The molecular pathways underlying early gonadal development

Journal of Molecular Endocrinology Y Yang et al. Mammalian early genital ridge development 62 :1 R47–R64 REVIEW The molecular pathways underlying early gonadal development Yisheng Yang, Stephanie Workman and Megan J Wilson Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand Correspondence should be addressed to M J Wilson: meganj.wilson@otago.ac.nz Abstract The body of knowledge surrounding reproductive development spans the fields of genetics, anatomy, physiology and biomedicine, to build a comprehensive understanding of the later stages of reproductive development in humans and animal models. Despite this, there remains much to learn about the bi-potential progenitor structure that the ovary and testis arise from, known as the genital ridge (GR). This tissue forms relatively late in embryonic development and has the potential to form either the ovary or testis, which in turn produce hormones required for the development of the rest of the reproductive tract. It is imperative that we understand the genetic networks underpinning GR development if we are to begin to understand abnormalities in the adult. This is particularly relevant in the contexts of disorders of sex development (DSDs) and infertility, two conditions that many individuals struggle with worldwide, with often no answers as to their aetiology. Here, we review what is known about the genetics of GR development. Investigating the genetic networks required for GR formation will not only contribute to our understanding of the genetic regulation of reproductive development, it may in turn open new avenues of investigation into reproductive abnormalities and later fertility issues in the adult. The distinction between sexes is one of the most obvious examples of dimorphism in the animal kingdom and highlights one of the most crucial fate decisions made in utero to develop as a male or a female. This fate decision to follow either developmental trajectory is an essential process that determines the reproductive success of all sexually reproducing animals (Fig. 1). There are a number of different mechanisms underlying sex development that can be broadly categorized as either genetically sex determined or environmentally determined (Capel 2017). Typically, mammals fall within the genetically sexdetermined category. Traditionally, sexual development has been viewed as two distinct processes, gonadal sex determination (decision of the gonad to become a testis or ovary) and sexual differentiation (establishment of https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Key Words f gonad f urogenital ridge f disorders of sex determination f infertility f molecular genetics Journal of Molecular Endocrinology (2019) 62, R47–R64 testicular/ovarian structures, accessory sex structures and secondary sexual characteristics), whereby genital ridge (GR) formation was included only as a step in gonadal sex determination or excluded from the broader picture all together. The focus of this review is formation of the GR in mice and humans, and consequences of failure of its development, rather than sex determination, which has been reviewed extensively elsewhere (Greenfield 2015, Capel 2017). Genital ridge formation The mammalian GR is derived from intermediate mesoderm as paired structures that lie on either side of the dorsal mesentery in the coelomic cavity (Fig. 2). Formation of Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. Figure 1 Summary of the origin of reproductive tissues within the mammalian embryo. The gonad region of the GR develops into either the testis or the ovary depending upon expression of the Sry gene. In XY gonads, the Leydig cells produce testosterone, required for the masculinization of the genitalia and maintenance of the mesonephric duct. Sertoli cells secrete AMH, resulting in the degradation of the paramesonephric (Müllerian) duct. In the absence of androgens, the paramesonephric duct develops as the uterus, uterine tubules and upper portion of the cervix. Female genitalia are also formed from the genital tubercle, folds and swellings. Post-natal oestradiol synthesis by the ovary is important for the final maturation of the oocytes and secondary sex characteristics. Other tissues of the body also show sex-dimorphic gene expression