X-linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation of hypothalamic neurons

Vol.:(0123456789) 1 3 Cellular and Molecular Life Sciences (2021) 78:7043–7060 https://doi.org/10.1007/s00018-021-03945-0 ORIGINAL ARTICLE X‑linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation ofhypothalamic neurons LucasE.CabreraZapata1 · CarlaD.Cisternas1,2 · CamilaSosa1 · LuisMiguelGarcia‑Segura3,4 · MariaAngelesArevalo3,4 · MaríaJuliaCambiasso1,2 Received: 31 May 2021 / Revised: 26 August 2021 / Accepted: 17 September 2021 / Published online: 11 October 2021 © The Author(s) 2021 Abstract Several X-linked genes are involved in neuronal differentiation and may contribute to the generation of sex dimorphisms in the brain. Previous results showed that XX hypothalamic neurons grow faster, have longer axons, and exhibit higher expression of the neuritogenic gene neurogenin 3 (Ngn3) than XY before perinatal masculinization. Here we evaluated the participation of candidate X-linked genes in the development of these sex differences, focusing mainly on Kdm6a, a gene encoding for an H3K27 demethylase with functions controlling gene expression genome-wide. We established hypothalamic neuronal cultures from wild-type or transgenic Four Core Genotypes mice, a model that allows evaluating the effect of sex chromosomes independently of gonadal type. X-linked genes Kdm6a, Eif2s3x and Ddx3x showed higher expression in XX compared to XY neurons, regardless of gonadal sex. Moreover, Kdm6a expression pattern with higher mRNA levels in XX than XY did not change with age at E14, P0, and P60 in hypothalamus or under 17β-estradiol treatment in culture. Kdm6a pharmacological blockade by GSK-J4 reduced axonal length only in female neurons and decreased the expression of neuritogenic genes Neurod1, Neurod2 and Cdk5r1 in both sexes equally, while a sex-specific effect was observed in Ngn3. Finally, Kdm6a downregulation using siRNA reduced axonal length and Ngn3 expression only in female neurons, abolishing the sex differences observed in control conditions. Altogether, these results point to Kdm6a as a key mediator of the higher axogenesis and Ngn3 expression observed in XX neurons before the critical period of brain masculinization. Keywords Kdm6a· H3K27 demethylation· Sex differences· Ngn3· Neuritogenesis· Hypothalamic neurons Introduction The brain is a sexually dimorphic organ, along with many other organs and tissues besides the gonads. These sex differences are found on a wide range of levels, both in the biochemical and architectural nature of certain brain regions and, consequently, in the physiological and behavioral responses controlled by such dimorphic regions, as well as in the susceptibility to certain neurodevelopmental, psychiatric, and neurodegenerative diseases, such as autism spectrum disorders, dyslexia, depression and anxiety disorders, attention deficit hyperactivity disorder, schizophrenia, and dementia, among others [1, 2]. Two major factors are currently known to contribute to the setting of sexual dimorphisms in the brain during development: (1) a sex-specific trophic environment due to differences in gonadal hormones secretion and (2) a distinct genetic and epigenetic pattern for males and females generated by differences in the expression of X and Y chromosomes-linked genes [3–5]. X and Y chromosomes have undergone a divergent evolutionary process, determining that they currently encode mostly dissimilar genetic information and are subject to different epigenetic regulations. As a result, these chromosomes are no longer capable of recombining during meiosis along most of their length except for the pseudoautosomal Cellular andMolecular Life Sciences * Maria Angeles Arevalo are[email protected] * María Julia Cambiasso [email protected].edu 1 Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC-CONICET, Universidad Nacional de Córdoba, 5016Córdoba, Argentina 2 Cátedra de Biología Celular, Facultad de Odontología, Universidad Nacional de Córdoba, 5000Córdoba, Argentina 3 Instituto Cajal, CSIC, 28002Madrid, Spain 