A genetic map of the mouse dorsal vagal complex and its role in obesity

Abstract

The brainstem dorsal vagal complex (DVC) is known to regulate energy balance and is the target of appetite-suppressing hormones, such as glucagon-like peptide 1 (GLP-1). Here we provide a comprehensive genetic map of the DVC and identify neuronal populations that control feeding. Combining bulk and single-nucleus gene expression and chromatin profiling of DVC cells, we reveal 25 neuronal populations with unique transcriptional and chromatin accessibility landscapes and peptide receptor expression profiles. GLP-1 receptor (GLP-1R) agonist administration induces gene expression alterations specific to two distinct sets of Glp1r neurons-one population in the area postrema and one in the nucleus of the solitary tract that also expresses calcitonin receptor (Calcr). Transcripts and regions of accessible chromatin near obesity-associated genetic variants are enriched in the area postrema and the nucleus of the solitary tract neurons that express Glp1r and/or Calcr, and activating several of these neuronal populations decreases feeding in rodents. Thus, DVC neuronal populations associated with obesity predisposition suppress feeding and may represent therapeutic targets for obesity.

Extended Data Fig. 1. Body weight and food intake for the single-nucleus RNA- and ATAC-seq in vivo study

a Daily body weight and b food intake in semaglutide-administered (n=9), ad libitum fed vehicle-administered (n=9) and weight-matched control mice (n=8). Mean ± SEM are shown. *P<0.05, **P<0.01, ***P<0.001 vs. vehicle and ^#P<0.05, ^##P<0.01, ^###P<0.001 semaglutide vs. weight-matched, linear mixed effects model, Bonferroni-adjusted least-squares means two-tailed t-test.

Extended Data Fig. 2. Expression of tanycyte-like cell marker genes

a Expression of marker genes for tanycyte-like cells. b Wt1 (n=1 mouse), c Wif1 (n=2 mice), d Slc22a3 (n=1 mouse), and e Cdon (n=1 mouse) in situ hybridization of sagittal brain sections (Allen Mouse Brain Atlas). Scale bar for panel b is representative for panels c-e, 100 μm. AP, area postrema; NTS, nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus nerve; OPCs, oligodendrocyte precursor cells; VLMCs, vascular leptomeningeal cells. Data to reconstruct panel a can be found in Supplementary Data 2.

Extended Data Fig. 3. Expression of DVC neuronal marker genes

a Expression of marker genes for different neuronal populations. From top to bottom, dendrogram illustrating the similarity of the neuronal populations computed based on their gene expression levels, heatmap depicting the gene expression specificity values (ES_μ) of the neuronal marker genes, the most likely DVC origin of the neuronal populations. b-z In situ hybridization of coronal brain sections (Allen Mouse Brain Atlas). N=1 mouse for all hybridizations except panels m and o (n=2 mice) and panels p (n=3 mice). Scale bar for panel b is representative for panels c-z, 100 μm. DVC, dorsal vagal complex; AP, area postrema; NTS, nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus nerve. Data to reconstruct panel a can be found in Supplementary Data 2.

Extended Data Fig. 4. Expression of appetite-supressing receptors in mice and non-human primates

a Representative image showing db-ISH of Casr (blue) and Glp1r (red) in non-human primates (n=2). Scale bar, 250 μm. b Representative image showing db-ISH of Gfral (blue) and Glp1r (red) in non-human primates (n=2). Scale bar, 100 μm. c Representative image showing db-ISH of Calcr (blue) and Glp1r (red) in non-human primates (n=2). Scale bar, 250 μm. d Representative image showing db-ISH of Grpr (blue) and Calcr (red) in mice (n=4). Scale bar, 100 μm. e Representative image showing db-ISH of Casr (blue) and Bdnf (red) in mice (n=4). Scale bar, 100 μm. f Representative image showing db-ISH of Grpr (blue) and Bdnf (red) in mice (n=4). Scale bar, 100 μm. g Representative image showing db-ISH of Casr (blue) and Mc4r (red) in mice (n=4). Scale bar, 100 μm. h Representative image showing db-ISH of Grpr (blue) and Mc4r (red) in mice (n=4). Scale bar, 100 μm. i-l Representative images showing TH (purple) or DDC immunoreactivity (purple) and GFP immunoreactivity (green) in Glp1r-Cre;GFP or Calcr-Cre;GFP mice (n=3). Scale bar, 150 μm. db-ISH; double in situ hybridization; AP, area postrema; NTS, nucleus of the solitary tract; GFP, green fluorescent protein.

