Figures and data
Single cell RNA sequencing of the mouse vomeronasal neuroepithelium
A) Uniform Manifold Approximation and Projection (UMAP) of 9185 cells from vomeronasal neuroepithelium with 18 identified clusters. Each point represents a cell that is color coded according to the cell type. Clusters were assigned a cell type identity based on previously known markers. Percentage of total cells corresponding each cluster is in supplementary table-3 B) Dot plot showing the expression of top 10 gene markers for each cluster identified by differential expression analysis. Gene expresion markers shared across multiple clusters are listed only once. The dot radius and dot color indicate the percentage of cells expressing the gene and level of expression in that cluster, respectively. Top 20 gene markers for each cluster with log2 fold change are listed in supplementary table-1. C) Feature plot showing the expression level of major known gene markers of neuronal and sustentacular cell types.
Gene markers of non-sensory cells in the vomeronasal organ identified from single cell RNAseq.
A) UMAP feature plot (top) of a single representative gene marker for non-sensory cell clusters and their spatial gene expression pattern by RNA in situ hybridization on VNO coronal sections (bottom, scale bar: 100μm). B) Three custers of macrophage-like cell types represented by UMAP projection (left) and a dot plot (right) showing differentially expressed genes between these clusters. The dot radius indicates percentage of cells expressing a gene, whose scaled expression level is represented by intensity of color as per the scale. Additional markers of each non-sensory cell type are in supplementary table-1 and differential expression between macrophage clusters is in supplementary table-4.
Gene expression dynamics during development of sensory neurons
A) UMAP of neuronal cell types with clusters (n1-n13) represented in different colors. The line on the UMAP plot shows the pseudotime developmental trajectory of neurons. B) Volcano pot showing the differentially expressed genes between Gap43+,Gnao1+ (cluster n6) and Gap43+,Gnai2+ (cluster n7) neurons. Genes that satisfy |log2 fold change normalized expression| > 1 (green) and -log10 p value > 6 (blue) are considered significant and coloured in red. Non-significant (NS) genes are colored in grey. Arrows point to transcription regulators enriched in Gnao1+ immature neurons. Complete list of differentially expression genes is in supplementary table 5. C) Feature plots showing the normalized expression levels of previosly known markers for Gnao1 neurons (Robo2, Tfap2e); Gnai2 neurons (Meis2) and transcription factor or related genes: Creb5, Prrxl1, Shisa8, Lmo4, Foxo1. Arrows highlight the limited expression of Creb5 and Prrxl1 to immature neurons (Gap43+,Gnao1+: cluster n6), but absent from mature (Gap43-,Gnao1+: cluster n1-n4) indicating transient expression during development of Gnao1 neurons.
Divergent expression profile of Gnao1 and Gnai2 neurons
A) Volcano plot showing differentially expressed genes between mature Gnao1 and Gnai2 neurons. Genes that satisfy |log2 fold change normalized expression| > 1 (green) and -log10 p value > 6 (blue) are considered significant and coloured in red. Non-significant (NS) genes are colored in grey. Complete list of differentially expressed genes is in supplementary table 6. B-D) Two color RNA in situ hybridization (ISH) of Gnao1 and Gnai2 markers of basal and apical neurons (B), along with genes enriched in Gnai2 neurons (C) and Gnao1 neurons (D) showing restricted expression. Scale bar:100 μm. E) Gene ontology (GO) annotation of biological processes that are significantly enriched from upregulated genes in Gnao1 neurons. GO terms related to ER processes are marked in red. p-value < 0.05 was used as cut-off.
Differential ER environment in Gnao1 and Gnai2 neurons
A) Two color fluorescent RNA in situ hybridization of endoplamic reticulum (ER) related gene probes selected from Figure 4A (green: Creld2, Pdia6, Dnajc3, Sdf2l1) and co-labelled with Gnaol probe (red) on coronal sections of VNO shows enrichement of these genes in basal neurons. Scale bar 100µm. B, C) Pseudocolored fluorescence images of VNO sections immuno-labelled with antibodies against ER chaperones - Bip/Hspa5, PDI, Calnexin, Grp94; SEKDEL; ER membrane protein-Sec61β (B) and ER structural proteins - Atlastinl, Reep5, NogoB, Ckap4/Climp-63 (C) showing differential labelling in Gnaol and Gnai2 neurons. Scale bar 50µm
Basal/Gnao1 neurons are densely packed with cubic membrane ER.
