Dermal white adipose tissue development and metabolism: The role of transcription factor Foxn1

Intradermal adipocytes form dermal white adipose tissue (dWAT), a unique fat depot localized in the lower layer of the dermis. However, recognition of molecular factors regulating dWAT development, homeostasis, and bioactivity is limited. Using Foxn1−/− and Foxn1+/+ mice, we demonstrated that epidermally expressed Foxn1 regulates dWAT development and defines the adipogenic capacity of dermal fibroblasts. In intact and post‐wounded skin, Foxn1 contributes to the initial stimulation of dWAT adipogenesis and participates in the modulation of lipid metabolism processes. Furthermore, Foxn1 activity strengthens adipogenic processes through Bmp2 and Igf2 signaling and regulates lipid metabolism in differentiated dermal fibroblasts. The results reveal the contribution of Foxn1 to dWAT metabolism, thus identifying possible targets for modulation and regulation of dWAT in physiological and pathological processes in the skin.


| INTRODUCTION
Dermal white adipose tissue (dWAT) is a unique and least understood population of adipocytes located in the dermis (commonly named intradermal adipocytes).][3] Intradermal adipocytes forming dWAT participate in thermoregulation, 3 play an important role in the proper course of the hair cycle, 4,5 and stimulate immune response in skin infections. 6Importantly, dWAT was found to be key for the wound healing process.Studies by Shook et al. demonstrated that fatty acids released from adipocytes in the process of lipolysis contribute to the activation of the immune cells and promote skin repair. 7Loss of intradermal adipocytes observed in AZIP mice (mice lacking white adipose tissue) or upon inhibition of adipogenesis regulators (e.g., Pparγ) affects the number and function of fibroblasts in the wound area that abrogates wound repair and contributes to pathogenesis of skin fibrosis. 8,9WAT, similar to other fat depots, is regulated by obesogenic environment, but its size and volume undergo changes also during the hair cycle, 4,5 cold exposure, 10 and bacterial skin infections. 6Furthermore, age and diet can also regulate skin homeostasis by influencing dWAT morphology, lipid content, and migratory capacity of dermal fibroblasts (DFs). 11The high degree of intradermal adipocytes plasticity is closely related to lipid accumulation and mobilization directed by lipogenesis and lipolysis processes. 12everal findings suggest that the intradermal adipocytes arise from pre-existing cells in the reticular dermis predisposed to adipogenic differentiation independent of subcutaneous adipose tissue. 1,13In rodents, adipocyte precursor cells were identified in the lower layer of the dermis during late stage of embryonic development (days E16-18), which were characterized by the expression of the transcription factor C/EBPα, one of the first markers of adipogenesis, and Fabp4, a marker of mature adipocytes. 1,14Furthermore, Driskell et al. demonstrated that cells within the reticular layer of the dermis that express the protein Delta homolog 1 can differentiate into fibroblasts and adipocytes, whereas cells of the upper dermis (papillary layer) only contribute to the dermal fibroblast population. 13Although intradermal adipocytes are involved in numerous processes in the skin, the specific molecular factors that regulate their expansion and function in the skin warrant further investigation.Donati et al. showed that the activation of the Wnt/β-catenin pathway correlates with dWAT thickness and stimulates adipogenic differentiation in vivo.9][20] In the epidermis, Foxn1 is expressed in the suprabasal layer of keratinocytes where it regulates their differentiation and stimulates the proliferation of neighboring epithelial cells in a paracrine manner 19,21 and is involved in hair follicle development. 21During wound healing process, Foxn1 has been recognized as an important factor that partakes in the re-epithelialization and epithelial-mesenchymal transition processes stimulating reparative (scar-forming) wound healing. 22,23Interestingly, mice lacking the activity of Foxn1 (Foxn1 −/− mice) heal skin injuries in a scarless manner that resembles scar-free healing characteristic for mammalian fetal skin. 24A comparison of the transcriptomes of mouse fetuses skin at 14 days of fetal life (capable of scarless healing) and the skin of adult Foxn1 −/− mice revealed similarities in the expression of numerous genes, implying that Foxn1 is an important factor in the redirection of scarless (regenerative) onto scar-forming (reparative) pathway during the skin wound healing process. 25lthough Foxn1 expression in the skin is limited to the epidermal layer, it can also influence the phenotype and function of DFs. 26,27DFs from Foxn1 −/− mice were found to have higher percentage of cells with stem cell markers (CD117 and Oct3/4) and higher expression of wound healing-related genes (COLI, COLIII, and MMP-3/9/13) than those of Foxn1 +/+ mice. 26][27] Further evidence showed that Foxn1 affects adiposity and adipogenesis in the skin.Partial inactivation of Foxn1 (Foxn1 +/− mice) results in resistance to dietinduced obesity, whereas Foxn1 loss (Foxn1 −/− mice) prevented diet-induced obesity when exposed to mildcold stress. 28,29Furthermore, partial inactivation of Foxn1 (Foxn1 +/− mice) abrogates the expression of adipogenesis markers in intact and post-wounded skin. 29otably, lipidome changes were observed in Foxn1 −/− mice skin, suggesting that Foxn1 regulates skin lipid profile. 30erein, we examined the consequences of Foxn1 activity (Foxn1 +/+ vs. Foxn1 −/− ) on dWAT development and homeostasis.Specifically, we examined the transcriptional regulation of the adipogenic process and lipid metabolism in intact and post-wounded skin.To our knowledge, this is the first study to investigate the role of the epidermally expressed transcription factor Foxn1 in the regulation of the dWAT developmental and differentiation program, as well as the analysis of the profiles of genes related to lipid metabolism during skin wound healing.

| Mice
Animal experiments were performed in accordance with the European Union Directive of the European Parliament and the Council on the protection of animals used for scientific purposes (2010/63/EU).The experimental animal procedures were approved by the Ethics Committee of the University of Warmia and Mazury, Olsztyn, Poland (Approval No. 68/2018).
The studies were performed on Foxn1 −/− (CBy.Cg-Foxn1<nu>/cmdb; nude mice) that are homozygous for the nude spontaneous mutation and genetically matched controls Foxn1 +/+ (Balb/c/cmdb).Because dWAT shows remarkable dynamics during the hair cycle, 4,5 current research was conducted on adult 7-to 11-week-old mice to exclude any differences related to the hair stage cycle between Foxn1 −/− and Foxn1 +/+ animals.
The animals were bred and housed in rooms with controlled temperature (22 to 23°C) and humidity (50 ± 10%), regulated 12-h day/night cycle, and given constant access to water and food at the Center of Experimental Medicine, Medical University of Bialystok, Poland.