4 CIBERFES, Instituto de Salud Carlos III, 28029Madrid, Spain 7044 L.E.Cabrera Zapata et al. 1 3 regions (PARs), the small regions of sequence identity at the termini of both X and Y. Therefore, the intermediary regions between the PARs that cannot recombine, called the nonPAR of the X and the male-specific region of the Y (MSY), encompass most of both chromosomes and encode genes that are Xand Y-specific, respectively [6, 7]. Although X-linked genes are present in both sexes, the existence of two X in every cell of females and only one in males generates a "dosage difference" in the copy number of virtually all of these genes between the sexes. This imbalance is compensated during development by the X chromosome inactivation mechanism (XCI), which involves the transcriptional silencing of one of the two Xs in each cell of an XX embryo, thus defining an inactive (Xi) and an active (Xa) X chromosome that will be inherited through the successive mitotic divisions to all the cells that finally shape that individual [8–11]. However, XCI does not lead to complete repression of all genes in the Xi, but some "escape" inactivation and are therefore expressed from both Xa and Xi [12–15]. Kdm6a is an XCI “escapee” located on the non-PAR of the X that encodes for the lysine demethylase 6a (Kdm6a) or Utx enzyme, a member of the Kdm6 subfamily of histone 3 (H3) demethylases that remove di- (me2) and trimethyl (me3) groups on lysine (K) at position 27 (H3K27me2/ me3) [16]. H3K27me2/me3 are epigenetic marks known to repress gene expression, so their removal by Kdm6 demethylases promotes transcription [17–19]. Along with Kdm6a, the other two members of the Kdm6 subfamily are lysine demethylase 6b (Kdm6b) or Jmjd3, encoded on autosome 11, and lysine demethylase 6c (Kdm6c) or Uty, a Kdm6a paralogue encoded on the MSY [16, 19, 20]. Kdm6a plays a key role in cell fate determination and cellular identity during development by controlling pluripotency and lineagespecific sets of genes [21, 22]. During brain development, Kdm6a is involved in neurogenesis promotion, determining the neural stem cell status and modulating the subsequent differentiation of these pluripotent cells into neurons and glia [23–26]. Regarding neuronal differentiation, it has been shown that Kdm6a deletion prevents the proper development and maturation of neurons, leading to a repression of genes required for neuritogenesis and synaptogenesis (such as CREB5, CAMK2A, CKB, ASIC1, and ASCL1, among many others), defects and immaturity in dendritic arborization and synapse formation, failures in electrophysiological activity, increased expression of anxious behaviors, and deficits in spatial learning and memory [27, 28]. Finally, Kdm6a constitutive mutations with loss of function lead to Kabuki syndrome, a congenital disorder characterized by intellectual disability accompanied by growth retardation and skeletal, cardiac, and facial abnormalities, among other manifestations [29–31]. The use of the Four Core Genotypes (FCG) transgenic mice allows independent evaluation of sex hormones and X and Y chromosomes effects on sexual differentiation. FCG model conjugates the deletion of the testis-determining Sry gene from the Y chromosome [32] with the reinsertion of this gene into autosome 3 [33]. This way, it was possible to unlink the inheritance of the Y chromosome from the inheritance of Sry and testis differentiation, obtaining four distinct genotypes: XX males (XXm) and females (XXf) and XY males (XYm) and females (XYf). Our previous results using FCG mice showed that sex chromosome complement (XY/XX) determines a sexually dimorphic expression of the proneural basic Helix-Loop-Helix transcription factor neurogenin 3 (Ngn3), with XX hypothalamic neurons presenting higher levels of Ngn3 mRNA than XY neurons, independently of gonadal sex [34]. In turn, XX neurons showed faster growth and maturation invitro than XY neurons, a characteristic that was dependent on the higher Ngn3 expression in XX cultures [34, 35]. These and other results [36–41] point to the importance of