A) Scanning electron micrographs of vomeronasal sensory epithelium at low magnification showing cell bodies of VSNs, sustentacular cells and basal lamina. Boxed regions in red or green mark cell bodies of basal/Gnao1 or apical/Gnai2 neurons respectively, that are displayed at higher magnification below. Nucleus (N) appears light and ER is dark. Cell bodies of basal/Gnao1 neurons are larger and occupied by substantial amount of ER, incomparison to apical/Gnai2 neurons. B, B’) Magnified micrographs show the cell body of a basal/Gnao1 neuron, packed with cubic ER membranes. White arrows point to dense membrane stacks that are better resolved in Figure supplement. Red arrow points to lamellar ER membrane that is contiguous with the cubic membrane. C, C’) Cell bodies of apical/Gnai2 neurons also seem to show dense cubic membrane ER, that is smaller in comparison to basal/Gnao1 neurons.
Subpopulation of Gnao1 neurons defined by V2R and H2-Mv expression
A) UMAP representation of Gnao1 neurons from Figure 3. Each dot represents a cell and four Gnao1 neuron clusters (n1-n4) are marked in different colours. B-D) Feature plot showing exclusive expression of Vmn2r1 (B), Vmn2r2 (C) and the most abundant H2-Mv, H2-M10. 3 (D) in Gnao1 neurons. E) Heat map showing the expression of V2R and H2-Mv genes in Gnao1 neurons. Cluster numbers are marked on the top with color coding as in (A) and gene families are labelled on the left. Each column represents a cell and the scaled gene expression in each row is colour coded as per the scale with red and blue indicating high and low expression respectively. Vmn2r1 is expressed in almost all cells of cluster-1 and cluster-4; Vmn2r2 is expressed is all cells of cluster 3; Cluster2 has mutually exclusive expression of Vmn2r1 and Vmn2r2. F-H) Bar plot showing number of cells expressing: 0-7 family-C V2Rs per cell (F), 0-5 family-ABD V2Rs per cell (G), 0-6 H2-Mv genes per cell (H) with composition of cells associated with family C1 (orange) or C2 (blue) V2R color coded on the bar. I) Box plots comparing the sum of normalised expression levels of family-C V2Rs and Gnao1 (Green) in a cell that expresses 1 to 5 family-C V2Rs. J) Box plot comparing the level of total ABD-V2R expression from cells with 1 and 2 ABD-VRs along with Gnao1 expression level (Green). K) Box plot comparing the level of total H2-Mv expression in cell that express 1-5 H2-Mv genes along with Gnao1 expression level (Green). Multiple combinations of family-C, family ABD V2Rs and H2-Mvs identified to be co-expressed in a single cell and their cell frequency are listed in supplementary table 7.
Onset of V2R expression coincides with the expression of ER chaperone genes
A) Feature Plot showing the expression of Gnao1, Vmn2r1, Sdf1l1 and Manf. Sdf2l1 and Manf are known ER chaperones and their upregulation in Gnao1 neurons coincides with Vmn2r1 expression, which is preceded by Gnao1 expression. B) Heatmap showing the expression of Gnao1, Vmn2r1, Vmn2r2 and several ER chaperone genes (red) in the clusters arranged as per their developmental trajectory. C) Cartoon summarizing major transcription factor expression during development leading to Gnao1 neurons with chaprone rich hypertropic ER compared to Gnai2 neurons.
Comparison of cell type composition and gene expression from male and female vomeronasal neuroepithelium.
A) Uniform Manifold Approximation Projection (UMAP) of cells from male and female vomeronasal neuroepithelium, with the cluster numbers corresponding to Figure 1A, 1B. Solitiary chemosensory neuron (Cluster 18) were seen only in female data. B) Scatter plots comparing average expression of genes accross each cluster from male and female with Pearson correlation co-efficient at the top of the plot. Each point in the plot represents a gene. Known sexually dimorphic genes: Eif2s3y, Ddx3y, Uty, Kdm5d are marked in red. Scatter plot of cluster 17 between male and female is not shown due to low cell count. The results of differential expression between each cluster of male and female are in supplementary table 2.
