# NR2F2 is required in the embryonic testis for fetal Leydig cell development

Aitana Perea-Gomez, Natividad Bellido Carreras, Magali Dhellemmes, Furong Tang, Coralie Le Gallo, Marie-Christine Chaboissier

► To cite this version:

Aitana Perea-Gomez, Natividad Bellido Carreras, Magali Dhellemmes, Furong Tang, Coralie Le Gallo, et al.. NR2F2 is required in the embryonic testis for fetal Leydig cell development. eLife, 2025, 14, 10.7554/elife.103783. hal-05164909

HAL Id: hal-05164909

https://hal.science/hal-05164909v1

Submitted on 16 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# NR2F2 is required in the embryonic testis for fetal Leydig cell development

Aitana Perea-Gomez*, Natividad Bellido Carreras, Magali Dhellemmes, Furong Tang, Coralie Le Gallo, Marie-Christine Chaboissier*

Université Côte d'Azur, CNRS, INSERM, iBV, Nice, France

## eLife Assessment

This **important** study, which has been improved further upon revision, reveals a critical role of the transcription factor NR2F2 in mouse fetal Leydig cell (FLC) differentiation. With elegantly carried out experiments, the authors provide **compelling** evidence that NR2F2 helps to initiate the differentiation of certain interstitial cells into FLC until these cells mature into functional secretory cells that produce androgen and insulin-like peptide 3 (INSL3). The particular importance of the work comes from the fact that NR2F2 affects FLCs without altering paracrine signals known to be involved in FLC differentiation. The work will be of interest to colleagues studying reproductive development in mammals including humans or the biological functions of the nuclear receptor family.

## Abstract
Male genital development in XY mammalian fetuses is triggered by the action of hormones, including testosterone, secreted by the developing testes. Defects in this process are a cause for differences in sex development (DSD), one of the most common congenital abnormalities in humans. Fetal Leydig cells (FLCs) play a central role in the synthesis of masculinizing hormones in the developing testes. Yet, the genetic cascade controlling their differentiation is poorly understood. Here, we investigate the role of the orphan nuclear receptor NR2F2 (COUP-TFII) in FLC development. We report that NR2F2 is expressed in interstitial progenitor cells of the mouse embryonic testes and is downregulated upon their differentiation into FLC. By using two mouse models for conditional mutation of *Nr2f2* in the developing testes, we demonstrate that NR2F2 is required for testis morphogenesis and FLC development. NR2F2 acts in interstitial progenitors to regulate the initiation and progression of FLC differentiation. These results establish NR2F2 as an essential regulator of FLC development and steroid hormone synthesis in the mouse fetal testis and provide an entry point in understanding the etiology of 46,XY DSD associated with pathogenic NR2F2 variants.

## Introduction

Sexual development in mammals is conditioned by the gonadal sex established during fetal life. In XY embryos, the initially undifferentiated gonads develop into testes that can produce testosterone which stimulates the differentiation of male internal and external genitalia (including epididymis, vas deferens, seminal vesicles, scrotum, and penis) leading to the masculinization of the fetus. In contrast, fetal ovaries do not produce testosterone, and female internal and external genitalia (oviducts, uterus, vagina, and vulva) develop in XX individuals. Abnormalities in the masculinization process are mainly associated with defects in androgen synthesis or signaling (Reyes et al., 2023). Although this molecular cascade is well defined, the differentiation of androgen-producing cells in the embryo is only partially understood.

Androgen production in the developing testis relies mainly on fetal Leydig cells (FLCs), which express all enzymes required for the biosynthesis of androstenedione from cholesterol (*Miyabayashi*

*For correspondence:
Aitana.PEREA-GOMEZ@univ-cotedazur.fr (AP-G);
marie-christine.chaboissier@univ-cotedazur.fr (M-ChristineC)

**Competing interest:** The authors declare that no competing interests exist.

**Funding:** See page 18

**Preprint posted**
25 September 2024

**Sent for Review**
19 November 2024

**Reviewed preprint posted**
03 January 2025

**Reviewed preprint revised**
10 June 2025

**Version of Record published**
10 July 2025

**Reviewing Editor:** Detlef Weigel, Max Planck Institute for Biology Tübingen, Germany

© Copyright Perea-Gomez et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
et al., 2017; Inoue et al., 2016; Shima et al., 2013; Ademi et al., 2022). The final step of conversion of androstenedione into testosterone, catalyzed by HSD17B3, takes place in a different cell type, the fetal Sertoli cells (Shima et al., 2013; O'Shaughnessy et al., 2000). In addition to their role in androgen synthesis, FLCs also produce INSL3, a hormone required for testis descent (Nef and Parada, 1999; Zimmermann et al., 1999). Defects in testis descent, regulated also by androgens, result in cryptorchidism, a condition which impacts fertility and constitutes a risk factor for testicular cancer (Hutson et al., 2015). While a fraction of FLCs persist after birth, others de-differentiate or involute, and adult testosterone production is ensured by a distinct population of steroidogenic cells, the adult Leydig cells (ALCs), which differentiate at puberty (Shima and Morohashi, 2017; Shima, 2019).

FLCs differentiate from embryonic day 12.5 (E12.5) in mice and increase in number during fetal life through the recruitment and differentiation of Wnt5a positive proliferative progenitors located in the interstitial space of the testes (Ademi et al., 2022; Shima and Morohashi, 2017; Shima, 2019; Rotgers et al., 2018). Interstitial progenitors also give rise to the contractile peritubular myoid cells (PTM) that will surround the future seminiferous tubules (Ademi et al., 2022). Despite being the most abundant cell population in the fetal testis (Ademi et al., 2022; Mayère et al., 2022), little is known about the genetic control of the proliferation, specification, and differentiation of the interstitial steroidogenic progenitor cells.

Lineage tracing and single-cell transcriptomic analyses have revealed that interstitial progenitors have a dual origin. The majority are derived from the coelomic epithelium of the undifferentiated gonad, which harbors early bipotential progenitors able to differentiate along the supporting (the future Sertoli cells) or the steroidogenic lineage (Ademi et al., 2022; Karl and Capel, 1998; Stévant et al., 2018; Stévant et al., 2019). In addition, Nestin positive cells migrate from the adjacent mesonephros into the gonad and differentiate along the steroidogenic lineage from E13.5 to give rise to up to a third of the FLC population by the end of gestation (Ademi et al., 2022; DeFalco et al., 2011; Kumar and DeFalco, 2018).

Positive and negative paracrine signals drive FLC differentiation by upregulating the transcription factors NR5A1, GATA4, and GATA6, which in turn regulate the expression of genes related to cholesterol metabolism and steroidogenesis (Rotgers et al., 2018; Wen et al., 2016; Morohashi et al., 2020; Baba et al., 2018; Baba et al., 2014; Shima et al., 2018; Padua et al., 2015; Bielinska et al., 2007; Viger et al., 2022). Desert Hedgehog (DHH) is secreted by Sertoli cells and acts on the interstitial progenitors expressing the Hedgehog receptor Patched1 (PTCH1) and the Hedgehog effectors GLI1, -2, and -3 to trigger FLC and PTM differentiation (Yao et al., 2002; Barsoum and Yao, 2011; Barsoum et al., 2009; Park et al., 2000; Kothandapani et al., 2020; Pierucci-Alves et al., 2001; Clark et al., 2000). In addition, FLC development requires the activation of signaling pathways downstream of PDGFRA in the steroidogenic progenitors (Brennan et al., 2003; Inoue et al., 2022; Schmahl et al., 2008). On the other hand, ligands present in vascular and perivascular cells activate the NOTCH2 receptor and the expression of the effectors HES1 and HEYL in interstitial progenitors to maintain their undifferentiated state and inhibit FLC differentiation (Kumar and DeFalco, 2018; Tang et al., 2008; Liu et al., 2016; Defalco et al., 2013). In addition to the paracrine signals from adjacent cell populations, FLC differentiation is also regulated by the cell-autonomous action of the transcription factors ARX, TCF21, PBX1, MAF, and MAFB expressed in the interstitial steroidogenic progenitors, although their precise roles remain elusive (Ademi et al., 2022; Miyabayashi et al., 2013; Li et al., 2021; Cui et al., 2004; Schnabel et al., 2003).

Nuclear receptor subfamily 2 group F member 2 (NR2F2, also known as COUP-TFII) is abundantly expressed in interstitial progenitors of the fetal and adult testis in rodents and humans (Inoue et al., 2016; Ademi et al., 2022; Mendoza-Villarroel et al., 2014b; Kilcoyne et al., 2014; van den Driesche et al., 2012; Lottrup et al., 2014; Qin et al., 2008; Taelman et al., 2024). NR2F2 activates or represses transcription depending on the cellular context by directly binding to DNA responsive elements or by interacting with other transcription factors. NR2F2 regulates cell differentiation during organogenesis, adult tissue homeostasis, and tumorigenesis (Polvani et al., 2019). NR2F2 function is essential for cardiac and vascular development so that Nr2f2 mutation in mouse leads to embryonic lethality at mid-gestation (Pereira et al., 1999). The study of mouse conditional mutants has shown that NR2F2 is essential for ALC differentiation in the postnatal testis before puberty (Qin et al., 2008). However, the function of NR2F2 in the developing testis during fetal life has not been addressed, and its role in the interstitial progenitors that give rise to the FLC lineage remains unknown. It was initially
proposed that NR2F2 could act as a negative regulator of steroidogenesis at fetal stages based on the inverse correlation between NR2F2 expression and steroidogenesis genes and testosterone levels in mouse and rat fetal testes treated with endocrine disruptors (van den Driesche et al., 2012). More recently, rare variants in NR2F2 have been associated with cryptorchidism, hypospadias, and defective penile growth in human patients (Zidoune et al., 2022; Ganapathi et al., 2023; Wankanit et al., 2024). These phenotypes can be attributed to defective testosterone and INSL3 production during gestation (Hutson et al., 2015; Amato et al., 2022), suggesting a positive role for NR2F2 in promoting FLC differentiation and/or function in the fetal testis.

In this study, we show that NR2F2 is expressed in interstitial progenitors of coelomic and mesonephric origin of the mouse fetal testes and is downregulated upon FLC differentiation. By using two Cre lines that drive *Nr2f2* deletion in mouse embryonic gonads, we show that NR2F2 is required for fetal mouse testis morphogenesis and for FLC differentiation. Absence of *Nr2f2* does not impair paracrine signals known to regulate FLC differentiation nor the proliferation or survival of the steroidogenic progenitor population. Our findings reveal that NR2F2 promotes the initiation of FLC differentiation as well as FLC maturation. Taken together, these results establish NR2F2 as an essential factor that positively regulates the development of steroidogenic cells in the mouse fetal testis.

## Results

### NR2F2 is expressed in steroidogenic progenitors of the developing testis

We analyzed the spatiotemporal distribution of NR2F2 in the developing testis by immunofluorescence. At E11.5 (18–21 tail somites [ts]), NR2F2 was detected in both the coelomic epithelium and the mesonephric mesenchyme adjacent to the gonads, two tissues that contribute to the population of interstitial steroidogenic progenitors (Ademi et al., 2022; Karl and Capel, 1998; Kumar and DeFalco, 2018; Figure 1A and C). The majority of mesenchymal cells in the gonad expressed the transcription factor RUNX1 (RUNX1+) (Nicol et al., 2019), indicating that most of the gonadal somatic cells at this stage belong to the supporting lineage (Figure 1A and D). Nevertheless, NR2F2+ RUNX1- cells were observed, revealing that interstitial progenitors were already present at this stage (Figure 1A). NR2F2+ cells were either GATA4+ or GATA4-, suggesting that interstitial progenitors of coelomic and mesonephric origins, respectively, were both present (Figure 1A–C).

In E12.5 testes, NR2F2+ cells were detected in the coelomic epithelium and in the interstitial space outside the developing testis cords (Figure 1E–H, Figure 1—figure supplement 1A–D). NR2F2+ cells co-expressed the steroidogenic progenitor marker ARX (Ademi et al., 2022; Miyabayashi et al., 2013) and were actively proliferating (Figure 1E–H, Figure 1—figure supplement 1E–H). At E14.5, NR2F2 expression was maintained in interstitial cells co-expressing PDGFRA (Brennan et al., 2003), as well as in the PTM lining the testis cords and in the cells beneath the surface of the testis that will contribute to the future tunica albuginea (Figure 1I and J, Figure 1—figure supplement 1M–P). NR2F2 expression was also found in NESTIN-expressing perivascular cells that correspond to the mesonephros-derived steroidogenic progenitors (Kumar and DeFalco, 2018; Figure 1K and L). In contrast, NR2F2 protein was either absent or detected at very low levels in FLC marked by the expression of the steroidogenic enzyme HSD3B (Figure 1M–P, Figure 1—figure supplement 1I–L).