such as the brain and liver, under the influence of steroid hormones and other gonadal factors such as AMH (Yang et al. 2006, Wang et al. 2009, Conforto & Waxman 2012, Wittmann & McLennan 2013, Maekawa et al. 2014). the mammalian GR begins with increased proliferation of coelomic epithelial cells on the ventromedial surface of the mesonephroi. Each mesonephros also contains the mesonephric duct (also known as the Wolffian duct), a primordial urogenital tissue that will give rise to the male epididymis, vas deferens and seminal vesicles following male sex determination (Hannema & Hughes 2007, Shaw & Renfree 2014). In addition, the paramesonephric duct (also known as the Müllerian duct) is also present in the mesonephros, running in parallel to the mesonephric duct (Fig. 2A). This is the female equivalent to the mesonephric duct, which will form the fallopian tubes, uterus and part of the vagina following female sex determination (Acien 1992). Together, the mesonephros and GR are known as the urogenital ridge (UGR). Proliferation of the coelomic epithelial cells on the ventromedial surface of the mesonephros creates a dense, pseudostratified epithelial cell layer (Gropp & Ohno 1966, Pelliniemi 1975, Wartenberg et al. 1991). Alongside the proliferation of the coelomic epithelium, the underlying basement membrane becomes fragmented, allowing many epithelial cells to ingress dorsally, towards the mesonephros (Fig. 2B) (Karl & Capel 1998, Kusaka et al. https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R48 2010). As these cells delaminate from the coelomic epithelium, they undergo an epithelial-to-mesenchymal transition (EMT) (Kusaka et al. 2010) and as mesenchymal cells, begin to populate the space between the coelomic epithelium and the mesonephros (Karl & Capel 1998, Schmahl et al. 2000). These mesenchymal cells of the GR are precursor cells that can differentiate into somatic support cells and interstitial/stromal cell lineages of the early sexually differentiated gonad (Karl & Capel 1998, Ito et al. 2006, Mork et al. 2012). In the testis, additional cells are also recruited into the GR region from the mesonephros to augment the population of mesenchymal cells. Cell-tracing and organ culture studies in mice revealed that these largely contribute to the endothelial cell population for the establishment of the male vascular system (Karl & Capel 1995, Martineau et al. 1997, Coveney et al. 2008). Migrating into the GR Figure 2 Schematic illustrations of the structures and components involved in early UGR development in mice. (A) (i) Side view of a E11.5 embryo indicating the location of the developing GR (orange bar). (ii) A ventral view of the abdominal region focuses on the mesonephros (white) and the gonadal ridge (grey) that develops on the ventromedial surface. The aorta is shown in red. (iii) In transverse sections the mesonephros, containing mesonephric and paramesonephric ducts, are often visible. The gonad appears as a bulge facing into the coelomic cavity. At this stage, the PGCs have migrated into the GR, from the hindgut via the dorsal mesentery. (B) Starting at E9.5, the coelomic epithelium (yellow) on the ventromedial surface of the mesonephros begins to proliferate, forming a pseudostratified epithelial layer. The basement membrane becomes fragmented, allowing GR progenitor cells that have undergone EMT to migrate inward. These cells continue to proliferate, just behind the coelomic epithelium to form the bi-potential gonad. Between E10.0 and 11.5, the PGCs (green) also migrate into the GR. CE, coelomic epithelium; DM, dorsal mesentery; GC, germ cells; GR, genital ridge; HG, hindgut; M, mesonephros; MD, mesonephric duct; MT, mesonephric tubule; PGC, primordial germ cell; PMD, paramesonephric duct. Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. just prior to gondal sex determination are primordial germ cells (PGC), the precursors to the germ cells that become sperm and oocytes later in life. PGCs arise near the yolk sac and travel, via the hindgut, to the GR as a result of chemotaxis (Doitsidou et al. 2002). Genital ridge formation in mice Most of the work investigating mammalian gonad development has been performed on mice. It is assumed that events in the human embryo, as well as other Eutherian mammals, follow the same basic pattern, albeit with some differences in the timing and anatomy. The process of GR formation, starting with the proliferation of the coelomic epithelium, begins at about E9.5 in mice (Hu et al. 2013), which equates to about 5 weeks of gestation in humans (Jost 1972). The GR itself is not morphologically evident until about E10.0– 10.5 where a clear distinction between the GR and the mesonephros can be seen under light microscopy. From E10.5 to 11.5, further proliferation of the GR coelomic epithelium and mesonephros expands the overall size on the ventral side of the mesonephros (Fig. 2A). The initiation, proliferation and expansion of the GR is indistinguishable between XX and XY embryos, and the size of the GR is determined by the length of the embryonic trunk (Wainwright et al. 2014). At around E11.5–12.0, the molecular events that determine gonad fate occur, prompting the gonad to follow either a testisor ovary-specific developmental trajectory from this time-point. The first sign of sexual dimorphism of the early differentiated gonad becomes evident in XY gonads at about E12.5, when testis cords can be seen (Hacker et al. 1995, Schmahl et al. 2000). The GR is not only a progenitor tissue for the gonad, but also the adrenal cortex, a critical endocrine tissue that synthesizes and secretes a variety of steroid hormones to maintain body homeostasis and regulate the stress response (Yates et al. 2013, Walczak & Hammer 2015). Consequently, several genes required for gonad development are also important for adrenal gland development (Luo et al. 1994, Gut et al. 2005, Katoh-Fukui et al. 2005, Bandiera et al. 2013, Tevosian et al. 2015). The medulla and the cortex of the adult adrenal gland have separate origins; the medulla is derived from neural crest cells, whereas the cortex is derived from cells located at the anterior-most region of the GR, indicating it has an intermediate mesoderm origin (Hatano et al. 1996). Beginning at E10.5, a small https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R49 cluster of cells at the anterior region of mesenchyme separates from the GR primordium and moves dorsomedially to form the adrenal anlage (Hatano et al. 1996, Val & Swain 2010). From E11.5 to 12.5, neural crest cells invade the adrenal anlage and aggregate in the centre to form the medulla. Mesenchyme cells, hypothesized to be of a coelomic epithelial origin, form a fibrous capsule around the composite adrenal primordium by E14.5 (Xing et al. 2015). Development of the PGC in the Genital ridge As previously mentioned, PGCs originate from a separate location near the yolk sac, away from the mesonephros and GR (Saga 2008). Mammalian PGCs are specified via an inductive system of signalling molecules, particularly BMP4. In mice, PGCs are induced around E6.5 (equating to approximately 2 weeks gestation in humans) from the proximal epiblast by BMP4 signalling (Lawson et al. 1999). These cells subsequently move to and cluster at the base of the allantois/yolk sac wall, near the forming hindgut, which can be seen in mouse embryos at about E7.0 (week 3–4 human gestation) as a small population of ~45 cells (Lawson et al. 1999). As development proceeds, the hindgut folds and the PGCs migrate into the embryo proper (Hara et al. 2009, Harikae et al. 2013). By E9.5 the PGCs begin to migrate away from the hindgut towards the UGR and colonize the gonad between E10.0 and 11.0 (~5-week gestation in humans) (Fig. 2A; Witschi 1948, Molyneaux et al. 2001). During this mass migration of PGCs, the hindgut descends into the coelomic cavity and the last PGCs to migrate must travel through the dorsal mesentery before entering the gonads (Molyneaux et al. 2001). The PGCs undergo several rounds of cell division to achieve a population of about ~3000 cells by ~E11.5 (Tam & Snow 1981). Around this time, PGCs begin to undergo a process known as licensing, undergoing a global change in gene expression, turning on genes required for gametogenesis, concurrently switching off their pluripotency genes over the course of the following days (Stebler et al. 2004, Gill et al. 2011, Rolland et al. 2011, Seisenberger et al. 2012). Following this transition, the PGCs are referred to as gametogenesis-competent cells and are poised to initiate either male or female differentiation, including meiosis, upon receiving cues from the somatic cells of either the forming testis or ovary and the nearby mesonephric tissue (McLaren & Southee 1997, Adams & McLaren 2002, Gill et al. 2011). Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. Mammalian early genital ridge development Genes essential for initial gonad formation Mutational analysis using the mouse model, with some additional evidence from human clinical cases, have brought to light a number of genes that are required to initiate the formation and proliferation of the GR, as well as testis/ovary differentiation (Table 1). These include Wilms’ tumour suppressor 1 (WT1) (Kreidberg et al. 1993, Hammes et al. 2001), LIM homeobox gene 9 (LHX9) (Birk et al. 2000), Nuclear Receptor Subfamily 5 Group A member 1 (NR5A1; also called steroidogenic factor 1 (SF1)) (Luo et al. 1994), empty spiracles homeobox gene 2 (EMX2) (Miyamoto et al. 1997) and GATA-binding protein 4 (GATA4) (Hu et al. 2013). In mouse embryos, a homozygous null mutation in any one of these genes causes gonadal agenesis. While the function of these five Table 1 62 :1 R50 factors have mainly been characterized in mice, mutations in three of these genes (NR5A1, WT1 and GATA4) have also been found in patients with DSD, indicating a conserved role in reproductive development (Bashamboo et al. 2010b, Kohler et al. 2011, Lourenco et al. 2011, Swartz et al. 2017). However, these genes are expressed and function in many developing organ systems, meaning the loss of function in both mice and humans produce a range of phenotypes beyond the reproductive tract (Ingraham et al. 1994, Klamt et al. 1998, Hammes et al. 2001, Tevosian et al. 2015). Genes known to be required for GR development often show altered expression in gene knockout lines, indicating a co-regulatory relationship exists between these critical factors (discussed further below). It is evident that many of these critical early factors discussed below have multiple roles in reproductive Summary of genes essential for GR formation. Gene Mouse UGR expression Mouse gonadal phenotype Human phenotype WT1 Expressed in the coelomic epithelium and mesonephros (Armstrong et al. 1993) Frasier syndrome (Klamt et al. 1998) WAGR syndrome (Le Caignec et al. 2007) Denys-Drash syndrome (Hastie 1992), Meacham syndrome Phenotypes include streak gonads, ambiguous genitalia, hypospadias, 46, XY sex reversal (Suri et al. 2007) Nr5a1 Initially expressed in the anterior region of the coelomic epithelium. Expression domain expands in an anterior–posterior direction (Hu et al. 2013) Degenerating gonad due to increased apoptosis (Kreidberg et al. 1993, Hammes et al. 2001) Male WT1-KTS mice exhibit complete sex reversal (Hammes et al. 2001) Following sex determination, roles in ovary and follicle development (Gao et al. 2014) Gonad regression due to increased apoptosis Loss of adrenal development, obesity and pituitary abnormalities XY KO mice show complete male-tofemale sex reversal (Luo et al. 1994, Shinoda et al. 1995) Emx2 Lhx9 Gata4 MAP3K1 Range of phenotypes including 46, XY sex reversal, gonad dysgenesis, male infertility, hypospadias, adrenal insufficiency, gonad dysgenesis, premature ovarian failure (reviewed in Ferraz-de-Souza et al. 2011) 46, XX sex reversal, ovotestis (Bashamboo et al. 2016, Swartz et al. 2017) Absent gonads Endometriosis (Daftary & Taylor 2004) Coelomic epithelium and cells that Reduced proliferation of the coelomic Endometrial cancer (Daftary & Taylor move into the underlying 2004) epithelium (Miyamoto et al. 1997, mesenchyme Incomplete Müllerian fusion (Liu et al. Mesonephric duct epithelia (Hu et al. Pellegrini et al. 1997) 2013) 2015) Coelomic epithelium and cells that Absent gonads None reported move into the underlying Reduced proliferation of the Limited evidence of involvement in mesenchyme (Birk et al. 2000, Hu coelomic epithelium (Birk et al. reproductive cancers (cervical cancer et al. 2013) 2000) promoter methylation (Bhat et al. 2017)) Absent gonads Testicular abnormalities, ambiguous Coelomic epithelium, expression No expansion of the coelomic genitalia (Lourenco et al. 2011) domain expands in an anterior– epithelium (Hu et al. 2013) Frequent mutation in ovarian cancers posterior direction, precedes Following sex determination, roles in (Cai et al. 2009) expression of Sf1 (Hu et al. 2013) ovarian and testicular development (Efimenko