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A molecular census of arcuate hypothalamus and median eminence cell types

Nature Neuroscience volume 20, pages 484496 (2017) | Download Citation

Abstract

The hypothalamic arcuate–median eminence complex (Arc-ME) controls energy balance, fertility and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a new leptin-sensing neuron population, multiple agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) subtypes, and an orexigenic somatostatin neuron population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type–specific responses to energy status, including distinct responses in AgRP and POMC neuron subtypes. Finally, integrating our data with human genome-wide association study data implicates two previously unknown neuron populations in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred.

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Acknowledgements

We gratefully acknowledge Z. Yang, J. Madara and C. Wu for technical assistance, A. Garfield for editorial advice and P. Kharchenko for advice on SCDE. Quantitative PCR and confocal imaging were done at BIDMC's Molecular Medicine Core and Confocal Imaging Core, respectively. Funding was provided by US National Institutes of Health grants to B.B.L. (R01 DK096010, R01 DK089044, R01 DK071051, R01 DK075632, R37 DK053477, BNORC Transgenic Core P30 DK046200, BADERC Transgenic Core P30 DK057521), E.D.R. (R01 DK102170, R01 DK085171, R01 DK102173), E.D.R. and L.T.T. (BNORC Functional Genomics Core P30 DK046200), L.T.T. (BADERC Pilot and Feasibility grant NIH 2P30DK057521-16) and J.M.R. (F32 DK103387); a Department of Defense grant to L.T.T. (Discovery Award W81XWH-15-1-0251); an American Heart Association Postdoctoral Fellowship to J.N.C. (14POST20100011); the Lundbeck Foundation and the Benzon Foundation (T.H.P.); the Stanley Center for Psychiatric Research (S.A.M.); and the Stanley-MGH Fellowship in Psychiatric Neuroscience (E.Z.M.).

Author information

Affiliations

  1. Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

    • John N Campbell
    • , Henning Fenselau
    • , Anna Lyubetskaya
    • , Danielle Tenen
    • , Anne M J Verstegen
    • , Jon M Resch
    • , Evan D Rosen
    • , Bradford B Lowell
    •  & Linus T Tsai
  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Evan Z Macosko
    • , Melissa Goldman
    •  & Steven A McCarroll
  3. Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

    • Evan Z Macosko
    • , Melissa Goldman
    •  & Steven A McCarroll
  4. Program in Medical and Population Genetics, Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

    • Evan Z Macosko
    •  & Steven A McCarroll
  5. The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.

    • Tune H Pers
  6. Department of Epidemiology Research, Statens Serum Institut, Copenhagen, Denmark.

    • Tune H Pers
  7. Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, USA.

    • Steven A McCarroll
    •  & Bradford B Lowell
  8. Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

    • Steven A McCarroll
    •  & Evan D Rosen

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Contributions

J.N.C., L.T.T., E.Z.M., S.A.M., E.D.R. and B.B.L. conceived the study. J.N.C., L.T.T., E.Z.M., E.D.R. & B.B.L. designed the study. J.N.C., A.M.J.V. and L.T.T. prepared samples for Drop-seq. L.T.T., D.T., J.N.C., E.Z.M. and M.G. did Drop-seq. D.T., M.G. and L.T.T. made Drop-seq libraries. J.N.C. did single-cell RNA-seq. L.T.T., J.N.C., A.L. and E.Z.M. analyzed transcriptomic data. J.M.R. did in situ hybridization. J.N.C. and H.F. did histology and imaging, with advice from B.B.L. H.F. did electrophysiology. H.F. and A.M.J.V. did stereotaxic injections and feeding studies. T.H.P. performed DEPICT analyses. J.N.C., L.T.T. and A.L. prepared figures. J.N.C., L.T.T., S.A.M., E.D.R. and B.B.L. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Evan D Rosen or Bradford B Lowell or Linus T Tsai.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8

  2. 2.

    Supplementary Methods Checklist

Excel files

  1. 1.

    Supplementary Table 1: The number and percentage representation of cells within each all-cell and neuron cluster parsed by experiment, age, sex, and dietary intervention.

    Number and percentage are presented in reference to total cells/neurons evaluated (top) or total Arc-ME cells/neurons (bottom) to account for dissection artifacts. Likely sex-of-origin of Fasted and Re-fed cells was determined using MLSeq package (see Online Methods). Red lettering indicates non-arcuate cell clusters.

  2. 2.

    Supplementary Table 2: For each all-cell cluster, a table of fold-change values for genes differentially expressed with false-discovery rate (FDR) <25% in at least one comparison, sorted based on specificity (i.e., number of positive fold-change values) and average expression.

    Fold-change values with FDR>25% are indicated by zeroes.

  3. 3.

    Supplementary Table 3: For each all-cell subcluster, a table of fold-change values for genes differentially expressed with false-discovery rate (FDR) <25% in at least one comparison, sorted based on specificity (i.e., number of positive fold-change values) and average expression.

    Fold-change values with FDR>25% are indicated by zeroes.

  4. 4.

    Supplementary Table 4: For each neuronal cluster, a table of fold-change values for genes differentially expressed with false-discovery rate (FDR) <25% in at least one comparison, sorted based on specificity (i.e., number of positive fold-change values) and average expression.

    Fold-change values with FDR>25% are indicated by zeroes.

  5. 5.

    Supplementary Table 5

    For each all-cell cluster from fed vs. fasted mice, tables of fold-change values, false-discovery rates (FDR), and fold-change values with at least one significant comparison (FDR<25%).

  6. 6.

    Supplementary Table 6

    For each all-cell cluster from low-fat diet vs. high-fat diet fed mice, tables of fold-change values, false-discovery rates (FDR), and fold-change values with at least one significant comparison (FDR<25%).

  7. 7.

    Supplementary Table 7

    For each neuronal cluster from fed vs. fasted mice, tables of fold-change values, false-discovery rates (FDR), and fold-change values with at least one significant comparison (FDR<25%).

  8. 8.

    Supplementary Table 8

    For each neuronal cluster from low-fat diet vs. high-fat diet fed mice, tables of fold-change values, false-discovery rates (FDR), and fold-change values with at least one significant comparison (FDR<25%).

  9. 9.

    Supplementary Table 9

    (DEPICT_GWAS Sources Tab) Overview of GWAS p value cutoffs used to define loci, the number of resulting DEPICT loci (defined by linkage disequilibrium r2>0.5), the number of genes in DEPICT loci, PubMed identifiers of the publications from which the GWAS summary statistics were sourced, and links to the GWAS summary statistics file. For the GWAS only waist hip ratio summary statistics, please contact the authors. (TRH_Lef1_CandidateObesityGenes Tab) Evidence in support of inclusion as candidate gene for obesity with increased expression in the n25.Trh/Lef1 neuron cluster, adapted from Vimaleswaran et al. DEPICT_Statistics tab. (Slc17a6_Trhr_CandidateObesityGen Tab) Evidence in support of inclusion as candidate gene for obesity with increased expression in the n32.Slc17a6/Trhr neuron cluster, adapted from Vimaleswaran et al. (DEPICT_Statistics Tab) Summary DEPICT p-values and FDR for included GWAS phenotypes represented in Figure 8 and Supplemental Figure 8.

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https://doi.org/10.1038/nn.4495

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