sex chromosome complement in the sexual differentiation of the brain, regulating the expression of autosomal genes involved in neurodevelopment and modulating neuronal differentiation in a sex-specific way independently of sex hormones. However, although it is clear that sex chromosomes determined the sexual dimorphisms observed in Ngn3 expression and neuronal growth and differentiation, the identity of particular X and Y-linked genes taking part in these processes remains unknown. Therefore, in the present study we aimed to evaluate specific X-linked genes participation in the sexually dimorphic differentiation of hypothalamic neurons, focusing on Kdm6a as a genomewide regulator of gene expression that escapes XCI and has a significant role in neurogenesis and neuritogenesis. Materials andmethods Animals FCG transgenic mice generated from MF1 strain (MF1-FCG mice, kindly donated by Dr. Paul Burgoyne, National Institute for Medical Research, London, UK), MF1 wild-type mice (Harlan Laboratories Inc., USA), and CD1 wild-type mice were used. MF1-FCG and MF1 wild-type mice were bred at the Instituto M. y M. Ferreyra (INIMEC-CONICETUniversidad Nacional de Córdoba, Córdoba, Argentina), whereas CD1 mice were obtained from colonies bred at both the Instituto M. y M. Ferreyra and the Instituto Cajal (CSIC, Madrid, Spain). Embryos used in FCG cultures were produced by breeding transgenic MF1-FCG XYm mice with wild-type MF1 females. Animals were kept in specific pathogen-free (SPF) conditions in individually ventilated cages until crosses were made to obtain pregnant females for experiments, at which time animals were placed in open cages. In all cases, the animals received water and food 7045 X‑linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation of… 1 3 adlibitum and were kept in controlled macroenvironmental conditions of temperature at 23°C and a 12h light/12h dark periodic cycle. Procedures for care, welfare and proper use of all experimental animals were approved and controlled by the Institutional Animal Care and Use Committee (CICUAL) of the Instituto M. y M. Ferreyra, following national and international regulations, and were in accordance with the Consejería del Medio Ambiente y Territorio (Comunidad de Madrid, Ref. PROEX 200/14), the European Commission (86/609/CEE and 2010/63/UE), and the Spanish Government Directive (R.D.1201/2005) guidelines. FCG animals genotyping was performed by Polymerase Chain Reaction (PCR) as previously described in Cisternas etal. [41]. Hypothalamic neuronal cultures andcell treatments FCG or CD1 wild-type embryos at 14days of gestation (E14, defining E0 as the day of the vaginal plug) were used to establish primary hypothalamic neuronal cultures. Donor embryos age was specifically selected with the purpose of avoiding exposure of neurons to the peak in gonadal testosterone secretion during in utero development, which occurs in male mice around E17 [42]. Pregnant mice were sacrificed by cervical dislocation under CO2 anesthesia, and embryos were dissected from the uterus. Neurons were cultured separately according to sex (by observing the presence/ absence of the spermatic artery in developing gonads) or genotype (by PCR) of embryos. The ventromedial hypothalamic region was dissected out and stripped off the meninges. Blocks of tissue were incubated for 15min at 37 ºC with 0.5% trypsin (Gibco, USA) and then washed three times with Ca2+/Mg2+-free Hank’s Buffered Salt Solution. Finally, tissue was mechanically dissociated to single cells in 37 ºC warm culture medium and cells were seeded. The medium was phenol red-free Neurobasal (Gibco) to avoid “estrogenlike effects” [43] and was supplemented with B-27, 0.043% L-alanyl-L-glutamine (GlutaMAX-I), 100 U/ml penicillin, and 100mg/ml streptomycin (Gibco). For gene expression analyses, cells were plated on 35mm × 10mm dishes (Corning, USA) or 6-wells plates (Falcon, USA) at a density of 500–1000 neurons/mm2. To study the effect of Kdm6 H3K27 demethylase activity inhibition on gene expression, after 2days invitro (DIV) some cultures