| Wound model
The day before the injury, the mice were anesthetized with isoflurane.After the dorsal hair of Foxn1 +/+ mice were removed with an electric razor and the shaved skin was cleaned with 70% ethanol, four full-thickness excisional wounds with a diameter of 4 mm were made on the back of the animals using biopsy punches (Miltex, Plainsboro, NJ, USA).Skin tissues obtained in the wounding process were collected as control tissue (intact; day 0).After wounds were performed, the animals were transferred to individual cages and observed until awakening.On days 3, 5, 7, and 14 after wounding, the animals were anesthetized with isoflurane and euthanized by decapitation.The post-injured tissues were harvested using 8 mm biopsy punches, frozen in liquid nitrogen, and stored at −80°C until further use.

| Isolation of dermal fibroblasts (DFs)
For DF isolation, skin samples were collected from the back of Foxn1 +/+ (Balb/c/cmdb) and Foxn1 −/− mice.Skin tissues were rinsed with a 70% ethanol solution and mechanically processed.The tissues were then digested with collagenase type I (3.68 mg/mL; Sigma-Aldrich Co., St. Louis, MO, USA) for 1.5 h at 37°C with agitation.The cell suspension obtained was filtered using a cell strainer (pore diameter, 100 μm) and centrifuged at 270× g for 5 min at room temperature.The supernatant was removed and the cell pellet was resuspended in DMEM/F-12 medium (Sigma-Aldrich Co.) with 15% FBS and gentamicin/amphotericin solution (Gibco, Thermo Fisher Scientific).Cells were then plated in 60or 100-mm Petri dishes.After reaching confluence, the primary cultured DFs were rinsed with PBS, trypsinized for 3-4 min at 37°C, counted, and frozen for further experiments.
After reaching 70% confluence in the culture inserts, keratinocytes were incubated for 4 h with Foxn1-GFP or GFP adenoviral vectors (multiplicity of infection: 200) with the addition of 0.5 mL of CnT basal medium (CELL-nTEC).After 4 h, 1 mL of CnT basal medium with supplements A, B, C (CELLnTEC) was added per insert.

| Coculture experiments
Transfected keratinocytes and DFs were seeded in the inserts and the lower well of 6-well plates, respectively.Every other day, the DF maintenance medium was replaced by an adipogenic medium until 7-9 days of differentiation.Every 2 days, the keratinocyte medium was replaced with fresh CnT basal medium with supplements.On days 2, 4, and 7 of the experiment, keratinocytes and fibroblasts were harvested and lysed in TRI Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) solution to isolate total RNA.

| Oil-Red O staining
Oil-Red O staining was performed as previously described. 11,27After 7 days of DF adipogenic stimulation in the monolayer or coculture model, differentiated DFs were fixed with 10% neutral-buffered formalin for 1 h and stained with a 60% solution of Oil-Red O.After 10 min of incubation, DFs were washed five times with water and visualized under a microscope (IX51, Olympus, Tokyo, Japan) equipped with an Olympus digital camera (XC50, Olympus).For the quantitative assessment of adipocyte differentiation in DFs, extraction of Oil-Red O from dyeretaining with isopropanol was performed.The accumulation of lipid concentration was measured based on absorbance at 500 nm.

| Histological and immunostaining analyses
Formalin-fixed tissues were embedded in paraffin and sectioned into 5 μm slices.The samples were deparaffinized by immersion in xylene, hydrated in an ethanol gradient, and rinsed with water and PBS.For immunohistochemical detection of perilipin 1 (PLIN1), adipose triglyceride lipase (ATGL), and glucose transporter 1 (GLUT1) (Table S1), slides were incubated in citrate buffer (pH 6.0) at 95°C for 25 min for heat-mediated antigen retrieval.After being cooled, the slides were washed with distilled water and PBS.Endogenous peroxidase inactivation was performed by incubating the slides in a solution of 3% hydrogen peroxide.Nonspecific antigenbinding sites were blocked with a 10% goat and horse serum mixture for 1 h at room temperature.Samples were incubated with primary antibody (Abcam) overnight at 4°C in a humid chamber.Subsequently, the slides were washed with PBS and incubated with biotinylated secondary antibodies and avidin-biotinylated horseradish peroxidase reagent included in a commercial detection kit (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA).To visualize the activity of horseradish peroxidase, the slides were incubated with 3,3′-diaminobenzidine tetrachloride.The slides were counterstained with hematoxylin.Images were acquired using an Olympus microscope (BX43; Olympus).
Based on the stain identification of the tissue localization of PLIN1, the percentage of adipocyte area in the skin, as well as the size and diameter of intradermal adipocytes, was measured.All measurements were made using Im-ageJ software (version v1.53k; https://imagej.nih.gov/ij/index.html).Quantification of adipocytes number, size, and diameter were measured in the representative adipocytes areas (n = 4-5 sections per mice per group) from images acquired at 40x magnification.Adipocyte size was measured as cell area (volume).Adipocyte diameter by ferret diameter was measured as a cell size along with a specific direction. 34,35 2.9 | Immunofluorescence staining Skin tissues were fixed in 4% paraformaldehyde, washed with PBS, and then stored in 18% sucrose with sodium azide at 4°C. Te immersed skin tissues were mounted in a medium (Tissue-Tek®), frozen in liquid nitrogen, and cryosectioned at 5-10 μm.After washing three times with PBS, slides were incubated in blocking solution (a mixture of 10% goat and horse serum in PBS solution) for 1 h and incubated with primary antibodies (Vimentin, BMP2, IGF2) (Table S1).After overnight incubation at 4°C, the slides were rinsed with PBS and then incubated with appropriate secondary antibodies (Alexa Fluor 594 or Alexa Fluor 680) for 1 h at room temperature and protected from light.The sections were additionally stained for lipid droplet detection using HCS LipidTOX Green neutral lipid staining (Life Technologies, Thermo Fisher Scientific).After incubation, the slides were washed several times in PBS and sealed using the ProLong Gold Antifade Mountant with DAPI (Invitrogen, Thermo Fisher Scientific).Stained sections were imaged with SLIDEVIEW VS200 Research Slide Scanner (Olympus).Neutral lipid accumulation was measured as the total fluorescence intensity of the lipid signal stained by HCS LipidTOX Green neutral lipid stain using ImageJ software (version v1.53k).

