Two layers of cell identity for the murine DVC cells from snRNA-seq data

A. Adult C57BL/6J mice were subjected to 10X Genomics-based snRNA barcoding and sequencing after dissection of their DVC (n=30). B. Labeled UMAP plot of the mouse DVC (cells n=99,740) at the lowest resolution (layer 1) including 4 cell identities, and C. the cell class (layer 2) including 18. D. Heatmap of the main marker genes (average log2FC expression >80 percentiles of upregulated genes) for each neuronal class of the mouse DVC (MAST algorithm; neurons n= 48,131; adj. p<0.05). Each column is one cell and each row is one marker gene. E. Stacked barplot of log-normalized counts of genes for 10 transcripts (i.e. Gad2, Gad1, Slc6a5, Slc17a6, Slc18a2, Addc, Sst, Npy, Cck and Slc5a7) related to transmission/release of 8 neurotransmitters and neuropeptides, by neuronal cell class. Each bar represents one neuron (n=48,131). snRNA-seq=single-nuclei RNA-sequencing; DVC=dorsal vagal complex; FACs=fluorescence-activated cell sorting; UMAP=uniform manifold approximation and projection; FC=fold-change; adj.=adjusted; GABA=gamma-aminobutyric acid

Third layer of non-neuronal murine DVC cell identities

A. Labeled UMAP plot of the mouse DVC with the 15 non-neuronal cell identities shown (total cells n=99,740; non-neuronal cells n=51,609). B. UMAP plot showing scaled expression of Gfap, Gria2 and Kcnj3 genes in Ca+-permeable astrocytes (circled; total cells n=99,740; Ca+-permeable astrocytes n=1,719). C. Barplot of log-normalized expression of six genes in oligodendrocytes (premyelinating n=267; myelinating intermediate n=25,692; myelinating n= 6,170). Only premyelinating oligodendrocytes lack expression of Mobp, involved in myelin formation. Reduction of Synpr and Opalin expression is observed as Anln and Aff3 increase in myelinating oligodendrocytes. D. Balloon plot of the main cell types of non-neuronal cells and neurons showing average log-normalized counts of their marker genes (MAST algorithm; adj. p<0.05). Markers shown are upregulated genes in >80% of cells per group with average log2FC>4, or upregulated in >70% of cells with average log2FC>8.

DVC=dorsal vagal complex; UMAP=uniform manifold approximation and projection; APNTS=Area postrema and nucleus of the solitary tract; OL=oligodendrocyte; OPC=oligodendrocyte precursor cell; Smc=smooth muscle cells

Neuronal populations of the murine DVC

A. Labeled UMAP plot with the neuronal layer 3 cell identities (neurons n=48,131). B. Monoamine class balloon plot showing average log-normalized expression of marker genes for each cell identity (MAST algorithm; M0 cells n=770, M1 cells n=551, M2 cells n=666 and M3 cells n=2,593; adj. p<0.05), and C. heatmap of expression of monoamine related genes. In the heatmap, each column represents one cell and each row, one gene. D. Barplot of Gcg average log-normalized expression by neuronal cell identity. E. UMAP plot showing the expression of leptin receptor and prolactin receptor genes in neurons. The GLP1 cluster is highlighted (GLP1 cells n=153). F. Stacked barplot of the proportion of cells expressing one or more GABA-related genes (i.e. Slc32a1, Gad1, Gad2) and glutamate-related genes (i.e. Slc17a6, Slc17a7). The proportion of cells per cell group co-expressing GABA and glutamate associated genes are shown in purple. Only cells with log-normalized counts >0 were considered to express the genes.