et al. 2013) Coelomic epithelium and gonad Minor testis abnormalities (Warr Ambiguous genitalia streak gonads mesenchyme (Warr et al. 2011) et al. 2011) (Pearlman et al. 2010, Granados et al. 2017) https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. development, from formation of the bi-potential gonad of both sexes, to sex-specific roles following gonadal sex determination. Currently, we are lacking a detailed gene network integrating both the roles of these transcription factors, along with the signalling pathways that regulate them, to gain a broader picture of how these components together regulate cellular processes like differentiation and proliferation to control formation of the bi-potential gonad. Future genome-wide approaches to study gene interactions will help to better define the regulatory interactions between these proteins, and others, required for GR formation Wilms tumour suppressor (Wt1) Wt1 mRNA is expressed during gonad development in the coelomic epithelium and mesonephros prior to gonadal sex determination and in the progenitor support cells of both the developing testis and ovary (Armstrong et al. 1993). Wt1−/− mice have a recognizable gonad primordium at E11.0, but this then degenerates due to apoptosis of somatic cells (Kreidberg et al. 1993, Hammes et al. 2001). Knockout mouse embryos, on a C57BL/6 genetic background, do not survive to parturition due to embryonic lethality from heart defects, while knockout mice generated on other genetic backgrounds, such as Balb/c, survive until birth (Kreidberg et al. 1993, Herzer et al. 1999). The Wt1 gene encodes for a zinc finger transcription factor that functions as either an activator or repressor of transcription; however, the structure and function of the WT1 protein varies depending on the cell type and promoter used to transcribe the gene, RNA editing, alternative usage of translational start sites and alternative splicing (Bruening & Pelletier 1996, Scharnhorst et al. 1999, Dallosso et al. 2007). Of particular interest to gonad development are two alternate splice forms of the WT1 protein named WT1–KTS and WT1+KTS, due to either the inclusion or exclusion of three amino acids located between the third and fourth zinc finger domains (Hammes et al. 2001). Mice lacking expression of the Wt1(−KTS) isoform have gonads markedly reduced in size and poorly differentiated (Hammes et al. 2001), suggesting that WT1(−KTS) is the isoform required for the proliferation and differentiation of GR cells. Knockout of the Wt1(+KTS) splice transcript leads to complete maleto-female sex reversal and reduced Sry gene expression, yet, ovarian development proceeds normally (Hammes et al. 2001; Fig. 3). For both knockout lines, the overall https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R51 Figure 3 The Wt1 gene has multiple roles in gonad development and differentiation. Two isoforms of WT1, WT1(+KTS) and WT1(−KTS) have differing roles in gonad development based on gene-knockout studies. Loss of WT1(+KTS) leads to reduced Sry gene expression and XY sex reversal, in contrast, deletion of WT1(−KTS) halts GR development for both sexes (Hammes et al. 2001). However, if both splice forms of WT1 are present until E10.5, the gonad primordium still develops but with altered cell fate, with most somatic cells now adopting a steroidogenic cell fate (Chen et al. 2017). expression of Wt1 mRNA was similar to WT animals, due to increased expression of the alternative splice form compensating for the loss of the targeted splice form (Hammes et al. 2001). Thus, this resulting shift in isoform ratio (the ratio of −KTS/+KTS), leading to overexpression of the alternative isoform, may also contribute to the observed knockout phenotype. However, this has not been investigated further in mice with transgenic mouse lines. In humans, an altered splice form ratio (increased WT1−KTS, reduced WT1+KTS isoform) does severely affect gonadal development resulting in Frasier syndrome with streak gonads (Barbaux et al. 1997, Klamt et al. 1998). More recently, Chen et al. (2017) showed that the conditional inactivation of Wt1 just prior to sex determination at E10.5 allows gonadogenesis to proceed with reduced differentiation of Sertoli and granulosa cells from somatic cell precursors. Thus, when Wt1 expression is lost from gonads at E13.5, most somatic cells develop into steroidogenic cell types (Chen et al. 2017; Fig. 3). In contrast, in traditional knockout animals where Wt1 is inactive throughout development, development of the GR is blocked (Kreidberg et al. 1993; (Fig. 3). Thus, the role of WT1 alters as gonad development progresses, from being required for initial cell proliferation and growth of the GR, predominately through the WT1(−KTS) splice form, to controlling cell fate of somatic cells, and this is likely to occur through direct regulation of Nr5a1 gene expression (Fig. 4, described further in the following section). Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. Figure 4 Summary of the molecular relationships during early gonad formation. (A) Summary of relationships between the core genes necessary for GR development. Relationships are based on previous studies using knockout lines where expression changes of genes were observed. Loss of expression from knockout indicated a positive regulatory relationship and increased expression suggests a negative relationship. If direct relationships are known, through studies such as ChIP-PCR and reporter assays (Wilhelm & Englert 2002, Katoh-Fukui et al. 2005, Franca et al. 2013, Chen et al. 2017), they have been indicated by a solid line. (B) Recent studies suggest a complex regulatory relationship exist between WT1 and Nr5a1 (Chen et al. 2017). Nr5a1 expressing cells (Nr5a1+) in the GR contribute to the Sertoli, interstitial and Leydig cell populations following sex determination. In the bi-potential GR, WT1 is required for Nr5a1 gene expression. In Sertoli cells WT1 binds directly to the Nr5a1 gene promoter and appears to reduce its expression. In Leydig cells (no Wt1 expression), Nr5a1 gene expression is significantly higher. Lhx9 is a candidate protein partner for WT1 (Wilhelm & Englert 2002), it is expressed in the GR, and later in interstitial cells. Steroidogenic factor 1 (Sf1), nuclear receptor subfamily 5 group A member 1 (Nr5a1) The Nr5a1 (Sf1) gene encodes a transcription factor that is expressed in the adrenal glands, coelomic epithelium, hypothalamus and anterior pituitary gland during development (Luo et al. 1994, Morohashi & Omura 1996). During UGR development, Nr5a1 gene expression is limited to the gonad primordium, where it is required for the proliferation and survival of progenitor somatic cells (Luo et al. 1994). After gonadal sex determination, Nr5a1 gene expression is restricted to testis-specific cells, namely Leydig and Sertoli cells (Luo et al. 1994). Nr5a1−/− mice show complete failure of adrenal and gonadal development, abnormalities of the pituitary and hypothalamus and obesity (Luo et al. 1994, Shinoda et al. 1995). The gonads of Nr5a1-knockout mice embryos do https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R52 not develop beyond the early GR stage, and as a result, XY mice show complete male-to-female sex reversal (Luo et al. 1994). Gonadal regression in Nr5a1−/− embryos is due to increased apoptosis of the somatic cells (Luo et al. 1994), also observed with Wt1 loss-of-function mice (Hammes et al. 2001). Recent single-cell RNA sequencing (scRNA-seq) of XY somatic cells, expressing Nr5a1 (Nr5a1-GFP labelled cells) prior to (E10.5) and following sex determination (E11.5–E16.5), confirmed the presence of a multipotent Nr5a1+ cell population in the GR that progressively forms both the supporting and steroidogenic cell lineages from E11.5 (Stevant et al. 2018). Regulation of Nr5a1 gene expression plays a critical role in gonad development, with all key transcription factors described to date in GR development function in regulating correct Nr5a1 expression (Fig. 4; Wilhelm & Englert 2002, Katoh-Fukui et al. 2005, Hu et al. 2013, Chen et al. 2017). LHX9 and WT1(−KTS) proteins both bind to the promoter region of Nr5a1 in vitro, and together they increase reporter gene expression in a Sertoli-like (TM4) cell line (Wilhelm & Englert 2002), a finding replicated in Leydig-like (TM3) and C2C12 (myoblast derived) cell lines (Val et al. 2007, Takasawa et al. 2014). However, in primary Leydig cells WT1 overexpression repressed Nr5a1 gene expression. Further experiments confirmed that WT1 directly binds in vivo to the Nr5a1 promoter in Sertoli cells obtained from 2-week-old mice (Chen et al. 2017). It was