Extended Data Fig. 2: Projection-seq analysis faithfully reveals the molecular architecture and organ projection of VSNs.
From: A multidimensional coding architecture of the vagal interoceptive system
a, VSNs retrogradely labelled from the stomach using Projection-seq AAV (UPB-stomach, top) or AAVrg-GFP (bottom). b, RT-PCR analysis of vagal ganglia cDNA from mice with stomach injection of Projection-seq AAV-UPB2 (red) or control (grey) mice using primers that recognize UPB2 (dark colour) or UPB4 (light colour) sequences. For gel source data, see Supplementary Fig. 1. c, Percentage of tdTomato+ neurons after acute VSN dissociation (blue) and UPB marked neurons after Projection-seq analysis (red). d, Dot plot of expression of indicated marker genes in 12 VSN subpopulations (A-L)/52 VSN clusters (A1-L2). e, Correlation scores for VSNs labelled with UPBs from indicated visceral organs. f, UMAP plots of 31,182 cells from control scRNA-seq, coloured by expression of indicated genes. Dashed circle indicates Syn1+/Slc17a6+ neuronal clusters. g, (top) UMAP plot of 16,476 neurons from neuronal clusters indicated in (f), coloured by expression of indicated genes, showing the two developmental origins of VSNs. Prdm12 (blue) labels neural crest derived VSNs in the jugular ganglia. Phox2b+ placode derived clusters (red, dashed circle) containing 13,210 VSNs in the nodose ganglia from control scRNA-seq data are re-clustered and plotted on the UMAP plot (bottom), coloured by VSN subpopulations. h, UMAP plot of 27,800 Phox2b+ placode-derived VSNs from integrated Projection-seq (cyan) and control scRNA-seq (red) data. i, Percentage of neurons in 49 VSN clusters from control scRNA-seq (blue) and Projection-seq (red) datasets. E-VSNs were excluded. j, UMAP plots of VSNs colour by expression of indicated marker genes from control scRNA-seq (top) and Projection-seq (bottom) data. k, RNAscope HiPlex Assay in the nodose/jugular ganglia for indicated marker genes identified from Projection-seq. l, Zoom-in images from the dashed regions in (k). The numbers of VSNs expressing one gene (red or green) or both genes (yellow) were counted. Consistent with Projection-seq data as shown in (d), Trpa1 and Tmc3 were largely expressed in non-overlapping VSNs, whereas Ut2sb, Runx3, Gabra1, and Gm765 each labelled a distinct VSN subset. m, VSN subpopulations determined from RNAscope HiPlex Assays using indicated genes identified by Projection-seq. One neuron per column as indicated in the bottom. X, unlabelled; Jg, jugular neurons. n, Cumulative percentage of neurons in 11 VSN subpopulations revealed by Projection-seq, control scRNA-seq, or RNAscope. X, unlabelled. o, Dot plot of expression of UPBs representing indicated organs in 12 VSN subpopulations (A-L)/52 clusters (A1-L2). p, RNAscope HiPlex Assays for indicated genes. VSNs were retrogradely labelled from indicated organs with AAVrgs and visualized using corresponding RNAscope probes. *, VSNs from oesophagus, lung, or colon. +, VSNs from stomach, heart, or duodenum. #, double-labelled VSNs. q, Cumulative percentage of neurons in 11 VSN subpopulations expressing organ UPBs from Projection-seq analysis (top) and retrogradely labelled from indicated organs from RNAscope analysis (bottom). X, unlabelled. Scale bars: 100 μm (a, k), 20 μm (l, p).