| Lipid analyses
The lipid measurements included analysis of total lipid content in the whole skin tissue (epidermal and dermal layers), enzymatically separated epidermis and dermis (trypsin separation), and iWAT (positive control).Skin samples were isolated by punch biopsies.Each sample had the same or similar weight.Lipids were extracted using the Folch method as previously described with minor modifications 36 that extract a broad range of lipid classes.Samples were weighed and homogenized with a chloroform: methanol (2:1) solution (4 mL of mixture per 0.05 g of tissue) and centrifuged at 15 000× g for 10 min.The supernatant obtained was transferred to new tubes, which were previously weighed.The samples were washed with 0.8 mL of distilled water, vortexed, and centrifuged at 2500× g for 15 min.The top layers were discarded, and the lower phases with lipid content were air-dried under nitrogen at 37°C for 35 min and weighed.The lipids's dry weight was calculated and deducted from the original weight of the tube.The percentage of lipids/total lipid content was calculated according to the following formula: (tube weight after drying [g] − weight of empty tube [g])/tissue weight [g]) × 100%.The triglycerides and cholesterol content were determined spectrophotometrically using commercially available reagents (Alpha Diagnostic Ltd., Warsaw, Poland).
The triglyceride concentration was also measured using commercially available kits (Abcam; ab65336).Whole skin, epidermis, dermis, and iWAT samples were washed with cold PBS and homogenized in liquid nitrogen with mortar and pestle in 5% NP-40/ddH2O solution.After three cycles of heating and cooling, the samples were centrifuged for 2 min at the highest speed and then diluted.The assay procedure was performed according to the manufacturer's instructions.

| Gene expression analysis
Total RNA was extracted from collected skin tissues and cell cultures using TRIzol reagent (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's recommendations.The concentration and quality of the RNA were determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and agarose gel electrophoresis, respectively.cDNA was synthesized from 500 ng of RNA using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific) according to the manufacturer's instructions.Single TaqMan Gene Expression probes (Thermo Fisher Scientific) were used to analyze the mRNA expression of all tested genes (Table S2).Realtime polymerase chain reaction (PCR) was performed according to the manufacturer's protocol.Hprt1 was used as a reference gene.The amplification reaction was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific) using the following conditions: initial denaturation for 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 60°C.Relative mRNA expression was determined using the standard curve-based method, normalized to the expression of Hprt1, and multiplied by 10.

| Western blotting
Uninjured skin tissues collected from Foxn1 +/+ and Foxn1 −/− mice were rinsed with PBS and incubated overnight at 4°C in dispase solution (6 U/mL).Afterward, the epidermis was mechanically separated from the dermis, and the samples were grounded in liquid nitrogen and homogenized in RIPA lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich Co.), phosphatase inhibitor cocktail (Sigma-Aldrich Co.), and phenylmethanesulfonyl fluoride (Sigma-Aldrich Co.).DFs isolated from Foxn1 +/+ and Foxn1 −/− mice were washed with PBS and then lysed in RIPA buffer with protease inhibitors.All samples, tissues, and the cell culture material were sonicated (three cycles of 5 s, 40% amplitude; Vibro-Cell VCX 130 PB sonicator) and then incubated for 45 min on ice.The lysates were centrifuged at 10 000× g for 15 min, and the supernatant was transferred to a new tube.Protein concentrations were measured using the Bradford method.The samples were adjusted to a concentration of 45 μg, combined with Laemmli reducing buffer supplemented with βmercaptoethanol, and boiled for 5 min at 95°C.The proteins were separated in 9% tricine gels and transferred to polyvinylidene fluoride membranes.The membranes were then blocked for 60 min at room temperature in Odyssey Blocking Buffer (LI-COR Biotechnology, Lincoln, NE, USA) diluted in PBS and then incubated overnight at 4°C with a primary antibody (Table S1).After washing, the membranes were incubated for 1 h at room temperature with a secondary antibody (Table S1).Each protein was visualized using the ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).
For FOXN1 analysis, powdered skin punches were homogenized in 500 μL of RIPA buffer supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich Co.).Cell lysates were normalized for protein concentration and separated on 12% sodium dodecyl sulfate-polyacrylamide gels.Proteins (40 μg) were transferred to polyvinylidene difluoride membranes (Millipore) and incubated with antibodies against FOXN1 (Table S1).Bands were visualized using the Odyssey imaging system (LI-COR Bioscience, Lincoln, NE, USA) with fluorescent (IRDye800TM or Cy5.5; Table S1)-labeled secondary antibodies according to the manufacturer's protocol.GAPDH or ACTB was used as internal controls.

| Flow cytometry analysis
Confluent DFs isolated from Foxn1 +/+ and Foxn1 −/− mice were washed with PBS, trypsinized for 3-4 min at 37°C, and counted.Approximately, 1-1.5 × 10 6 cells were suspended in PBS and incubated with the indicated fluorescence-labeled antibodies for 30 min (Table S3).Cells were rinsed with PBS, fixed with a 1% paraformaldehyde solution for 20 min, and stored at 4°C.The following day, stained cells were analyzed on a BD LSR Fortessa flow cytometer equipped with BD FACSDiva v6.2 software (Becton Dickinson, Franklin Lakes, NJ, USA).

| BrdU incorporation and flow cytometry analysis
Keratinocytes isolated from newborn Foxn1 +/+ mice were transduced with adenovirus carrying Foxn1-GFP or GFP, as mentioned before.Upon reaching 50% confluency, DFs from Foxn1 +/+ and Foxn1 −/− mice were cocultured with keratinocytes.After 16 h of coculture, the fibroblasts were incubated with BrdU at a final concentration of 10 μM in cell culture medium for 16 h.The DFs were then trypsinized, counted, and stained according to the manufacturer's protocol (BD Pharmingen BrdU Flow Kit; Becton Dickinson).The next day, stained cells were analyzed using a BD LSR Fortessa Cell Analyzer flow cytometer (Becton Dickinson) and BD FACS Diva v6.2 software (Becton Dickinson).Data are expressed as a percentage of BrdU-positive cells in the gated population.

| Lipogenesis and lipolysis rate
DFs isolated from Foxn1 +/+ and Foxn1 −/− mice were stimulated with adipogenic media.After 7 to 9 days of differentiation, DFs were subjected to glucose uptake or lipolysis analysis using commercially available kits (ab136955 and ab185433, Abcam) according to the manufacturer's instructions.For glucose uptake analysis, cells were washed with PBS and then serum and glucose were starved for 2 h.Subsequently, the DFs were rinsed with PBS and incubated for 40 min in Krebs-Ringer-Phosphate-Hepes buffer containing 2% bovine serum albumin.The cells were further stimulated with the medium in the presence or absence of 1 nM insulin and incubated with 2-deoxyglucose (1 mM) for 20 min at 37°C.After incubation, cells were washed with PBS and lysed.Absorbance was measured using a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific) in kinetic mode at 37°C according to the manufacturer's instructions.For measurements of lipolysis rate, differentiated DFs were washed with lipolysis wash buffer and stimulated with isoproterenol (100 nM) for 3 h at 37°C.The glycerol content in the conditioned medium was measured by colorimetric absorbance read at 570 nm and calculated from a standard curve, according to the manufacturer's protocol.