Extended Data Fig. 5. Expression of appetite-supressing receptors and peptides in mice and non-human primates

a Representative image showing db-ISH of Calcr (blue) and Ramp3 (red) in non-human primates (n=2). Scale bar, 100 μm. b Representative image showing db-ISH of Ccbe1 (blue) and Gipr (red) in mice (n=4). Scale bar, 100 μm. c Representative image showing db-ISH of Pax5 (blue) and Gipr (red) in mice (n=4). Scale bar, 100 μm. d Representative image showing db-ISH of Gipr (blue) and Glp1r (red) in mice (n=4). Scale bar, 100 μm. e Representative image showing db-ISH of Asb4 (blue) and Gcg (red) in mice (n=4). Scale bar, 100 μm. db-ISH; double in situ hybridization; AP, area postrema; NTS, nucleus of the solitary tract.

Extended Data Fig. 6. Additional DVC neuronal populations with previously-defined functions

Expression of genes defining DVC neurons previously implicated in metabolic control. Top, expression specificity (ES_μ) of selected genes. Bottom, the most likely DVC origin of the neuronal populations. Mc4r cholinergic (Chat3) DMV neurons regulate circulating insulin, Hsd11b2 and Nr3c2 glutamatergic (Glu2) NTS neurons drive sodium appetite, and Lepr and Gal glutamatergic (Glu3) NTS neurons module breathing. DVC, dorsal vagal complex; AP, area postrema; NTS, nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus nerve. Data to reconstruct figure can be found in Supplementary Data 2.

Extended Data Fig. 7. GLP-1RA-downregulated modules

a Most enriched Gene Ontology terms for modules M18–20. Bonferroni-adjusted g:Profiler P-value. b Top 10 genes for modules M18–20. Data to reconstruct figure can be found in Supplementary Data 1, Supplementary Data 4, and through the NCBI Gene Expression Omnibus.

Extended Data Fig. 8. Expression of Bdnf and Mc4r following GLP-1RA administration

a Representative image showing db-ISH of Casr (blue) and Mc4r (red) in mice administered with Exendin-4 (IV, 200 μg/kg; n=2). Scale bar, 100 μm. b Representative image showing db-ISH of Casr (blue) and Bdnf (red) in mice administered with Exendin-4 (IV, 200 μg/kg; n=2). Scale bar, 100 μm. db-ISH; double in situ hybridization; AP, area postrema; NTS, nucleus of the solitary tract; IV, intravenous.

Extended Data Fig. 9. FOS immunoreactivity in Calcr AP and NTS cells following GLP-1RA or salmon calcitonin administration

a Representative image showing FOS immunoreactivity (purple) and GFP immunoreactivity (green) in Calcr-Cre;GFP mice administered with Exendin-4 (IP, 150 μg/kg; n=3) or vehicle (n=3). Scale bar, 150 μm. b Representative image showing FOS immunoreactivity (purple) and GFP immunoreactivity (green) in Calcr-Cre;GFP mice administered with sCT (IP, 150 μg/kg; n=3) or vehicle (n=3). Scale bar, 150 μm. AP, area postrema; NTS, nucleus of the solitary tract; GFP, green fluorescent protein; GLP-1RA, GLP-1 receptor agonist; sCT, salmon calcitonin; IP, intraperitoneal.

Extended Data Fig. 10. Activation of Calcr NTS neurons suppresses feeding

a Representative image showing mCherry immunoreactivity (pseudo-colored green) and FOS immunoreactivity (purple) after CNO treatment in Calcr-Cre mice injected with hM3Dq-mCherry in the NTS (n=7). Scale bar, 150 μm. b Long-term chow food intake and c body weight in control (n=6) and Calcr-Cre mice injected with hM3Dq-mCherry in the NTS (n=7) measured during 1 day of saline, 2 days of CNO (IP, 1 mg/kg) followed by 1 day of saline treatment. d Short-term HFD food intake in Calcr-Cre mice injected with hM3Dq-mCherry in the NTS and treated with saline (n=7) or CNO (n=7; IP, 1 mg/kg). e Long-term HFD food intake and f body weight in control (n=5) or Calcr-Cre mice injected with hM3Dq-mCherry in the NTS (n=7) measured during 3 days of saline, 2 days of CNO (IP, 1 mg/kg) followed by 2 days of saline treatment. g Short-term chow food intake in control mice injected with hM3Dq-mCherry in the NTS and treated with saline (n=6) or CNO (n=6; IP, 1 mg/kg). Mean ± SEM are shown. P<0.05 are specified, linear mixed effects model, Bonferroni-adjusted least squares means two-tailed t-test. h Long-term chow food intake in control mice injected with hM3Dq-mCherry in the NTS and treated with saline (n=6) or CNO (n=6; IP, 1 mg/kg. Mean ± SEM are shown. Linear model, least squares means two-tailed t-test. AP, area postrema; NTS, nucleus of the solitary tract; HFD, high-fat diet; CNO, Clozapine-N-oxide; IP, intraperitoneal.