were treated with 1.8µM GSK-J4 (Sigma-Aldrich, USA) or vehicle for 24h. To study the effect of 17β-estradiol (E2) on Kdm6a expression, after 3 DIV the medium of some cultures was replaced for 2h by a fresh medium devoid of B-27 and GlutaMAXI supplement and then cells were incubated for 2h with 10–10M E2 (Sigma-Aldrich) or vehicle. For morphometric analysis, cells were plated on 10mm glass coverslips (Assistent, Germany) at a density of 800 cells/mm2. After 3 DIV, some cultures were treated with 1.8µM GSK-J4 or vehicle for 24h. The surfaces of glass coverslips and plates were pre-coated with 1μg/μl poly-L-lysine (Sigma-Aldrich). RNA isolation andquantitative Real‑Time PCR (qPCR) Total RNA was extracted from cell cultures and hypothalamic tissue using TRIzol reagent (Invitrogen, USA), purified, and quantified by spectrophotometry on NanoDrop 2000 (Thermo Fisher Scientific, USA) as previously described [41]. 1µg of RNA per sample was reverse transcribed to cDNA in a 20µl reaction using M-MLV reverse transcriptase (Promega, USA) and random primers (Invitrogen), following manufacturer’s instructions. qPCR reactions were performed on a StepOne Real-Time PCR System using TaqMan or SYBR Green Universal PCR Master Mix (Applied Biosystems, USA) and Rn18s (18S rRNA) as control housekeeping gene. TaqMan probes and primers for Ngn3 were assay-on-demand gene expression products (Applied Biosystems). All other primers (Table1) were designed using the on-line Primer-Basic Local Alignment Search Tool (Primer-BLAST; National Institutes of Health, USA), selecting primer pairs spanning an exon-exon junction to restrict amplification specifically to mRNA. Primers were verified to amplify with a 95–100% efficiency by performing 4-point calibration curves. Relative quantification of mRNA expression was determined with the ΔΔCt method. Control XYm or control male samples were used as a reference group for experiments with FCG or wild-type mice, respectively. Small interfering RNA (siRNA) transfection A mixture of 4 different siRNA sequences specific to Kdm6a was used at a final concentration of 40nM total RNA during transfection (1: AGU UAG CAG UGG AAC GUU A, 2: GGA CUU GCA GCA CGA AUU A, 3: GGU ACG GCC UAC UGG AAU U, 4: CCA CGU UGG UC A UAC UAU A; ON-TAR GE Tplus Mouse Kdm6a Set of 4 siRNA, Dharmacon, UK). A non-targeting siRNA sequence (ntRNA; Dharmacon) was used as control and co-transfection with pmaxGFP (Lonza, Switzerland) was performed in all cases for transfected neurons identification. The efficacy of siRNA targeting Kdm6a was assessed by electroporation of neurons using a 4D-Nucleofector X Unit and the corresponding P3 Primary Cell nucleofection kit (Lonza) according to the manufacturer’s instructions, followed by qPCR for Kdm6a after 16h of knockdown. The same experimental design was used to analyze the effect of Kdm6a knockdown on Ngn3 gene expression. The transfection efficacy by electroporation was 15%, calculated as the percentage of GFP-expressing cells over total number. For morphometric analysis, neurons were transfected at 3 DIV with siRNA targeting Kdm6a or ntRNA 7046 L.E.Cabrera Zapata et al. 1 3 using Effectene Transfection Reagent (Qiagen, Germany) according to the manufacturer’s instructions. After 16h of knockdown, cultures were processed for axonal length measurement. The transfection efficacy by this method was 0.1%. Western blot 2 DIV hypothalamic neurons were treated with 0.5, 1, 1.8µM GSK-J4 (Sigma-Aldrich) or vehicle for 24h and then washed, harvested at 4°C in RIPA buffer with protease and phosphatase inhibitors, and proteins were resolved and transferred onto nitrocellulose membranes (GE Healthcare, UK) as previously described [44]. Membranes were blocked 90min at room temperature (RT) in Tris-buffered saline containing 0.1% Tween 20 and 5% BSA, and then incubated overnight at 4°C with 1:1000 anti-H3K27me3 primary antibody (Cell Signaling Technology, USA). After that, membranes were incubated 1h at RT with 1:10000 infrared dyeconjugated secondary antibody (LI-COR, USA) and proteins were visualized by Odyssey