| In silico detection of transcription factor-binding sites
8][39] Identification of the Foxn1-binding site (GACGC) 40,41 in the promoter regions of each gene was performed using the FIMO tool from MEME Suite v.5.4.1 with a p-value threshold of 0.001. 42 2.17 | Quantification and statistical analysis Statistical analyses were performed with Prism 9 (Version 9.2.0; GrphPad Software, San Diego, CA, USA).All data were checked for normality using the Shapiro-Wilk test.Normally distributed datasets were further evaluated for outliers using the Grubbs test.The differences between groups were evaluated using twoway analysis of variance (ANOVA), one-way ANOVA followed by Tukey's multiple comparisons, and paired Student's t-tests.Data are presented as mean ± standard deviation (SD).p < .05 was considered statistically significant.All statistically significant results are presented in Supplementary Tables.

| Skin injury stimulates intradermal adipogenic processes
Intradermal adipocytes are necessary for efficient repair process at early skin wound healing stages. 7,8The findings suggested that mature adipocytes present at the wound edge rapidly (16 h post-injury) loose adipocyte-specific characteristics and require Atgl-mediated lipolysis to migrate into wound beds. 7Considering the regulatory role of Foxn1 in reparative (scar-forming) skin wound healing, 22,23 we hypothesized that Foxn1 regulates skin wound repair through modulation of adipogenesis and metabolic processes during healing.Therefore, we analyzed skin tissue samples collected from Foxn1 +/+ (Balb/c/cmdb; scar-forming healing) and genetically matched Foxn1 −/− (CBy.Cg-Foxn1; scarless/ regenerative healing) mice at day 0 (control, intact skin) and days 1, 3, 5, 7, and 14 after injury (Figure 1A).
To assess whether the alterations in the expression of adipogenic marker genes in the skin of Foxn1 +/+ versus Foxn1 −/− mice at early post-wounding days could be due to differences in macrophages content, 44,45 we performed qRT-PCR for macrosialin, a murine homolog of the human macrophage marker CD68. 46No differences were detected in mRNA expression of CD68 between Foxn1 +/+ and Foxn1 −/− mice at early stage of wound healing process (days 1-3, Figure S1F).Higher levels of CD68 mRNA were noted in the skin of Foxn1 +/+ mice at the day 5 after injury (Figure S1F).Therefore, observed differences in the profile of adipogenic and lipid metabolism-related genes between Foxn1 +/+ and Foxn1 −/− mice seem to be independent of the presence of macrophages in wounds at 24 h after injury.
Furthermore, at a later stage of wound healing (days 5-7), a gradual increase of Mest (the regulator of the expansion of white adipose tissue) was detected in the skin of Foxn1 +/+ mice (Figure 1Q and Table S4).These data indicate that the injury stimulates adipogenic skin response exclusively in mice with active Foxn1.Foxn1 −/− mice respond to injury with unaltered (e.g., Fabp4, Mest, Fasn, and Atgl) or slightly changed (e.g., Pparγ) levels of adipogenesis and lipid metabolism-related genes expression at initial stage of wound healing (Figure 1D-K and Table S4).mRNA levels of Zfp423 and Abhd5 demonstrated a gradual decrease in expression (from postwounded day 1 to day 14) exclusively in Foxn1 −/− mice (Figure 1D,R and Table S4).Further analysis revealed that Glut1, Srebp-1c, and Abhd6 expression increased significantly in Foxn1 −/− mice compared to Foxn1 +/+ during the late phase of the wound healing process (Figure 1N,S,T and Table S4).
These results indicate that injury-related changes in Foxn1 activity affect the expression of adipogenesis, lipogenesis, and lipolysis markers during the early phase of skin healing.In particular, after skin injury, the increase in Foxn1 activity may contribute to the initial stimulation of adipogenesis through modulating the metabolic properties of dWAT (lipogenesis and lipolysis).

| Epidermal signaling modulates the morphology and lipid storage of intradermal adipocytes
Different patterns of genes/proteins involved in adipogenesis and lipid metabolism between Foxn1 +/+ mice and Foxn1 −/− mice that correlate with Foxn1 surge during skin wound healing in Foxn1 +/+ mice prompted us to examine dWAT in the context of Foxn1 activity in intact skin.
First, we examined the morphological characteristics of dWAT in the skin of Foxn1 −/− and Foxn1 +/+ mice.The histomorphometry analysis based on immunohistochemical staining for perilipin 1 (a lipid droplet-associated protein) showed that the dermal skin layer of Foxn1 +/+ mice had more and larger adipocytes with a diameter greater than Foxn1 −/− mice (Figure 2A).
To better understand whether these differences in dWAT morphology were related to lipid deposition in adipocytes, we evaluated the neutral lipid content in the skin.Immunofluorescence staining with HCS LipidTOX dye revealed lipid accumulation in the lower layer of the dermis, which was particularly abundant in the skin of Foxn1 +/+ mice (Figure 2B).However, the measurement of intensity of neutral lipid staining and lipid content analysis did not reveal statistically significant differences between Foxn1 +/+ and Foxn1 −/− mice (Figure S1G; Figure 2C; Table S5).We also did not detect statistically significant differences in cholesterol and triglyceride content between Foxn1 +/+ and Foxn1 −/− mice regardless of the sample type (skin, epidermis, dermis, or inguinal white adipose tissue [iWAT]; Figure S1H,I) though cholesterol levels were slightly, but consistently, elevated in total skin, dermis, and particularly in the epidermis of Foxn1 −/− mice (Figure S1H).
The above-mentioned modulation in dWAT characteristics due to Foxn1 activity/non-activity supports our previous data established on Foxn1 +/− mice with partial inactivation of Foxn1. 29Moreover, the large differences in the expression of adipogenic genes related to lipolysis (Abhd5 and Abhd6), commitment to the adipo-lineage (Zfp423 and Zfp521), and the adipocyte differentiation process (Mest) between Foxn1 +/+ and Foxn1 −/− mice that were detected applying next-generation high-throughput DNA sequencing analysis 25 (Figure S2A) suggest Foxn1 as a relevant molecular regulator of adipogenesis, lipogenesis, and/or lipolysis.Here, using qRT-PCR analysis, we compared the mRNA expression levels of the de novo lipogenesis markers: Srebp-1c and Glut1 that were similar between Foxn1 +/+ and Foxn1 −/− mice or slightly increased (Glut4 and Fasn) in the skin of Foxn1 −/− mice (Figure 2D).The expressions of lipolysis markers: Abhd5 and Abhd6 were upregulated in the skin of Foxn1 −/− as compared with Foxn1 +/+ mice (Figure 2E).Western blot analysis revealed higher ABHD5 protein abundance in intact skin of Foxn1 −/− mice in comparison with Foxn1 +/+ mice (Figure 2G).Genes related to the adipogenic commitment process: Zfp423, Pparγ, Fabp4, and Mest expressions showed no significant differences (Figure 2F).However, PPARγ, the master adipogenic regulator, displayed a tendency toward consistent upregulation of protein levels, particularly PPARγ2 isoform, in the skin of Foxn1 +/+ mice (Figure 2H).Moreover, in silico analysis of the promoter region of the mouse adipogenesis-related genes demonstrated that it comprehends putative Foxn1binding sites for Pparγ (four), Mest (one), Abhd5 (five), and Abhd6 (two) (Figure S2B).
Altogether, these results showed that Foxn1 modulates the morphology of dWAT and indicates the possible role of Foxn1 in regulating dWAT metabolism processes particularly lipolysis in intact skin.