suggested that these conflicting results were most likely due to the use of cell lines vs primary cells in these studies (Chen et al. 2017). The TM4 and TM3 cell lines were derived from juvenile BALB/c mouse testis (Mather 1980) and both cell lines express a similar combination of cell type gene markers (Beverdam et al. 2003). It is also worth noting that Chen et al. (2017) used primary Leydig cells obtained from adult and juvenile mice on a mixed background (C57Bl/6:129/SvEv), given that strain background can strongly influence the resulting phenotypes (Herzer et al. 1999, Meeks et al. 2003, Brennan & Capel 2004, Munger et al. 2009). WT1 also may have differing roles in the regulation of Nr5a1 gene expression, as the gonad develops into a testis, perhaps being required for an initial activation of Nr5a1, with protein partner LHX9 in the early gonad and, following gonadal sex determination, WT1 reduces Nr5a1 expression in those NR5A1+ cells that are fated to become Sertoli cells. In this case, the presence or absence of certain protein partners, for instance LHX9, would impact on how WT1 regulates Nr5a1 gene expression. Previous studies have shown that WT1 is converted from an activator to a repressor protein by the protein partners such as BASP1 (McKay et al. 1999, Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. Carpenter et al. 2004). Given that NR5A1+ cells contribute to both cell populations, it maybe that Nr5a1 expression levels determine which fate, with Sertoli cells expressing significantly lower levels of Nr5a1 compared to Leydig cells (Fig. 4B; Chen et al. 2017). LIM homeobox 9 (Lhx9) The Lhx9 gene encodes a transcription factor expressed in a variety of regions within the developing mouse embryo, including the brain, heart, kidney, limb buds and the coelomic epithelium (Retaux et al. 1999, Birk et al. 2000, Failli et al. 2000, Molle et al. 2004, Oshima et al. 2007, Smagulova et al. 2008, Tzchori et al. 2009, Yang & Wilson 2015). Lhx9 gene expression in the UGR is first seen in the coelomic epithelium at E9.5 and later in the gonad primordium, until sexual differentiation where its expression becomes restricted to the interstitial/ mesothelial regions of testes and the cortical regions of ovaries (Birk et al. 2000). Lhx9−/− mutant mice exhibit a similar gonadal phenotype to that of Wt1(−KTS) −/− mice, whereby normal GR development and PGC migration is observed but discrete gonads fail to form and genetically male mice show complete male-to-female sex reversal of their secondary sex characteristics (Birk et al. 2000). The observed underdevelopment of the GR results from disrupted proliferation of the gonad primordium (Birk et al. 2000), as opposed to increased apoptosis observed in the Wt1−/− and Nr5a1−/− mice (Kreidberg et al. 1993, Luo et al. 1994). In addition, male- and female-knockout offspring are infertile, with atrophic uteri, vaginas and oviducts, no male accessory sex organs, increased folliclestimulating hormone levels, and undetectable levels of testosterone and oestrogen (Birk et al. 2000). Interestingly, Lhx9−/− mice show no other phenotypic abnormalities outside of gonad agenesis and male-to-female sex reversal (Birk et al. 2000, Tzchori et al. 2009), despite expression in other tissues, such as the limbs, nervous system and pancreas, during development. This may be a result of the functional redundancy of Lhx9 with its closely related paralogue, LIM homeobox gene, Lhx2 (Jurata & Gill 1998, Birk et al. 2000, Tzchori et al. 2009). Empty spiracles homeobox 2 (Emx2) Emx2 encodes a transcription factor that is the mouse homolog of the Drosophila head gap gene empty spiracles (ems). The Emx2 transcript is expressed in the dorsal telencephalon, mesonephros and coelomic epithelium (Miyamoto et al. 1997, Yoshida et al. 1997). Emx2−/−-knockout https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R53 mice embryos exhibit normal PGC migration into the UGR region, but the initial thickening of the coelomic epithelium is not prominent and the GR soon degenerates (Miyamoto et al. 1997). These mutants completely lack gonads, genital tracts and kidneys (Miyamoto et al. 1997). The gonadal dysgenesis phenotype in Emx2−/− mice is a result of impaired cell migration from the coelomic epithelium through the basement membrane, as well as increased apoptosis (Kusaka