Regulation of physical activity and energy expenditure through Phf6 in the medial preoptic area

Domain: biology
Published: 2026-03-24
Editorial outcome: minor_revision

Abstract
Obesity arises from disrupted energy homeostasis, yet the neural mechanisms linking transcriptional regulation to energy expenditure remain unclear. Here, we identify plant homeodomain finger protein 6 (Phf6), a gene mutated in Börjeson-Forssman-Lehmann syndrome (BFLS), as a pivotal regulator of energy balance. Phf6 is enriched in a subset of estrogen receptor 1 (Esr1)-expressing neurons within the hypothalamic medial preoptic area (MPOA). Knockout of Phf6 in the MPOA leads to obesity in a sex-dependent manner by reducing physical activity and energy expenditure, independent of food intake. In female mice, MPOAPhf6 neurons respond to physical activity. Activation and inhibition of MPOAPhf6 neurons increases and decreases physical activity and energy expenditure, respectively. Phf6 sustains the intrinsic excitability of MPOAPhf6 neurons and their responsiveness to estrogen. Circuit mapping identified an MPOAPhf6-VMHvlEsr1 pathway mediating Phf6’s effect on metabolism. These findings reveal a neurobiological basis for BFLS-associated obesity and highlight potential therapeutic targets.

Introduction
Obesity arises from disrupted energy homeostasis caused by imbalances between energy intake and expenditure1,2,3. Physical activity-associated thermogenesis, including non-exercise activity thermogenesis and exercise-induced thermogenesis, constitutes a major dynamic contributor to total energy expenditure and body weight regulation in both humans and rodents4,5,6. Within the central nervous system, the hypothalamus integrates metabolic and hormonal signals to orchestrate physical activity and energy metabolism, involving nuclei such as the ventrolateral subdivision of ventromedial hypothalamus (VMHvl), lateral hypothalamus (LH), paraventricular nucleus of the hypothalamus (PVH), arcuate nucleus (ARC), and medial preoptic area (MPOA)7,8,9,10,11,12,13. These regions form interconnected circuits modulated by neuropeptides, hormones, and transcription factors that regulate physical activity and energy expenditure14,15,16.

The medial preoptic area (MPOA) plays a crucial role in regulating thermoregulation and monitoring metabolic state17,18. The MPOA is enriched with estrogen receptor 1 (Esr1)-expressing neurons, which are of molecular heterogeneity and contribute to diverse functions, including thermogenesis, sexual behavior, and metabolic control12,19,20,21. Recent studies have implicated estrogen signaling through Esr1 in modulating physical activity7,8,22,23. However, it remains unclear which subpopulations of Esr1+ neurons regulate physical activity and energy expenditure, and how their excitability is molecularly tuned by transcriptional or epigenetic factors.

Plant homeodomain finger protein 6 (Phf6) is a chromatin-binding protein featuring two plant homeodomain (PHD)-like zinc fingers. Mutations in Phf6 cause Börjeson-Forssman-Lehmann syndrome (BFLS), an X-linked neurodevelopmental disorder characterized by intellectual disability and a high prevalence of obesity24,25,26. As a component of nucleosome remodeling complexes27,28,29, Phf6 plays critical roles in transcriptional and epigenetic regulation30,31,32,33. Clinical reports show that obesity affects 37% of female and 72% of male BFLS patients34,35,36,37, underscoring a strong link between Phf6 dysfunction and metabolic disturbances. Despite this, the role of Phf6 in regulating energy metabolism and the mechanisms underlying BFLS-associated obesity remain poorly understood.

In this study, we demonstrate that Phf6 is selectively enriched in a subgroup of Esr1+ neurons in the MPOA. Knockout of Phf6 in the MPOA induces obesity in a sex-dependent manner through reducing physical activity and energy expenditure. Phf6-deficient MPOAPhf6 neurons exhibit reduced intrinsic excitability and impaired estrogen responsiveness in female mice. Moreover, MPOAPhf6-VMHvlEsr1 circuit mediates the effect of Phf6 on promoting physical activity and energy expenditure. Our findings establish a neurobiological link between Phf6 dysfunction and metabolic imbalance and suggest potential therapeutic strategies for BFLS-associated and other forms of obesity.

Results
Phf6 marks a subset of Esr1-expressing neurons in the MPOA.
In the 8-week-old wild-type C57BL/6J mouse hypothalamus, Phf6 expression was highly enriched in the MPOA, extending from bregma +0.13 mm to −0.11 mm (Fig. 1a). Phf6 expression in the MPOA exhibited dynamic changes during early postnatal development but stabilized after P14, with no differences observed between sexes (Supplementary Fig. 1a, b). Co-immunofluorescence staining with anti-Phf6 and anti-Esr1 antibodies revealed that Phf6 expression was confined to Esr1-positive neurons in the MPOA, with 99.6% ± 0.3% of Phf6⁺ neurons expressing Esr1 and 45.1% ± 0.8% of Esr1+ neurons expressing Phf6 in the MPOA of 8-week-old female mice (Fig. 1b, c). These findings demonstrate that Phf6 is selectively expressed in a subset of Esr1+ neurons in the MPOA, hereafter referred to as MPOAPhf6 neurons.

To determine whether MPOAPhf6 neurons are glutamatergic excitatory neurons or GABAergic inhibitory neurons, we used the vGlut2-Cre::LSL-H2B-GFP and vGat-Cre::LSL-H2B-GFP reporter mice, in which the glutamatergic and GABAergic neurons are labeled by histone-GFP. In female mice, 31.6% ± 4.7% of Phf6-positive MPOAPhf6 neurons were vGlut2-positive, whereas 69.2% ± 4.2% were vGat-positive (Fig. 1d). In male mice, 18.0% ± 2.9% of Phf6-positive MPOAPhf6 neurons were vGlut2-positive, whereas 82.7% ± 2.0% were vGat-positive (Fig. 1e). Thus, the proportion of glutamatergic versus GABAergic MPOAPhf6 neurons is approximately 3:7 in female mice and 2:8 in male mice. Given that H2B-GFP-labeled cells in genetic reporter mice reflect developmental lineage history, they may not fully represent glutamatergic or GABAergic neuronal identity in adulthood. To selectively label adult glutamatergic and GABAergic neurons, an adeno-associated virus (AAV) expressing Cre-dependent histone-EGFP (AAV2/9-Ef1a-DIO-H2B-EGFP) was injected into the MPOA of 8-week-old female vGlut2-Cre and vGat-Cre mice. We observed that 28.7% ± 1.1% of Phf6-positive MPOAPhf6 neurons were vGlut2-positive and that 71.5% ± 2.1% were vGat-positive in the adult female MPOA (Supplementary Fig. 2a-d). Notably, the proportions of glutamatergic and GABAergic MPOAPhf6 neurons identified by virus-based labeling were indistinguishable from those obtained using genetic reporter mouse lines.

To further define the subpopulations of MPOAEsr1 neurons that include Phf6-positive cells, we analyzed previously published single-cell RNA sequencing (scRNA-seq) datasets that profile Esr1+ neurons in the MPOA20,21,38,39. Uniform manifold approximation and projection (UMAP) clustering revealed 10 distinct clusters of MPOAEsr1 neurons, with Phf6 mainly enriched in cluster #c0, and weakly expressed in clusters #c5, #c6 and #c7 (Supplementary Fig. 2e). MPOAPhf6 neurons positive for both Phf6 and Esr1 were further resolved into 4 distinct subclusters (Supplementary Fig. 2f, g). The excitatory neuronal marker Slc17a6 (vGlut2) was enriched in 2 subclusters of MPOAPhf6 neurons, whereas inhibitory neuronal markers Gad1 and Slc32a1 (vGat) were enriched in the other 2 subclusters of MPOAPhf6 neurons (Supplementary Fig. 2h-l). These scRNA-seq analyses confirm that MPOAPhf6 neurons comprise molecularly distinct glutamatergic and GABAergic subgroups, with proportions comparable to those observed by immunostaining using genetic reporter mouse lines and virus-based labeling.

Loss of Phf6 in MPOAPhf6 neurons induces obesity.
Given that Phf6 is selectively expressed in a subset of Esr1+ neurons in the MPOA, we next investigated the function of Phf6 in the MPOA by a loss-of-function strategy. We employed an AAV-Cre-mediated conditional knockout approach to selectively knock out Phf6 in MPOAPhf6 neurons. Phf6 conditional knockout (cKO) and control mice were obtained by bilaterally injecting AAVs expressing Cre recombinase, AAV2/9-CMV-Cre (or AAV2/9-CMV-GFP as the control), into the MPOA of 8-week-old female Phf6flox/flox mice, which carry loxP sites flanking exons 4 and 5 on the Phf6 gene40 (Fig. 1f). Post-hoc immunostaining confirmed efficient Phf6 deletion in AAV-Cre-injected mice, with no detectable changes in controls (Fig. 1g).

Compared to littermate controls, we found that female Phf6 cKO mice exhibited a significant obesity phenotype starting at 4 weeks post-viral injection (pvi), which persisted through 12 weeks (Fig. 1h-j). By 12 weeks pvi, body weight increased by 95.3% ± 10.1% in female Phf6 cKO mice compared to 24.8% ± 1.9% in controls (Fig. 1k). Inguinal white adipose tissue (iWAT) mass was also significantly elevated in female Phf6 cKO mice (Fig. 1l, m), indicating a pivotal role of Phf6 in regulating body weight through MPOAPhf6 neurons.

To assess whether the obesity phenotype in female Phf6 cKO mice was due to altered food intake, we measured feeding behavior under ad libitum chow diet. Compared to littermate controls, female Phf6 cKO mice showed no difference in total amount of food intake, feeding duration and feeding frequency during 24-hour periods at 4, 8, and 12 weeks pvi (Supplementary Fig. 3a-d), indicating that the Phf6-cKO-induced obesity results from mechanisms independent of food intake. Female Phf6 cKO mice also showed no difference in body temperature, as measured via interscapular brown adipose tissue (iBAT) temperature using thermal imaging camera (FLIR) (Supplementary Fig. 3e). In addition, plasma levels of hypothalamic–pituitary–gonadal (HPG) axis hormones—including estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH)—remained unchanged in Phf6 cKO females (Supplementary Fig. 4a-d). These results rule out changes in food intake, thermogenesis for body temperature, and HPG-axis hormones as contributors to the obesity observed in female Phf6 cKO mice.

Loss of Phf6 in MPOAPhf6 neurons reduces energy expenditure and physical activity.
To determine whether reduced energy expenditure underlies the Phf6-cKO-induced obesity phenotype, we analyzed metabolic and behavioral parameters using Comprehensive Laboratory Animal Monitoring System (CLAMS) metabolic cages at 4, 8, and 12 weeks pvi (Fig. 2a). We found that physical activity, measured by voluntary wheel running in CLAMS metabolic cages, was significantly reduced in female Phf6 cKO mice compared to controls (Fig. 2b-d). Female Phf6 cKO mice showed a significant reduction in counts of wheel running during the active phase (dark period: 8 PM-8 AM), in contrast to no difference during the inactive phase (Fig. 2d and Supplementary Fig. 3f). We also used the ClockLab circadian system with wheel running cages to monitor physical activity for 10 consecutive days in 12:12 light/dark conditions. Compared to littermate controls, female Phf6 cKO mice exhibited a significant decrease in the levels of physical activity, as measured by the counts of voluntary wheel running, during the active phase (dark period: 8 PM-8 AM) (Fig. 2e). These results demonstrate that conditional knockout of Phf6 in MPOAPhf6 neurons suppresses physical activity.

Compared to littermate controls, female Phf6 cKO mice exhibited significantly decreased energy expenditure (EE) at 8 and 12 weeks pvi (Fig. 2g-i), accompanied by decreased oxygen consumption (VO2) and carbon dioxide production (VCO2), while the respiratory exchange ratio (RER) remained unchanged (Supplementary Fig. 3g-i). Notably, energy expenditure reduction occurred primarily during the active phase (dark period: 8 PM-8 AM), in contrast to no difference during the inactive phase (light period: 8 AM-8 PM) (Fig. 2i and Supplementary Fig. 3j). A regression-based analysis of covariance (ANCOVA), with body mass as a covariate, confirmed a true mass-independent reduction in energy expenditure in female Phf6 cKO mice (Fig. 2f). Predicted energy expenditure normalized to 40g body weight was also significantly decreased in female Phf6 cKO mice compared to controls (Fig. 2f). These results demonstrate that loss of Phf6 in MPOAPhf6 neurons reduces energy expenditure.

Physical activity related thermogenesis includes exercise-induced thermogenesis (EIT) and non-exercise activity thermogenesis (NEAT). EIT during the active phase was significantly reduced in female Phf6 cKO mice compared to controls (Fig. 2j), whereas NEAT and basal metabolic rate (BMR) showed a slight decrease (Fig. 2k, l). These results suggest that the profound reduction in physical activity and EIT is the primary contributor to the lower total energy expenditure observed in female Phf6 cKO mice, whereas changes in BMR and NEAT play a minor role.

Considering that MPOAPhf6 neurons are a subset of MPOAEsr1 neurons, we ablated MPOAEsr1 neurons by injecting AAV2/9-CAG-DIO-taCaspase3 (or AAV2/9-Ef1a-DIO-mCherry) into 8-week-old female Esr1-Cre mice (Supplementary Fig. 4e, f). We found that ablation of MPOAEsr1 neurons also significantly reduced physical activity and energy expenditure (Supplementary Fig. 4g-k), providing supporting evidence of MPOAPhf6 neurons’ role in promoting energy expenditure and physical activity.

Phf6 regulates energy expenditure and physical activity in a sex-dependent manner.
To characterize Phf6-cKO-induced phenotypes in male mice, we knocked out Phf6 in the MPOA of 8-week-old male Phf6flox/y mice by bilaterally injecting AAV2/9-CMV-Cre (or AAV2/9-CMV-GFP as the control) into the MPOA. The efficiency of Phf6 deletion was measured by post-hoc immunostaining (Supplementary Fig. 5a-c). In contrast to the profound obesity phenotype observed in female Phf6 cKO mice, males exhibited no difference in body weight from 2 to 8 weeks pvi, yet showed a significant increase after 10 weeks pvi (Supplementary Fig. 5d). The extent of obesity in males was smaller than that observed in females (Supplementary Fig. 5h). Consistently, male Phf6 cKO mice exhibited a moderate decrease in energy expenditure and physical activity during the active period (Supplementary Fig. 5f, g). The extent of reduction in energy expenditure and physical activity in males was also smaller than that observed in females (Supplementary Fig. 5i, j). In addition, male Phf6 cKO mice showed no difference in feeding behavior or body temperature (Supplementary Fig. 5k, l). These results indicate that Phf6 functions in the MPOA in a sex-dependent manner, with females showing greater susceptibility to Phf6 loss–induced obesity and reduced physical activity and energy expenditure.

MPOAPhf6 neurons are activated by physical activity.
To genetically label MPOAPhf6 neurons, we generated a Phf6-CreER knock-in mice by inserting an IRES-CreERT2 cassette downstream of the TAG stop codon in exon 10 of the Phf6 locus (Fig. 3a). The inducible CreER enabled temporal control of recombination via tamoxifen. For validation, we injected AAV2/9-hEf1a-DIO-EGFP into the MPOA of 8-week-old female Phf6-CreER mice, followed by tamoxifen administration (100 mg/kg, i.p. for 4 days) 7 days later (Fig. 3a). Immunostaining confirmed that 96.3% ± 0.2% of EGFP+ neurons in the MPOA co-expressed Phf6 (Fig. 3b, c), verifying the targeting specificity of CreER for MPOAPhf6 neurons.

To assess whether MPOAPhf6 neurons are activated by physical activity, we performed fiber photometry to record calcium signals from MPOAPhf6 neurons in vivo. AAV2/9-hSyn-DIO-GCaMP7s was unilaterally injected into the MPOA of 8-week-old female Phf6-CreER mice and the optic fiber was implanted immediately above the MPOA. We recorded the temporally demodulated 470-nm calcium-dependent and 410-nm calcium-independent fluorescence signals from GCaMP7s-expressing MPOAPhf6 neurons in freely behaving mice (Fig. 3d). Significant increases in calcium signals from MPOAPhf6 neurons (as reflected in △F/F) were detected following the onset of the physical activity (i.e., walking and running in the horizontal plane or climbing vertically) (Fig. 3e-h). Following the onset of the physical activity, the calcium signals from MPOAPhf6 neurons showed peak responses reaching approximately 10% △F/F, averaged from three female mice (Fig. 3e-h). The 410-nm control signals showed no changes, excluding the possibility that 470-nm calcium signals recorded were artifacts. These results demonstrate that MPOAPhf6 neurons are markedly activated by physical activity.

We further validated this observation via c-Fos immunostaining. A 30-minute forced wheel running session significantly increased the proportion of c-Fos+ MPOAPhf6 neurons in 8-week-old wild-type C57BL/6J females compared to the baseline (Fig. 3i, j). In addition, more c-Fos+ MPOAPhf6 neurons were detected during the dark phase (10 PM) than the light phase (10 AM) (Fig. 3k, l), suggesting that more MPOAPhf6 neurons are activated when the mice are awake than asleep. Together, these results indicate that MPOAPhf6 neurons are selectively activated when the mice are engaged in physical activity.

Activation of MPOAPhf6 neurons increases physical activity and energy expenditure.
Given that MPOAPhf6 neurons respond to physical activity, we next tested whether activation of MPOAPhf6 neurons promotes physical activity and energy expenditure. For optogenetic activation, AAV2/9-hEf1a-DIO-Channelrhodopsin-2 (ChR2)-mCherry (or AAV2/9-Ef1a-DIO-mCherry as the control) was unilaterally injected into the MPOA of 8-week-old female Phf6-CreER mice. Optic fibers were implanted above the MPOA (Fig. 4a). Electrophysiological recordings in acute brain slices confirmed that ChR2-expressing neurons reliably fired action potentials upon 20-Hz blue light stimulation (Supplementary Fig. 6a). Physical activity was assessed in the open field of 40-cm square arena with or without light stimulation (473 nm, 5.7 mW, 20 Hz, 20 ms pulses) (Fig. 4a). Compared to the mCherry-expressing control mice, optogenetic activation of MPOAPhf6 neurons significantly increased the levels of physical activity, as shown by increased open-field movement distance (Fig. 4b, c). The ratio of time spent in the center area remained unchanged (Fig. 4o). These results suggest that optogenetic activation of MPOAPhf6 neurons is sufficient to promote physical activity without affecting anxiety level.

For chemogenetic activation of MPOAPhf6 neurons, AAV2/9-Ef1a-DIO-hM3D(Gq)-mCherry (or AAV2/9-Ef1a-DIO-mCherry as the control) was bilaterally injected into the MPOA of 8-week-old female Phf6-CreER mice. Electrophysiological recordings in acute brain slices confirmed that clozapine N-oxide (CNO, 50 μM) effectively depolarized hM3D-expressing MPOAPhf6 neurons (Supplementary Fig. 6b). Physical activity was assessed using a 15-minute open field test (OFT). The hM3D- or mCherry-expressing mice were i.p. administered with CNO (3 mg/kg) 40 minutes before the physical activity assessment (Fig. 4d). Compared to the control mice, chemogenetic activation of MPOAPhf6 neurons significantly increased the levels of physical activity, as shown by increased open-field movement distance (Fig. 4e, f) and locomotor activity in CLAMS (Fig. 4l). Time spent in the center area of the open field (Fig. 4p) and time spent in the open arms of the elevated plus maze (EPM) were unaltered upon activation of MPOAPhf6 neurons (Fig. 4r, s), suggesting no changes in anxiety. These results demonstrate that chemogenetic activation of MPOAPhf6 neurons is sufficient to promote physical activity without affecting anxiety level.

To evaluate effects on metabolism, we measured energy expenditure using CLAMS metabolic cages for a period of 2 hours. The hM3D- or mCherry-expressing mice were administered with CNO (3 mg/kg) 40 minutes before the energy expenditure assessment (Fig. 4j). Chemogenetic activation of MPOAPhf6 neurons significantly increased energy expenditure (Fig. 4k), accompanied by increased oxygen consumption and carbon dioxide production, with no difference in RER or iBAT temperature (Supplementary Fig. 7a-f). These findings demonstrate that activation of MPOAPhf6 neurons is sufficient to promote both physical activity and energy expenditure.

