Single cell transcriptomic profile of the whole vomeronasal organ

A. UMAP visualization of integrated cell-type clusters for whole-VNO single-cell RNA-seq. B. Cell-type marker-gene normalized expression across the cell clusters. C. UMAP of cell-type clusters split by age. D. UMAP of cell-type clusters split by sex. E. A representative image of transcript distribution for 9 genes in a VNO slice using the Molecular Cartography© platform Resolve Biosciences. Insets (a and b) show the magnified image of areas identified in the main panel. Individual cell shapes can be determined from the transcript clouds. F. Spatial location of individual VNO cells color-coded according to cell type prediction based on the spatial transcriptomic analysis. G. Location of cell belonging to HBC, GBC, INP, and LP cell types, respectively. Heat indicates confidence of predicted values. BL: basal lamina; MZ: marginal zone.

Novel neuronal lineage

A. UMAP visualization of cell-type clusters for the neuronal lineage. B. Expression of Gnai2 and Gnao1 in the neuronal lineage. C. Expression of Cnga2 and Gnal in the OSN lineage. D-E. Location of mOSNs (D) and sVSNs (E) in a VNO slice. Color indicates prediction confidence. F. Heatmap of normalized expression for a select set of mutually differentially expressed genes between sVSNs and mature V1Rs, V2Rs, and OSNs. G. Enriched gene ontology (GO) terms in sVSNs when compared with V1R and V2R VSNs, respectively. H. Box plots of normalized expression for Muc2, Obp2a, Obp2b, and Lcn3 across mature sensory neurons.

Molecular cascades separating the neuronal lineages

A. PCA plot of V1R and V2R pseudotime principal curves across cell types. B. Cell density plots across pseudotime for P14 and P56 mice. C. Heatmap showing expression in pseudotime for genes that differentially expressed between the V1R and V2R lineages. Heat indicates Z-score values. D. Zoomed in UMAP of cell types early in the neuronal lineage. E-I. Feature plots for select genes expressed early in the neuronal lineage. J. UMAP of INP, iVSN, iOSN, and mOSN cell types. K-U. Normalized expression of candidate genes for V1R/V2R/OSN lineage determination. V. A simplified model for lineage determination by transcription factors in VNO sensory neurons.

Receptor expression in the VNO

A-D. Expression level of individual receptor (total raw counts) plot against the number of cells expressing the receptor for V1R (A), V2R (B), VNO-Olfr (C), and MOE-Olfr (D). E-H. Ranked distribution of average expression per cell for the four receptor classes. Inset pie charts show the number of cells expressing a receptor at the specified range. I and J. Heatmaps showing the correlation of transcription factor expression among the V1Rs (I) or V2Rs (J). K. A simplified model of transcription factor selection in mVSNs.

Co-expression among VNO receptor classes

A. Shannon Indices showing the specificity of receptor expression. High values indicate more co-expressions. B-C. Prevalence and level of receptor co-expression by age and cell-type for the V1R (B) and V2R (C) lineages, respectively. D-F. Circos plot of genomic loci for significantly co-expressed receptor pairs in the V1R (D), V2R (E), and across-type populations (F). G-K. Detection of receptor gene co-expression using Molecular Cartography©. Individual dots represent single molecules. Colors represent different receptor genes. DAPI stain is shown as gray. Scale bar: 10 µm.

Axon guidance molecules associated with receptor genes.

A. Distribution density plot showing relationship between similarity of axon guidance (AG) gene expression profiles and receptor sequence similarity. The distribution of receptor similarity (x-mean and x-median) remains constant over the range of AG similarity. The AG similarity (y-mean and y-median) as a function of receptor similarity shows a strong correlation at the dense part of the curve. B. Heatmap showing the correlation among VRs in their AG expression. C and D. Heatmaps showing the V1R-AG (C) and V2R-AG (D) associations. Heat shows average expression level for AG genes for a given receptor type. E. A simplified model of hierarchical distribution of AG in the mVSNs.

Transcriptional determinants of axon guidance molecules for individual receptor types.

A. Correlation heatmap between receptor types calculated from co-expressed AG and TF genes. Receptor types are color-coded. B. 3-D heatmap showing the Jaccard Indices between AG and TF genes for each of the VRs in the dataset. C-G. Heatmaps showing Jaccard Indices of TF-AG associations for V1R (C and D) and V2R types (E-G). The lists of TF and AG genes here are abridged from the full list to enhance visualization. (C), these receptors share Meis2 expression but different AG genes. Note that Vmn1r185 and vmn1r69 both recognize female identify pheromones. (D), similarity and distinction of AG/TF expression for three V1R types that are located in the same genomic location and with high sequence homology. (E and F), shared TFs and AG genes for broadly (E) and uniquely (F) expressed V2R types. (G), distinct TFs and AGGs for uniquely expressed V2R types.