. 2015 Jan 6;10(1):62-74.

doi: 10.1016/j.celrep.2014.12.011. Epub 2014 Dec 24.

An estrogen-responsive module in the ventromedial hypothalamus selectively drives sex-specific activity in females

Affiliations

¹ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA.
² Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
³ Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA.
⁴ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94143, USA.
⁵ Liver Center, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: john.rubenstein@ucsf.edu.
⁸ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: holly.ingraham@ucsf.edu.

PMID: 25543145
PMCID: PMC4324838
DOI: 10.1016/j.celrep.2014.12.011

An estrogen-responsive module in the ventromedial hypothalamus selectively drives sex-specific activity in females

Stephanie M Correa et al. Cell Rep. 2015.

. 2015 Jan 6;10(1):62-74.

doi: 10.1016/j.celrep.2014.12.011. Epub 2014 Dec 24.

Authors

Affiliations

¹ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA.
² Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
³ Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA.
⁴ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94143, USA.
⁵ Liver Center, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: john.rubenstein@ucsf.edu.
⁸ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: holly.ingraham@ucsf.edu.

PMID: 25543145
PMCID: PMC4324838
DOI: 10.1016/j.celrep.2014.12.011

Abstract

Estrogen-receptor alpha (ERα) neurons in the ventrolateral region of the ventromedial hypothalamus (VMHVL) control an array of sex-specific responses to maximize reproductive success. In females, these VMHVL neurons are believed to coordinate metabolism and reproduction. However, it remains unknown whether specific neuronal populations control distinct components of this physiological repertoire. Here, we identify a subset of ERα VMHVL neurons that promotes hormone-dependent female locomotion. Activating Nkx2-1-expressing VMHVL neurons via pharmacogenetics elicits a female-specific burst of spontaneous movement, which requires ERα and Tac1 signaling. Disrupting the development of Nkx2-1(+) VMHVL neurons results in female-specific obesity, inactivity, and loss of VMHVL neurons coexpressing ERα and Tac1. Unexpectedly, two responses controlled by ERα(+) neurons, fertility and brown adipose tissue thermogenesis, are unaffected. We conclude that a dedicated subset of VMHVL neurons marked by ERα, NKX2-1, and Tac1 regulates estrogen-dependent fluctuations in physical activity and constitutes one of several neuroendocrine modules that drive sex-specific responses.

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Figures

**Figure 1. Activating *Nkx2-1* neurons increases movement in female but not male mice**
**(A)** Schematic of the postnatal medial basal hypothalamus (coronal view, sagittal view in inset, modified from Allen Brain Atlas). SF-1⁺ neurons are found in the dorsomedial and central VMH subregions (grey). NKX2-1⁺ (red) and ERα⁺ (green) neurons are confined to the VMH_VL. **(B)** SF-1 (green) and NKX2-1 (red) immunoreactivity in coronal sections from E11.5 or E13.5 (*Nkx2-1^f/f* or *Nkx2-1^f/+/Sf1Cre*) mice. **(C)** NKX2-1 (red) and *Cr-*-dependent βGAL (green) expression in P0 *Sf1Cre*-expressing mice. **(D)** NKX2-1 (red) and GFP (green) immunoreactivity in P0 *Sf1:TauGFP* reporter mice. **(E)** mCherry signal (no antibody, red) and NKX2-1 (green) in *Nkx2-1Cre* and *Sf1Cre* female VMH_VL. **(F)** NKX2-1 immunoreactivity (white) shown for reference and mCherry signal (dsRed immunoreactivity, red) in coronal sections from a wild-type (WT) female, *Nkx2-1Cre* female, and *Nkx2-1Cre* male mice. VMH and VMH_VL outlined in dashed lines, DMH = dorsomedial hypothalamus, ARC = arcuate nucleus, 3V = third ventricle, scale bars = 100 μM. **(G)** Metabolic chamber analysis of adult *Nkx2-1Cre* females (n = 8), *Nkx2-1Cre* males (n = 7), and *Sf1Cre* females (n=3). Oxygen consumption (VO₂) normalized to grams lean mass and total movement (total beam breaks,×axis) shown 1 h prior to and 4 h following IP injection (arrow) of saline (black circles) or CNO (red squares). Responses to CNO also shown for wild-type female (n = 5) and male (n = 3) littermates (blue triangles). Holm-Sidak multiple comparison tests were used for pairwise comparisons following significant effect of CNO by two-way repeated measures ANOVA (effect of time not shown, effect of CNO in wild-type mice NS). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. See also Figures S1 and S2.