Inhibition of MPOAPhf6 neurons reduces physical activity and energy expenditure.
To determine whether MPOAPhf6 neuronal activity is necessary for maintaining energy homeostasis, we employed chemogenetic inhibition of MPOAPhf6 neurons by bilaterally injecting AAV2/9-Ef1a-DIO-hM4D(Gi)-mCherry (or AAV2/9-Ef1a-DIO-mCherry as the control) into the MPOA of 8-week-old female Phf6-CreER mice (Fig. 4g). Electrophysiological recordings in acute brain slices confirmed that CNO (50 μM) effectively hyperpolarized hM4D-expressing MPOAPhf6 neurons (Supplementary Fig. 6c). Physical activity was evaluated using a 15-minute OFT and a 2-hour voluntary wheel running test. The hM4D- or mCherry-expressing mice were i.p. administered with CNO (3 mg/kg) 40 minutes before behavioral testing. Compared with mCherry controls, CNO/hM4D-mediated inhibition of MPOAPhf6 neurons significantly reduced physical activity, as indicated by decreased open-field movement distance (Fig. 4h, i) and reduced voluntary wheel running in CLAMS (Fig. 4n), with no difference in the time spent in the center zone (Fig. 4q).

Energy expenditure was further assessed using CLAMS metabolic cages. Mice were i.p. administered with CNO (3 mg/kg) 40 minutes prior to the energy expenditure assessment. Chemogenetic inhibition of MPOAPhf6 neurons significantly decreased energy expenditure (Fig. 4m), accompanied by reduced oxygen consumption and carbon dioxide production (Supplementary Fig. 7i-l). Together, these findings demonstrate that MPOAPhf6 neurons are necessary for maintaining both physical activity and energy expenditure.

Phf6 sustains the intrinsic excitability of MPOAPhf6 neurons and their responsiveness to estrogen.
To elucidate how Phf6 regulates MPOAPhf6 neuronal function, we examined the electrophysiological properties of MPOAPhf6 neurons in female Phf6-deficient and control mice. To simultaneously knock down Phf6 and label MPOAPhf6 neurons with EGFP, we employed a CRISPR/Cas9-mediated genome-editing strategy to delete Phf6. AAV vector carrying triple Phf6-targeting single guide RNAs (AAV2/9-hU6-sgRNA (Phf6)-hEf1a-DIO-EGFP) was bilaterally co-injected with AAV2/9-CAG-DIO-spCas9 into the MPOA of 8-week-old female Phf6-CreER mice. The AAV vector lacking sgRNAs (AAV2/9-hU6-sgRNA(NC)-hEF1a-DIO-EGFP) was used as the control (Fig. 5a). The knockdown efficiency of Phf6 in MPOAPhf6 neurons was confirmed by post-hoc immunostaining (Fig. 5b).

Whole-cell patch-clamp recordings revealed that Phf6-deficient MPOAPhf6 neurons exhibited reduced spontaneous action potential firing rates (Fig. 5f, g), and decreased evoked-action potential firing rates in response to step-rise depolarizing current injections (Fig. 5h, i). Phf6-deficient MPOAPhf6 neurons exhibited no difference in resting membrane potential (Fig. 5d), but a significant increase in input resistance (Fig. 5e), implicating that loss of Phf6 alters voltage-gated ion channel properties that may underlie the reduced intrinsic excitability. These results suggest that Phf6 sustains the intrinsic excitability of MPOAPhf6 neurons.

Upon exposed to 100 nM estradiol (E2), a potent estrogen41,42, Phf6-deficient MPOAPhf6 neurons displayed significantly attenuated E2-induced increases in firing rate compared to control neurons (Fig. 5j, k), indicating reduced estrogen responsiveness in Phf6-deficient MPOAPhf6 neurons. To assess whether this reduced responsiveness to estrogen was due to altered Esr1 receptor expression, we performed single-molecule fluorescent in situ hybridization (smFISH) with Esr1 mRNA probe. Compared to controls, Phf6-deficient MPOAPhf6 neurons exhibited a significant decrease in the level of Esr1 mRNA (Fig. 5l, m), suggesting that decreased Esr1 expression contributes to the reduced estrogen responsiveness in Phf6-deficient MPOAPhf6 neurons. No difference in plasma level of E2 was observed in Phf6 cKO mice (Supplementary Fig. 4b), excluding altered estrogen level as a cause of reduced estrogen responsiveness in Phf6-deficient MPOAPhf6 neurons.

Characterizing downstream targets of MPOAPhf6 neurons on promoting physical activity.
To map the downstream targets of MPOAPhf6 neurons, we unilaterally injected AAV expressing Cre-dependent membrane GFP (AAV2/9-hEf1a-DIO-mGFP) into the MPOA of 8-week-old female Phf6-CreER mice (Fig. 6a). Membrane GFP expression enabled visualization of full MPOAPhf6 neuronal morphology, including axonal projections. We observed several trajectories of MPOAPhf6 axons, including the VMHvl, tuberal nucleus (TN), nucleus accumbens (NAc) and PVH (Fig. 6b).

To investigate the effects of activating each of the MPOAPhf6 neuron axon terminals on physical activity, we applied optogenetic stimulation to each of the axon projection field of MPOAPhf6 neurons. AAV2/9-hEf1a-DIO-ChR2-mCherry was unilaterally injected into the MPOA of 8-week-old female Phf6-CreER mice, and the optic fiber was implanted above each axon projection field at the NAc, PVH, and the closely adjacent VMHvl/TN (Fig. 6c). We examined the combined functional outputs of the VMHvl and TN projections because these two regions are positioned in close proximity. Physical activity was assessed using the open field arena with or without blue light stimulation (473 nm, 5.7 mW, 20 Hz, 20 ms pulses). Optogenetic activation of axon terminals at the VMHvl/TN significantly increased physical activity (Fig. 6d), as evidenced by increased movement distance without changes in center time ratio (Fig. 6e-h). In contrast, optogenetic activation of axon terminals at the NAc or PVH had no effect on physical activity (Fig. 6c, d). These results suggest that the effect of MPOAPhf6 neurons on promoting physical activity is primarily mediated by the axon projection to the VMHvl/TN.

MPOAPhf6 neurons and VMHvlEsr1 neurons are mutually innervated and form reciprocal connections.
Previous studies have linked VMHvlEsr1 neurons to the regulation of physical activity in female mice7,8,43. We therefore hypothesized that VMHvlEsr1 neurons mediate the effect of MPOAPhf6 neurons on promoting physical activity. To determine whether MPOAPhf6 neurons directly innervate VMHvlEsr1 neurons, we employed AAV serotype 1 expressing recombinase Cre as an anterograde transsynaptic tracer by unilaterally injecting AAV1-hSyn-Cre into the MPOA and AAV2/9-Ef1a-DIO-mCherry into the VMHvl of 8-week-old wild-type C57BL/6J female mice (Fig. 6i). We detected mCherry-expressing neurons in the VMHvl, and Esr1 immunostaining revealed that 61.0% ± 0.5% of Esr1-positive neurons expressed mCherry (Fig. 6j, k), indicating that a subset of VMHvlEsr1 neurons received monosynaptic input from MPOA neurons. To confirm this anatomical link, AAV2/9-hEf1a-DIO-mGFP was injected into the MPOA of 8-week-old female Phf6-CreER mice, labeling MPOAPhf6 neurons with mGFP. Subsequent Esr1 and GFP immunostaining in the VMHvl revealed close apposition of mGFP-labeled MPOAPhf6 boutons to VMHvlEsr1 cell bodies (Fig. 6l, m). Together, these findings support a direct anatomical connection from MPOAPhf6 to VMHvlEsr1 neurons.

To determine whether MPOAPhf6 neurons form monosynaptic connection with VMHvlEsr1 neurons, we performed monosynaptic retrograde tracing using the pseudorabies virus (RV). The AAVs expressing Cre dependent avian-specific retroviral receptor (AAV2/9-Ef1a-DIO-His-EGFP-2A-TVA) and rabies virus glycoprotein (AAV2/9-Ef1a-DIO-RVG) were unilaterally delivered into the VMHvl of 8-week-old female Esr1-Cre mice, followed by RV-EnvA-ΔG-tdTomato injection into the same site. Neurons expressing both tdTomato and EGFP in the VMHvl were identified as starter cells (Fig. 6n). TdTomato-positive neurons were detected across multiple brain regions (Supplementary Fig. 8a-c). Among diverse upstream inputs to VMHvlEsr1 neurons, we observed direct monosynaptic connections from MPOAPhf6 neurons, as 16.1% ±1.4% of tdTomato-expressing neurons in the MPOA were positive for Phf6 (Fig. 6o, p), demonstrating direct monosynaptic MPOAPhf6→VMHvlEsr1 connectivity.

Given that VMHvlEsr1 neurons also project to the MPOA44, we further investigated whether MPOAPhf6 neurons and VMHvlEsr1 neurons are mutually innervated and make reciprocal connections. We employed RV-based retrograde tracing to label the upstream neurons of MPOAPhf6 neurons, by unilaterally injecting AAV2/9-Ef1a-DIO-RVG and AAV2/9-Ef1a-DIO-His-EGFP-2A-TVA into the MPOA of 8-week-old female Phf6-CreER mice, followed by tamoxifen induction and RV-EnvA-ΔG-tdTomato injected into the same site. Among diverse upstream inputs to MPOAPhf6 neurons (Supplementary Fig. 8d-f), we observed that 22.7% ± 2.0% of tdTomato-expressing neurons in the VMHvl are positive for Esr1 (Fig. 6q-s). This result reveals the monosynaptic VMHvlEsr1→MPOAPhf6 connectivity. Combined with the finding from RV-based retrograde tracing from both VMHvlEsr1 neurons and MPOAPhf6 neurons, our results demonstrate that MPOAPhf6 neurons and VMHvlEsr1 neurons are mutually innervated and form reciprocal connections.

VMHvlEsr1 neurons serve as the primary downstream target of MPOAPhf6 neurons in promoting physical activity.
Previous research reveals the function of VMHvlEsr1 neurons in sex-specific regulation of physical activity7,8. To examine whether VMHvlEsr1 neurons serve as the primary downstream effector of MPOAPhf6 neurons in promoting physical activity, we targeted hM3D to the VMHvlEsr1 neurons by bilaterally injecting AAV2/9-Ef1a-DIO-hM3D(Gq)-mCherry (or AAV2/9-Ef1a-DIO-mCherry as the control) into the VMHvl of 8-week-old female Esr1-Cre mice (Fig. 7a). Physical activity was assessed using a 15-minute OFT. The hM3D- or mCherry-expressing mice were administered with CNO (1 mg/kg, i.p.) 40 minutes before the OFT. Compared with controls, chemogenetic activation of VMHvlEsr1 neurons significantly increased movement distance in the OFT (Fig. 7b, c). Notably, activation of VMHvlEsr1 neurons also significantly decreased the ratio of time spent in the center area (Fig. 7d), suggesting that increased physical activity upon VMHvlEsr1 neuron activation is accompanied by an increase level of anxiety.

Next, we examined whether suppressing the VMHvl while activating MPOAPhf6 neurons would block MPOAPhf6-activation-induced increase in physical activity. AAV2/9-hSyn-hM4D(Gi)-GFP (or AAV2/9-hSyn-GFP as the control) were bilaterally injected into the VMHvl and AAV2/9-DIO-ChR2-mCherry was unilaterally injected into the MPOA of 8-week-old female Phf6-CreER mice. The optic fiber was implanted above the MPOA (Fig. 7e). We found that suppressing the VMHvl while activating MPOAPhf6 neurons profoundly inhibited the MPOAPhf6-activation-induced increase in physical activity (Fig. 7f). This result suggests that VMHvlEsr1 neurons serve as the primary downstream target of MPOAPhf6 neurons in promoting physical activity.

Given the reciprocal MPOAPhf6-VMHvlEsr1 connectivity, we also examined whether suppressing the MPOA while activating VMHvlEsr1 neurons would block VMHvlEsr1-activation-induced increase in physical activity. AAV2/9-hSyn-hM4D(Gi)-GFP (or AAV2/9-hSyn-GFP as the control) were bilaterally injected into the MPOA and AAV2/9-hEf1a-DIO-ChR2-mCherry was unilaterally into the VMHvl of 8-week-old female Esr1-Cre mice. The optic fiber was implanted above the VMHvl (Fig. 7g). We found that suppressing the MPOA while activating VMHvlEsr1 neurons only moderately inhibited VMHvlEsr1-activation-induced increase in physical activity (Fig. 7h). This result suggests that projection from VMHvlEsr1 to the MPOA also contribute to promoting physical activity. Taken together, our results demonstrate that VMHvlEsr1 neurons serve as the primary downstream target of MPOAPhf6 neurons in promoting physical activity, and the reciprocal MPOAPhf6-VMHvlEsr1 connections create a feedback loop enhancing the effect of MPOAPhf6 neurons on promoting physical activity.

MPOAPhf6→VMHvlEsr1 pathway comprises both excitatory and disinhibitory connections.
Considering that MPOAPhf6 neurons comprise both glutamatergic excitatory neurons and GABAergic inhibitory neurons, we investigated whether MPOAPhf6→VMHvlEsr1 circuitry comprises both excitatory and disinhibitory synaptic connections by ChR2-assisted circuit mapping using electrophysiology45. We targeted ChR2 to MPOAPhf6 neurons by injecting AAV2/9-hEf1a-DIO-ChR2-mCherry into the MPOA of 8-week-old female Phf6-CreER mice. Neurons were recorded at the VMHvl of acute slices by whole-cell patch clamp recording (Fig. 7i). Light-evoked EPSCs (oEPSCs) were recorded at -70 mV; light-evoked IPSCs (oIPSCs) were recorded at 0 mV. We recorded oEPSCs in 24 out of 69 neurons, which were inhibited by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 40 μM) (Fig. 7j, k and m); we recorded oIPSCs in 45 out of 69 neurons, which were inhibited by GABAa receptor antagonist picrotoxin (PTX, 100 μM) (Fig. 7j, k and n). Sodium channel blocker tetrodotoxin (TTX, 2 μM) inhibited the oEPSCs and oIPSCs, while potassium channel blocker 4-aminopyridine (4-AP, 100 μM) reversed the TTX-inhibited oEPSCs and oIPSCs (Fig. 7o, p), confirming monosynaptic excitatory and inhibitory inputs from MPOAPhf6 neurons to VMHvl neurons.

We further examined whether MPOAPhf6 neurons form a disinhibitory circuit with VMHvlEsr1 neurons through local inhibitory neurons. Prior evidence showed that VMHvlEsr1 neurons receive inhibitory inputs from local GABAergic inhibitory neurons in and surrounding the VMHvl45. Notably, a substantial population of somatostatin-positive (SST+) inhibitory neurons in the TN (TNSST neurons)46,47 are located close to the VMHvl. We sought to determine whether the disinhibitory circuitry from MPOAPhf6 neurons to VMHvlEsr1 neurons is through nearby TNSST neurons. Using 8-week-old female Phf6-CreER::SST-Flpo mice, we labeled MPOAPhf6 neurons with mGFP and TNSST neurons with mCherry. Anatomical analysis of mGFP-labeled MPOAPhf6 neuron axon terminals revealed close apposition of mGFP-labeled MPOAPhf6 axon terminal boutons to TNSST cell bodies (Supplementary Fig. 9a, b), while RV-based retrograde tracing confirmed MPOAPhf6 neurons as monosynaptic upstream inputs to TNSST neurons (Supplementary Fig. 9c-e). These results indicate that MPOAPhf6 neurons likely form a disinhibitory circuit with VMHvlEsr1 neurons via TNSST inhibitory neurons. Taken together, the MPOAPhf6→VMHvlEsr1 circuitry comprises both excitatory and disinhibitory connections.

Methods
Animals
All mouse experiments received approval from the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine and the Institutional Animal Care and Use Committee at Department of Laboratory Animal Science (JUMC2023-096-A). The mice were housed under standard conditions with a temperature of 25 °C, humidity levels of 40-60%, and an 8:00 AM-8:00 PM 12-hour light/dark cycle with water and mouse chow diet ad libitum. The following mouse lines were used in this study: wild-type C57BL/6J mice were purchased from SLAC laboratory (Shanghai); vGlut2-IRES-Cre (Stock number: 016963) and vGat-IRES-Cre (Stock number: 016962) were purchased from the Jackson Laboratory (USA); Phf6flox/flox (Phf6flox/y) mice on a C57BL/6J background were acquired from Tim Thomas’s laboratory at the Walter and Eliza Hall Institute of Medical Research (Australia); LSL-H2B-GFP and SST-Flpo mice were acquired from Miao He’s lab at Fudan university (Shanghai); Esr1-2A-Cre mice were acquired from Xiaohong Xu’s laboratory at Fudan University (Shanghai); Phf6-CreERT2 knock-in mice on a C57BL/6J background were generated by Cyagen Biosciences (Shanghai). Age balanced littermate female and male mice were randomly assigned to experimental groups. The sex and age of all experimental mice were shown in results section, figure legends and source data file. Mice genotyping primers are listed in Supplementary Table 2.

Generation of Phf6-CreERT2 knock-in mice
Phf6-CreERT2 knock-in mice were generated on a C57BL/6J background using CRISPR/Cas9-mediated genome editing. The targeting strategy inserted an IRES-CreERT2 cassette immediately downstream of the TAG stop codon in exon 10 of the Phf6 gene (NCBI RefSeq: NM_027642.2; Ensembl Transcript: ENSMUST00000078944) located on the X chromosome. Homology arms for the targeting vector were PCR-amplified from BAC clone RP23-133E22. The targeting vector containing the IRES-CreERT2 cassette, along with Cas9 mRNA and gRNA targeting the mouse Phf6 gene (sequence: AGCTTAATGGAAACTAGGTATGG), were co-injected into fertilized C57BL/6J eggs. Founder pups were genotyped by PCR amplification of the targeted region, followed by Sanger sequencing to confirm correct insertion and absence of off-target mutations. Positive founders were subsequently backcrossed to the C57BL/6J mice for at least five generations before use in experiments.

Tamoxifen induction
Tamoxifen (Sigma-Aldrich, T5648) was dissolved at 20 mg/mL in corn oil (ABCONE). Seven days post viral injection, 8-week-old Phf6-CreER mice were administered tamoxifen intraperitoneally at 100 mg/kg. The injections were administered every other day for four doses. Following the treatment, the animals were allowed a recovery period of at least 3 weeks in their home cages to ensure proper recovery and induction of CreER activity.

Stereotaxic viral injection
Standard stereotactic procedures were applied to mice under anesthesia. Adult 8-week-old mice were anesthetized with isoflurane (5% induction and 1.5% maintenance). Body temperature was maintained stable using a temperature controller (RWD Life Science). Virus was injected at a volume of 200-300 nL/site at a flow rate of 25 nL/min using a Nanofil syringe injector (World Precision Instruments) and a Quintessential stereotaxic injector controller (Stoelting CO.). After viral injection, the glass pipette was left in place for 7-10 minutes and withdrawn slowly to avoid the back-flow of virus. The mice were injected with ketoprofen (5 mg/kg, i.p.) for postoperative analgesia after surgery and put on a heating blanket before returning to their home cages. The animals were allowed at least 3 weeks in their home cage to recover from surgery and express virus before the behavioral tests were administered. In experiments for Phf6 conditional knockout, post-hoc Phf6 immunostaining was performed to confirm the effect of AAV-Cre-induced Phf6 deletion. In experiments for optogenetic stimulation and fiber photometry recording, an optic fiber (200 μm core, NA = 0.37) was implanted in places as indicated. The fiber was secured to the skull using dental cement. We performed post-hoc verification of the accuracy of viral infection and optic fiber implantation. Data were excluded in cases of absent viral expression or incorrect optic fiber positioning.