Fig. 1:. Overview of transcriptional changes in response to GLP-1 receptor agonist administration

a Overview of bulk RNA-seq study design. Diet-induced obese mice were randomized into four groups; 1) semaglutide-administered (SQ, day 0, 0.02 mg/kg; day 1, 0.04 mg/kg; day 2–6, 0.1 mg/kg; n=15), 2) liraglutide-administered (SQ, day 0, 0.2 mg/kg; day 1, 0.4 mg/kg; day 2–6, 1.0 mg/kg; n=15), 3) ad libitum fed vehicle-administrated (n=15), and 4) weight-matched controls (n=15) that were dosed once daily for seven days. Six brain areas were isolated with LCM, dissociated, and subjected to RNA-seq. b Daily body weight and c food intake following GLP-1RA administration (n=15 mice/group). Mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. vehicle and ^#P<0.05, ^##P<0.01, ^###P<0.001 semaglutide and liraglutide vs. weight-matched, linear mixed effects model, Bonferroni-adjusted least-squares means two-tailed t-test. d PCA plot of samples colored by brain area. e Number of differentially expressed genes compared to weight-matched animals. Benjamini-Hochberg-adjusted DESeq2 P<0.05. f Log₂ fold-changes of differentially expressed genes for semaglutideand liraglutide-administered animals vs. weight-matched animals. g AP volcano plot for semaglutide-administered vs. weight-matched animals. Dark red indicates log₂ fold-change>0.5 and Benjamini-Hochberg-adjusted DESeq2 P<0.05, top 10 significant genes are labelled. HFD, high-fat diet; LCM, laser capture microscopy; LS, lateral septum; PVH, paraventricular nucleus of the hypothalamus; DMH, dorsomedial hypothalamic nucleus; ARH, arcuate nucleus of the hypothalamus; AP, area postrema; NTS, nucleus of the solitary tract; SQ, subcutaneous; GLP-1RA, GLP-1 receptor agonist; PCA, principal component analysis; PC, principal component; RNA-seq, RNA sequencing. Data to reconstruct panels d-g can be found in Supplementary Data 1 and/or through the NCBI Gene Expression Omnibus.

Fig. 2:. Transcriptional atlas of DVC cell populations

a Overview of single-nucleus RNA-seq study design. Diet-induced obese mice were randomized into three groups; 1) semaglutide-administered (n=7), 2) ad libitum fed vehicle-administered (n=6), and 3) weight-matched controls (n=7) that were dosed once a day for seven days as described for the bulk RNA-seq in vivo study. The AP, NTS, and DMV were dissociated and subjected to single-nucleus RNA-seq. b UMAP plot of 72,128 cells colored by cell type. c Non-neuronal marker genes. Top, number of cells per cluster. Bottom, violin plot showing the normalized transcript counts for marker genes. A black label indicates that the gene is also a marker of the cell type in the hypothalamus, a red label indicates lack of specificity in the hypothalamus. d UMAP plot of 49,392 neurons colored by population. e Enrichment of AP and NTS marker genes. Left, dots indicate the significance level of overlap between AP and NTS marker genes and cell population marker genes (ES_μ>0). Bonferroni-adjusted one-tailed Fisher’s exact test. Right, the most likely DVC origin of the cell populations. f Neuronal marker genes. From top to bottom, number of cells per cluster, violin plots of the normalized transcript count for marker genes, the most likely DVC origin of the neuronal populations. Chat, Slc32a1, Slc17a6, Th, Ddc, and Dbh were used as markers for neurotransmitter subtypes. DVC, dorsal vagal complex; AP, area postrema; NTS; nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus nerve; UMAP, uniform manifold approximation and projection; OPCs, oligodendrocyte precursor cells; RNA-seq, RNA sequencing; VLMCs, vascular leptomeningeal cells. Data to reconstruct panels b-f can be found in Supplementary Data 2–3 and/or through the NCBI Gene Expression Omnibus.