**Figure 2. Responses to CNO are greater in *Nkx2-1Cre* mice than wild-type and *Sf1Cre* controls**
**(A)** cFOS immunoreactivity in the VMH_VL of *Nkx2-1Cre* female, Nkx2-1Cre male, and wild-type female mice 70–75 min after CNO injection. cFOS⁺ cell numbers in one VMH_VL from *Nkx2-1Cre* females (n=8) and wild-type females (n=5) analyzed 15–100 minutes after CNO injection, difference significant by two-tailed t-test and by Mann Whitney U test, scale bar = 100 μm. **(B)** Average oxygen consumption normalized to lean mass and cumulative total movement, ambulatory movement, and food intake per animal (24–148 minutes, ~1.5 h) after saline (black) and CNO (grey) in wild-type females (n=5), *Nkx2-1Cre* females (n=8), wild-type males (n = 3), and *Nkx2-1Cre* males (n = 7). Fold-changes comparing the effects of CNO (Nkx2-1Cre(CNO/Saline) / WT (CNO/Saline)) are shown for significant pairwise comparisons. Pairwise comparisons of wild-type and *Nkx2-1Cre* mice treated with CNO were performed using Holm-Sidak multiple comparison tests following significant effects of genotype in a two-way ANOVA (p<0.05 for all comparisons shown except food intake), * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. See also Figure S3.

**Figure 3. *Nkx2-1* ablation in *Nkx2-1^f/f/Sf1Cre* mice is specific to the VMH but marker gene expression is largely normal**
**(A)** NKX2-1 immunoreactivity is absent in the VMH of E13.5 or P0 *Nkx2-1^f/f/Sf1Cre* mice but unchanged in the periventricular area and adjacent nuclei, DMH = dorsomedial hypothalamus, ARC = Arcuate, 3V = third ventricle; scale bars = 500 μm. **(B)** RNA in situ hybridization analysis of VMH marker expression in E13.5 *Nkx2-1^f/f* and *Nkx2-1^f/f/Sf1Cre* mice. Coronal sections show expression of *Sf1, Fezf1* (FEZ family zinc finger 1)*, VglI1* (Vestigial like 1 homolog)*, Nkx2-2* (Nk2 homeobox 2), and *Nkx5-1* (Nk5 homeobox 1, also H6 homeobox 3, *Hmx3*). See also Figure S4.

**Figure 4. *Nkx2-1* ablation in the VMH results in female specific obesity but does not affect fertility**
**(A)** Photographs of adult *Nkx2-1^f/f* and *Nkx2-1^f/f/Sf1Cre* female mice. **(B)** Body weight curves from *Nkx2-1f/f/Sf1Cre* (grey open circles, dashed lines) males and females compared to respective control littermates (black triangles, solid lines, n = 8–11/group). **(C, D)** Mean body length, lean mass, and fat mass as determined by DEXA for *Nkx2-1^f/f/Sf1Cre* (n = 7) or *Nkx2-1^f/f* (n = 8) females at 22 weeks of age. **(E)** Mean weights of dissected visceral, subcutaneous and BAT fat depots standardized to total body weight from *Nkx2-1^f/f/Sf1Cre* (n = 5) or *Nkx2-1^f/f* (n = 6) females at 22 weeks of age. Plasma leptin (ng/mL) from females analyzed during estrus (n = 2/group). **(F)** Representative images of H&E staining of gonadal WAT (gWAT) and BAT in 30 week-old *Nkx2-1^f/f/Sf1Cre* (n = 3) or *Nkx2-1^f/f* (n = 2) females. **(G)** Transcript expression in BAT from 10 week-old *Nkx2-1^f/f/Sf1Cre* or *Nkx2-1^f/f* females (n = 4/group). **(H)** Pups per litter produced from mating male and female controls (*Nkx2-1^f/f*), *Nkx2-1^f/f/Sf1Cre* females with a control males or *Nkx2-1^f/f/Sf1Cre* males with control females (n ≥ 14 litters/group). Plasma estradiol during estrus (pg/mL, n = 2/group), ovarian histology, and number of corpora lutea per section per ovary (n = 4 mice/group). See also Figure S5.

