Hypothalamic oestrogen receptor alpha establishes a sexually dimorphic regulatory node of energy expenditure

Abstract

Oestrogen receptor alpha (ERα) signalling in the ventromedial hypothalamus (VMH) contributes to energy homeostasis by modulating physical activity and thermogenesis. However, the precise neuronal populations involved remain undefined. Here, we describe six neuronal populations in the mouse VMH by using single-cell RNA transcriptomics and in situ hybridization. ERα is enriched in populations showing sex-biased expression of reprimo (Rprm), tachykinin 1 (Tac1) and prodynorphin (Pdyn). Female-biased expression of Tac1 and Rprm is patterned by ERα-dependent repression during male development, whereas male-biased expression of Pdyn is maintained by circulating testicular hormone in adulthood. Chemogenetic activation of ERα-positive VMH neurons stimulates heat generation and movement in both sexes. However, silencing Rprm gene function increases core temperature selectively in females and ectopic Rprm expression in males is associated with reduced core temperature. Together, these findings reveal a role for Rprm in temperature regulation and ERα in the masculinization of neuron populations that underlie energy expenditure.

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Fig. 1: Sf1 lineage tracing allows for targeted scRNA-seq of the VMH.
Fig. 2: scRNA-seq reveals nonoverlapping gene expression signatures in the VMH.
Fig. 3: Tac1, Rprm and Pdyn are sexually dimorphic genes in the adult VMHvl.
Fig. 4: Sex-biased transcripts are restricted to the ventrolateral VMH.
Fig. 5: Female-biased gene expression is established organizationally during development in the VMH.
Fig. 6: Specific activation of Esr1+ neurons in the VMHvl causes enhanced movement and thermogenesis in male and female mice.
Fig. 7: Temperature is dysregulated in female but not male mice lacking Rprm.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The single-cell RNA-sequencing data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE143818. The Cellprofiler pipeline used to quantify fluorescent images and source data for Fig. 2e are presented with the paper.

Code availability

Custom R scripts written for single-cell analyses and for FISH quantification, statistics and plotting are available at https://github.com/jevanveen/vanveenkammel.

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Acknowledgements

The research was supported by UCLA Division of Life Sciences funds to S.M.C., NIH grant no. K01 DK098320 to S.M.C., NIH grant no. UL1TR001881 and Iris Cantor-UCLA Women’s Health Center/UCLA National Center of Excellence in Women’s Health Pilot Awards to S.M.C. and Z.Z., UCSD/UCLA Diabetes Research Center NIH grant no. P30 DK063491 Pilot and Feasibility awards to S.M.C. and M.L., NIH grant nos. DK104363 and DK117850 to X.Y., NIH grant nos. HD076125 and HL131182 to A.P.A., UCLA Department of Medicine Chair Commitment and NIH grant no. AA026914 to M.L., predoctoral NRSA (grant no. F31 AG051381) and Hyde Fellowship to L.G.K., UCLA Dissertation Year Fellowships to L.G.K. and D.A., Canadian Diabetes Association Postdoctoral fellowship to M.S., American Heart Association Postdoctoral Fellowship (grant no. 18POST33960457) to Z.Z. and NSF Graduate Research Fellowship to M.G.M. We thank C. De La Cruz for technical assistance. This work was supported by the following facilities at UCLA: the UCLA Translational Pathology Core Laboratory, the MCDB/BSCRC Microscopy Core, the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Flow Cytometry Core Resource, and the TCGB Technology Center for Genomics and Bioinformatics (supported by grant no. P30 CA016042).

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J.E.V., L.G.K. and S.M.C. conceived and designed the studies. J.E.V., L.G.K., P.C.B., M.S., M.S.R., J.W.P., Z.Z., M.G.M., A.M.J., H.H. and S.M.C. acquired and analysed data. J.E.V., L.G.K., P.C.B., M.S., D.A., M.L., A.P.A., X.Y. and S.M.C. contributed to data interpretation. J.E.V., L.G.K. and S.M.C. wrote the manuscript with substantial input from M.S., Z.Z., M.G.M., D.A., M.L., A.P.A. and X.Y.

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Correspondence to Stephanie M. Correa.

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Extended data

Extended Data Fig. 1 FACS strategy for the isolation of Sf1 lineage cells.

Related to Fig. 1. a, Gating of all events using forward scatter area (FSC-A) and side scatter area (SSC-A) to select for probable cellular objects (gate p1) based on size and internal complexity. b, FSC and SSC gating to remove doublets (gate p2,p3). c, Gating using live cell permeable (Alexa 700,DRAQ5) and live cell impermeable (DAPI) DNA dyes. Objects displaying high DRAQ5 and low DAPI are nucleated and alive (gate live-1). d, Gating to select nucleated single cells displaying red fluorescence (gate p4). TdTomato+ cells were sorted into 96 well plates for downstream scRNA-Seq. e, Hierarchy of populations demonstrating that tdTomato+ (p4) cells comprise ~4% of all live, nucleated objects obtained from rough dissection and dissociation of hypothalami. f, graphs comparing FSC-A and SSC-A of live, nucleated tdTomato- objects to live, nucleated tdTomato+ objects. FACS plots are representative of two separate experiments comprised of n = 3 female mice and n = 3 male mice.