Fig. 3:. Neuronal expression of receptors and peptides involved in body weight control

a Overview of Glp1r gene reannotation. The transcript counts for Glp1r were computed following manual re-annotation to include alternative polyadenylation signals. b Cell population expression specificities of genes previously linked to body weight control. Top, CELLEX expression specificity values (ES_μ) of selected genes. Bottom, the most likely DVC origin of the neuronal populations. c Representative image showing combined IHC of GLP-1R (green) and ISH of Gfral (magenta; n=3). d Representative image showing db-ISH of Glp1r (blue) and Calcr (red; n=3). e Representative image showing combined IHC of GLP-1R (green) and ISH of Casr (magenta; n=3). f Representative image showing db-ISH of Grpr (blue) and Glp1r (red; n=4). g Representative image showing db-ISH of Ramp3 (blue) and Calcr (red; n=3). h Representative image showing db-ISH of Olfr78 (blue) and Calcr (red; n=4). Scale bar for panels c-h, 100μm. DVC, dorsal vagal complex; poly(A), polyadenylation; pol II, RNA polymerase II; AP, area postrema; NTS, nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus nerve; IHC, immunohistochemistry; ISH, in situ hybridization; db, double; ES_μ, expression specificity. Data to reconstruct panel b can be found in Supplementary Data 2.

Fig. 4:. Transcriptional changes induced by GLP-1RA administration in glutamatergic Glp1r neurons

a Modules correlated with GLP-1RA administration. The bulk RNA-seq data was clustered into modules of co-regulated genes. Median, first and third quartiles, and whiskers with minimum and maximum 1.5 interquartile ranges are shown. P<0.05 are specified, semaglutide (n=14 mice) vs. weight-matched (n=13 mice), logistic regression with Bonferroni-adjusted likelihood ratio test. b Cell population enrichment of module genes. Top, dot size indicates the significance level of overlap between module genes and cell population marker genes (ES_μ>0). Bonferroni-adjusted one-tailed Fisher’s exact test. Bottom, the most likely DVC origin of the cell populations. c Five top-most enriched Gene Ontology terms for module M1. Bonferroni-adjusted g:Profiler P-value. d Top 30 genes for module M1. Dot size indicates log₂ fold-change between semaglutide and weight-matched animals. All genes were upregulated in the AP following semaglutide administration (Benjamini-Hochberg-adjusted DESeq2 P<0.05). e Differential expression of top 10 M1 genes in Glu4^AP neurons. Mean ± SEM. P<0.05 are specified, semaglutide (784 neurons, n=7 mice) vs. weight-matched (734 neurons, n=7 mice), Bonferroni-adjusted pseudo-bulk DESeq2 P-value. f Representative images showing FOS immunoreactivity (purple) and GFP immunoreactivity (green) in Glp1r-Cre;GFP mice treated with Exendin-4 (IP, 150 μg/kg; n=3) or vehicle (n=3). Scale bar, 150μm. DVC, dorsal vagal complex; AP, area postrema; NTS; nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus nerve; GLP-1RA, GLP-1 receptor agonist; GFP, green fluorescent protein; IP, intraperitoneal. Data to reconstruct panel d can be found in Supplementary Data 1, Supplementary Data 4, and through the NCBI Gene Expression Omnibus.