Together, our results show that NR2F2 is expressed in the coelomic epithelium and the mesonephros, as well as in the interstitial progenitors derived from both sources, and is downregulated upon FLC differentiation in the developing testis.

### NR2F2 is required for fetal testicular morphogenesis and FLC development

In order to investigate the function of NR2F2 in the developing mouse testis, we used an *Nr2f2 flox/flox* conditional allele (Hutson et al., 2015), and a knock-in *Wt1 CreERT2* line, in which tamoxifen-inducible CreERT2 is produced by *Wt1*-expressing cells (Shima and Morohashi, 2017; Figure 2A). *Wt1* is expressed from E9.5 in the coelomic epithelium of the gonadal ridge and in the adjacent mesonephros (*Armstrong et al., 1993*), and *Wt1 CreERT2*-mediated recombination can be induced in all somatic gonadal cells upon tamoxifen administration at E9.5 and E10.5 (*Manuylov et al., 2008; Figure 2—figure supplement 1A*).
<figure><img src="image_3.png" /><figcaption>**Figure 1.** NR2F2 is expressed in steroidogenic progenitors of the fetal testis. (A–D) Immunodetection of NR2F2, RUNX1, and GATA4 on embryonic day 11.5 (E11.5) (18 tail somites) XY gonad. NR2F2 is detected in coelomic epithelium (ce), mesonephros (me), and RUNX1 negative cells that are either GATA4 positive (arrows in A–C) or GATA4 negative (arrowheads in A–C). (E–H) Immunodetection of NR2F2 and ARX on E12.5 XY gonad. NR2F2 is co-expressed with ARX in the coelomic epithelium (ce, dotted lines in F–H) and in interstitial cells between the testis cords (arrows in F–H). (I,J)</figcaption></figure>

*Figure 1 continued on next page*
Figure 1 continued

Immunodetection of NR2F2 and PDGFRA on E14.5 XY gonad. NR2F2 is detected in PDGFRA positive cells, including interstitial progenitors (arrowheads in J), peritubular myoid cell surrounding testis cords (arrows in J), and cells of the future tunica albuginea (yellow arrow in J). (K,L) Immunodetection of NR2F2, NESTIN, and PECAM-1 on E14.5 XY gonad. NR2F2 is detected in NESTIN positive interstitial progenitors, including perivascular cells (arrowheads in L) and peritubular myoid cells (arrow in L). (M-P) Immunodetection of NR2F2 and HSD3B on E14.5 XY gonad. NR2F2 is absent from the majority of HSD3B positive fetal Leydig cells (arrowheads in N-P) and is only detected at low levels in a few HSD3B positive elongated cells (arrows in N-P). Data are representative of triplicate biological replicates. Scale bar = 50 µm in A-D, F-H, J, L, and N-P. Scale bar = 100 µm in E, I, K, M.

The online version of this article includes the following figure supplement(s) for figure 1:

**Figure supplement 1.** NR2F2 is expressed in steroidogenic progenitors of the fetal testis.

NR2F2 is co-expressed with WT1 in the gonadal coelomic epithelium, in the mesonephros, and in interstitial cells (Yu et al., 2012; **Figure 2—figure supplement 1C–F**). Tamoxifen treatment at E9.5 and E10.5 triggered an efficient NR2F2 deletion in gonadal and mesonephric tissues of Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> embryos analyzed at E12.5 and E14.5 (**Figure 2A–E, Figure 2—figure supplement 1B**). NR2F2 expression was completely absent in gonadal interstitial cells, including NESTIN+ cells, demonstrating that all interstitial steroidogenic progenitors were targeted in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> embryos (**Figure 2D and E**). Morphological examination of the urogenital system of E16.5 Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/ flox</sup> embryos revealed hypoplastic undescended testes, as well as hypoplastic kidneys (**Figure 2H and I**), indicating that NR2F2 function is required for testicular and kidney development.

Sertoli cells expressing SOX9 (Figure 2B and C) and interstitial progenitors marked by ARX (Figure 2F and G) were present in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> embryos at E12.5. However, differentiated steroidogenic FLC (marked by HSD3B expression) were almost completely absent in the mutant gonads (Figure 2J, K, and N). This phenotype was not due to a delay in the initiation of FLC differentiation, as the number of HSD3B positive cells remained strongly reduced in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> mutant gonads at E14.5 (60% reduction, Figure 2L–N). In addition, the transcripts of Cyp11a1 and Cyp17a1, two genes encoding steroidogenic enzymes expressed in FLC, and of Ins13 were strongly reduced (Figure 2O), providing a possible explanation for the observed undescended testes phenotype (Figure 2—figure supplement 1G and H). The mutant gonads showed fewer testis cords that were enlarged and exhibited irregular shapes at E14.5 (Figure 2E, Figure 2—figure supplement 1J and M). Moreover, the expression of ACTA2 was strongly reduced both in the periphery of the gonad and in cells lining the testis cords, indicating that tunica cell and PTM development were impaired in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> mutants (Figure 2—figure supplement 1G and H). Together, these results demonstrate that NR2F2 function is not essential for the initial specification of the interstitial and supporting cells of the testis but is required for testicular morphogenesis and for FLC development.

## Sertoli cell development is impaired in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> testes

FLC differentiation relies on signals produced by Sertoli cells such as DHH and PDGFA acting on interstitial cells (Rotgers et al., 2018; Wen et al., 2016). The number of Sertoli cells expressing SOX9 and the levels of Sox9 transcripts were not altered in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> mutants at E14.5 (Figure 2N and O). However, the expression of Dhh, Pdgfa, and Amh, another marker of differentiated Sertoli cells, was reduced in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> mutants compared to Nr2f2<sup>flox/+</sup> control gonads (Figure 2—figure supplement 1K), indicating that Sertoli cell differentiation is abnormal in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> gonads. WT1 is required for Sertoli cell development (Gao et al., 2006), and previous work suggested that Wt1 heterozygosity in Wt1<sup>CreERT2/+</sup> gonads results in delayed testicular development (Carré et al., 2018). To discriminate between the effects of Nr2f2 loss of function and Wt1 heterozygosity in the phenotype of Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> mutants, we analyzed gene expression in Wt1<sup>CreERT2/+</sup> testes compared to wild-type littermates. We found that the expression of the FLC markers Cyp11a1 and Cyp17a1 was not significantly different in Wt1<sup>CreERT2/+</sup> testes compared to wild-type littermates (Figure 2—figure supplement 1L). In contrast, transcript levels of Dhh, Pdgfa, and Amh were significantly reduced (Figure 2—figure supplement 1L). While these results indicate that impaired FLC development in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> mutants is associated with the loss of Nr2f2 function, the potential contribution of Sertoli cell defects caused by Wt1 heterozygosity to the FLC phenotype cannot be ruled out in this model.
<figure><img src="image_4.png" /><figcaption>**Figure 2.** NR2F2 deletion by Wt1<sup>CreERT2</sup> impairs Sertoli cell differentiation and fetal Leydig cell (FLC) development. (A) Generation of Wt1<sup>CreERT2</sup>, Nr2f2<sup>flox/flox</sup> mutants. Tamoxifen was administered at embryonic day 9.5 (E9.5) and E10.5, and embryos were recovered at E12.5, E14.5, and E16.5. (B,C) Immunodetection of NR2F2 and SOX9 on E12.5 Nr2f2<sup>flox/+</sup> and Wt1<sup>CreERT2</sup>, Nr2f2<sup>flox/flox</sup> testes (outlined by dotted lines) after tamoxifen treatment at E9.5 and E10.5. NR2F2 is efficiently deleted in the gonad and mesonephros. (D,E) Immunodetection of NR2F2 and NESTIN on E14.5 Nr2f2<sup>flox/+</sup> and *Figure 2 continued on next page*</figcaption></figure>
**Figure 2 continued**

Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> testes. NR2F2 is efficiently deleted in NESTIN1 positive cells. (F,G) Immunodetection of ARX on E12.5 Nr2f2<sup>flox/+</sup> and Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> testes (outlined by dotted lines). Interstitial cells are generated in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> mutants. (H,I) Macroscopic view of the urogenital tract of XY E16.5 Nr2f2<sup>flox/+</sup> and Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> testes dissected after tamoxifen treatment at E9.5 and E10.5. Testes (t) and kidneys (k) are hypoplastic in Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> mutants. bl: bladder. vd: vas deferens. (J,K) Immunodetection of HSD3B on E12.5 Nr2f2<sup>flox/+</sup> and Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> testes (outlined by dotted lines). ad: adrenal. (L,M) Immunodetection of HSD3B on E14.5 Nr2f2<sup>flox/+</sup> and Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> testes. ad: adrenal. (N) Quantification of the number of HSD3B positive cells per surface unit and of the number of SOX9 positive cells per surface unit in control (wild-type or Nr2f2<sup>flox/+</sup>) and Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> testes. Each triangle represents the mean number of HSD3B or SOX9 positive cells per surface unit of one individual measured on at least two sections per gonad. Data are shown as means ± SEM. Statistical significance was assessed by Mann-Whitney U two-tailed test. * indicates p-value≤0.05; ns indicates p-value>0.05. (O) Quantification of Cyp11a1, Cyp17a1, Ins13, and Sox9 transcripts in Nr2f2<sup>flox/+</sup> and Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> testes treated with tamoxifen at E9.5 and E10.5 and dissected at E14.5 after normalization to Sdha and Tbp by RT-qPCR. Data are shown as means ± SEM. Statistical significance was assessed by Mann-Whitney U two-tailed test. * indicates p-value≤0.05; ns indicates p-value>0.05. Immunodetection data are representative of triplicate biological replicates. Scale bar = 100 µm in B–G, J–M. Scale bar = 500 µm in H, I.

The online version of this article includes the following source data and figure supplement(s) for figure 2:

**Source data 1.** Source data for cell counts and RT-qPCR data in **Figure 2**.

**Figure supplement 1.** NR2F2 deletion by Wt1<sup>CreERT2</sup> impairs Sertoli cell differentiation and fetal Leydig cell (FLC) development.

**Figure supplement 1—source data 1.** Source data for testis cord measurements and RT-qPCR data in **Figure 2—figure supplement 1**.

## NR2F2 is required in the steroidogenic lineage for FLC development

In order to elucidate the specific function of NR2F2 in the steroidogenic lineage for FLC development, we used the transgenic *Nr5a1-Cre* line which drives robust recombination in somatic gonadal cells from E11.5, after the supporting and steroidogenic lineages have been specified (**Figure 3A**, **Figure 3—figure supplement 1A and B; Bingham et al., 2006; Manuylov et al., 2011**).

At E11.5, NR5A1 is co-expressed with NR2F2 in the gonadal coelomic epithelium and in interstitial cells, but is absent from the mesonephros and mesonephros-derived cells (*Stévant et al., 2018; Kumar and DeFalco, 2018; Figure 3—figure supplement 1D–G*). Nr5a1-Cre; Nr2f2<sup>flox/flox</sup> mutants show efficient Nr2f2 deletion in gonadal interstitial cells at E12.5 and E14.5 (*Figure 3B–E*, **Figure 3—figure supplement 1C**). Consistent with previous reports on the activity of Nr5a1-Cre (*Manuylov et al., 2011*), NR2F2 was still detected in the coelomic epithelium layer and in interstitial cells just beneath it, particularly in the anterior part of the gonad of *Nr5a1-Cre; Nr2f2<sup>flox/flox</sup>* mutants (*Figure 3B–E*). NESTIN+ NR2F2+ cells were still present, confirming that steroidogenic progenitors of mesonephric origin were not targeted by *Nr5a1-Cre* (*Figure 3D and E*, **Figure 3—figure supplement 1H and I; Kumar and DeFalco, 2018*). Together, these results demonstrate that in *Nr5a1-Cre; Nr2f2<sup>flox/flox</sup>* mutants, NR2F2 is deleted after E11.5 in interstitial cells derived from the coelomic epithelium, except in the outermost layer of the testis.

Nr2f2 deletion by *Nr5a1-Cre* did not affect the initial formation of the supporting and steroidogenic lineages as evidenced by SOX9 (*Figure 3B, C, and H*, **Figure 3—figure supplement 1N and O**) and ARX expression (*Figure 3F and G*), nor the differentiation of Sertoli cells as shown by normal expression levels of *Dhh*, *Pdgfa*, and *Amh* (*Figure 3H, Figure 3—figure supplement 1N*). In addition, ACTA2 was detected in PTM and was only slightly reduced in the tunica albuginea of the posterior region in the mutant testes (*Figure 3—figure supplement 1J and K*). In contrast, the FLC population marked by HSD3B or CYP11A1 was decreased (40% reduction, **Figure 3I–M, Figure 3—figure supplement 1L and M**), and the expression of the FLC markers *Cyp11a1*, *Cyp17a1*, and *Ins13* was strongly downregulated in *Nr5a1-Cre; Nr2f2<sup>flox/flox</sup>* mutant testes (*Figure 3N*). We conclude that Nr2f2 deletion after E11.5 by *Nr5a1-Cre* leads to FLC reduction without Sertoli cell defects, suggesting that NR2F2 is required cell-autonomously in the interstitial cells for FLC development.

