The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion

Domain: biology
Published: 2026-01-11
Editorial outcome: minor_revision

Abstract
Glucocorticoid-producing cells of the adrenal cortex (i.e. zona fasciculata, zF) constitute the critical effectors of the hypothalamic-pituitary-adrenal axis, mediating the mammalian stress response. With glucocorticoids being essential for life, zF dysfunction perturbs multiple organs that participate in optimizing cardiometabolic fitness. The zF forms a dynamic and heterogenous cell population endowed with the capacity to remodel through the engagement of both proliferative and differentiation programs that enable the adrenal to adapt and respond to diverse stressors. However, the mechanisms that sustain such differential responsiveness remain poorly understood. In this study, we resolve the transcriptome of the steroidogenic lineage by scRNA-seq using Sf1-Crehigh; Rosa mT/mG reporter mice. We identify HHEX, a homeodomain protein, as the most enriched transcription factor in glucocorticoid-producing cells. We utilize genetic mouse models to demonstrate that Hhex deletion causes glucocorticoid deficiency in male animals. Molecularly, we demonstrate that HHEX is an androgen receptor (AR) target gene, shaping the sexual dimorphism of the adrenal gland by repressing the female transcriptional program at puberty, while also maintaining zF cholesterol ester content by protecting lipid droplets from androgen-induced-lipophagy. Moreover, our study reveals that, in both sexes, HHEX is crucial for maintaining the identity of the innermost adrenocortical cell subpopulation. Specifically, loss of HHEX impairs the expression of Abcb1b (P-glycoprotein/MDR1), an efflux pump regulating steroid export and cellular levels of xenobiotics. Together, these data demonstrate that HHEX serves as a multi-functional regulator of postnatal adrenal maturation that is potentiated by androgens.

Introduction
Glucocorticoids (GC) are cholesterol-derived steroid hormones widely known for maintaining glucose homeostasis1, modulating the immune system2, and orchestrating the circadian rhythm established by the suprachiasmatic nucleus3. Cortisol in humans, and corticosterone in rodents, hold central importance for the body’s response to stress by promoting energy mobilization. The precise control of GC production and secretion is vital for restoring homeostasis after stress exposure, primarily through feedback regulation of the hypothalamo-pituitary-adrenal axis (HPA). However, an imbalance in GC production is a characteristic of chronic stress exposure and is associated with adverse outcomes such as depression, cognitive dysfunction4, susceptibility to infections5, elevated cardiometabolic morbidity, and increased mortality6.

Within the adrenal gland, the zona fasciculata (zF) is the major site of GC production. It is characterized by a high content of the steroid precursor, cholesterol, and the expression of CYP11B1 that encodes the enzyme required for the final step of GC synthesis7–12. At the molecular level, the elevation of circulating Adrenocorticotropic Hormone (ACTH) during stress exposure drives adrenal GC production by mobilizing cholesterol stored in lipid droplets in the form of cholesterol esters. The metabolic conversion of free cholesterol moieties into steroid hormones is accomplished by increasing steroidogenic enzyme expression and activity in the mitochondria of zF cells13,14. Historically, the zF has been defined as a homogeneous cell population based on the expression of CYP11B1 in all zF cells. However, recent findings have revealed unanticipated heterogeneity within the zF, including differences in proliferative capacities, sex-specific features, and spatial gradients of signaling activity. Refined sequencing techniques have identified discrete populations within the zF, marked by genes such as Abcb1b15, coding a GC exporter, suggesting that GC production and release may be governed by molecularly and functionally specialized cellular subpopulations.

The mechanisms underlying this cellular heterogeneity are intricately linked to signaling gradients, whereby their disruption perturbs steroidogenic cell differentiation and zonal identity resulting in hormonal disorders16. Furthermore, pronounced sex differences in mammalian adrenal biology are well documented, mediated in large part by androgen-mediated transcriptional activation of the nuclear androgen receptor (AR). Nevertheless, the molecular programs coordinating sex-dependent and independent zF cellular diversity and their relevance in disease states remain to be deciphered.

Among the potential regulators of adrenal zonation and function, the transcription factor HHEX (Hematopoietically Expressed Homeobox), also known as HEX and Proline-Rich Homeodomain protein, has emerged as a candidate with possible endocrine function. Initially described in the hematopoietic lineage, where it acts as a transcriptional repressor necessary for the maturation and proliferation of definitive hematopoietic progenitors17–19, HHEX has since been revealed to function as a transcriptional activator in differentiation processes across various organs20–24, including the pancreas, where it controls the maintenance of somatostatin-secreting delta cell differentiation25. Consistent with this role, polymorphisms in the HHEX gene have been associated with type 2 diabetes in Genome-Wide Association Studies (GWAS)26–33. Meta-analyses have also suggested associations between HHEX single-nucleotide polymorphism (SNP, rs2497306) and circulating levels of adrenal-derived dehydroepiandrosterone sulfate (DHEAS)34,35, suggesting a role for HHEX in adrenal steroidogenesis. Nevertheless, direct functional evidence for HHEX in adrenal cortex biology is lacking.

In this study, we combine single-cell RNA sequencing (scRNA-seq) and in vivo knockout (KO) of candidate genes to uncover factors that orchestrate cellular diversity in the zF and regulate GC production. We demonstrate that HHEX is uniquely expressed in the zF of rodent and human adrenals. Using the mouse as a model organism, we show that loss of HHEX leads to GC deficiency in males. Importantly, in the male adrenal, we find that HHEX protects the inner zF from androgen-induced lipophagy (lipid depletion) at puberty. Molecularly, HHEX exerts both androgen-dependent and independent functions. Using CUT&Tag technology36, we demonstrate that HHEX is a bona fide AR target gene that shapes the AR-driven sexual dimorphism of the adrenal gland in the zF. Finally, in both male and female mice, HHEX maintains the expression of GC exporter Abcb1b in the inner zF at baseline and is necessary for the expansion of the Abcb1b-positive cell population during chronic stress.

Results
scRNA-seq reveals heterogeneity within the steroidogenic lineage of the adrenal cortex
To achieve single-cell resolution of the adrenocortical transcriptome, we selectively labeled steroidogenic adrenocortical cells using a transgenic Cre-LoxP approach. We combined Sf1-Crehigh37 mice with Rosa mT/mG38 animals to obtain mice expressing fluorescent reporter proteins at the cell membrane (mT/mG) of cells that express or have expressed the transcription factor SF-1 (Steroidogenic Factor 1, also AdBP4, encoded by NR5A1) (Fig. 1A). Consistent with previous results using these transgenic mice, only the adrenal cortex was labeled with green fluorescence (mGFP), leaving the capsule and the medulla fluorescent in the red channel (mTomato)39–41. We prepared a single-cell suspension from male adrenals by combining mechanical and enzymatic dissociation at low temperature to preserve the viability of steroidogenic cells and their transcriptional state. Fluorescence-activated cell sorting (FACS) was then used to isolate GFP-positive cells representing the steroidogenic lineage (Fig. 1B). GFP-positive cells include the progenitor populations as well as differentiated cells (zona glomerulosa (zG) and zF cells). Libraries for scRNA-seq were generated using the 10× Genomics platform. Following quality control and filtering out poor-quality cells, a total of 6497 cells from two replicates were retained for subsequent analyses (Supplementary Fig. 1a). A principal component analysis (PCA), considering a gene set of the most variably expressed genes, distinctly separated cells according to their identity. We then annotated 9 clusters according to the expression of known feature genes (Fig. 1C). All cells expressed GFP, demonstrating the successful enrichment of cells belonging to the steroid lineage. As expected, no medulla cells were found in our dataset. Across the dataset, we identified the mineralocorticoid-producing zG based on the expression of Cyp11b2 (Fig. 1D) and the glucocorticoid-producing zF based on the expression of Cyp11b1 (Fig. 1E). Among the cells, 35% expressed Cyp11b2, 43% expressed Cyp11b1, and 13% expressed both. We confirmed the presence of cells co-expressing Cyp11b2 and Cyp11b1 transcripts in both sexes by performing RNAscope on serial sections (Fig. 1F and Supplementary Fig. 1b). We identified the proliferative population based on the expression of mKi67 (Supplementary Fig. 1c) and we found Shh-positive cells partially clustering with the zG and the zF cells (Supplementary Fig. 1d). This finding was validated by RNAscope, where we found a portion of Shh transcripts in the upper zF cells, specifically in male adrenals (Supplementary Fig. 1e). We confirmed the presence of a recently described unique cell population defined by high expression of Abcb1b, Sbsn, Mgst2, and Srd5a2 identified by Lopez and colleagues15, representing 3–5% of the Cyp11b1-positive population (Supplementary Fig. 1f). To provide a user-friendly access to this dataset to the scientific community, we used the 10× Genomics’ LoupeR package to generate a CLOUPE file that can be easily imported into the 10× Genomics Loupe Browser for data visualization and further exploration (See below “Methods” and “Data availability” sections).

To gain insights into transcripts that underly differences in zonation (zG versus zF) of the cortex and provide a thorough understanding of the zF transcriptome, we analyzed the most enriched genes in zG and zF cell populations (Fig. 1G). Our dataset revealed specific enrichment of previously unrecognized markers in the zG, such as Ppp2r2b (a Wnt pathway antagonist), and Pcdh19 (a protocadherin involved in cell-to-cell adhesion) (Supplementary Fig. 1g). Besides the bona fide marker Cyp11b1, we found the expression of genes encoding proteins, such as Mmd2 (a membrane progestin receptor), Acsbg1 (an acyl-CoA synthetase), and lipid membrane transporters such as Abca1 (Supplementary Fig. 1h) were enriched in the zF. We then focused our analysis on factors that could directly control gene expression, such as DNA-binding proteins. This analysis identified 9 zG-enriched and 8 zF-enriched transcription factors (Tables 1 and 2). To summarize, our scRNA-seq dataset provides an advanced atlas of the adrenal steroid lineage in the male mouse adrenal, which can be used to identify potential regulators of steroid function.

HHEX is a highly conserved marker of glucocorticoid-producing cells
Hhex was identified as the top enriched transcription factor in the zF with an expression pattern mirroring Cyp11b1 (Fig. 2A). To validate these results, we performed immunohistochemistry and confirmed that HHEX expression was absent from DAB2-positive cells marking the zG (Supplementary Fig. 2a), and was limited to the zF in adult male mouse adrenal (Fig. 2B) and the zF and zR in adult male rat adrenals (Fig. 2C). Using publicly available datasets, we found that human adrenal tissue is among the organs that express the highest levels of HHEX (Supplementary Fig. 2b). We also confirmed that HHEX is expressed in the human adrenocortical cell line H295R by immunofluorescence (Fig. 2D) and the zF of the human adrenal cortex (Fig. 2E) by immunohistochemistry. In conclusion, we were able to define HHEX as a marker of the zF that is conserved across species, which supports a potential role in zF function.

Genetic inactivation of Hhex in the mouse adrenal cortex impairs glucocorticoid levels in males
To gain insights into HHEX function in the adrenal cortex, in vivo, we generated a series of Hhex KO models. First, Hhexflox/flox mice were crossed with mice expressing Cre driven by the Sf1 promoter37 to allow for Hhex genetic ablation in the entire adrenal cortex from embryonic day E10.5 (Sf1-Crehigh; Hhexflox/flox) (Fig. 3A). To analyze the Sf1-Crehigh Hhex KO phenotype, we collected tissue and plasma samples from 6-, 15-, and 50/55-week-old mice for adrenal histology, transcriptomic analysis, and glucocorticoid measurements. Genetic ablation of Hhex was validated in 6-week-old mice both by RT-qPCR from mRNAs extracted from whole adrenals and by immunohistochemistry. As anticipated, we observed a significant decrease in Hhex expression by RT-qPCR in 6-week-old males (Fig. 3B), and no HHEX staining was observed in Sf1-Crehigh Hhex KO animals by immunohistochemistry (Fig. 3C). Since SF-1 is also expressed in somatic cells of the gonads37, we assessed HHEX expression in both testis and ovaries. No staining was discernible (Supplementary Fig. 3a), ruling out a major effect of gonadal Hhex deletion on the adrenal. At 6 weeks of age the overall zonation of the adrenal cortex was conserved in Sf1-Crehigh Hhex KO, despite cells of the inner zF appearing eosinophilic as illustrated by hematoxylin and eosin staining (Supplementary Fig. 3b). Some cells also displayed hypertrophy, without changes in the adrenal body weight ratio at this age (Supplementary Fig. 3c). At 15-week-old, the hypertrophy was evident and was confirmed by a 32% and 36% decrease in nuclear density in the zF and inner zF respectively (Supplementary Fig. 3d). At 19-week-old, the adrenal body weight ratio was increased by 32% in Sf1-Crehigh Hhex KO (Supplementary Fig. 3e).

To determine if HHEX contributes to zF function, we measured plasma GC levels by mass spectrometry in WT (Wild Type) and Sf1-Crehigh Hhex KO mice. We observed a significant 55% decrease in baseline corticosterone in adult males (Fig. 3D). We then cultivated adrenal explants in the presence of dibutyryl-cAMP (Bt2-cAMP) for 2.5h to stimulate GC production, and observed a 28% decrease in corticosterone levels in media of Sf1-Crehigh Hhex KO compared to WT explants (Fig. 3E). These results show that HHEX is required to maintain normal GC production and/or secretion. Despite the fact that the adrenal gland’s main function is to produce steroids, adrenal cells do not store GC. Instead, they rapidly metabolize cholesterol and engage the steroidogenic pathway to produce steroids on demand42–47. To further interrogate steroidogenic potential in the Sf1-Crehigh Hhex KO adrenals, we assessed the expression of steroidogenic enzymes such as Cyp21a1 and Cyp11b1 by RT-qPCR (Fig. 3F, G and Supplementary Fig. 3f–i), and immunohistochemistry for in situ localization (Fig. 3H, I). Surprisingly, we observed an increase in expression and the entire zF was positive for both enzymes. This result made us consider the possibility that GC deficiency was likely a consequence of an upstream defect leading to compensatory upregulation of steroidogenic enzyme gene expression. In support of this hypothesis, corticosteroid precursors such as Progesterone and 11-Deoxycorticosterone plasma levels were indeed significantly decreased (63% and 82% respectively) at baseline in adult males (Supplementary Fig. 3j, k).

To produce steroid hormones on demand, adrenocortical cells metabolize cholesterol (the precursor to all steroids48,49). Storage of cholesterol, in the form of lipid droplets (LD), allows for a reservoir of this precursor to be available when needed. Thus, we evaluated cholesterol content in Sf1-Crehigh Hhex KO by detecting neutral lipids such as cholesterol esters (CE) and triacylglycerol (TG) stored in lipid droplets using Oil Red O staining. By comparing WT and Sf1-Crehigh Hhex KO stained adrenal sections, we observed a striking reduction of neutral lipids stored in LD in the inner part of the cortex in 15-week-old Sf1-Crehigh Hhex KO male mice (Fig. 3J). This observation was confirmed by the quantification of intra-adrenal cholesterol esters by colorimetric assay (Fig. 3K), which demonstrated a 61% decrease of CE in the adrenal of 45-week-old Sf1-Crehigh Hhex KO compared to their littermate WT controls. Taken together, these results suggest that HHEX is a critical transcription factor that serves to maintain glucocorticoid levels in vivo in males.

Lipid depletion observed in Hhex KO male mice results in part from activation of lipophagy in the inner zF
To better understand the mechanisms by which HHEX protects the sterol content of the zF, we interrogated cholesterol ester uptake, lipid droplet catabolism, and de novo synthesis. In rodents, adrenocortical cells obtain extracellular cholesterol mainly through the import of High Density Lipoprotein particles from the circulation50. Analysis by immunohistochemistry demonstrated that the inner cortex of Sf1-Crehigh Hhex KO strongly maintained the expression of SR-B1, the receptor for CE-laden HDL particles (Supplementary Fig. 4a), suggesting cholesterol uptake is not perturbed in the inner zF cells of Sf1-Crehigh Hhex KO adrenals. The expression of Hmgcr, the rate-controlling enzyme of the mevalonate pathway was significantly upregulated (Supplementary Fig. 4b), suggesting de novo synthesis was not impaired. Taken together, these results indicated that defects in cholesterol uptake and/or synthesis were unlikely the driving mechanism of lipid depletion in Sf1-Crehigh Hhex KO.

To identify potential alternative mechanisms of lipid depletion, we explored the differentially expressed genes in the bulk RNA seq (see below) from male Sf1-Crehigh Hhex KO versus WT adrenals, searching for factors involved in lipid catabolism (Supplementary Data 1). The gene Lipa (p-value=2.46E-5, LogFC=1.56) particularly drew our attention for its involvement in the process of acidic lipolysis. Also called “lipophagy”, it is defined as the selective engulfment of lipid droplets by the autophagic machinery (Fig. 4A). In brief, this process has been shown to involve the sequestration of lipid droplets by LC3-II positive membranes, where the macroautophagy cargo receptor p62/SQSTM1 bridges LD with the autophagic membrane, the phagophore51. The recruitment of p62/SQSTM1 to the LDs is in part mediated by the perilipin PLIN1, an LD-associated protein52. Upon fusion with the lysosomes, where the lysosomal acid lipase (LAL) resides, lipid droplets are degraded and cholesterol content recycled. Of note, it has been suggested that Perilipins and p62/SQSTM1 are co-degraded with lipid droplets in lysosomes, whereby their expression is inversely correlated with autophagic activity51,53,54. We therefore evaluated the integrity of lipophagy in WT and Sf1-Crehigh Hhex KO adrenals. In WT adrenals, PLIN1 (Fig. 4B, left) and p62/SQSTM1 staining (Fig. 4C, left) was robust throughout the cortex, while absent in the capsular cells. In Sf1-Crehigh Hhex KO, both PLIN1 and p62/SQSTM1 were markedly decreased in the inner zone of the cortex (Fig. 4B, C, right), mirroring the lipid-depleted area. Specifically, PLIN1 staining was quantified using image processing analysis (Supplementary Fig. 4c). The inner zF of Sf1-Crehigh Hhex KO exhibited significantly lower PLIN1 when compared to WT adrenals. The upper cortex was not affected in either genotypes (Fig. 4D). Despite the drastic reduction of lipid droplets in the inner zF cells, the mitochondria content was not affected, suggesting that other selective autophagy processes, such as mitophagy, were not activated (Supplementary Fig. 4d). We ruled out the persistence of the X-zone to Hhex KO phenotype by deleting Hhex, in vivo, using the AS-Cre, which targets the definitive cortex and excludes the X-zone55. Similarly to Sf1-Crehigh Hhex KO, the inner zF of AS-Cre Hhex KO was devoid of lipid droplets at 15-week-old (Supplementary Fig. 4e). We also performed RT-PCR for Pik3c2g, a robust X-zone marker56, and could not detect Pik3c2g cDNAs in male adrenals from either WT and Sf1-Crehigh Hhex KO genotypes (Supplementary Fig. 4f), indicating that, as expected, the X-zone was absent from both genotypes after puberty.

