Journal: Nature

Article Title: A multidimensional coding architecture of the vagal interoceptive system

doi: 10.1038/s41586-022-04515-5

Figure Lengend Snippet: a , Schematic illustration of Projection-seq analysis of VSNs innervating the lung, heart, oesophagus, stomach, duodenum, transverse colon and pancreas. Organ illustrations were adapted from BioRender.com. b , UMAP plot from Projection-seq of 14,590 Phox2b + VSNs (30 mice divided into 4 samples) showing 52 clusters (A1–L2) in 12 VSN subpopulations (A–L) (top) or VSNs expressing UPBs representing 7 visceral organs (colour-coded) (bottom). c , Two-dimensional (2D) (top) and three-dimensional (3D) (bottom) UMAP plots of VSNs innervating different physiological systems. E-VSNs were excluded. The three heart VSN groups (red, arrowheads) are clustered together away from other gut VSNs (green) in the 3D UMAP plot. d , Dot plot showing transcription factors that are differentially expressed in lung, heart, gut and pancreas VSNs. e , UMAP plot of VSN clusters, coloured by target preference (weighted organ position score), showing a ‘visceral organ’ trajectory (arrow) coding visceral organs along the body’s rostral–caudal axis. f , Correlation between the normalized position of the indicated organs along the body’s rostral–caudal axis (mean; n = 4) and the position of VSNs expressing indicated organ UPBs along the ‘visceral organ’ trajectory (organ trajectory score; mean ± s.e.m.; n as indicated). Linear regression R 2 = 0.7547. g , Histograms showing the distributions of UPB-labelled VSNs (colour-coded) along the identified ‘visceral organ’ trajectory. The bars underneath indicate normalized organ positions along the body’s rostral–caudal axis (beginning–end; mean ± s.e.m.; n = 4).

Article Snippet: Transcriptomic data were aligned to the mm10 mouse genome reference (control scRNA-seq) or a custom mouse genome reference with additional sequence information of UPBs (Projection-seq) using the Cell Ranger software v.3.0.2 (10X Genomics).

Techniques: Expressing

Journal: Nature

Article Title: A multidimensional coding architecture of the vagal interoceptive system

doi: 10.1038/s41586-022-04515-5

Figure Lengend Snippet: 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. . 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 ).

Article Snippet: Transcriptomic data were aligned to the mm10 mouse genome reference (control scRNA-seq) or a custom mouse genome reference with additional sequence information of UPBs (Projection-seq) using the Cell Ranger software v.3.0.2 (10X Genomics).

Techniques: Reverse Transcription Polymerase Chain Reaction, Injection, Control, Expressing, Marker, Derivative Assay, RNAscope

Journal: Nature

Article Title: A multidimensional coding architecture of the vagal interoceptive system

doi: 10.1038/s41586-022-04515-5

Figure Lengend Snippet: a , UMAP plots of VSNs showing that Sprr1a (top) and Ecel1 (bottom), genes upregulated in damaged sensory neurons, are selectively expressed in E-VSNs (dashed circles, control scRNA-seq data). b , Percentage of E-VSNs in label-free control scRNA-seq data (blue) and Projection-seq (red) data. c , Detection of Sprr1a using RNAscope in vagal ganglia from control (top) and AAVrg stomach injected (bottom) mice. Dashed lines indicate the shape of vagal ganglia. Sprr1a + neurons are indicated in red dashed circle. Scale bar: 100 μm. d , Percentage of Sprr1a + VSNs in control versus AAVrg stomach injected mice. mean ± SEM, n = 4. *p < 0.05, p = 0.0204, two-tailed t -test. e , Heat map of genes differentially expressed in VSNs innervating indicated organs. f , Percentage of VSNs marked by UPBs from different gut regions in indicated clusters, coloured per cluster. E (oesophageal)-Sphincter VSNs express both UPB-oesophagus and UPB-stomach. P (pyloric)-Sphincter VSNs express both UPB-stomach and UPB-duodenum. g , Differential expression of transcription factors in UPB-marked VSNs innervating indicated organs. From left: Atf3 , Terf1 , Runx1 , Sox4 , Tshz2 , Pou4f1 , Cebpb , Irf6 , Esr1 , Tbx3 , Id3 , Hoxb5 , Carhsp1 , Hoxb6 , Mef2c , Zfhx3 , Tcf4 , Egr1 , Klf2 , Klf5 , Casz1 , Etv1 , Epas1 , Nfkbia , Scrt2 , St18 , Scx , Klf4 , and Nhih2 . h , IPA predicted regulatory network of Pou4f1 in lung VSNs. Orange arrows, predicted activation; grey arrow, unknown. i , Dot plots of cell-cell signalling between organ-UPB labelled VSNs and various cell types in corresponding organs, predicted by CellPhoneDB. (top left), HVSN, heart-VSN; CM, cardiomyocyte; EDC, endothelial cell; EP, epicardial cell; FB, fibroblast. (top right), LVSN, lung-VSN; ATI, alveolar epithelial type I cell; ATII, alveolar epithelial type II cell; B, B cell; C&S, ciliated and secretory cell; DC, dendritic cell; EDC, endothelial cell; FB, fibroblast; MO, monocyte; Mac, macrophage; NK, natural kill cell; Neutro, neurophil; Peri, pericyte; T, T cell. (bottom left), CVSN, colon-VSN; Endo, endothelial cell; Immu, immune cell; EN, enteric neuron; Glia, glial cell; Entero, enteroendocrine cell; Mus/Fb, muscle cell and fibroblast. (bottom middle), DVSN, duodenum-VSN; EXMN, excitatory motor neuron; INMN, inhibitory motor neuron; IN, inter neuron; IPAN, intrinsic primary afferent neuron. (bottom right), PVSN, pancreas-VSN; ISL, islet cell; ACI, acinar cell; DUCT, duct cell; MES, mesenchymal cell; IMVS, immune and vascular cell. j , Scatterplots of expression measures vs tissue layer trajectory scores for indicated DEGs along the tissue layer trajectory. Bars: 0–3. k , Fraction of DEG + VSN endings characterized in Gpr65 tdT , Drd2 tdT , and Agtr1a tdT mice in indicated tissue layers of indicated organs. mean ± SEM, number of mice: Gpr65-Oesophagus (3), Gpr65-Stomach (7), Gpr65-Duodenum (4), Drd2-Oesophagus (3), Drd2-Stomach (6), Drd2-Duodenum (3), Drd2-Colon (2), Drd2-Heart (6), Agtr1a-Oesophagus (4), Agtr1a-Stomach (12), Agtr1a-Duodenum (4), Agtr1a-Colon (3), Agtr1a-Heart (6). l , Expression of Gpr65 and Trpv1 in F-VSNs (left) and maximum depths of duodenal MEs, normalized to the thickness of the sample, in Trpv1 tdT and Gpr65 tdT mice (right). mean ± SEM, 15 endings from 3 mice for Trpv1, 26 endings from 4 mice for Gpr65. ***p < 0.001, p = 6.6 × 10 −6 , two-tailed t -test. m , Expression of Sst and Trpv1 in F1-VSNs (left) and the number of stomach gland tips innervated in the antrum and corpus, normalized to the innervation area, in Trpv1 tdT and Sst tdT mice (right). mean ± SEM, 11 endings from 5 mice for Trpv1, 10 endings from 5 mice for Sst, **p < 0.01, p = 0.0031, two-tailed t -test.

