title: Investigating CNS synaptogenesis at single-synapse resolution by combining reverse genetics with correlative light and electron microscopy.
abstract: Determining direct synaptic connections of specific neurons in the central nervous system (CNS) is a major technical challenge in neuroscience. As a corollary, molecular pathways controlling developmental synaptogenesis in vivo remain difficult to address. Here, we present genetic tools for efficient and versatile labeling of organelles, cytoskeletal components and proteins at single-neuron and single-synapse resolution in Drosophila mechanosensory (ms) neurons. We extended the imaging analysis to the ultrastructural level by developing a protocol for correlative light and 3D electron microscopy (3D CLEM). We show that in ms neurons, synaptic puncta revealed by genetically encoded markers serve as a reliable indicator of individual active zones. Block-face scanning electron microscopy analysis of ms axons revealed T-bar-shaped dense bodies and other characteristic ultrastructural features of CNS synapses. For a mechanistic analysis, we directly combined the single-neuron labeling approach with cell-specific gene disruption techniques. In proof-of-principle experiments we found evidence for a highly similar requirement for the scaffolding molecule Liprin-α and its interactors Lar and DSyd-1 (RhoGAP100F) in synaptic vesicle recruitment. This suggests that these important synapse regulators might serve a shared role at presynaptic sites within the CNS. In principle, our CLEM approach is broadly applicable to the developmental and ultrastructural analysis of any cell type that can be targeted with genetically encoded markers.
authors.affiliation: Neuronal Wiring Laboratory, Vesalius Research Center, VIB, Herestraat 49 box 912, Leuven 3000, Belgium Neuronal Wiring Laboratory, Vesalius Research Center, Department of Oncology, KU Leuven, Herestraat 49 box 912, Leuven 3000, Belgium olivier.urwyler@vib-kuleuven.be dietmar.schmucker@vib-kuleuven.be.
authors.forename: Olivier
authors.initials: O
authors.lastname: Urwyler
authors.affiliation: Neuronal Wiring Laboratory, Vesalius Research Center, VIB, Herestraat 49 box 912, Leuven 3000, Belgium Neuronal Wiring Laboratory, Vesalius Research Center, Department of Oncology, KU Leuven, Herestraat 49 box 912, Leuven 3000, Belgium.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Izadifar
authors.affiliation: Neuronal Wiring Laboratory, Vesalius Research Center, VIB, Herestraat 49 box 912, Leuven 3000, Belgium Neuronal Wiring Laboratory, Vesalius Research Center, Department of Oncology, KU Leuven, Herestraat 49 box 912, Leuven 3000, Belgium.
authors.forename: Dan
authors.initials: D
authors.lastname: Dascenco
authors.affiliation: Neuronal Wiring Laboratory, Vesalius Research Center, VIB, Herestraat 49 box 912, Leuven 3000, Belgium Neuronal Wiring Laboratory, Vesalius Research Center, Department of Oncology, KU Leuven, Herestraat 49 box 912, Leuven 3000, Belgium.
authors.forename: Milan
authors.initials: M
authors.lastname: Petrovic
authors.affiliation: Neuronal Wiring Laboratory, Vesalius Research Center, VIB, Herestraat 49 box 912, Leuven 3000, Belgium Neuronal Wiring Laboratory, Vesalius Research Center, Department of Oncology, KU Leuven, Herestraat 49 box 912, Leuven 3000, Belgium.
authors.forename: Haihuai
authors.initials: H
authors.lastname: He
authors.affiliation: Neuronal Wiring Laboratory, Vesalius Research Center, VIB, Herestraat 49 box 912, Leuven 3000, Belgium Neuronal Wiring Laboratory, Vesalius Research Center, Department of Oncology, KU Leuven, Herestraat 49 box 912, Leuven 3000, Belgium.
authors.forename: Derya
authors.initials: D
authors.lastname: Ayaz
authors.affiliation: VIB, Bio Imaging Core Gent, Technologiepark 927, Zwijnaarde 9052, Belgium Department of Biomedical Molecular Biology, University of Gent, Technologiepark 927, Zwijnaarde 9052, Belgium.
authors.forename: Anna
authors.initials: A
authors.lastname: Kremer
authors.affiliation: VIB, Bio Imaging Core Gent, Technologiepark 927, Zwijnaarde 9052, Belgium Department of Biomedical Molecular Biology, University of Gent, Technologiepark 927, Zwijnaarde 9052, Belgium.
authors.forename: Saskia
authors.initials: S
authors.lastname: Lippens
authors.affiliation: VIB, Center for the Biology of Disease, Herestraat 49 box 602, Leuven 3000, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Baatsen
authors.affiliation: VIB, Bio Imaging Core Gent, Technologiepark 927, Zwijnaarde 9052, Belgium Department of Biomedical Molecular Biology, University of Gent, Technologiepark 927, Zwijnaarde 9052, Belgium VIB, Inflammation Research Center Microscopy and Cytometry Core, Technologiepark 927, Zwijnaarde 9052, Belgium.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Guérin
authors.affiliation: Neuronal Wiring Laboratory, Vesalius Research Center, VIB, Herestraat 49 box 912, Leuven 3000, Belgium Neuronal Wiring Laboratory, Vesalius Research Center, Department of Oncology, KU Leuven, Herestraat 49 box 912, Leuven 3000, Belgium olivier.urwyler@vib-kuleuven.be dietmar.schmucker@vib-kuleuven.be.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Schmucker
mesh_terms: D000818:Animals; D002490:Central Nervous System / Q000254:growth & development*; D004330:Drosophila; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007150:Immunohistochemistry; D008465:Mechanoreceptors / Q000166:cytology*; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D034622:RNA Interference; D059386:Reverse Genetics / Q000379:methods*; D013569:Synapses / Q000502:physiology* / Q000648:ultrastructure*
keywords: 3D CLEM; CNS; Drosophila; Sensory neuron; Synapse formation