We confirmed by RT-qPCR the expression of key effectors of lipophagy and observed a significant increase in Map1lc3a (encoding the autophagic protein LC3), Lipa (encoding the acidic lipase LAL) (Fig. 4E, F) and Npc2 (encoding a lysosomal protein responsible for cholesterol removal from lysosomes) (Supplementary Fig. 4g) coincident with a marked increase in Abca1 (encoding a transporter known to limit the intracellular free concentration by promoting cholesterol efflux referred to as reverse cholesterol transport) (Fig. 4G). To test the hypothesis that lipophagy activation contributes to the absence of lipid droplets, we temporally and pharmacologically inhibited autophagy in vivo by treating WT and Sf1-Crehigh Hhex KO mice with hydroxychloroquine sulfate (HCQ-S), which prevents the fusion of the autophagosomes with lysosomes57. In WT, HCQ-S treatment did not affect the domain of expression of PLIN1 compared to vehicle-treated mice (Supplementary Fig. 4h, i). In contrast, we observed a partial lipid rescue specifically in the inner part of the cortex of Sf1-Crehigh Hhex KO male mice (Fig. 4H and Supplementary Fig. 4j), which was quantified using image processing analysis (Fig. 4I). However, this partial rescue was not sufficient to restore GC production by adrenal explants, when stimulated with Bt2-cAMP (Supplementary Fig. 4k). Together, these results indicate a crucial role for HHEX in protecting inner zF cells from lipophagy-mediated lipid depletion.

HHEX-dependent lipid depletion and HHEX expression are androgen-mediated
To characterize the temporal dynamic of the LD observed in Sf1-Crehigh Hhex KO adrenals, we assessed PLIN1 expression by immunohistochemistry at multiple time points (in 2-week-old, 6-week-old, and >1-year-old mice) (Fig. 5A). The results revealed that the genetic loss of Hhex prompted a decrease in lipid droplets in Sf1-Crehigh Hhex KO male adrenals that became visible at 6 weeks of age. This lipid depletion was noted primarily in the inner zF and expanded over time. With age, the entire zF was substantially lipid-depleted. However, in 2-week-old prepubertal mice, WT and Sf1-Crehigh Hhex KO male adrenals were indiscernible, coincident with intense staining for PLIN1 throughout the cortex in both genotypes. These results indicate that lipid depletion in the Sf1-Crehigh Hhex KO adrenals begins near puberty and suggest that HHEX is required to actively maintain LD stores from this stage onward. Contrary to what was observed in aged Sf1-Crehigh Hhex KO male adrenals, we did not observe a drastic LD depletion in females over 1 year of age (Fig. 5A, right). Consistently, when stimulated with Bt2-cAMP, WT, and Sf1-Crehigh Hhex KO female adrenal explants released an equivalent amount of corticosterone into the media (Supplementary Fig. 5a). Of note, we observed that the X-zone was devoid of lipid droplets, which complicated the analysis of the lipid depletion phenotype in females and gonadectomized male mice. To circumvent this issue, we also performed immunohistochemistry for PLIN1 on aged Sf1-Crehigh Hhex KO females who carried one or more pregnancies resulting in the well-characterized complete regression of the X-zone. We did not observe any loss of lipid droplets in Sf1-Crehigh Hhex KO primiparous females (Supplementary Fig. 5b).

To explain the decrease of lipid stores observed only in Sf1-Crehigh Hhex KO males after puberty, we hypothesized an interplay between lipid droplet catabolism and androgen signaling in the adrenal zF. We postulated that the androgenic milieu is the driving force of lipophagy and that HHEX acts as a protective mechanism to maintain LD integrity. To test this hypothesis, we gonadectomized WT and Sf1-Crehigh Hhex KO males at 6 weeks of age, when lipid depletion is first observed, and analyzed the adrenal glands at 15 weeks of age, when LD depletion is observed in the majority of the zF cells in non-gonadectomized Sf1-Crehigh Hhex KO males. In accordance with our hypothesis, we observed a rescue of lipid droplets in the zF of gonadectomized Sf1-Crehigh Hhex KO, becoming visually indistinguishable from their gonadectomized WT littermates (Fig. 5B). Consistently, p62/SQSTM1 staining was restored in the inner cortex of previously gonadectomized Sf1-Crehigh Hhex KO (Fig. 5C). Together, these data support the hypothesis that lipid depletion in Sf1-Crehigh Hhex KO results from an increase in autophagic flux that is specifically the result of androgen-driven lipophagy.

The adrenal glands rely on high cholesterol ester content to support steroidogenesis, which suggests that the catabolic action of the androgens on LD content needs to be tightly controlled. Therefore, we questioned the possible signaling pathways controlling Hhex expression. We hypothesized that to protect the LDs at puberty, Hhex expression in the male itself might be regulated by androgens. We first looked at the expression of Hhex at different time points from birth through aging past 1-year, in both males and females. At the mRNA level, we observed a dramatic increase at puberty, specifically in males (between 3 and 4 weeks of age), resulting in a nine-fold difference between sexes at 6 weeks of age (Fig. 5D). We then proceeded to assess the expression of HHEX in the absence of circulating androgens. We analyzed adrenals from adult gonadectomized male mice and observed a striking decrease in HHEX staining compared to their non-gonadectomized littermates (Fig. 5E). Next, we confirmed the direct contribution of androgens to the upregulation of Hhex expression ex vivo while ruling out the participation of gonadotrophins released by the central system. We incubated female adrenal explants with 5a-Androstan-17b-ol-3-one (Dihydrotestosterone, DHT) (an androgen, non-aromatizable into estrogens58,59) to activate androgen signaling. Adrenal explants incubated with DHT for 48h led to a significant 2.2-fold increase in Hhex expression compared to the control-treated explants (Supplementary Fig. 5c). This result confirm the direct role of androgens in promoting Hhex expression in adrenocortical cells.

We then tested whether Hhex expression is directly regulated through AR signaling, so we proceeded to look at HHEX expression following adrenal-specific AR deletion in the adrenal cortex (ARKO) by breeding ARflox/Y60 mice with AS-Cre55 mice. Similar to our findings upon gonadectomy in WT mice, HHEX expression assessed by immunohistochemistry was dramatically reduced in the adrenals of adult adrenal-specific ARKO mice (Fig. 5F). Finally, we employed CUT&Tag, a technique based on the same principles as Chromatin Immunoprecipitation (ChIP) that offers the advantage of being more sensitive and requiring less material36, which makes it suitable to study binding profiles of transcription factors in vivo in the mouse adrenal gland. Using an antibody targeting AR to map genomic AR binding sites in the mouse adrenal, we identified 106726 peaks genome-wide. The distribution profiles showed enrichment of AR binding at transcription start sites (TSS), extending over a 2kb interval (Supplementary Fig. 5d). At the genome level, AR was mainly distributed in the intergenic and promoter regions (Supplementary Fig. 5e). This finding underscores the significant role that AR plays in transcriptional regulation in the male mouse adrenal. Next, we conducted motif analysis using HOMER, which showed significant enrichment for Jun-AP1, Fosl2, and Nr5a2 motif (similar to Nr5a1) (Supplementary Data 1). Importantly, AR peaks exhibited significant enrichment for the PGR motif, which carries similarities to steroid receptor binding motifs, including AR. In exploring a possible connection between AR and Hhex expression, we identified in WT male adrenals an AR peak in the Hhex promoter that overlaps with a H3K27ac peak (Fig. 5G). H3K27ac is a histone modification associated with active chromatin, typically found at active enhancers and promoters, indicating potential transcriptional activation. The sexually dimorphic expression of HHEX in the adrenal cortex led us to examine other HHEX-expressing, sexually dimorphic tissues, such as the liver and the pancreas61,62. Contrary to the adrenal gland, HHEX was strongly expressed in both male and female hepatocytes (liver), somatostatin-secreting delta cells (pancreas), and hematopoietic cells from the bone marrow, where it was initially identified (Supplementary Fig. 5f). Altogether, these results demonstrate that HHEX is a bona fide AR-target gene in the adrenal zF, which coordinates the precise timing of protecting the integrity of lipid droplets at puberty.

Adrenal sexual dimorphism is partially mediated by HHEX
Recent studies have highlighted the influence of androgens, rather than sex chromosomes, on differentiation, proliferation, and disease development in the adrenal cortex63–65. Given the sex differences observed in HHEX expression and the direct transcriptional dependency of HHEX expression on AR, we postulated that HHEX is a mediator of AR actions in the zF, which contributes to sex-biased transcriptional programs in the adrenal cortex. To decipher the transcriptional programs driven by HHEX in the adrenal cortex, we performed bulk RNA seq on adrenals from WT and Sf1-Crehigh Hhex KO mice at 6 weeks of age in both males and females. For a global analysis of gene expression, we performed PCA, a common unsupervised dimensionality-reduction method to visualize and assess the clustering of the data66. PCA revealed that over 80% of the variance between the studied mice was explained by the first and second principal components (PCs) that separated individual mice according to sex on the first component (X-axis), and according to genotype on the second component (Y-axis) (Fig. 6A). The clustering suggested that Hhex deletion in the steroidogenic lineage drives significant changes at the transcriptome level in the adrenal as soon as 6 weeks of age. To further explore the basis of this change, we examined the differentially expressed genes (DEGs). Using a cut-off of 0.05 for the adjusted p-value, the analysis revealed that 3607 genes were differentially expressed between genotypes in males, with only 23 DEGs in females. By analyzing the top DEGs in 6-week-old Sf1-Crehigh Hhex KO males versus their WT littermates (Supplementary Data 1), we observed an enrichment in well-known sexually dimorphic genes63,67–69, suggesting a feminization of the male transcriptome when Hhex is deleted. We confirmed these observations by assessing the expression of “female-biased” Nr0b1 (known as Dax1) (Fig. 6B), Frzb (Fig. 6C), and Cyp2f2 (Supplementary Fig. 6a, b) in WT and Sf1-Crehigh Hhex KO, male and female, adrenals by RT-qPCR. The sexual dimorphism was completely or at least partially abolished for these three genes in Sf1-Crehigh Hhex KO male adrenals. In situ, Nr0b1-positive cells assessed by RNAscope were observed in the zF of Sf1-Crehigh Hhex KO male adrenals (similar to WT female), while restricted to the zG in WT male adrenals, as previously described69 (Fig. 6D). The expression of Frzb was mostly found in cells of the zF in Sf1-Crehigh Hhex KO (similar to the WT female), while absent from the WT male zF (Fig. 6E). By quantifying the expression of Frzb and Nr0b1 by RT-qPCR during postnatal life, we observed a striking decrease in males at puberty, which corresponds to the time when Hhex expression increases, supporting the role of HHEX in repressing female programs and promoting male programs (Fig. 6F, G). Since testosterone levels (assessed by LC-MSMS) were unchanged in Sf1-Crehigh Hhex KO compared to WT plasma (Supplementary Fig. 6c), we ruled out a contribution of the gonads to the reversal of the sexual dimorphism observed in the adrenal cortex of Sf1-Crehigh Hhex KO mice. Therefore, we performed immunohistochemistry, and staining revealed high expression of AR in the zF (and to a lesser extent in the zG) (Fig. 6H), which was confirmed by scRNA-seq (Supplementary Fig. 6d). This suggested that the feminization of Sf1-Crehigh Hhex KO male adrenals could be a result of the loss of androgen signaling directly in the zF. However, AR expression assessed by RT-qPCR was not abolished in males Sf1-Crehigh Hhex KO compared to their littermate controls (Supplementary Fig. 6e), suggesting that HHEX is unlikely upstream of the androgen signaling. To provide a comprehensive analysis, we compared the transcriptome of 6-week-old Sf1-Crehigh Hhex KO male adrenals and 25-week-old ARKO male adrenals and found a significant overlap. Nearly 70% of genes upregulated in Sf1-Crehigh KO male adrenals belong to the list of upregulated genes in ARKO male adrenals, and 55% belong to the list of downregulated genes (Fig. 6I and Supplementary Data 1). Taken together, these results suggest that HHEX is a mediator of AR action and that the female transcriptome in the zF is a common program in both sexes before puberty. During the rise of systemic androgen levels concomitant with testis maturation, HHEX expression emerges throughout the zF, which participates to establishing the mature male adrenal transcriptome and consequently institutes an AR:HHEX-dependent sexual dimorphism in the adrenal cortex.

HHEX is crucial for maintaining the identity of the innermost adrenocortical cell subpopulation and promoting Abcb1b expression in both sexes
The lower expression of Hhex in females compared to males after puberty and the lack of evident LD depletion in female Sf1-Crehigh Hhex KO mice led to question whether HHEX contributes to transcriptional programs in the female mouse adrenal cortex. Analysis of HHEX expression by immunohistochemistry in WT male and female adrenals revealed a striking lower staining intensity in zF cells of female adrenals (Fig. 7A). Nonetheless, Hhex transcript levels were higher in WT female adrenals than their Sf1-Crehigh Hhex KO littermates, demonstrating the existence of a baseline expression (albeit lower than male) in female adrenal cells (Fig. 7B). Additionally, bulk RNA seq analysis of 6-week-old female adrenals revealed 23 significant DEGs between Sf1-Crehigh Hhex KO and WT mice. Accordingly, the PCA plot demonstrated the separation of female samples based on genotype on the second component of the PCA plot (Fig. 6A), suggesting that HHEX contributes to transcriptional programs in the female adrenal as well. Despite the low number of genes differentially expressed between WT and Sf1-Crehigh Hhex KO females, 21 out of 23 were common to DEGs in male WT and Sf1-Crehigh Hhex KO. This included 16 upregulated genes and 5 downregulated genes in Sf1-Crehigh Hhex KO compared to their respective controls (Fig. 7C). Among them, Abcb1b was the most downregulated gene upon Hhex deletion, and was further studied based on its established role in sterol flux and stress adaptation in the adrenal cortex15,70. Decreased Abcb1b expression was further confirmed in larger cohorts, at two time points, in both sexes, using RT-qPCR (35–80% decrease) and RNAscope (Fig. 7D–F). At baseline, we observed that the Abcb1b population was localized to the inner zF in WT adult mouse adrenals, consistent with previous reports15. In females, Abcb1b expression was also found in cells of the X-zone (see below). In both sexes, the intensity of the Abcb1b staining was visibly reduced in Sf1-Crehigh Hhex KO adrenals. Based on the decreased expression of Abcb1b upon Hhex deletion, we thus hypothesized that the expression of other genes uniquely expressed in the inner zF subpopulation might be downregulated with Hhex loss. In agreement with this hypothesis, we performed RT-qPCR on WT and Sf1-Crehigh Hhex KO male adrenals and observed a decrease of mRNA levels for other markers of these cells, including Srd5a2 and Mgst2 at 6 weeks of age (Supplementary Fig. 7a), suggesting HHEX is required for a unique gene expression profile in this population.

To clarify when HHEX is required for Abcb1b expression, we used Cyp11b1-CreERT2 Hhexflox/flox and Cyp11b1-CreERT2 Hhexwt/wt or flox/wt (WT and Heterozygotes) mice fed a tamoxifen chow for 4 weeks after puberty (Supplementary Fig. 7b). This strategy restricted the recombination to the fasciculata lineage upon tamoxifen administration and discriminated defects affecting embryogenesis and/or homeostasis. Similar to the Sf1-Crehigh model, we observed a significantly decreased expression of Abcb1b in the adrenal of Cyp11b1-CreERT2 Hhexflox/flox males and females compared to littermate controls (Supplementary Fig. 7c), suggesting that HHEX is necessary for Abcb1b expression during homeostasis. This also confirmed that the function of HHEX in promoting Abcb1b expression was specific to the adrenal and not to the brain or gonad.

Based on our finding that HHEX promotes Abcb1b expression, we hypothesized that the previously described expansion of the Abcb1b population in response to chronic stress15 would be impaired in Sf1-Crehigh Hhex KO mice. To test this hypothesis, we used long-term ACTH administration as a surrogate for chronic stress exposure71. As expected, ACTH induced hyperplasia and hypertrophy in WT mice (male and female) and led to significant increases in adrenal body weight ratios in both sexes and both genotypes (Supplementary Fig. 7d, e). Consistent with other in vivo models of chronic stress, we observed by RNAscope a centrifugal expansion of the Abcb1b expression domain in the adrenals of both male and female WT mice, demonstrating the robustness of this response to HPA axis activation (ACTH response). Under chronic ACTH administration, the Abcb1b staining was also visibly reduced in Sf1-Crehigh Hhex KO compared to WT adrenals (Fig. 7F and Supplementary Fig. 7f, right panels). Surprisingly, chronic ACTH administration led to an increased adrenal weight in Sf1-Crehigh Hhex KO in both sexes despite a failure to increase Abcb1b expression, suggesting that expansion of the Abcb1b expression domain and zF hyperplasia are two partially uncoupled mechanisms. While the adrenal-specific role of Abcb1b is unknown in mice, ABCB1 has been shown to mediate GC release from human adrenal cells15 and ABCB1 polymorphism (rs2032582) has been associated with higher cortisol secretion in cortisol-producing adrenal adenomas in humans72. Therefore, to assess whether HHEX is contributing to GC release during a chronic stress response, we harvested female WT and Sf1-Crehigh Hhex KO adrenals upon chronic ACTH administration (when Abcb1b expression is increased in both sexes). We measured corticosterone released into media by explants and found similar levels between female ACTH-treated WT and ACTH-treated Sf1-Crehigh Hhex KO adrenal explants (Supplementary Fig. 7g). This observation suggests that in females, unlike in males, despite Hhex loss and a decrease in Abcb1b expression of 46%, residual Abcb1b expression may be sufficient to sustain corticosterone release under the conditions tested. Taken together, these results demonstrate that in both sexes, HHEX is required to maintain optimal Abcb1b expression in the inner zF, at baseline and upon chronic stress.

Methods
Sex as a biological variable
This study examined both male and female mice since sexual dimorphism was observed in the results and therefore studied in the current manuscript.

