DOI: 10.5061/dryad.bzkh189qm
title: Data from: Sex-specific thermoregulatory effects of estrogen signaling in <em>Reprimo</em> lineage cells

description: Estrogens have considerable impact on energy homeostasis and metabolic
 health. In mice, signaling through estrogen receptor alpha (ERα) alters
 energy intake and expenditure, effects that may be mediated by specific
 regions or cellular sub-populations of the hypothalamus. This study
 investigates the function of ERα signaling in the lineage that expresses
 Rprm (Reprimo), a gene we previously linked to thermoregulation in
 females. Here, we engineered a novel ReprimoCre mouse to selectively knock
 out ERα in Rprm lineage cells (Reprimo-specific ERα KO; RERKO). We report
 modest changes in core temperature, higher brown adipose tissue (BAT)
 mass, elevated BAT temperature during the light phase, and lower tail
 temperature during the light phase in females relative to controls. RERKO
 females also exhibited a subtle difference in locomotion and no
 differences in feeding or body mass. These phenotypes suggest sex-specific
 effects on the patterns of body temperature instead of overall increases
 or decreases in heat generation or dissipation. Labeling of the Rprm
 lineage was detected in the brain, but not in BAT or white adipose,
 suggesting that temperature changes may be mediated by the nervous system.
 To test for centrally mediated effects on temperature, we ablated Rprm
 expressing cells in the mediobasal hypothalamus. Although this approach
 eliminates the cells entirely instead of selectively eliminating ERα in
 Rprm expressing cells, we observed a phenotype similar to RERKO mice, with
 effects on core temperature and BAT mass. Together, these results indicate
 that estrogen signaling in the Rprm lineage is important for
 thermoregulation in female, but not male, mice. This file shares the
 supplementary data relevant to this publication. 
description: # Data from: Sex-specific thermoregulatory effects of estrogen signaling
 in *Reprimo* lineage cells Dataset DOI:
 [10.5061/dryad.bzkh189qm](https://doi.org/10.5061/dryad.bzkh189qm) ##
 Description of the data and file structure Body mass, body composition
 measurements, and estrous cycle staging are shared in CSV files from
 control and RERKO mice (Reprimo-specific Esr1 KO). We also share
 supplementary figures and statistical tables.  ### Files and variables
 #### File: Table_1._Statistical_details_of_all_tests.csv (on Zenodo)
 **Description:**  results of statistical tests for each figure in the
 manuscript.   #### File: Supplementary_Figures_and_Legends.docx (on
 Zenodo) **Supplemental Figure 1:** ***Rprm*** **lineage tracing reveals
 robust expression in the brain and peripheral tissues.** Heterozygous
 *Rprm^Cre^* mice were bred with *Ai14*^f/f^ mice to produce
 *Rprm^Cre^;Ai14* ^f/+^ mice expressing tdTomato in *Rprm*lineage tissues.
 Cre-negative littermates (WT*;Ai14* ^f/+^ *mice*) were used to establish
 baseline tdTomato levels. (A-C) Representative images of tissues harvested
 from adult (13-15 weeks old) *Rprm^Cre^;Ai14* ^f/+^ and WT*;Ai14* ^f/+^
 mice. (A) Left – images highlighting tdTomato expression throughout the
 whole brain in male mice. Right – 30 µm DAPI-stained coronal brain
 sections emphasize the expression of tdTomato in the cortex, thalamus, and
 hypothalamus across representative bregma levels. Scale bars = 1 mm. (B)
 Peripheral tissues exhibiting tdTomato expression include the uterus,
 ovary, pancreas, and pituitary in female mice. (C) Peripheral tissues that
 do not express tdTomato include adipose tissue depots (BAT, iWAT, and
 gWAT) in female mice. Brightfield images displayed on top, fluorescent
 images below. Scale bars = 200 µm. BAT = brown adipose tissue, iWAT =
 inguinal white adipose tissue, gWAT = gonadal white adipose tissue.