The corresponding coordinates of the target area were as follows (calculated from bregma): MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm), NAc (AP, +0.50 mm; ML, ±0.75 mm; DV, -4.50 mm), PVH (AP, -0.90 mm; ML, ±0.25 mm; DV, -4.80 mm), VMHvl (AP, -1.43 mm; ML, ±0.90 mm; DV, -5.75 mm), TN (AP, -1.55 mm; ML, ±1.00 mm; DV, -5.75 mm). The virus and titer used in this study were as follows: AAV2/9-CMVbGl-Cre-eGFP (4.15E+12v.g./mL); AAV2/9-CMVbGl-eGFP (3.40E+12v.g./mL); AAV2/9-hEf1a-DIO-H2B-EGFP-WPRE-pA (5.19E+12v.g./mL); AAV2/9-hEf1a-DIO-EGFP-WPRE-pA (5.40E+12v.g./mL); AAV2/9-hSyn-DIO-GCaMP7s-WPRE-pA (2.5E+12v.g./mL); AAV2/9- hEf1a-DIO-ChR2(H134R)-mCherry-WPRE-pA (4.24E+12v.g./mL); AAV2/9-Ef1a-DIO-hM3D(Gq)-mCherry-WPRE-pA (6.91E+12v.g./ml); AAV2/9-Ef1a-DIO-hM4D(Gi)-mCherry-WPREs-pA (5.18E+12v.g./mL); AAV2/9-Ef1a-DIO-mCherry-WPRE-pA (2.55E+12v.g./mL); AAV2/9-CAG-DIO-taCaspase3-TEVp-WPRE-pA (1.99E+13v.g./mL); AAV2/9-hU6-sgRNA (Phf6)-hEf1a-DIO-EGFP-WPRE-pA (1.13E+13v.g./ml); AAV2/9-hU6-sgRNA(NC)-hEF1a-DIO-EGFP-WPRE-pA (2.08E+13v.g./ml); AAV2/9-CAG-DIO-spCas9(wt)-WPRE-pA (1.05E+13v.g./ml); AAV2/9-hEf1a-DIO-mGFP-WPRE-pA (2.50E+12v.g./mL); AAV2/9-Ef1a-DIO-RVG-WPRE-pA (2.00E+12v.g./mL); AAV2/9-Ef1a-DIO-His-EGFP-2A-TVA-WPRE-pA (2.00E+12v.g./mL); RV-EnvA-ΔG-tdTomato (7.00E+08 IFU/mL); AAV1-hSyn-Cre (1.3E+13v.g./ml); AAV2/9-hEF1a-fDIO-mCherry-WPRE-pA (5.45E+12v.g./mL); AAV2/9-hSyn-hM4D(Gi)-GFP-ER2-WPRE-pA (4.02E+12v.g./mL); AAV2/9-hSyn-GFP-ER2-WPRE-pA (5.05E+12v.g./mL).

Histology and immunostaining
Mice were anesthetized with isoflurane and perfused with saline followed by 2% paraformaldehyde (PFA). The brain was postfixed for 2 hours at 4 ℃ and cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) overnight. 30 μm frozen sections were cut. Tissue sections were blocked in 5% goat serum, 0.4% Triton X-100, and 3% bovine serum albumin (BSA) in 1x PBS for 2 hours at room temperature. We applied the rabbit anti-Phf6 antibody (NOVUS, NB100-68262, 1:400), mouse anti-ERα(D-12) antibody (Santa Cruz, SC-8005, 1:200), rat anti-mCherry antibody (Thermo Fisher Scientific, M11217, 1:500), chicken anti-GFP antibody (Abcam, ab13970, 1:500) or rat anti-c-Fos antibody (Synaptic Systems, 226017, 1:1000) overnight at 4 ℃. After washing in 1x PBS three times, the slices were incubated with goat anti-rabbit 488 (Thermo Fisher Scientific, A11008, 1:500), goat anti-rabbit 555 (Thermo Fisher Scientific, A21428, 1:500), goat anti-rabbit 633 (Thermo Fisher Scientific, A21070, 1:500), goat anti-mouse 488 (Thermo Fisher Scientific, A11001, 1:500), goat anti-mouse 555 (Thermo Fisher Scientific, A21422, 1:500), goat anti-rat 555 (Thermo Fisher Scientific, A21434, 1:500), goat anti-chicken 488 (Thermo Fisher Scientific, A11039, 1:500), goat anti-rat 488 (Thermo Fisher Scientific, A11006, 1:500), and DAPI (1:10,000 of 5 mg/mL) for 2 hours at room temperature. Confocal images were acquired using the Leica STED system, Olympus IFV3000, or Olympus VS200. For each staining (e.g., Phf6, Esr1, c-Fos), immunopositive cells were manually quantified using FIJI (ImageJ) software.

For running-induced c-Fos activation, the wild-type C57BL/6J female mice were placed in the forced running wheel (YLS-10B, Yuyan Instruments) at a speed of 10 m/min for 30 minutes. One hour after wheel running, mice were perfused for immunostaining.

Single-cell RNA sequencing data analyses
The single-cell RNA sequencing datasets (GSE113576 and GSE183093)38,39 from previous studies (Knoedler et al.; Moffitt et al.20,21) were utilized for the analysis. Prior to analysis, quality control was performed to ensure the reliability of the datasets. Cells were filtered based on the following criteria: (i) the percentage of mitochondrial gene counts was required to be less than 10%; (ii) the number of detected gene counts per cell was between 200 and 4,000; and (iii) RNA counts per cell were required to be less than 10,000. Following these filtering steps, the data from different experimental batches were integrated and analyzed using Seurat (version 5.3.0). To focus on cells expressing Esr1 and cells co-expressing Esr1 and Phf6, we extracted cells with expression levels of Esr1 or Esr1/Phf6 greater than 0. Integration of the datasets was performed using the canonical correlation analysis (CCA) method, implemented in the Seurat package. The integrated data were subsequently used for dimensionality reduction and UMAP was performed by RunUMAP using the first 20 principal components (dims = 1:20), with the CCA-based reduction method. Next, cell clustering was performed by calculating nearest neighbors using the FindNeighbors function, and then clustering the cells using the FindClusters function. We adopted a resolution of 0.13 (for Esr1 positive cells) and 0.08 (for Esr1/Phf6 double positive cells) to optimize the clustering of cells. To visualize the expression of the Phf6 gene, a density plot was generated using the plot density function in Nebulosa. The plot visualizes the distribution of the Phf6 expression across the single-cell dataset.

Metabolic analysis
For metabolic analysis, mice were measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments) metabolic cages that consist of individual live-in cages for mice that allow automated, noninvasive data collection49. All mice for metabolic analysis were maintained in an animal facility under a 12 h light–dark cycle (lights on at 8:00 AM, lights off at 8:00 PM) at a temperature of 22 ℃ and allowed free access to water and food. The mice were individually acclimated to the metabolic chamber cage for at least 3 days. Oxygen consumption, carbon dioxide production, voluntary wheel-running, locomotion in the horizontal plane, and food intake were measured at 10- or 11-minute intervals over a 48-hour period. The RER was calculated as the quotient of VCO2/VO2, with 100% carbohydrate oxidation giving a value of 1, and 100% fat oxidation giving a value of 0.750,51. Energy expenditure (kcal heat produced) was calculated as Calorific Value (CV) x VO2, where CV is 3.815 + 1.232 x RER52. Physical activity was measured by infrared technology (OPT-M3, Columbus Instruments).

Basal metabolic rate (BMR), non-exercise activity thermogenesis (NEAT), and exercise-induced thermogenesis (EIT) analyses
Female Phf6 cKO and control mice were individually acclimated to the metabolic cages for at least 3 days at 12 weeks pvi. On the test day, female Phf6 cKO mice were first fasted overnight to eliminate diet-induced thermogenesis effects and placed in CLAMS metabolic cages. Total energy expenditure (TEE) was recorded every 8 minutes. TEE data were analyzed using two-sided regression-based analysis-of-covariance ANCOVA with body weight as a covariate53,54. Predicted EE normalized to 40 g body weight was calculated from the regression line equation, y = ax + b55,56. BMR was defined as TEE during the 1 hour of minimal activity in the inactive phase (light period: 8:00 AM-8:00 PM); NEAT was calculated by subtracting BMR from the TEE when wheel running = 0 during the active phase (dark period: 8:00 PM-8:00 AM), and EIT was determined by subtracting BMR from the TEE when wheel running > 0 during the dark phase5,57.

Circadian behavioral experiments (ClockLab, Actimetrics)
Female Phf6 cKO and control mice were individually housed in light-tight, ventilated cabinets with controlled temperature and humidity. Mice were entrained to a 12-h light/dark cycle (lights on at 8:00 AM, lights off at 8:00 PM) for seven days with water and mouse chow ad libitum. Voluntary wheel-running activity was recorded continuously for 10 days using the ClockLab data collection system (Actimetrics, Wilmette, IL)58,59. In the ClockLab system, a sensor was connected to a wheel-running cage, which transmits wheel rotations to the computer. The ClockLab software subsequently elaborated the data. Black vertical bars were the readout of the number of wheel revolutions every 5 minutes. The height of each vertical black bar cluster indicated the sum of wheel revolutions. Each horizontal line represented one day.

Measurement of body temperature
Mice were housed individually under a 12-hour light/dark cycle at 22 °C with ad libitum food and water. Surface temperature of the mice was imaged using a high-resolution infrared camera (FLIR A655sc). iBAT temperature was measured along the midline of the interscapular region following hair removal. Thermal images were captured at specified time points and quantified using FLIR Tools software. The maximum value was taken for analysis.

ELISA
Blood samples were collected exclusively from diestrus females, identified via vaginal smear cytology. Vaginal cytology samples were collected before collecting blood samples. We gently flushed 20 μL saline into the vagina using a pipette three to four times to collect vaginal lavage. Attention was paid to not touch the vaginal wall to avoid cervical stimulation. Smears were dried, stained with Giemsa (60529-A, Yeasen Biotechnology), and examined. Diestrus was defined by the predominance of neutrophils with few nucleated epithelial cells.

Mice were euthanized by decapitation, and blood samples were collected into EDTA-coated EP tubes. Samples were immediately centrifuged at 9,400 g for 15 minutes at 4 °C, and the resulting supernatants (plasma) were collected, aliquoted, and stored at -80 °C until assay. Estradiol and follicle stimulating hormone (FSH) were measured using the Mouse 17 beta Estradiol (XL-EM0478, Xinle) and the Mouse FSH (XL-EM0427, Xinle) ELISA kit, respectively. LH was measured using the Mouse LH ELISA kit (E-EL-M3035, Elabscience). Hormone levels in plasma were measured according to the manufacturers’ instructions. Briefly, 50 μL of sample/standard, 25 μL of primary antibody, and 25 μL of biotinylated peptide were added per well and incubated for 2 hours at room temperature. After washing, 100 μL of streptavidin–horseradish peroxidase was added and incubated for 1 hour. Following additional washing, 100 μL of tetramethylbenzidine (TMB) substrate solution was added, and the plate was incubated in the dark for color development. Reactions were stopped with 100 μL 2N HCl, and absorbance was measured at 450 nm using a BioTek Synergy HTX microplate reader (Agilent Technologies, USA). Hormone concentrations were determined via standard curves fitted with a four-parameter logistic regression model.

Maternal behavior of pup retrieval
Maternal behavior was assessed by scattering three pups (P1-4) away from the nest and recording for ~15 min, repeated 2–3 times. Virgin females were exposed to pups for 20 minutes/day over five consecutive days. Virgin females were group-housed until 8 weeks, then individually housed with nesting material. During exposure, home cages were not cleaned. Pups (1–4 days old) were placed in the cage and removed after 20 minutes. Pup retrieval was scored only when pups were fully returned to the nest; partial transport did not count. Latency to approach (defined as first nose contact within 1 cm) and the number of pups retrieved were analyzed. Recordings were made using EthoVision 3.0 (Noldus).

Fiber photometry recording
AAV2/9-hSyn-DIO-GCaMP7s-WPRE-pA (2.5E+12v.g./mL) was unilaterally injected at a volume of 300 nL into the MPOA of 8-week-old female Phf6-CreER mice. An optic fiber with a 200-μm outer diameter was implanted approximately 200 μm above the viral injection site. Fiber photometry experiments were performed using a two-color fiber photometry system (Inper Ltd.) to record the in vivo calcium signals of MPOAPhf6 neurons. The GCaMP7s fluorescence was excited at 470-nm and the internal control at 410-nm was acquired at a sampling rate of 100 Hz. LED light power at the fiber tip was 30-40 μW to minimize bleaching. Mouse behavior was recorded via video (Logitech). Calcium signals were acquired in freely moving mice in their home cage, with both calcium and control signals recorded simultaneously. Fluorescence dynamics before and after walking and climbing onset were calculated as ΔF/F = (F − F₀)/F₀, where F₀ is the baseline averaged over a 5-second window. Data are presented as the mean ± standard error of the mean (SEM).

Open field test (OFT)
Mice in the OFT were placed in an open field area (40-cm length, 40-cm width, 30-cm height) and allowed to explore for 15 minutes. A video-tracking system (EthoVision 3.0, Noldus) was used to measure physical activity of the animal. The movement distance traveled in the whole open field arena was used to assess locomotor activity, and time spent in the center zone (20 × 20 cm) was used as an anxiety index.

Elevated plus maze (EPM) test
The maze comprised a central platform (7-cm length, 7-cm width), two open arms without walls (30-cm length, 7-cm width) and two closed arms with walls (30-cm length, 7-cm width, 15-cm height). Mice were placed on the central platform facing one closed arm and allowed to explore for 5 minutes. The apparatus was cleaned with 75% ethanol between trials. Time spent in open arms was recorded by the video-tracking system (EthoVision 3.0, Noldus) served as an anxiety index.

Optogenetic activation
For optogenetic activation of MPOAPhf6 neurons, 250 nL of AAV2/9-hEf1a-DIO-ChR2(H134R)-mCherry (4.24E+12v.g./mL) or AAV2/9-Ef1a-DIO-mCherry virus (2.55E+12v.g./mL) was unilaterally injected into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) of adult female Phf6-CreER mice with optic fiber implantation above the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.05 mm). For terminal activation, the same viral constructs were injected into the MPOA and fibers implanted above downstream targets: NAc (AP, +0.50 mm; ML, ±0.75 mm; DV, -4.50 mm), PVH (AP, -0.90 mm; ML, ±0.25 mm; DV, -4.80 mm), or VMHvl/TN (AP, -1.43 mm; ML, ±0.90 mm; DV, -5.75 mm). Mice were injected with ketoprofen (5 mg/kg, i.p.) for postoperative analgesia after surgery. Tamoxifen (100 mg/kg, i.p., 4 days, 20 mg/mL in corn oil) was administered on day 7. Three weeks were allowed for animal recovery and viral expression. OFT consisted of three 5-minute sessions (Laser OFF-ON-OFF). A 473-nm blue laser (20-ms pulses at 20 Hz) was applied at 5.7 mW to activate the MPOAPhf6 neurons or axon terminals. The movement distance traveled in the whole open field arena and time spent in the center zone were calculated. In these experiments, post-hoc verification confirmed viral expression and fiber placement; animals with absent expression or incorrect fiber positioning were excluded.

For optogenetic activation of VMHvlEsr1 neuron axonal terminals in the MPOA, 250 nL of AAV2/9-hEf1a-DIO-ChR2(H134R)-mCherry (4.24E+12v.g./mL) was unilaterally injected into the VMHvl (AP, -1.43 mm; ML, ±0.90 mm; DV, -5.75 mm) of adult female Esr1-Cre mice, and the optic fiber was implanted above the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.05 mm). Ketoprofen (5 mg/kg, i.p.) was administered postoperatively. We waited 3 weeks to allow animal recovery and viral expression. OFT consisted of two 5-minute sessions (Laser OFF-ON). A 473-nm blue laser (20-ms pulses at 20 Hz) was applied at 5.7 mW to activate the axon terminals of VMHvlEsr1 neurons. The movement distance traveled in the whole open field arena and time spent in the center zone were calculated. In these experiments, post-hoc verification confirmed viral expression and fiber placement; animals with absent expression or incorrect fiber positioning were excluded.

Chemogenetic activation
For chemogenetic activation of MPOAPhf6 neurons, 250 nL of AAV2/9-Ef1a-DIO-hM3D(Gq)-mCherry (6.91E+12v.g./ml) or AAV2/9-Ef1a-DIO-mCherry virus (2.55E+12v.g./mL) was bilaterally injected into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) of adult female Phf6-CreER mice. Ketoprofen (5 mg/kg, i.p.) was administered postoperatively. Tamoxifen (100 mg/kg, i.p., 4 days) was injected on day 7. We waited 3 weeks to allow animal recovery and viral expression. On the test day, the mice were intraperitoneally injected with clozapine N-oxide (CNO, 3 mg/kg, Sigma, C0832). The 15-minute OFT was conducted 40 minutes after CNO or saline administration. The movement distance and time spent in the center zone were calculated. The 5-minute EPM was conducted 40 minutes after CNO or saline administration. Time spent in the open arms was calculated. For metabolic assessment, the mice were habituated in the metabolic cages for at least 3 days. Then, the mice were intraperitoneally injected with CNO (3 mg/kg) 40 minutes before behavioral testing. Oxygen consumption, carbon dioxide production, and horizontal locomotor activity were recorded every 10-11 minutes for 2 hours. Post-hoc verification ensured accurate viral expression; animals with absent expression were excluded.

For chemogenetic activation of VMHvlEsr1 neurons, 250 nL of AAV2/9-Ef1a-DIO-hM3D(Gq)-mCherry (6.91E+12v.g./ml) or AAV2/9-Ef1a-DIO-mCherry virus (2.55E+12v.g./mL) was bilaterally injected into the VMHvl (AP, -1.43 mm; ML, ±0.90 mm; DV, -5.75 mm) of adult female Esr1-Cre mice. The mice were injected with ketoprofen (5 mg/kg, i.p.) for postoperative analgesia after surgery. We waited 3 weeks to allow animal recovery and viral expression. On the test day, the mice were intraperitoneally injected with CNO (1 mg/kg). The 15-minute OFT was conducted 40 minutes after CNO administration. The movement distance and time spent in the center zone were calculated. Post-hoc verification excluded animals with absent viral expression.

Chemogenetic inhibition
For chemogenetic inhibition of MPOAPhf6 neurons, 250 nL of AAV2/9-Ef1a-DIO-hM4D(Gi)-mCherry (5.18E+12v.g./mL) or AAV2/9-Ef1a-DIO-mCherry virus (2.55E+12v.g./mL) was bilaterally injected into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) of adult female Phf6-CreER mice. The mice were injected with ketoprofen (5 mg/kg, i.p.) for postoperative analgesia after surgery. On the seventh day, mice were intraperitoneally injected with tamoxifen (100 mg/kg for 4 days) dissolved in corn oil at a concentration of 20 mg/mL. We waited 3 weeks to allow animal recovery and viral expression. For OFT, the mice were intraperitoneally injected with CNO (3 mg/kg). The 15-minute OFT was conducted 40 minutes after CNO administration. The movement distance traveled in the whole open field arena and time spent in the center zone were calculated. For metabolic analysis, the mice were first habituated in the metabolic cages for at least 3 days. Then, the mice were intraperitoneally injected with CNO (3 mg/kg) 40 minutes before behavioral testing. Oxygen consumption, carbon dioxide production, and voluntary wheel running activity were measured at 10- or 11-minute intervals over a 2-hour period. We performed post-hoc verification of the accuracy of viral infection. Data were excluded in cases of absent viral expression.

Optogenetic and chemogenetic manipulations
For simultaneous optogenetic activation of MPOAPhf6 neurons and chemogenetic inhibition of the VMHvl neurons, 250 nL of AAV2/9-hEf1a-DIO-ChR2(H134R)-mCherry (4.24E+12v.g./mL) was unilaterally injected into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) and 250 nL of AAV2/9-hSyn-hM4D(Gi)-GFP-ER2-WPRE-pA (4.02E+12v.g./mL) was bilaterally injected into the VMHvl (AP, -1.43 mm; ML, ±0.90 mm; DV, -5.75 mm) of adult female Phf6-CreER mice. Control animals received AAV2/9-hEf1a-DIO-ChR2(H134R)-mCherry into the MPOA and AAV2/9-hSyn-GFP-ER2-WPRE-pA (5.05E+12v.g./mL) into the VMHvl. The optic fiber was implanted above the MPOA. Postoperative analgesia was provided with ketoprofen (5 mg/kg, i.p.). On the seventh day, mice were intraperitoneally injected with tamoxifen (100 mg/kg for 4 days). We waited 3 weeks to allow animal recovery and viral expression. Mice were intraperitoneally injected with CNO (3 mg/kg). The 5-minute OFT was conducted 40 minutes after CNO administration, accompanied by 473-nm blue laser light (20 ms pulses, 20 Hz, 5 minutes) at 5.7 mW to activate MPOAPhf6 neurons. The movement distance traveled and time spent in the center zone were calculated. Viral expression and fiber placement were confirmed post hoc, and animals with absent expression or misplacement were excluded.