Article Snippet: Transcriptomic data were aligned to the mm10 mouse genome reference (control scRNA-seq) or a custom mouse genome reference with additional sequence information of UPBs (Projection-seq) using the Cell Ranger software v.3.0.2 (10X Genomics).

Techniques: Control, RNAscope, Injection, Two Tailed Test, Quantitative Proteomics, Activation Assay, Expressing

Journal: Nature

Article Title: A multidimensional coding architecture of the vagal interoceptive system

doi: 10.1038/s41586-022-04515-5

Figure Lengend Snippet: a , Cartoon depiction of the 11 regions along the gastrointestinal tract with injection sites indicated. Cartoon illustration adapted with permission from ref. . b , Innervation intensity of various vagal afferent ending types (innervated area/total area for mucosal endings and IMAs, number of terminals/total area for IGLEs) along indicated regions on the gastrointestinal tract in Vglut2 tdT mice. (mean ± SEM, n = 3-4). E.S., oesophageal sphincter; P.S., pyloric sphincter; D, duodenum; C, transverse colon. Regions 1–3, upper, middle, and lower oesophagus. Regions 4–8 correlate with stomach regions as shown in ( a ). c , 9 primary VSN clusters for stomach UPB-labelled neurons, visualized on the UMAP plot (top, red) or column graph (bottom, red stars). d , Innervation density of vagal ending types around the oesophageal (top) and pyloric (bottom) sphincters, normalized to the density across the entire stomach (mean ± SEM, n = 4). e , UMAP plots of VSN clusters enriched for the oesophageal (top, VSNs dual labelled with oesophagus and stomach UPBs) and pyloric (bottom, VSNs dual labelled with stomach and duodenum UPBs) sphincters over other stomach regions. Light red indicates clusters containing more than 4% of dual UPB labelled VSNs. Red indicates clusters enriched (>1.05 fold) in dual UPB labelled VSNs over stomach UPB labelled VSNs. f , Fraction of DEG + VSNs among indicated clusters, calculated as the number of DEG + VSNs in the indicated cluster normalized by the total number of DEG + VSNs in all shown clusters. g , Representative stomach endings formed by DEG + VSNs around the oesophageal or pyloric sphincter in corresponding Cre tdT mice. h , UMAP plots of VSN clusters (red) enriched for the stomach regions 5, 6, and 7 as shown in ( a ). Light red indicates clusters containing more than 4% of stomach/colon (region 7) or stomach/pancreas (region 6) dual UPB labelled, or stomach UPB single labelled (region 5) VSNs. Red indicates clusters enriched (>1.05 fold) in dual UPB labelled VSNs over stomach UPB labelled VSNs (for region 6 and 7), or in stomach UPB labelled VSNs over all other 4 groups of dual UPB labelled VSNs (for region 5). i , Simulation results for VSN clusters I2, I4–7, J2, and J4 (see ). Arrow indicates the trial with the lowest variation (red dots, ending types listed on the right). j , Innervation density of four VSN ending types in different stomach regions normalized to the intensity across the entire stomach. Quantified in Vglut2 tdT mice (blue, mean ± SEM, n = 4) or predicted by Projection-seq (red). k , UMAP plots of VSNs showing Agtr1a and Oxtr expression in I-VSNs. l , Representative stomach IGLE endings formed by I-VSNs in Agtr1a tdT mice. m , Innervation intensity (innervated area/total area for ME, pIMA, and cIMA; number/total area for IGLE, normalized to Vglut2 tdT ) of stomach afferent ending types formed by indicated VSNs in corresponding Cre tdT mice (mean ± SEM, n = 3–12). Scale bars: 100 μm.

Article Snippet: Transcriptomic data were aligned to the mm10 mouse genome reference (control scRNA-seq) or a custom mouse genome reference with additional sequence information of UPBs (Projection-seq) using the Cell Ranger software v.3.0.2 (10X Genomics).

Techniques: Injection, Expressing