Mice
Mice were bred in-house and maintained on a C57Bl/6 background at the University of Michigan, USA. They were housed on a 12h light/12h dark cycle (light on at 6 am), at 22.2°C (72°F), 35–40% humidity, and fed the commercial rodent chow 5L0D (PicoLab® Laboratory Rodent Diet). Exceptions were made during breeding and before weaning when the Formula b Diet 5008 chow (LabDiet) was used, and during CreERT2 activation experiments, when mice were placed in a new cage and exclusively fed Teklad Custom TAM diet (400 TC, 2016 - TD.130859, Envigo, expected to provide ~40 mg tamoxifen per kg of body weight per day, assuming 20–25 g body weight and 3–4 g intake). In all experiments (P0 to >1 year old), mice were provided water and food ad libitum. At weaning (around 3 weeks of age), mice were separated from parents and kept with same sex littermates at a maximum of five animals per cage. Hhexflox/flox mice were purchased at Jackson Laboratory (Stock 025396; B6N.129S1(Cg)-Hhex<tm2Cwb>/J; https://www.jax.org/strain/025396) and initially donated by Dr. Clifford Bogue, M.D., Yale University, USA112. They contained loxP sequences flanking exon 2–3 of the Hhex mouse gene (ENSMUSG00000024986). Hhexflox/flox mice have been previously described and used to successfully delete Hhex in the liver112, pancreas25,113, osteoclast114, and myeloid lineage115. After cryo recovery, Hhexflox/flox mice were crossed with Sf1-Crehigh; mT/mG mice, AS-Cre; mT/mG, and Cyp11b1-CreERT2; mT/mG lines. Sf1-Crehigh mice were provided by the late Dr. Keith Parker, M.D, Ph.D, University of Texas, USA37. Sf1-Crehigh allele harbors five copies of a transgene containing 111 kb of the Sf1 locus to target Cre expression in various steroidogenic tissues, including the adrenal cortex. This mouse line has been described multiple times to successfully delete various floxed alleles in the adrenal steroidogenic lineage39–41,64,116–125. AS-Cre (Cyp11b2-Cre) mice were kindly shared by Dr. David Breault, M.D., Ph.D., Harvard, USA55, and were similarly used to delete various floxed alleles in the definitive cortex39,41,63. Cyp11b1-CreERT2 mice were developed and kindly shared by Dr. Felix Beuschlein, M.D, Zurich, Switzerland. The Cyp11b1-CreERT2 mouse line was generated by PolyGene AG (Rümlang, Switzerland) to establish a Cyp11b1-KO mouse model with a tamoxifen-inducible Cre recombinase (Cre-ERT2) under the gene’s control. Exon 2 was selected for inserting the expression cassette, preserving potential regulatory elements in intron 1. To prevent alternative translation initiation, all five ATG start codons in exon 1 were mutated to CTG. The targeting vector included homology arms (4.97 kb and 1.83 kb) generated by PCR from C57Bl/6N BAC DNA, as well as Cre-ERT2 and β-globin poly(A) sequences assembled via overlap-extension PCR. Additionally, a 1.3 kb fragment containing mutated exon 1, intron 1, and part of exon 2 was synthesized and cloned into the short arm – Cre-ERT2 construct. The final targeting vector, H052.4 TV, incorporated an FRT-flanked neomycin selection cassette and was validated through sequencing. The following primers were used to select positive embryonic stem cell clones H052.17: binds to the elongated short arm of homology 5′-CACGCTGAACTAGCATAGCC-3′ H052.19: binds to the Cre-ERT2 5′-CTACACCAGAGACGGAAATCCATC-3′. Rosa mT/mG reporter line38 has been described multiple times and successfully used to track recombined cells, including steroidogenic cells39,40,55,63,64,122. ARflox mice60 were made at the Catholic University of Leuven, Belgium, and kindly shared by Dr. Frank Claessens and Dr. Johan Swinnen.

Rats
Naïve rats were obtained from the animal/rodent redistribution program in place at the University of Michigan and euthanized with CO2 according to ULAM protocol. Rat adrenals were harvested, fixed, processed, and embedded similarly to the mouse adrenals.

Human adrenal glands
Human adrenal samples were obtained from renal transplantation donors at the University of Michigan. Formalin-fixed paraffin-embedded human adrenal samples without overt pathology based on histological analysis were used to prepare 5-µm sections that were used for immunohistochemistry. Bulk human tissue gene expression for HHEX were obtained from the GTEx portal (GTEx Analysis Release V10 (dbGaP Accession phs000424.v10.p2).

Cells
NCI-H295R cells (ATCC, #CRL-2128, STR profiling) were grown in DMEM/F-12 (GIBCO, #11330032) supplemented with 10% NuSerum (BD Bioscience, #355100), 1% insulin-transferrin-selenium-ethanolamine (GIBCO, #51500056), and 1% Penicillin-Streptomycin (GIBCO, #15140122).

Genotyping
Mouse tail clips of 2 to 3 mm of length were harvested at weaning, and genomic DNA was extracted using the HotSHOT method126. Briefly, tissue was digested in 75 µl of Alkaline Lysis Reagent (25 mM NaOH, 0.2 mM disodium EDTA, pH of 12 without adjusting) for 30 min at 95°C. Samples were then chilled on ice for 2–3 min, and 75 µl of Neutralization Buffer (40 mM Tris-HCl, pH of 5 without adjusting) were added (Supplementary Data 2). 20 µL PCR reactions were set up in TempAssure 0.2 mL PCR 8 strips color (USA scientific, #1402-4708). For each PCR reaction, 2 µL of genomic DNA were added to a mix comprised of 10 µL of 2× GoTaq® Green Master Mix (Promega, #M7123), combined with 1 µL of each primer (10 µM) and milliQ water for a final volume of 20 µL. Genotyping primer pairs used are listed in Supplementary Data 2. After amplification, PCR products were separated on 2% agarose gels dyed with SYBR Safe (Thermo Fischer Scientific, #S33102).

Hormonal manipulation, treatment, and surgery
Chronic ACTH treatment was performed using Synachten 1 mg/mL diluted in PBS 1× (Gibco, #10010023) to 0.25 mg/mL. 13 to 15-week-old male and female mice were injected twice a day intraperitoneally (50 µL) for 10 days. Adrenal enlargement was confirmed at dissection. Testicular gonadectomy was performed using 6-week-old WT and Hhex KO males. Animals were anesthetized with isoflurane (Fluriso, VetOne) and simultaneously injected with Carprofen (Rimadyl, diluted to 1 mg/mL) at 5 mg/kg for pain relief. Testes were surgically removed, and animals were placed individually in new clean cages after surgery, monitored twice a day for 7–10 days for recovery until suture clips were removed under brief isoflurane anesthesia. At dissection, the regression of seminal vesicles (androgen-sensitive organ) was confirmed, indicating the successful withdrawal of androgen production.

In vivo hydroxychloroquine treatment
Hydroxychloroquine sulfate (HCQ-S, Tokyo Chemical Industry, #H1306) was first resuspended in di-ionized water at a concentration of 6 mg/mL, aliquoted, and stored at −20°C. HCQ-S was then diluted to a concentration of 0.6 mg/mL with room temperature deionized water (similar to control mice). The solution was administered ad libitum in clean water bottles every week. No other liquid source was available during the treatment duration. Based on average water intake, mice were delivered a daily oral dose of 2–4 mg of HCQ-S. Mice were monitored at least weekly.

Tissue harvesting
The day prior to euthanasia, each individual mouse was briefly put on a scale, and the body weight (in grams) was recorded to calculate the adrenal to body weight ratio after euthanasia. At dissection, left adrenal glands were placed in PBS 1× on ice until a precise dissection was performed under a stereo microscope (Nikon, #SMZ800) to remove surrounding fat. Weighing (in milligrams) and imaging (Olympus, #DP21) were quickly recorded to prevent tissue dehydration and lysis. Right adrenals were briefly harvested, dissected from surrounding fat, and immediately snap frozen in liquid nitrogen for long-term storage at −80°C.

Mouse adrenal single-cell dissociation and single-cell RNA seq
Adrenal glands were harvested from 15-week-old Sf1-Crehigh; Rosa mT/mG male mice following rapid decapitation to reduce stress-induced transcriptional changes. 14 adrenals (31 mg of tissue) were processed for the first replicate and 20 (49 mg of tissue) for the second. Adrenals were placed immediately into ice-cold 1× Hank’s Balanced Salt Solution (HBSS 1×, ThermoFisher, #14025092) containing calcium and magnesium. 30–50 mg of tissues were then finely chopped in 100–200 µL of HBSS 1× using single-edge razor blades (Persona GEM, #62-0179). From this point, all tips and tubes were precoated with 3% BSA (Roche, #3116956001) in PBS 1× (w/v) to prevent cell loss. Tissue pieces were then transferred to 5 mL tubes (Eppendorf, #0030119487) containing 2 mL of cold HBSS 1× supplemented with 10% Fetal Bovine Serum (Corning, #35-010-CV) and centrifuged for 5 min at 4°C at 500×g and supernatant was pipetted out and discarded. Single cell suspension was then obtained at low temperature (4 to 10°C) by combining enzymatic and mechanic dissociation according to the following steps. Mix 1, containing 1900 µL of Buffer X (Mitenyi Biotec, #130-092-628), 50 µL of Papain (Mitenyi Biotec, #130-092-628), and 20 µL of collagenase (Sigma-Aldrich, #C2139) prepared at a concentration of 100 mg/mL in DMEM-12, was added to the tissue preparation and incubated for 15 min at low temperature. Mix 2 (10 µL of enzyme A and 20 µL of Buffer Y, Mitenyi Biotec, #130-092-628) was added to the digestive solution for 1 h from this step. During the dissociation, the cells suspension was gently agitated with mechanical pipetting every 10 min and visually assessed under a microscope for 1 h until the tissue was fully digested. The suspension was then filtered through 70 μm filters (PluriSelect USA, #43-10070-40) to obtain a single cell suspension, and enzymes were neutralized using HBSS 1× containing 10% Fetal Bovine Serum. Red blood cells were removed using Red Blood Cell Lysis buffer (Roche, #11814389001) according to manufacturer guidelines (15 min on orbital shaker in a cold room), and the cells were washed twice in HBSS containing 2% FBS before counting. After final centrifugation, cells were resuspended in 1 mL of HBSS 1×, FBS 2%. Following dissociation, cells were stained with 2 μg/ml DAPI and 5 μM Vybrant DyeCycle Ruby (Invitrogen, #V10309). Ruby-positive, DAPI-negative single cells were sorted based on fluorescent properties using the Thermofisher Bigfoot Cell Sorter at the University of Michigan Flow Cytometry Core. Gating strategy is described in Supplementary Data 1. Cells were collected in BSA-precoated tubes containing HBSS 1× with 2% FBS and kept on ice. Single-cell droplets with a target capture of 10,000 cells were immediately prepared on the 10× Chromium system at the Advanced Genomic Core. Single-cell libraries were prepared using the Chromium Next GEM Single Cell 3′ Gene Expression Library Construction Kit version 3.1 according to manufacturer instructions. Sequencing was performed on an Illumina NovaSeq (S4) 300-cycle. Raw and processed data have been deposited in NCBI’s GEO database (GSE291343).

scRNA-seq analysis, cluster identification, and gene marker determination
Data of each individual experiment was analyzed according to the following steps: Raw sequencing data was aligned to the mouse reference genome (GRCm39) and quantified using cellranger count (10× Genomics). The resulting count matrices were processed in R (version 4.3.1) using Seurat (version 5.0.1)127. Low-quality cells, defined as those exhibiting a high percentage of mitochondrial reads or a low number of detected features, were identified and removed using miQC128 (version 1.10.0). Doublets were identified and removed using DoubletFinder (version 2.0.4)129. Datasets from individual experiments were merged and normalized using the SCTransform function from Seurat. To account for technical variability, we regressed out the percentage of reads mapped to mitochondria and ribosomal genes during normalization. We performed integration of the datasets using Harmony (version 1.2.0)130. Cell clusters were identified using the FindClusters function in Seurat using the Leiden algorithm (version 0.10.0)131. Uniform Manifold Approximation and Projection (UMAP) dimensional reduction was performed on the harmony-corrected embeddings using RunUMAP function in Seurat. Smoothed visualizations of gene expression in UMAP space was generated using Nebulosa (version 1.12.1), which employs kernel density estimates for enhanced visualization and interpretation132. Marker genes for each cluster were identified using the FindAllMarkers function in Seurat. Statistical significance was inferred using the Wilcoxon rank-sum test and adjusted for multiple testing using the Benjamini–Hochberg method. Cortical cell clusters (defined by the expression of cortical cell-specific marker Nr5a1) were further classified as ZG- or ZF-biased based on the expression of known ZG and ZF markers such as Vsnl1, Cyp11b2 (ZG), and Cyp11b1 (ZF). Grouped differential expression analysis between ZG- and ZF-biased clusters was performed using the FindMarkers function from Seurat. A volcano plot displaying the top 20 DEG genes was built using EnhancedVolcano (version 1.18.0). A CLOUPE file was generated from the Seurat-processed single cell gene expression dataset using the 10× Genomics’ LoupeR (version 1.1.3) for interactive and visualization and exploration (see below Data availability).

Hormonal measurements
When hormonal measurements were expected to be performed, mice were euthanized by decapitation in compliance with IACUC protocol between 8:30 and 10 am, within 30 s of handling to minimize stress-induced ACTH secretion. Core trunk blood was collected using sodium heparin-coated tubes (BD Vacutainer® Heparin Tubes, #367871) and centrifuged at 1800×g for 20 min at 4°C to obtain plasma. Samples were divided into two aliquots for ACTH and steroid measurements and stored at −80°C prior to analysis. Corticosterone, Progesterone, 11-Deoxycorticosterone, and Testosterone concentration were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Intra-adrenal cholesterol esters and free cholesterol measurement
For each mouse, both adrenals were harvested and placed in cold PBS 1×, removed fat and weighed for downstream calculation and homogenized in 600 µL of lipid extraction solution (Chloroform, Isopropanol, Igepal) as described by manufacturer (Abcam, #ab65359) with slight modifications. Adrenal tissues were homogenized using a BeadBug™ Microtube Homogenizer (2× 30 s 400 (×10 speed)). Lysate was transferred into new 1.5 mL Eppendorf tubes and evaporated for 4 h at 50°C in a chemical hood, then vacuum centrifuged for 30 min. Lipids were finally resuspended in 200 µL of assay buffer. Cholesterol levels were measured using two volumes that were preliminary determined to be in the range of the standard curve (5 and 10 µL for total cholesterol and 25 and 50 µL for Free cholesterol). The average quantity of cholesterol ester in WT adrenals was 9.6 µg/mg of adrenal gland, similar to published literature133.

Ex vivo mouse adrenal explant culture
DHT stimulation: Seven-month-old female adrenals were quickly harvested after euthanasia and put in ice-cold PBS 1×. They were then cut in half under a tissue culture hood, in a tissue culture dish, using sterile single-edge razor blades (Persona GEM, #62-0179). In a 24-well plate (USA Scientific, #CC7682-7524), two half adrenals were placed in each well in 1 mL of DMEM/F-12 (GIBCO, #11330032) supplemented with 1% insulin-transferrin-selenium-ethanolamine (GIBCO, #51500056) and 1% Penicillin-Streptomycin (GIBCO, #15140122). No serum was added to avoid exogenous steroid contamination. After 24 h, explants were incubated with Vehicle or 5α-dihydrotestosterone (DHT) at 0.1 μM (Cerilliant, #D-073-1ML) for 48 h. RNA was extracted from explants using the Mini RNA extraction kit (RNeasy Mini Kit Qiagen, #74104).
Dibutyryl cAMP stimulation (Bt2-cAMP): Dibutyryl-cAMP (sodium salt) (Sigma-Aldrich, #D0260 and Cell Signaling #35857) was resuspended in sterile water at a concentration of 0.1 mg/µL and used at a final concentration of 2.5 mM. After dissection, two half-adrenals were placed in each well in 1 mL of prewarmed DMEM/F-12 (GIBCO, #11330032) supplemented with 1% insulin-transferrin-selenium-ethanolamine (GIBCO, #51500056) and 1% Penicillin-Streptomycin (GIBCO, #15140122) for 1 h. No serum was added to avoid exogenous steroid contamination. Adrenals were then transferred into new wells and incubated with Vehicle (water) or Bt2-cAMP for the time indicated in the figures. Media was harvested at the end, and 2.5 µL of culture media was diluted 100 times in DMEM F12 and used for ELISA assays. Corticosterone levels (ENZO, #ADI-900-097) were assayed in duplicate according to manufacturer instructions. In this experiment, samples were not treated with the steroid displacement reagent.

