Altered energetic homeostasis in female Esr1 Nkx2-1Cre mice

In Esr1Nkx2-1Cre mice, ERα is efficiently eliminated in the entire MBH in both male and female brains, including the ARC and VMHvl, but is largely maintained in the anteroventral periventricular nucleus (AVPV), preoptic area, nucleus tractus solitarius, and medial amygdala (Fig. 1a and Supplementary Figure 1A). Peripheral tissues including the lung, thyroid, BAT, and pituitary maintained Esr1 expression in mutant females compared to wild-type females (Supplementary Figure 1B). Deleting ERα in the MBH depleted primordial follicles, and led to female infertility, and uterine imbibition (Supplementary Figures 1C–E). Mutant females displayed a small but significant increase in body weight, which was less robust than reported for Esr1POMC-Cre12, whereas body weights for mutant males decreased (Fig. 1b). Food intake was unchanged in both sexes (Fig. 1c).

Fig. 1 Ablating ERα in MBH impairs energy expenditure and increases bone density. a Immunohistochemistry of ERα (green) or native TdTomato (TdT (Ai14); red) on coronal brain sections (scale bar = 100 µm) of Esr1fl/+; Ai14fl/+; Nkx2-1Cre (control) or Esr1fl/fl; Ai14fl/+; Nkx2-1Cre (mutant) from females or males (bottom panels). Lp lateroposterior. Fl/+ = heterozygous for Esr1 floxed allele. b Body weight curves of control (black line) or mutant females (red line) (F 1,250 = 57.01, p < 0.0001) and males (grey line) (F 3,316 = 25.39, p < 0.0001) fed on standard chow from 3 weeks of age. c Daily food intake per animal over 24 h determined in CLAMS (controls (black bars) mutant females (red bars) and mutant males (grey bars)). d Lean mass and e averages of ambulatory movement per animal over 12 h determined by metabolic chamber analyses for Esr1fl/fl and Esr1Nkx2-1Cre female and male cohorts, female ambulatory movement (F 1,36 = 10.14, p = 0.003). c–e Animals are 8–9-week old. f Representative images of hematoxylin and eosin (H&E) staining of BAT Esr1fl/fl and Esr1Nkx2-1Cre females housed at 22 °C (8–16 weeks). Scale bar = 100 μm. g Serum leptin levels in 9-week-old control (open bars bar, n = 5) and mutant females (red bar, n = 5) (center line = median; bounds extend minimum to maximum). h BMD measured by DEXA in Esr1fl/fl (black circles) and Esr1Nkx2-1Cre females (red squares, 16–23 weeks) and males (grey squares, 11–18 weeks). Unless otherwise indicated, number per group for female controls (n = 11) and mutants (n = 9) and for male controls (n = 4) and mutants (n = 5). Error bars are SEM. Two-way ANOVA (b, e). Unpaired Student’s t tests (c, d, g, h). For all figures, p values = *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. NS = p > 0.05 Full size image

Esr1Nkx2-1Cre females exhibited a sex-dependent change in energy balance that was entirely absent in male mice. The lean mass of mutant females was significantly higher than control floxed (Esr1fl/fl) littermates (Fig. 1d) and was accompanied by decreased physical activity during the dark phase (Fig. 1e and Supplementary Figure 2C). Although highly significant, increased lean mass observed in mutant females failed to change their overall mobility and muscle strength as measured by rotarod and grip strength assays, respectively (Supplementary Figure 2D, E). Blunted BAT thermogenesis was observed in mutant females as evidenced by whitening of BAT and decreased Ucp1 levels; circulating catecholamines were not lower (Fig. 1f and Supplementary Figures 2F, G). Serum leptin levels were also unchanged (Fig. 1g). Thus, these data reveal that central estrogen signaling in this brain region promotes a sex-dependent negative energy state in females in the absence of any change in feeding behavior. This unexpected finding implies that the hyperphagia reported for Esr1POMC-Cre mice might result from selective or ectopic activity of POMC-Cre in non-ARC neurons8.