Infrared Imaging System (LICOR). After H3K27me3 visualization, blots were stripped and then re-probed with 1:2000 anti-total H3 primary antibody (Cell Signaling Technology) to ensure equal protein loading. Densitometric analyses were performed with the ImageJ software (National Institutes of Health; freely available at https:// imagej. nih. gov). Data are presented as a ratio of H3K27me3/total H3 of 3–4 independent cultures. Immunocytochemistry After treatment, neurons were fixed for 20min at RT in 4% paraformaldehyde prewarmed to 37°C. Transfected neurons were rinsed and mounted on glass slides immediately after, while neurons treated with GSK-J4 were rinsed, permeabilized for 6min with 0.12% Triton-X plus 0.12% gelatin in phosphate-buffered saline (PBS), blocked 1h at RT in PBS/ gelatin, and incubated for 1h at RT with anti-microtubule associated protein 2 (MAP2) mouse monoclonal antibody Table 1 Primer sequences used for gene expression assays by qPCR Gene Forward sequence 5′–3′ Reverse sequence5′–3′ Cdk5r1 GAT GCT GCA GAT CAA TGC CG GGA GTC GCT TCT TGT CCT CC Ddx3x ACT GCA CAA GGT GTC AAT TCTG TCC CAG TGG CAT AGG CAT TT Dll1 GCG ACT GAG GTG TAA GAT GGAA TCT CAG CAG CAT TCA TCG GG Eif2s3x GGT GAG GGT GGA GTG ACT CT TTC CCA TGA GCT ACG TGA CCA Hes1 CCA GCC AGT GTC AAC ACG A AAT GCC GGG AGC TAT CTT TCT Hes5 GGA GAA AAA CCG ACT GCG GA AGC TGC TCT ATG CTG CTG TT Kdm5c AAC CTT GTG CAG TGT AAC ACACG GGT TCC GGA TCA GGC TGT AG Kdm6a GCT GGA ACA GCT GGA AAG TC GAG TCA ACT GTT GGC CCA TT Mecp2 ACA GCG GCG CTC CAT TAT C CCC AGT TAC CGT GAA GTC AAAA Neurod1 CTG CGA GAT CCC CAT AGA CAAC CCT CTA ATC GTG AAA GAT GGC ATT Neurod2 CCA GAG GCA GTT GGT AAG GG GCG GAG ATT CGT GTT AGG GT Notch1 ACA GTG CAA CCC CCT GTA TG TCT AGG CCA TCC CAC TCA CA Syp AGT ACC CAT TCA GGC TGC AC CCG AGG AGG AGT AGT CAC CA Usp9x TCG CCA TAT TGA GGCTG TTG CAT AAC CGA GAG CTT GC Uty GAA AAG GCT AGA GGC GAG GG AAC CCG AAG AAG CTG CTA TCTAA Rn18s CGC CGC TAG AGG TGA AAT TCT CAT TCT TGG CAA ATG CTT TCG 7047 X‑linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation of… 1 3 (diluted 1:200 in PBS/gelatin; Sigma-Aldrich) and with anti-Tau rabbit polyclonal antibody (diluted 1:500 in PBS/ gelatin; Abcam, UK). After rinsing with PBS, cells were incubated for 1h at RT with the secondary antibodies Alexa 594 goat anti-mouse, for the detection of MAP2, and Alexa 488 goat anti-rabbit, for the detection of Tau (diluted 1:1000 in PBS/gelatin; Jackson ImmunoResearch, USA). Finally, neurons were mounted on glass slides using gerbatol (0.3g/ ml glycerol, 0.13g/ml Mowiol, 0.2M Tris–HCl, pH 8.5) plus 1:5000 DAPI for nuclei staining. Morphometric analysis Morphometric evaluation consisted of the assessment of neuritic arborization and axonal length, using in all cases digitized images of the immunostained neurons obtained at 20 × magnification with a standard Leica DMI 6000 fluorescence microscope (Leica, Germany) equipped with a digital camera of the same firm. At 4 DIV, the minor processes/ dendrites were identified as MAP2 immunoreactive neurites with acute-angled branching, whereas the axon was recognized as a single, thinner neurite of homogeneous caliber along its entire length, at least three times longer than the other processes, with right-angled branching and Tau selective immunoreactivity. Evaluation of neuritic arborization was carried out by a Sholl analysis [45] performed with the CellTarget software ([46]; freely available at https:// www3. uah. es/ biolo gia_ celul ar/ JPM/ CellT arg et/ CellT arget. html). A grid of 6 concentric circles with an increasing radius of 5μm one respect to the previous one was placed centered on the cell soma, and the number of times neurites intersected any circle was counted in 30 neurons per experimental condition and culture (4 independent cultures). The total number of intersections per neuron was obtained and neuritic arborization complexity was estimated by the Branching Index (BI; [46]). Axonal length was randomly measured in 50–80 neurons per experimental condition and culture (4 independent cultures) using