Molecular, Cellular, and Developmental Organization of the Mouse Vomeronasal organ at Single Cell Resolution

We have generated single cell transcriptomic atlases of vomeronasal organs (VNO) from juvenile and adult mice. Combined with spatial molecular imaging, we uncover a distinct, previously unidentified class of cells that express the vomeronasal receptors and a population of canonical olfactory sensory neurons in the VNO. High resolution trajectory and cluster analyses reveal the lineage relationship, spatial distribution of cell types, and a putative cascade of molecular events that specify the V1r, V2r, and OR lineages from a common stem cell population. The expression of vomeronasal and olfactory receptors follow power law distributions, but there are high variabilities in average expression levels between individual receptor and cell types. Substantial co-expression is found between receptors across clades, from different classes, and between olfactory and vomeronasal receptors, with nearly half from pairs located on the same chromosome. Interestingly, the expression of V2r, but not V1r, genes is associated with various transcription factors, suggesting distinct mechanisms of receptor choice associated with the two cell types. We identify association between transcription factors, surface axon guidance molecules, and individual VRs, thereby uncovering a molecular code that guides the specification of the vomeronasal circuitry. Our study provides a wealth of data on the development and organization of the accessory olfactory system at both cellular and molecular levels to enable a deeper understanding of vomeronasal system function. Overall design: Mice VNOs were dissected in cold oxygenated ACSF following Ma et al, 2011.?Dissected epitheliums were dissociated in papain solution (20mg/mL papain and 3mg/mL L-cysteine in HBSS) with RNase-free DNase I (10unit) at 37°C for 15-20 minutes. 0.01% BSA in PBS was added to the digestion solution before filtering with 70µm and 30µm filters (pluriSelect). Dissociated cells were washed twice in 0.01% BSA with final volume 1mL, followed by Draq5 (25µM) and DAPI (0.5µg/mL) staining 5min on ice. Draq5+/DAPI- cells (live/nucleated cells) were sorted on BD Influx cytometer (BD Bioscience) with 100µm nozzle. Dissociated sorted cells were assessed for concentration and viability via Luna-FL cell counter (Logos Biosystems). Cells deemed to be at least 90% viable were loaded on a Chromium Single Cell Controller (10x Genomics), based on live cell concentration.