Elevated bone density in female Esr1 Nkx2-1Cre mice

Strikingly, bone mineral density (BMD), as determined by dual X-ray absorptiometry (DEXA), was significantly elevated in Esr1Nkx2-1Cre females, but not males (Fig. 1h), consistent with the sex-dependent significant increases in lean mass. Further analyses of femoral bone, using three-dimensional high resolution micro-computed tomography (µCT), confirmed a striking increase in trabecular bone mass and microarchitecture in older Esr1Nkx2-1Cre females compared to control littermates (Fig. 2a). Mutant females exhibited a ~500% increase in fractional bone volume in the distal femur, rising from 11 to 52 bone volume/tissue volume (BV/TV) (%) (Fig. 2a). A similar trend was found for vertebral bone (Supplementary Figure 3A). Accompanying structural changes included increases in trabecular number and thickness and reduced trabecular separation (Fig. 2a). Mutant females also exhibited a significant increase in cortical thickness but a modest decrease in tibial and femoral length (Supplementary Figure 3B). This striking skeletal phenotype is sex-dependent, as no changes in bone mass were observed in Esr1Nkx2-1Cre males (Fig. 2b). Further, unlike the 20% increase in femoral bone mass reported for Esr1POMC-Cre and Esr1Nestin-Cre mice that vanishes in OVX females, bone parameters in Esr1Nkx2-1Cre females remained elevated 5 weeks following ovariectomy (Fig. 2c). In fact, no significant changes in serum sex steroids (E2, T) were detected in 4–5-week-old mutant females when the high bone mass phenotype is clearly present (Fig. 2d, f). The percentage of bone loss in OVX female mutants is higher than their wild-type littermates (72% versus 43%), consistent with the finding by others that a higher starting %BV/TV at baseline results in higher OVX-mediated bone loss26. Pituitary and thyroid hormones in mutant females were also unchanged at 7–8 weeks of age (Supplementary Figure 3C). Removing circulating androgens in juvenile Esr1Nkx2-1Cre males by castration failed to elevate their bone mass, implying that male gonadal hormones are unable to account for the lack of high bone mass in Esr1Nkx2-1Cre males (Supplementary Figure 3D). These data imply that while the high BMD in Esr1Nkx2-1Cre females is partially maintained by ovarian steroids, elevated levels of circulating E2 or pituitary hormones are not the primary drivers of this sex-dependent bone phenotype.

Fig. 2 Sex-dependent increase in bone mass and strength in Esr1Nkx2-1Cre females. Representative μCT 3D reconstruction images of distal femurs in ∼24-week-old Esr1fl/fl and Esr1Nkx2-1Cre a females, b males, and c OVX females (15–21 weeks). Right panels show quantitative morphometric properties of distal femurs showing fractional bone volume (BV/TV (%)); trabecular number (Tb. N) trabecular thickness (Tb. Th), and separation (Tb. Sp). For females (n = 11) controls and (n = 8) mutants and for males (n = 3, 3). For OVX (n = 11) controls and (n = 8) mutants. Esr1fl/fl (black boxes), Esr1Nkx2-1Cre females (red bars) and males (grey bars). d LC–MS/MS of plasma E2 and T for Esr1fl/fl (n = 9, open box) and Esr1Nkx2-1Cre (n = 11, red box) juvenile females at 4.5 weeks of age (center line = median; bounds extend minimum to maximum). e Scatter plot of mechanical testing of distal femurs and L5 vertebral bodies (33 weeks) Esr1fl/fl (black squares), Esr1Nkx2-1Cre (red squares). f BV/TV (%) of the distal femur generated by either μCT or 2D histomorphometric analysis over time from 4.5 weeks of age to 54–74 weeks of age, for genotype (F 1,50 = 172.1, p < 0.0001), animal number in each Esr1fl/fl and Esr1Nkx2-1Cre group for 4.5 weeks (n = 3, 4), 12 weeks (n = 5, 6), 22.5 weeks (4, 5), 33 weeks (7, 9), and 54+ weeks (9, 7). g Representative μCT images of age-dependent changes in femoral bone mass, as well as image of cortical bone at the tibial fibular joint (TFJ) in females (20 weeks), and L5 vertebral bodies (33 weeks) in Esr1fl/fl and Esr1Nkx2-1Cre females. h Representative H&E staining of female femurs from Esr1fl/fl and Esr1Nkx2-1Cre juvenile females at 4.5 weeks. Error bars are ±SEM (standard error of the mean). Two-way ANOVA (e), Unpaired Student’s t test (a–d, f). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bars = 100 μm Full size image

Mechanical bone strength tests established that femora and L5 vertebrae in older Esr1Nkx2-1Cre females were substantially stronger than controls (Fig. 2e). The dense skeletal phenotype in Esr1Nkx2-1Cre females observed in femoral and vertebral trabecular bone emerged early and continued to persist in older females (54–74 weeks), exceeding values found for OVX mutant females (Fig. 2c, f, g). Thus, trabecular bone, which becomes porous and more fragile in osteoporosis, is remarkably dense and durable in older Esr1Nkx2-1Cre females. Upregulation of bone metabolism in Esr1Nkx2-1Cre females was not associated with ectopic Cre expression in femoral bone (Supplementary Figure 3F). Representative H&E stained femoral bone sections from juvenile female mice illustrate the striking increase in bone density accompanied by a marked decrease in bone marrow space (Fig. 2h). Despite a narrowing of the bone marrow cavity, no differences in spleen weights were observed in mutant females compared to controls at all ages examined (Supplementary Figure 3E).