Histology
Left adrenals were quickly transferred to be fixed in Formalin (FischerBrand, #427-098) for 24 h at room temperature under gentle agitation on an orbital shaker. The next day, adrenals were rinsed in PBS 1× for 10 min at room temperature and transferred in 70% ethanol until processing. Tissue processing was performed on a Leica ASP 300S Tissue Processor at the UMICH Orthopaedic Research Laboratories (ORL) Histology Core (15 min per station for adrenal, 1 h for larger tissues). Tissues were embedded in paraffin (Leica Paraplast, #39601006), and 5 μm sections were obtained using a HistoCore BIOCUT microtome (Leica) on Superfrost™ Plus Microscope Slides (Fischer Scientific, #12-550-15). A minimum of two serial sections were placed on each slide, thus allowing the inclusion of negative controls for each biological replicate. Sections were first baked for 45 min-1 h in an incubator at 55°C, then deparaffinized in histoclear (National Diagnostics, #HS-200) (2 times-5 min) and rehydrated in graded ethanol (2 times-5 min in absolute ethanol; 2 times-5 min in 95%; 1 time-5 min in 70%; 1 time-5 min in 50%). After 5 min under running tap water, slides were immersed in antigen retrieval according to the Supplementary Data 2. After 3 times-5 min washes in PBS 1×, slides were incubated in hydrogen peroxide (Sigma-Aldrich, #H1009, 0.3% v/v in water), for 30 min at room temperature to inhibit endogenous peroxidase. Hydrophobic barriers were drawn around each section with an ImmEdge pen (Vector Laboratories, #H-4000). After 3 times-5 min washes in PBS 1×, slides were blocked using 2.5%, ready to use, Horse or Goat Serum (Vector Laboratories, #S-2012-50, #S-1012-50) depending on the host specie of the secondary antibody. When antibody made in mouse was used, the M.O.M.® (Mouse on Mouse) ImmPRESS® HRP (Peroxidase) Polymer Kit (MP-2400) was used according to manufacturer instructions. Adrenal sections were then incubated overnight at 4°C in a humid chamber with primary antibodies Supplementary Data 2. The following day, sections were rinsed 3 times-5 min in PBS 1× and incubated with appropriate secondary antibody (Vector Laboratories, Immpress HRP) for 30 or 10 min according to the manufacturer instructions (details in Supplementary Data 2). After 3 times-5 min washes in PBS 1×, staining was developed with ImmPACT® DAB EqV Substrate Kit, Peroxidase (HRP) (Vector Laboratories, #SK4103). Sections were then rinsed 3 times-5 min in PBS 1× and 1 time-5 min under running tap water. Hematoxylin staining (Sigma-Aldrich, #GHS132-1L) was then performed to visualize nuclei before mounting with Permount™ Mounting Medium. Images were acquired on a Nikon Optiphot-2 with an Olympus DP70 camera and the DP manager software (version 3.3.1.292). For PLIN1 quantification of integrated density, we used Fiji/ImageJ software (2.14.0/1.54f/Java 1.8.0_66)134,135 with the following parameters (Hue: 0–250, Saturation: 0–255, Brightness: 150–255, Thresholding method: Default, Threshold color: B&W, Color Space: HSB, Dark Background). For nuclear density, the adrenal cortex was divided into 3 equal tiers, and the number of nuclei was counted in each tier and divided by the area. Results are reported as number of nuclei per mm2. Costaining. After HHEX staining was developed and sections rinsed 3 times-5 min in PBS 1×, slides were incubated in HCL 0.02 N in water for 20 min to inhibit peroxidase from the Immpress HRP secondary antibody. Sections were then rinsed 3 times-5 min in PBS 1× and blocked as described above, and incubated with DAB2 antibody overnight at 4°C in a humid chamber. The following day, the secondary antibody was applied as described above, and staining was developed using the ImmPACT® VIP Substrate Kit, Peroxidase (HRP) (Vector Laboratories, #SK-4605). Visualization of endogenous fluorescence: All steps were performed using dark tube or foil-covered plates to minimize exposure to light. At dissection, left and right adrenals were quickly harvested, dissected from surrounding fat, and incubated for 2 h in 4% PFA at 4°C, then incubated in Sucrose 30% (Sigma-Aldrich, #84097) diluted in PBS 1× overnight at 4°C. The next day, adrenals were transferred into new tubes and incubated in Tissue-Plus™ O.C.T. Compound (Fisher Healthcare, #23-730-571) for 1 h at 4°C. Finally, adrenals were transferred to cryomolds (Tissue-Tek, #4565) and embedded in O.C.T. on a dry ice ethanol bath. The blocks were stored at −80°C until cryosection. The day of imaging, 5–7 μm thick sections were made on a cryostat (Leica CM3050S) on Superfrost™ Plus Microscope Slides (Fischer Scientific, #12-550-15), incubated for 10 min in Hoechst (1:10 000 in PBS 1×) for nuclei visualization, then mounted in a PBS/glycerol 50% (v/v).

In situ single-molecule hybridization, RNAscope
Adrenals were fixed in 10% normal buffered formalin (VWR) for 24 h at room temperature, rinsed in PBS 1× for 10 min, and paraffin-embedded, and cut into 5 μm sections. The RNAscope™ 2.5 HD Reagent Kit-BROWN (Advanced Cell Diagnostics, #322300) was used according to the manufacturer’s instructions. Probes are listed in Supplementary Data 2. Images were acquired on a Nikon Optiphot-2 with an Olympus DP70 camera.

In situ neutral lipids staining, Oil red O
Oil red O stock solution was prepared by adding 1.25 g of ORO (Sigma-Aldrich, #00625) to 200 mL of 99% (vol/vol) isopropyl alcohol (Sigma-Aldrich, #I9516), and the solution was mixed with magnetic stirring for 2 h at room temperature. Snap-frozen adrenals were embedded in O.C.T. compound directly in mold in the cryostat’s chamber. Organs were kept frozen during the entire process, and 5–7 μm thick sections were made on a cryostat (Leica CM3050S) on Superfrost™ Plus Microscope Slides (Fischer Scientific, #12-550-15). Prior to storage at −80°C, sections were dried at room temperature for 10 min to prevent detachment from the slides. Oil Red O working solution was prepared the day of the staining by adding 1.5 parts of ORO stock solution to one part of distilled water (30 mL of ORO to 20 mL of water). The solution was left at 4°C for 10 min to thicken before filtration through a 45 μM filter (Corning, #430627) to remove precipitates. In the meantime, slides were equilibrated for 10 min at room temperature, then incubated for 5 min in Oil Red O working solution before being rinsed for 30 min with running tap water. The slides were finally mounted in PBS: Glycerol (Fischer chemical, #G33) solution (1:1), let sit for 10 min at room temperature and sealed using dots of Permount™ Mounting Medium on the edge of the coverslip (Corning, #2980-225).

RNA expression analysis
The right adrenals were quickly harvested after euthanasia, fat was quickly removed, and adrenal tissues were transferred into a tube, snap frozen, and stored at −80°C. At the time of RNA extraction, each adrenal was quickly transferred into a Lysing Matrix D 2 mL tube (MP Biochemicals, #6913100) containing 600 μL of RLT lysis Buffer (RNeasy Mini Kit Qiagen, 74104) supplemented with 6 μL of 2-mercaptoethanol (Sigma-Aldrich, M7522). Lysis was performed using a BeadBug™ Microtube Homogenizer (2× 30 s 400 (×10 speed)). Lysate was transferred into new 1.5 mL tubes and an equal volume of 70% ethanol was added and mixed by pipetting. RNA was extracted according to manufacturer instructions with few modifications as follow to avoid contamination with Thiocyanate Guanidine in the final eluted sample. New collection tubes were used after each step of centrifugation so that the exterior of the column never came in contact with previously discarded flowthrough. Additional collection tubes were made by cutting the lids of 2 mL centrifuge tubes (USA Scientific, #1620-2700). An additional step of centrifugation was added after the RW1 step to ensure complete removal from the column. When rinsing the column with 500 μL of RPE, the inside of the lid and the inner wall from the top of the column were rinsed during both washes. An additional centrifugation step was performed for 1 min at full speed (12,000×g) to completely dry the membrane before elution. RNAs from male adrenals were eluted in 20 μL of water and 30 μL for female adrenals. RNA quality and concentration were assessed using a nanodrop (expected 260/280 ratio >1.8 and 260/230 ratio 2-2.2). RNAs were then stored at −80°C until retrotranscription or library preparation for RNA seq. For RT-qPCR, 500 ng of RNA were treated with DNAse (Invitrogen, #18068-015) according to manufacturer instructions, and retrotranscribed into cDNAs at a final volume of 20 μL using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368814). cDNAs were diluted 10 times in water, and 2 μL were used for qPCR, which was performed in MicroAmp EnduraPlate Optical 96 well fast clear (Applied Biosystems, #4483485), using the Power SYBR Green PCR Master Mix (Applied Biosystems, # 4367659) in a final volume of 10 μL. Relative gene expression was determined using the ΔΔCt method using Actb (Actin beta) as a housekeeping gene and averaging duplicates for each biological replicates. Primer pairs are listed in Supplementary Data 2. Adrenal gene expression profiles for four 6-week-old Sf1-Crehigh; Hhexflox/flox and four WT littermates were analyzed by bulk RNA sequencing. RNA samples were similarly treated with DNAse, and quality was evaluated by Bioanalyzer 2100 (Eukaryote Total RNA Nano chip, Agilent) at the Advanced Genomic Core at the University of Michigan (RIN>=7.50 and free of genomic DNA contamination). Libraries were prepared from total RNA using standard poly(A) capture-based protocols and sequenced in paired-end mode on a NovaSeq (S4) 300. Sequencing quality metrics, including base quality scores and read count, were assessed using FastQC. Adapter sequences and low-quality bases were trimmed using the bbduk tool from BBTools. Processed paired-end reads were aligned to the mouse transcriptome sequence (GENCODE release M28, obtained from https://www.gencodegenes.org) using Kallisto136. Transcript-level abundance estimates were summarized to gene-level expression values using the lengthScaledTPM method in tximport137. Downstream analyses were performed in R using Bioconductor packages. To account for differences in library size, gene-level expression data were normalized using the Trimmed Mean of M-values (TMM) method implemented in edgeR138. Lowly expressed genes were filtered out using the filterByExpr function from edgeR to retain only genes with biologically meaningful expression levels. Unwanted and hidden sources of variation, such as batch effects, were removed using the sva package139. PCA was performed using the standard prcomp function in R, and biplots were constructed using ggplot2. Differential gene expression analysis was performed using limma140. Raw and processed data have been deposited in NCBI’s GEO database (GSE291472). Common differentially expressed genes between 6-week-old male Sf1-Crehigh Hhex KO compared to their respective littermate WT and 25-week-old male ARKO compared to their respective littermate WT are available in Supplementary Data 1.

Genome-wide profiling of DNA-binding proteins, cleavage under targets and tagmentation (CUT&Tag-IT™)
CUT&Tag-IT™ Assay Kit–Tissue (Active Motif, #53170) was used according to manufacturer instructions. Briefly, fresh adrenals from 6 to 9-week-old WT males were quickly harvested after euthanasia, cleaned from fat and transferred into ice-cold PBS 1× until being weighed. 10 mg of tissue (4 to 6 adrenals) was used for nuclei preparation, including the following conditions: no primary antibody, H3K27ac, and AR. For each condition, 20 µL of concanavalin A beads were prepared at room temperature according to manufacturer instructions. Briefly, 10 mg of tissue was lysed in 1 mL of Lysis buffer, and nuclei were extracted using a 1 mL dounce homogenizer. Lysate was filtered through 40 μm strainer, and nuclei were pelleted by centrifugation at 4°C. A range of 127,000–170,000 nuclei per mg of male mouse adrenals were obtained, and 280,000 nuclei per conditions were used. Nuclei were then bound to concanavalin A beads and incubated at 4°C overnight with primary antibodies Supplementary Data 2. The following day, guinea pig anti-rabbit secondary antibody was bound to primary antibodies, followed by an incubation with assembled pA-Tn5 transposomes. Tagmentation was then achieved by incubating samples at 37°C for 1 h. DNA was then solubilized, purified, and eluted. 30 µL of tagmented DNA was used to prepare libraries. Libraries were sequentially purified using SPRI Bead cleanup from the kit, and AMPure XP Reagent (Beckman Coulter, #A63880) according to the manufacturer’s instructions. The quality of the library was assessed on a Bioanalyser at the Advanced Genomic Core at the University of Michigan. Libraries were sequenced on an Illumina NextSeq 500 platform using the P1 100-cycle kit in paired-end mode. Raw sequencing reads were processed using the following bioinformatics pipeline. Read preprocessing: Paired-end reads were interleaved using seqtk merge (https://github.com/lh3/seqtk). Adapter sequences and low-quality bases were trimmed using fastp (https://github.com/OpenGene/fastp) with default parameters. Quality control reports, including read length distribution, base quality, and adapter content, were generated in HTML format. Alignment: Preprocessed reads were aligned to the mm10 mouse reference genome using Bowtie2 (version 2.3.5.1) with the following parameters: –local –very-sensitive –no-mixed –no-discordant -I 10 -X 700 to ensure high sensitivity for local alignment while preventing discordant and mixed alignments. Peak calling: Peaks were called using Genrich (https://github.com/jsh58/Genrich), with the -j option to enable ATAC-seq mode, which adjust for the insertion bias caused by the Tn5 transposase. To visualize genome-wide signal distributions, BigWig files were generated from the aligned BAM files using the bam_to_bigwig function from the GenomicAlignments package in R. Signal to noise was assessed by calculating the Fraction of Reads in Peaks (FRiP) and the TSS enrichment score. For H3K27ac, the FRiP score was 39% and the TSS enrichment score was 11.34231. For AR, the FriP score was 15% and the TSS enrichment score was 12.92894. Raw and processed data have been deposited in NCBI’s GEO database (GSE291344).

Software and applications
Graphs were generated using GraphPad Prism (10.2.2). Illustrations and Figures were created using PowerPoint (Version 2411 Build 16.0.18277.20082), the free life science icon library from Biorender.com, and R for the UMAP, the Inferno plots and the Volcano plot. The plugin Grammarly for Microsoft Word was used for grammar and spell-checking purposes at writing and editing stages of the manuscript. Literature references were added using the Zotero app (7.0.13).

Statistics, scientific rigor, and reproducibility
GraphPad Prism (10.2.2), R (version 4.3.1), and G*Power (Version 3.1.9.7) were used for statistical analysis. Details related to the statistical test, number of biological replicates, mean, standard deviation, t-values, degree of freedom, F-value, power, and p-value are available in Source Data and Supplementary Data 3. No data was excluded from the analyses. A p-value<0.05 was considered significant. Exact p-values of interest are reported on each graphs together with appropriate star symbols (*<0.05, **<0.01, ***<0.001, ****<0.0001). When comparing two independent groups, we assessed the normality of the data by performing a Shapiro–Wilk test. If passed, a two-tailed unpaired t-test was performed to compare the means with a Welch correction to address issues related to unequal variances (F value ≠ 1), and differences in sample size between groups. When data were not passing the normality test, a two-tailed Mann–Whitney test was applied to compare the ranks. Sample size was determined based on preliminary data and power calculations performed using G*Power to obtain >80% power with 5% type 1 error. When the p-value was <0.05, but 80% power could not be reached, an orthogonal approach was employed (detailed in Supplementary Data 3). For more than two-groups, we performed a two-way or three-way ANOVA, followed by a Šídák’s multiple comparison test. For histology, samples were blinded before analysis. When possible, orthogonal methods were used to ensure reproducibility of the results. For example, genes of interest identified by scRNAseq and bulk RNA seq were confirmed by RT-qPCR and/or immunohistochemistry on larger cohorts.

Nomenclature
Gene symbols were italicized, with only the first letter in upper-case for mouse species. Protein symbols were set in roman type, and all letters in upper case according to the following resources, https://www.genenames.org and https://www.informatics.jax.org.