| Foxn1 activity determines pro-adipogenic characteristics of DFs
Adipocytes comprised in the dWAT are developmentally different from those in subcutaneous fat. 1 Accordingly, several studies suggest that intradermal adipocytes and DFs are derived from common precursor cells, 1, 13 indicating that DFs may contribute to the pool of intradermal adipocytes.Our recent evidence suggests that Foxn1 expressed in epidermis influences cells phenotype beyond its place of expression (i.e., dermal fibroblasts).Given the role of Foxn1 in modulating DF properties/characteristics, 26,27 we hypothesized that Foxn1 activity impacts DF developmental process.Therefore, to establish the role of Foxn1 in DFs toward dWAT, modulation/differentiation comparison of DFs isolated from Foxn1 −/− and Foxn1 +/+ mice was performed.
To further investigate the impact of Foxn1 on the DFs adipogenic capacity, the expression of adipogenesisrelated genes was analyzed in cultured (passage 0) DFs isolated from the skin of Foxn1 +/+ or Foxn1 −/− mice.DFs isolated from the skin of Foxn1 +/+ mice revealed higher levels of transcripts characteristic for the initial stages of adipogenic differentiation (Zfp423 and Pparγ) (Figure 3D).Furthermore, the analysis of protein levels showed tendency toward elevated levels of PPARγ2 in Foxn1 +/+ DFs (Figure 3D).
The observed discrepancies between Foxn1 +/+ and Foxn1 −/− DFs in the expression of adipogenic-related markers prompted us to examine the markers of lipogenesis and lipolysis processes.Lipid metabolism-related genes revealed higher mRNA expression levels and higher protein abundance of basal glucose transporter -GLUT1 in DFs isolated from Foxn1 −/− in comparison to DFs originated from Foxn1 +/+ mice (Figure 3E).These data point to potential increase in insulin-independent glucose transport in cells derived from mice that lack Foxn1 activity that is similar to the cells during fetal/ neonatal period. 50To further explore this notion, we performed a glucose uptake and glycerol release assays in DFs isolated from Foxn1 +/+ versus Foxn1 −/− mice that were stimulated with adipogenic medium (Figure 3F).Differentiated DFs (days 7-9 of adipogenic differentiation) originated from Foxn1 −/− mice were characterized by a significantly higher intensity of basal lipogenesis and insulin-stimulated lipogenesis than DFs isolated from Foxn1 +/+ mice (Figure 3G).These results correspond with elevated levels of Glut1 in Foxn1 −/− DFs (compare Figure 3E with Figure 3G).The levels of glycerol release under both control and isoproterenol stimulated conditions did not differ between differentiated DFs originating from Foxn1 +/+ or Foxn1 −/− mice (Figure 3H).
These results imply the direct association between Foxn1 activity and skin adipogenesis.DFs origin (Foxn1 +/+ vs. Foxn1 −/− mice) defines adipogenic characteristics pointing to the crucial role of Foxn1 in the DFs adipogenic capacity and metabolic properties.

| DFs originated from Foxn1 +/+ mice display increased potential for adipogenic differentiation
It has been shown that factors present in the epidermis affect the differentiation of DFs into adipocytes. 15,51o comprehensively explore the involvement of Foxn1 (expressed in keratinocytes) on DFs adipogenic differentiation potential, we performed in vitro coculture experiments.Keratinocytes isolated from Foxn1 +/+ mice were engineered to overexpress Foxn1 (Ad-Foxn1 transduced), then set up, and cocultured with DFs isolated from the skin of Foxn1 +/+ or Foxn1 −/− mice (day 0) (Figure 4A).DFs isolated from Foxn1 +/+ or Foxn1 −/− mice were stimulated with an adipogenic or control medium for 9 days.
Initially, we validated Foxn1 expression in keratinocytes and DFs (Figure S4A).The highest Foxn1 levels in Ad-Foxn1 transduced keratinocytes were detected on day 2 post-transduction that remained relatively stable at days 4-7 of differentiation (Figure S4A).Keratinocytes transduced with Ad-GFP (control) displayed low and stable levels of Foxn1 expression, regardless of days of culture.No Foxn1 mRNA expression was detected in DFs isolated from the skin of Foxn1 +/+ or Foxn1 −/− mice (day 0) or during adipogenic differentiation (Figure S4A).The BrdU incorporation assays showed no differences in DFs proliferation rate regardless of coculture with keratinocytes transduced with Ad-Foxn1 or Ad-GFP (Figure S4B).
DFs cocultured with keratinocytes and stimulated by adipogenic media gradually (days 0-9) changed their shape from long, fibroblastic cells toward filled by lipid droplets round in shape adipocytes (Figure 4B).However, the adipogenic potential of DFs depended on the DF origin (Foxn1 +/+ vs. Foxn1 −/− mice).DFs from the skin of Foxn1 +/+ mice regardless of coculture with Ad-Foxn1 or Ad-GFP transduced keratinocytes showed robust adipogenic differentiation, marked by a higher number of lipid-filled adipocyte-like cells and an increased accumulation of lipid droplets as demonstrated by Oil-Red O quantification in comparison to DFs isolated from Foxn1 −/− mice (Figure 4B).It demonstrates intrinsic, exclusively dependent on origin (Foxn1 +/+ mice) DFs adipogenic potential and adipocyte traits characteristics.Interestingly, exclusively Foxn1 +/+ DFs that were cocultured with Ad-Foxn1 transduced keratinocytes displayed spontaneous (without adipogenic media stimulation) ability to accumulate lipids after 9 days of culture in control media.It indicates not only intrinsic differences between Foxn1 +/+ and Foxn1 −/− DFs origin but also sensitivity of Foxn1 +/+ DFs to Foxn1 stimulation (Ad-Foxn1 in cocultured keratinocytes) (Figure 4C).
Analysis of adipogenic-related genes revealed that the most evident differences in adipogenic DFs potential depended on Foxn1 origin (Foxn1 +/+ vs. Foxn1 −/− mice) (Figure 5A-J; Tables S7 and S8).Upon adipogenic stimulation (at day 2), the expression of early (Zfp423 and Pparγ) and late (Fabp4 and Lep) adipogenesis markers was significantly higher in Foxn1 +/+ DFs than in Foxn1 −/− mice, regardless of coculture with Ad-Foxn1 or Ad-GFP transduced keratinocytes (Figure 5A-C; Figure S4C and Table S7).Conversely, Foxn1 −/− DFs were characterized by higher levels of Zfp521 (a negative regulator of adipogenesis) (Figure 5D, Tables S7 and S8) and Mest (a regulator of white adipose tissue expansion) during the initial days of differentiation (day 2) (Figure S4D, Tables S7 and S8).These findings suggest that Foxn1 developmental activity shapes DFs intrinsic properties stimulating enhanced adipocyte differentiation propensities and as a result affects dWAT development.
Altogether, these data demonstrate Foxn1 as a transcriptional regulator of dWAT.Foxn1 developmental activity (Foxn1 +/+ vs. Foxn1 −/− DFs) shapes the basal adipogenic and metabolic properties of differentiated DFs and the presence of Foxn1 in keratinocytes (overexpression) directly regulates/modulates lipogenesis and lipolysis processes in differentiated DFs.