For simultaneous optogenetic activation of VMHvlEsr1 neurons and chemogenetic inhibition of the MPOA neurons, 250 nL of AAV2/9-hEf1a-DIO-ChR2(H134R)-mCherry (4.24E+12v.g./mL) was unilaterally injected into the VMHvl (AP, -1.43 mm; ML, ±0.90 mm; DV, -5.75 mm) and 250 nL of AAV2/9-hSyn-hM4D(Gi)-GFP-ER2-WPRE-pA (4.02E+12v.g./mL) was bilaterally injected into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) of adult female Esr1-Cre mice. Control animals received AAV2/9-hEf1a-DIO-ChR2(H134R)-mCherry into the VMHvl and AAV2/9-hSyn-GFP-ER2-WPRE-pA (5.05E+12v.g./mL) into the MPOA. The optic fiber was implanted above the VMHvl. Postoperative analgesia was provided with ketoprofen (5 mg/kg, i.p.), and three weeks were allowed for recovery and viral expression. Mice were intraperitoneally injected with CNO (3 mg/kg). The 5-minute OFT was conducted 40 minutes after CNO administration, accompanied by 473-nm blue laser light (20 ms pulses, 20 Hz, 5 minutes) at 5.7 mW to activate VMHvlEsr1 neurons. The movement distance traveled and time spent in the center zone were calculated. Viral expression and fiber placement were confirmed post hoc, and animals with absent expression or misplacement were excluded.

Neuronal ablation
For Caspase-3-mediated neuronal ablation experiment, we bilaterally injected 250 nL of AAV2/9-CAG-DIO-taCaspase3 (1.99E+13v.g./mL) or AAV2/9-Ef1a-DIO-mCherry virus (2.55E+12v.g./mL) into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) of female Esr1-Cre mice. The mice were injected with ketoprofen (5 mg/kg, i.p.) for postoperative analgesia after surgery. We waited 3 weeks to allow animal recovery and viral expression. For metabolic analysis, the mice were first habituated in the metabolic cage for at least 3 days. Oxygen consumption, carbon dioxide production, and voluntary wheel running activity were measured at 10- or 11-minute intervals over a 48-hour period. After these experiments, we validated that Esr1+ neurons in the MPOA were effectively ablated. Data were excluded if the Caspase-3-mediated Esr1 ablation failed.

CRISPR/Cas9-mediated gene knockout
CRISPR/Cas9-mediated gene knockout methods were used to knockout Phf6 in MPOAPhf6 neurons. The sgRNA target sequences were first selected by submitting the exon sequences of the Phf6 gene to the CRISPR design tool (http://zlab.bio/guide-design-resources). Then, we performed additional Cas9 target selection from the top 6 candidates obtained from the CRISPR design tool. We transfected both the Cas9- and Phf6 sgRNAs-expressing plasmids into Neuro2A cells. Three days after transfection, we harvested the cells for genomic DNA extraction followed by sequencing. Based on the analyses of CRISPR-mediated gene editing efficiency in Neuro2A cells, we selected the top 3 sgRNAs for Phf6. The three target sequences for the Phf6 gene are as follows: GCCACAGGACATACCACTAC; GGTATCATTAGGACTGCTCC; CGCTTAATCTCCTGAAGTAC. The sgRNAs were then cloned into the construct according to the CRISIPR-Cas9 mouse Toolbox. An AAV vector backbone without sgRNA (AAV2/9-hU6-sgRNA(NC)-hEF1a-DIO-EGFP, 2.08E+13v.g./ml) was used as the control. We bilaterally co-injected AAV2/9-hU6-sgRNA (Phf6)-hEf1a-DIO-EGFP (1.13E+13v.g./ml) and AAV2/9-CAG-DIO-spCas9 virus (1.05E+13v.g./ml) at a 2:1 volume ratio into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) of adult female Phf6-CreER mice (250 nL). Postoperative analgesia was provided with ketoprofen (5 mg/kg, i.p.). After 7 days of recovery, the mice were intraperitoneally injected with tamoxifen (100 mg/kg) for four times. Three weeks after tamoxifen induction, we performed whole-cell patch clamp recording or single-molecule fluorescent in situ hybridization. Post-hoc Phf6 immunostaining was performed to validate the knockout efficiency of endogenous Phf6 in the MPOA. Data were excluded if the viral-mediated Phf6 knockout failed.

Electrophysiology
For functional characterization of MPOAPhf6 neurons, we bilaterally co-injected AAV2/9-hU6-sgRNA (Phf6)-hEf1a-DIO-EGFP (1.13E+13v.g./ml) and AAV2/9-CAG-DIO-spCas9 virus (1.05E+13v.g./ml) at a 2:1 volume ratio into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) of adult female Phf6-CreER mice (250 nL). An AAV vector backbone without sgRNA (AAV2/9-hU6-sgRNA(NC)-hEF1a-DIO-EGFP, 2.08E+13v.g./ml) was used as the control. After 7 days of recovery, the mice were intraperitoneally injected with tamoxifen (100 mg/kg) for four times. Three weeks after tamoxifen induction, the female Phf6 sgRNAs and control mice were sacrificed at 8:00 PM (dark period). ChR2-assisted circuit mapping (CRACM) was performed to evaluate synaptic input from MPOAPhf6 neurons onto VMHvl neurons. AAV2/9-hEf1a-DIO-ChR2(H134R)-mCherry-WPRE-pA (4.24E+12v.g./mL) was bilaterally injected into the MPOA of adult female Phf6-CreER mice (250 nL). After 7 days of recovery, the mice were intraperitoneally injected of tamoxifen (100 mg/kg) for four times. Three weeks after tamoxifen induction, we performed whole-cell patch clamp recording.

Acute 250 μm-thick brain slices were prepared by vibratome (Leica, VT1200S) in cold high-Mg2+ and low-Ca2+ slicing solution containing 83 mM NaCl, 2.4 mM KCl, 0.5 mM CaCl2, 6.8 mM MgCl2, 24 mM NaHCO3, 1.61 mM NaH2PO4, 24 mM D-glucose, and 65 mM sucrose. Then, acute brain slices were recovered for 1 hour at 37 °C in artificial cerebrospinal fluid (ACSF). The ACSF contained 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 26 mM NaHCO3, 1.3 mM MgCl2, 2.5 mM CaCl2, and 10 mM D-glucose bubbled with 95% O2/5% CO2. During recording, the acute brain slices were transferred to a recording chamber that was perfused continuously with O2-saturated ACSF at a rate of 2 mL/min. Unless otherwise indicated, the intracellular solution used for whole-cell patch clamp recording contained 147 mM K-gluconate, 3 mM KCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4 mM ATP-Mg, 0.3 mM GTP-Na2, 10 mM Na2-phosphocreatine, and 0.3 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). For light-evoked EPSC/IPSC recordings, the recording pipettes were filled with a cesium-based internal solution (125 mM CsMeSO4, 2 mM CsCl, 10 mM HEPES, 0.5 mM EGTA, 4 mM MgATP, 0.3 mM Na2GTP, 10 mM Na2-phosphocreatine, 5 mM TEACl, 3.5 mM QX-314, pH 7.3, adjusted with CsOH, 290-300 mOsm) and isolated by clamping the membrane potential of the recorded neurons at the reversal potential of inhibitory (0 mV) and excitatory (-70 mV) synaptic currents, respectively, with 2-ms blue laser pulses every 30 s. Recordings were acquired using a MultiClamp 700B amplifier digitized at 10 kHz by a Digidata 1550B D-A converter and Bessel-filtered at 2 kHz. The recording pipette resistance was 3-4 MΩ, and the series resistance was < 30 MΩ. The cells were excluded if the series resistance exceeded 30 MΩ. Light stimulation (20-ms blue LED) was delivered via an Olympus 40× water-immersion lens. The whole-cell patch current clamp was engaged to test neural firing rate at the baseline and after puff application of 100 nM estradiol. Percent change in mean firing frequency was averaged over 1-minute bins. Drugs were applied at the following concentrations: Estradiol (Sigma, E8875-1G), 100 nM; PTX (Tocris, 1128), 100 μM; CNQX (Tocris, 0190), 40 μM; D-APV (Tocris, 0106), 100 μM; TTX (Tocris, 1078), 2 μM; 4-AP (Sigma, 275875), 100 μM; and CNO (Sigma, C0832), 50 μM.

Single-molecule fluorescent in situ hybridization (smFISH)
For CRISPR/Cas9-mediated Phf6 gene knockout, we bilaterally co-injected AAV2/9-hU6-sgRNA (Phf6)-hEf1a-DIO-EGFP (1.13E+13v.g./ml) and AAV2/9-CAG-DIO-spCas9 virus (1.05E+13v.g./ml) at a 2:1 volume ratio into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) of adult female Phf6-CreER mice (250 nL). An AAV vector backbone without sgRNA (AAV2/9-hU6-sgRNA(NC)-hEF1a-DIO-EGFP, 2.08E+13v.g./ml) was used as the control. After 7 days of recovery, tamoxifen (100 mg/kg for 4 times) was administered. Three weeks later, mice were sacrificed. smFISH was performed on fresh-frozen sections (20 μm) using RNAScope Multiplex Reagent Kits (323110, ACDBio, USA). Sections of 20 μm thickness were cut and air-dried inside the cryostat for less than 20 minutes. Then, the frozen sections were fixed in ice-cold 4% PFA for 15 minutes, followed by dehydration in an ethanol series. The slices were treated with H2O2 for 10 minutes and washed in PBS. Protease Ⅲ was added and incubated for 20 minutes at room temperature. Hybridization with the Esr1 (Mm-Esr1-C1, 478201-C1) and EGFP (Mm-EGFP-C3, 400281-C3) probes was performed in a HybEZ humidified incubator at 40 °C for 2.5 hours. After washing in ACD buffer, slices were sequentially incubated with AMP1-FL and AMP2-FL (30 minutes each), AMP3-FL (15 minutes), and counterstained with DAPI. Images were acquired using an Olympus FV3000 confocal microscope.

Monosynaptic retrograde tracing
To map upstream inputs to VMHvlEsr1 neurons, we unilaterally co-injected AAV2/9-Ef1a-DIO-RVG-WPRE-pA (2.00E+12v.g./mL) and AAV2/9-Ef1a-DIO-His-EGFP-2A-TVA-WPRE-pA (2.00E+12v.g./mL) at a 1:1 volume ratio into the VMHvl (AP, -1.43 mm; ML, ±0.90 mm; DV, -5.75 mm) of adult female Esr1-Cre mice (120 nL). Postoperative analgesia was provided via ketoprofen (5 mg/kg, i.p.). Following two weeks of recovery and viral expression, 120 nL of RV-EnvA-ΔG-tdTomato (7.00E+08 IFU/mL) was injected into the VMHvl under biosafety level 2 conditions. The mice were sacrificed 7 days after rabies virus injection. Whole-brain imaging was performed using the Leica STED confocal system and Olympus VS200.

For tracing inputs to MPOAPhf6 neurons, we unilaterally co-injected AAV2/9-Ef1a-DIO-RVG-WPRE-pA (2.00E+12v.g./mL) and AAV2/9-Ef1a-DIO-His-EGFP-2A-TVA-WPRE-pA (2.00E+12v.g./mL) at a 1:1 volume ratio into the MPOA (AP, -0.10 mm; ML, ±0.25 mm; DV, -5.25 mm) of adult female Phf6-CreER mice (120 nL). Postoperative analgesia was provided with ketoprofen (5 mg/kg, i.p.). After 7 days of recovery, the mice were intraperitoneally injected with tamoxifen (100 mg/kg) for four times. One week after tamoxifen induction, 120 nL of RV-EnvA-ΔG-tdTomato (7.00E+08 IFU/mL) was injected into the MPOA under biosafety level 2 conditions. The mice were sacrificed 7 days after rabies virus injection. Whole-brain imaging was performed using the Leica STED confocal system and Olympus VS200.

To identify inputs to local SST⁺ inhibitory neurons in the TN, we unilaterally co-injected AAV2/9-Ef1a-DIO-RVG-WPRE-pA (2.00E+12v.g./mL) and AAV2/9-Ef1a-DIO-His-EGFP-2A-TVA-WPRE-pA (2.00E+12v.g./mL) at a 1:1 volume ratio into the TN (AP, -1.55 mm; ML, ±1.00 mm; DV, -5.75 mm) of female SST-Cre mice (120 nL). Postoperative analgesia was provided via ketoprofen (5 mg/kg, i.p.). After two weeks of recovery and AAV expression, 120 nL RV-EnvA-ΔG-tdTomato (7.00E+08 IFU/mL) was injected into the TN under biosafety level 2 conditions. Mice were sacrificed 7 days after rabies virus injection, and brains were fixed and sectioned. We obtained images using the Leica STED confocal system.