Elevated bone formation rate in Esr1 Nkx2-1Cre female femurs

Young Esr1Nkx2-1Cre females showed a significant increase in bone formation rate (BFR) and mineralized surface (Fig. 3a, b), demonstrating robust osteoblast function. Both the mineral apposition rate (MAR) and normalized osteoclast number were unaffected in mutant bone, implying that significant decreases in osteoclast number and function are unable to account for the high bone mass phenotype (Fig. 3b and Supplementary Figure 3G). A similar trend was observed in older Esr1Nkx2-1Cre females after maximal bone density is achieved (Supplementary Figure 3G). Based on transcriptional profiling, gene changes in mutant bone marrow included upregulation of BMP signaling and osteoblast differentiation/ossification (Supplementary Dataset 1) with a concomitant elevation of Sp7 (Osterix), Wnt10b, Bglap (Osteocalcin), Sost, and osteoclast markers in mutant bone (Fig. 3c, d). While Runx2 was unchanged in mutant bone chips harvested from long bones minus endplates, this osteoblast precursor marker was modestly increased in female bone marrow when examined at 4.5 weeks of age (Fig. 3d). Markers for chondrocyte differentiation (Sox927) and those that would indicate a change in sympathetic tone28,29, were unchanged (Supplementary Table 1 and Dataset 1), whereas markers of interferon (IFN) signaling (Oas2, Oas3, Itga11, Gbp6, and Gbp4) and cartilage (Cola2) were elevated (Fig. 3c, Supplementary Figure 3H and Dataset 1), consistent with the estrogen-independent increases in bone mass following interferon-gamma treatment30. Collectively, these data suggest that ablating ERα in the MBH leads to an expansion of bone marrow stromal cells or adult skeletal stem cells31, fated for osteoblast differentiation/proliferation that give rise to mature bone and cartilage.

Fig. 3 Increased bone formation in Esr1Nkx2-1Cre females. a Representative images of labeled mineralized surface of distal femur with calcein (green) and demeclocycline (orange) over a period of 1 week in a 12 week old female. Scale bars = 50 μm. b Dynamic histomorphometric results for Esr1fl/fl (n = 4, black bars) and Esr1Nkx2-1Cre (n = 4, red bars) 12–14 week females showing bone formation rate (BFR), mineralized surface (MS), and mineralized apposition rate (MAR). Number of active osteoclasts normalized to bone surface quantified by TRAP-positive staining determined in distal femurs from 5- to 7-week-old Esr1fl/fl (n = 5) and Esr1Nkx2-1Cre (n = 6) females. c Heat map of top 50 differentially expressed genes (DEGs) up and down in 4.5-week-old Esr1fl/fl and Esr1Nkx2-1Cre bone marrow Esr1fl/fl (n = 4) and Esr1Nkx2-1Cre (n = 4) females. BMP regulated genes (red) and IFN regulated genes (blue). d Quantification of indicated transcripts marking pre-osteoblasts, osteocytes, and osteoclasts in 4.5–7-week female control (n = 10) and mutant (n = 7) flushed bone marrow (BM), or in female control (n = 13) and mutant (n = 8) femur bone chips. Error bars are ±SEM. Unpaired Student’s t test (b, d). *p < 0.05; ****p < 0.0001. NS = p > 0.05 Full size image

Elevated bone mass after stereotaxic deletion of ERα in ARC

To unequivocally establish that the high bone mass phenotype in Esr1Nkx2-1Cre females arises specifically from loss of ERα signaling in the brain, stereotaxic delivery of AAV2-Cre was used to eliminate ERα in either the VMHvl or the ARC (referred to as ERαKOVMHvl or ERαKOARC, respectively, Fig. 4a). Adult Esr1fl/fl females injected with either AAV2-GFP (control) or AAV2-Cre were evaluated for ERα expression (Supplementary Figures 4A, B). Successful hits were defined as partial or full loss of ERα on one or both sides of the VMHvl or ARC. As noted for Esr1Nkx2-1Cre females, eliminating ERα in the ARC but not the VMHvl fully recapitulated the significant increase in BMD without changing food intake or E2, T, Leptin, and uterine weights (Fig. 4b–d and Supplementary Figure 5A), disentangling the high bone mass phenotype in ERαKOARC females from changes in these circulating hormones. Strikingly, just 12 weeks postinfection (PI), ERαKOARC females showed a similar massive elevation in fractional femoral bone volume, which was accompanied by an increase in trabecular number and thickness and decreased bone marrow space (Fig. 4e–g), as well as a modest increase in osteoprotegerin and elevated SOST (Supplementary Figure 5A). Cortical bone thickness was also enhanced without affecting the cortical perimeter (Fig. 4f and Supplementary Figure 5B).

