COUP-TFII regulates early bipotential gonad signaling and commitment to ovarian progenitors

Abstract

Background: The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored.

Results: By differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells.

Conclusions: Our findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.

Keywords: 46,XX DSD; Bipotential gonad; COUP-TFII; Sex development; Supporting gonadal cells.

Fig. 1

NR2F2 expression during early human gonadal development and in COV434 and NT2/D1 cell lines. A The expression pattern of NR2F2 projected on the UMAP plot showing cell lineages in the scRNA-seq datasets of male and female somatic cells obtained from human gonads [24]. The color scale represents NR2F2 gene expression levels. Arrows indicate the predicted developmental trajectory of cell lineages based on evidence from the literature. B The monolayer differentiation protocol for human embryonic bipotential gonad described by Knarston et al. [25]. C, D RT-qPCR data of gene expression of NR2F2 transcript variants in CRL1502 (female) and PCS_201 (male) iPSCs (C), and of the bipotential gonad markers GATA4, ZFPM2, EMX2, and WT1, and the steroidogenic cell marker STAR (D) during the differentiation protocol. NR2F2 v3 was not detected under these experimental conditions. Data were normalized as a percentage of GAPDH (reference gene) expression. Mean ± SEM (n = 3). E RT-qPCR assay comparing the expression of NR2F2 transcript variants (v1–4) in COV434, NT2/D1, and HepG2 cell lines. Data is represented as a percentage of S8 (reference gene) expression. Mean ± SEM (n = 4). One-Way ANOVA followed by Tukey test (v1, v2, and v4) and t-test (v3). Asterisks indicate statistical significance in relation to COV434 expression. ***p < 0.001, ****p < 0.0001

Fig. 2

Generation and validation of a COV434 cell line carrying a knockout of the NR2F2 gene. A Schematic representation of the human NR2F2 locus on chromosome 15. Four mRNA variants generated by alternative transcription start sites, which encode three protein isoforms, are depicted. Filled boxes indicate coding sequences (CDS), empty boxes indicate untranslated regions and lines represent introns. Arrows indicate transcriptional start sites. The position of the guide RNA (gRNA) targeting exon 2 is indicated. PAM, protospacer adjacent motif. B Genotyping of representative isolated alleles from two selected single-cell clones by Sanger sequencing revealed potential wild-type (WT) and NR2F2-knockout (KO) clones. The KO clone presented the homozygous mutation c.484delG (NM_021005), p.Gln163fs*4 (NP_066285). C Western blotting assays using total protein extracts from NT2/D1, COV434 non-transfected (NT), WT, and NR2F2-KO cell clones. The anti-COUP-TFII used recognizes the human isoform A, expressed by the NR2F2 variant 1. Alpha-tubulin was used as endogenous control. The molecular weight (kDa) is indicated. D, E RT-qPCR assay comparing relative mRNA expression of the four NR2F2 transcript variants (D) and known COUP-TFII regulated genes (E) between WT and NR2F2-KO COV434 cell clones. S8 was used as a reference gene. Values are mean ± SEM (n = 3–4). Student’s t-test with Welch’s correction, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Cytomorphology examination of WT and the NR2F2-KO COV434 cells. A Light microscopy images of WT and NR2F2-KO COV434 cell clones stained with H&E. Magnifications of the areas outlined by the dashed boxes are shown on the right panels. B Fluorescence images of cells stained with phalloidin (yellow), DAPI (cyan), and Anti-Alpha-Tubulin (magenta). Merged images are shown

Fig. 4

Transcriptome analysis of WT and the NR2F2-KO COV434 cells. A Volcano plot representing downregulated (log₂FC < − 1) and upregulated (log₂FC > 1) differentially expressed genes (DEGs) between NR2F2-KO and WT COV434 cells. Absolute theta > 2 was used as a cutoff. B Heatmap representing gene expression levels of cell markers associated with early gonadal somatic cells, extragonadal mesonephros, and endothelial cells in the RNA-seq of WT and NR2F2-KO COV434 cells. The values of fragments per million mapped fragments (FPM) are expressed relative to the WT mean. C Gene ontology analysis of biological processes for downregulated and upregulated DEGs (NR2F2-KO versus WT COV434 cells). D RT-qPCR assay comparing relative mRNA expression of transcripts related to early gonadal somatic cells (LHX9 and SOX4), testis (SOX9 and PDGFB), and ovary development (WNT4, CTNNB1, and FST) between WT and NR2F2-KO COV434 cell clones. S8 was used as a reference gene. Values are mean ± SEM (n = 3–4). Student’s t-test with Welch’s correction, *p < 0.05, **p < 0.01. Source data for this figure are available in Additional file 2

Fig. 5

Comparison of the unique gene signatures of WT and the NR2F2-KO COV434 cells to the transcriptome of cell types from fetal organ systems and female fetal gonads. A Venn diagram depicting the adopted strategy to obtain the unique gene signatures for WT and NR2F2-KO COV434 cells from RNA-seq data. FPM, fragments per million mapped fragments. B The unique gene signatures for WT and NR2F2-KO cells were compared with tissue-cell type expression signatures of different human fetal organs using the online platform WebCSEA. Data show the − log₁₀(p-value) generated for each query of the cell-type specificity enrichment analysis. Each dot represents one tissue-cell type characterized by scRNA-seq experiments. The red dashed line indicates the Bonferroni-corrected significance by 1355 tissue-cell types. The grey solid line indicates the nominal significance. C UMAP of cell lineages in the scRNA-seq datasets of developing ovary and mesonephros obtained from human female fetuses [24]. The color scale represents the shared gene signature enrichment for WT and NR2F2-KO COV434 cells. CoelEpi coelomic epithelium, OSE ovarian surface epithelium, preGC pre-granulosa cell, Gi gonadal interstitial, Oi ovarian interstitial, SMC smooth muscle cell. Source data for this figure are available in Additional file 3

Fig. 6

Proposed model for COUP-TFII function during early gonadal development in humans. DSD differences of sex development