| Foxn1 regulates pro-adipogenic signals of Bmp2 and Igf2
As proposed by Donati et al., epidermal Wnt/β-catenin signaling contributes to skin adipogenesis by triggering the BMP and insulin pathways. 15Our previous data suggest that Foxn1 as epidermally expressed factor stimulates the adipogenic signals of BMP2 and IGF2. 11,29o further demonstrate pro-adipogenic role of Foxn1 in the skin, herein we analyzed Igf2 (Figure 6A-C) and Bmp2 (Figure 6E-G) localization and expression in intact and post-wounded skin of Foxn1 +/+ and Foxn1 −/− mice.IGF2 localization was limited to the epidermis of both: Foxn1 +/+ and Foxn1 −/− mice (Figure 6A).Western blot analysis showed the higher levels of IGF2 protein in the epidermis of mice with active (Foxn1 +/+ ) than non-active Foxn1 (Foxn1 −/− ) mice (Figure 6B).In post-wounded skin, the pattern of Igf2 mRNA levels of expression was similar between Foxn1 +/+ and Foxn1 −/− mice with a surge on day 5, followed by a gradual decrease until day 14 (Figure 6C and Table S9).Furthermore, we examined Igf2 expression in Ad-Foxn1-transduced keratinocytes (in vitro conditions; Figure 6D).Foxn1 surge dramatically increased Igf2 expression particularly at day 2 (p < .0001)and day 4 (p < .001)after transduction.Ad-GFP-transduced keratinocytes showed low and unchanged levels of Igf2 mRNA (Figure 6D).The increase in Igf2 expression in keratinocytes correlates with the levels of Foxn1 expression in Ad-Foxn1-transduced keratinocytes (Figures 6D and Figure S4A).
The comparison of skin transcriptomic profile between Foxn1 −/− and Foxn1 +/+ mice 25 revealed that Foxn1 activity was associated with large differences in the expression of BMP genes (Bmp2 and Bmp4) and components of the IGF family (Igf2), including IGF-binding proteins (Igfbps) and surface receptors (Igf2r) (Figure S4G).Accordingly, performed in silico analysis of the promoter region of mouse Bmp2 and Igf2 revealed four complete putative FOXN1-binding sites for each of the pro-adipogenic pathway genes (Figure 6I).
Overall, these findings indicate that Foxn1 regulates Igf2 expression in the epidermis and is involved in the adipogenic Bmp2 signals in the dermis.Considering the documented contribution of BMP ligands in the stimulation of adipogenic differentiation 15,55,56 and in cell differentiation during hair development, 54,[57][58][59][60] these findings may suggest that skin adipogenesis and hair shaft differentiation depend on Foxn1 activity.