Statistical analysis
In Figures 1m, 2f (Predicted EE), 2j–l, 3j, 3l, 4i, 4l, 4n, 4o–q, 5d, 5e, 5g, 5k, 5m, 6h, 7c, 7d, 7f, 7h, 7l, S4b–d, S4g–i, S5e–k, S7a–d, and S7h–l, data were analyzed using two-tailed unpaired Student’s t test to compare the means of two independent groups. In Figures 6d, 7m–p, S9g, and S9h, data were analyzed using two-tailed paired Student’s t test to compare the means of the same samples subjected to different treatments. In Figures 1i–k, 2c–e, 2h, 2i, 4c, 4f, 4k, 4m, 4s, 5i, 6g, S3a–m, S4j, S4k, S5d, S5l, and S7f, data were analyzed using two-way repeated-measures ANOVA with genotype as a between-subject factor and age as a within-subject factor, followed by Bonferroni’s multiple-comparisons test to compare genotypes at each time point. In Figure 2f, data were analyzed using ANCOVA. Data are presented as mean ± SEM in all figure legends. Statistical difference is displayed as *p < 0.05, **p < 0.01, and ***p < 0.001, whereas n.s. indicates no significant difference. Statistical analyses were performed using GraphPad Prism 8. Details of sex, age, number, statistical methods, and corresponding statistical values are provided in the Source Data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Obesity is prevalent among BFLS patients, who have mutations in the Phf6 gene. In this study, Wang et al. investigated the role of MPOA Phf6 neurons in female mice regarding exercise and energy expenditure. They found that knockout or inhibition of MPOA Phf6 led to decreased activity and energy expenditure, resulting in obesity, whereas activation promoted exercise. Electrophysiological studies further confirmed that Phf6 is crucial for maintaining neuronal intrinsic excitability and estrogen sensitivity. Finally, downstream targets of MPOA Phf6 were explored, with VMHvl Esr1 chosen, as its activation facilitated exercise. Overall, this study provides theoretical support for treating BFLS-related obesity. This paper is of great importance; however, it still lacks novelty, which is why I prefer to decline. Below are my questions:\nMajor Issues\n1.In humans, BFLS is found mainly in boys, why was only female mice selected for the experiments? Research by Joseph R. Knoedler et al. (2022) indicates that MPOA Esr1 neurons exhibit sexual dimorphism, with males and females showing different hormone levels and sensitivities.\n2.Does chronic knockout of the Phf6 gene in the MPOA lead to hormonal changes? The MPOA plays a crucial role in the hypothalamic-pituitary-gonadal (HPG) axis, suggesting that alterations in Phf6 could impact hormonal levels.\n3.How are non-exercise activity thermogenesis (NEAT) and exercise-induced thermogenesis regulated separately in your working model? During the inactive phase of the mice, there was no difference in heat production between the control and knockout groups, with differences observed only during the active phase. Does this indicate that basal metabolism remains unaltered, and that the differences in heat production result solely from variations in activity levels?\n4. Did the increase in activity in the open field after activating MPOA Phf6 affect anxiety level?\n5. The experiment involving caspase ablation of Esr1 cells in the MPOA seems out of place. It may be more appropriate to include this in the Results section, specifically in the subsection “Loss of Phf6 in MPOA Phf6 neurons induces obesity.”\n6. In electrophysiological experiments, did the knockout of Phf6 reduce cellular sensitivity to estradiol due to decreased Esr1 receptors? Furthermore, does this suggest that the knockout of Phf6 could alter hormonal levels?\n7. Besides changes in the F-I curve, was there any variation in the resting membrane potential following the knockout of Phf6?\n8. Since 80% of MPOA Esr1 neurons are GABAergic, are Phf6 neurons also GABAergic or glutamatergic? The text mentions that MPOA Phf6 projects to VMHvl Esr1 with monosynaptic projections, and since both MPOA Phf6 and VMHvl Esr1 activation increases activity, does this imply that the MPOA Phf6 to VMHvl Esr1 projection might be excitatory? Or dis-inhibition?\n9. When using rabies virus to demonstrate projections from MPOA Phf6 to VMHvl, how many upstream neurons of VMHvl Esr1 are Phf6-positive? Statistical data is needed here. Additionally, the authors could use electrophysiology to activate the upstream neurons and record downstream EPSCs/IPSCs to further validate the projection relationship.\n10. Given the numerous downstream targets of MPOA Phf6, all related to activity and metabolism, why focus solely on the projection to VMHvl Esr1? Additionally, since VMHvl provides excitatory input to MPOA, why not investigate the projection from VMHvl to MPOA Phf6 in relation to activity?\n11.Furthermore, the last experiment in the study, using chemical genetic activation of VMHvl Esr1 alone, does not prove that VMHvl is a downstream effector of MPOA Phf6. The authors should suppress upstream MPOA Phf6 while activating VMHvl Esr1; or activate upstream and inhibit the downstream. if increased activity is observed, it would support the idea that VMHvl acts downstream of MPOA Phf6, rather than projecting to MPOA Phf6.\n12.Why do you generate Phf6 CreER mice rather than classical Cre mouse line?\nMinor Issues:\n1. Incorrect full name of VMH in Line 46.\n2. Please systematically quantify rabies virus tracing data.\n3. Why were different CNO doses used for chemical genetic activation and inhibition?\n4. In Line 195, the CRISPR-Cas9 experiment should refer to Phf6 knockout (KO), not knockdown (KD), and subsequent references to KD should be changed to KO.", "reviewer_2": "This is an interesting study that identified a role for Phf6 in the POA on locomotion and related impacts on obesity development. A combination of novel animal models, viral tracing, opto and chemogenetic approaches was used to generate evidence supporting a specific role of POA Phf6 neurons promotes locomotion through specific projections to VMH Esr+ neurons via Phf6 mediated activation of neuron activity. The experiments were well designed and analyzed, and the results were cleanly presented. However, a few major and minor concerns were noted.\nMajor:\n1) Locomotion vs temperature: Since POA is well known to regulate body temperature, which is known to modify energy expenditure (EE), the presented effect on EE may also be due to an effect on body temperature. Data on body temperature need to be included in all panels on locomotion.\n2) The POA area is known to be made of both Glu+ and GABA+ neurons, both of which are implicated in body temperature, although some discrepancy has been reported. Given this, the authors need to verify whether Phf6-expressing neurons are Glu+ or GABA+ neurons. This point is also related to the data presented in Fig. 6 on the projection.\n3) Data in Fig. 6 generated with optogenetic stimulation of VMH terminals will not provide sufficient evidence supporting a role of VMH neurons mediating the effect as terminal stimulation may cause collateral activation through back-propagation. Further analysis is required to confirm the functional connection.\nMinor:\n1) Fig. 2c-e data on no changes in food intake lack resolution. Real time feeding data from CLAMS data will be very helpful to provide feeding patterns, which is important in this case as feeding is tightly related to EE and it is important to see whether no changes in feeding is caused by an increased feeding in day but reduced feeding in night.\n2) Fig. 2f the presented EE is normalized to body weight is problematic as body weight is different in all time points measured, which will cause distortion in comparison in both day and night periods. From the curves shown, it is likely that EE would be reduced in night but increased in day.", "reviewer_3": "This study by Jingjie Wang et al. addresses an important topic regarding the role of Phf6+ neurons in the medial preoptic area (mPOA) in regulating physical activity and energy expenditure, which may be relevant to the human disorder Börjeson–Forssman–Lehmann syndrome (BFLS). The authors present data showing the effects of Phf6 deletion on feeding behavior, physical activity, and energy expenditure. They further evaluate the necessity and sufficiency of Phf6+ mPOA neurons in driving physical activity, investigate the role of Phf6 in neuronal excitability, and identify a downstream circuit projecting to the ventrolateral portion of the ventromedial hypothalamus (VMHvl) that may contribute to these behavioral effects. Overall, the manuscript is informative and well-structured. I am enthusiastic about its publication in Nature Communications, provided that the following points are clarified and revised.\nMajor comments\n1. In Figure 1e, the authors show that the majority of Phf6+ cluster neurons are Slc17a6-negative. Given that approximately 60% of Esr1+ neurons in the mPOA are GABAergic, this suggests that Phf6+ neurons in the mPOA are likely to be inhibitory. Moreover, electrophysiological data presented in Figure 4 indicate that Phf6 is important for maintaining the excitability of these neurons. In Figure 5, the authors propose that mPOA Phf6+ neurons directly project to Esr1+ neurons in the VMHvl, which implies a potentially inhibitory input onto this downstream population. However, Figure 6d to f shows that activation of Esr1+ VMHvl neurons increases physical activity.\nThis raises an important mechanistic question: how can a potentially inhibitory projection from Phf6+ mPOA neurons to Esr1+ VMHvl neurons facilitate increased locomotor activity, given that activation of the target neurons also promotes this behavior? A discussion or experiment clarifying the circuit-level mechanism by which Phf6+ mPOA neurons influence Esr1+ VMHvl neurons, and how this connectivity supports the observed behavioral effects, would strengthen the manuscript.\n2. The data from acute activation (Figures 3g–i and 6) are somewhat less convincing in establishing sufficiency. I have two specific concerns:\nA) In Figure 3g–i, the authors use the open field test (OFT) to assess locomotor activity following acute activation of Phf6+ neurons. While the primary interpretation focuses on physical activity, OFT is also widely used to assess anxiety-like behavior. From the representative movement traces, it appears that center time and distance may also be altered by the manipulation. This raises the possibility that Phf6+ neuronal activity could influence anxiety-related behaviors in addition to locomotion. Although changes in overall locomotor activity can complicate the interpretation of anxiety-related OFT measures, the conclusions would be strengthened by including data from additional anxiety-specific assays, such as the elevated plus maze or light-dark box test, to help disentangle locomotion-specific effects from potential anxiety-related changes.\nB) The data shown in Figure 3m–o, demonstrating that chemogenetic inhibition of Phf6+ neurons reduces both physical activity and energy expenditure, together with reduced locomotion in Figure 3j–l, provide compelling evidence for the necessity of these neurons in regulating physical activity. In contrast, the data from acute activation (Figure 3g–i) only show change in locomotion in OFT seems less robust in establishing sufficiency. To more fully support the model proposed in Figure 6g, it would be helpful to determine whether chemogenetic activation of Phf6+ neurons also promotes voluntary activity or increases energy expenditure.\nAddressing point A and/or B would help to strengthen the overall conclusions of the study.\nMinor comments\n3. While the authors note in the Introduction (line 66) that Börjeson–Forssman–Lehmann syndrome (BFLS) affects both males and females, all experiments in the current study were conducted using female mice. Given that Esr1 expression is present in the mPOA of both sexes, it would be helpful for the authors to clarify the rationale for focusing exclusively on females. A brief discussion of the reasons for this choice would improve the interpretability and generalizability of the findings.\n4. Do Phf6+ mPOA neurons specifically modulate physical activity and energy expenditure? Previous studies have demonstrated that both Esr1+ neurons in the mPOA and VMHvl play key roles in regulating sexual behavior. Given that the current study shows projections from Phf6+ (and Esr1+) mPOA neurons to Esr1+ VMHvl neurons, it would be helpful if the authors could discuss whether this circuit might also participate in the regulation of reproductive behaviors, and how such potential functions might relate to the observed modulation of physical activity and energy expenditure.\n5. The manuscript does not report the laser power used for optogenetic stimulation. For the sake of reproducibility and proper interpretation of the results, the authors should include this information in the Methods section.", "reviewer_4": "Wang et al discovered a unique role of Phf6 in maintaining body weight homeostasis in female mice through modulating the neuronal excitability of mPOA neurons to regulate locomotor activity. They also proposed the VMHvl as a potential downstream target for the locomotion change. The mutation of Phf6 gene was reported in BFLS, a rare human disease. It is intriguing how the authors linked Phf6 with obesity, which was of a high percentage in BFLS, and further demonstrated how it modulates the neuronal excitability with the hypothalamus. I believe this paper will be a good example of how basic scientific research links with bench work. However, there are a few key experiments that must be performed for the authors to make a clear conclusion.\n1. Phf6 is located on the X-chromosome and the prevalence of obesity does have a sex difference in BFLS, and the percentage is higher in men compared to women. Yet the authors did not explain why only female animals were used in the study. Key functional experiments should be repeated and shown in the paper and clear clarification of why female mice were mainly studied should be made.\n2. The authors showed that mPOA Phf6 neurons send monosynaptic projections to the VMHvl Esr1 neurons, and optogenetic activation of the circuit as well as VMHvl Esr1 neurons increased locomotor activity, consistent with the function of mPOA Phf6 neurons. However, based on the trajectory tracing from Figure 5a-5c, there are also other downstream targets including NAc, PVH, and TMN in addition to the VMHvl. All of these regions have been reported to be involved in changes of locomotor activities in mice. Key experiments should be done to further prove that VMHvl is the main downstream target.\n3. Authors utilized slice recordings to demonstrate the potential role of Phf6 in the electrophysiology properties of mPOA Phf6 neurons and showed that loss of Phf6 leads to reduced excitability and estradiol-induced firing increase. However, it remains unclear about the physiological significance of these cells in vivo. It will be helpful to show when these neurons will be recruited.\nI also have some minor issues that need to be addressed:\n1. The change of body weight became significantly different after 2-4 weeks post surgery (Figures 1h-1i), and the change of energy expenditure was not significant at 4 weeks post surgery (Figures 1g-1h). As there was no change of food intake (Figures 1c-1e), potential mechanisms leading to the early phase of body weight change need to be clarified or discussed. mPOA neurons are known to regulate body temperature, do these Phf6 neurons also modulate body temperature?\n2. Line 52: “which are molecular heterogeneity”   “which are of molecular heterogeneity”\n3. Line 66: where is the sampling rate from? The obesity rate in the US is around 40% in females.\n4. Line 106: “increase body weight gain”, “increase” and “gain” are redundant.\n5. Line 195: “knockdown” should be verb “knock down”\n6. Line 237: AAV1-based tracing is not monosynaptic.\n7. Line 251: if one hyphen is used, the rest should also use hyphen."}, "author_response": "Response to Reviewers’ Comments\nWe would like to thank the reviewers for their insightful and constructive comments.\nWe have effectively addressed reviewers’ comments in the revised manuscript and\nincorporated changes where appropriate. The changes are highlighted in yellow in the\nrevised manuscript.\nReviewer #1:\nWe thank the 1st reviewer for stating, “Obesity is prevalent among BFLS patients, who\nhave mutations in the Phf6 gene. In this study, Wang et al. investigated the role of MPOA Phf6\nneurons in female mice regarding exercise and energy expenditure. They found that knockout or\ninhibition of MPOA Phf6 led to decreased activity and energy expenditure, resulting in obesity,\nwhereas activation promoted exercise. Electrophysiological studies further confirmed that Phf6 is\ncrucial for maintaining neuronal intrinsic excitability and estrogen sensitivity. Finally, downstream\ntargets of MPOA Phf6 were explored, with VMHvl Esr1 chosen, as its activation facilitated exercise.\nOverall, this study provides theoretical support for treating BFLS-related obesity. This paper is of\ngreat importance; however, it still lacks novelty, which is why I prefer to decline. Below are my\nquestions:”\nComments:\n1. “In humans, BFLS is found mainly in boys, why was only female mice selected for the experiments?\nResearch by Joseph R. Knoedler et al. (2022) indicates that MPOA Esr1 neurons exhibit sexual\ndimorphism, with males and females showing different hormone levels and sensitivities.”\nRESPONSE:\nWe thank the reviewer for the helpful comment. The reason for selecting female mice\nover male mice for the experiments in the original manuscript was that male Phf6 cKO mice\nexhibited a weaker obesity phenotype than female Phf6 cKO mice. As guided by the\nreviewer, we have now completed new experiments to examine the body weight, feeding\nbehavior, physical activity, energy expenditure, and body temperature in male Phf6 cKO\nmice.\nWe knocked out Phf6 in the MPOA by injecting AAV-CMV-Cre (or AAV-CMV-GFP as\nthe control) into the MPOA of 8-week-old male Phf6flox/y mice. The efficiency of Phf6 deletion\nwas measured by post hoc immunostaining. Compared to controls, male Phf6 cKO mice\nshowed no difference in body weight 2-8 weeks post viral injection, yet showed a significant\nincrease in body weight at 10 and 12 weeks post viral injection (new Supplementary Fig.\n4d). However, it was notable that the extent of obesity was much smaller in male Phf6 cKO\nmice than in female Phf6 cKO mice (new Supplementary Fig. 4h).\nphysical activity compared to littermate controls (new Supplementary Fig. 4f, g). Similarly,\nthe extent of decreased energy expenditure and physical activity was also smaller in male\nPhf6 cKO mice than in female Phf6 cKO mice (new Supplementary Fig. 4i, j).\nNo differences were observed in feeding behavior and body temperature in both male\nand female Phf6 cKO mice (new Supplementary Fig. 4k, l).\nThe differences in the extents of Phf6-cKO-induced obesity and reduction in energy\nexpenditure and physical activity between male and female mice indicates that Phf6\nregulates physical activity and metabolism in a sex-dependent manner.\nWe have summarized the Phf6-cKO-induced phenotypes in both female and male\nmice in the following table:\nFemale Phf6 cKO mice Male Phf6 cKO mice\nBody weight ↑↑↑ ↑\nEnergy expenditure ↓↓↓ ↓\nPhysical activity ↓↓↓ ↓\nFeeding Behavior n.s. n.s.\nBody temperature n.s. n.s.\n2. “Does chronic knockout of the Phf6 gene in the MPOA lead to hormonal changes? The MPOA\nplays a crucial role in the hypothalamic-pituitary-gonadal (HPG) axis, suggesting that alterations in\nPhf6 could impact hormonal levels.”\nRESPONSE:\nGuided by reviewer, we have now conducted new experiments to investigate whether\nknockout of Phf6 in the MPOA leads to changes in HPG-axis hormones in adult female mice.\nWe knocked out Phf6 in the MPOA by injecting AAV-CMV-Cre (or AAV-CMV-GFP as\nthe control) into the MPOA of 8-week-old female Phf6flox/flox mice. The efficiency of Phf6\ndeletion was evaluated by post hoc immunostaining. At 8 weeks post viral injection, we\n2\nexamined the plasma levels of follicle stimulating hormone (FSH), luteinizing hormone (LH),\nand estradiol (E2) using ELISA kits. No differences were detected for the plasma levels of\nFSH, LH, and E2 between adult female control and Phf6 cKO mice (new Supplementary\nFig. 3a-d), suggesting that knockout of Phf6 in the MPOA of adult female mice does not\naffect the plasma levels of HPG-axis hormones. These findings exclude that the observed\nphenotypes in the female Phf6 cKO mice result from changes in HPG-axis hormones.\n3. “How are non-exercise activity thermogenesis (NEAT) and exercise-induced thermogenesis\nregulated separately in your working model? During the inactive phase of the mice, there was no\ndifference in heat production between the control and knockout groups, with differences observed\nonly during the active phase. Does this indicate that basal metabolism remains unaltered, and that\nthe differences in heat production result solely from variations in activity levels?”\nRESPONSE:\nWe thank the reviewer for this insightful guidance. We have now assessed the\nchanges of basal metabolic rate (BMR), non-exercise activity thermogenesis (NEAT) and\nexercise-induced thermogenesis (EIT) in control and Phf6 cKO female mice.\nWe knocked out Phf6 in the MPOA by injecting AAV-CMV-Cre (or AAV-CMV-GFP as\nthe control) into the MPOA of 8-week-old female Phf6flox/flox mice. The efficiency of Phf6\ndeletion was evaluated by post hoc immunostaining. At 12 weeks post viral injection, the\nmice were first fasted overnight to eliminate the effect of diet-induced thermogenesis. Then,\nthe mice were subjected to metabolic cages (CLAMS) and the metabolic parameters were\nreported every 8 minutes.\nBMR was calculated as the total energy expenditure (TEE) during the 1 hour of least\nactivity in sleep period; NEAT was assessed by subtracting BMR from the TEE when wheel\nrunning = 0 during active phase (dark period: 8 PM- 8 AM); EIT was assessed by subtracting\nBMR from the TEE when wheel running > 0 during active phase (dark period: 8 PM- 8 AM).\nCompared to the littermate controls, we found that EIT was profoundly reduced in\nfemale Phf6 cKO mice (new Fig. 2j), whereas BMR and NEAT showed slight decreases\n(new Fig. 2k, l). These results suggest that the reduction in EIT is the primary contributor to\nthe lower total energy expenditure observed in female Phf6 cKO mice, whereas changes in\nBMR and NEAT play a minor role.\n4. “Did the increase in activity in the open field after activating MPOA Phf6 affect anxiety level?”