the NeuronJ ImageJ plugin ([47]; freely available at https:// image scien ce. org/ meije r ing/ sof tw are/ neuro nj/). Statistical analysis Data are presented as mean ± SEM and were statistically evaluated by one-, twoor three-way analysis of variance (ANOVA) with treatment, gonadal sex and/or sex chromosome complement as independent variables. In twoand three-way ANOVAs, the statistical significance of the effects of each independent variable and their interactions was tested. Post hoc comparisons of means by Fisher’s Least Fig. 1 Relative mRNA levels of X-linked genes Kdm6a, Eif2s3x, Ddx3x, Kdm5c, Mecp2, Usp9x, and Syp in FCG hypothalamic neurons at 3 DIV. Kdm6a, Eif2s3x, and Ddx3x show higher expression levels in XX compared to XY cultures, regardless of gonadal sex (XXf and XXm > XYf and XYm). Data are mean ± SEM. n = 5–7 independent cultures for each genotype. *p < 0.05; **p < 0.01 7048 L.E.Cabrera Zapata et al. 1 3 Significant Difference (LSD) test were performed for those variables/interactions for which ANOVA p values were statistically significant. Statistical analysis was performed entirely with Statistica 8 software (StatSoft Inc., USA). p < 0.05 was considered statistically significant. Sample size (n) for all experiments was 3–9 individuals/independent cultures and it is indicated in the figure legends. The number of independent cultures corresponds to the number of pregnant mothers or the number of embryos of each genotype and treatment for wild-type or FCG cultures, respectively. FCG embryos were obtained from 3–5 pregnant mothers per experiment. Results Kdm6a, Ddx3x, andEif2s3x expression ishigher inXX hypothalamic neurons regardless ofgonadal sex First, we studied the expression of several X-linked genes particularly interesting for their involvement in neuronal growth and differentiation, to identify those with higher expression levels in XX than in XY neurons. Primary cultures of hypothalamic neurons from E14 FCG mice were established, maintained during 3 DIV, and then processed to determine the relative mRNA levels of seven X-linked genes by qPCR: Kdm6a, Eif2s3x, Ddx3x, Kdm5c, Mecp2, Usp9x, and Syp (Fig.1). Two-way ANOVA revealed no effect of gonadal sex, but a significant main effect of sex chromosome complement in Kdm6a, Ddx3x and Eif2s3x, presenting these genes significantly higher expression levels in hypothalamic neurons carrying the XX chromosome complement compared to those carrying the XY, regardless of the gonadal sex of the donor embryos (XX > XY; Kdm6a: F(1, 18) = 6. 58, p = 0.0194; Ddx3x: F(1,19) = 14.14, p = 0.0013; Eif2s3x: F(1,17) = 5.79, p = 0.0278). There was no interaction of gonadal sex and sex chromosome complement effects. On the other hand, no differences by either sex chromosome complement, or gonadal sex were found in Kdm5c, Mecp2, Usp9x, and Syp mRNA levels. Higher Kdm6a expression levels inXX neurons donotchange withE2 treatment orage Given the role of Kdm6a as a key epigenetic regulator of gene transcription and considering the results showing Fig. 2 17β-estradiol (E2) effect on Kdm6a mRNA levels in FCG hypothalamic neurons. Incubation with 10–10 M E2 did not change Kdm6a gene expression levels, remaining higher in XX vs. XY cultures, regardless of sex and treatment. Data are mean ± SEM. n = 4–5 independent cultures for each genotype and treatment. ***p < 0.001 Fig. 3 Relative Kdm6a mRNA levels in hypothalamic tissue of FCG mice at E14, P0, and P60. Gene expression was higher in XX than XY animals at all ages and independently of gonadal sex. Data are mean ± SEM. n = 5–6 individuals for each genotype and age. **p < 0.01; ***p < 0.001 7049 X‑linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation of… 1 3 its sexually dimorphic expression determined by the sex chromosome complement (XX > XY), we decided to focus our study henceforth on its involvement in the sex-specific differentiation of hypothalamic neurons. First, knowing the importance of E2 as the main sex hormone mediating organizational processes in the rodent brain, we treated cultures with this estrogen