| DISCUSSION
Despite the ever-expanding knowledge on dWAT development and function, the underlying molecular events involved in the regulation of intradermal adipocytes remain unexplored.In this study, we investigated the molecular regulation of dWAT in intact and post-wounded skin by transcription factor Foxn1.Experiments in which we used mice models with Foxn1 activity (Foxn1 +/+ mice) and Foxn1 inactivity (Foxn1 −/− mice) strongly confirmed the contribution of Foxn1 in dWAT adipogenesis that initially was observed for mice with partial Foxn1 inactivity (Foxn1 +/− mice). 29The most significant findings that emerge from this study are that Foxn1 activity shapes DFs adipogenic capacity and metabolic properties that contribute to dWAT developmental process.Furthermore, observed differences between Foxn1 +/+ and Foxn1 −/− DFs further support the postulated role of Foxn1 in the skin development program, 25 which includes dWAT development as the current data show.We also demonstrated that Foxn1 activity in the skin is associated with an increase in the expression of genes involved not only in adipogenic commitment but also in genes related to lipid metabolism, particularly during skin wound healing process.Together, these results provide insights into Foxn1's role in the development and functional properties of dWAT and skin wound healing through dWAT regulation.It further extends our knowledge of the potential stimulatory differentiation role of Foxn1 beyond the epidermis, reaching the adipogenic program of DFs/ dWAT and the association between Foxn1 activity and skin adiposity.
Comparison of dWAT morphology in intact skin revealed consistent differences between Foxn1 +/+ versus Foxn1 −/− mice.DFs derived from the skin of Foxn1 +/+ or Foxn1 −/− mice displayed basic differences attributed to the adipogenic potential.Foxn1 +/+ DFs contained a twofold larger population of cell with low adipogenic but high pro-scaring potential (CD26 positive cells) than the Foxn1 −/− DFs.However, DFs originated from Foxn1 +/+ displayed higher levels of genes expression related to preadipocyte markers of commitment (Zfp423) and adipogenic differentiation (Pparγ and Fabp4) when stimulated with adipogenic media that resulted in robust adipogenic differentiation.Taken together, Foxn1 activity in Foxn1 +/+ mice shapes the characteristics of DFs that predispose them to adipocyte lineage when stimulated by favorable environmental cues.In the view of the fact that intradermal adipocytes and DFs derive from common precursor cells, 1,13 DFs' ability to differentiate into lipid-filled adipocytes under adipogenic stimuli suggests that DFs may contribute to intradermal adipocytes pool in dermal layer.This may be important aspect in the regulation of skin homeostasis in processes like skin wound healing, thermoregulation, or during skin infections.Identification of Foxn1 as molecular regulator of dWAT may accelerate recognition of Foxn1-mediated signaling involved in dWAT formation and modulation which may be one of the therapeutically relevant target genes that can improve physiological and pathological processes in the skin.
In our previous studies, we proposed Foxn1 as a key molecular factor involved in skin development and maturation, with a strong "imprinting" effect on DFs. 25 Here, we revealed the role of Foxn1 in dWAT development/ modulation.It is conceivable that Foxn1 activity during embryonic development (initiated at days 16-17 of gestation) 21 triggers the adipogenic potential of distinctive population of DFs that continued throughout postnatal life.The present data showed that the consequences of Foxn1 activity (Foxn1 +/+ vs. Foxn1 −/− mice) in the skin (in vivo conditions) are so prominent and stable that alternations in the adipogenic process are still observed in isolated and cultured DFs differentiated into adipocytes (in vitro conditions).Based on the literature review, we deduced that the initial expression of Foxn1 in the skin during fetal development overlaps in time with the dWAT differentiation program in the dermis, suggesting that Foxn1 may act as an important element in directing the development and expansion of dermal adipose tissue. 1,14,21Although additional studies are required to establish the contribution of Foxn1 to the dermis-associated adipocyte differentiation program during embryonic and postnatal development, our current analysis of Foxn1 +/+ versus Foxn1 −/− DF with previous transcriptome data 25 firmly supports the important part of Foxn1 in the dWAT developmental and differentiation program.
The key behavior of bona fide adipocytes is de novo lipogenesis through insulin-stimulated glucose uptake and lipolysis characterized by glycerol release.In in vitro settings, we detected increased basal glucose transport and enhanced Glut1-mediated glucose uptake in Foxn1 −/− DFs, but a reduced capability of lipid accumulation.This unexpected finding was additionally manifested by inhibition of Fasn expression in differentiated DFs cocultured with keratinocytes overexpressing Foxn1.These observations suggest that Foxn1 activity regulates the ability of dWAT to accumulate lipids with increased glucose uptake acting as a compensatory mechanism in the absence of Foxn1.Conversely, Foxn1 inactivation is associated with the upregulation of lipolysis markers and downregulation of adipogenesis regulators, which resembles the epididymal and subcutaneous white adipose tissue phenotype observed in obesity and metabolic disorders. 61,62These results support the hypothesis that Foxn1 activity may be associated with the genetic regulation of whole-body metabolism, including the development of obesity, insulin sensitivity, and energy balance.
Although several studies have revealed the key role of dWAT in skin wound healing and dermal fibrosis, 8,9,63 the transcriptional regulation of adipogenic and lipid-related processes occurring in post-wounded skin remains poorly understood.The first investigations into dWAT's role in wound healing process found that adipocyte precursor cells present in the dermis proliferate and differentiate during the early stage of wound healing (days 5-7), leading to colonization of the injury site with mature adipocytes along with migrating fibroblasts to support dermal reconstruction. 8Presented here in vivo wound healing experiments showed that only mice with active Foxn1 presented activation of adipogenic differentiation, evidenced by increased expression of adipogenesis-specific genes (Zfp423, Pparγ, and Fabp4), immediately after injury.These findings are in line with the study by Chen et al. that demonstrated increased PPAR-γ protein levels in both the subcutaneous and dermal layers of post-wounded skin and in macrophages present in wound area. 64The ongoing adipogenic process during wound healing in Foxn1 +/+ mice was further confirmed by higher expression (days 3-7 after injury) of the recently identified marker of fat mass and expansion of adipose tissue Mest. 65,66Importantly, increased Foxn1 levels were associated with increased expression of adipogenic-related genes in the skin on post-wounded days 1-5.Hence, Foxn1 activity affects skin adipogenesis and may participate in the molecular activation of intradermal adipocyte reconstruction after injury.
Skin of Foxn1 +/+ mice was characterized by higher levels of lipid metabolism-related genes (Atgl, Abhd5 and Fasn, Glut4) in comparison to Foxn1 −/− mice during initial stage of wound healing that suggest induced lipid metabolism.Recent evidence stressed that intradermal adipocytes undergo ATGL-dependent lipolysis shortly after injury and release fatty acids (FA) that promote macrophages infiltration and ensure proper wound healing. 7Blocking lipolysis process after injury by depletion of ATGL activity resulted in lower quantity and amount of FA released from adipocytes that support the repair process by activating macrophages recruitment and/or monocyte differentiation during the inflammatory and proliferative phases. 7n example of a new marker of ongoing lipolysis in dWAT after injury is ABHD5.As showed by Grond et al., ABHD5 is found in the epidermal layer, particularly in suprabasal keratinocytes and dermis.Thus far, available evidence suggests the key role of ABHD5 in keratinocyte development and differentiation. 67As ABHD5 interacts with perilipins (PLINs) and fatty acid-binding proteins (FABPs), both of which are key regulators of lipid homeostasis in adipose tissues, our data showing stimulated by injury an increase in Abhd5 particularly in association to Foxn1 (Foxn1 +/+ mice; Foxn1 +/+ DFs) suggest that ABHD5 is involved in dWAT lipolytic challenges in response to the injury.9][70] In basal conditions, insulin stimulates GLUT4 translocation and activity in skeletal muscle and adipose tissue. 71Whether transcriptional activation of lipid metabolism pathways occurs in adipocytes present in wound beds or in a population of newly differentiated cells after injury should be elucidated in future studies.
Based on present data, it can be hypothesized that the scarless healing process observed in Foxn1 −/− mice exhibits stable and unchanged expression of genes related to adipogenic and lipid metabolism processes after injury.This finding stresses the role of intradermal adipocytes and their metabolic properties in rapid initiation of the healing process including inflammation, migration of keratinocytes, and fibroblasts to close the wound.Such mechanism, however, may not be associated with scarless healing and raises the question of the role of dWAT in the initial phase of skin regeneration.Furthermore, since mutation in Foxn1 gene has been linked to T-cell immunodeficiency, it may have also effect on inflammatory microenvironment and activation of proinflammatory macrophages in wounds.
Surprisingly, we detected alterations in the expression of lipogenic and lipolytic markers in Foxn1-deficient mice at a later wound healing stage (day 14).It is possible that transcriptional activation of adipocyte metabolic processes may be explained by the presence of regenerated hair follicles that appear at the wound site 14-19 days after injury with adjacent newly differentiated adipocytes characterized by metabolic and physiological features of mature adipose cells. 52,53To date, numerous examples of reciprocal crosstalk between dWAT and hair follicles have been demonstrated. 4,5,53Considering the role of Foxn1 in the regulation of hair shaft differentiation 54 and the involvement of dWAT in hair regeneration, 4,5 additional research aiming to explore the relationship between the Foxn1 activity and dWAT layer is mandatory.
The observed increase in the expression of genes related to lipid metabolism in Foxn1 active mice at later stages of wound healing could also be attributed to thermogenesis.The lack of Foxn1 activity (Foxn1 −/− mice) is characterized by, i.e., the lack of hair growing above the skin surface, which results in increased energy expenditure and reduced individual susceptibility to diet-induced obesity. 28,29,72Recent literature data indicate dWAT as an isolation layer that protects the body against heat loss, yet remains independent of thermogenin signaling (UCP-1), and is insensitive to βadrenergic agonists. 73The demonstrated relationship between the activity of the Foxn1 factor and weight gain, the role of Foxn1 in the process of adipogenesis, and the participation of intradermal adipocytes in response to exposure to cold raise the question of the relationship between the Foxn1 factor and dWAT in thermoregulation mechanisms.As proposed by Sampath et al. in the transgenic mouse model (Scd1 lox/lox mice), due to alopecia, the expression of lipolysis-related markers increases to maintain a constant body temperature (energy). 74Therefore, further studies are required that take these variables into account.
6][77] Study by Nowinski et al. showed that keratinocytes regulate the expression of fibroblast genes through keratinocyte-derived IL-1α implying paracrine "loops" between keratinocytes-dermal fibroblasts communications. 75Our studies revealed link between Foxn1 activity and IGF2 and BMP2 expression based on both in vitro (keratinocytes transduced with Ad-Foxn1) and in vivo (post-wounded skin) settings.Transduction of Foxn1 into keratinocytes resulted in large increase in Igf2 mRNA levels.The effect of Foxn1 activity on the increase in Igf2 expression observed in in vitro experiment was also detected in post-wounded skin of Foxn1 +/+ mice.Furthermore, our recent in vivo studies showed that lentivirus carrying Foxn1 transgene introduced into post-wounded skin of Foxn1 −/− mice increases Igf2 expression. 780][81] Interestingly, our recent proteomic analysis revealed that Foxn1 upregulates and stimulates the release of CREG1 protein into conditioned media collected from keratinocytes transduced with Ad-Foxn1. 39Considering that the expression of IGF family members in dermal fibroblasts can be regulated by keratinocyte-mediated changes, 75 it is conceivable that Foxn1 appears as an initiator of the IGF2 signaling cascade that stimulates the process of adipogenesis in the skin.
][84][85] In particular, three members of BMPs family, BMP2, BMP4, and BMP7, are considered as functional regulators of adipose tissue.BMP2 expression was detected in abdominal and gluteal adipose tissue as well in preadipocytes. 82As observed in various types of cells i.a.4][85] Our results indicate a possible interaction between Foxn1 activity and BMP2 adipogenesis-stimulating pathway.We are aware that these results need to be interpreted with caution.A future study examining the involvement of Foxn1 in the BMP2 pathway promoting adipocyte differentiation needs to be focused on crosstalk between dermal white adipose tissue and hair follicles.
Collectively, our data show that Foxn1 expressed in the epidermal region affects the skin adipogenesis process through modulation of the developmental and adipogenic differentiation programs of DFs.Furthermore, Foxn1 activity (Foxn1 +/+ vs. Foxn1 −/− ) modulates the metabolic processes of dWAT (lipogenesis and lipolysis) in intact and post-wounded skin.Because the knowledge regarding dWAT homeostasis and molecular pathways is still scarce, our findings establish the role of Foxn1 in skin physiology.It also identifies possible targets for modulating and regulating dWAT homeostasis under physiological and pathological conditions.