In agreement with reduced *Ins13* expression, the testes of *Nr5a1-Cre; Nr2f2<sup>flox/flox</sup>* embryos were undescended and exhibited an abnormal abdominal position at postnatal day (P) 3 (*Figure 3O and P*). In addition, the anogenital distance, a readout for androgen levels (*Schwartz et al., 2019*), was reduced in P0 and P1 *Nr5a1-Cre; Nr2f2<sup>flox/flox</sup>* males when compared to male controls (*Figure 3—figure supplement 1P*), consistent with the reduced expression of steroidogenic enzymes in the mutant testes. We conclude that defective FLC development in *Nr2f2* mutants results in cryptorchidism and impaired masculinization of the external genitalia.
<figure><img src="image_5.png" /><figcaption>**Figure 3.** NR2F2 deletion by Nr5a1-Cre impairs fetal Leydig cell (FLC) development. (A) Generation of Nr5a1-Cre; Nr2f2 flox/flox mutants. (B,C) Immunodetection of NR2F2 and SOX9 on embryonic day 12.5 (E12.5) Nr2f2 flox/+ and Nr5a1-Cre; Nr2f2 flox/flox testes (outlined by dotted lines). NR2F2 is deleted in interstitial cells but is still present in the outermost layer of the testis. (D,E) Immunodetection of NR2F2 and NESTIN on E14.5 Nr2f2 flox/+ and Nr5a1-Cre; Nr2f2 flox/flox testes. NR2F2 is still detected in NESTIN1 positive cells. (F,G) Immunodetection of ARX on E12.5 Nr2f2 flox/+ and Nr5a1-Cre; Nr5a1-Cre; Nr2f2 flox/flox testes. NR2F2 is still detected in NESTIN1 positive cells. (F,G) Immunodetection of ARX on E12.5 Nr2f2 flox/+ and Nr5a1-Cre; Nr5a1-Cre; Nr2f2 flox/flox testes. Figure 3 continued on next page</figcaption></figure>
**Figure 3 continued**

Nr2f2 flox/flox testes (outlined by dotted lines). Interstitial cells are generated in Nr5a1-Cre; Nr2f2 flox/flox mutants. (H) Quantification of Sox9, Dhh, Pdgfa, and Amh transcripts after normalization to Sdha and Tbp in control (wild-type or Nr2f2 flox/+) and Nr5a1-Cre; Nr2f2 flox/flox by RT-qPCR at E14.5. Data are shown as means ± SEM. Statistical significance was assessed by Mann-Whitney U two-tailed test. * indicates p-value≤0.05; ns indicates p-value>0.05. (I,J) Immunodetection of HSD3B on E12.5 Nr2f2 flox/+ and Nr5a1-Cre; Nr2f2 flox/flox testes (outlined by dotted lines). ad: adrenal. (K,L) Immunodetection of HSD3B on E14.5 Nr2f2 flox/+ and Nr5a1-Cre; Nr2f2 flox/flox testes. (M) Quantification of the number of HSD3B positive cells per surface unit in control (wild-type or Nr2f2 flox/+) and Nr5a1-Cre; Nr2f2 flox/flox testes at E12.5 and E14.5. Each circle represents the mean number of HSD3B positive cells per surface unit of one individual measured on at least two sections per gonad. Data are shown as means ± SEM. Statistical significance was assessed by Mann-Whitney U two-tailed test. * indicates p-value≤0.05; ns indicates p-value>0.05. (N) Quantification of Cyp11a1, Cyp17a1, and Ins13 after normalization to Sdha and Tbp in control (wild-type or Nr2f2 flox/+) and Nr5a1-Cre; Nr2f2 flox/flox by RT-qPCR at E14.5. Data are shown as means ± SEM. Statistical significance was assessed by Mann-Whitney U two-tailed test. * indicates p-value≤0.05; ns indicates p-value>0.05. (O,P) Macroscopic view of the urogenital tract of XY P3 Nr2f2 flox/+ and Nr5a1-Cre; Nr2f2 flox/flox mutants. The testes are in abdominal position in Nr5a1-Cre; Nr2f2 flox/flox mutants. k: kidney, t: testis, bl: bladder. vd: vas deferens. Immunodetection data are representative of triplicate biological replicates. Scale bar = 100 µm in B–G, I–L. Scale bar = 500 µm in O, P.

The online version of this article includes the following source data and figure supplement(s) for figure 3:

**Source data 1.** Source data for cell counts and RT-qPCR data in **Figure 3**.

**Figure supplement 1.** NR2F2 deletion by Nr5a1-Cre impairs fetal Leydig cell (FLC) development.

**Figure supplement 1—source data 1.** Source data for cell counts, anogenital distance measurements and RT-qPCR data in **Figure 3—figure supplement 1**.

## NR2F2 is required for the initiation of FLC differentiation

FLCs differentiate from proliferating interstitial progenitors that progressively lose their mitotic ability, downregulate the transcription factors ARX and NR2F2, upregulate the master regulator of steroidogenesis NR5A1, and activate the expression of steroidogenesis-related genes (Inoue et al., 2016; Ademi et al., 2022; Miyabayashi et al., 2013). We wanted to determine which of these steps of FLC differentiation are NR2F2 dependent.

The transcription factor ARX is required in the pool of proliferating interstitial progenitors for FLC development (Miyabayashi et al., 2013). We first examined whether NR2F2 regulates the survival, proliferation, or identity of the ARX+ cell population. We found that the expression of Arx mRNA was not modified in Nr5a1-Cre; Nr2f2 flox/flox mutants (Figure 4—figure supplement 1A). The total number of gonadal cells, the percentage of ARX+ cells among the total number of gonadal cells, and the fraction of proliferating cells among the ARX+ population were similar to controls at E12.5 and E14.5 (Figure 4A, B, and E, Figure 4—figure supplement 1B–E). In addition, we did not find evidence of increased cell death in Nr5a1-Cre; Nr2f2 flox/flox mutants (Figure 4—figure supplement 1F–H). Together, these results indicate that ARX+ steroidogenic progenitor cells are present and proliferate at normal rates in Nr5a1-Cre; Nr2f2 flox/flox mutants.

As the ARX+ steroidogenic progenitors adopt an FLC identity, NR2F2 is progressively lost, and the nuclear receptor NR5A1 is strongly upregulated (Inoue et al., 2016; Ademi et al., 2022; van den Driesche et al., 2012). NR5A1 directs FLC differentiation by regulating the expression of genes associated with cholesterol metabolism and steroidogenesis (Morohashi et al., 2020; Shima et al., 2018; Jeyasuria et al., 2004; Buaas et al., 2012). In Nr5a1-Cre; Nr2f2 flox/flox mutants, the cells expressing high levels of NR5A1+ in the interstitial compartment were reduced compared to controls (Figure 4C–E, Figure 4—figure supplement 1I and J). This observation indicates that NR2F2 function is required for NR5A1 upregulation at the initial step of steroidogenic cell differentiation.

## DHH, PDGFRA, and NOTCH pathways are not impaired in Nr5a1-Cre; Nr2f2 flox/flox mutants

We next investigated the impact of Nr5a1-Cre; Nr2f2 flox/flox mutation on the activation of the signaling pathways involved in FLC differentiation. DHH secreted by Sertoli cells binds to its receptor PTCH1 expressed in interstitial progenitors and activates its target genes, including Gli1, to promote FLC differentiation (Yao et al., 2002; Barsoum and Yao, 2011). The expression levels of Dhh and of its target Gli1, a readout of Hedgehog pathway activation, were not modified in Nr5a1-Cre; Nr2f2 flox/flox mutants (Figure 3H, Figure 4F, Figure 3—figure supplement 1N). Gli1 transcripts exhibited a similar distribution in interstitial cells of control and Nr5a1-Cre; Nr2f2 flox/flox mutants at E14.5 (Figure 4G and H). PDGFA produced by Sertoli cells binds to the PDGFRA receptor expressed in interstitial progenitors to activate downstream signaling required for FLC development (Brennan et al., 2003; Schmahl
<figure><img src="image_6.png" /><figcaption>**Figure 4.** NR2F2 function is required for the initiation of fetal Leydig cell (FLC) differentiation. (A,B) Immunodetection of ARX and BrdU (arrows) on embryonic day 14.5 (E14.5) XY Nr2f2 flox/+ and Nr5a1-Cre; Nr2f2 flox/flox testes. (C,D) Immunodetection of NR5A1 on E14.5 XY Nr2f2 flox/+ and Nr5a1-Cre; Nr2f2 flox/flox testes. Arrows indicate strong expression of NR5A1 positive FLC. (E) Quantification of the percentage of ARX positive cells (number of ARX positive nuclei relative to the total number of nuclei labeled by DAPI), of the percentage of ARX positive cells labeled by BrdU (number of nuclei positive

Figure 4 continued on next page</figcaption></figure>
Figure 4 continued

for ARX and BrdU relative to the number of ARX positive nuclei) and of the number of NR5A1 positive cells per surface unit in E14.5 XY control (wild-type or *Nr2f2 flox/+*) and *Nr5a1-Cre; Nr2f2 flox/flox* testes. Each circle represents the mean percentage of ARX+ or ARX+/BrdU+ or NR5A1+ cells per surface unit of one individual measured on at least two sections per gonad. Data are shown as means ± SEM. Statistical significance was assessed by Mann-Whitney U two-tailed test. \* indicates p-value≤0.05; ns indicates p-value>0.05. (F) Quantification of *Gli1*, *Pdgfra*, *Pdgfb*, and *Sgpl1* transcripts after normalization to *Sdha* and *Tbp* in control (wild-type or *Nr2f2 flox/+*) and *Nr5a1-Cre; Nr2f2 flox/flox* by RT-qPCR at E14.5. Statistical significance was assessed by Mann-Whitney U two-tailed test. \* indicates p-value≤0.05; ns indicates p-value>0.05. (G,H) In situ hybridization detection of *Gli1* transcripts and immunodetection of HSD3B protein on E14.5 XY *Nr2f2 flox/+* and *Nr5a1-Cre; Nr2f2 flox/flox* testes. (I,J) Immunodetection of PDGFRA on E14.5 XY *Nr2f2 flox/flox* and *Nr5a1-Cre; Nr2f2 flox/flox* testes. (K,L) Immunodetection of PECAM-1 and NR2F2 on E12.5 XY *Nr2f2 flox/+* and *Nr5a1-Cre; Nr2f2 flox/flox* testes. PECAM-1 is expressed in germ cells and in endothelial cells (white arrows). (M) Quantification of Cdh5, Notch2, Heyl, and Hes1 transcripts after normalization to *Sdha* and *Tbp* in control (wild-type or *Nr2f2 flox/+*) and *Nr5a1-Cre; Nr2f2 flox/flox* by RT-qPCR at E14.5. Statistical significance was assessed by Mann-Whitney U two-tailed test. \* indicates p-value≤0.05; ns indicates p-value>0.05. Immunodetection data are representative of triplicate biological replicates. Scale bar = 100 µm.

The online version of this article includes the following source data and figure supplement(s) for figure 4:

**Source data 1.** Source data for cell counts and RT-qPCR data in **Figure 4**.

**Figure supplement 1.** NR2F2 function is required for the initiation of fetal Leydig cell (FLC) differentiation.

**Figure supplement 1—source data 1.** Source data for cell counts and RT-qPCR data in **Figure 4—figure supplement 1**.

et al., 2008). The expression levels of *Pdgfra* and *Pdgfb*, coding for two PDGFRA ligands expressed in developing testes (Brennan et al., 2003), were not altered in *Nr5a1-Cre; Nr2f2 flox/flox* mutants (Figures 3H and 4F). Transcript levels of *Pdgfra* were unchanged in *Nr5a1-Cre; Nr2f2 flox/flox* mutants (Figure 4F), and PDGFRA protein was detected at the plasma membrane of interstitial cells in both control and *Nr5a1-Cre; Nr2f2 flox/flox* mutant testes at E14.5 (Figure 4I and J). In addition, the expression of *Sgpl1*, a PDGFRA signaling target involved in steroidogenic differentiation (Schmahl et al., 2008), was not reduced in *Nr5a1-Cre; Nr2f2 flox/flox* mutants (Figure 4F). These observations indicate that the activities of Hedgehog and PDGFRA signaling, two pathways that positively regulate FLC differentiation, are not impaired in *Nr5a1-Cre; Nr2f2 flox/flox* mutants.

