An Atlas of Vagal Sensory Neurons and Their Molecular Specialization Jussi Kupari, Martin Häring, Eneritz Agirre, Gonçalo Castelo-Branco, Patrik Ernfors  Cell Reports  Volume 27, Issue 8, Pages 2508-2523.e4 (May 2019) DOI: 10.1016/j.celrep.2019.04.096 Copyright © 2019 The Authors Terms and Conditions

Cell Reports 2019 27, 2508-2523.e4DOI: (10.1016/j.celrep.2019.04.096) Copyright © 2019 The Authors Terms and Conditions

Figure 1 Cellular Heterogeneity in the Vagal Ganglion Complex (A) Schematic illustration of the vagal sensory system and the workflow. (B) Heatmap showing the five most-selective genes (by lowest padj) in each cluster. Clusters were hierarchically ordered in PCA space. (C) tSNE visualization. Clusters were numbered according to (B). Clusters 4–27 are sensory neurons. (D) Violin plots showing major cell type-specific marker expression across the clusters. Values on the y axis represent raw UMI counts. (E) Violin plots showing the numbers of total genes and UMIs detected in each cluster. Endo, endothelial cells; SGC, satellite glial cells; Symp, sympathetic neurons. Cell Reports 2019 27, 2508-2523.e4DOI: (10.1016/j.celrep.2019.04.096) Copyright © 2019 The Authors Terms and Conditions

Figure 2 Major Molecular Differences between Jugular and Nodose Ganglion Neurons (A) tSNE illustrating the two different neuron populations: Prdm12+ jugular neural crest- and Phox2b+ nodose placode-derived neurons. (B) ISH images showing the mutually exclusive expression of Prdm12 and Phox2b. Scale bar is 20 μm. Stacked bar illustrates the proportions of jugular and nodose cell profiles per section (469 cells, n = 3 animals) (C) Heatmap showing the 330 differentially expressed genes between the nodose and jugular clusters. (D) Violin plots showing the expression of genes with highly selective enrichment in all or most nodose neurons. (E) Violin plots showing the expression of genes with highly selective enrichment in all or most jugular neurons. Blue and red colors in (D and E) indicate nodose and jugular clusters, respectively; y axes in (D and E) indicate the number of raw UMIs detected. (F) GO terms enriched in neurons derived from the different ganglia. (G) STRING interaction map for nodose neurons. Cell Reports 2019 27, 2508-2523.e4DOI: (10.1016/j.celrep.2019.04.096) Copyright © 2019 The Authors Terms and Conditions

Figure 3 Jugular Ganglion Comprises Neurons with General Somatosensory Neuron Features (A) Dot plot showing selected genes that define splits in the major branches of the jugular ganglion dendrogram. (B) Pie chart showing the proportional abundance of the jugular neuron types. (C) tSNE visualization of the jugular clusters. (D) Heatmap showing the top five most-specific genes for individual jugular clusters (by lowest padj). (E) Single-cell bar plots showing the expression of genes used in the in vivo validation of the jugular-clustering data. Numbers on the right indicate the maximum number of UMIs detected. (F) Images from the ISH validation identifying the existence of each jugular cluster in vivo. Scale bar indicates 10 μm. Cell Reports 2019 27, 2508-2523.e4DOI: (10.1016/j.celrep.2019.04.096) Copyright © 2019 The Authors Terms and Conditions

Figure 4 Jugular-DRG Comparison Reveals Shared Identities between Neuronal Types (A) Heatmap showing the results from the unsupervised MetaNeighbor analysis between jugular and DRG clusters. Vertical names refer to DRG neuron types, and horizontal names refer to jugular neuron types. Numbers inside the cells indicate the mean AUROC score for the comparison. (B) Heatmap showing the expression of top jugular markers in the DRG neuron types (10 genes by lowest padj); note that the DRG clusters represent the main branches of neuron types (see Emery and Ernfors, 2018). (C) Heatmaps showing the expression of top DRG markers in the jugular clusters (30 genes by lowest padj value). (D) Heatmap showing the segregation of NP2- and NP3-specific DRG cluster markers into separate subsets of JG3 neurons. (E) Heatmap showing the segregation of Aβ-LTMR (NF2/3)- and Aδ-nociceptor (PEP2)-specific markers into separate subsets of JG5 neurons. Cell Reports 2019 27, 2508-2523.e4DOI: (10.1016/j.celrep.2019.04.096) Copyright © 2019 The Authors Terms and Conditions

Figure 5 Broad Diversity of Nodose Ganglion Neuron Types (A) Heatmap showing the five most-specific genes for each of the 18 nodose neuron types (five genes by lowest padj). (B) tSNE plot of the nodose clusters. (C) Proportional abundance of the nodose neuron types. (D) Single-cell bar plots showing the expression of genes used in the in vivo validation of the nodose clustering data. Numbers on the right indicate the maximum number of UMIs detected. (E) Images from the ISH validation for in vivo existence of predicted nodose neuron types. Scale bars indicate 10 μm. In (A) and (D), NG1–NG11 and NG12–NG18 are indicated by blue and red color, respectively. Cell Reports 2019 27, 2508-2523.e4DOI: (10.1016/j.celrep.2019.04.096) Copyright © 2019 The Authors Terms and Conditions

Figure 6 Unique Features Define Nodose Sensory Neuron Types (A) Heatmap showing the results from the unsupervised MetaNeighbor analysis between nodose and DRG clusters. Numbers inside the cells indicate the mean AUROC score for the comparison. Vertical names refer to DRG neuron types, and horizontal names refer to nodose neuron types. (B) Pie charts showing the distribution of GO terms equally shared between, or enriched, in the two major branches of nodose neurons (NG1–NG11 and NG12–NG18). Enrichment was defined as >60% of the term genes significantly upregulated in the group in question. (C) Dot plot showing the 20 most-specific genes for NG1–NG11 and NG12–NG18 (by lowest padj). (D) Violin plots showing selected genes differentially expressed between the two major groups (NG1–NG11 versus NG12–NG18); y axis indicates detected, raw, UMI counts. (E) Heatmaps showing the expression profiles of different categories of genes within the different nodose neuron types. In (A), (C), and (D), NG1–NG11 and NG12–NG18 are indicated by blue and red color, respectively. Cell Reports 2019 27, 2508-2523.e4DOI: (10.1016/j.celrep.2019.04.096) Copyright © 2019 The Authors Terms and Conditions

Figure 7 Proposed Vagus Nerve Sensory Neuron Classification and Their Relation to Function (A) Jugular ganglion neuron types and their functional relations based on shared neuronal identity with functionally characterized DRG neurons. (B) NG1–NG11 nodose ganglion neuron types showing similarity to LTMRs. Predicted fiber type, selected relevant genes, and proposed functions are indicated. (C) NG12–NG18 nodose ganglion neurons showing similarity to polymodal nociceptors. Predicted fiber type, selected relevant genes, and proposed functions are indicated. IGLEs, intraganglionic laminar endings. Cell Reports 2019 27, 2508-2523.e4DOI: (10.1016/j.celrep.2019.04.096) Copyright © 2019 The Authors Terms and Conditions