 **Supplemental Figure 2:**  **Knock-in construct does not alter**
 ***Rprm*** **expression in the MPO and VMH.** *Rprm*+ cell counts were
 compared between *Rprm^Cre^**^^*** and wildtype C57BL/6J female mice
 (n=5-9 per group) in the medial preoptic area (MPO) (A) and ventromedial
 hypothalamus (VMH) (B). Statistical analysis performed using Welch’s
 t-test. **Supplementary Figure 3. Longitudinal body weight analysis of
 RERKO mice.** (A) Body mass measurements of control (black) and RERKO
 (blue) male mice from 5 to 16 weeks of age (n = 16 WT, n = 18 RERKO). (B)
 Body mass measurements of control (black) and RERKO (pink) female mice
 from 5 to 16 weeks of age (n = 13 WT, n = 13 RERKO). In females, we only
 detected an effect of age (F~11,238~ = 61, *p* &lt; 0.0001). Mean ± SEM is
 plotted at each time point. A mixed-effects model using Restricted Maximum
 Likelihood (REML) estimation was used for statistical analysis due to
 missing values. Pairwise comparisons at each hourly timepoint were not
 significant using Šídák's multiple comparisons test. **Supplementary
 Figure 4. RERKO female mice have disrupted reproduction.** (A) Images
 depict vaginal lavages from WT (left) and RERKO (right) mice stained with
 Giemsa. While the WT mice display expected cellular morphological changes
 across the estrous cycle, swabs from RERKO mice mostly contain leukocytes.
 (B) The percent of days across 14 days spent in diestrus [D], proestrus
 [P], estrus [E], and metestrus [M] in WT (left) and RERKO (right) mice. C)
 Dissected ovarian and uterine mass from control (black) and RERKO (gray)
 mice. (D) Ovarian histology visualized with hematoxylin & eosin stain.
 Follicular cysts are present and the corpus luteum is absent in the ovary
 from RERKO mice. CL = corpus luteum, F = ovarian follicles, Cy = cyst.
 Scales are 100 µm (ovary) and 500 µm (uterus). T-test used to compare
 groups. The mean ± SEM are depicted. Individual data points are shown in
 Figure D.  *p* &lt; 0.05, *p* &lt; 0.0001. **Supplementary Figure 5. The
 estrous cycle in controls and mice with** ***Reprimo*****+ cells in the
 MBH ablated. The percent of days across 7 days spent in diestrus [D],
 proestrus [P], estrus [E], and metestrus [M] in controls expressing GFP
 (left) or caspase (right). n = 2 GFP, n = 6 caspase. #### File:
 RERKO_longitudinal_bodymass_males_females.csv First column contains the
 mouse ID.  Second column contains the sex of the mouse (Male or female; M
 or F) and Genotype (control or RERKO; WT or KO).  Rest of the columns
 contains body mass (in grams) for each mouse for that week of age
 (starting from 5 weeks until 16 weeks of age).  Empty cells indicate
 measurements were not taken at that time point for these mice.  #### File:
 RERKO_EchoMRI_data.csv A separate cohort of mice were weighed and placed
 into an EchoMRI^tm^ (Houston, TX, USA) to record fat and lean mass at 16
 weeks of age. First column contains "Label" (mouse ID).  Second
 column reports fat mass (grams), third column reports lean mass (grams),
 fourth reports "free water" (grams), and fifth "total
 water" (grams). Weight of mouse is reported in the sixth column
 (grams). "Gene" column contains the sex of the mouse (Male or
 female; M or F) and Genotype (control or RERKO; WT or KO).  Last two
 columns show percent fat and lean mass per mouse ([depot mass / total body
 mass] x 100).  #### File: RERKO_estrouscycle.csv For estrous cycling,
 vaginal lavages were deposited onto slides, stained with Giemsa (0.6% in
 1x phosphate buffered saline;) to visualize nucleic acid and distinguish
 cell morphology, and imaged using light microscopy. The estrous stage was
 determined by the relative amounts of leukocytes and cornified and
 nucleated epithelial. First column contains the mouse ID. Rest of the
 columns estrous stage calls for each day for 14 consecutive days. D =
 diestrus, P = proestrus, E = estrus, M = metestrus.