In addition to the positive signals, FLC differentiation is also negatively modulated by NOTCH signaling triggered by ligands expressed in vascular and perivascular cells. An increase in testicular endothelial cells results in a reduction in FLC numbers (Kumar and DeFalco, 2018; Tang et al., 2008; Liu et al., 2016; Defalco et al., 2013). The distribution and abundance of endothelial cells marked by PECAM-1 was not altered in *Nr5a1-Cre; Nr2f2 flox/flox* mutants (Figure 4K and L). In addition, Cdh5 transcript levels (a readout of the abundance of endothelial cells) were similar in controls and *Nr5a1-Cre; Nr2f2 flox/flox* mutants (Figure 4M). These observations indicate that the reduction in FLCs in *Nr5a1-Cre; Nr2f2 flox/flox* mutants was not associated with an increased population of endothelial cells. The receptor NOTCH2 expressed in interstitial cells is involved in restricting FLC differentiation (Kumar and DeFalco, 2018; Tang et al., 2008; Liu et al., 2016; Defalco et al., 2013). Notch2 mRNA levels were not altered in *Nr5a1-Cre; Nr2f2 flox/flox* mutants (Figure 4M). NOTCH signaling activates the expression of its target genes (that also act as effectors of the pathway), including Heyl, specifically expressed in interstitial cells and strongly upregulated upon vascular depletion (Kumar and DeFalco, 2018) and Hes1, expressed in interstitial cells and involved in restricting FLC differentiation (Tang et al., 2008; Liu et al., 2016). Transcripts of the NOTCH pathway targets Hes1 and Heyl were detected at similar levels in controls and *Nr5a1-Cre; Nr2f2 flox/flox* mutants (Figure 4M), indicating that the observed decrease in FLC numbers cannot be attributed to an increase in NOTCH signaling.

Together, these results indicate that NR2F2 deficiency does not impact the activity of the signaling pathways known to induce or repress FLC formation, suggesting that NR2F2 acts downstream or in parallel to these pathways to regulate FLC differentiation from interstitial progenitors.

## NR2F2 is required for FLC maturation

A small fraction of HSD3B positive cells formed in the absence of NR2F2 function, even in the case of *Wt1 CreERT2; Nr2f2 flox/flox* mutant gonads that exhibit widespread NR2F2 deletion in steroidogenic progenitors of coelomic and mesonephric origin. One possibility is that these cells are ALCs that have prematurely differentiated in *Nr2f2* mutant fetal testes. *Bhmt* (*Betaine-homocysteine methyltransferase*) is specifically expressed in ALC and absent in FLC (*Sararols et al., 2021*). *Bhmt* transcripts were not detected in control or *Nr2f2* mutant testes by RT-qPCR at E14.5, while they were present in
adult testes (Figure 5—figure supplement 1A), indicating that the steroidogenic cells of the mutants do not have adult characteristics.

To evaluate the steroidogenic capacities of the remaining FLC in *Nr2f2* mutant gonads, we analyzed the transcript levels of *Cyp11a1* and *Cyp17a1* normalized to the FLC number as quantified by HSD3B immunofluorescence (Figure 2N, Figure 3M; Kothandapani *et al.*, 2020). Normalized data showed reduced steroidogenic gene expression in *Nr5a1-Cre; Nr2f2 flox/flox* and in *Wt1<sup>CreERT2</sup>; *Nr2f2 flox/flox* mutant testes (Figure 5A and B), in agreement with the reduced HSD3B expression levels detected by immunofluorescence in *Nr2f2* mutant testes (Figures 2J–M and 3I–L). These observations suggest that FLC formed in the mutant testes have reduced steroidogenic function.

FLC differentiation is accompanied by a change in cell shape from spindle-shaped progenitors to round-shaped FLC and an increase in cytoplasmic volume (van den Driesche et al., 2012; Haider, 2004). HSD3B positive FLC present in *Nr5a1-Cre; Nr2f2 flox/flox* and in *Wt1<sup>CreERT2</sup>; *Nr2f2 flox/flox* mutants were smaller and more elongated than those in control littermates (Figure 5C–H, Figure 5—figure supplement 1). These cellular characteristics have been associated with immature FLC at the initial stages of FLC differentiation before the formation of large and round testosterone-producing FLC (van den Driesche et al., 2012; Haider, 2004).

Taken together, our results show that NR2F2 is required in interstitial steroidogenic progenitor cells for initiation of FLC differentiation but also for the subsequent step of FLC maturation leading to robust steroid production (Figure 5I).

Discussion

NR2F2 protein is expressed in interstitial cells of coelomic epithelium and mesonephric origin and is absent or detected at very low levels in FLC as soon as they are formed at E12.5. Our observations are consistent with NR2F2 positive cells being progenitors for FLC (Inoue et al., 2016; Ademi et al., 2022; Estermann et al., 2025) and indicate that NR2F2 is quickly downregulated upon differentiation of the fetal steroidogenic lineage, when the cells begin to synthesize steroid hormones.

We used two Cre lines to address the role of NR2F2 in the mouse fetal testis. *Nr2f2* mutants exhibit reduced FLC numbers together with a strong reduction in *Insl3* and steroidogenic gene expression, resulting in cryptorchidism and reduced anogenital distance. Importantly, the reduction of *Insl3* expression is sufficient to impair testis descent even when a fraction of FLC is still present. Our findings identify NR2F2 as an essential regulator of FLC and male reproductive system development in the mouse. This conclusion is confirmed by an independent *Nr2f2* conditional mutation resulting in a similar phenotype of FLC hypoplasia, cryptorchidism, and hypospadias (Estermann et al., 2025).

*Nr2f2* deletion using *Nr5a1-Cre* does not impair the survival or proliferation of the interstitial steroidogenic progenitor population, yet the FLC population is reduced in *Nr5a1-Cre; Nr2f2 flox/flox* mutant testes. We found that the expression of the master regulator of steroidogenic differentiation of NR5A1 is not upregulated in *Nr2f2* mutants, which is sufficient to account for the decrease in FLC numbers (Morohashi et al., 2020; Baba et al., 2018; Baba et al., 2014; Shima et al., 2018). ChIP-seq analysis of whole E14.5 testes has identified NR2F2 binding peaks in the regulatory region of *Nr5a1* (Estermann et al., 2025). In addition, NR2F2 regulates gene expression by directly interacting with NR5A1 in MA-10 cells, an in vitro model for immature ALCs (Mendoza-Villarroel et al., 2014b; Mendoza-Villarroel et al., 2014a; Di-Luoffo et al., 2022). It would be interesting to explore whether an interaction between NR2F2 and NR5A1 contributes to the strong upregulation of *Nr5a1* itself in differentiating FLCs and/or whether NR2F2 cooperates with other factors for the regulation of *Nr5a1*.

The activity of the main paracrine pathways regulating FLC differentiation was not significantly altered in *Nr5a1-Cre; Nr2f2 flox/flox* mutant testes, as shown by the expression of target genes of the DHH, PDGFRA, and NOTCH signaling. These observations indicate that NR2F2 is a permissive factor in steroidogenic progenitors acting downstream or in cooperation with the signaling pathways regulating FLC differentiation. NR2F2 expression has been found to be modulated by Hedgehog and/or NOTCH signaling in other cell types, and whether a similar regulation of *Nr2f2* expression by DHH and NOTCH signaling occurs in the steroidogenic progenitors remains unknown (Lee et al., 2006; Swift et al., 2014).

A fraction of FLC differentiates in *Nr2f2* mutants, including when progenitors of both coelomic epithelium and mesonephric origins are targeted. This observation indicates that additional factors cooperate with NR2F2 to regulate the transition from the steroidogenic progenitor state to the
<figure><img src="image_7.png" /><figcaption>**Figure 5.** NR2F2 function is required for fetal Leydig cell (FLC) maturation. (A) RT-qPCR quantification of *Cyp11a1* and *Cyp17a1* transcripts in control (wild-type or *Nr2f2 flox/+*) and *Nr5a1-Cre; Nr2f2 flox/flox* testes after normalization to *Sdha* and *Tbp* and to the number of FLC as quantified by HSD3B immunofluorescence at embryonic day 14.5 (E14.5). Data are shown as means ± SEM. Statistical significance was assessed by Mann-Whitney U two-tailed test. \* indicates p-value≤0.05; ns indicates p-value>0.05. (B) RT-qPCR quantification of *Cyp11a1* and *Cyp17a1* transcripts in *Nr2f2 flox/+* and *Figure 5 continued on next page*</figcaption></figure>
**Figure 5 continued**

Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> gonads after normalization to *Sdha* and *Tbp* and to the number of FLC as quantified by HSD3B immunofluorescence at E14.5. Data are shown as means ± SEM. Statistical significance was assessed by Mann-Whitney U two-tailed test. * indicates p-value≤0.05; ns indicates p-value>0.05. (C,D) Immunodetection of HSD3B on E14.5 XY Nr2f2<sup>flox/+</sup> and Nr5a1-Cre; Nr2f2<sup>flox/flox</sup> testes. (E,F) Immunodetection of HSD3B on E14.5 XY Nr2f2<sup>flox/+</sup> and Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> testes after tamoxifen was administered at E9.5 and E10.5. (G) Quantification of the area and circularity of HSD3B positive cells in two E14.5 control (wild-type or Nr2f2<sup>flox/+</sup>, 737 cells, gray violin plot) and three Nr5a1-Cre; Nr2f2<sup>flox/flox</sup> (486 cells, red violin plot) testes. Statistical significance was assessed by Mann-Whitney U two-tailed test. * indicates p-value≤0.05; ns indicates p-value>0.05. (H) Quantification of the area and circularity of HSD3B positive cells in three E14.5 control (wild-type or Nr2f2<sup>flox/+</sup>, 1485 cells, gray violin plot) and three Wt1<sup>CreERT2</sup>; Nr2f2<sup>flox/flox</sup> (474 cells, blue violin plot) testes. Statistical significance was assessed by Mann-Whitney U two-tailed test. * indicates p-value≤0.05; ns indicates p-value>0.05. (I) Summary figure: NR2F2 is expressed in spindle-shaped interstitial steroidogenic progenitors together with ARX and is progressively downregulated upon FLC differentiation. NR2F2 is required for the initiation of FLC differentiation (marked by the upregulation of NR5A1) and for FLC maturation (characterized by the increase in cytoplasmic volume and the high expression of steroidogenic enzymes and *Ins13*). Immunodetection data are representative of triplicate biological replicates. Scale bar = 10 µm.

The online version of this article includes the following source data and figure supplement(s) for figure 5:

**Source data 1.** Source data for cell measurements and RT-qPCR data in **Figure 5**.

**Figure supplement 1.** NR2F2 function is required for fetal Leydig cell (FLC) maturation.

**Figure supplement 1—source data 1.** Source data for RT-qPCR data in **Figure 5—figure supplement 1**.

differentiating FLC. The homeodomain protein ARX is expressed in *Nr2f2* mutant steroidogenic progenitors and is a good candidate to be such a factor. Arx mutants exhibit decreased FLC numbers without defects in paracrine signals driving their differentiation, similar to *Nr2f2* mutants (Miyabayashi et al., 2013).

FLC and ALC are morphologically, transcriptionally, and functionally distinct (Shima, 2019; Sararols et al., 2021), yet NR2F2 function is required for the differentiation of both lineages. In contrast to the situation in the fetal testis, NR2F2 is maintained in cells that have started to express steroidogenesis genes in the postnatal testis (Inoue et al., 2016; Ademi et al., 2022; Mendoza-Villarroel et al., 2014b). NR2F2 regulates the transition from the adult progenitor Leydig cell (characterized by their elongated shape, their ability to proliferate, and a low level of testosterone synthesis) to the immature ALC (characterized by their round shape, low mitotic activity, and increased testosterone production) (Qin et al., 2008; Haider, 2004). In agreement with its in vivo role promoting maturation along the ALC lineage, NR2F2 cooperates with NR5A1 and GATA4 to activate the transcription of *Ins13*, *Star* (encoding the cholesterol transporter), and *Amhr2* (Mendoza-Villarroel et al., 2014b; Mendoza-Villarroel et al., 2014a; Di-Luoffo et al., 2022; Mehanovic et al., 2021; Mehanovic et al., 2022) in MA-10 cells.

Here, we found that the steroidogenic cells that differentiate in *Nr2f2* mutant fetal testes exhibit a small size, an elongated shape, and reduced steroidogenic gene expression, features of the earliest stages of FLC differentiation (Wen et al., 2016; van den Driesche et al., 2012; Haider, 2004). A similar phenotype is obtained when *Nr5a1* is deleted after the onset of FLC differentiation (Buaas et al., 2012). This suggests that in addition to controlling the initial engagement of steroidogenic progenitors into the FLC lineage and similar to the situation in the postnatal testis, NR2F2 promotes FLC maturation possibly by directly regulating the initial expression of genes involved in steroidogenesis. In agreement with this hypothesis, NR2F2 binding peaks are found in the regulatory regions of genes involved in lipid metabolism and cholesterol biosynthesis in ChIP-seq analysis of whole E14.5 testes (Estermann et al., 2025).