\nWe thank the reviewer for this comment. We have now conducted new analyses on\nthe levels of anxiety using the open field (OF) test and the elevated plus maze (EPM) test.\nIn the optogenetic activation experiment, we targeted ChR2 to the MPOAPhf6 neurons\nby injecting AAV-DIO-ChR2-mCherry (or AAV-DIO-mCherry as the control) into the MPOA\nof 8-week-old female Phf6-CreER mice. We found that optogenetic activation of MPOAPhf6\nneurons did not alter the ratio of time spent in the center zone of the OF tests, suggesting\nthat activating MPOAPhf6 neurons has no effect on anxiety (new Fig. 4o).\nIn the chemogenetic activation experiment, we targeted hM3D to the MPOAPhf6\nneurons by injecting AAV-DIO-hM3D-mCherry (or AAV-DIO-mCherry as the control) into the\nMPOA of 8-week-old female Phf6-CreER mice. CNO was i.p. administered 40 minutes\nbefore assessment. Chemogenetic activation MPOAPhf6 neurons did not alter the ratio of time\nspent in the center zone of the OF test and the time spent in the open arms of the EPM test\n(new Fig. 4p, r and s), suggesting that activating MPOAPhf6 neurons has no effect on anxiety.\nThese results from OF and EPM tests exclude changes in anxiety as an explanation\nfor the observed increases in physical activity upon activating MPOAPhf6 neurons.\n5. “The experiment involving caspase ablation of Esr1 cells in the MPOA seems out of place. It may\nbe more appropriate to include this in the Results section, specifically in the subsection “Loss of Phf6\nin MPOA Phf6 neurons induces obesity.”\nRESPONSE:\nWe thank the reviewer for this comment, we have moved the results related to the\nexperiment involving caspase ablation of Esr1 cells in the MPOA from the subsection\n“Inhibition of mPOAPhf6 neurons reduces energy expenditure and physical activity.” to the\nsubsection “Loss of Phf6 in MPOAPhf6 neurons reduces energy expenditure and physical\nactivity.”.\n6. “In electrophysiological experiments, did the knockout of Phf6 reduce cellular sensitivity to\nestradiol due to decreased Esr1 receptors? Furthermore, does this suggest that the knockout of Phf6\ncould alter hormonal levels?”\nRESPONSE:\nexperiment to assess the expression of Esr1 mRNA in MPOAPhf6 neurons using single-\nmolecule fluorescent in situ hybridization (smFISH). We used the same strategy to knock\ndown Phf6 in this experiment as in the electrophysiological experiment. To knock down Phf6\nand simultaneously label MPOAPhf6 neurons with EGFP, we employed a CRISPR/Cas9-\nmediated genome editing approach by bilaterally injecting AAV vector carrying triple Phf6-\ntargeting single-guide RNAs, AAV-hU6-sgRNA (Phf6)-hEf1a-DIO-EGFP, co-injected with\n1-3\nAAV2/9-CAG-DIO-spCas9 into the MPOA of adult female Phf6-CreER mice. The AAV\nvector without sgRNAs was used as the control.\nUsing smFISH with Esr1 and EGFP mRNA probe, we found that knockout of Phf6\nled to a significant decrease in the level of Esr1 mRNA in Phf6-deficient MPOAPhf6 neurons\ncompared to the control (new Fig. 5l, m). This result suggests that the reduced cellular\nsensitivity and responsiveness to estradiol in Phf6-deficient MPOAPhf6 neurons very likely\nstem from decreased expression of estrogen receptors Esr1.\nWe have also evaluated the plasma levels of HPG-axis hormones, including follicle\nstimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) using ELISA kits.\nWe found that knockout of Phf6 in the MPOA of adult female mice had no effect on the\nplasma levels of FSH, LH, or E2 compared to the controls (new Supplementary Fig. 3a-d),\nruling out changes in HPG-axis hormones as an explanation for the observed reduction in\nsensitivity and responsiveness to estradiol in Phf6-deficient MPOAPhf6 neurons.\n7. “Besides changes in the F-I curve, was there any variation in the resting membrane potential\nfollowing the knockout of Phf6”\nRESPONSE:\nGuided by the reviewer, we have now performed new experiments to measure the\nresting membrane potential and input resistance. In electrophysiological experiments, we\nknocked down Phf6 in MPOAPhf6 neurons by Phf6-sgRNAs using CRISPR/Cas9-mediated\ngenome editing. We performed whole-cell patch clamp recording on Phf6-deficient and\ncontrol MPOAPhf6 neurons that labelled with EGFP.\nCompared to the control neurons, knockdown of Phf6 did not change the resting\nmembrane potential as (new Fig. 5d). However, knockdown of Phf6 significantly increased\nthe input resistance (new Fig. 5e). The unchanged resting membrane potential but\nincreased input resistance suggest that voltage-gated ion channels expression or function\nare likely altered in Phf6-deficient MPOAPhf6 neurons, which provides an explanation for the\nPhf6-knockdown-induced decrease in intrinsic excitability.\n5\n8. “Since 80% of MPOA Esr1 neurons are GABAergic, are Phf6 neurons also GABAergic or\nglutamatergic? The text mentions that MPOA Phf6 projects to VMHvl Esr1 with monosynaptic\nprojections, and since both MPOA Phf6 and VMHvl Esr1 activation increases activity, does this imply\nthat the MPOA Phf6 to VMHvl Esr1 projection might be excitatory? Or dis-inhibition?”\nRESPONSE:\nGuided by the reviewer, we have conducted new experiments to investigate:\n• the proportion of MPOAPhf6 neurons that are glutamatergic vs. GABAergic.\n• the involvement of excitation and dis-inhibition circuitry from MPOAPhf6 neurons\nto VMHvlEsr1 neurons.\nTo label the glutamatergic and GABAergic neurons, we crossed the Vglut2-Cre mice\nand Vgat-Cre mice with a reporter line expressing Cre-dependent histone-GFP (LSL-H2B-\nGFP). In Vglut2-Cre::LSL-H2B-GFP mice, we found that 31.6% ± 4.7% of MPOAPhf6 neurons\nin females and 18.0% ± 2.9% in males were Vglut2-positive glutamatergic neurons (new\nFig. 1d, e); in Vgat-Cre::LSL-H2B-GFP mice, 69.2% ± 4.2% of MPOAPhf6 neurons in females\nand 82.7% ± 2.0% in males were Vgat-positive GABAergic neurons (new Fig. 1d, e). The\nratio of glutamatergic vs. GABAergic MPOAPhf6 neurons is approximately 3:7 in female mice\nand 2:8 in male mice. The proportion of glutamatergic MPOAPhf6 neurons was slightly higher\nin female mice than in male mice.\nIn addition, we have also analyzed gene expression profiles of MPOAPhf6 neurons\n(Phf6+/Esr1+ double positive) using previously published scRNA-seq datasets (Knoedler et\nal., Cell 2022; Moffitt et al., Science 2018). MPOAPhf6 neurons were identified as 4 distinct\nsubclusters by UMAP analysis. Excitatory neuronal marker Slc17a6 (Vglut2) was expressed\nin 2 clusters of MPOAPhf6 neurons; inhibitory neuronal markers Gad1 and Slc32a1(Vgat)\nwere expressed in the other 2 clusters (new Supplementary Fig. 1d-f). Similar proportion\nof glutamatergic vs. GABAergic MPOAPhf6 neurons were identified in scRNA-seq analyses\nas that observed in immunostaining experiments (new Supplementary Fig. 1h).\nBy ChR2-assisted circuit mapping using electrophysiology, we have investigated\nwhether the projections from MPOAPhf6 to the VMHvl comprise both excitation and dis-\ninhibition circuitry. We targeted ChR2 to MPOAPhf6 neurons by injecting AAV-DIO-ChR2-\nmCherry into the MPOA of 8-week-old female Phf6-CreER mice. Neurons were recorded at\nthe VMHvl of acute slices by whole-cell patch clamp recording. Light-evoked EPSCs\n(oEPSCs) were recorded at -70 mV; light-evoked IPSCs (oIPSCs) were recorded at 0 mV.\noEPSCs were recorded in 24 out of 69 neurons, which were inhibited by the glutamate\nreceptor antagonist CNQX (new Fig. 7k, m); oIPSCs were recorded in 45 out of 69 neurons,\nwhich were inhibited by GABA receptor antagonist PTX (new Fig. 7k, n). Sodium channel\nA\nblocker TTX inhibited the oEPSCs and oIPSCs, while potassium channel blocker 4-AP\nreversed the TTX-inhibited oEPSCs and oIPSCs (new Fig. 7o, p). These results\ndemonstrate that MPOAPhf6 neurons form both excitatory and inhibitory synapses with the\nVMHvl neurons.\nVMHvlEsr1 neurons through a dis-inhibition circuitry. A prior study showed that VMHvlEsr1\nneurons received inhibitory synaptic inputs from local GABAergic inhibitory neurons in and\nsurrounding the VMHvl (Wei et al., Nat Neurosci 2023). We also note that there are substantial\nnumber of SST+ inhibitory neurons in the TMN (TMNSST neurons) surrounding the VMHvl\n(Luo et al., Science 2018). We sought to determine whether the dis-inhibition circuitry from\nMPOAPhf6 neurons to VMHvlEsr1 neurons is through TMNSST inhibitory neurons. Using Phf6-\nCreER::SST-Flpo mice, we labeled MPOAPhf6 neuron with mGFP and TMNSST neurons with\nmCherry by injecting AAV-DIO-mGFP into the MPOA and AAV-fDIO-mCherry into the TMN.\nAnatomical analysis of mGFP-labeled MPOAPhf6 neuron axon terminals revealed close\napposition of MPOAPhf6 boutons to TMNSST cell bodies (new Supplementary Fig. 8a, b).\nWe also performed rabies virus-based retrograde tracing from the TMNSST neurons\nby injecting AAV-DIO-RVG and AAV-DIO-His-EGFP-2A-TVA into the TMN of adult female\nSST-Cre mice, followed by injecting RV-EnvA-ΔG-tdTomato into the same site. We found\nthat 31.4% ± 1.6% of tdTomato-positive upstream neurons of TMNSST in the MPOA were\npositive for Phf6 (new Supplementary Fig. 8c-e), suggesting that MPOAPhf6 neurons are\nthe monosynaptic upstream of TMNSST neurons. We infer that MPOAPhf6 neurons form a dis-\ninhibitory circuitry with VMHvlEsr1 neurons through local TMNSST inhibitory neurons.\nTaken together, we reveal that the ratio of glutamatergic vs. GABAergic MPOAPhf6\nneurons is approximately 3:7 in female mice and 2:8 in male mice. The MPOAPhf6→VMHvlEsr1\npathway comprise both excitation and dis-inhibition circuitry.\n9. “When using rabies virus to demonstrate projections from MPOA Phf6 to VMHvl, how many\nupstream neurons of VMHvl Esr1 are Phf6-positive? Statistical data is needed here? Additionally,\nthe authors could use electrophysiology to activate the upstream neurons and record downstream\nEPSCs/IPSCs to further validate the projection relationship.”\nRESPONSE:\nWe thank the reviewer for this helpful comment. We have conducted new analyses\nand experiments to investigate:\n• percentage of VMHvlEsr1 upstream neurons in the MPOA that are positive for Phf6.\n• EPSCs/IPSCs recorded in the VMHvl in response to activating MPOAPhf6 neuron\naxon terminals.\n• the excitation and dis-inhibition circuitry involved in the projection relationship\nfrom MPOAPhf6→VMHvlEsr1.\n7\n(1) In the rabies virus-based retrograde tracing experiment, we have quantified the\npercentage of upstream neurons of VMHvlEsr1 that were positive for Phf6 in the\nMPOA. Briefly, AAV-DIO-RVG and AAV-DIO-His-EGFP-2A-TVA were injected\ninto the VMHvl of adult female Esr1-Cre mice, followed by injecting RV-EnvA-\nΔG-tdTomato into the same site. We found that 16.1% ±1.4% of tdTomato-\nexpressing neurons in the MPOA were positive for Phf6 (new Fig. 6p). This result\nshowed that approximately 20% of VMHvlEsr1 upstream neurons in the MPOA are\npositive for Phf6.\n(2) By ChR2-assisted circuit mapping using electrophysiology, we have performed\nwhole-cell patch clamp recording to record neurons at the VMHvl in acute slices\nupon activating the axon terminals of MPOAPhf6 neurons. We targeted ChR2 to\nMPOAPhf6 neurons by injecting AAV-DIO-ChR2-mCherry into the MPOA of 8-week-old female Phf6-CreER mice. Light-evoked EPSCs (oEPSCs) were\nrecorded in 24 out of 69 neurons, which were inhibited by the glutamate receptor\nantagonist CNQX (new Fig. 7k, m); light-evoked IPSCs (oIPSCs) were recorded in 45 out\nof 69 neurons, which were inhibited by GABA receptor antagonist PTX (new Fig.\nA\n7k, n). Sodium channel blocker TTX inhibited the oEPSCs and oIPSCs, while\npotassium channel blocker 4-AP reversed the TTX-inhibited oEPSCs and\noIPSCs (new Fig. 7o, p). These results demonstrate that MPOAPhf6 neurons form\nboth excitatory and inhibitory synapses with the VMHvl neurons.\n(3) Besides excitation circuitry, we further investigated whether MPOAPhf6 neurons\nform a dis-inhibition circuitry with VMHvlEsr1 neurons through a dis-inhibition\ncircuitry. A prior study showed that VMHvlEsr1 neurons received inhibitory synaptic\ninputs from local GABAergic inhibitory neurons in and surrounding the VMHvl\n(Wei et al., Nat Neurosci 2023). We also note that there are substantial number of\nSST+ inhibitory neurons in the TMN (TMNSST neurons) surrounding the VMHvl\n(Luo et al., Science 2018).\nWe sought to determine whether the dis-inhibition circuitry from MPOAPhf6 neurons to\nVMHvlEsr1 neurons is through TMNSST inhibitory neurons. Using Phf6-\nCreER::SST-Flpo mice, we labeled MPOAPhf6 neuron with mGFP and TMNSST\nneurons with mCherry by injecting AAV-DIO-mGFP into the MPOA and AAV-\nfDIO-mCherry into the TMN. Anatomical analysis of mGFP-labeled MPOAPhf6\nneuron axon terminals revealed close apposition of MPOAPhf6 boutons to TMNSST\ncell bodies (new Supplementary Fig. 8a, b).\nWe also performed rabies virus-based retrograde tracing from the TMNSST\nneurons by injecting AAV-DIO-RVG and AAV-DIO-His-EGFP-2A-TVA into the\nTMN of adult female SST-Cre mice, followed by injecting RV-EnvA-ΔG-tdTomato\ninto the same site. We found that 31.4% ± 1.6% of tdTomato-positive upstream\nneurons of TMNSST in the MPOA were positive for Phf6 (new Supplementary\nFig. 8c-e), suggesting that MPOAPhf6 neurons are the monosynaptic upstream of\nwith VMHvlEsr1 neurons through local TMNSST inhibitory neurons.\nTaken together, these results demonstrate that ~20% of VMHvlEsr1 upstream\nneurons in the MPOA are positive for Phf6; projections from MPOAPhf6 to VMHvlEsr1 comprise\nboth excitation and dis-inhibition circuitry.\n10. “Given the numerous downstream targets of MPOA Phf6, all related to activity and metabolism,\nwhy focus solely on the projection to VMHvl Esr1? Additionally, since VMHvl provides excitatory\ninput to MPOA, why not investigate the projection from VMHvl to MPOA Phf6 in relation to activity?”\nRESPONSE:\nWe thank the reviewer for this insightful comment. We have conducted new\nexperiments to investigate:\n• the effects of activating each of the MPOAPhf6 axon terminals on physical activity.\n• whether MPOAPhf6 neurons and VMHvlEsr1 neurons form reciprocal connections.\n• whether the projection from VMHvlEsr1→ MPOAPhf6 also promote physical activity.\n(1) To investigate the effects of activating each of the MPOAPhf6 axon terminals on\nphysical activity, we targeted ChR2 to MPOAPhf6 neurons by injecting AAV-DIO-ChR2-\nmCherry into the MPOA of 8-week-old female Phf6-CreER mice; and the optic fiber\nwas implanted above each axon projection field at the NAc, the PVH, and the\nVMHvl/TMN (new Fig. 6c). Practically, we examined the combined functional outputs\nof the VMHvl and TMN projections because these two regions are positioned in close\nproximity. We found that optogenetic activation of the axon terminals at the NAc and\nthe PVH did not affect the levels of physical activity. By contrast, optogenetic\nactivation of the axon terminals at the VMHvl/TMN significantly increased the levels\nof physical activity (new Fig. 6d). These results suggest that the effect of MPOAPhf6\nneurons on promoting physical activity is primary mediated by the axon projection to\nthe VMHvl/TMN.\nRegarding metabolism, it seems impossible to measure energy expenditure\nsimultaneously with optogenetic stimulation, as the experimental approach we used\nfor optogenetic stimulation is not compatible with use of metabolic cages.\n(2) Next, we have investigated whether MPOAPhf6 neurons and VMHvlEsr1 neurons are\nmutually innervated and form reciprocal connections.\n9\ni) We have determined MPOAPhf6 → VMHvlEsr1 monosynaptic connection by RV-\nbased retrograde tracing. Briefly, AAV2/9-Ef1a-DIO-His-EGFP-2A-TVA and\nAAV2/9-Ef1a-DIO-RVG were injected into the VMHvl of 8-week-old female Esr1-\nCre mice, followed by RV-EnvA-ΔG-tdTomato into the same site. Among diverse\nupstream inputs to VMHvlEsr1 neurons, we observed that a substantial number\n(16.1% ± 1.4%) of tdTomato-expressing neurons in the MPOA were positive for\nPhf6 (new Fig. 6n-p), providing direct evidence of the MPOAPhf6 → VMHvlEsr1\nmonosynaptic connectivity.\nii) We have also determined VMHvlEsr1 → MPOAPhf6 monosynaptic connection by RV-\nbased retrograde tracing. Briefly, AAV2/9-Ef1a-DIO-RVG and AAV2/9-Ef1a-DIO-\nHis-EGFP-2A-TVA were injected into the MPOA of 8-week-old female Phf6-\nCreER mice, followed by tamoxifen induction and RV-EnvA-ΔG-tdTomato\ninjected into the same site. Among diverse upstream inputs to MPOAPhf6 neurons,\nwe observed that a substantial number (22.7% ± 2.0%) of tdTomao-expressing\nneurons in the VMHvl were positive for Esr1 (new Fig. 6q-s), providing direct\nevidence of the VMHvlEsr1 → MPOAPhf6 monosynaptic connectivity.\n(3) Given the finding that MPOAPhf6 neurons and VMHvlEsr1 neurons are mutually\ninnervated and form reciprocal connections, we investigated whether the projection\nfrom VMHvlEsr1 → MPOA also promotes physical activity. AAV-DIO-ChR2-mCherry\nwas injected into the VMHvl of 8-week-old female Esr1-Cre mice. The optic fiber was\nplanted immediately above the MPOA to activate the axon terminals of VMHvlEsr1\nneurons in the MPOA. We found that optogenetic activation of axon terminals of\nVMHvlEsr1 neurons in the MPOA also significantly increased physical activity, as well\nas the level of anxiety (new Supplementary Fig. 8f-h). In contrast to the effect of\nactivating MPOAPhf6 → VMHvl projection on increasing physical activity without\naffecting anxiety (new Fig. 6e-h), activating VMHvlEsr1→ MPOA projection increased\nboth physical activity and anxiety. These results suggest that the reciprocal\nprojections between MPOAPhf6 neurons and VMHvlEsr1 neurons form a functional\nfeedback loop that enhances physical activity.\n11. “Furthermore, the last experiment in the study, using chemical genetic activation of VMHvl Esr1\nalone, does not prove that VMHvl is a downstream effector of MPOA Phf6. The authors should\nsuppress upstream MPOA Phf6 while activating VMHvl Esr1; or activate upstream and inhibit the\ndownstream. if increased activity is observed, it would support the idea that VMHvl acts downstream\nof MPOA Phf6, rather than projecting to MPOA Phf6.”\nRESPONSE:\nthe new finding that MPOAPhf6 neurons and VMHvlEsr1 neurons mutually innervate each other\nto make reciprocal connections. Guided by reviewer, we have conducted new experiments\nto assess the contribution of these mutual projections to physical activity.\nTo investigate whether the VMHvl serves as the primary downstream target of\nMPOAPhf6 neurons in promoting physical activity, we suppressed the VMHvl neurons by\nhM4D while optogenetically activating MPOAPhf6 neurons by ChR2. Briefly, AAV-hSyn-\nhM4D-GFP (or AAV-hSyn-EGFP as the control) were bilaterally injected into the VMHvl and\nAAV-DIO-ChR2-mCherry was unilaterally injected into the MPOA of the 8-week-old Phf6-\nCreER mice. The optic fiber was implanted above the MPOA. We found that\nchemogenetically suppressing VMHvl neurons while optogenetically activating MPOAPhf6\nneurons profoundly inhibited the level of MPOAPhf6-activation-induced physical activity (new\nFig. 7e, f). This finding suggests that the VMHvl serves as the primary downstream target\nof MPOAPhf6 neurons in promoting physical activity.\nTo investigate whether projection from VMHvlEsr1 → MPOA also contributes to physical\nactivity, we suppressed the MPOA neurons by hM4D while optogenetically activating\nVMHvlEsr1 neurons by ChR2. Briefly, AAV-hSyn-hM4D-GFP (or AAV-hSyn-EGFP as the\ncontrol) were bilaterally injected into the MPOA and AAV-DIO-ChR2-mCherry was\nunilaterally into the VMHvl of the 8-week-old Esr1-Cre mice. The optic fiber was implanted\nabove the VMHvl. We found that chemogenetically suppressing MPOA neurons while\noptogenetically activating VMHvlEsr1 neurons just moderately inhibited the level of VMHvlEsr1-\nactivation-induced physical activity (new Fig. 7g, h). This finding suggests that projection\nfrom VMHvlEsr1 → MPOA contributes to physical activity.\nTaken together, our results demonstrate that the VMHvl serves as the primary\ndownstream target of MPOAPhf6 neurons in promoting physical activity; MPOAPhf6 neurons\nand VMHvlEsr1 neurons make reciprocal connections to create a feedback loop that enhances\nthe role of MPOAPhf6 neurons in promoting physical activity.\n12. “Why do you generate Phf6 CreER mice rather than classical Cre mouse line?”\nRESPONSE:\nThe reason why we chose to generate Phf6-CreER mice rather than classical Cre\nmouse line is based on the known differences in Phf6 expression during development. For\nexample, Phf6 is ubiquitously expressed in the cortex, hippocampus, and hypothalamus in\nnew natal mice (Zhang et al., Neuron 2013; Cheng et al., Cell reports 2018), whereas Phf6\ndisappears from the cortex and hippocampus and is restricted to certain hypothalamic\nneurons after the second postnatal week (Gan et al., Cell reports 2020). In the present study,\n11\nwe also detected changes in Phf6 expression in the cortex, the hippocampus, and the\nhypothalamus on P3, P14, and P28 (new Supplementary Fig. 5a). Owing to the changing\npattern of Phf6 expression during development, we generated Phf6-CreER mice, in which\nCreER-mediated recombination can be precisely controlled by the timing of tamoxifen\nadministration.\nMinor points:\n1. “Incorrect full name of VMH in Line 46.”\nWe have replaced the ‘ventromedial nucleus’ with ‘ventromedial hypothalamus’ in\nLine 46.\n2. “Please systematically quantify rabies virus tracing data.”\nWe thank the reviewer for this guidance. In the revised manuscript, we have\nsystematically quantified rabies virus tracing data (new Supplementary Fig. 7a-c).\n3. “Why were different CNO doses used for chemical genetic activation and inhibition?”\nThe CNO doses we used are based on literature references (Campbell et al., Br J\nPharmacol 2018; Mahler et al., Nat Neurosci 2014).\n4. “In Line 195, the CRISPR-Cas9 experiment should refer to Phf6 knockout (KO), not knockdown\n(KD), and subsequent references to KD should be changed to KO.”\nWe have corrected this in the revised manuscript.\nWe would again like to thank the reviewer for the helpful guidance for improving our\nstudy and manuscript.\nWe thank the reviewer for stating, “This is an interesting study that identified a role for\nPhf6 in the POA on locomotion and related impacts on obesity development. A combination of novel\nanimal models, viral tracing, opto and chemogenetic approaches was used to generate evidence\nsupporting a specific role of POA Phf6 neurons promotes locomotion through specific projections to\nVMH Esr+ neurons via Phf6 mediated activation of neuron activity. The experiments were well\ndesigned and analyzed, and the results were cleanly presented. However, a few major and minor\nconcerns were noted.”