Ethical approval declaration
All experiments were carried out in accordance with protocols approved by the Institutional Animal Care & Use Committee (IACUC) at the University of Michigan (protocol #00010217), the guidelines set by the National Institutes of Health (NIH), and the three R rule (Replacement, Reduction and Refinement). All efforts were made to minimize animals suffering and distress.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study uncovers a major role for HHEX in mediating zona fasciculata function, elegantly demonstrated through a well-designed series of experiments using appropriate mouse models. The authors take the reader on a clear and compelling discovery journey, from initial observations (single cell data) to functional validation (androgen mediated protection, via various mouse models), highlighting the critical importance of HHEX in adrenal cortical biology.\nI believe that, once finalized, this manuscript has the potential to be highly cited and represents a significant milestone in advancing our understanding of adrenal zona fasciculata function. I encourage the authors to address the comments below with thoughtful revisions, and where applicable, additional experimental validation or analysis to further strengthen the manuscript.\n1. Although the reader could infer this by analyzing the repositories associated with the manuscript, it would significantly strengthen the manuscript to explicitly show where SHH⁺ cells localize within the single-cell UMAP data. This addition would nicely complement panels 1D and 1E. It is anticipated that SHH⁺ cells would partially cluster in the CYP11B2-expressing region and exhibit low HHEX expression and visualizing this would provide additional context for interpreting the spatial and functional dynamics discussed.\n2. It is intriguing that the authors identify a small (13%) but significant population of cells co-expressing CYP11B1 and CYP11B2. To my knowledge, this co-expression is not typically observed in mouse adrenal tissue based on existing literature, where double staining for CYP11B1 and CYP11B2 generally reveals mutually exclusive expression patterns (even more so in rats ‘adrenals, where the two zone are “physically” separated by the so called undiffertiated zone). Happy to be proven wrong. It would be valuable for the authors to comment on this apparent discrepancy and, if possible, provide supporting evidence to confirm this finding—doing so would represent a novel and potentially important insight into adrenal zonation/plasticity.\n3. In assessing HHEX expression in HUMAN the adrenal gland, it would be helpful to clarify whether HHEX is detected in CYP11B2 and/or aldosterone-producing cell clusters (alternatively, DAB2+ cells) or in the more undifferentiated DLK1⁺ cell populations. This information would significantly strengthen the manuscript by extending the observed similarities between mouse and human adrenal biology. Demonstrating conservation of HHEX expression patterns across species would enhance the translational relevance of the findings and broaden their appeal to a wider readership.\n4. Why, in the authors ‘opinion, does residual HHEX expression following ablation in SF1⁺ cells differ between male (99%) and female (89%) adrenals? Do the authors attribute this sex-specific difference to varying levels of HHEX expression in local non-steroidogenic cells between male and female mice? Wouldn’t the authors be able to observe such expression in Cre negative, non-steroidogenic cells in immunohistochemistry experiments (right panel of Fig 2C)?\n5. Although the authors provide numerous histological images of HHEX KO adrenals, a brief description of observed differences in the zona fasciculata (ZF) area, overall adrenal zonation would be a welcome addition, and complement subsequent Oil Red O analysis.\n6. In HHEX KO mice, there is a significant reduction in serum corticosterone levels. Given that this measurement was performed using LC-MS/MS, it would be valuable to know whether their LC-MS/MS panel included other relevant steroid metabolites that could complement the corticosterone data and provide additional physiological insights. Are these metabolites all down or perhaps some accumulate?\n7. What is the effect of HCQ-S on corticosterone serum level in HHEX KO mice?", "reviewer_2": "In the present manuscript the authors performed single cell sequencing analysis on GFP sorted steroidogenic cells of the adrenal cortex and discovered a set of genes expressed in a zone-specific manner. Focusing on the transcription factor Hex, they show that this protein is specifically expressed in the zF and that loss of Hex results in reduced glucocorticoid production in males. This loss seems to be linked to a loss of lipid droplets due to increased lipophagy. Treatment with hydroxychloroquine, a drug that suppresses lipophagy, appears to partly restore lipid droplets in the inner zF. The authors next concentrated on the sexual dimorphism of Hex expression, which is higher in males and appears to be under control of androgen signalling. Female adrenals and glucocorticoid production on the other hand seem to be only mildly affected by the loss of Hex. RNA-Seq experiments indicate that male specific expression of HEX mediates a proportion of the sexual dimorphism of the adrenal cortex and demonstrate that this TF is required for the expression of Abcb1b.\nThe identification of a gene that contributes to adrenocortical sex bias is in principal of significant interest to the scientific community. However, I find the paper to be somewhat strangely organised, which makes the authors line of thoughts sometimes difficult to follow. Moreover, the text is very long with the introduction reading more like a review and the results section often containing unnecessary information that confuses rather than helps the reader. Shortening would make the manuscript more concise and the message crispier.\nPaper flow:\nThe authors first concentrate on male adrenals and describe the sexually dimorphic expression in females only in the second part of the manuscript. To me this is counterintuitive. Why not describe the sexually dimorphic expression first and then compare the effect of deletion in both sexes? Also, consider changing the title of the manuscript to include the role of Hhex in repressing the female program.\nSeveral omics data sets have been generated and analyzed in this study (bulk RNAseq, single-cell RNA seq and CUT&tag sequencing). While the authors provide an interactive single-cell dataset in a supplementary file, which is highly useful and appreciable, other analysis regarding the different dataset should be provided as supplementary tables. For instance :\n- Lines 99-100 « we focused our analysis on factors that could directly control gene expression, such as DNA-binding proteins » : could the authors provide the data of this analysis ? Such a list of zF-enriched TF would be useful for the scientific community.\n- I can not find the supplementary table 7 with the complete list of marker genes of the single-cell clusters (Suppl. Table 7 instead contains the statistics of each experiment).\n- Can the authors provide a supplementary file with all the DEG of their bulk transcriptomic analysis (with fold change instead of alphabetical order)?\n- AR- and H3K27ac-bound peaks and motif enrichment analysis : could the authors show/provide the data ?\n- Lines 167-168 « we performed a detailed analysis of the bulk RNAseq from male Hhex KO versus WT adrenals. This revealed activation of acidic lipolysis by ‘lipophagy’ ». Do they refer to an Ontology enrichment analysis ? Could the authors provide the data for this analysis ?\n- Scripts for the bioinformatic analysis performed should be made available.\nThe first part of the results section focuses on the « heterogeneity of the steroidogenic lineage », with scRNAseq clustering suggesting various subpopulation of the zG (zG1,2,3) and the zF (zG/zF, zF, inner zF), but in fact relatively little new information are provided regarding this aspect. The analysis presented here is a global zG versus zF comparison with identification of a new zF candidate. The authors do provide a list of the Top 100 marker genes in Suppl. Fig. 1a, but none seemed to be confirmed by expression pattern analysis ? Do they differ in term of localization, state of differentiation, … ? I understand this is perhaps not the scope of the present manuscript, but in this case I suggest the title of the first paragraph to be changed, or alternatively, to shorten the first paragraph and merge it with the next paragraph (« HHEX is a novel and highly conserved marker of glucocorticoid-producing cells »).\nLines 75-76 « 97% highly expressed GFP » : does this mean that 200 cells don’t express GFP ? Do these cells cluster together on the UMAP ? Can the authors show data of GFP expression (if relevant regarding a unique GFP- population)?\nAlso, I encourage the authors to provide data on the distribution of the replicates in their clustering (UMAP or table) to rule-out possible batch-effect, further supporting the heterogeneity they observe. What are the characteristics (cell types) of the « non-steroidogenic » and the two « cycling cell clusters » ?\nLines 103-104 : « confirmed that HHEX expression […] is limited to the zF in mouse (Figure 1H) and adult male rat adrenals (Figure 1J)» but the Figure 1J shows expression also in the innermost zone, which would correspond to the zR. Could the authors provide higher magnification and/or depict the additional zones that are present in rat cortex and/or provide double immunostaining as it has been done on mouse sample in figure S1F ?\nFigure 1J : Is the human adrenal sample used for HHEX immunostaining also male ? Could the author provide human expression data from both sexes ?\nLines 144-146 « This result made us consider the possibility that GC insufficiency was unlikely to be due to a defect in steroidogenesis, but was instead a consequence of a proximal upstream defect leading to compensatory upregulation of steroidogenic enzyme gene expression. » This statement is very strong given that the expression of only 2 enzymes has been measured. What about the expression of other steroidogenic enzymes ? Also, the following lines regarding cholesterol content would fit better in the next paragraph (« Lipid depletion observed in Hhex KO males results in part from activation of lipophagy in the inner zF »).\nFigure 3B-C : Could the authors please draw the representative zones of upper and inner zF on the IHC images that were taken for image quantification ? IHC in Fig3C suggests weaker staining of PLIN1 in lower zF compared to upper zF in WT, but the quantification in Fig3D indicates similar levels.\nHCQ-S treatment : in addition to PLIN1 IHC, can the authors provide Oil red O staining or quantification of intra-adrenal cholesterol esters? This is important as they state that they « observed a partial lipid rescue » (line 189). Same applies for the next subsection, Oil red O staining (or cholesterol measurement) should be used instead of PLIN1 IHC when statements are made about « dynamic of the LD » or « decrease in LD », etc.\nCan HCQ-S treatment (and therefore LD rescue) also restore GC production (in vivo or in vitro on adrenal explants + or – Bt2-cAMP) ?\nLines 275-276 « The sexual dimorphism was completely or at least partially abolished for all the genes tested in Hhex KO male adrenals », does that mean between KO males versus KO females ? or KO males versus WT females ? This statement should be supported by calculating p-values between these groups for the qPCR results. Also, in Figure 5B, why not comparing the transcriptomics data between the four genotypes and group the genes to provide an exhaustive list of genes whose sexual dimorphism is lost (or not) upon Hhex deletion ? As well as genes whose expression differs between KO and WT regardless of the genetic sex ?\nHHEX is a downstream mediator of AR action in the adrenal gland, and its loss leads to feminization of the transcriptome: can the authors rule-out the persistence of an X-zone in Hhex KO males (AKR1C18 IHC or transcriptomic data) ? This is important as the lipophagy-mediated lipid depletion in KO is affecting the inner zF.\nAre the female-biased genes (Nr0b1, Frzb, Cyp2f2) affected by HCQ-S treatment in Hhex KO males?\nIn the text, the description of Abcb1b expression pattern at baseline is redundant and sometimes figure panels that are referred to are mixed up, please proof read.\nLine 340-341 « At baseline the intensity of the Abcb1b staining was visibly reduced in both male and female Hhex KO adrenals ». Figure S6e (female) does not seem to support this and should be replaced by more representative images.\nDefects in ACTH-induced expression of Abcb1b : how does it translates in GC production in both sexes ?\nMinor points\n- Lines 131: The authors introduce the Cyp11b1-CreERT2 ko model ( Suppl. Figure 2b), but then refer to Suppl. Figure 6b for the actual analysis. This is confusing and figures should be labelled according to the citation within the text. Alternatively, I suggest removing these sentences as it is distracting from the message of the paragraph (impaired GC levels inf Sf1:Cre;Hhexflox/flox males).\nTt would be good to specify within the figures which knockout approach has been used (Nr5a1-Cre vs Cyp11b1-CreERT2)\n-The gene nomenclature needs to be double-checked. Example :\nLine 105 and Figure S1G, HHEX should be written in italic.\nLine 226 : « HHEX » mouse gene should be written « Hhex ».\n-Please correct the following typos :\nLine 104 : « Figure 1J » should be « 1I ».\nLegend of Figure S1F : remove « and SF1-Cre Hhex KO ».\n-Legend of the gene expression on the UMAPs : I was confused by the unit « density » at a first glance. To improve clarity, I suggest replacing « density » by « expression » on the color scale of the UMAP, and add « density plot expression » or equivalent in the text of the legend.\n-62 references are listed in the introduction, perhaps this list could be shortened by keeping the most relevant ones or by replacing with reviews if they exist (example : line 43 « necessary for the maturation and proliferation of definitive hematopoietic progenitors » justified by 6 references).\n-Line 114 : please replace « E9.5 » by E10.5 » (as shown in the original paper + other sources).\n-How many animal/adrenals per replicate were used for the single cell experiment?\n-In the legends of the figures, the abbreviations or the genotypes are not always mentionned. For instance :\nFigure 3A : « LAL », « p62 », etc ?\nFigure S6c-d : is it Sf1:Cre or Cyp11b1:CreER ?\nFig S4c : please indicate these are gonadectomized males.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The transcription factor HHEX maintains glucocorticoid levels and protects adrenals from androgen-induced lipid depletion\nThis manuscript presents a compelling and technically rigorous body of work establishing the homeobox transcription factor HHEX as a central regulator of zona fasciculata (zF) identity, steroidogenic capacity, and adrenal sexual dimorphism. Using a combination of single-cell RNA-sequencing, CUT&Tag, genetically engineered mouse models, and biochemical assays, the authors dissect both cell-specific and sexually dimorphic roles of HHEX in adrenal homeostasis. The integration of transcriptomic, physiological, and imaging data is particularly impressive, and the focus on androgen-driven lipophagy adds novel mechanistic depth to our understanding of lipid regulation in the adrenal cortex. The study’s major strengths lie in its biological insight, technical breadth, and well-supported mechanistic claims. By linking HHEX to both transcriptional regulation and protection against lipid droplet degradation, the authors present a clear and compelling model of how zF identity and glucocorticoid production are maintained. The figures are well-executed and supported by extensive supplementary data. That said, several points require clarification to enhance the clarity, quantitative interpretation, and broader relevance of the findings.\nPoints for Revision\n1. The manuscript would benefit from a more cohesive framing of how HHEX links lipid metabolism, sex hormones, and steroidogenesis. Currently, multiple themes (zF identity, sexual dimorphism, lipid storage, ABC transporters) are well-developed but not clearly integrated.\n2. The link between androgen signaling, lipophagy activation, and lipid droplet loss is compelling but could be more directly demonstrated. Consider additional evidence or discussion on whether autophagic flux (LC3-II turnover assays) is increased. Is lipophagy specifically increased, or is there general autophagy activation?\n3. Lines 243–248: The authors report 106,726 AR peaks genome-wide, but do not provide information on background levels or non-specific binding. Including a figure or discussion of signal-to-noise ratios and CUT&Tag peak enrichment would help establish confidence in peak calling. Additionally, while the CUT&Tag data are presented clearly, the specificity of AR binding to the HHEX promoter would benefit from further validation. It would strengthen the claim to assess whether this peak is conserved in other androgen-responsive tissues or to support the finding with approaches such as ChIP-qPCR.\n4. Lines 258–298: Claims that HHEX is downstream of AR and establishes male-specific transcriptional profiles are compelling. Since AR levels are not changed in KO adrenals (line 289), how exactly is this downstream transcriptional feminization established? Could HHEX act in parallel to AR rather than strictly downstream? Expand on whether this is a feedforward loop or necessary mediator, and temper definitive causality language unless supported by rescue.\n5. The manuscript depends heavily on quantitative imaging data (PLIN1, p62/SQSTM1, RNAscope). Quantified figures were not provided with the main document, so it is difficult to evaluate image quality or quantification rigor. Throughout, ensure all quantified imaging data include scale bars, number/age of animals, and whether analysis was blinded. Clarify these details in figure legends or the Methods.\n6. In Figure 6A, the male HHEX IHC panel appears to be duplicated from Figure 1H. If this is intentional, it should be clearly indicated in the figure legend. Additionally, the age of the sample is not provided in the figure or caption. If this image is indeed reused from Figure 1H, it should be stated that the tissue is from a 15-week-old animal. Including age information is important for interpreting potential developmental or context-specific differences in HHEX expression.\n7. Lines 330–355: The data show that HHEX regulates Abcb1b, but it is unclear whether this directly contributes to impaired steroid export or just serves as a marker of inner zF identity. Consider discussing this or including a validation experiment (Abcb1b knockdown).\n8. Line 351–353: The ACTH-induced GC output in females with Abcb1b loss is interesting. Consider a brief discussion on redundancy or compensation by other transporters.\n9. P-values and test types are summarized well in the supplement, but many figures likely require post hoc multiple testing correction. Clarify which tests used corrections. Add a note in Methods or legends for transparency.\n10. Line 62–73: Clarify if both sexes were included in scRNA-seq, or only males.\n11. Line 270: Only 23 DEGs in females is a striking difference. Consider adding power analysis or coverage statistics to rule out under-sampling."}, "author_response": "September 26th, 2025\nThis document constitutes the point-by-point response to reviewer’s comments for our\nmanuscript entitled \"The transcription factor HHEX maintains glucocorticoid levels and\nprotects adrenals from androgen-induced lipid depletion”.\nWe would like to thank the reviewers for their thoughtful and constructive feedback on our\nmanuscript. We have carefully considered each of the comments and suggestions and have\nrevised our manuscript accordingly. We appreciated the overall positive reception of our\nmanuscript and the reviewer’s engagement. Below, we provide a point-by-point response to the\nreviewer’s comments, accompanied by 24 new data panels added to the results section and 11\nadditional panels for the reviewers (noted #R.a to #R.k). We hope that the following revisions\nmade to our manuscript adequately address the requirements necessary for publication.\nFurthermore, we would like to express our gratitude to the reviewer who involved a young\nresearcher to co-review our manuscript as part of the Nature Communications initiative to facilitate\ntraining in peer-review.\nReviewer #1 (Remarks to the Author)\nThis study uncovers a major role for HHEX in mediating zona fasciculata function,\nelegantly demonstrated through a well-designed series of experiments using appropriate\nmouse models. The authors take the reader on a clear and compelling discovery journey,\nfrom initial observations (single cell data) to functional validation (androgen mediated\nprotection, via various mouse models), highlighting the critical importance of HHEX in\nadrenal cortical biology.\nI believe that, once finalized, this manuscript has the potential to be highly cited and\nrepresents a significant milestone in advancing our understanding of adrenal zona\nfasciculata function. I encourage the authors to address the comments below with\nthoughtful revisions, and where applicable, additional experimental validation or analysis\nto further strengthen the manuscript.