NR2F2 is required for additional aspects of fetal testis morphogenesis and differentiation. Testis cords are enlarged and abnormally shaped in *Nr2f2* mutants. Testis cord development involves the formation of Sertoli-germ cell masses after E11.5 and their subsequent partition by growing wedges of interstitial cells and associated vascular branches at E12.5 (Cool et al., 2012). How NR2F2-dependent regulation of interstitial cell adhesion or migration contributes to this process will be the aim of future research.

Pathogenic variants in NR2F2 have been associated with congenital malformations, including congenital heart disease, congenital diaphragmatic hernia, and syndromic 46,XX testicular or ovo-testicular difference/disorder in sex development (DSD) (Polvani et al., 2019; Bashamboo et al., 2018). More recently, defects in the external genitalia (micropenis, hypospadias) and cryptorchidism have been associated with rare heterozygous variants in NR2F2 in 46,XY patients (Zidoune et al.,
2022; Ganapathi et al., 2023; Wankanit et al., 2024). These phenotypes can be attributed to defects in testosterone-dependent masculinization and INSL3-dependent testis descent during gestation and could be explained by a failure of FLC differentiation in the fetal testis. NR2F2 is abundantly expressed in interstitial cells of fetal human testes, a population that likely contains the progenitors for FLC (Kilcoyne et al., 2014; van den Driesche et al., 2012; Lottrup et al., 2014; Taelman et al., 2024). The present work demonstrating that NR2F2 is required in the steroidogenic progenitors of the murine fetal testis for the initiation and progression of FLC differentiation provides an entry point in understanding the etiology of 46,XY DSD associated with pathogenic NR2F2 variants.

## Materials and methods

### Key resources table

<table><thead><tr><th>Reagent type (species) or resource</th><th>Designation</th><th>Source or reference</th><th>Identifiers</th><th>Additional information</th></tr></thead><tbody><tr><td>Gene (Mus musculus)</td><td>Nr2f2</td><td>MGI</td><td>MGI:1352452</td><td></td></tr><tr><td>Genetic reagent (Mus musculus)</td><td>Nr2f2<sup>tm1Vc</sup></td><td>Dr. M. Vasseur-Cognet</td><td>MGI:3578106</td><td></td></tr><tr><td>Genetic reagent (Mus musculus)</td><td>Wt1<sup>tm2(cre/ERT2)Wtp</sup></td><td>Dr. William T Pu</td><td>MGI:7528785</td><td></td></tr><tr><td>Genetic reagent (Mus musculus)</td><td>Tg(Nr5a1-cre)2Klp</td><td>Dr. Keith L Parker</td><td>MGI:5493455</td><td></td></tr><tr><td>Chemical compound</td><td>Tamoxifen</td><td>Sigma-Aldrich</td><td>T5648</td><td>200 mg/kg body weight</td></tr><tr><td>Chemical compound</td><td>5-Bromo-2'-deoxy-uridine</td><td>Sigma-Aldrich</td><td>B5002</td><td>50 mg/kg body weight</td></tr><tr><td>Chemical compound</td><td>Paraformaldehyde</td><td>EMS</td><td>15710-S</td><td>4% in PBS</td></tr><tr><td>Antibody</td><td>Anti-ACTA2 (Mouse monoclonal)</td><td>Gift from Dr. Chaponnier</td><td></td><td>IF (1:500)</td></tr><tr><td>Antibody</td><td>Anti- Activated Caspase 3 (Rabbit polyclonal)</td><td>R&D Systems</td><td>AF835 (RRID:AB_2243952)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti- AMH (Mouse monoclonal)</td><td>Bio-Rad</td><td>MCA2246 (RRID:AB_2226471)</td><td>IF (1:50)</td></tr><tr><td>Antibody</td><td>Anti- ARX (Rabbit polyclonal)</td><td>Gift from Pr. Morohashi and Dr. Inoue</td><td></td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-COL4A1 (Rabbit polyclonal)</td><td>Abcam</td><td>ab19808 (RRID:AB_445160)</td><td>IF (1:400)</td></tr><tr><td>Antibody</td><td>Anti-CYP11A1 (Rabbit polyclonal)</td><td>Gift from Dr. Wilhelm</td><td></td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-GATA4 (Goat polyclonal)</td><td>Santa Cruz Biotechnology</td><td>Sc-1237 (RRID:AB_2108747)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-GFP (Chicken polyclonal)</td><td>Abcam</td><td>Ab13970 (RRID:AB_300798)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-HSD3B (Goat polyclonal)</td><td>Santa Cruz Biotechnology</td><td>Sc-30820 (RRID:AB_2279878)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-HSD3B (Rabbit polyclonal)</td><td>Invitrogen</td><td>PA5-76669 (RRID:AB_2720396)</td><td>IF (1:500)</td></tr><tr><td>Antibody</td><td>Anti-Ki67 (Rabbit monoclonal)</td><td>Spring Bioscience</td><td>M3062 (RRID:AB_11219741)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-LAMA1 (Rabbit polyclonal)</td><td>Sigma-Aldrich</td><td>L9393 (RRID:AB_477163)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-NESTIN (Rabbit)</td><td>BioLegend</td><td>839801 (RRID:AB_2565443)</td><td>IF (1:1000)</td></tr><tr><td>Antibody</td><td>Anti-NR2F2 (Mouse monoclonal)</td><td>R&D Systems</td><td>PP-H7147-00 (RRID:AB_2155627)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-NR5A1 (Rabbit polyclonal)</td><td>Cosmo Bio</td><td>KO611(RRID:AB_2861370)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-PDGFRA (Rabbit polyclonal)</td><td>Santa Cruz Biotechnology</td><td>SC-338 (RRID:AB_631064)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-PECAM-1 (Goat polyclonal)</td><td>Santa Cruz Biotechnology</td><td>Sc-1506 (RRID:AB_2161037)</td><td>IF (1:200)</td></tr><tr><td>Antibody</td><td>Anti-RUNX1 (Rabbit monoclonal)</td><td>Abcam</td><td>ab92336 (RRID:AB_2049267)</td><td>IF (1:500)</td></tr></tbody></table>

Continued on next page
Continued

<table><thead><tr><th>Reagent type (species) or resource</th><th>Designation</th><th>Source or reference</th><th>Identifiers</th><th>Additional information</th></tr></thead><tbody><tr><td>Antibody</td><td>Anti-SOX9 (Rabbit polyclonal)</td><td>Sigma-Aldrich</td><td>HPA001758 (RRID:AB_1080067)</td><td>IF (1:250)</td></tr><tr><td>Antibody</td><td>Anti-WT1 (Goat polyclonal)</td><td>R&D Systems</td><td>AF5729 (RRID:AB_2216239)</td><td>IF (1:200)</td></tr><tr><td>Commercial assay or kit</td><td>BrdU detection kit</td><td>Roche</td><td>11 296 736 001</td><td></td></tr><tr><td>Commercial assay or kit</td><td>RNAscope Multiplex Fluorescent Reagent Kit v2</td><td>Bio-Techne</td><td>323110</td><td></td></tr><tr><td>Sequence-based reagent</td><td>Gli1 probe</td><td>Bio-Techne</td><td>311001</td><td></td></tr><tr><td>Commercial assay or kit</td><td>RNeasy Micro Kit</td><td>QIAGEN</td><td>74004</td><td></td></tr><tr><td>Commercial assay or kit</td><td>SYBR Green I Master</td><td>Roche</td><td>04887352001</td><td></td></tr><tr><td>Software, algorithm</td><td>RefFinder</td><td>https://www.ciidirsinaloa.com.mx/RefFinder-master/?type=reference</td><td></td><td></td></tr><tr><td>Software, algorithm</td><td>OMERO</td><td>https://www.openmicroscopy.org/omero/</td><td></td><td></td></tr><tr><td>Software, algorithm</td><td>GraphPad Prism</td><td>Graphpad Software, Inc, La Jolla, CA, USA</td><td>GraphPad Prism 10.2.1</td><td></td></tr></tbody></table>

## Mouse strains and genotyping

The experiments described herein were carried out in compliance with the guidelines of the French Regulations for Animal Care and with the approval of the local Ethical Committee (APAFIS APAFIS#12789-2017121515109323 v1 and APAFIS#44072-2023061915491990 v5). Mouse lines were kept on a mixed background B6CBAF1/JRj. The *Nr2f2*<sup>tm1Vc</sup> line where *Nr2f2* exon 1 sequences (encoding the DNA binding domain) are deleted upon CRE-mediated recombination (referred to as *Nr2f2*<sup>flox</sup>), the knock-in *Wt1*<sup>tm2(cre/ERT2)Wtp</sup> line where tamoxifen-inducible Cre<sup>ERT2</sup> is produced by WT1 expressing cells (referred to as *Wt1*<sup>CreERT2</sup>), and the transgenic *Tg(Nr5a1-cre)2KIp* line where Cre expression is driven by *Nr5a1* regulatory sequences (referred to as *Nr5a1-Cre*) were genotyped as previously described (Bardoux et al., 2005; Zhou et al., 2008; Bingham et al., 2006). *Wt1*<sup>CreERT2</sup>; *Nr2f2*<sup>flox/+</sup> or *Nr5a1-Cre<sup>tg/0</sup>*; *Nr2f2*<sup>flox/+</sup> males were crossed with *Nr2f2*<sup>flox/+</sup> females to obtain mutant embryos at different stages. Embryos were named controls (*Nr2f2*<sup>+/+</sup> or *Nr2f2*<sup>flox/+</sup>) or mutants (*Wt1*<sup>CreERT2</sup>; *Nr2f2*<sup>flox/flox</sup> or *Nr5a1-Cre<sup>tg/0</sup>*; *Nr2f2*<sup>flox/flox</sup>). Genotypes of mice and embryos were determined using PCR assays on lysates from ear biopsies or tail tips. Genotyping primers are listed in Appendix 1. To activate the Cre<sup>ERT2</sup> recombinase in embryos, tamoxifen (TAM, T5648, Sigma-Aldrich) was directly diluted in corn oil to a concentration of 40 mg/mL, and two TAM treatments (200 mg/kg body weight) were administered to pregnant females by oral gavage at E9.5 and E10.5. For proliferation assays, 5-bromo-2'-deoxy-uridine (BrdU) (B5002, Sigma-Aldrich) diluted to a concentration of 10 mg/mL in sterile H<sub>2</sub>O was administered to the pregnant females (50 mg/kg body weight) by intraperitoneal injection, and pregnant females and their embryos were humanely killed after 3 hr and 30 min. The day when a vaginal plug was found was designated as E0.5. E11.5–E12.5 embryos were staged by counting the number of ts with 18 ts corresponding to E11.5.

## Immunofluorescence staining and in situ hybridization

Embryos were fixed in 4% (wt/vol) paraformaldehyde (PFA, 15,710-S, EMS) overnight, processed for paraffin embedding, and sectioned into 5-µm-thick sections. Immunofluorescence and DAPI staining were performed as described in Tang et al., 2020. Proliferation analysis was performed by using a BrdU detection kit (11 296 736 001, Roche). Gli1 mRNA was detected with the RNAscope technology (probe 311001) according to Advanced Cell's instructions using the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay. Images were obtained on a motorized Axio Imager Z1 microscope (Zeiss) coupled with an AxioCam MRm camera (Zeiss) and processed with Fiji (Bethesda, MD, USA). The DAPI staining marking the nuclei was adjusted to visualize the tissues and may vary between samples.
However, for the immunofluorescence analysis, the exposure time of the acquisition of the fluorescent signal was identical in the same experiment to allow comparison between controls and mutants. Images were assembled using the open-source software platform OMERO (https://www.openmicroscopy.org/omero/). Antibodies are listed in the Key resources table. At least three embryos of each genotype were analyzed for each marker.

## Cell quantifications

The gonadal area for each section was measured by creating a gonadal region of interest (ROI) drawn manually in Fiji. The number of HSD3B positive cells (2–493 cells per section depending on the stages and genotypes), SOX9 positive cells (231–882 cells per section depending on the stages and genotypes), NR5A1 positive cells (3–256 cells per section depending on the stages and genotypes), ARX positive cells (666–2487 cells per section depending on the stages and genotypes), ARX positive cells that had incorporated BrdU (232–656 cells per section depending on the stages and genotypes), activated caspase 3 positive cells (0–6 cells per section depending on the stages and genotypes), or the number of nuclei labeled by DAPI (1582–4279 cells per section depending on the stages and genotypes) were counted manually in the entire gonadal section using the Cell Counter Plugin from Fiji. For each genotype, gonads of three or four biological replicates were analyzed. Two to three coronal sections spaced by at least 30 µm in the medial regions of the gonads were analyzed for each individual. Statistical significance was assessed by Mann-Whitney U two-tailed test. * indicates p-value≤0.05; ns indicates p-value>0.05.

