Project description:Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues, and thus govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally-resolved approach to profile cell surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed a global downregulation of wiring molecules and an up-regulation of synaptic molecules in the transition from developing to mature PNs. To compare the RNA and protein dynamics of PN surface molecules in the developing-to-mature transition, we also profiled the PN transcriptomes from 36hAPF pupae and 5d adults. We performed bulk RNA-sequencing of PNs with 3 samples for each stage and 2k cells for each sample.
Project description:Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological features. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage—neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.
Project description:To validate different projection targets of already molecularly-defined olfactory bulb projection neurons we used viral targeting specifically into anterior or posterior cortical areas, Fluorescence Activated Nuclei Sorting (FANS) to enrich for olfactory bulb projection neurons, and single-nuclei RNA sequencing (sn-RNA seq) To isolate GFP-labelled nuclei, 1 individual replicate of AON or PCx-injected mice was used. Ipsilateral and controlateral sides were minced separately and placed into two different tubes. The minced tissue was gently homogenized in Nuclei PURE Lysis Buffer and 10% Triton X-100 using an ice-cold dounce and pestle, and filtered two times through a 40 μm cell strainer on ice. After centrifuging at 500 rpm for 5 min at 4 °C, the supernatant was aspirated and gently resuspended in 500 μl of cold buffer (1x of cold Hanks' Balanced Salt Solution HBSS, 1% nuclease-free BSA, RNasin Plus and 1/2000 DRAQ5). Our study identifies molecularly distinct subtypes of mitral cells projecting to anterior or posterior olfactory cortices.
Project description:To characterize the molecular diversity of olfactory bulb projection neurons we used viral targeting and Fluorescence Activated Nuclei Sorting (FANS) to enrich for piriform cortex-projecting or AON-projecting neurons, and bulk RNA deep sequencing (bulk RNA deep seq) to comprehensively characterize their transcriptomes.
Project description:To characterize the molecular diversity of olfactory bulb projection neurons we used viral targeting and Fluorescence Activated Nuclei Sorting (FANS) to enrich for olfactory bulb projection neurons, and single-nuclei RNA sequencing (sn-RNA seq) to comprehensively characterize their transcriptomes. To isolate GFP-labelled nuclei, 3 individual replicates of AON and PCx-injected mice were used. Ipsilateral and controlateral sides were minced separately and placed into two different tubes. The minced tissue was gently homogenized in Nuclei PURE Lysis Buffer and 10% Triton X-100 using an ice-cold dounce and pestle, and filtered two times through a 40 μm cell strainer on ice. After centrifuging at 500 rpm for 5 min at 4 °C, the supernatant was aspirated and gently resuspended in 500 μl of cold buffer (1x of cold Hanks' Balanced Salt Solution HBSS, 1% nuclease-free BSA, RNasin Plus and 1/2000 DRAQ5). Our study identifies molecularly distinct subtypes of mitral and tufted cells.
Project description:How a neuronal cell type is defined and how this relates to its transcriptome are still open questions. The Drosophila olfactory projection neurons (PNs) are among the bestcharacterized neuronal types: Different PN classes target dendrites to distinct olfactory glomeruli and PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA-sequencing, we comprehensively characterized the transcriptomes of 40 PN classes and unequivocally identified transcriptomes for 6 classes. We found a new lineage-specific transcription factor that instructs PN dendrite targeting. Transcriptomes of closely-related PN classes exhibit the largest difference during circuit assembly, but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Genes encoding transcription factors and cell-surface molecules are the most differentially expressed, indicating their central roles in specifying neuronal identity. Finally, we show that PNs use highly redundant combinatorial molecular codes to distinguish subtypes, enabling robust specification of cell identity and circuit assembly.