| CONCLUSIONS
In this study, we demonstrated that Foxn1 activity regulate/modulate dWAT morphology and functional as well as metabolic properties (Figure 7).The results establish the role of Foxn1 on skin dWAT/DFs physiology and identify possible targets for modulating and regulating dWAT homeostasis in physiological and pathological conditions.
Particularly, Foxn1-dependent molecular alterations in expression of genes participating in lipogenesis and lipolysis shed a light on mechanism underlying wound healing process.

| LIMITATIONS
Foxn1 −/− mice displayed a significantly enlarged dWAT layer, which contrasts with our findings that suggested that Foxn1 may have a stimulatory role in adipogenic processes due to hair cycle-associated plasticity of intradermal adipocytes. 4,5Although Foxn1-deficient mice exhibit a regular hair cycle along with well-defined phases of hair growth, only a few hair follicles grow above the surface of the skin, leading to the hairless phenotype. 73,86,87Moreover, histologic analysis indicates numerous hair follicles deeply anchored within the dermis of Foxn1 −/− mice, which enlarges the area of the dWAT compartment.As Foxn1 regulates keratin in the hair follicle 20,88 and dWAT is involved in hair regeneration processes, 4,5 further studies focusing on understanding the relationship between Foxn1 activity on dWAT regulation during hair cycle are warranted.
During fetal development, the initiation of Foxn1 expression in the skin overlaps in time with the so-called transitional point between scar-free (regenerative) to scarpresent (reparative) skin healing that occurs at days 16.5-18.5 of gestation for mice. 24,89Studies by Wojciechowicz et al. indicated that in mice, the presence of adipocyte precursor cells in the reticular dermis is already observed at day 16 of embryonic development. 1Cells from lower dermis corresponding to intradermal adipocytes express the transcription factor Cebpα, which is one of the first markers of adipogenesis.During the following days of development, cells acquire the expression of Pparγ, Fabp4, and adiponectin, indicating that the status of mature fat cells has been reached.Considering that intradermal adipocytes and DFs originate from common precursor cells and given Foxn1's function in altering DF properties/characteristics, it can be hypothesized that Foxn1 can be one of the factors involved in determination of intradermal adipocytes fate during skin development and maturation.As present data demonstrate, the increase in Foxn1 activity following skin injury supports the early activation of adipogenesis by modifying the metabolic characteristics of dWAT (lipogenesis and lipolysis).Further studies are warranted.