\n1. Although the reader could infer this by analyzing the repositories associated with the\nmanuscript, it would significantly strengthen the manuscript to explicitly show where SHH cells\nlocalize within the single-cell UMAP data. This addition would nicely complement panels 1D and\n⁺\n1E. It is anticipated that SHH cells would partially cluster in the CYP11B2-expressing region and\nexhibit low HHEX expression and visualizing this would provide additional context for interpreting\n⁺\nthe spatial and functional dynamics discussed.\nR1.1: Localization of Shh+ cells in the Sf1 mTmG single cell data set. We have now added\na new Supplementary Figure showing the localization of Shh+ cells across the UMAP plot\n(Supplementary Figure 1d).\ncluster with Cyp11b2+ cells (dotted blue line) and\nmore surprisingly Cyp11b1+ cells (dotted red line).\nTo strengthen this result (and provide additional\ndata regarding the existence of\nCyp11b2+/Cyp11b1+ co-expressing cells, see\nR1.2), we performed RNAscope on serial sections\nfor Shh, Cyp11b2 and Cyp11b1 (in this order, each\nsection 5μm apart) on 10 biological replicates (5\nmales/5 females). While Shh and Cyp11b1 sections\nwere not technically serial sections, they were only\n10μm apart, so we included RNAscope images of\nthe same area for Cyp11b1 next to Shh to confirm the localization of the zF.\nWe updated the results section accordingly: Line 66 ‘We identified the proliferative population\nbased on the expression of mKi67 (Supplementary Figure 1c) and we found Shh-positive cells\npartially clustering with the zG and the zF cells (Supplementary Figure 1d). This finding was\nvalidated by RNAscope, where we found a portion of Shh transcripts in the upper zF cells,\nspecifically in male adrenals (Supplementary Figure 1e).’ While the biological significance of these\nfindings remains unclear, we are not exploring further this result in the current manuscript since it\nfocuses on HHEX and lipid biology. However, we updated the discussion as follows Line 358:\n‘In our data set, the expression domain of Shh included some Cyp11b2-positive cells. This is\nconsistent with results obtained using methods with enhanced sensitivity such as RNAscope\n(Hammer and Basham 2021) and observed in other species (Guasti et al. 2011). More surprisingly\nwas the existence of Shh transcripts in the zF cells, which we confirmed using RNAscope.\nmethods and reflective of differences in read out used (transcript versus reporter line). Age and\nsex differences could also contribute to discrepancies found across multiple studies. Indeed, we\nfound that Shh transcripts were easily visualized in the zF cells in male adrenals at 15 weeks of\nage and were rarely present in the zF of female adrenals. Taken together these observations\nrepresent a novel and potentially important insight into adrenal zonation and plasticity’.\n2. It is intriguing that the authors identify a small (13%) but significant population of cells co-\nexpressing CYP11B1 and CYP11B2. To my knowledge, this co-expression is not typically\nobserved in mouse adrenal tissue based on existing literature, where double staining for\nCYP11B1 and CYP11B2 generally reveals mutually exclusive expression patterns (even more so\nin rats ‘adrenals, where the two zone are “physically” separated by the so called undiffertiated\nzone). Happy to be proven wrong. It would be valuable for the authors to comment on this\napparent discrepancy and, if possible, provide supporting evidence to confirm this finding—doing\nso would represent a novel and potentially important insight into adrenal zonation/plasticity.\nR1.2: Co-expression of Cyp11b1 and Cyp11b2 in the mouse adrenal gland. We appreciate\nthe reviewer’s insight regarding the co-expression of CYP11B1 and CYP11B2, especially in\nregards of the rat adrenal zonation which slightly differs from the mouse adrenal zonation. At the\nprotein level some co-expression has been reported in the literature:(Freedman et al. 2013):\n‘…co-immunostaining for CYP11B1 and CYP11B2 led to the identification of occasional\ndouble positive cells present at the zG-zF boundary (Figure S2D, arrows), suggesting that\nan intermediate transition state may exist’.\nIn our hands, at the transcript level, we have observed on multiple occasions the presence of\nCyp11b1 transcripts in cells localized in the subcapsular region and some at the zG-zF boundary\nin mice using RNAscope. This was illustrated in Figure 2I (now 3I) and we are providing here\ndetailed images from that experiment. (n=6 WT male 15 week).\n#R.a. Cyp11b1 RNAscope details\nperformed RNAscope on serial sections as detailed in the previous comments (see R1.1). In both\nmales and females, some Cyp11b1 transcripts are found clustering with Cyp11b2+ cells in the\nsubcapsular region. We are now including pictures representative of 1 male and 1 female in a\nnew Figure 1F, and the additional 4 males and 4 females as a new Supplementary Figure 1e.\nBlue and red stars indicate blood vessels in the capsule used as a reference point to image the\nsame area on serial sections.\nWe have now added the following sentence to the results section, Line 65: ‘We confirmed the\npresence of cells co-expressing Cyp11b2 and Cyp11b1 transcripts in both sexes by performing\nRNAscope on serial sections (Figure 1F and Supplementary Figure 1b).’\n4\n3. In assessing HHEX expression in HUMAN the adrenal gland, it would be helpful to clarify\nwhether HHEX is detected in CYP11B2 and/or aldosterone-producing cell clusters (alternatively,\nDAB2+ cells) or in the more undifferentiated DLK1 cell populations. This information would\nsignificantly strengthen the manuscript by extending the observed similarities between mouse\n⁺\nand human adrenal biology. Demonstrating conservation of HHEX expression patterns across\nspecies would enhance the translational relevance of the findings and broaden their appeal to a\nwider readership.\nR1.3: HHEX expression in the human adrenal gland. Extending our results to human samples\nin more detail will be of critical importance. However, at this stage, we cannot provide staining on\nnormal human serial sections or co-staining for CYP11B2, DLK1 and HHEX. Given the significant\nrole of the androgens on HHEX expression in mice, we believe that exploring this further in human\nwarrants a separate, dedicated study that would include a representative number of male and\nfemale samples at various pubertal stages. We had initially mentioned this aspect in the\nparagraph dedicated to the limitations of our study, now Line 565: ‘We also demonstrated that\nHHEX is expressed in the normal human adrenal of both women and men by\nimmunohistochemistry, but the number of samples was limited and may not allow the accurate\nassessment of important differences in HHEX expression between sexes, ages, or hormonal\nstatus in humans.’\nNevertheless, to provide additional insights for the reviewer, we explored a single cell data set\nfrom a studies recently published by (Iwahashi et al. 2024), which includes over 35,000 cells from\nthree patients (a 27-year old woman, and 64- and 74-year-old men). In this data set, HHEX\nexpression was found to be the highest in endothelial cells and macrophages as expected based\non previous studies. The capsule expressed low levels of HHEX transcripts, consistent with our\nresults obtained by immunohistochemistry and some expression was found in the zF cluster.\nPossible expression in the zG is not excluded but in the absence of validation with an orthogonal\napproach such as costaining, we are not commenting on this aspect in the current manuscript.\n#R.b. Human adrenal single cell RNAseq - clusters\n5\n#R.c. Human adrenal single cell RNAseq – Marker expression\nTo avoid overstating this conclusion, we are restricting our comment to HHEX being expressed in\nthe human adrenal cortex (zF/zR) in 3 normal human samples (1 female / 2 males).\nWe updated Figure 2D to include 2 additional male samples. Additionally, to enhance the\ntranslational relevance of our results, we are also providing HHEX immunostaining on human\nadrenocortical cell line H295R. We updated the results section accordingly Line 92. ‘We also\nconfirmed that HHEX is expressed in the human adrenocortical cell line H295R by\nimmunofluorescence (Figure 2D), and the human adrenal cortex by immunohistochemistry\n(Figure 2E).’\n6\n4. Why, in the authors ‘opinion, does residual HHEX expression following ablation in SF1 cells\ndiffer between male (99%) and female (89%) adrenals? Do the authors attribute this sex-specific\n⁺\ndifference to varying levels of HHEX expression in local non-steroidogenic cells between male\nand female mice? Wouldn’t the authors be able to observe such expression in Cre negative, non-\nsteroidogenic cells in immunohistochemistry experiments (right panel of Fig 2C)?\nR1.4: Residual Hhex expression in the Hhex KO. We would like to thank the reviewer for\npointing out a potential misunderstanding regarding the residual Hhex expression in the adrenal\ngland of Sf1-Cre; Hhex KO male and female mice. Briefly, the adrenal is a composite organ,\ncomprised of steroidogenic cells, capsular cells, medulla cells, endothelial cells, and\nmacrophages. When performing qPCR on the adrenal gland, RNAs are extracted from all the cell\npopulations previously described, therefore when using Sf1-Cre, residual expression of Hhex\nmight represent expression in other cell types. Therefore, we would like to clarify that the residual\nHhex expression does not inherently differ between male Hhex KO and female Hhex KO. The\napparent discrepancy is due to the initial levels of Hhex expression in WT mice, which are\nsignificantly lower in females compared to males starting from puberty (results highlighted in\nFigure 5D - Timeline of Hhex expression). Thus causes the percentage reduction to appear less\npronounced in females (89%), not due to differences in the residual expression per se, but rather\nbecause of the variation in initial expression levels.\nWe acknowledge that our decision to present male and female data in separate ways (Figure 3B\nand Figure 7B respectively) may have contributed to this misunderstanding. This separation was\nmade to serve the narrative flow of our manuscript (in regards of the sexual dimorphism observed\nin the phenotype of Hhex KO mice,) but inadvertently made it challenging to compare directly\nHHEX expression in male HHEX KO and female Hhex KO.\nAs a solution, we are providing the mean Ct value for Hhex in Hhex KO which is 26.7 in both male\nand female Hhex KO. Given this data, we have no reason to think that HHEX expression is as\nsexually dimorphic in local, non-steroidogenic cells of the adrenal, as it is in the steroidogenic\ncells of the adrenals, at the time point assessed. We thank the reviewer for raising this point and\nappreciate the interest in understanding the nuances of our findings and hope this clarifies the\nissue. However, to accommodate the request from reviewer #3 point regarding “the results section\noften containing unnecessary information” and journal word limitations, we have decided to\nremove these details from the main result in the text.\n#R.d. Hhex expression\nHhex\n1.5\n1.0\n0.5\n0.0\nWT KO WT KO\n7\nslevel\nANRm\nlanerda\n)elamef\nTWsv\negnahc\ndloF(\n98%\n89%\nfemale 6 w male 6 w\n5. Although the authors provide numerous histological images of HHEX KO adrenals, a brief\ndescription of observed differences in the zona fasciculata (ZF) area, overall adrenal zonation\nwould be a welcome addition, and complement subsequent Oil Red O analysis.\nR1.5: Description of adrenal zonation and differences observed in the zF. As suggested by\nthe reviewer, we added a few sentences to briefly describe the histologic differences observed in\nmale Hhex KO. We are now providing pictures of Hematoxylin and Eosin staining and adrenal\nbody weight ratio at 6-week-old, nuclei density at 15-week-old and adrenal body weight ratio at\n19-week-old, demonstrating hypertrophy (new Supplementary Figure 3b,3c and 3c).\nLine 106: ‘At 6-week-old the overall zonation of the adrenal cortex was conserved in Sf1-Crehigh\nHhex KO, despite cells of the inner zF appearing eosinophilic as illustrated by hematoxylin and\neosin staining (Supplementary Figure 3b). Some cells also displayed hypertrophy, without\nchanges in the adrenal body weight ratio at this age (Supplementary Figure 3c). At 15-week-old,\nthe hypertrophy was evident and was confirmed by a 32% and 36% decrease in nuclear density\nin the zF and inner zF respectively (Supplementary Figure 3d). At 19-week-old, the adrenal body\nweight ratio was increased by 32% in Sf1-Crehigh Hhex KO (Supplementary Figure 3e).’\nTo assess nuclear density in the adrenal cortex, we divided the adrenal cortex into 3 equal tiers\n(black lines), counted the nuclei in each tier and divided by the area. Results are reported as\nnumber of nuclei per mm2. The larger the cells are the less nuclei are found per surface area.\nmethods accordingly.\n6. In HHEX KO mice, there is a significant reduction in serum corticosterone levels. Given that\nthis measurement was performed using LC-MS/MS, it would be valuable to know whether their\nLC-MS/MS panel included other relevant steroid metabolites that could complement the\ncorticosterone data and provide additional physiological insights. Are these metabolites all down\nor perhaps some accumulate?\nR1.6: Steroid precursor levels in Hhex KO: We have revised the manuscript to include data on\nadditional precursors measured using LC-MS/MS. Our results show a significant downregulation\nof progesterone and 11-DOC plasma levels. This information is now detailed within the results\nsection and the new supplemental figures S3j and S3k. This is consistent with a the decrease in\ncholesterol esters, the precursor of steroid hormones.\nLine 123: ‘This result made us consider the\npossibility that GC deficiency was likely a\nconsequence of an upstream defect leading to\ncompensatory upregulation of steroidogenic\nenzyme gene expression. In support of this\nhypothesis, corticosteroid precursors such as\nProgesterone and 11-Deoxycorticosterone\nplasma levels were also significantly decreased\n(63% and 82% respectively) at baseline in adult\nmales (Supplementary Figure 3j and 3k).’\n7. What is the effect of HCQ-S on corticosterone serum level in HHEX KO mice?\nR1.7 & R2.18: We believe this comment is similar to a comment made by reviewer 2: ‘Can HCQ-\nS treatment (and therefore LD rescue) also restore GC production (in vivo or in vitro on adrenal\nexplants + or – Bt2-cAMP) ?\nTo answer these two comments, we assessed GC production by explants. To provide as many\nbiological replicates as possible we gathered a total of 20 WT and Hhex KO male mice ranging\nfrom 27 to 38-week-old. 12 animals were placed on HCQ-S treatment, and 8 on vehicle for 9\nweeks. At dissection, 40 adrenals were retrieved and cultured in media with Vehicle or Bt2-cAMP.\nMedia was collected after 2.5h of incubation and corticosterone concentration was measured by\nELISA.\naddition to the media\nincreased GC production by\nexplants in all conditions\n(positive control of the\nexperiment). Similarly, as\nexpected, in Bt2 conditions\nwe observed a decreased of\nGC production from Hhex\nKO explants (right side, blue\nconditions). However, the\nHCQ-S treatment did not\nrestore GC production by Hhex KO explants (last two bars on the right). This is not surprising\ngiven the rescue of lipid droplets was not complete. We have now included this result in a new\nSupplementary Figure 4k and updated the result section:\nLine 180: In contrast, we observed a partial lipid rescue specifically in the inner part of the cortex\nof Sf1-Crehigh Hhex KO male mice (Figure 4H and Supplementary Figure 4j), which was quantified\nusing image processing analysis (Figure 4I). However, this partial rescue was not sufficient to\nrestore GC production by adrenal explants, when stimulated with Bt2-cAMP (Supplementary\nFigure 4k). Unfortunately, given the known variability of glucocorticoid levels in plasma, we were\nnot able to reach sufficient statistical power to comment with rigor on this aspect in the current\nmanuscript.\nIn the present manuscript the authors performed single cell sequencing analysis on GFP sorted\nsteroidogenic cells of the adrenal cortex and discovered a set of genes expressed in a zone-\nspecific manner. Focusing on the transcription factor Hex, they show that this protein is specifically\nexpressed in the zF and that loss of Hex results in reduced glucocorticoid production in males.\nThis loss seems to be linked to a loss of lipid droplets due to increased lipophagy. Treatment with\nhydroxychloroquine, a drug that suppresses lipophagy, appears to partly restore lipid droplets in\nthe inner zF. The authors next concentrated on the sexual dimorphism of Hex expression, which\nis higher in males and appears to be under control of androgen signalling. Female adrenals and\nglucocorticoid production on the other hand seem to be only mildly affected by the loss of Hex.\nRNA-Seq experiments indicate that male specific expression of HEX mediates a proportion of the\nsexual dimorphism of the adrenal cortex and demonstrate that this TF is required for the\nexpression of Abcb1b. The identification of a gene that contributes to adrenocortical sex bias is in\nprincipal of significant interest to the scientific community. However, I find the paper to be\nsomewhat strangely organised, which makes the authors line of thoughts sometimes difficult to\nfollow. Moreover, the text is very long with the introduction reading more like a review and the\nresults section often containing unnecessary information that confuses rather than helps the\nreader. Shortening would make the manuscript more concise and the message crispier.\nPaper flow:\nThe authors first concentrate on male adrenals and describe the sexually dimorphic expression\nin females only in the second part of the manuscript. To me this is counterintuitive. Why not\ndescribe the sexually dimorphic expression first and then compare the effect of deletion in both\nsexes?\nR2.1 Paper flow: While we considered both ways to present the manuscript - i.e lipid and GC\nphenotype first or sexual dimorphism first – (even wrote the first draft of the manuscript as\nsuggested by the reviewer), we finally elected to first describe the consequences of HHEX loss\non the fasciculata and glucocorticoid levels for 2 main reasons.\nDespite a long-lasting interest in aspects related to adrenal sexual dimorphism, we felt that first\ndefining the functional defect and lipid biology perhaps has more relevance for a broader scientific\naudience and for Nature Communications. Indeed, dysregulation of GCs is pertinent to many\nendocrine and metabolic disorders. Perhaps most importantly, this manuscript is submitted as\npart of an active call for collection called ‘lipid in cell biology’. Nevertheless, we hope that the\nchanges made throughout the manuscript to streamline the story will help address the reviewer’s\nsuggestion.\nAlso, consider changing the title of the manuscript to include the role of Hhex in repressing the\nfemale program.\nR2.2 Title: Nature Communications allows for a maximum of 15 words for the title and given the\nfocus on the paper on lipid biology, it is difficult to include additional HHEX roles in adrenocortical\ncells. Given the endocrine phenotype of HHEX KO mice (GC deficiency) and the identification of\nlipophagy as a participating mechanism to the maladaptive intracellular lipid droplets handling,\nwe remain with in the impression that the current title is a good fit to capture the novelty and\nimpact of our study.\ncell RNA seq and CUT&tag sequencing). While the authors provide an interactive single-cell\ndataset in a supplementary file, which is highly useful and appreciable, other analysis regarding\nthe different dataset should be provided as supplementary tables. For instance :\n- Lines 99-100 « we focused our analysis on factors that could directly control gene expression,\nsuch as DNA-binding proteins » : could the authors provide the data of this analysis ? Such a list\nof zF enriched TF would be useful for the scientific community.\nR2.3: Transcription factors expressed in the zF. We are now providing the zG-enriched and\nzF-enriched transcription factors lists as new tables (Table 1 and Table 2).\nTo do so, we aggregated the zG population and zF populations and obtained a list of differentially\nexpressed genes that were initially plotted in the Volcano Plot in Figure 1F. From this list we\nfiltered out the Differentially Expressed genes (DEG) with an adjusted p-value >0.05 and with a\nfold change between -1 and +1. We then use the following databases to identify transcription\nfactors.\nhttps://humantfs.ccbr.utoronto.ca/allTFs.php\nhttps://esbl.nhlbi.nih.gov/Databases/KSBP2/Targets/Lists/TranscriptionFactors/\n- I can not find the supplementary table 7 with the complete list of marker genes of the single-cell\nclusters (Suppl. Table 7 instead contains the statistics of each experiment).\nR2.4 Complete list of marker genes: We apologize for the confusion. For clarity, we have now\nremoved the Supplementary Figures that included tables in the manuscript and provided a\nsupplementary file (Supplementary Data 1) including the different lists of DEG mentioned in the\nmanuscript.\n- Can the authors provide a supplementary file with all the DEG of their bulk transcriptomic\nanalysis (with fold change instead of alphabetical order)?