\nComments:\n1. “Locomotion vs temperature: Since POA is well known to regulate body temperature, which is\nknown to modify energy expenditure (EE), the presented effect on EE may also be due to an effect on\nbody temperature. Data on body temperature need to be included in all panels on locomotion.”\nRESPONSE:\nWe thank the reviewer for this insightful guidance. We have completed new\nexperiments to monitor body temperature and assess its potential impact on the observed\nreduction in energy expenditure and obesity phenotype of Phf6 cKO mice.\nWe injected AAV-CMV-Cre (or AAV-CMV-GFP as the control) into the MPOA of 8-\nweek-old female Phf6flox/flox mice to knock out Phf6 in the MPOA. At six weeks post viral\ninjection, we assessed the interscapular brown adipose tissue (iBAT) temperature once\nevery hour over a 24-hour monitoring period using a thermal imaging camera (FLIR). There\nwas no difference in the iBAT temperature between female Phf6 cKO and control mice (new\nSupplementary Fig. 2e).\nWe also investigated whether chemogenetic activation of MPOAPhf6 neurons affects\nbody temperature. We injected AAV-DIO-hM3D-mCherry (or AAV-DIO-mCherry as the\ncontrol) into the MPOA of 8-week-old female Phf6-CreER mice. At three weeks post viral\ninjection. CNO was administrated 40 minutes before the assessment of iBAT temperature\nfor 3 hours. We found that chemogenetic activation of MPOAPhf6 neurons did not alter the\niBAT temperature compared to the controls (new Supplementary Fig. 6f).\nThese results suggest that body temperature is not affected by knockout of Phf6 in\nMPOAPhf6 neurons or by activation of MPOAPhf6 neurons, ruling out changes in body\ntemperature as an explanation for the observed reduction in energy expenditure and obesity\nphenotype in Phf6 cKO mice.\n13\n2. “The POA area is known to be made of both Glu+ and GABA+ neurons, both of which are\nimplicated in body temperature, although some discrepancy has been reported. Given this, the\nauthors need to verify whether Phf6-expressing neurons are Glu+ or GABA+ neurons. This point is\nalso related to the data presented in Fig. 6 on the projection.”\nRESPONSE:\nWe thank the reviewer for this helpful comment. We have conducted new\nexperiments to investigate:\n• the proportion of MPOAPhf6 neurons that are glutamatergic vs. GABAergic in both\nmale and female mice.\n• whether MPOAPhf6→VMHvlEsr1 circuitry comprises both excitatory and disinhibitory\nconnections.\n(1) To label the glutamatergic and GABAergic neurons, we crossed the Vglut2-Cre mice\nand Vgat-Cre mice with a reporter line expressing Cre-dependent histone-GFP (LSL-\nH2B-GFP). In Vglut2-Cre::LSL-H2B-GFP mice, we found that 31.6% ± 4.7% of\nMPOAPhf6 neurons in females and 18.0% ± 2.9% in males were Vglut2-positive\nglutamatergic neurons (new Fig. 1d, e); in Vgat-Cre::LSL-H2B-GFP mice, 69.2% ±\n4.2% of MPOAPhf6 neurons in females and 82.7% ± 2.0% in males were Vgat-positive\nGABAergic neurons (new Fig. 1d, e). The ratio of glutamatergic vs. GABAergic\nMPOAPhf6 neurons is approximately 3:7 in female mice and 2:8 in male mice. The\nproportion of glutamatergic MPOAPhf6 neurons was slightly higher in female mice than\nin male mice.\nIn addition, we have also analyzed gene expression profiles of MPOAPhf6 neurons\n(Phf6+/Esr1+ double positive) using previously published scRNA-seq datasets\n(Knoedler et al., Cell 2022; Moffitt et al., Science 2018). MPOAPhf6 neurons were identified\nas 4 distinct subclusters by UMAP analysis. Excitatory neuronal marker Slc17a6\n(Vglut2) was expressed in 2 clusters of MPOAPhf6 neurons; inhibitory neuronal\nmarkers Gad1 and Slc32a1(Vgat) were expressed in the other 2 clusters (new\nSupplementary Fig. 1d-f). Similar proportion of glutamatergic vs. GABAergic\nMPOAPhf6 neurons were identified in scRNA-seq analyses as that observed in\nimmunostaining experiments (new Supplementary Fig. 1h).\n(2) Considering that MPOAPhf6 neurons comprise both glutamatergic excitatory neurons\nand GABAergic inhibitory neurons, we have conducted new experiments to\ninvestigate whether MPOAPhf6→VMHvlEsr1 circuitry comprises both excitatory and\ndisinhibitory synaptic connections. By ChR2-assisted circuit mapping using\nelectrophysiology, we performed whole-cell patch clamp recording to record neurons\nat the VMHvl in acute slices upon activating the axon terminals of MPOAPhf6 neurons.\nWe targeted ChR2 to MPOAPhf6 neurons by injecting AAV-DIO-ChR2-mCherry into\nthe MPOA of adult female Phf6-CreER mice. Light-evoked EPSCs (oEPSCs) were\nrecorded in 24 out of 69 neurons, which were inhibited by the glutamate receptor\nantagonist CNQX (new Fig. 7k, m); light-evoked IPSCs (oIPSCs) were recorded in 45 out\nof 69 neurons, which were inhibited by GABA receptor antagonist PTX (new\nA\nFig. 7k, n). Sodium channel blocker TTX inhibited the oEPSCs and oIPSCs, while\npotassium channel blocker 4-AP reversed the TTX-inhibited oEPSCs and oIPSCs\n(new Fig. 7o, p). These results demonstrate that MPOAPhf6 neurons form both\nexcitatory and inhibitory synapses with the VMHvl neurons.\nVMHvlEsr1 neurons through local inhibitory neurons. Previous studies showed that\nVMHvlEsr1 neurons receive inhibitory inputs from local GABAergic inhibitory neurons\nin and surrounding the VMHvl, and that a substantial population of SST+ inhibitory\nneurons in the TMN (TMNSST neurons) are located close to the VMHvl (Wei et al., Nat\nNeurosci 2023; Luo et al., Science 2018). We sought to determine whether the\ndisinhibitory circuitry from MPOAPhf6 neurons to VMHvlEsr1 neurons is through nearby\nTMNSST neurons. Using Phf6-CreER::SST-Flpo mice, we labeled MPOAPhf6 neurons\nwith mGFP and TMNSST neurons with mCherry. Anatomical analysis of mGFP-labeled\nMPOAPhf6 neuron axon terminals revealed close apposition of mGFP-labeled\nMPOAPhf6 axon terminal boutons to TMNSST cell bodies (new Supplementary Fig. 8a,\nb), while RV-based retrograde tracing confirmed MPOAPhf6 neurons as monosynaptic\nupstream inputs to TMNSST neurons (new Supplementary Fig. 8 c-e). These results\nindicate that MPOAPhf6 neurons likely form a disinhibitory circuit with VMHvlEsr1 neurons\nvia TMNSST inhibitory neurons. Taken together, the MPOAPhf6→VMHvlEsr1 circuitry\ncomprises both excitatory and disinhibitory connections.\n3. “Data in Fig. 6 generated with optogenetic stimulation of VMH terminals will not provide\nsufficient evidence supporting a role of VMH neurons mediating the effect as terminal stimulation\nmay cause collateral activation through back-propagation. Further analysis is required to confirm\nthe functional connection.”\nRESPONSE:\nGuided by reviewer, we have conducted new experiments to investigate the\nfunctional connection by addressing:\n• the effect of activating each of the MPOAPhf6 axon terminals on physical activity.\n• whether the VMHvl is the primary downstream target of MPOAPhf6 neurons to\npromote physical activity.\nphysical activity, we targeted ChR2 to MPOAPhf6 neurons by injecting AAV-DIO-ChR2-\nmCherry into the MPOA of 8-week-old female Phf6-CreER mice; and the optic fiber was\nimplanted above each axon projection field at the NAc, the PVH, and the VMHvl/TMN (new\nFig. 6c). Practically, we examined the combined functional outputs of the VMHvl and TMN\nprojections because these two regions are positioned in close proximity. We found that\noptogenetic activation of the axon terminals at the NAc and the PVH did not affect the levels\nof physical activity (new Fig. 6d). By contrast, optogenetic activation of the axon terminals\nat the VMHvl/TMN significantly increased the levels of physical activity (new Fig. 6d). These\nresults suggest that the effect of MPOAPhf6 neurons on promoting physical activity is primary\nmediated by the axon projection to the VMHvl/TMN.\nTo determine whether the VMHvl is the primary downstream target of MPOAPhf6\nneurons to promote physical activity, we suppressed the VMHvl neurons by hM4D while\noptogenetically activating MPOAPhf6 neurons by ChR2. AAV-hSyn-hM4D-GFP (or AAV-\nhSyn-EGFP as the control) were bilaterally injected into the VMHvl and AAV-DIO-ChR2-\nmCherry was unilaterally injected into the MPOA of the 8-week-old Phf6-CreER mice. The\noptic fiber was implanted above the MPOA. We found that chemogenetically suppressing\nVMHvl neurons while optogenetically activating MPOAPhf6 neurons profoundly inhibited the\nlevel of MPOAPhf6-activation-induced physical activity (new Fig. 7e, f). These findings\nsuggest that VMHvl is the primary downstream target of MPOAPhf6 neurons to promote\nphysical activity.\nMinor points:\n1. “Fig. 2c-e data on no changes in food intake lack resolution. Real time feeding data from CLAMS\ndata will be very helpful to provide feeding patterns, which is important in this case as feeding is\ntightly related to EE and it is important to see whether no changes in feeding is caused by an\nincreased feeding in day but reduced feeding in night.”\nGuided by reviewer, we have added the real time feeding data from CLAMS in the\nrevised manuscript. The amount of food intake was presented once every hour for 48 hours.\nNo changes in the amount of food intake were overserved between Phf6 cKO and control\nmice (new Supplementary Fig. 2a).\n2. “Fig. 2f the presented EE is normalized to body weight is problematic as body weight is different\nin all time points measured, which will cause distortion in comparison in both day and night periods.\nFrom the curves shown, it is likely that EE would be reduced in night but increased in day.”\nWe thank the reviewer for pointing out the problem of ratio-based analysis by\nnormalizing EE to body weight. In the revised manuscript, according to two prior studies\n16\n(Tschöp et al., Nat Methods 2012; Müller et al., Nat Metab 2021), we have employed a\nregression-based analysis-of-covariance (ANCOVA), with body mass as a covariate, to\ndiscriminate between mass-dependent and mass-independent metabolic rate differences.\nWe found that knockout of Phf6 in the MPOA of female mice led to a true mass-\nindependent decrease in EE as compared to controls (p < 0.05), using two-sided regression-\nbased ANCOVA with body weight as a covariate (new Fig. 2f).\nPredicted EE normalized to the same body weight was calculated by regression line\nequation y = ax + b in which a is determined by the ANCOVA (Wang et al., Neuron 2025; Sa\net al., Nat Metab 2023). We found that predicted EE with 40g body weight showed a significant\ndecrease in female Phf6 cKO mice compared to controls (new Fig. 2f), indicating a true\ndecrease in EE in Phf6 cKO mice.\nWe would again like to thank the reviewer for the helpful guidance for improving our\nstudy and manuscript.\nWe thank the reviewer for stating, “This study by Jingjie Wang et al. addresses an\nimportant topic regarding the role of Phf6+ neurons in the medial preoptic area (mPOA) in\nregulating physical activity and energy expenditure, which may be relevant to the human disorder\nBörjeson–Forssman–Lehmann syndrome (BFLS). The authors present data showing the effects of\nPhf6 deletion on feeding behavior, physical activity, and energy expenditure. They further evaluate\nthe necessity and sufficiency of Phf6+ mPOA neurons in driving physical activity, investigate the role\nof Phf6 in neuronal excitability, and identify a downstream circuit projecting to the ventrolateral\nportion of the ventromedial hypothalamus (VMHvl) that may contribute to these behavioral effects.\nOverall, the manuscript is informative and well-structured. I am enthusiastic about its publication in\nNature Communications, provided that the following points are clarified and revised.”\nComments:\n1. “In Figure 1e, the authors show that the majority of Phf6+ cluster neurons are Slc17a6-negative.\nGiven that approximately 60% of Esr1+ neurons in the mPOA are GABAergic, this suggests that\nPhf6+ neurons in the mPOA are likely to be inhibitory. Moreover, electrophysiological data\npresented in Figure 4 indicate that Phf6 is important for maintaining the excitability of these neurons.\nIn Figure 5, the authors propose that mPOA Phf6+ neurons directly project to Esr1+ neurons in the\nVMHvl, which implies a potentially inhibitory input onto this downstream population. However,\nFigure 6d to f shows that activation of Esr1+ VMHvl neurons increases physical activity. This raises\nan important mechanistic question: how can a potentially inhibitory projection from Phf6+ mPOA\nneurons to Esr1+ VMHvl neurons facilitate increased locomotor activity, given that activation of the\ntarget neurons also promotes this behavior? A discussion or experiment clarifying the circuit-level\nmechanism by which Phf6+ mPOA neurons influence Esr1+ VMHvl neurons, and how this\nconnectivity supports the observed behavioral effects, would strengthen the manuscript.”\nRESPONSE:\nWe thank the reviewer for the insightful comment. We have conducted new\nexperiments to assess:\n• The proportion of MPOAPhf6 neurons that are glutamatergic vs. GABAergic.\n• The ratio of EPSCs vs. IPSCs recorded in the VMHvl neurons in response to\nactivating MPOAPhf6 neuron axon terminals.\n• The involvement of both excitation and dis-inhibition circuitry from MPOAPhf6 to\nVMHvlEsr1 neurons.\n(1) To label the glutamatergic and GABAergic neurons, we crossed the Vglut2-Cre mice\nand Vgat-Cre mice with a reporter line expressing Cre-dependent histone-GFP (LSL-\nH2B-GFP). In Vglut2-Cre::LSL-H2B-GFP mice, we found that 31.6% ± 4.7% of\nglutamatergic neurons (new Fig. 1d, e); in Vgat-Cre::LSL-H2B-GFP mice, 69.2% ±\n4.2% of MPOAPhf6 neurons in females and 82.7% ± 2.0% in males were Vgat-positive\nGABAergic neurons (new Fig. 1d, e). The ratio of glutamatergic vs. GABAergic\nMPOAPhf6 neurons is approximately 3:7 in female mice and 2:8 in male mice. The\nproportion of glutamatergic MPOAPhf6 neurons was slightly higher in female mice than\nin male mice.\nIn addition, we have also analyzed gene expression profiles of MPOAPhf6 neurons\n(Phf6+/Esr1+ double positive) using previously published scRNA-seq datasets\n(Knoedler et al., Cell 2022; Moffitt et al., Science 2018). MPOAPhf6 neurons were identified\nas 4 distinct subclusters by UMAP analysis. Excitatory neuronal marker Slc17a6\n(Vglut2) was expressed in 2 clusters of MPOAPhf6 neurons; inhibitory neuronal\nmarkers Gad1 and Slc32a1(Vgat) were expressed in the other 2 clusters (new\nSupplementary Fig. 1d-f). Similar proportion of glutamatergic vs. GABAergic\nMPOAPhf6 neurons were identified in scRNA-seq analyses as that observed in\nimmunostaining experiments (new Supplementary Fig. 1h).\n(2) By ChR2-assisted circuit mapping using electrophysiology, we have performed\nwhole-cell patch clamp recording to record neurons at the VMHvl in acuate slices\nupon activating the axon terminals of MPOAPhf6 neurons. We targeted ChR2 to\nMPOAPhf6 neurons by injecting AAV-DIO-ChR2-mCherry into the MPOA of adult\nfemale Phf6-CreER mice. Light-evoked EPSCs (oEPSCs) were recorded in 24 out of\n69 neurons, which were inhibited by the glutamate receptor antagonist CNQX (new\nFig. 7k, m); light-evoked IPSCs (oIPSCs) were recorded in 45 out of 69 neurons,\nwhich were inhibited by GABA receptor antagonist PTX (new Fig. 7k, n). Sodium\nA\nchannel blocker TTX inhibited the oEPSCs and oIPSCs, while potassium channel\nblocker 4-AP reversed the TTX-inhibited oEPSCs and oIPSCs (new Fig. 7o, p).\nThese results demonstrate that MPOAPhf6 neurons form both excitatory and inhibitory\nsynapses with the VMHvl neurons.\n(3) Besides excitation circuitry, we further investigated whether MPOAPhf6 neurons form\na dis-inhibition circuitry with VMHvlEsr1 neurons through a dis-inhibition circuitry. A prior\nstudy showed that VMHvlEsr1 neurons received inhibitory synaptic inputs from local\nGABAergic inhibitory neurons in and surrounding the VMHvl (Wei et al., Nat Neurosci\n2023). We also note that there are substantial number of SST+ inhibitory neurons in\nthe TMN (TMNSST neurons) surrounding the VMHvl (Luo et al., Science 2018).\nWe sought to determine whether the dis-inhibition circuitry from MPOAPhf6 neurons to\nVMHvlEsr1 neurons is through TMNSST inhibitory neurons. Using Phf6-CreER::SST-\nFlpo mice, we labeled MPOAPhf6 neuron with mGFP and TMNSST neurons with\nmCherry by injecting AAV-DIO-mGFP into the MPOA and AAV-fDIO-mCherry into\nthe TMN. Anatomical analysis of mGFP-labeled MPOAPhf6 neuron axon terminals\n19\nrevealed close apposition of MPOAPhf6 boutons to TMNSST cell bodies (new\nSupplementary Fig. 8a, b).\nWe also performed rabies virus-based retrograde tracing from the TMNSST neurons\nby injecting AAV-DIO-RVG and AAV-DIO-His-EGFP-2A-TVA into the TMN of adult\nfemale SST-Cre mice, followed by injecting RV-EnvA-ΔG-tdTomato into the same\nsite. We found that 31.4% ± 1.6% of tdTomato-positive upstream neurons of TMNSST\nin the MPOA were positive for Phf6 (new Supplementary Fig. 8c-e), suggesting that\nMPOAPhf6 neurons are the monosynaptic upstream of TMNSST neurons. We infer that\nMPOAPhf6 neurons form a dis-inhibitory circuitry with VMHvlEsr1 neurons though local\nTMNSST inhibitory neurons.\nTaken together, the ratio of glutamatergic vs. GABAergic MPOAPhf6 neurons is\napproximately 3:7 in female mice and 2:8 in male mice; projections from MPOAPhf6 to\nVMHvlEsr1 comprise both excitatory and disinhibitory circuitry. We have now clarified the\nrelated results in the discussion section of the revised manuscript (Lines 509-515 on Page\n16).\n2. “The data from acute activation (Figures 3g–i and 6) are somewhat less convincing in establishing\nsufficiency. I have two specific concerns:\nA) In Figure 3g–i, the authors use the open field test (OFT) to assess locomotor activity following\nacute activation of Phf6+ neurons. While the primary interpretation focuses on physical activity,\nOFT is also widely used to assess anxiety-like behavior. From the representative movement traces, it\nappears that center time and distance may also be altered by the manipulation. This raises the\npossibility that Phf6+ neuronal activity could influence anxiety-related behaviors in addition to\nlocomotion. Although changes in overall locomotor activity can complicate the interpretation of\nanxiety-related OFT measures, the conclusions would be strengthened by including data from\nadditional anxiety-specific assays, such as the elevated plus maze or light-dark box test, to help\ndisentangle locomotion-specific effects from potential anxiety-related changes.”\nB). “The data shown in Figure 3m–o, demonstrating that chemogenetic inhibition of Phf6+ neurons\nreduces both physical activity and energy expenditure, together with reduced locomotion in Figure\n3j–l, provide compelling evidence for the necessity of these neurons in regulating physical activity.\nIn contrast, the data from acute activation (Figure 3g–i) only show change in locomotion in OFT\nseems less robust in establishing sufficiency. To more fully support the model proposed in Figure 6g,\nit would be helpful to determine whether chemogenetic activation of Phf6+ neurons also promotes\nvoluntary activity or increases energy expenditure.” Addressing point A and/or B would help to\nstrengthen the overall conclusions of the study.\nWe thank the reviewer for this helpful comment. We have conduced new experiments\nto address both points A and B.\n• whether activating MPOAPhf6 neurons increases anxiety.\n• whether chemogenetic activation of MPOAPhf6 neurons increases physical activity\nand energy expenditure.\nA) We assessed anxiety-like behaviors using the open field (OF) test and the elevated\nplus maze (EPM) test. In OF tests, we found that both optogenetic activation and\nchemogenetic activation of MPOAPhf6 neurons did not alter the time ratio spent in the\ncenter area (new Fig. 4o, p). In EPM tests, chemogenetic activation of MPOAPhf6\nneurons did not alter the time spent in the open arms (new Fig. 4r, s). These results\ndemonstrate that activation of MPOAPhf6 neurons does not increase the level of\nanxiety, excluding changes in anxiety as an explanation for the observed increase in\nphysical activity upon MPOAPhf6 neuron activation.\nB) To determine whether chemogenetic activation of MPOAPhf6 neurons increases\nphysical activity and energy expenditure, we targeted hM3D to the MPOAPhf6 neurons\nby bilaterally injecting AAV-DIO-hM3D-mCherry (or AAV-DIO-mCherry as the control)\ninto the MPOA of 8-week-old female Phf6-CreER mice. Physical activity was\nassessed using a 15-minute open field test. The hM3D- or mCherry-expressing mice\nwere i.p. administered with CNO (3 mg/kg) 40 minutes before the physical activity\nassessment. Compared to the control mice, chemogenetic activation of MPOAPhf6\nneurons significantly increased the levels of physical activity, as shown by increased\nopen-field movement distance (new Fig. 4f). Chemogenetic activation of MPOAPhf6\nneurons did not alter the time spent in the center area of the open field and the time\nspent in the open arms of the elevated plus maze (new Fig. 4p, r and s), suggesting\nno changes in anxiety. These results demonstrate that chemogenetic activation of\nMPOAPhf6 neurons is sufficient to promote physical activity without affecting anxiety.\nEnergy expenditure was assessed using CLAMS metabolic cage for a period of 2\nhours. The hM3D- or mCherry-expressing mice were administered with CNO (3 mg/kg)\n40 minutes before CLAMS assessment. We found that chemogenetic activation of\nMPOAPhf6 neurons significantly increased energy expenditure (new Fig. 4k),\naccompanied by increased oxygen consumption (VO ) and carbon dioxide production\n2\n(VCO ) compared to the controls (new Supplementary Fig. 6a-c). In addition, we\n2\nfound that chemogenetic activation of MPOAPhf6 neurons significantly increased the\ncounts of locomotion in CLAMS (new Fig. 4l), recapitulating the increased physical\nactivity observed in the open field upon MPOAPhf6 neuron activation.\nTaken together, these findings demonstrate that chemogenetic activation of MPOAPhf6\nneurons increases both physical activity and energy expenditure.\n1. “While the authors note in the Introduction (line 66) that Börjeson–Forssman–Lehmann syndrome\n(BFLS) affects both males and females, all experiments in the current study were conducted using\nfemale mice. Given that Esr1 expression is present in the mPOA of both sexes, it would be helpful for\nthe authors to clarify the rationale for focusing exclusively on females. A brief discussion of the\nreasons for this choice would improve the interpretability and generalizability of the findings.”