A) UMAP projection of neurons with 13 clusters (n1-n13). B) Feature plot showing expression of neuronal markers that mark different stages of differentiation: progenitors cells-Neurod1, Neurog1 (n5); immature neurons-Gap43 (n6,n7); mature neurons: Gap43-,Gnao1+ (n1-n4) and Gap43-,Gnai2+ neurons (n8-n13). Arrows highlight the expression of Gnao1 and Gnai2 in Gap43+ immature neurons (n6, n7).
Expression pattern of enriched genes in Gnai2 or Gnao1 neurons.
RNA-ISH showing expression of Gnai2 enriched genes: Nsg1, Rtp1, Dner, Qpct, Pcdh7, Prph (A) and Gnao1 enriched genes: Apmap, Selenof, Hspa5 (Bip/Grp78), Itm2b, Agpat5, Sncg (B). Sncg and Prph are expressed in a scattered pattern amongst few neurons. Scale bar: 100 µm
Comparison of ER gene expression between Gnai2 and Gnao1 neurons.
Violin plots showing the expression of genes in mature Gnao1 and Gnai2 neurons whose protein levels are elevated in Gnao1 neurons as shown in Figure 5B, 5C. Log2 fold change value for Gnao1 vs Gnai2 calculated from pseudobulk differential gene expression analysis is mentioned on top of the line and Bonferroni-adjusted p-value is mentioned below the line (ns - not significant). RNA levels of Hspa5, Calnexin, Hsp90b1, Sec61b, Reep5 are significantly upregulated in Gnao1 neurons while PDI and Atlastin1 do not differ significantly between Gnao1 and Gnai2 neurons.
A, B) Scanning electron micrographs showing cell bodies of basal/Gnao1 neurons with sinusoidal ER membranes interspersed with stacked membranes (arrows).
Bar plot showing the number of cells expressing top 20 V1Rs (A), V2Rs (B) and H2-Mvs (C). Normalized gene expression values were extracted for all V1Rs (D) from Gnai2 neurons, V2Rs (E) and H2-Mvs (F) from Gnao1 neurons and density was plotted to identiy the distribution of gene expression. The red dashed line represents a normalized gene expression value that was used as threshold to call the expression of respective genes.
Characteristics of H2-Mv expression
A -B) Feature plot showing the expression H2-M10 family genes (A) and limited expression of phylogentically divergent H2-Mv members H2-M1, H2-M9 and H2-M11 (B) in Gnao1 neurons. C) Feature plot showing the expression of Vmn2r81 and Vmn2r82 in few cells of cluster 2 and 4 of Gnao1 neurons that express H2-M1, M9 and M11. D) Scatter plot showing the normalized expression level per cell of rarely expressed H2-Mv genes (H2-M1, M9 and M11) on x-axis and Vmn2r1 or Vmn2r2 on y-axis indicating that H2-M1, M9 and M11 co-express with Vmn2r1 unlike other H2-Mv genes. E) Two-color RNA in situ hybridization of H2-M1,H2-M9 and H2-M11 with Vmn2r81/82 confirming the co-expression. The ISH probe was common for Vmn2r81 and 82; H2-M1/M9/M11 indicates pooling ofprobes. Scale bar: 100 µm.
Co-expression of V1Rs in Gnai2 neurons
A) Heatmap showing the expression of selected V1Rs in Gnai2 neurons. Each column represents a cell and the scaled expression value of each VR is color coded in each row with red and blue indicating high and low expression respectively. A continuos red line in two rows of a single column indicates the expression of two receptors in a single cell. B) Bar plot showing number of cells expressing 0-3 V1Rs per cell indicating the V1R co-expression is limited to small subset of cells. C) Box plot comparing the sum of normalised expression levels of V1Rs and Gnai2 (Green) in a cell that expresses 1 or 2 V1Rs. Multiple combinations of V1Rs co-expressed in single cell and their cell frequency are listed in supplementary table 8.