\nR2.5: Differentially expressed genes. We are now including a supplementary file with all the\nDEG of the bulk RNAseq presented in the manuscript (Supplementary Data 1).\n12\n- AR- and H3K27ac-bound peaks and motif enrichment analysis : could the authors show/provide\nthe data ?\nR2.6: Motif enrichment analysis was performed using HOMER, the result of the analysis can be\nfound in Supplementary Data 1). Please also see our response to reviewer 3.3 for additional\nmetrics related to the CUT&Tag experiments.\n- Lines 167-168 « we performed a detailed analysis of the bulk RNAseq from male Hhex KO\nversus WT adrenals. This revealed activation of acidic lipolysis by ‘lipophagy’ ». Do they refer to\nan Ontology enrichment analysis ? Could the authors provide the data for this analysis ?\nR2.7. Bulk RNAseq analysis. We acknowledge that the terms ‘detailed analysis’ might have\nbeen misleading here. We were not referring to Ontology enrichment analysis, but to a ‘manual’\ncuration of the differentially expressed genes that could be involved in lipid metabolism. We have\nupdated the text Line 148, which reads now as ‘To identify potential alternative mechanisms of\nlipid depletion, we explored the differentially expressed genes in the bulk RNAseq (see below)\nfrom male Sf1-Crehigh Hhex KO versus WT adrenals searching for factors involved in lipid\ncatabolism (Supplementary Data). The gene Lipa (p-value=2.46E-5, Log FC:1.56) particularly\ndrew our attention for its involvement in the process of acidic lipolysis.\n- Scripts for the bioinformatic analysis performed should be made available.\nR2.8 Script. No new codes have been developed by the bioinformatic team. The packages used\nare described in the material and methods. Therefore, per Nature Communications guidelines,\nwe will provide the script upon request. Raw and processed data have been deposited in NCBI’s\nGEO database (GSE291472). Lists of differentially expressed genes mentioned in the manuscript\nare all provided in Supplementary Data, and we made accessible the single cell data set by\ngenerating a CLOUPE file that can be easily imported into the 10X Genomics Loupe Browser for\ndata visualization. Line 824: The CLOUPE file related to the scRNAseq dataset presented in this\nmanuscript are accessible at the following address\nhttps://doi.org/10.6084/m9.figshare.28612406.\nThe first part of the results section focuses on the « heterogeneity of the steroidogenic lineage »,\nwith scRNAseq clustering suggesting various subpopulation of the zG (zG1,2,3) and the zF\n(zG/zF, zF, inner zF), but in fact relatively little new information are provided regarding this aspect.\nThe analysis presented here is a global zG versus zF comparison with identification of a new zF\ncandidate. The authors do provide a list of the Top 100 marker genes in Suppl. Fig. 1a, but none\nseemed to be confirmed by expression pattern analysis ? Do they differ in term of localization,\nstate of differentiation, … ? I understand this is perhaps not the scope of the present manuscript,\nin this case, I suggest the title of the first paragraph to be changed, or alternatively, to shorten\nthe first paragraph and merge it with the next paragraph (« HHEX is a novel and highly conserved\nmarker of glucocorticoid-producing cells »).\nR2.9 Expression pattern analysis. Since the initial submission, we have now expanded Figure\n1 into two figures and two tables. For the single cell RNAseq data set, we have added new\ninformation regarding the localization Cyp11b1 and Cyp11b2 transcripts (See reviewer R1.2) and\n13\nprovided the list of transcription factors that are enriched in the zG and zF as suggested by\nreviewer 2 (R2.3). We provide an expression pattern analysis coupled with a deep functional\ninvestigation for HHEX. Given our scientific interest in the transcriptional programs regulating\nadrenal function, additional target genes are being examined, but providing such exhaustive data\nwould be indeed beyond the scope of the present manuscript.\nLines 75-76 « 97% highly expressed GFP » : does this mean that 200 cells don’t express GFP ?\nDo these cells cluster together on the UMAP ? Can the authors show data of GFP expression (if\nrelevant regarding a unique GFP- population)?\nR2.10. GFP expression in the single cell data set. We apologize for an error on our part. When\nwe initially received the raw data from the 10X genomic pipeline (Cell Ranger), some cells\nappeared to be GFP negative. We did not filter out these cells on purpose, however, as described\nin the material and methods, we performed filtering steps. ‘Low-quality cells, defined as those\nexhibiting a high percentage of mitochondrial reads or a low number of detected features were\nidentified and removed using miQC147 (version 1.10.0)’\nAs a result, none of the ‘GFP-negative’ cells passed the quality control steps, suggesting that\nthese cells are unlikely to represent a biologically relevant cell population, rather poor-quality cells.\nAs seen in the loupe cell browser, 100% of the cells represented in the final data set express\neGFP. We have updated the result section accordingly\nLine 60: ‘All cells expressed GFP, demonstrating the successful enrichment of cells belonging to\nthe steroid lineage.’\n#R.e. GFP expression in the scRNAseq dataset\nAlso, I encourage the authors to provide data on the distribution of the replicates in their clustering\n(UMAP or table) to rule-out possible batch-effect, further supporting the heterogeneity they\nobserve.\nR2.11. Distribution of the replicates for the single cell RNAseq data set. We agree with the\nreviewer and have provided this information in the 10X cloupe file that was deposited on the\nfigshare repository at submission. In case the reviewers were not given access to this element by\nthe editor, we are now providing this information as a new supplementary figure 1a demonstrating\nthat both replicates contribute to each cluster identified.\nIn the loupe browser, the replicates can be viewed under the tab ‘orig.ident’.\nclusters » ?\nR2.12. Non-steroidogenic cell and cycling clusters: Given the expression of the top genes\nsuch as Ccr2 (monocytes), Trbc2 (T cells), Il1b (macrophages, monocytes), the ‘non-\nsteroidogenic cells’ likely represent immune cells. This is consistent with recent papers published\nby us and others demonstrating the presence of immune cells in the adrenal cortex (Dolfi et al.\n2022; Warde et al. 2023). In our hands, we observed the presence of CD68-positive cells\n(monocytes and macrophages) in the adrenal cortex in 15-week-old male, the time point and sex\nrepresentative of the single cell data set. We provide here for the reviewers the immunostaining\nfor CD68 for 4 biological replicates.\n#R.f. CD68 expression\nHowever, the apparent expression of the eGFP by the immune cells in the single cell data set is\npredicted to be an artifact due to membrane remodeling during immune cell phagocytosis of\nsteroidogenic cells and less likely indicative of an origin from the steroidogenic lineage. We have\nnot formally confirmed this hypothesis and are not commenting on this topic in the current\nmanuscript.\nRegarding the cycling cells, they are mKi67-positive, SF1-positive with some expression of Wnt\ntarget genes. Interestingly, we observed that Znrf3 is enriched in Cycling 1 cluster and Bub1 in\nCycling 2 cluster (See list of DE genes provided, now Supplementary Data 1, tab 1). Given that\nthese two genes are known as tumor suppressor/prognostic factors in adrenocortical carcinoma,\nadditional studies are necessary to determine the functional differences between these two\ncycling populations.\nand adult male rat adrenals (Figure 1J)» but the Figure 1J shows expression also in the innermost\nzone, which would correspond to the zR.\nCould the authors provide higher magnification and/or depict the additional zones that are present\nin rat cortex and/or provide double immunostaining as it has been done on mouse sample in figure\nS1F ?\nR2.13 HHEX expression in the rat adrenal. We thank the reviewer for highlighting the wrongful\nmislabeling of the rat inner adrenal cortex. We updated the result section accordingly. We were\nnot able to provide additional immunostaining to comment on HHEX expression in CYP11B2-\npositive zone, the zU or the zona intermedia found in rats, but we are now providing higher\nmagnification of the subcapsular region and the inner cortex that shows HHEX expression in the\nzF and zR.\nFigure 1J : Is the human adrenal sample used for HHEX immunostaining also male ? Could the\nauthor provide human expression data from both sexes ?\nR2.14. HHEX expression in human samples. The human adrenal sample was female; we\nupdated the legends accordingly. Unfortunately, we cannot rigorously comment on any differences\nof expression between the sexes given the limited number of samples. We have now added two\nmale samples. For additional explanation please refer to R1.3.\nLines 144-146 « This result made us consider the possibility that GC insufficiency was unlikely to\nbe due to a defect in steroidogenesis, but was instead a consequence of a proximal upstream\ndefect leading to compensatory upregulation of steroidogenic enzyme gene expression. » This\nstatement is very strong given that the expression of only 2 enzymes has been measured. What\nabout the expression of other steroidogenic enzymes ? Also, the following lines regarding\ncholesterol content would fit better in the next paragraph (« Lipid depletion observed in Hhex KO\nmales results in part from activation of lipophagy in the inner zF »).\nR2.15 Expression of steroidogenic enzymes. Our conclusion was based on more than two\nsteroidogenic enzymes presented, but we had chosen to only include the two proximal enzymes\nCyp11b1 and Cyp21a1. We are now providing the results for Mc2r, Hsd3b1, Nr5a1, and StAR as\nnew Supplementary Figures 3f-3i. We would like to keep the sentences related to cholesterol\n16\ncontent together with the steroid analysis in Figure 3, since the subsequent section (Figure 4) is\nfocused on the molecular mechanisms responsible for lipid droplet depletion during HHEX\ndeficiency.\nFigure 3B-C : Could the authors please draw the representative zones of upper and inner zF on\nthe IHC images that were taken for image quantification ? IHC in Fig3C suggests weaker staining\nof PLIN1 in lower zF compared to upper zF in WT, but the quantification in Fig3D indicates similar\nlevels.\nR2.16 Quantification of PLIN1 in inner and upper zF (Figure 3B-C, now 4B-C). We revised\nthe supplementary Figure related to the workflow applied to quantify PLIN1 staining using Fiji. We\nhave now drawn the representative zones used for quantification and revised the labeling in\nFigure 4D. The left graph now displays the ‘upper cortex’ rather than upper zF, based on the\nabsence of co-staining with zonal marker.\nWe are also providing additional pictures for the reviewer regarding the ‘staining of PLIN1 in lower\nzF compared to upper cortex in WT’. We do not observe any differences between the upper cortex\nand inner zF, both visually or when quantified with Fiji (n=3 WT). Please see also R3.5 for\nadditional comments regarding quantified images.\nquantification of intra-adrenal cholesterol esters? This is important as they state that they «\nobserved a partial lipid rescue » (line 189). Same applies for the next subsection, Oil red O\nstaining (or cholesterol measurement) should be used instead of PLIN1 IHC when statements are\nmade about « dynamic of the LD » or « decrease in LD », etc.\nR2.17 Partial lipid droplet rescue: We acknowledge that the use of Oil Red O is one of the\nstandard methods for visualizing neutral lipids in the adrenal cortex. We assessed Oil Red O and\nPLIN1 staining on right and left adrenals, respectively, on adrenal sections from Hhex KO treated\nwith HCQ-S (n=2). We are now providing these additional pictures as a new Supplementary\nFigure 4j.\nCan HCQ-S treatment (and therefore LD rescue) also restore GC production (in vivo or in vitro on\nadrenal explants + or – Bt2-cAMP) ?\nR2.18 HCQ treatment and GC production: Please see response to reviewer 1.7\nLines 275-276 « The sexual dimorphism was completely or at least partially abolished for all the\ngenes tested in Hhex KO male adrenals », does that mean between KO males versus KO females\n? or KO males versus WT females ? This statement should be supported by calculating p-values\nbetween these groups for the qPCR results.\nR2.19 qPCR sexual dimorphism Nr0b1, Frzb and Cyp2f2: We apologized for any confusion in\nthe original statement. To clarify, our objective was to highlight the loss or the reduction of the\nsexually dimorphic expression of Nr0b1, Frzb and Cyp2f2 in Hhex KO males, when compared to\nthe typical sexual dimorphism observed in WT animals.\nIn brief, in WT animals, female expressions of these genes are significantly higher than in male\n(negative delta: female>male). In contrast, in KO males, gene expression is higher than WT males\n(p-values originally shown) and even higher than both WT and Hhex KO females (for Nr0b1 and\nCyp2f2) resulting in a reversal or significant reduction of the sexual dimorphism (delta become\npositive or less negative). These differences are now indicated in the updated figures.\ngroup the genes to provide an exhaustive list of genes whose sexual dimorphism is lost (or not)\nupon Hhex deletion ? As well as genes whose expression differs between KO and WT regardless\nof the genetic sex ?\nR2.20. Transcriptomics of four genotypes. For clarity we removed the heatmap and all the DE\ngenes can be found in the Supplementary Data file.\nHHEX is a downstream mediator of AR action in the adrenal gland, and its loss leads to\nfeminization of the transcriptome: can the authors rule-out the persistence of an X-zone in Hhex\nKO males (AKR1C18 IHC or transcriptomic data) ? This is important as the lipophagy-mediated\nlipid depletion in KO is affecting the inner zF.\nR2.21 X-zone in Hhex KO. We had provided data in our initial submission regarding the lack of\na role of the X-zone in the adrenal phenotype of the Hhex KO, but we acknowledge that the data\ncould have been presented more clearly. To this end, we added new data to rule out the\npersistence of the X-zone and updated the results section:\nLine 166: ‘We ruled out the persistence of the X-zone to Hhex KO phenotype by deleting Hhex,\nin vivo, using the AS-Cre, which targets the definitive cortex and excludes the X-zone(Freedman\net al. 2013). Similarly to Sf1-Crehigh Hhex KO, the inner zF of AS-Cre Hhex KO was devoid of lipid\ndroplets at 15-week-old (Supplementary Figure 4e). We also performed RT-PCR for Pik3c2g, a\nrobust X-zone marker(Pihlajoki et al. 2013), and could not detect Pik3c2g cDNAs in males from\nboth genotypes (Supplementary Figure 4f), indicating that, as expected, the X-zone was absent\nfrom both WT and Sf1-Crehigh Hhex KO after puberty.\n2013). Its expression is absent in the adrenal of adult Hhex KO males. While we also measured\nAkr1c18 (mentioned by the reviewer), some studies suggest that it is not solely an X-zone marker\n(Sahut-Barnola et al. 2010; Dumontet et al. 2018; Dumontet and Martinez 2021). Furthermore,\nthe X-zone expresses low levels of Cyp11b1 transcripts (see supplementary figure 1e), which is\nnot the case of the inner zone of Hhex KO males, still strongly expressing Cyp11b1 (see Figure\n3I). Taken together, these results suggest that the X-zone regresses in Hhex KO males similar to\nWT.\nAre the female-biased genes (Nr0b1, Frzb, Cyp2f2) affected by HCQ-S treatment in Hhex KO\nmales?\nR2.22: Female biased genes under HCQ-S treatment. To address this question, we performed\nqPCR but did not observe significant changes for Nr0b1 and Frzb (see graphs below). To maintain\nthe clarity of the results presented in the current manuscript, we do not include these results in\nthe actual manuscript, but they are provided below for the reviewer.\n20\n#R.g-#R.i. Nr0b1, Frzb, Cyp2f2 qPCR\nIn the text, the description of Abcb1b expression pattern at baseline is redundant and sometimes\nfigure panels that are referred to are mixed up, please proof read.\nR2.23: We have proof read the figure panels and edited the text to avoid redundancy.\nLine 340-341 « At baseline the intensity of the Abcb1b staining was visibly reduced in both male\nand female Hhex KO adrenals ». Figure S6e (female) does not seem to support this and should\nbe replaced by more representative images.\nR2.24. RNAscope Abcb1b female. We apologize for the quality of the image provided in Figure\nS6e (now Figure S7f). We have reimaged the corresponding adrenal sections and areas at greater\nmagnification and updated the manuscript accordingly.\nDefects in ACTH-induced expression of Abcb1b : how does it translates in GC production in both\nsexes ?\nR2.25 GC production in ACTH-treated mice. We have not directly assessed the glucocorticoid\nlevels in plasma after ACTH because it would require a considerable number of biological samples\nto achieve appropriate power and statistical significance in both males and females. We reasoned\nthat at baseline, glucocorticoid levels are already impaired in males both when measured in\nplasma (Figure 3D) or in media after Bt2 stimulation of adrenal explants (Figure 3E). In females,\nas described in Figure S7g, there was no change in glucocorticoid production by explants after\nACTH treatment.\nMinor points\n- Lines 131: The authors introduce the Cyp11b1-CreERT2 ko model ( Suppl. Figure 2b), but then\nrefer to Suppl. Figure 6b for the actual analysis. This is confusing and figures should be labelled\naccording to the citation within the text. Alternatively, I suggest removing these sentences as it is\ndistracting from the message of the paragraph (impaired GC levels inf Sf1:Cre;Hhexflox/flox\nmales).\nR2.26. We removed these sentences as suggested by the reviewer.\nTt would be good to specify within the figures which knockout approach has been used (Nr5a1-\nCre vs Cyp11b1-CreERT2)\nR2.27. Specify KO approach. We proofread all the figures and legends and edited where\nnecessary.\n21\n-The gene nomenclature needs to be double-checked. Example :\nLine 105 and Figure S1G, HHEX should be written in italic.\nLine 226 : « HHEX » mouse gene should be written « Hhex ».\nR2.28. Gene nomenclature. We have updated the gene nomenclature where necessary.\n-Please correct the following typos :\nLine 104 : « Figure 1J » should be « 1I ».\nLegend of Figure S1F : remove « and SF1-Cre Hhex KO ».\nR2.29. Typos. We edited these typos.\n-Legend of the gene expression on the UMAPs : I was confused by the unit « density » at a first\nglance.\nTo improve clarity, I suggest replacing « density » by « expression » on the color scale of the\nUMAP, and add « density plot expression » or equivalent in the text of the legend.\n#2.30. As described in the material and methods ‘smoothed visualizations of gene expression in\nUMAP space was generated using Nebulosa (version 1.12.1), which employs kernel density\nestimates for enhanced visualization and interpretation (Alquicira-Hernandez and Powell 2021).’\nTo preserve the original copyright related to the ‘Nebulosa’ package, we would like to keep the\nunit as ‘density’, but we edited the legends of the figure as ‘visualization of Gene X expression’.\n-62 references are listed in the introduction, perhaps this list could be shortened by keeping the\nmost relevant ones or by replacing with reviews if they exist (example : line 43 « necessary for\nthe maturation and proliferation of definitive hematopoietic progenitors » justified by 6 references).\n#2.31. References. We edited the introduction and kept only the most relevant references. 35\nreferences are now listed in the introduction.\n-Line 114 : please replace « E9.5 » by E10.5 » (as shown in the original paper + other sources).\nR2.32. Edited.\n-How many animal/adrenals per replicate were used for the single cell experiment?\nR2.33. Single cell experiment details: We used 7 animals (14 adrenals, 31 mg of tissue) for the\nfirst replicate and 10 animals (20 adrenals, 49 mg of tissue) for the second replicate, hence the\nhigher number of cells sequenced in replicate #2. We updated the material and methods to include\nthis information Line 587.\n-In the legends of the figures, the abbreviations or the genotypes are not always mentionned.\nFor instance :\nFigure 3A : « LAL », « p62 », etc ?\nFigure S6c-d : is it Sf1:Cre or Cyp11b1:CreER ?\nR2.34. We revised the legends to detail abbreviations and genotypes as suggested.\nFig S4c : please indicate these are gonadectomized males.\nwhen Hhex deletion was driven by the AS-Cre. They are not gonadectomized males. This Cre\nwas originally described in Freedman et al 2013 and exclude the X-zone, therefore confirming the\nnon-contribution of the X-zone to the lipid depletion observed in the Hhex KO. Please see also\nresponse R2.21 for additional details.\nReviewer #3 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe are glad to hear our manuscript provided an opportunity for a young researcher to co-review\nand would like to thank you for your detailed report.\nThe transcription factor HHEX maintains glucocorticoid levels and protects adrenals from\nandrogen-induced lipid depletion.\nThis manuscript presents a compelling and technically rigorous body of work establishing the\nhomeobox transcription factor HHEX as a central regulator of zona fasciculata (zF) identity,\nsteroidogenic capacity, and adrenal sexual dimorphism. Using a combination of single-cell RNA-\nsequencing, CUT&Tag, genetically engineered mouse models, and biochemical assays, the\nauthors dissect both cell-specific and sexually dimorphic roles of HHEX in adrenal homeostasis.