and analyzed the effect on Kdm6a mRNA levels. Remarkably, three-way ANOVA showed that E2 in the culture milieu did not alter Kdm6a gene expression levels, maintaining higher values in XX than in XY neurons, independently of gonadal sex and hormonal treatment (Fig.2; F(1, 28) = 14.14, p = 0.00079). Moreover, Kdm6a expression pattern did not change either with age, remaining higher in the hypothalamic tissue of XX than XY animals at E14 (two-way ANOVA: F(1,19) = 87.74, p = 0.000000015), postnatal day 0 (P0, newborns; two-way ANOVA: F(1,13) = 12.49, p = 0.0037), and postnatal day 60 (P60, gonadally intact adults; two-way ANOVA: F(1,15) = 11.15, p = 0.0045), regardless of whether individuals carried testes or ovaries (Fig.3). Kdm6 H3K27 demethylase activity isrequired forthedifferentiation oflonger axons inXX hypothalamic neurons To analyze the requirement for Kdm6 H3K27 demethylase activity in the sexually dimorphic differentiation of hypothalamic neurons, we employed the specific cell-permeable small-molecule inhibitor GSK-J4 [48]. First, we validated the effectiveness of GSK-J4 blocking Kdm6 demethylase activity in primary cultures of hypothalamic neurons from wild-type CD1 mice, performing a dose/response curve and evaluating the H3K27me3 levels by Western blot. In agreement with times and concentrations previously reported by others [49], treatment of cultures for 24h with GSK-J4 1.8µM showed the highest H3K27me3 levels (Fig.4a; oneway ANOVA: F(3, 10) = 10.45, p = 0.002) without affecting cell viability, proving to be effective in inhibiting the Kdm6 enzymatic activity. Besides, treatment did not affect the gene expression levels of Kdm6a or its paralogue on the Y chromosome, Uty, presenting neuronal cultures derived from female embryos higher Kdm6a mRNA levels than those from male, both in control and treated conditions (Fig.4b; two-way ANOVA: F(1, 13) = 10.25, p = 0.007). Considering these results, we selected this condition for all further experiments using GSK-J4 to inhibit Kdm6 demethylase activity. Thereafter, we evaluated the effect of Kdm6 H3K27 demethylase activity inhibition by GSK-J4 over neuronal growth and differentiation invitro, analyzing neuritic arborization complexity and axonal length as parameters of neuronal morphology. Assessment of neuritic arborization was performed by a Sholl analysis, counting per cell the number of times neurites intersected any line of the concentric circle grid (Fig.5a). At this level, significant differences were only observed in the number of intersections due to sex, with no effect of GSK-J4 or sex-treatment interaction: neurons derived from female embryos presented a higher mean number of intersections than those from male embryos, independently of GSK-J4 blockade (Fig.5b; two-way ANOVA: F(1, 12) = 5.15, p = 0.0425). Accordingly, calculation of the BI estimated a higher complexity in the neuritic arborization of female (BI = 14.91 ± 0.85) than male (BI = 11.26 ± 1.25) Fig. 4 Validation of GSK-J4 treatment as an inhibitor of Kdm6 demethylase activity in wild-type hypothalamic neurons. a Quantification and a representative immunoblot showing the increase in histone 3 lysine 27 trimethylation (H3K27me3) levels as a result of Kdm6 demethylation blockade for 24h at increasing concentrations of GSK-J4. Total histone 3 (H3) was used as a loading control. b GSK-J4 1.8µM treatment for 24h inhibited demethylation without affecting Kdm6a or Uty gene expression. Kdm6a mRNA levels were higher in female than male cultures, regardless of treatment. Data are mean ± SEM. n = 3–6 independent cultures for each sex and treatment. *p < 0.05; **p < 0.01; ***p < 0.001 7050 L.E.Cabrera Zapata et al. 1 3 neurons (two-way ANOVA: F(1, 12) = 6.067, p = 0.03), without GSK-J4 treatment or sex-treatment interaction effects. Regarding axonal length analysis, in agreement with previous results [34, 35], a clear sexual dimorphism was observed in control conditions, showing female neurons significantly longer axons on average than male neurons. Remarkably, two-way ANOVA revealed a significant