 is composed of two globular regions separated by a flexible linker. The distal globular domain serves as a ligand-binding module, and FLRTs and TENs interact with different portions therein.