DVC=dorsal vagal complex; UMAP=uniform manifold approximation and projection; Res.=resolution; GABA=gamma-aminobutyric acid

Th and Cck co-expression in the DVC

A. UMAP plot of neuronal Th and Cck scaled expression and cells with co-expression of both genes (neurons n=48,131; Th-expressing neurons n=764; Cck-expressing neurons n=3,821; Th/Cck co-expressing neurons n=80). On the right plot, cells highlighted in magenta are the co-expressing neurons. The three neuronal cell identities in which the majority of nuclear co-expression of Th and Cck mRNA is found, as well as the percentage of total co-expressing neurons, are shown. B. In-situ hybridization of Cck and Th mRNA in coronal mouse DVC sections corresponding to -7.2mm and -7.56mm relative to bregma showing overlap of Th/Cck. Some of the cells with overlapping signal are highlighted with an arrow in the enhanced merged image.

DVC=dorsal vagal complex; UMAP=uniform manifold approximation and projection

The murine DVC cell hierarchy

A. Dendrogram of the harmonized hierarchy which incorporates cells from Ludwig and our datasets. Orange cell identities are a fourth layer of cell identity resolution obtained as some of the Ludwig dataset identities are subgroups of our original cell identities at their highest resolution. Magenta labels represent layer 3 of cellular granularity. These two layers are considered high-resolution. B. UMAP plot of the integration between our and Ludwig datasets using treeArches (cells n=171,868). C. Pairwise heatmap showing the proportion of cells originally labeled in this study and in Ludwig dataset (y-axis), predicted to belong to each identity group (x-axis) by treeArches using our learned harmonized hierarchy. In blue are the labels considered non-specific for a high-resolution cell identity (n=2,324; 1.3%). In pink are the cell labels for rejected cells, therefore not assigned any identity by treeArches (n=3,108; 1.8%). D. UMAP plot of the Dowsett dataset labeled through treeArches using the learned harmonized hierarchy representation from our murine DVC atlas. The ‘unlabeled’ cells include those rejected by the algorithm and thus without an assigned identity, and those with an unspecific layer-3/4 label. For example, some were labeled ‘neuron’ or ‘monoamine-class’ but without a high-resolution cell identity.

UMAP=uniform manifold approximation and projection; APNTS=Area postrema and nucleus of the solitary tract; Lnc=long non-coding; OL=oligodendrocyte; OPC=oligodendrocyte precursor cell; Neur=neuronal; COE=collier/Olf1/EBF transcription factor

The snRNA-seq derived cell identities for the rat DVC

A. Schematic of the pipeline for snRNA-seq of the rat DVC. B. Labeled UMAP plots of the low resolution (i.e. layers 1 and 2 of granularity) and C. high resolution (i.e. layer 3) cell identities in our rat DVC dataset (cells n=12,167). We labeled four layer-1, nineteen layer-2 and fifty-two layer-3 cell identities. Those labels novel to this dataset and not present in the murine data are highlighted with a pink • symbol. We found a small cluster (cells n=35) that we could not corroborate its identity at any layer of cellular granularity that we named ‘unspecific’. snRNA-seq=single-nuclei RNA-sequencing; DVC=dorsal vagal complex; FACs=fluorescence-activated cell sorting; UMAP=uniform manifold approximation and projection; APNTS=Area postrema and nucleus of the solitary tract; Lnc=long non-coding; OL=oligodendrocyte; OPC=cursor cell; Neur=neuronal; COE=collier/Olf1/EBF transcription factors