Fig. 4 Bone volume in intact and OVX female mice after acute loss of ERα in ARC. a Schematic of stereotaxic delivery of AAV2-GFP or AAV2-Cre-GFP to 16-week-old Esr1fl/fl females to either the VMHvl or ARC regions to delete ERα 5-week postinfection (PI). b Food intake for AAV2-GFP (black bars), AAV2-Cre-VMHvl (blue bars) and AAV2-Cre-ARC (red bars). c LC–MS/MS plasma E2 and uterine weight for control (n = 6, open box) and for ERαKOARC (n = 9–11, red box) (center line = median; bounds are minimum to maximum). d Scatter plot of BMD for controls, ERαKOVMHvl, and ERαKOARC females. e Representative images of distal femur (H&E) in control and ERαKOARC females. f μCT images with morphometric properties of distal femur for controls (n = 4) and ERαKOARC (n = 5) and tibio-fibular joint in control (n = 4) and ERαKOARC (n = 4) females. Scale bar = 100 μm. g Representative μCT images of distal femur in OVX females 5- and 12-week postinfection, showing AAV2-Cre hit to ARC (Cre-Hits, Red line), AAV2-Cre miss to ARC (Cre-Miss, black line and AAV2-GFP to ARC (blue line)). Schematic of time line for in vivo bone imaging from 5- to 12-week PI, with graph showing percent change in volumetric bone normalized to 5 week PI. Cre-Hits (n = 5), Cre-Miss (n = 4), and GFP (n = 5), AAV2-Cre Hit versus GFP or Miss (F 2,40 = 56.9, p < 0.0001). Error bars are ±SEM. Student’s unpaired t test (d, f). Two-way ANOVA (g). *p < 0.05; **p < 0.01; ***p < 0.001 Full size image

The impact of viral-mediated deletion of ERα in the ARC was assessed over time in older OVX females that partially model postmenopausal bone loss. As expected, in vivo imaging showed that volumetric bone in OVX females dropped rapidly after ovariectomy, dipping by half from ~11 to 5.6 ± 1.3 SEM %BV/TV for all cohorts. Bone mass declined further to ~3.1 ± 0.9 SEM %BV/TV 5-week PI—the time period required to achieve complete ERα deletion in the ARC32. While bone density continued to deteriorate in control groups where ERα remained intact (GFP or Miss), complete or partial loss of ERα in the ARC (Hits) resulted in a remarkable ~50% increase in bone volume 12-week PI despite the mature age (38 weeks) of these older females (Fig. 4g and Supplementary Figure 4C). In sum, these data using both intact and older estrogen-depleted females suggest that the increased bone formation observed in Esr1Nkx2-1Cre females is central in origin, thus supporting the existence of a robust estrogen-sensitive neuroskeletal circuit.

Altered Kiss1 neuron transcripts in Esr1 Nkx2-1Cre females

To assess molecular changes in the ARC that are associated with upregulation of bone metabolism in Esr1Nkx2-1Cre females, transcriptional profiling was performed. Using microdissected female ARC tissue from controls and mutants, we defined ~180 DEG significantly changed in Esr1Nkx2-1Cre mutants (Fig. 5a, b). Of those transcripts, 83% overlapped with genes that are known to be regulated by estrogen (Fig. 5c) as illustrated by loss of Greb1, a highly responsive ERα gene target33. Strikingly, however, more than 100 differentially regulated transcripts were distinct from the well-characterized markers of either POMC or AgRP neurons (Fig. 5c and Supplementary Dataset 2). Among significantly downregulated genes, four transcripts are associated with dopaminergic neurons: the dopamine transporter Slc6a3 (DAT), the synaptic vesicle glycoprotein Sv2c, the transcription factor Nr4a2, and the prolactin receptor Prlr (Fig. 5a). After loss of ERα, Slc6a3 is downregulated in the ARC consistent with Slc6a3 upregulation by E2 in cultured cells34 (Fig. 5d, e). Accordingly, we find that the majority of DAT-positive neurons in the dorsal medial ARC coexpress ERα, by means of an Slc6a3Cre;Tdtomato reporter line (Fig. 5e). Another triad of DEGs is Kiss1, Pdyn, and Tac2 (Fig. 5b) that together with the glutamate transporter Slc17a6 define KNDy ( K isspeptin, N eurokinin B, D ynorphin) ARC neurons35,36. As expected and based on the dynamic transcriptional repression of KNDy markers by estrogen20, both Kiss1 and Pdyn are elevated in Esr1Nkx2-1Cre mutants (Fig. 5d).