\nWe thank the reviewer for this helpful comment. The reason for selecting female\nmice over male mice for the experiments in the original manuscript was that male Phf6 cKO\nmice exhibited a weaker obesity phenotype than female Phf6 cKO mice. As guided by the\nreviewer, we have now completed new experiments to examine the body weight, feeding\nbehavior, physical activity, energy expenditure, and body temperature in male Phf6 cKO\nmice (new Supplementary Fig. 4). Here, we summarized the phenotypes between female\nand male Phf6 cKO mice in the following table:\nFemale Phf6 cKO mice Male Phf6 cKO mice\nBody weight ↑↑↑ ↑\nEnergy expenditure ↓↓↓ ↓\nPhysical activity ↓↓↓ ↓\nFeeding Behavior n.s. n.s.\nBody temperature n.s. n.s.\n2. “Do Phf6+ mPOA neurons specifically modulate physical activity and energy expenditure?\nPrevious studies have demonstrated that both Esr1+ neurons in the mPOA and VMHvl play key roles\nin regulating sexual behavior. Given that the current study shows projections from Phf6+ (and Esr1+)\nmPOA neurons to Esr1+ VMHvl neurons, it would be helpful if the authors could discuss whether\nthis circuit might also participate in the regulation of reproductive behaviors, and how such potential\nfunctions might relate to the observed modulation of physical activity and energy expenditure.”\nWe thank the reviewer for the insightful comment. Given that MPOAEsr1 neurons\nprimarily regulate maternal behavior instead of sexual behavior in female mice (Lawal et al.,\nAnnu Rev Neurosci 2025; Ribeiro et al., PNAS 2012) and that the population of MPOAPhf6 neurons\n22\nis a subset of MPOAEsr1 neurons, we have conducted a new experiment to investigate\nwhether activating MPOAPhf6 neurons would influence female maternal behavior.\nBriefly, we targeted hM3D to the MPOAPhf6 neurons by injecting AAV-DIO-hM3D-\nmCherry (or AAV-DIO-mCherry as the control) into the MPOA of 8-week-old female Phf6-\nCreER mice. We measured pup retrieval as the female maternal behavior in a 20-minute\ntest (Wei et al., Nat Commun 2018). We found that chemogenetic activation of MPOAPhf6\nneurons in female mice had no significant effect on the number of pups retrieved and latency\nto approach first pup compared to the controls (new Supplementary Fig.6h), suggesting\nthat MPOAPhf6 neurons do not regulate female maternal behavior.\n3. “The manuscript does not report the laser power used for optogenetic stimulation. For the sake\nof reproducibility and proper interpretation of the results, the authors should include this information\nin the Methods section.”\nGuided by reviewer, we have added the laser power used for optogenetic stimulation\ninto the Methods section, as well as the figures and figure legends of the revised manuscript.\nWe would again like to thank the reviewer for the helpful guidance for improving our\nstudy and manuscript.\nWe thank the reviewer for stating, “Wang et al discovered a unique role of Phf6 in\nmaintaining body weight homeostasis in female mice through modulating the neuronal excitability of\nmPOA neurons to regulate locomotor activity. They also proposed the VMHvl as a potential\ndownstream target for the locomotion change. The mutation of Phf6 gene was reported in BFLS, a\nrare human disease. It is intriguing how the authors linked Phf6 with obesity, which was of a high\npercentage in BFLS, and further demonstrated how it modulates the neuronal excitability with the\nhypothalamus. I believe this paper will be a good example of how basic scientific research links with\nbench work. However, there are a few key experiments that must be performed for the authors to\nmake a clear conclusion.”\nComments:\n1. “Phf6 is located on the X-chromosome and the prevalence of obesity does have a sex difference in\nBFLS, and the percentage is higher in men compared to women. Yet the authors did not explain why\nonly female animals were used in the study. Key functional experiments should be repeated and shown\nin the paper and clear clarification of why female mice were mainly studied should be made.”\nRESPONSE:\nWe thank the reviewer for the helpful comment. The reason for selecting female mice\nover male mice for the experiments in the original manuscript was that male Phf6 cKO mice\nexhibited a weaker obesity phenotype than female Phf6 cKO mice. As guided by the\nreviewer, we have now completed new experiments to examine the body weight, feeding\nbehavior, physical activity, energy expenditure, and body temperature in male Phf6 cKO\nmice.\nWe knocked out Phf6 in the MPOA by injecting AAV-CMV-Cre (or AAV-CMV-GFP as\nthe control) into the MPOA of 8-week-old male Phf6flox/y mice. The efficiency of Phf6 deletion\nwas measured by post hoc immunostaining. Compared to controls, male Phf6 cKO mice\nshowed no difference in body weight 2-8 weeks post viral injection, yet showed a significant\nincrease in body weight at 10 and 12 weeks post viral injection. However, it was notable that\nthe extent of obesity was much smaller in male Phf6 cKO mice than in female Phf6 cKO\nmice (new Supplementary Fig. 4d, h).\nIn male Phf6 cKO mice, we detected a moderate decrease in energy expenditure and\nphysical activity compared to littermate controls (new Supplementary Fig. 4f, g). Similarly,\nthe extent of decreased energy expenditure and physical activity was also smaller in male\nPhf6 cKO mice than in female Phf6 cKO mice (new Supplementary Fig. 4i, j).\nNo differences were observed in feeding behavior and body temperature in both male\nand female Phf6 cKO mice (new Supplementary Fig. 4k, l).\nexpenditure and physical activity between male and female mice indicates that Phf6\nregulates physical activity and metabolism in a sex-dependent manner.\nWe have summarized the phenotypes in female and male Phf6 cKO mice in the\nfollowing table:\nFemale Phf6 cKO mice Male Phf6 cKO mice\nBody weight ↑↑↑ ↑\nEnergy expenditure ↓↓↓ ↓\nPhysical activity ↓↓↓ ↓\nFeeding Behavior n.s. n.s.\nBody temperature n.s. n.s.\n2. “The authors showed that mPOA Phf6 neurons send monosynaptic projections to the VMHvl Esr1\nneurons, and optogenetic activation of the circuit as well as VMHvl Esr1 neurons increased\nlocomotor activity, consistent with the function of mPOA Phf6 neurons. However, based on the\ntrajectory tracing from Figure 5a-5c, there are also other downstream targets including NAc, PVH,\nand TMN in addition to the VMHvl. All of these regions have been reported to be involved in changes\nof locomotor activities in mice. Key experiments should be done to further prove that VMHvl is the\nmain downstream target.”\nRESPONSE:\nWe thank the reviewer for this insightful comment. We have conducted new\nexperiments to assess:\n• the effects of activating each of the MPOAPhf6 axon terminals on physical activity.\n• whether the VMHvl is the primary downstream target of MPOAPhf6 neurons to\npromote physical activity.\nactivity, we targeted ChR2 to MPOAPhf6 neurons by injecting AAV-DIO-ChR2-mCherry into\nthe MPOA of 8-week-old female Phf6-CreER mice; and the optic fiber was implanted above\neach axon projection field at the NAc, the PVH, and the VMHvl/TMN. Practically, we\nexamined the combined functional outputs of the VMHvl and TMN projections because\nthese two regions are positioned in close proximity. We found that optogenetic activation of\nthe axon terminals at the NAc and the PVH did not affect the levels of physical activity (new\nFig. 6d). By contrast, optogenetic activation of the axon terminals at the VMHvl/TMN\nsignificantly increased the levels of physical activity (new Fig. 6d). These results suggest\nthat the effect of MPOAPhf6 neurons on promoting physical activity is primary mediated by the\naxon projection to the VMHvl/TMN.\nTo determine whether the VMHvl is the primary downstream target of MPOAPhf6\nneurons to promote physical activity, we suppressed the VMHvl neurons by hM4D while\noptogenetically activating MPOAPhf6 neurons by ChR2. AAV-hSyn-hM4D-GFP (or AAV-\nhSyn-EGFP as the control) were bilaterally injected into the VMHvl and AAV-DIO-ChR2-\nmCherry was unilaterally injected into the MPOA of the 8-week-old Phf6-CreER mice. The\noptic fiber was implanted above the MPOA. We found that chemogenetically suppressing\nVMHvl neurons while optogenetically activating MPOAPhf6 neurons profoundly inhibited the\nlevel of MPOAPhf6-activation-induced physical activity (new Fig. 7f). These findings suggest\nthat the VMHvl is the primary downstream target of MPOAPhf6 neurons to promote physical\nactivity.\n3. “Authors utilized slice recordings to demonstrate the potential role of Phf6 in the electrophysiology\nproperties of mPOA Phf6 neurons and showed that loss of Phf6 leads to reduced excitability and\nestradiol-induced firing increase. However, it remains unclear about the physiological significance\nof these cells in vivo. It will be helpful to show when these neurons will be recruited.”\nRESPONSE:\nWe thank the reviewer for the insightful comments. Guided by reviewer, we have\nconducted new experiments to assess what physiological contents activate MPOAPhf6\nneurons, i.e., whether MPOAPhf6 neurons are activated by physical activity.\nWe employed fiber photometry to record calcium signals from MPOAPhf6 neurons in\nvivo to determine whether MPOAPhf6 neurons are activated by physical activity. AAV-DIO-\nGCaMP7s was unilaterally injected into the MPOA of 8-week-old female Phf6-CreER mice\nand the optic fiber was implanted immediately above the MPOA. We recorded the temporally\ndemodulated 470-nm calcium-dependent and 410-nm calcium-independent fluorescence\nsignals with fiber photometry from GCaMP7s-expressing MPOAPhf6 neurons in freely\nbehaving mice (new Fig. 3d). Significant increases in calcium signals from MPOAPhf6\nneurons (as reflected in △F/F) were detected following the onset of the physical activity (i.e.,\n26\nwalking and running horizontally or climbing vertically) (new Fig. 3e-h). Following the onset\nof the physical activity, the calcium signals from MPOAPhf6 neurons showed peak responses\nreaching approximately 10% △F/F, averaged from three female mice (new Fig. 3e-h). The\ninternal control of 410-nm signals showed no changes, excluding the possibility that 470-nm\ncalcium signals recorded were artifacts. These results suggest that MPOAPhf6 neurons are\nmarkedly activated by physical activity.\nWe also performed c-Fos immunostaining to examine whether MPOAPhf6 neurons are\nactivated by physical activity. We found that a 30-min forced wheel running significantly\nincreased the percentage of c-Fos+ MPOAPhf6 neurons in 8-week-old C57BL/6 female mice\ncompared to the baseline (new Fig. 3j), suggesting that MPOAPhf6 neurons are activated by\nphysical activity. In addition, we observed an increased number of c-Fos+ MPOAPhf6 neurons\nwhen the mice were examined at 10 PM than at 10 AM (new Fig. 3l), suggesting that more\nMPOAPhf6 neurons are activated when the mice are awake than asleep.\nThese three lines of evidence all support that MPOAPhf6 neurons are activated by\nphysical activity.\nMinor points:\n1. “The change of body weight became significantly different after 2-4 weeks post surgery (Figures\n1h-1i), and the change of energy expenditure was not significant at 4 weeks post surgery (Figures\n1g-1h). As there was no change of food intake (Figures 1c-1e), potential mechanisms leading to the\nearly phase of body weight change need to be clarified or discussed. mPOA neurons are known to\nregulate body temperature, do these Phf6 neurons also modulate body temperature?”\nWe thank the reviewer for pointing out this problem. In the original manuscript, we\nhad made a mistake to use two-way mixed ANOVA for the statistical analyses of these\nexperiments. In the revised manuscript, we have made a correction; we now use two-way\nrepeated measures ANOVA to analyze body weight and energy expenditure between\ngroups across various time points. After this correction in our statistics, now our data showed\nthat the change in body weight became significant at the 4 weeks post viral injection time\npoint (new Fig. 1j);\nHowever, as we measured the body weight once a week but measured energy\nexpenditure only at three time points (at 4, 8, 12 weeks post viral injection), we were unable\nto ascertain whether there was a difference in energy expenditure had already shown\ndifference before the 4 weeks post viral injection time point.\nRegarding the body temperature, Phf6 cKO mice exhibited no significant difference\nin body temperature compared to the control mice, as measured by interscapular brown\nadipose tissue (iBAT) temperature (new Supplementary Fig. 2e), suggesting that the\n27\nreduced energy expenditure seen in Phf6 cKO mice does not result from a decrease in body\ntemperature.\n2. “Line 52: “which are molecular heterogeneity” (cid:0) “which are of molecular heterogeneity””\nGuided by reviewer, we have replaced the text with “which are of molecular\nheterogeneity” in the revised manuscript.\n3. “Line 66: where is the sampling rate from? The obesity rate in the US is around 40% in females.”\nThe obesity rates of BFLS patients we calculated in our manuscript were sampled\nfrom the following literatures:\nReferences Female BFLS patients Male BFLS patients\nJain et al., Eur J Hum Genet. 2023 6/7 obesity 7/8 obesity\nGerber et al., Clin Gent. 2023 5/25 obesity 44/48 obesity\nZhang et al., J Clin Res Pediatr Endocrinol.\n7/19 obesity 23/32 obesity\nZweier et al., Am J Med Genet C 2014 4/12 obesity /\nTotal 25/67 (37.3%) 94/114 (82.5%)\n4. “Line 106: “increase body weight gain”, “increase” and “gain” are redundant.”\nGuided by reviewer, we have replaced the text with “increase body weight” in the\nrevised manuscript.\n5. “Line 195: “knockdown” should be verb “knock down””\nrevised manuscript.\n6. “Line 237: AAV1-based tracing is not monosynaptic.\nThank you for bringing this mistake to our attention. We have replaced “AAV1-based\nmonosynaptic tracing” to “AAV serotype 1 mediated-anterograde transsynaptic tracing” in\nthe revised manuscript.\n7. “Line 251: if one hyphen is used, the rest should also use hyphen.”\nWe thank the reviewer for point out this problem. We have completed the appropriate\nchanges.\nWe would again like to thank the reviewer for the helpful guidance for improving our\nstudy and manuscript.\nWe would like to thank the reviewers for their constructive comments. We have\naddressed each comment below and incorporated the corresponding changes into the\nrevised manuscript (highlighted in yellow).\nReviewer #1:\nWe thank the reviewer for stating, “This revision has addressed most of my questions. I\nagree to the publication.”\nComments:\n1. “There are some technical concerns, though I know our field mostly still accepts them; Given that\nyou used Phf6-Cre-ER because there are developmentally transient cells in the mPOA, why did you\nuse vGlut2-Cre::H2B-GFP and vGat-Cre::H2B-GFP to reflect the expression of GABAergic and\nglutamatergic cells? Do you believe these two markers do not have transient expression during\ndevelopment? (Fig. 1d)”\nRESPONSE:\nWe thank the reviewer for focusing our attention on developmental expression and\nlabeling strategies. We agree with the reviewer that vGlut2-Cre::LSL-H2B-GFP and vGat-\nCre::LSL-H2B-GFP are constitutive Cre driver lines that permanently label cells that have\nexpressed vGlut2 or vGat at any point during development. Therefore, GFP-labeled cells\nreflect developmental lineage history and may not fully represent the glutamatergic or\nGABAergic neuronal identity in adulthood.\nTo address this concern, we performed an additional experiment to selectively label\nadult vGlut2+ and vGat+ cells by injecting AAV2/9-Ef1a-DIO-H2B-EGFP into the MPOA of\n8-week-old female vGlut2-Cre and vGat-Cre mice. Using this virus-based labeling approach,\nwe found that, in the adult female MPOA, 28.7% ± 1.1% of Phf6-positive neurons were\nvGlut2-positive, whereas 71.5% ± 2.1% were vGat-positive (new Supplementary Fig. 2a-\nd). Notably, the proportions of glutamatergic and GABAergic MPOAPhf6 neurons identified by\nvirus-based labeling were indistinguishable from those obtained using the genetic reporter\nmouse lines shown in Fig. 1d, indicating an approximate 3:7 ratio of glutamatergic to\nGABAergic MPOAPhf6 neurons in female mice, regardless of the labeling method.\nIn addition, we examined Phf6 expression in the MPOA across multiple postnatal\ndevelopmental stages and into adulthood in both female and male mice at the following\nages: P3, P14, P28, and P42, as well as 2, 4, and 6 months of age. Briefly, we found that\nPhf6 expression in the MPOA exhibited dynamic changes during early postnatal\ndevelopment but stabilized after P14 and did not differ between sexes (new Supplementary\nassessed the proportions of glutamatergic versus GABAergic MPOAPhf6 neurons using both\ngenetic reporter mice and the virus-based labeling approach.\nSpecifically, we added the following text to the Results in the revised manuscript:\n“Phf6 expression in the MPOA exhibited dynamic changes during early postnatal\ndevelopment but stabilized after P14, with no differences observed between sexes\n(Supplementary Fig. 1a, b).” (Lines 88-90 in the revised manuscript)\n“Given that H2B-GFP-labeled cells in genetic reporter mice reflect developmental\nlineage history, they may not fully represent glutamatergic or GABAergic neuronal\nidentity in adulthood. To selectively label adult glutamatergic and GABAergic\nneurons, an adeno-associated virus (AAV) expressing Cre-dependent histone-\nEGFP (AAV2/9-Ef1a-DIO-H2B-EGFP) was injected into the MPOA of 8-week-old\nfemale vGlut2-Cre and vGat-Cre mice. We observed that 28.7% ± 1.1% of Phf6-\npositive MPOAPhf6 neurons were vGlut2-positive and that 71.5% ± 2.1% were vGat-\npositive in the adult female MPOA (Supplementary Fig. 2a-d). Notably, the\nproportions of glutamatergic and GABAergic MPOAPhf6 neurons identified by virus-\nbased labeling were indistinguishable from those obtained using genetic reporter\nmouse lines.” (Lines 104-113 in the revised manuscript)\n2. “The margin of the VMH is incorrect; please refer to established papers in this field. (Fig. 6j).”\nRESPONSE:\nWe thank the reviewer for this guidance. We have corrected the margin of the VMH\nin the revised manuscript (new Fig. 6j) in accordance with established literature in the field\n(e.g., Lin et al., Nature 2011; Hashikawa et al., Nat. Neurosci. 2017; Karigo et al., Nature 2021).\nReviewer #2:\nWe thank the reviewer for stating, “The authors have performed substantially more\nexperiments and provided necessary results to effectively address the concerns raised. Recommend\nacceptance.”\nWe thank the reviewer for stating, “I found the revised manuscript is clearly written and\nwell organized, with new experiments and analyses that effectively address the previous concerns.\nThese additions considerably strengthen the study and provide solid evidence for the critical role of\nthe Phf6-expressing mPOA–VMH circuit in regulating physical activity. The clarity and presentation\nof the work have been greatly improved, and I believe the manuscript is now suitable for publication.”\nReviewer #4:\nWe thank the reviewer for stating, “The authors have implemented significant amount\nof work and have addressed most of the comments.\nComments:\n1. “However, I do think the authors failed to explain why there is a major difference between human\nand mice in the prevalence of obesity in BFLS in different sex although they added experiments on\nmale mice. The authors should add more discussion about it. Also, I added a few minor editorial\nsuggestions. Otherwise, I think the article has no major issues.”\nRESPONSE:\nWe thank the reviewer for this comment. We have expanded the Discussion to\naddress the discrepancy in sex bias between BFLS patients (in whom obesity is reported to\nbe more prevalent in males) and our mouse model (in which a stronger obesity phenotype\nis observed in female MPOA-specific Phf6 cKO mice).\nSpecifically, we added the following text to the Discussion in the revised manuscript：\n“It should be noted that the female-biased obesity phenotype in our MPOA-specific\nPhf6 cKO mouse model differs from clinical reports in BFLS, where obesity is more\nprevalent in male patients than in female patients. BFLS is caused by germline Phf6\nmutations that act throughout development and across multiple brain regions and\nperipheral tissues, and therefore Phf6 dysfunction in BFLS patients extends beyond\nthe MPOA and is not restricted to the circuits targeted in our Phf6 cKO mice. Our\ndata reveal that loss of Phf6 in MPOAPhf6 neurons has a larger effect on energy\nexpenditure and physical activity in female mice. In contrast, in males, Phf6-\ndependent circuits in other hypothalamic or extra-hypothalamic regions as well as\nin peripheral tissues may make a larger contribution to metabolic disturbances and\nobesity-related phenotypes. The apparent difference between the sex bias in BFLS-\nassociated obesity and the sex-biased phenotype in our mouse model does not\nnecessarily reflect separate mechanisms, but rather points to Phf6-dependent\n3\npathways that influence energy metabolism in a sex-dependent manner. Follow-up\nwork could include combined measurements of energy expenditure, physical activity,\nand food intake in male and female Phf6 mutant mice relevant to BFLS patient\nmutations and/or experiments that test Phf6 function in other hypothalamic or extra-\nhypothalamic regions; such studies may help clarify how additional Phf6-dependent\ncircuits contribute to the higher prevalence of obesity in males with BFLS.” (Lines\n512-528 in the revised manuscript)\nWe believe that this expanded discussion more clearly defines the scope of our\nmouse model and appropriately contextualizes our circuit-level findings with respect to the\nclinical presentationof BFLS.\nMinor points:\n1. “Line 85: in figure 1a, there is no panel showing bregma level +0.25 mm, you could change the\ntext or add an extra panel.”\nRESPONSE:\nWe thank the reviewer for pointing out this error. In the revised manuscript, we have\ncorrected the bregma level in the text to “+0.13 mm” (Line 88 in the revised manuscript)\nfor consistency with Fig. 1a.\n2. “Line 288: should be “Fig. 4h, i, l and n.”\nRESPONSE:\nWe thank the reviewer for raising this point. We carefully rechecked the relevant\nfigure panels and confirmed that Fig. 4l corresponds to data from the preceding hM3D\nactivation experiment, rather than the hM4D inhibition experiment discussed here. The\napparent confusion likely arose from an inconsistency in the figure citations in the original\ntext.\nIn the revised manuscript, we have corrected the figure citations in the Results\nsection as follows:\n“, as indicated by decreased open-field movement distance (Fig. 4h, i) and reduced\nvoluntary wheel running in CLAMS (Fig. 4n), with no difference in the time spent in\nthe center zone (Fig. 4q).” (Lines 299-301 in the revised manuscript)\n4"}]

Decision letter