Two novel neuronal classes specific to the rat DVC

A. Balloon plots comparing the expression of the marker genes of the two novel neuronal classes found in rats (cells n=12,167): immunity-akin and the ortus-akin classes (framed in gray). Expression is shown across the layer-2 rat cell identities. Based on the expression of two DVC neuronal markers Mtus2 and Syt1, we corroborated those cell classes contain neurons, and not microglial or vascular/endothelial cells (In orange and red, respectively). The immunity-akin class (n=48 neurons) shares high expression of Cst3, Inpp5d, Csf1r, Slco2b1 and Lyn with microglial cells. Meanwhile, the ortus-akin class (n=34 neurons) shows higher overlap of gene expression markers (i.e. Arhgap31, Pdgfra, Bcas1, Ppfibp1 and Itga9) with OPCs (highlighted with a • symbol). Mouse cell identities (cells n=99,740) do not show these expression patterns (framed in black). Average expression was calculated using log-normalized counts. B. Violin plots of scaled expression of 10 genes (i.e. Glp1r, Gfral, Calcr, Ramp3, Gipr, Lepr, Cckar, Cckbr,Mc4r and Prlr) coding for metabolism-associated receptors in the Pdgfra and immunity-related rat neurons (Pdgfra neurons n=34; immunity-related neurons n=48). Pdgfra neurons are the only layer-3 identity within the ortus-akin class. An overlapping dotplot shows one dot per cell. C. Immunofluorescent detection of PDGFRA in mouse and rat DVC. Pink arrowheads point to cells with OPC morphology, white arrowheads point to cells with fibroblast morphology and the yellow arrowhead points to cells with neural morphology. D. Co-staining for PDGFRA and HuC/D in rat DVC with high magnification (right images) of area postrema PDGFRA-expressing cells . Yellow arrowheads indicate colocalization of HuC/D and PDGFRA.

DVC=dorsal vagal complex; OPC=oligodendrocyte precursor cell; AP=area postrema; NTS=nucleus of the solitary tract; PDGFRA= Platelet-derived growth factor receptor A; CC=central canal

The rodent DVC cell hierarchy

A. UMAP plot of the integration between the murine hierarchy and our rat dataset using treeArches (cells n=184,035). B. Dendrogram of the harmonized hierarchy of our labeled rat dataset and the murine DVC hierarchy. We highlight the incorporated rat cell identities (blue and magenta). Those in magenta are the novel rat identities established by us. The immunity-akin and the ortus-akin classes were not found in the murine datasets. The M3 and M5 rat identities are subgroups of a Ludwig layer 4 cell identity, therefore yields a fifth layer within the monoamine neuronal class.

UMAP=uniform manifold approximation and projection; DVC=dorsal vagal complex; APNTS=Area postrema and nucleus of the solitary tract; Lnc=long non-coding; OL=oligodendrocyte; OPC=oligodendrocyte precursor cell; Neur=neuronal; COE=collier/Olf1/EBF transcription factors

Meal-related transcriptional changes in the mouse DVC

A. Horizontal barplots of the number of differential genes between refed and ad libitum fed, and refed and fasted mice in glial cells and neurons per layer-2 cell identity (MAST algorithm on log-normalized counts; adj. p<0.05; refed neurons n=14,396; refed glial cells n=18,019; ad libitum fed neurons n= 9,885; ad libitum fed glial cells n=4,610; fasted neurons n=8,829; fasted glial cells n=12,496). B. Venn Diagrams of the differentially expressed genes in magnaclass 1 and magnaclass 2 neurons between refed and ad libitum fed, and between refed and fasted treatments (MAST algorithm on log-normalized counts). The number of overlapping log2FC≥1 upregulated genes between the two magnaclasses per treatment are highlighted. The treatment-induced changes in only one cell class are shown as non-overlapping. The percentage is based on the total differential genes surveyed per comparison. C. Volcano plots of the differential genes in neurons belonging to the magnaclass 1 and the magnaclass 2 between refed and ad libitum fed, and D. refed and fasted treatments (MAST algorithm on log-normalized counts; adj. p<0.05). Each point represents one gene. Only genes upregulated or downregulated with a log2FC≥2 are labeled. Since we considered a minimum log fold-change of 0.1 between treatments per cell group, those genes with very low variance (i.e. log fold-change<0.1) were excluded from the differential expression analysis, therefore a variable number of genes are shown per comparison per identity, in the volcano plots.

DVC=dorsal vagal complex; vr=versus; OPC=oligodendrocyte precursor cell; FC=fold-change; adj.=adjusted; NS=non-significant