Fig. 5 Bone formation correlates with changes in KNDy and DAT ARC neurons. a Heat map of top 25 most significant DEGs in ARC of Esr1fl/fl (n = 3) and Esr1Nkx2.1Cre females (n = 3) (15 weeks). b Volcano plot of data set with highlighted genes (red). Dashed red line represents significance cutoff with adjusted p value < 0.05). c Overlap of DEGs with adjusted p value < 0.05 and |log2 fold change| > 0.6 between ARC from Esr1fl/fl and Esr1Nkx2.1Cre mice (red) with identified ERα agonist-responsive transcripts (white)61,62 and with identified markers of AgRP and POMC neurons (grey)63. d Expression of Esr1, Kiss1, Pdyn, and Slc6a3 measured by qPCR (n = 4–6 per genotype with controls as open boxes and Esr1Nkx2.1Cre females as red boxes; (center line = median; bounds are minimum to maximum). e Representative ISH of Slc6a3 in ARC and confocal image of ARC co-labeled with Slc6a3 reporter (Ai9, tdT), ERα and DAPI. Image scale bars for top and bottom panels = 100 µm. Student’s unpaired t test (d). Error bars are ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 Full size image

Deleting ERα signaling in Kiss1 cells elevates bone mass

Given that the gene signature of KNDy neurons is altered in Esr1Nkx2-1Cre females, we deleted ERα in Kiss1 cells (Esr1Kiss1-Cre) using a Kiss1-Cre-GFP knockin allele to ask if we might identify which estrogen-responsive ARC neurons drive the robust female bone phenotype. As the majority of Kiss1 neurons share a common lineage with POMC neurons in development37, we also deleted ERα in POMC neurons (Esr1POMC-Cre). While ERα was partially ablated in the Esr1POMC-Cre ARC, we failed to detect a trabecular or cortical bone phenotype previously reported for Esr1POMC-Cre females7 (Fig. 6a and Supplementary Figures 6A, B); our negative results could stem from strain, dosage or transmission (paternal versus maternal) differences. In stark contrast, after confirming loss of ERα in all Kiss1 ARC neurons in Esr1Kiss1-Cre females (Fig. 6b and Supplementary Figure 6C), both juvenile and older mutant females displayed a striking increase in bone mass that was easily visualized by the naked eye (Fig. 6c), with values reaching 88% BV/TV for the distal femur; mutant L5 vertebrae and cortical bone mass were similarly affected (Fig. 6d and Supplementary Figure 6D). The striking elevation in bone density in Esr1Kiss1-Cre females exceeded the observed bone mass in Esr1Nkx2-1Cre females at all ages, perhaps reflecting the narrow versus broad expression of these two different Cre drivers. Similar to Esr1Nkx2-1Cre mice the bone phenotype is limited to females and appears to be independent of high E2 levels (Fig. 6d and Supplementary Figure 6E), consistent with the findings that deleting ERα with other Kiss1-Cre knockin alleles accelerates pubertal onset in female mice without altering negative feedback19,38. Although E2 levels were unchanged in juvenile Esr1Kiss1-Cre at 4.5–19 weeks, it is possible that the higher average volumetric bone mass for the distal femur observed in Esr1Kiss1-Cre compared to Esr1Nkx2-1Cre females (79 ± 4.8 versus 52 ± 4.5 %BV/TV) results from the premature postnatal LH surge, as noted by others19,39. As might be predicted with extremely dense bone and probable bone marrow failure, spleen weights and markers of extramedullary hematopoiesis (indicated by megakaryocytes) increase significantly in females (Fig. 6e and Supplementary Figure 6F). Taken together, the contrasting bone phenotypes observed in Esr1Kiss1-Cre and Esr1POMC-Cre infer that this female-specific brain-to-bone pathway is mediated by a subset of Kiss1 neurons that arise independently of the POMC lineage37. Collectively, our data also suggest that disrupting the transcriptional output and activity of KNDy neurons breaks a brain–bone homeostatic axis that would normally restrain anabolic bone metabolism.