effect of sex-GSK-J4 interaction (F(1, 12) = 22.86, p = 0.0004), with GSK-J4 treatment decreasing axonal length only in female derived neurons and thus nullifying the sex differences observed in control conditions (Fig.5c, d). Kdm6 H3K27 demethylase activity regulates theexpression ofNgn3 inasex‑specific manner determined bythesex chromosome complement Since in previous work we have demonstrated that higher expression of Ngn3 in XX hypothalamic neurons is required for faster maturation and longer axons of these neurons invitro [34], we investigated whether Kdm6a regulates the expression of Ngn3 and other neuritogenic genes such as Neurod1, Neurod2, and Cdk5r1. Cultures from E14 wildtype mice segregated by sex were maintained 2 DIV and then treated with GSK-J4 for 24h before processing for gene expression analysis by qPCR. As in Scerbo etal. [34], Fig. 5 Sexually dimorphic neuritogenesis of hypothalamic neurons depends on Kdm6 H3K27 demethylase activity. a Representative fluorescence image of female control neurons showing the grid used for Sholl analysis. b Number of intersections of neurites with Sholl’s grid of female and male neurons treated (GSK-J4) or not (Control) at 3 DIV with GSK-J4 1.8 µM for 24 h. Female neurons showed higher values than male neurons, irrespective of treatment. c Representative fluorescence images of female (F) and male (M) neurons treated (GSK-J4) or not (Control) at 3 DIV with GSK-J4 1.8µM for 24h. Arrows indicate the axon of a female control neuron and a male control neuron along their full length. d Mean axonal length for each experimental condition. GSK-J4 treatment abolished sex differences decreasing axonal length only in female neurons. Data are mean ± SEM. n = 4 independent cultures for each sex and treatment. *p < 0.05; ***p < 0.001 7051 X‑linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation of… 1 3 neurons derived from female embryos presented higher Ngn3 mRNA levels than those from males under control conditions. Interestingly, inhibition of Kdm6 demethylase activity significantly reduced Ngn3 expression in female cultures, while increasing it in males (Fig.6a; two-way ANOVA, sex-GSK-J4 interaction effect: F(1, 24) = 14.75, p = 0.0008). Moreover, GSK-J4 treatment significantly reduced Neurod1, Neurod2, and Cdk5r1 gene expression equally in both sexes (Fig.6a; two-way ANOVA, GSK-J4 treatment main effect: Neurod1: F(1, 25) = 13.5, p = 0.001; Neurod2: F(1, 24) = 10.15, p = 0.0039; Cdk5r1: F(1, 18) = 4.41, p = 0.049). Next, we evaluated the effect of the Kdm6 demethylase activity inhibition over the expression of some of the main Notch signaling elements, knowing that Ngn3 transcription is downregulated when this pathway is activated [50, 51]. Statistical analysis showed no significant differences due to gonadal sex, GSK-J4 treatment or sex-treatment interaction in Dll1, Hes1, and Hes5 relative mRNA levels, while Notch1 expression increased with treatment both in female and male neurons (Fig.6b; two-way ANOVA: F(1, 18) = 5.68, p = 0.028). Finally, neuronal cultures derived from FCG transgenic mice were treated with the GSK-J4 inhibitor and processed for Ngn3 relative expression analysis. Three-way ANOVA indicated a significant effect of sex chromosome complement-GSK-J4 interaction (F(1, 21) = 29.646, p = 0.00002), with inhibition of Kdm6 H3K27 demethylase activity by GSK-J4 inducing a decrease in Ngn3 levels in XX neurons and an increase in XY neurons, in line with the results observed in wild-type cultures and irrespective of gonadal type (Fig.7). Fig. 6 Effect of Kdm6 H3K27 demethylase activity inhibition on neuritogenesis-related genes expression in wild-type hypothalamic neurons. a Effect of GSK-J4 treatment on neuritogenic genes expression. Kdm6 demethylases blockade reduced Neurod1, Neurod2 and Cdk5r1 mRNA levels in both sexes equally, while a sex-specific effect was observed on Ngn3 levels, with a decrease in female and an increase in male neurons. b Effect of GSK-J4 treatment on Notch signaling genes expression. 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