**Figure 5. Obese *Nkx2-1* mutant females exhibit reduced physical activity**
**(A)** Daily (24 h) food intake for 8–22 week old singly-housed *Nkx2-1^f/f/Sf1Cre* (n = 7) or *Nkx2-1^f/f* female mice (n = 9) over 6 days after 7 days of acclimation, shown relative to total body mass. Food spillage was accounted for in food weight measurements. **(B)** VO₂ (mL/h) and heat (kcal/h) per mouse in the light phase (12 h, left panels) or dark phase (12 h, right panels) for 22-week old singly housed *Nkx2-1^f/f/Sf1Cre* (n = 6) or *Nkx2-1^f/f* (n = 8) females. Averages (± SE) represent 2 days of acquisition following 2 days of acclimation in metabolic chambers. Linear relationships were analyzed by ANCOVA, achieving better fit with lean mass for VO₂ and body mass for heat generated. **(C)** Total movement and Ambulatory movement in 22-week old *Nkx2-1^f/f/Sf1Cre* (n = 6) or *Nkx2-1^f/f* (n = 8) mice. **(D)** Wheel running in 7-week old *Nkx2-1^f/f/Sf1Cre* (n = 3) or *Nkx2-1^f/f* (n = 5) mice over 2 days of acquisition following 9 days of acclimation. Pairwise comparisons performed with Holm-Sidak multiple comparison tests, * p<0.05, ** p<0.01, **** p<0.0001.

**Figure 6. Obesity in *Nkx2-1^f/f/Sf1Cre* females is associated with fewer NKX2-1⁺, ERα⁺ and *Tac1*⁺ neurons in the VMH_VL**
**(A)** NKX2-1 (red) and ERα (green) immunoreactivity in the VMH and ARC of P10 *Nkx2-1^f/f/Sf1Cre* and *Nkx2-1^f/f* mice. Images show coronal brain sections, VMH, VMH_VL, and ARC bordered by dashed lines, scale bars = 100 µm, 3V = third ventricle. **(B)** Quantification of ERα⁺ nuclei in the VMH_VL or ARC from P10 *Nkx2-1^f/f/Sf1Cre* (green) or *Nkx2-1^f/f* female mice (black) (n = 2 mice/group, 3 sections/brain). Bottom panel shows double-labeling of NKX2-1 (green) and ERα (red) in the VMH_VL of P110 control females. ERα-negative/NKX2-1-positive (arrows) and ERα-positive/NKX2-1-negative nucleus (asterisk). **(C)** Quantification of cells derived from the *Sf1* lineage determined for the ventrolateral (VMH_VL), central (VMH_c), and dorsomedial (VMH_dm) subregions of *Nkx2-1^f/f/Ai14^f/+/Sf1Cre* mutant (red) and *Nkx2-1^f/+/Ai14^f/+/Sf1Cre* control (black) P10 female mice (n = 5 animals/group, 3 sections/brain). **(D)** Relative expression by qPCR of *Nkx2-1, Nmu, Tac1, Esr1,* and *Gal* from microdissected VMH of P10 *Nkx2-1^f/f/Sf1Cre* (grey) or *Nkx2-1^f/f* (black) females (n = 3–6/group). **(E)** Double-labeling of NKX2-1 immunoreactivity (green, nuclear) and *Tac1* transcripts (red, cytoplasmic) analyzed by confocal microscopy in the VMH_VL of P10 wild-type female mice. **(F)** Patterns of *Esr1* and *Tac1* transcripts by ISH in *Nkx2-1^f/f/Sf1Cre* or *Nkx2-1^f/f* P10 females. Pairwise comparisons performed with Holm-Sidak multiple comparison tests following two-way ANOVA, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. See also Figure S6.

Figure 7. The burst of physical activity after activating *Nkx2-1* VMH_VL neurons is dependent on ERα and *Tac1*
**(A, B)** ERα immunoreactivity or *Tac1* transcript in situ hybridization in *Esr1^f/f/Nkx2-1Cre* or *Tac1^−/− Nkx2-1Cre* adult female mice (n = 3–4/group). VMH_VL and ARC indicated with dashed lines, scale bars = 100 µm. **(C)** Total movement (total beam breaks, × axis) in Sham *Nkx2-1Cre* (n = 4), OVX *Nkx2-1Cre* (n=10), *Esr1^f/f/Nkx2-1Cre* (n = 4), and *Tac1^−/−/Nkx2-1Cre* (n = 6) female mice 1 h prior to and 4 h following saline or CNO injection (arrow) in a randomized balanced design. **(D)** cFOS immunoreactivity in Sham *Nkx2-1Cre*, OVX *Nkx2-1Cre, Esr1^f/f/Nkx2-1Cre*, and *Tac1^−/−/Nkx2-1Cre* female mice analyzed 1.5 h after CNO injection. **(E)** Quantification of cFOS⁺ nuclei in all subjects 1.5–3 h after injection, sample sizes as in C. **(F)** Average total movement (24–148 minutes) after saline (black bars) and CNO (colored bars), data from C for direct comparison. Pairwise comparisons performed with Holm-Sidak multiple comparison tests following two-way repeated measures ANOVA, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. See also Figure S7.

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An estrogen-responsive module in the ventromedial hypothalamus selectively drives sex-specific activity in females

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An estrogen-responsive module in the ventromedial hypothalamus selectively drives sex-specific activity in females

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