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.
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The source code used to analyse the data and produce the statistical figures is available at https://github.com/perslab/Ludwig-2021/.
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Novo Nordisk Foundation Center for Basic Metabolic Research is an independent research centre, based at the University of Copenhagen, and partially funded by an unconditional donation from the Novo Nordisk Foundation (https://www.cbmr.ku.dk/; grant no. NNF18CC0034900). We acknowledge the Novo Nordisk Foundation (grant no. NNF16OC0021496 to T.H.P.) and the Lundbeck Foundation (grant no. R190-2014-3904 to T.H.P.). We acknowledge J. J. Thompson from the Single-Cell Omics Platform at the Novo Nordisk Foundation Center for Basic Metabolic Research for help with uploading the data to the GEO database. Furthermore, this work was supported by a research grant from the Danish Diabetes Academy, which is funded by the Novo Nordisk Foundation (grant no. NNF17SA0031406). Studies at the University of Michigan were funded by the NIH (P01DK117821 to M.G.M.), BioPharmaceuticals R&D, AstraZeneca (to M.G.M.), the American Diabetes Association (1-16-PDF-021 to W.C.) and supported by the Michigan Diabetes Research Center Molecular Genetics Core (P30DK020572).
P.B. is employed by Gubra. S.J.P., S.N.H., A.S., L.B.K. and C.P. are employed by Novo Nordisk A/S. C.J.R. is employed by AstraZeneca and holds stock in the company. All other authors declare no competing interests.
Peer review information Nature Metabolism thanks Zachary Knight, Karolina P. Skibicka and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Body weight and food intake for the single-nucleus RNA- and ATAC-seq in vivo study.
a,b, Daily body weight (a) and food intake (b) in semaglutide-administered (n=9), ad libitum-fed vehicle-administered (n=9) and weight-matched control mice (n=8). Values are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001 versus vehicle and #P<0.05, ##P<0.01, ###P<0.001 semaglutide versus weight-matched, linear mixed effects model, Bonferroni-adjusted least-squares means two-tailed t-test.
a, Expression of marker genes for tanycyte-like cells. b-e, ISH of sagittal brain sections (Allen Mouse Brain Atlas) of Wt1 (n=1 mouse) (b), Wif1 (n=2 mice) (c), Slc22a3 (n=1 mouse) (d) and Cdon (n=1 mouse) (e). Scale bar for panel b is representative for panels c-e, 100 μm. Data for reconstruction of a are available in Supplementary Data 2.
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 (ESμ) of the neuronal marker genes, the most likely DVC origin of the neuronal populations. b-z, ISH of coronal brain sections (Allen Mouse Brain Atlas). N=1 mouse for all hybridizations except panels m and o (n=2 mice) and panel p (n=3 mice). Scale bar for panel b is representative for panels c-z, 100 μm. Data for reconstruction of a are available in Supplementary Data 2.
a, Representative image showing db-ISH of GFRALASR (blue) and GLP1R (red) in non-human primates (n=2). Scale bar, 250 μm. b, Representative image showing db-ISH of CALCR (blue) and GLP1R (red) in non-human primates (n=2). Scale bar, 100 μm. c, Representative image showing db-ISH of CASR (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.
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.
Expression of genes defining DVC neurons previously implicated in metabolic control. Top, expression specificity (ESμ) of selected genes. Bottom, the most likely DVC origin of the neuronal populations. Mc4r cholinergic (Chat3) DMV neurons regulate circulating insulin39, Hsd11b2 and Nr3c2 glutamatergic (Glu2) NTS neurons drive sodium appetite40 and Lepr and Gal glutamatergic (Glu3) NTS neurons module breathing41. Data for reconstruction of figure are available in Supplementary Data 2.
a, Representative ic of Casr (blue) and Mc4r (red) in mice administered with Ex4 (i.v., 200 μg kg−1; n=2). Scale bar, 100 μm. b, Representative image showing db-ISH of Casr (blue) and Bdnf (red) in mice administered with Ex4 (i.v., 200 μg kg−1; n=2). Scale bar, 100 μm. i.v., 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 Ex4 (i.p., 150 μg kg−1; 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 (i.p., 150 μg kg−1; n=3) or vehicle (n=3). Scale bar, 150 μm.
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,c, Long-term chow food intake (b) and body weight (c) 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 (i.p., 1 mg kg−1) 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 (i.p., 1 mg kg−1; n=7). e,f, Long-term HFD food intake (e) and body weight (f) 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 (i.p., 1 mg kg-1) 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 (i.p., 1 mg kg-1; n=6). Values are the mean ± s.e.m. 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 (i.p., 1 mg kg−1; n=6). Values are the mean ± s.e.m. Linear model, least squares means two-tailed t-test.
Supplementary Figs. 1–3
Differentially expressed genes in bulk RNA-seq data.
CELLEX scores for cell populations identified in the snRNA-seq data.
AP and NTS marker genes.
Gene modules and their association with treatment.
Motif enrichment for cell populations identified in the snATAC–seq data.
Meta-analysis of CELLECT results.
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Ludwig, M.Q., Cheng, W., Gordian, D. et al. A genetic map of the mouse dorsal vagal complex and its role in obesity. Nat Metab 3, 530–545 (2021). https://doi.org/10.1038/s42255-021-00363-1
Nature Metabolism (2021)