Fig. 5:. Chromatin accessibility atlas of DVC cell populations

a Overview of single-nucleus ATAC-seq study design. Diet-induced obese mice were randomized into three groups; 1) semaglutide-administered (n=5), 2) ad libitum fed vehicle-administered (n=5), and 3) weight-matched controls (n=5) that were dosed once daily for seven days as described for the bulk RNA-seq in vivo study. The AP, NTS, and DMV were dissociated and subjected to single-nucleus ATAC-seq. b Fragment size distribution for single-nucleus ATAC-seq data. c UMAP plot of the 22,545 cells colored by cell type. d UMAP plot of the 11,651 neurons colored by population. e Motif enrichment in non-neuronal cells. From top to bottom, dendrogram showing the cell type similarity in motif accessibility, number of cells per cluster, most enriched motifs colored by the expression specificity values (ES_μ) of the corresponding transcription factor genes. Enriched motifs were identified using logistic regression with Bonferroni-adjusted likelihood ratio test. f Motif enrichment in neuronal populations. From top to bottom, dendrogram showing the neuronal population similarity in motif accessibility, number of cells per cluster, most enriched motifs colored by the ES_μ values of the corresponding transcription factor genes, the most likely DVC origin of the neuronal populations. g Differential motif accessibility in Glu4^AP neurons for GLP-1RA-induced transcription factors. Mean ± SEM. **P=0.007, semaglutide (182 neurons, n=5 mice) vs. weight-matched (239 neurons, n=5 mice), pseudo-bulk logistic regression with Bonferroni-adjusted likelihood ratio test. DVC, dorsal vagal complex; AP, area postrema; NTS; nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus nerve; ATAC, assay for transposase-accessible chromatin using sequencing; RNA-seq, RNA-sequencing; bp, base pairs; UMAP, uniform manifold approximation and projection; OPCs, oligodendrocyte precursor cells; VLMCs, vascular leptomeningeal cells; GLP-1RA, GLP-1 receptor agonist Data to reconstruct panels b-f can be found in Supplementary Data 5 and/or through the NCBI Gene Expression Omnibus.

Fig. 6:. Glp1r and Calcr neurons enrich for genetic variants associated with BMI

a Overview of BMI GWAS integration. The expression specificity profile (top) or the motif enrichment (bottom) for each cell population was integrated with BMI GWAS data to compute the genetic enrichment in each cell population. b BMI GWAS integration with gene expression and c motif accessibility. Top, genetic enrichment. Bonferroni-adjusted CELLECT P-value. Dashed line indicates significance threshold. Bottom, the most likely DVC origin of the neuronal populations. d Representative image showing tdTomato immunoreactivity (red) in Calcr-Cre;tdTomato reporter rats (n=5). e Representative image showing mCherry immunoreactivity (red) in Calcr-Cre injected with hM3Dq-mCherry in the AP (n=4). Scale bar for panels d-e, 200μm. f Food intake in Calcr-Cre rats injected with hM3Dq-mCherry in the AP following treatment with saline (n=4) or CNO (IP, 1 mg/kg; n=4). g Food intake in Calcr-Cre rats following treatment with saline (n=10) or CNO (IP, 1 mg/kg; n=10). Mean ± SEM are shown. P<0.05 are specified, linear mixed effects model, Bonferroni-adjusted two least squares means two-tailed t-test. BMI, body mass index; GWAS, genome-wide association study; OPCs, oligodendrocyte precursor cells; VLMCs, vascular leptomeningeal cells; DVC, dorsal vagal complex; AP, area postrema; NTS; nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus nerve; CNO, Clozapine-N-oxide; IP, intraperitoneal.

Fig. 7:. Overview of DVC neuronal populations and their role in metabolism

Single-nucleus RNA-seq profiling of 49,392 neurons in the DVC identified a total of 25 neuronal populations distributed across the AP (n=5), NTS (n=15), and DMV (n=5). Integrating the single-cell data with GWAS data for BMI showed that Glu4^AP, Glu9^NTS, Glu10^AP, and Glu11^NTS neurons expressed marker genes with human orthologs that non-randomly co-localized with BMI-associated genetic variants. Glu10^AP, Glu11^NTS, and Glu14^NTS[,38] neurons suppress feeding when activated by DREADDs. Glu4^AP neurons are activated by GLP-1RAs, Glu10^AP neurons by sCT, and Glu11^NTS neurons by GLP-1RAs and sCT. Chat3^DMV neurons regulate circulating insulin, Glu2^NTS neurons drive sodium appetite, and Glu3^NTS neurons modulate breathing via leptin-mediated mechanisms. We note that functions for other DVC populations remain to be determined. Within each of the three DVC nuclei, the spatial placement of the neuronal populations is arbitrary. Magnified neuronal shapes denote neuronal populations with genetic enrichments, potential or proven utility as targets for therapeutic intervention in obesity, or roles in central regulation of feeding or other metabolic processes. Shapes and colors indicate neurotransmitter type (circle, glutamatergic; square, GABAergic; diamond, cholinergic). DVC, dorsal vagal complex; BMI, body mass index; AP, area postrema; NTS, nucleus of the solitary tract; DMV, dorsal motor nucleus of the vagus nerve; GWAS, genome-wide association study; BMI, body mass index; DREADD, designer receptors exclusively activated by designer drugs; GLP-1RA, GLP-1 receptor agonist; sCT, salmon calcitonin.