\nThe integration of transcriptomic, physiological, and imaging data is particularly impressive, and\nthe focus on androgen-driven lipophagy adds novel mechanistic depth to our understanding of\nlipid regulation in the adrenal cortex. The study’s major strengths lie in its biological insight,\ntechnical breadth, and well-supported mechanistic claims. By linking HHEX to both transcriptional\nregulation and protection against lipid droplet degradation, the authors present a clear and\ncompelling model of how zF identity and glucocorticoid production are maintained. The figures\nare well-executed and supported by extensive supplementary data. That said, several points\nrequire clarification to enhance the clarity, quantitative interpretation, and broader relevance of\nthe findings.\nPoints for Revision\n1. The manuscript would benefit from a more cohesive framing of how HHEX links lipid\nmetabolism, sex hormones, and steroidogenesis. Currently, multiple themes (zF identity, sexual\ndimorphism, lipid storage, ABC transporters) are well-developed but not clearly integrated.\nR4.1. Integration of lipid metabolism, sex hormones and steroidogenesis. We have edited\nthe introduction to improve the narrative and have also expanded the first paragraph of the\ndiscussion: Line 355. ‘Therefore, our findings position HHEX as a central integrator of multiple\nregulatory axes, that synchronizes lipid metabolism, sex hormone signaling and steroidogenic\ndifferentiation’.\n2. The link between androgen signaling, lipophagy activation, and lipid droplet loss is compelling\nbut could be more directly demonstrated. Consider additional evidence or discussion on whether\nautophagic flux (LC3-II turnover assays) is increased. Is lipophagy specifically increased, or is\nthere general autophagy activation?\nR4.2: Autophagic flux. Assessing autophagic flux in Hhex KO is important but presents\nsignificant technical challenges in vivo and in our experimental set-up. The most common method\nis to assess LC3-II turnover by immunoblotting. However, the lipid-depleted cell population (i.e.\nthe inner zF) only represents a portion of the cortex and the adrenal gland itself includes other\ncell types. When we performed immunohistochemistry for LC3 on WT animals using two different\nantibodies, the medulla was strongly positive (see attached), which suggested that performing\nimmunoblotting on adrenal protein extracts would not be representative of the molecular process\ninvolved in the inner zF.\n24\n#R.j. LC3 IHC antibodies test\nFor this reason, we chose an in-situ method to account for spatial visualization. However,\nimmunohistochemistry for LC3 would not distinguish between LC3-I and LC3-II, and our attempt\nto localize LC3 puncta has been inconsistent across antibodies. Therefore, we did not feel\ncomfortable presenting results using this method. We instead assessed p62/SQSTM1 which is\ncommonly used as an alternative to LC3 (Sánchez-Martín and Komatsu 2018; Zhang et al. 2016).\nRegarding whether lipophagy is specifically increased, the expression of multiple enzymes in the\ninner cortex such as CYP21 (Figure 3H) and SR-B1 (Figure S4a) did not suggest that general\nautophagy is activated. Additionally, the lipid depletion occurred in the inner cortex of Hhex KO\nwithout affecting the presence of mitochondria suggesting mitophagy is not activated. We are now\nincluding the immunostaining for mitochondria marker TOMM20 as a new Supplementary Figure\nand updated the result section accordingly. Line 164 “Despite the drastic reduction of lipid droplets\nin the inner zF cells, the mitochondria content was not affected, suggesting that other selective\nautophagy processes such as mitophagy were not activated (Supplementary Figure 4d).\n25\n3. Lines 243–248: The authors report 106,726 AR peaks genome-wide, but do not provide\ninformation on background levels or non-specific binding. Including a figure or discussion of\nsignal-to-noise ratios and CUT&Tag peak enrichment would help establish confidence in peak\ncalling. Additionally, while the CUT&Tag data are presented clearly, the specificity of AR binding\nto the HHEX promoter would benefit from further validation. It would strengthen the claim to\nassess whether this peak is conserved in other androgen-responsive tissues or to support the\nfinding with approaches such as ChIP-qPCR.\nR4.3. Specificity of CUT&Tag experiments. Please see also response R2.6.\nTo provide more information on background levels, we performed a FriP analysis (Fraction of\nReads in Peaks) which is a metric calculated as the percentage of reads that falls within identified\npeak regions. We obtained a FriP score of 39% for H3K27ac and 15% for AR. This information is\nnow provided in the material and methods. We also determined the TSS (Transcription Start Site)\nenrichment score, which compares the average read depth in a region centered on the TSS to\nthe average read depth in the flanking regions and therefore indicates how well the Cut and Tag\nassay captured the transcriptionally machinery at active promoters. The scores were calculated\nusing ATACseqQC v.1.28.0 (PMID 29490630). For H3K27ac, we obtained a score of 11.34231\nand 12.92894 for AR. We updated the method section accordingly and added a new\nSupplementary Figure 5d showing the TSS enrichment plots. We are also now including pie charts\nshowing peak distribution as a new Supplementary Figure 5e. Taken together, those metrics\nindicates specificity of our Cut&Tag results.\nWe appreciate the suggestion of performing ChIP-qPCR approaches. Given the extensive\nnumber of additional experiments recommended, we were not able to provide additional data in\nthe current manuscript. We hope that the in vivo epigenomic profiling supported by genetic,\npharmacologic, and transcriptomic approaches collectively support the hypothesis that HHEX is\na bona fide target gene of AR. However, we looked at previous literature providing ChIP seq data\nin prostate cell lines. Interestingly, HHEX was found to be bound by AR and containing ARE full\nsites. We now mention this information in the discussion, Line 454: ‘Moreover, studies performed\nin prostate cancer cell lines indicates that HHEX locus is also found to be bound by AR and\ncontaining androgen response elements (Wilson et al. 2016), raising the possibility that HHEX\nmight mediate the function of other androgen-sensitive cell type’.\n26\n4. Lines 258–298: Claims that HHEX is downstream of AR and establishes male-specific\ntranscriptional profiles are compelling. Since AR levels are not changed in KO adrenals (line 289),\nhow exactly is this downstream transcriptional feminization established? Could HHEX act in\nparallel to AR rather than strictly downstream? Expand on whether this is a feedforward loop or\nnecessary mediator, and temper definitive causality language unless supported by rescue.\nR4.4. AR-HHEX transcriptional profiles. We thank the reviewer for this insightful comment. The\ncausality was established based on the decreased expression of HHEX in ARKO and the stable\nexpression of AR in the Hhex KO. Therefore, we believe it rules out the possibility that HHEX is\nacting solely as a feedforward loop of AR action since Hhex deletion alone feminizes the\ntranscriptome. However, the opposite statement can’t be made, since ARKO implies some ‘Hhex-\nlike KO’ effects due to the dependance of HHEX expression on AR. Our results do not rule out\nthe possibility that HHEX might also act in parallel with AR and even independently of AR. Indeed,\nthe comparison of Hhex KO and ARKO transcriptomes support the existence of ‘AR-restricted’\nand ‘Hhex-restricted’ transcriptional program as shown in Figure 6I. We were in the impression\nthat the title of the result section ‘Adrenal sexual dimorphism is partially mediated by HHEX’ was\nreflecting this notion.\nThe exact molecular mechanism of the establishment of the female program in Hhex KO are not\nfully understood, but our results suggest that HHEX is actively and constantly repressing the\nfemale transcriptome in the zF which is common in both sexes before puberty. This particular\nnotion is supported by the extensive adrenal transcriptome analysis recently published\ndemonstrating that the ‘male and female transcriptome are initially very similar from P0 to P15’\n(Wang et al. 2025).\nWe tempered the conclusion of the result section as follows Line 286: Taken together, these\nresults suggest that HHEX is a mediator of AR action and that the female transcriptome in the zF\nis a common program in both sexes before puberty. During the rise of systemic androgen levels\nconcomitant with testis maturation, HHEX expression emerges throughout the zF, which\nparticipates to establishing the mature male adrenal transcriptome and consequently institutes an\nAR:HHEX-dependent sexual dimorphism in the adrenal cortex.\n5. The manuscript depends heavily on quantitative imaging data (PLIN1, p62/SQSTM1,\nRNAscope). Quantified figures were not provided with the main document, so it is difficult to\nevaluate image quality or quantification rigor. Throughout, ensure all quantified imaging data\ninclude scale bars, number/age of animals, and whether analysis was blinded. Clarify these\ndetails in figure legends or the Methods.\nR4.5. Imaging and quantification: We have proofread all the figures with images to ensure they\nwere including scale bar, number of animals and age. Indeed, histology techniques (IHC and\nRNAscope) were performed throughout the manuscript, but the conclusion does not depend\nheavily on quantification of these images, solely PLIN1 images were quantified in Figure 4D and\nFigure 4H. We provided RNAscope images for Cyp21, Cyp11b1, Abcb1b, Frzb and Nr0b1, and\nIHC for HHEX, but quantifications were based on qPCR, which has the advantage of providing\nan orthogonal and direct, unbiased quantitative approach. The information was provided in the\ncaption. For example, legend of Figure 3F ‘Quantification of Cyp21a1 transcripts by RT-qPCR’.\nfor quantification of PLIN1 in the updated Supplementary Figure 4c. See also Response to\nreviewer R2.16.\nThe analysis was blinded, as stated in the material and methods (line 815 in the initial submission)\nnow Line 819 “For histology, samples were blinded before analysis”.\n6. In Figure 6A, the male HHEX IHC panel appears to be duplicated from Figure 1H. If this is\nintentional, it should be clearly indicated in the figure legend. Additionally, the age of the sample\nis not provided in the figure or caption. If this image is indeed reused from Figure 1H, it should be\nstated that the tissue is from a 15-week-old animal. Including age information is important for\ninterpreting potential developmental or context-specific differences in HHEX expression.\nR4.6 Figure 6A and Figure 1H clarification: Indeed, Figure 1H was a representative picture of\nHHEX expression obtained when validating HHEX expression in both male and female and\ntherefore was used in Figure 6 relative to\ncomparing HHEX expression in adrenals of both\nsexes. We agree it should have been clearly\nindicated in the figure legend. To avoid duplication\nof the exact image, we chose a different\nrepresentative picture (from a separate experiment\nand biological replicate) to show HHEX expression\nand we kept the original picture in now Figure 7A.\nThis will allow the reviewer and the reader to\nappreciate the consistency of the staining across\nmultiple biological replicates.\nsimilar to other figures, and in the caption.\n7. Lines 330–355: The data show that HHEX regulates Abcb1b, but it is unclear whether this\ndirectly contributes to impaired steroid export or just serves as a marker of inner zF identity.\nConsider discussing this or including a validation experiment (Abcb1b knockdown).\nR4.7. Abcb1b role: Although a direct genetic tissue-specific approach to knock out Abcb1b in\nvivo would provide valuable insights regarding the potential roles of ABCB1B in steroid export\nversus its function as a marker of the inner zF, it is currently beyond the scope of our study due\nto the lack of available and validated floxed allele. Generating such a model would drastically\ndelay the revision of the current manuscript. Additionally, prior research has demonstrated that\ninhibition of ABCB1B activity using the pharmacological inhibitor Tariquidar decreases\nglucocorticoid secretion in both mouse and human adrenocortical cells (Lopez et al. 2021). These\nresults nicely complement an earlier study from 1993, demonstrating that ACTH-stimulated\nsteroid secretion is markedly decreased in the mdr1 mutant Y1 cell line (mdr1=mutation found in\nABCB1 gene) (Altuvia et al. 1993). Nevertheless, the observation that glucocorticoid output is\nmaintained in female Hhex KO, despite reduced Abcb1b expression raises interesting questions\nabout potential compensatory mechanisms. We have now added the following sentence Line 419:\n‘These findings raise the question as to whether HHEX variants account for physiological or\nsubclinical variation in the stress response. However, despite the reduced expression of Abcb1b\nobserved in the adrenals of female Sf1-Crehigh Hhex KO, we found similar corticosterone levels\nreleased, which could be indicative of redundant or compensatory mechanisms. Indeed, others\nATP transporter, such as ABCC1, have been shown to transport glucocorticoids.’\n8. Line 351–353: The ACTH-induced GC output in females with Abcb1b loss is interesting.\nConsider a brief discussion on redundancy or compensation by other transporters.\nR4.8.GC output: Please see response to response to previous comment R4.7\n9. P-values and test types are summarized well in the supplement, but many figures likely require\npost hoc multiple testing correction. Clarify which tests used corrections. Add a note in Methods\nor legends for transparency.\nR4.9. P-values. We agree with the reviewer that many figures present comparison between more\nthan two groups and therefore require post hoc multiple testing. The figures in question were listed\nin the supplementary Table 7, column B, related to Statistics provided at submission. The updated\nstatistic file is now found in Supplementary Data. We proofread the legends of the figures to\nensure that test and post hoc type were mentioned.\nTo be transparent regarding our statistics strategy, we have already provided information in the\nmaterial and methods section, now Line 818: ‘For more than two-groups, we performed an\nin column N (see snapshot below).\n10. Line 62–73: Clarify if both sexes were included in scRNA-seq, or only males.\nR4.10 Clarification of sex in scRNA-seq: In our original submission, we have specified that\nmale adrenals were used for single cell sequencing in the Materials and Methods (Line 586), but\nthis info was omitted in the main text. We have now clarified in the results section that male\nadrenals were used for single cell transcriptomics, Line 53. ‘We prepared a single-cell suspension\nfrom male adrenals by combining mechanical and enzymatic dissociation at low temperatures to\npreserve the viability of steroidogenic cells and their transcriptional state.’\n11. Line 270: Only 23 DEGs in females is a striking difference. Consider adding power analysis\nor coverage statistics to rule out under-sampling.\nR4.11 DEG in females. We performed bulk RNA sequencing on 4 males WT, 4 males Sf1-Crehigh\nHhex KO, 4 females WT and 4 females Sf1-Crehigh Hhex KO, and indeed found a striking\ndifference in the number of differentially expressed genes in each sex. This is consistent with the\nminimal expression of Hhex in females compared to males, and the attenuated phenotype in\nfemales Hhex KO. When we performed qPCR on larger cohorts, in each sex, we found a similar\npattern where most genes of interest were differentially expressed in males, but not in females.\nThis is well illustrated in Figure 6B and 6C for example.\nHowever, to rule out under-sequencing as an explanation for the higher number of\ndifferentially expressed genes in males versus females, we first compared library sizes using\nPicard’s CollectRnaseqMetrics. The mean number of mapped reads was 1.06×10^8 for males\nand 7.54×10^7 for females. Next, we performed an empirical rarefaction analysis on the raw count\nmatrix. For each sample, we subsampled 10%–100% of its total reads (in 10% increments) by\ndrawing from a multinomial distribution proportional to the original gene-count vector. Each depth\nwas resampled 10 times to mitigate stochastic variation, and we recorded the mean number of\ngenes with ≥1 read per replicate. We then plotted “genes detected” versus “reads sampled” curves\nfor every sample and overlaid the group means for males and females (see below). Both sex-\nspecific curves reached a clear plateau by ~30 million reads, indicating that the female\n30\nlibraries were not under-sequenced and that sequencing depth did not drive the observed\nsex bias in differential expression.\n#R.k. Saturations curves\nReferences included in the rebuttal\nAlquicira-Hernandez, Jose, and Joseph E. Powell. 2021. “Nebulosa Recovers Single-Cell Gene\nExpression Signals by Kernel Density Estimation.” Bioinformatics (Oxford, England) 37\n(16): 2485–87. https://doi.org/10.1093/bioinformatics/btab003.\nAltuvia, S, W D Stein, S Goldenberg, S E Kane, I Pastan, and M M Gottesman. 1993. “Targeted\nDisruption of the Mouse Mdr1b Gene Reveals That Steroid Hormones Enhance Mdr\nGene Expression.” Journal of Biological Chemistry 268 (36): 27127–32.\nhttps://doi.org/10.1016/S0021-9258(19)74226-4.\nDolfi, Bastien, Alexandre Gallerand, Maria M. Firulyova, et al. 2022. “Unravelling the Sex-\nSpecific Diversity and Functions of Adrenal Gland Macrophages.” Cell Reports 39 (11):\n110949. https://doi.org/10.1016/j.celrep.2022.110949.\nDumontet, Typhanie, and Antoine Martinez. 2021. “Adrenal Androgens, Adrenarche, and Zona\nReticularis: A Human Affair?” Molecular and Cellular Endocrinology 528 (May): 111239.\nhttps://doi.org/10.1016/j.mce.2021.111239.\nDumontet, Typhanie, Isabelle Sahut-Barnola, Amandine Septier, et al. 2018. “PKA Signaling\nDrives Reticularis Differentiation and Sexually Dimorphic Adrenal Cortex Renewal.” JCI\nInsight 3 (2). https://doi.org/10.1172/jci.insight.98394.\nFreedman, Bethany D., Petra Bukovac Kempna, Diana L. Carlone, et al. 2013. “Adrenocortical\nZonation Results from Lineage Conversion of Differentiated Zona Glomerulosa Cells.”\nDevelopmental Cell 26 (6): 666–73. https://doi.org/10.1016/j.devcel.2013.07.016.\nGuasti, Leonardo, Alex Paul, Ed Laufer, and Peter King. 2011. “Localization of Sonic Hedgehog\nSecreting and Receiving Cells in the Developing and Adult Rat Adrenal Cortex.”\nMolecular and Cellular Endocrinology 336 (1–2): 117–22.\nhttps://doi.org/10.1016/j.mce.2010.11.010.\nHammer, Gary D., and Kaitlin J. Basham. 2021. “Stem Cell Function and Plasticity in the Normal\nPhysiology of the Adrenal Cortex.” Molecular and Cellular Endocrinology 519 (January):\n111043. https://doi.org/10.1016/j.mce.2020.111043.\nIwahashi, Norifusa, Hironobu Umakoshi, Masamichi Fujita, et al. 2024. “Single-Cell and Spatial\nTranscriptomics Analysis of Human Adrenal Aging.” Molecular Metabolism 84 (June):\n101954. https://doi.org/10.1016/j.molmet.2024.101954.\nLopez, Juan Pablo, Elena Brivio, Alice Santambrogio, et al. 2021. “Single-Cell Molecular\nProfiling of All Three Components of the HPA Axis Reveals Adrenal ABCB1 as a\nhttps://doi.org/10.1126/sciadv.abe4497.\nPihlajoki, Marjut, Elisabeth Gretzinger, Rebecca Cochran, et al. 2013. “Conditional Mutagenesis\nof Gata6 in SF1-Positive Cells Causes Gonadal-Like Differentiation in the Adrenal\nCortex of Mice.” Endocrinology, ahead of print, March 7. Chevy Chase, MD.\nhttps://doi.org/10.1210/en.2012-1892.\nSahut-Barnola, I., C. De Joussineau, P. 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Filipp. 2016. “Refinement of the Androgen Response\nElement Based on ChIP-Seq in Androgen-Insensitive and Androgen-Responsive\nProstate Cancer Cell Lines.” Scientific Reports 6 (September): 32611.\nhttps://doi.org/10.1038/srep32611.\nZhang, Ziyan, Rajat Singh, and Michael Aschner. 2016. “Methods for the Detection of\nAutophagy in Mammalian Cells.” Current Protocols in Toxicology 69 (August): 20.12.1-\n20.12.26. https://doi.org/10.1002/cptx.11.\n32"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am pleased that the authors have thoroughly addressed all reviewer comments. They have provided additional analyses and, in most cases, new experimental evidence that substantially strengthen the main conclusion regarding the role of HHEX as a regulator of adrenocortical function. The revised manuscript is now significantly improved and presents a more compelling and comprehensive dataset supporting their findings.", "reviewer_2": "The authors have performed an impressive amount of additional experiments and addressed all of my major concerns. This has always been an important paper and I fully support publication in Nature communication", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have fully addressed my previous concerns, and the revisions significantly strengthen the manuscript. The data are clearly presented, the analyses are rigorous, and the conclusions are well supported. I have no remaining concerns and consider the study suitable for publication in Nature Communications."}, "author_response": ""}]

Decision letter