## Quantification of area and circularity of HSD3B positive cells

Gonadal (ROI) was drawn manually, and HSD3B positive cells were segmented using the Stardist Deep Learning plugin of Fiji with a minimum area of 20 µm² to remove small particles. The area and circularity of each segmented cell were measured with Fiji. Circularity = $4\pi*area/perimeter^2$. A value of 1.0 indicates a perfect circle. As the value approaches 0.0, it indicates an increasingly elongated shape. For each genotype, two to three biological replicates were analyzed. The data are shown as violin plots (with median and quartiles) for control and mutant samples.

## RNA extraction and quantitative PCR analysis

Individual gonads were dissected from the mesonephros in PBS, snap-frozen in liquid nitrogen, and kept at –80°C. RNA was extracted by RNeasy Micro Kit (74004, QIAGEN) and reverse-transcribed by M-MLV reverse transcriptase (M170A, Promega). The cDNA was used as a template for quantitative PCR analysis using the SYBR Green I Master (04887352001, Roche) and a LightCycler 480 System (Roche). Primer sequences are listed in Appendix 1.

All biological replicates of different genotypes (N=3–9) were run in the same plate and run as duplicate technical replicates. Relative gene expression of each gonad was normalized to the expression of the housekeeping genes *Shda* and *Tbp* (*Yokoyama et al., 2018*) by the 2-ΔΔCt calculation method. GeNorm, BestKeeper algorithms, and the comparative delta-Ct method provided through the online tool RefFinder (https://www.ciiidsrinaloa.com.mx/RefFinder-master/?type=reference#) were used to confirm reference gene stability in the experimental datasets. Fold change in gene expression was obtained by dividing the normalized gene expression in gonads of a given genotype by the mean of the normalized gene expression in control gonads. Data are shown as means ± SEM. Statistical significance was assessed by Mann-Whitney U two-tailed test (GraphPad Prism 10.2.1). * indicates p-value≤0.05; ns indicates p-value>0.05.

## Acknowledgements

We acknowledge the help from members of the Experimental Histopathology Platform, the PRISM Imaging Platform, and the Animal House at iBV (Institut de Biologie Valrose, Université Côte d'Azur, CNRS, Inserm, iBV, France). We are grateful to members of the A Schedl, MC Chaboissier, and S Nef groups for helpful discussions and to D Wilhelm for critical reading of the manuscript. We are indebted to Dr. Chaponnier, Dr. Inoue, Pr Morohashi, Dr. M Vasseur-Cognet, and Dr. Wilhelm for sharing mouse lines and reagents. This research was funded by Agence Nationale de la Recherche ANR-23-CE14-0012, Heterosex, and by a scholarship from the China Scholarship Council (to FT).
## Additional information

### Funding

<table><thead><tr><td>Funder</td><td>Grant reference number</td><td>Author</td></tr></thead><tbody><tr><td>Agence Nationale de la Recherche</td><td>ANR-23-CE14-0012</td><td>Marie-Christine Chaboissier</td></tr><tr><td>China Scholarship Council</td><td></td><td>Furong Tang</td></tr><tr><td>Agence Nationale de la Recherche</td><td>Heterosex</td><td>Marie-Christine Chaboissier</td></tr></tbody></table>

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

### Author contributions

Aitana Perea-Gomez, Conceptualization, Formal analysis, Supervision, Investigation, Methodology, Writing - original draft, Writing - review and editing; Natividad Bellido Carreras, Magali Dhellemmes, Furong Tang, Coralie Le Gallo, Investigation; Marie-Christine Chaboissier, Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

### Author ORCIDs

Aitana Perea-Gomez https://orcid.org/0000-0002-7788-038X

Natividad Bellido Carreras http://orcid.org/0000-0003-1207-0711

Magali Dhellemmes https://orcid.org/0000-0001-7983-3221

Furong Tang http://orcid.org/0000-0001-7546-7500

Coralie Le Gallo http://orcid.org/0009-0000-5079-4791

Marie-Christine Chaboissier https://orcid.org/0000-0003-0934-8217

### Ethics

The experiments described herein were carried out in compliance with the relevant institutional and French animal welfare laws, guidelines, and policies (APAFIS APAFIS#12789-2017121515109323 v1 and APAFIS#44072-2023061915491990 v5).

### Peer review material

Reviewer #1 (Public review): https://doi.org/10.7554/eLife.103783.3.sa1

Reviewer #2 (Public review): https://doi.org/10.7554/eLife.103783.3.sa2

Author response https://doi.org/10.7554/eLife.103783.3.sa3

## Additional files

### Supplementary files

MDAR checklist

### Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

## References

* Ademi H, Djari C, Mayère C, Neirijnck Y, Sararols P, Rands CM, Stévant I, Conne B, Nef S. 2022. Deciphering the origins and fates of steroidogenic lineages in the mouse testis. *Cell Reports* **39**:110935. DOI: https://doi.org/10.1016/j.celrep.2022.110935, PMID: 35705036

* Amato CM, Yao HHC, Zhao F. 2022. One tool for many jobs: Divergent and conserved actions of androgen signaling in male internal reproductive tract and external genitalia. *Frontiers in Endocrinology* **13**:910964. DOI: https://doi.org/10.3389/fendo.2022.910964, PMID: 35846302

* Armstrong JF, Pritchard-Jones K, Bickmore WA, Hastie ND, Bard JB. 1993. The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo. *Mechanisms of Development* **40**:85-97. DOI: https://doi.org/10.1016/0925-4773(93)90090-k, PMID: 8382938

* Baba T, Otake H, Sato T, Miyabayashi K, Shishido Y, Wang C-Y, Shima Y, Kimura H, Yagi M, Ishihara Y, Hino S, Ogawa H, Nakao M, Yamazaki T, Kang D, Ohkawa Y, Suyama M, Chung B-C, Morohashi K-I. 2014. Glycolytic
genes are targets of the nuclear receptor Ad4BP/SF-1. *Nature Communications* **5**:3634. DOI: https://doi.org/10.1038/ncomms4634, PMID: 24727981

Baba T, Otake H, Inoue M, Sato T, Ishihara Y, Moon J-Y, Tsuchiya M, Miyabayashi K, Ogawa H, Shima Y, Wang L, Sato R, Yamazaki T, Suyama M, Nomura M, Choi MH, Ohkawa Y, Morohashi K-I. 2018. Ad4BP/SF-1 regulates cholesterol synthesis to boost the production of steroids. *Communications Biology* **1**:18. DOI: https://doi.org/10.1038/s42003-018-0020-z, PMID: 30271905

Bardoux P, Zhang P, Flamez D, Perilhou A, Lavin TA, Tanti J-F, Hellemans K, Gomas E, Godard C, Andreelli F, Buccheri MA, Kahn A, Le Marchand-Brustel Y, Burcelin R, Schuit F, Vasseur-Cognet M. 2005. Essential role of chicken ovalbumin upstream promoter-transcription factor II in insulin secretion and insulin sensitivity revealed by conditional gene knockout. *Diabetes* **54**:1357-1363. DOI: https://doi.org/10.2337/diabetes.54.5.1357, PMID: 15855320

Barsoum IB, Bingham NC, Parker KL, Jorgensen JS, Yao HHC. 2009. Activation of the Hedgehog pathway in the mouse fetal ovary leads to ectopic appearance of fetal Leydig cells and female pseudohermaphroditism. *Developmental Biology* **329**:96-103. DOI: https://doi.org/10.1016/j.ydbio.2009.02.025, PMID: 19268447

Barsoum I, Yao HHC. 2011. Redundant and differential roles of transcription factors Gli1 and Gli2 in the development of mouse fetal Leydig cells. *Biology of Reproduction* **84**:894-899. DOI: https://doi.org/10.1095/biolreprod.110.088997, PMID: 21209421

Bashamboo A, Eozenou C, Jorgensen A, Bignon-Topalovic J, Siffroi J-P, Hyon C, Tar A, Nagy P, Sólyom J, Halász Z, Paye-Jaouen A, Lambert S, Rodriguez-Buritica D, Bertalan R, Martinerie L, Rajpert-De Meyts E, Achermann JC, McElreavey K. 2018. Loss of function of the nuclear receptor NR2F2, encoding COUP-TF2, causes testis development and cardiac defects in 46,XX children. *American Journal of Human Genetics* **102**:487-493. DOI: https://doi.org/10.1016/j.ajhg.2018.01.021, PMID: 29478779

Bielinska M, Seehra A, Toppari J, Heikinheimo M, Wilson DB. 2007. GATA-4 is required for sex steroidogenic cell development in the fetal mouse. *Developmental Dynamics* **236**:203-213. DOI: https://doi.org/10.1002/dvdy.21004, PMID: 17096405

Bingham NC, Verma-Kurvari S, Parada LF, Parker KL. 2006. Development of a steroidogenic factor 1/Cre transgenic mouse line. *Genesis* **44**:419-424. DOI: https://doi.org/10.1002/dvg.20231, PMID: 16937416

Brennan J, Tilmann C, Capel B. 2003. Pdgfr-alpha mediates testis cord organization and fetal Leydig cell development in the XY gonad. *Genes & Development* **17**:800-810. DOI: https://doi.org/10.1101/gad.1052503, PMID: 12651897

Buaas FW, Gardiner JR, Clayton S, Val P, Swain A. 2012. In vivo evidence for the crucial role of SF1 in steroid-producing cells of the testis, ovary and adrenal gland. *Development* **139**:4561-4570. DOI: https://doi.org/10.1242/dev.087247, PMID: 23136395

Carré G-A, Siggers P, Xipolita M, Brindle P, Lutz B, Wells S, Greenfield A. 2018. Loss of p300 and CBP disrupts histone acetylation at the mouse Sry promoter and causes XY gonadal sex reversal. *Human Molecular Genetics* **27**:190-198. DOI: https://doi.org/10.1093/hmg/ddx398, PMID: 29145650

Clark AM, Garland KK, Russell LD. 2000. Desert hedgehog (Dhh) gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules. *Biology of Reproduction* **63**:1825-1838. DOI: https://doi.org/10.1095/biolreprod63.6.1825, PMID: 11090455

Cool J, DeFalco T, Capel B. 2012. Testis formation in the fetal mouse: dynamic and complex de novo tubulogenesis. *Wiley Interdisciplinary Reviews. Developmental Biology* **1**:847-859. DOI: https://doi.org/10.1002/wdev.62, PMID: 23799626

Cui S, Ross A, Stallings N, Parker KL, Capel B, Quaggin SE. 2004. Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout mice. *Development* **131**:4095-4105. DOI: https://doi.org/10.1242/dev.01266, PMID: 15289436

DeFalco T, Takahashi S, Capel B. 2011. Two distinct origins for Leydig cell progenitors in the fetal testis. *Developmental Biology* **352**:14-26. DOI: https://doi.org/10.1016/j.ydbio.2011.01.011, PMID: 21255566

Defalco T, Saraswathula A, Briot A, Iruela-Arispe ML, Capel B. 2013. Testosterone levels influence mouse fetal Leydig cell progenitors through notch signaling. *Biology of Reproduction* **88**:91. DOI: https://doi.org/10.1095/biolreprod.112.106138, PMID: 23467742

Di-Luoffo M, Pierre KJ, Robert NM, Girard MJ, Tremblay JJ. 2022. The nuclear receptors SF1 and COUP-TFII cooperate on the Insl3 promoter in Leydig cells. *Reproduction* **164**:31-40. DOI: https://doi.org/10.1530/REP-22-0109, PMID: 35666805

Estermann MA, Grimm SA, Kitakule AS, Rodriguez KF, Brown PR, McClelland K, Amato CM, Yao HH-C. 2025. NR2F2 regulation of interstitial cell fate in the embryonic mouse testis and its impact on differences of sex development. *Nature Communications* **16**:3987. DOI: https://doi.org/10.1038/s41467-025-59183-6, PMID: 40295478

Ganapathi M, Matsuoka LS, March M, Li D, Brokamp E, Benito-Sanz S, White SM, Lachlan K, Ahimaz P, Sewda A, Bastarache L, Thomas-Wilson A, Stoler JM, Bramswig NC, Baptista J, Stals K, Demurger F, Cogne B, Isidor B, Bedeschi MF, et al. 2023. Heterozygous rare variants in NR2F2 cause a recognizable multiple congenital anomaly syndrome with developmental delays. *European Journal of Human Genetics* **31**:1117-1124. DOI: https://doi.org/10.1038/s41431-023-01434-5, PMID: 37500725

Gao F, Maiti S, Alam N, Zhang Z, Deng JM, Behringer RR, Lécureuil C, Guillou F, Huff V. 2006. The Wilms tumor gene, Wt1, is required for Sox9 expression and maintenance of tubular architecture in the developing testis. *PNAS* **103**:11987-11992. DOI: https://doi.org/10.1073/pnas.0600994103, PMID: 16877546