Extended Data Fig. 2 The transcriptional architecture of the VMH is similar in males and females.

Related to Fig. 2. a, tSNE showing all clusters identified by bioinformatic analyses including those predicted to be from the arcuate nucleus of the hypothalamus (Pomc) and rare non-neuronal cells (Apoe) b, tSNE showing that male and female neurons are present in all clusters identified.

Extended Data Fig. 3 Clustering and expression of broadly expressed VMH markers and markers outside of the VMH.

Related to Fig. 3. a, Hpcal1 expression appears diffusely in the adult male (image from Allen Brain Atlas), top, and female VMH, below (representative of images from n = 5 mice). b, Gal expression is restricted to scattered cells in the adult male (image from Allen Brain Atlas), top, and female VMH, below (representative of images from n = 1 mouse). c, expression of Tac1, Rprm, Pdyn, and Sst in the retrochiasmatic area (RCH) and ventral premammillary nucleus (PMv), adjacent to the VMH along the rostral-caudal axis in males (n = 3 mice) and females (n = 4 mice). Scalebars = 100μm, DAPI shown in cyan.

Extended Data Fig. 4 Limited overlap in expression of Sst with Tac1 or Rprm.

Related to Fig. 4. a, Sst (yellow) and Rprm (magenta) transcripts in the VMHvl (images representative of n = 5 female mice). b, Sst (yellow) and Tac1 (magenta) transcripts in the VMHvl (images representative of n = 5 female mice). c, Sst transcripts are sparse but often associated with ERα expression (images representative of n = 6 female mice). d, quantification of Sst transcripts and ERα immunoreactivity confirms that while the majority of ERα expressing cells do not co-express Sst, the majority of Sst expressing cells co-express ERα (n = 6 female mice).

Extended Data Fig. 5 Activational effects of hormones maintain male-biased expression of Pdyn.

Related to Fig. 5. a, four core genotypes of mice (n = 2 animals for all GDX panels, n = 3 animals for sham panel) analysis showing that Pdyn expression is maintained in adult males by circulating testicular hormone. b, four core genotypes analysis confirming that there is no sex difference in Sst expression(n = 2 animals for all GDX panels, n = 3 animals for sham panel). Dashed line shows boundary of VMH and VMHvl, in blue for male and magenta for female. Scalebars = 200μm.

Extended Data Fig. 6 Chemogenetic activation of Esr1+ VMHvl neurons enhances BAT thermogenesis.

cFOS immunoreactivity in wild-type (Esr1Cre-negative, n = 2 animals) or Esr1Cre (Esr1Cre-positive, n = 3 animals) littermate female mice perfused 90 minutes after CNO injection. Scalebar = 200 μm. b, Image quantification, mean±SEM shown. c, Infrared thermography of male and female Esr1Cre mice (n = 6: 4 male mice + 2 female mice) injected with AAV-DIO-hM3Dq-mCherry, 30 minutes before (Pre-Tx) and 60 minutes after injection with CNO or saline (Post-Tx). Dashed line indicates interscapular region directly above BAT. d, quantification of shows a rise in intrascapular temperature following treatment with CNO compared to saline treatment in the same animals on a different day. Two-way RM ANOVA: pre vs post: F(1,10) = 6.331, p = .0306. Sidak’s multiple comparisons test: pre vs post CNO: t = 2.763, p = .0397; pre vs post saline: t = .7954, p = .6918.

Extended Data Fig. 7 Depletion of Rprm in the VMH enhances BAT thermogenesis.

Representative thermal images of female mice injected with either Rprm targeting or non-targeting siRNA pools. b, Quantification of thermography shows a significant increase in skin temperature above the interscapular BAT depots in ovariectomized (OVX) female mice injected with Rprm targeting siRNA pools (n = 6 animals) compared to OVX female mice injected with non-targeting siRNA pools (n = 6 animals) (Two-way RM ANOVA: siRNA type (F(1,5) = 16.16, p= 0.0101); hormone treatment (F(1,5) = 0.2471, p = 0.6402); interaction (F(1,5) = 0.0005832, p = 0.9817). The effect of Rprm depletion on BAT is not changed by estrogen replacement (mean±SEM shown). c, Quantification of thermography shows no significant difference in tail skin temperature in ovariectomized (OVX) female mice injected with Rprm targeting siRNA pools (n = 6 animals) compared to OVX female mice injected with non-targeting siRNA pools (n = 6 animals) (mean±SEM shown). d, representative images of BAT histology showing a slight decrease in lipid content in female mice injected with Rprm targeting siRNA pools (n = 8 animals) as compared to female mice injected with non-targeting siRNAs (n = 8 animals). BAT was collected 14 days after siRNA injection. e, Representative images of BAT histology in male mice with developmental ablation of hypothalamic ERα (Esr1fl/fl; Nkx2-1Cre, n = 5 animals) or littermate controls (Esr1fl/fl, n = 5 animals). Scalebars = 50μm.

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van Veen, J.E., Kammel, L.G., Bunda, P.C. et al. Hypothalamic oestrogen receptor alpha establishes a sexually dimorphic regulatory node of energy expenditure. Nat Metab 2, 351–363 (2020). https://doi.org/10.1038/s42255-020-0189-6

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