Haider SG. 2004. Cell biology of Leydig cells in the testis. *International Review of Cytology* **233**:181-241. DOI: https://doi.org/10.1016/S0074-7696(04)33005-6, PMID: 15037365
Hutson JM, Li R, Southwell BR, Newgreen D, Cousinery M. 2015. Regulation of testicular descent. *Pediatric Surgery International* **31**:317–325. DOI: https://doi.org/10.1007/s00383-015-3673-4, PMID: 25690562

Inoue M, Shima Y, Miyabayashi K, Tokunaga K, Sato T, Baba T, Ohkawa Y, Akiyama H, Suyama M, Morohashi K. 2016. Isolation and characterization of fetal leydig progenitor cells of male mice. *Endocrinology* **157**:1222–1233. DOI: https://doi.org/10.1210/en.2015-1773, PMID: 26697723

Inoue M, Baba T, Takahashi F, Terao M, Yanai S, Shima Y, Saito D, Sugihara K, Miura T, Takada S, Suyama M, Ohkawa Y, Morohashi K-I. 2022. Tmsb10 triggers fetal Leydig differentiation by suppressing the RAS/ERK pathway. *Communications Biology* **5**:974. DOI: https://doi.org/10.1038/s42003-022-03941-5, PMID: 36109592

Jeyasuria P, Ikeda Y, Jamin SP, Zhao L, De Rooij DG, Themmen APN, Behringer RR, Parker KL. 2004. Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function. *Molecular Endocrinology* **18**:1610–1619. DOI: https://doi.org/10.1210/me.2003-0404, PMID: 15118069

Karl J, Capel B. 1998. Sertoli cells of the mouse testis originate from the coelomic epithelium. *Developmental Biology* **203**:323–333. DOI: https://doi.org/10.1006/dbio.1998.9068, PMID: 9808783

Kilcoyne KR, Smith LB, Atanassova N, Macpherson S, McKinnell C, van den Driesche S, Jobling MS, Chambers TJG, De Gendt K, Verhoeven G, O'Hara L, Platts S, Renato de Franca L, Lara NLM, Anderson RA, Sharpe RM. 2014. Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells. *PNAS* **111**:E1924–E1932. DOI: https://doi.org/10.1073/pnas.1320735111, PMID: 24753613

Kothandapani A, Lewis SR, Noel JL, Zacharski A, Krellwitz K, Baines A, Winske S, Vezina CM, Kaftanovskaya EM, Agoulnik AI, Merton EM, Cohn MJ, Jorgensen JS. 2020. GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation. *PLOS Genetics* **16:e1008810. DOI: https://doi.org/10.1371/journal.pgen.1008810, PMID: 32497091

Kumar DL, DeFalco T. 2018. A perivascular niche for multipotent progenitors in the fetal testis. *Nature Communications* **9**:4519. DOI: https://doi.org/10.1038/s41467-018-06996-3, PMID: 30375389

Lee K, Jeong J, Kwak I, Yu C-T, Lanske B, Soegiarto DW, Toftgard R, Tsai M-J, Tsai S, Lydon JP, DeMayo FJ. 2006. Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus. *Nature Genetics* **38**:1204–1209. DOI: https://doi.org/10.1038/ng1874, PMID: 16951680

Li S-Y, Gu X, Heinrich A, Hurley EG, Capel B, DeFalco T. 2021. Loss of Mafb and Maf distorts myeloid cell ratios and disrupts fetal mouse testis vascularization and organogenesis†. *Biology of Reproduction* **105**:958–975. DOI: https://doi.org/10.1093/biolre/ioab098, PMID: 34007995

Liu C, Rodriguez K, Yao HHC. 2016. Mapping lineage progression of somatic progenitor cells in the mouse fetal testis. *Development* **143**:3700–3710. DOI: https://doi.org/10.1242/dev.135756, PMID: 27621062

Lottrup G, Nielsen JE, Maroun LL, Møller LMA, Yassin M, Leffers H, Skakkebæk NE, Rajpert-De Meyts E. 2014. Expression patterns of DLK1 and INSL3 identify stages of Leydig cell differentiation during normal development and in testicular pathologies, including testicular cancer and Klinefelter syndrome. *Human Reproduction* **29**:1637–1650. DOI: https://doi.org/10.1093/humrep/deu124, PMID: 24908673

Manuylov NL, Smagulova FO, Leach L, Tevosian SG. 2008. Ovarian development in mice requires the GATA4-FOG2 transcription complex. *Development* **135**:3731–3743. DOI: https://doi.org/10.1242/dev.024653, PMID: 18927154

Manuylov NL, Zhou B, Ma Q, Fox SC, Pu WT, Tevosian SG. 2011. Conditional ablation of Gata4 and Fog2 genes in mice reveals their distinct roles in mammalian sexual differentiation. *Developmental Biology* **353**:229–241. DOI: https://doi.org/10.1016/j.ydbio.2011.02.032, PMID: 21385577

Mayère C, Regard V, Perea-Gomez A, Bunce C, Neirijnck Y, Djari C, Bellido-Carreras N, Sararols P, Reeves R, Greenaway S, Simon M, Siggers P, Condrea D, Kühne F, Gantar I, Tang F, Stévant I, Batti L, Ghyselinck NB, Wilhelm D, et al. 2022. Origin, specification and differentiation of a rare supporting-like lineage in the developing mouse gonad. *Science Advances* **8:eabm0972. DOI: https://doi.org/10.1126/sciadv.abm0972, PMID: 35613264

Mehanovic S, Mendoza-Villarroel RE, de Mattos K, Talbot P, Viger RS, Tremblay JJ. 2021. Identification of novel genes and pathways regulated by the orphan nuclear receptor COUP-TFII in mouse MA-10 Leydig cells†. *Biology of Reproduction* **105**:1283–1306. DOI: https://doi.org/10.1093/biolre/ioab131, PMID: 34225363

Mehanovic S, Pierre KJ, Viger RS, Tremblay JJ. 2022. Chicken ovalbumin upstream promoter transcription factor type II interacts and functionally cooperates with GATA4 to regulate anti-Müllerian hormone receptor type 2 transcription in mouse MA-10 Leydig cells. *Andrology* **10**:1411–1425. DOI: https://doi.org/10.1111/andr.13266, PMID: 35973717

Mendoza-Villarroel RE, Di-Luoffo M, Camiré E, Giner XC, Brousseau C, Tremblay JJ. 2014a. The INSL3 gene is a direct target for the orphan nuclear receptor, COUP-TFII, in Leydig cells. *Journal of Molecular Endocrinology* **53**:43–55. DOI: https://doi.org/10.1530/JME-13-0290, PMID: 24780841

Mendoza-Villarroel RE, Robert NM, Martin LJ, Brousseau C, Tremblay JJ. 2014b. The nuclear receptor NR2F2 activates star expression and steroidogenesis in mouse MA-10 and MLTC-1 Leydig cells. *Biology of Reproduction* **91**:26. DOI: https://doi.org/10.1095/biolreprod.113.115790, PMID: 24899578

Miyabayashi K, Katoh-Fukui Y, Ogawa H, Baba T, Shima Y, Sugiyama N, Kitamura K, Morohashi K. 2013. Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells. *PLOS ONE* **8:e68050. DOI: https://doi.org/10.1371/journal.pone.0068050, PMID: 23840809

Miyabayashi K, Shima Y, Inoue M, Sato T, Baba T, Ohkawa Y, Suyama M, Morohashi K-I. 2017. Alterations in fetal Leydig cell gene expression during fetal and adult development. *Sexual Development* **11**:53–63. DOI: https://doi.org/10.1159/000453323, PMID: 27960177
**Morohashi KI**, Inoue M, Baba T. 2020. Coordination of multiple cellular processes by NR5A1/NR5A1. *Endocrinology and Metabolism* **35**:756–764. DOI: https://doi.org/10.3803/EnM.2020.402, PMID: 33397036

**Nef S**, Parada LF. 1999. Cryptorchidism in mice mutant for Insl3. *Nature Genetics* **22**:295–299. DOI: https://doi.org/10.1038/10364, PMID: 10391220

**Nicol B**, Grimm SA, Chalmel F, Lecluze E, Pannetier M, Pailhoux E, Dupin-De-Beyssat E, Guiguen Y, Capel B, Yao HH-C. 2019. RUNX1 maintains the identity of the fetal ovary through an interplay with FOXL2. *Nature Communications* **10**:5116. DOI: https://doi.org/10.1038/s41467-019-13060-1, PMID: 31712577

**O'Shaughnessy PJ**, Baker PJ, Heikkilä M, Vainio S, McMahon AP. 2000. Localization of 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase isoform expression in the developing mouse testis--androstenedione is the major androgen secreted by fetal/neonatal leydig cells. *Endocrinology* **141**:2631–2637. DOI: https://doi.org/10.1210/endo.141.7.7545, PMID: 10875268

**Padua MB**, Jiang T, Morse DA, Fox SC, Hatch HM, Tevosian SG. 2015. Combined loss of the GATA4 and GATA6 transcription factors in male mice disrupts testicular development and confers adrenal-like function in the testes. *Endocrinology* **156**:1873–1886. DOI: https://doi.org/10.1210/en.2014-1907, PMID: 25668066

**Park HL**, Bai C, Platt KA, Matise MP, Beeghly A, Hui CC, Nakashima M, Joyner AL. 2000. Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation. *Development* **127**:1593–1605. DOI: https://doi.org/10.1242/dev.127.8.1593, PMID: 10725236

**Pereira FA**, Qiu Y, Zhou G, Tsai MJ, Tsai SY. 1999. The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development. *Genes & Development* **13**:1037–1049. DOI: https://doi.org/10.1101/gad.13.8.1037, PMID: 10215630

**Pierucci-Alves F**, Clark AM, Russell LD. 2001. A developmental study of the Desert hedgehog-null mouse testis. *Biology of Reproduction* **65**:1392–1402. DOI: https://doi.org/10.1095/biolreprod65.5.1392, PMID: 11673255

**Polvani S**, Pepe S, Milani S, Galli A. 2019. COUP-TFII in health and disease. *Cells* **9**:101. DOI: https://doi.org/10.3390/cells9010101, PMID: 31906104

**Qin J**, Tsai MJ, Tsai SY. 2008. Essential roles of COUP-TFII in Leydig cell differentiation and male fertility. *PLOS ONE* **3:e3285. DOI: https://doi.org/10.1371/journal.pone.0003285, PMID: 18818749**

**Reyes AP**, León NY, Frost ER, Harley VR. 2023. Genetic control of typical and atypical sex development. *Nature Reviews. Urology* **20**:434–451. DOI: https://doi.org/10.1038/s41585-023-00754-x, PMID: 37020056

**Rotgers E**, Jørgensen A, Yao HHC. 2018. At the crossroads of fate-somatic cell lineage specification in the fetal gonad. *Endocrine Reviews* **39**:739–759. DOI: https://doi.org/10.1210/er.2018-00010, PMID: 29771299

**Sararols P**, Stévant I, Neirijnck Y, Rebourcet D, Darbey A, Curley MK, Kühne F, Dermitzakis E, Smith LB, Nef S. 2021. Specific transcriptomic signatures and dual regulation of steroidogenesis between fetal and adult mouse Leydig cells. *Frontiers in Cell and Developmental Biology* **9**:695546. DOI: https://doi.org/10.3389/fcell.2021.695546, PMID: 34262907

**Schmahl J**, Rizzolo K, Soriano P. 2008. The PDGF signaling pathway controls multiple steroid-producing lineages. *Genes & Development* **22**:3255–3267. DOI: https://doi.org/10.1101/gad.1723908, PMID: 19056881

**Schnabel CA**, Selleri L, Cleary ML. 2003. Pbx1 is essential for adrenal development and urogenital differentiation. *Genesis* **37**:123–130. DOI: https://doi.org/10.1002/gene.10235, PMID: 14595835

**Schwartz CL**, Christiansen S, Vinggaard AM, Axelstad M, Hass U, Svingen T. 2019. Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. *Archives of Toxicology* **93**:253–272. DOI: https://doi.org/10.1007/s00204-018-2350-5, PMID: 30430187

**Shima Y**, Miyabayashi K, Haraguchi S, Arakawa T, Otake H, Baba T, Matsuzaki S, Shishido Y, Akiyama H, Tachibana T, Tsutsui K, Morohashi K. 2013. Contribution of Leydig and Sertoli cells to testosterone production in mouse fetal testes. *Molecular Endocrinology* **27**:63–73. DOI: https://doi.org/10.1210/me.2012-1256, PMID: 23125070

**Shima Y**, Morohashi K. 2017. Leydig progenitor cells in fetal testis. *Molecular and Cellular Endocrinology* **445**:55–64. DOI: https://doi.org/10.1016/j.mce.2016.12.006

**Shima Y**, Miyabayashi K, Sato T, Suyama M, Ohkawa Y, Doi M, Okamura H, Suzuki K. 2018. Fetal Leydig cells dedifferentiate and serve as adult Leydig stem cells. *Development* **145**:dev169136. DOI: https://doi.org/10.1242/dev.169136, PMID: 30518625

**Shima Y.** 2019. Development of fetal and adult Leydig cells. *Reproductive Medicine and Biology* **18**:323–330. DOI: https://doi.org/10.1002/rmb2.12287, PMID: 31607792

**Stévant I**, Neirijnck Y, Borel C, Escoffier J, Smith LB, Antonarakis SE, Dermitzakis ET, Nef S. 2018. Deciphering cell lineage specification during male sex determination with single-cell RNA sequencing. *Cell Reports* **22**:1589–1599. DOI: https://doi.org/10.1016/j.celrep.2018.01.043, PMID: 29425512

**Stévant I**, Kühne F, Greenfield A, Chaboissier M-C, Dermitzakis ET, Nef S. 2019. Dissecting cell lineage specification and sex fate determination in gonadal somatic cells using single-cell transcriptomics. *Cell Reports* **26**:3272–3283. DOI: https://doi.org/10.1016/j.celrep.2019.02.069, PMID: 30893600

**Swift MR**, Pham VN, Castranova D, Bell K, Poole RJ, Weinstein BM. 2014. SoxF factors and Notch regulate nr2f2 gene expression during venous differentiation in zebrafish. *Developmental Biology* **390**:116–125. DOI: https://doi.org/10.1016/j.ydbio.2014.03.018, PMID: 24699544

**Taelman J**, Czukiewska SM, Moustakas I, Chang YW, Hillenius S, van der Helm T, van der Meeren LE, Mei H, Fan X, Chuva de Sousa Lopes SM. 2024. Characterization of the human fetal gonad and reproductive tract by single-cell transcriptomics. *Developmental Cell* **59**:529–544. DOI: https://doi.org/10.1016/j.devcel.2024.01.006, PMID: 38295793
Tang H, Brennan J, Karl J, Hamada Y, Raetzman L, Capel B. 2008. Notch signaling maintains Leydig progenitor cells in the mouse testis. *Development* **135**:3745-3753. DOI: https://doi.org/10.1242/dev.024786, PMID: 18927153

Tang F, Richardson N, Albina A, Chaboissier MC, Perea-Gomez A. 2020. Mouse gonad development in the absence of the pro-ovary factor WNT4 and the pro-testis factor SOX9. *Cells* **9**:1103. DOI: https://doi.org/10.3390/cells9051103, PMID: 32365547

van den Driesche S, Walker M, McKinnell C, Scott HM, Eddie SL, Mitchell RT, Seckl JR, Drake AJ, Smith LB, Anderson RA, Sharpe RM. 2012. Proposed Role for COUP-TFII in regulating fetal Leydig cell steroidogenesis, perturbation of which leads to masculinization disorders in rodents. *PLOS ONE* **7**:e37064. DOI: https://doi.org/10.1371/journal.pone.0037064

Viger RS, de Mattos K, Tremblay JJ. 2022. Insights Into the roles of GATA factors in mammalian testis development and the control of fetal testis gene expression. *Frontiers in Endocrinology* **13**:902198. DOI: https://doi.org/10.3389/fendo.2022.902198, PMID: 35692407

Wankanit S, Zidoune H, Bignon-Topalovic J, Schlick L, Houzelstein D, Fusée L, Boukri A, Nouri N, McElreavey K, Bashamboo A, Elzaiat M. 2024. Evidence for NR2F2/COUP-TFII involvement in human testis development. *Scientific Reports* **14**:17869. DOI: https://doi.org/10.1038/s41598-024-68860-3, PMID: 39090159

Wen Q, Cheng CY, Liu Y-X. 2016. Development, function and fate of fetal Leydig cells. *Seminars in Cell & Developmental Biology* **59**:89-98. DOI: https://doi.org/10.1016/j.semcdb.2016.03.003

Yao HHC, Whoriskey W, Capel B. 2002. Desert Hedgehog/Patched 1 signaling specifies fetal Leydig cell fate in testis organogenesis. *Genes & Development* **16**:1433-1440. DOI: https://doi.org/10.1101/gad.981202, PMID: 12050120

Yokoyama T, Omotehara T, Hirano T, Kubota N, Yanai S, Hasegawa C, Takada T, Mantani Y, Hoshi N. 2018. Identification of reference genes for quantitative PCR analyses in developing mouse gonads. *The Journal of Veterinary Medical Science* **80**:1534-1539. DOI: https://doi.org/10.1292/jvms.18-0417, PMID: 30175754

Yu C-T, Tang K, Suh JM, Jiang R, Tsai SY, Tsai M-J. 2012. COUP-TFII is essential for metanephric mesenchyme formation and kidney precursor cell survival. *Development* **139**:2330-2339. DOI: https://doi.org/10.1242/dev.076299, PMID: 22669823

Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, Rivera-Feliciano J, Jiang D, von Gise A, Ikeda S, Chien KR, Pu WT. 2008. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. *Nature* **454**:109-113. DOI: https://doi.org/10.1038/nature07060, PMID: 18568026

Zidoune H, Ladjouze A, Chellat-Rezgoune D, Boukri A, Dib SA, Nouri N, Tebibel M, Sifi K, Abadi N, Satta D, Benelmadani Y, Bignon-Topalovic J, El-Zaiat-Munsch M, Bashamboo A, McElreavey K. 2022. Novel genomic variants, atypical phenotypes and evidence of a Digenic/Oligogenic contribution to disorders/differences of sex development in a large North African cohort. *Frontiers in Genetics* **13**:900574. DOI: https://doi.org/10.3389/fgene.2022.900574, PMID: 36110220

Zimmermann S, Steding G, Emmen JM, Brinkmann AO, Nayernia K, Holstein AF, Engel W, Adham IM. 1999. Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. *Molecular Endocrinology* **13**:681-691. DOI: https://doi.org/10.1210/mend.13.5.0272, PMID: 10319319
# Appendix 1

## Primers

<table><thead><tr><td>Primer name</td><td>Application</td><td>Sequence (5'-3')</td></tr></thead><tbody><tr><td>CRE 1</td><td>Genotyping Cre</td><td>CAGGATATACGTAATCTGGC</td></tr><tr><td>CRE 4</td><td>Genotyping Cre</td><td>CACGGGCACTGTGTCCAGACCAG</td></tr><tr><td>CCR5mR</td><td>Genotyping internal control</td><td>ATGTGGATGGAGAGGAGTCG</td></tr><tr><td>CCR5mL</td><td>Genotyping internal control</td><td>CAACCGAGACCTTCCTGTTC</td></tr><tr><td>TF2.1</td><td>Genotyping Nr2f2</td><td>TGCCCACACTTTCCCTACTCC</td></tr><tr><td>TF2.5</td><td>Genotyping Nr2f2</td><td>TTTCTGCAAGGAATGGGTTG</td></tr><tr><td>Amh-Fwd</td><td>RT-qPCR primers</td><td>GGGGAGACTGGAGAACAGC</td></tr><tr><td>Amh-Rev</td><td>RT-qPCR primers</td><td>AGAGCTCGGGCTCCCATA</td></tr><tr><td>Arx-Fwd</td><td>RT-qPCR primers</td><td>GCACCACGTTCCACCAGTTAC</td></tr><tr><td>Arx-Rev</td><td>RT-qPCR primers</td><td>GCACCACGTTCCACCAGTTAC</td></tr><tr><td>Bhmt-Fwd</td><td>RT-qPCR primers</td><td>CGTCAGCTTCATCGGGAGTT</td></tr><tr><td>Bhmt-Rev</td><td>RT-qPCR primers</td><td>CTTGCCGTGCAATGTCACAA</td></tr><tr><td>Cdh5-Fwd</td><td>RT-qPCR primers</td><td>TCCTCTGCATCCTCACCATCACA</td></tr><tr><td>Cdh5-Rev</td><td>RT-qPCR primers</td><td>GTAAGTGACCAACTGCTCGTGAAT</td></tr><tr><td>Cyp11a1-Fwd</td><td>RT-qPCR primers</td><td>TGGCCCCATTTACAGGGAGAA</td></tr><tr><td>Cyp11a1-Rev</td><td>RT-qPCR primers</td><td>GGCATCTGAACTCTTAAACAGGA</td></tr><tr><td>Cyp17a1-Rev</td><td>RT-qPCR primers</td><td>CAGAGAAGTGCTCGTGAAGAAG</td></tr><tr><td>Cyp17a1Fwd</td><td>RT-qPCR primers</td><td>CAGAGAAGTGCTCGTGAAGAAG</td></tr><tr><td>Dhh-Fwd</td><td>RT-qPCR primers</td><td>GGACCTCGTACCCAACTACAA</td></tr><tr><td>Dhh-Rev</td><td>RT-qPCR primers</td><td>CGATGGCTAGAGCGTTCACC</td></tr><tr><td>Gli1-Fwd</td><td>RT-qPCR primers</td><td>TGGTACCATGAGCCCTTCTT</td></tr><tr><td>Gli1-Rev</td><td>RT-qPCR primers</td><td>GTGGTACACAGGGCTGGACT</td></tr><tr><td>Hes1-Fwd</td><td>RT-qPCR primers</td><td>ATAGCTCCCGGCATTCCAAG</td></tr><tr><td>Hes1-Rev</td><td>RT-qPCR primers</td><td>ATAGCTCCCGGCATTCCAAG</td></tr><tr><td>HeyL-Fwd</td><td>RT-qPCR primers</td><td>CAGCCCTTCGCAGATGCAA</td></tr><tr><td>HeyL-Rev</td><td>RT-qPCR primers</td><td>CCAATCGTCGCAATTCAGAAAG</td></tr><tr><td>Ins13-Fwd</td><td>RT-qPCR primers</td><td>ATTGCTCCCCACCTCCTGGCTATG</td></tr><tr><td>Ins13-Rev</td><td>RT-qPCR primers</td><td>GGTCATGATGGGGCTTCTTG<br>GGGA</td></tr><tr><td>Notch2-Fwd</td><td>RT-qPCR primers</td><td>ATATCGACGACTGCCCCAAC</td></tr><tr><td>Notch2-Rev</td><td>RT-qPCR primers</td><td>CCATAGCCTCCGTTTCGGTT</td></tr><tr><td>Nr2f2-Fwd</td><td>RT-qPCR primers</td><td>CGGAGGAACCTGAGCTACAC</td></tr><tr><td>Nr2f2-Rev</td><td>RT-qPCR primers</td><td>CGGAGGAACCTGAGCTACAC</td></tr><tr><td>Pdgfa-Fwd</td><td>RT-qPCR primers</td><td>CAGTGTCAAGGTGGCCAAAGT</td></tr><tr><td>Pdgfa-Rev</td><td>RT-qPCR primers</td><td>CAGTGTCAAGGTGGCCAAAGT</td></tr><tr><td>Pdgfb-Fwd</td><td>RT-qPCR primers</td><td>GATCTCTCGGAACCTCATCG</td></tr><tr><td>Pdgfb-Rev</td><td>RT-qPCR primers</td><td>GATCTCTCGGAACCTCATCG</td></tr></tbody></table>

Continued on next page
Continued

<table><thead><tr><td>Primer name</td><td>Application</td><td>Sequence (5'-3')</td></tr></thead><tbody><tr><td>Pdgfra-Fwd</td><td>RT-qPCR primers</td><td>TCCATGCTAGACTCAGAAGTCA</td></tr><tr><td>Pdgfra-Rev</td><td>RT-qPCR primers</td><td>TCCCGGTGGACACAATTTTT</td></tr><tr><td>Sdha-Fwd</td><td>RT-qPCR primers</td><td>TGTTCAGTTCCACCCCACA</td></tr><tr><td>Sdha-Rev</td><td>RT-qPCR primers</td><td>TCTCCACGACACCCTTCTG</td></tr><tr><td>Sgpl1-Fwd</td><td>RT-qPCR primers</td><td>CTGAAGGACTTCGAGCCTTATTT</td></tr><tr><td>Sgpl1-Rev</td><td>RT-qPCR primers</td><td>CTGAAGGACTTCGAGCCTTATTT</td></tr><tr><td>Sox9-Fwd</td><td>RT-qPCR primers</td><td>GCGGAGCTCAGCAAGACTCTG</td></tr><tr><td>Sox9-Rev</td><td>RT-qPCR primers</td><td>ATCGGGGTGGTCTTTCTTGTG</td></tr><tr><td>Tbp-Fwd</td><td>RT-qPCR primers</td><td>GCTCTGGAATTGTACCGCAG</td></tr><tr><td>Tbp-Rev</td><td>RT-qPCR primers</td><td>TGACTGCAGCAAATCGCTTG</td></tr></tbody></table>