et al. 2010). Interestingly, Nr5a1 gene expression is also significantly affected in Emx2−/− embryos (Kusaka et al. 2010), suggesting that Emx2 acts upstream of Nr5a1 in the developmental cascade, but how Emx2 regulates Nr5a1 transcription, whether directly or indirectly remains unclear. GATA-binding protein 4 (Gata4) GATA4 is a transcription factor that is essential for the development of multiple organs such as the heart, foregut, liver, ventral pancreas and the UGR (Kuo et al. 1997, Molkentin et al. 1997, Viger et al. 1998, Hu et al. 2013). Gata4 gene expression in the GR was originally linked to a role in testis differentiation through activating transcription of Sry together with WT1 (Tevosian et al. 2002). Later studies revealed that Gata4 is expressed prior to any other gonadal factor, initially in the anterior half of the coelomic epithelium at E9.5 and expanding to the posterior region by E10.2 (Hu et al. 2013). Gata4-knockout mice show no signs of gonadal initiation, with the coelomic epithelium remaining as an undifferentiated monolayer, indicating that Gata4 is required for the initial thickening of the coelomic epithelial layer (Hu et al. 2013). In these knockout mice, Lhx9 and Nr5a1 gene expression is lost, but Wt1 and Emx2 gene expression is unaffected (Hu et al. 2013). This suggests that GATA4 acts not only upstream of the Lhx9 gene, but also Nr5a1, possibly even directly regulating both genes, as binding sites for GATA4 have been identified in the proximal promoter regions of these two genes (Tremblay & Viger 2001, Smagulova et al. 2008, Hu et al. 2013). Genes with minor but essential roles in Genital ridge development Other genes have been implicated in early GR development, although mice-knockout strains for these genes exhibit a less severe phenotype than that of the null phenotype of the key genes described earlier. While the loss of function of the following genes does not cause termination of gonad development, it does result in the underdevelopment of the gonads, often in combination with sex reversal of the secondary sex characteristics. Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. Transcription factor 21 (Tcf21 or Pod1) TCF21, also known as epicardin, capsuling or transcription factor 21, belongs to the basic helix-loop-helix family of transcription factors. It is expressed by mesodermal cell types including heart proepicardial cells, kidney and visceral smooth muscle as well as the endodermal gastrointestinal tract (Quaggin et al. 1998, 1999, Lu et al. 2000, Miyagishi et al. 2000). Tcf21-knockout mice die in the perinatal period due to lung, kidney and cardiac defects, but also display gastric and splenic defects (Cui et al. 2003, Funato et al. 2003, Plotkin & Mudunuri 2008). In addition, Tcf21 XX and XY KO mice have irregular shaped gonads, the urogenital tracts of both XX and XY mice are indistinguishable and XY pups had feminized genitalia (Cui et al. 2004). Consistent with this phenotype, Tcf21 gene expression is initially detected throughout the GR at the bi-potential stage and continues to be expressed in the gonads of both sexes, with expression slightly higher in the testes following sex determination (Tamura et al. 2001). While the gonad does form in the Tcf21−/− embryo, morphological defects such as a shorted length were observed by E11, and vascular abnormalities were observed by E12.5 (Cui et al. 2004), suggesting a defect early in gonadal development. Further studies focused on expression analyses, these suggested a negative regulatory relationship exists between Tcf21 and Nr5a1, as Tcf21 KO results in increased Nr5a1 expression and an expanded Leydig cell population (Miyagishi et al. 2000). Despite an increase in Leydig cell numbers, the XY KO genitalia was feminized and the gonads fail to descend from an abdominal position, suggesting that testosterone levels were low, either due to later apoptosis of the gonadal tissue or a not all steroidogenic enzymes were expressed correctly (Cui et al. 2004). TCF21 also appears to repress Nr5a1 gene expression in the adrenal gland, with in vitro studies revealing that TCF21 directly represses Nr5a1 gene expression, through binding at E-box sequences located in the Nr5a1 promoter (Franca et al. 2013, 2015). Interestingly, the Sertoli cells do differentiate in the Tcf21 KO line, but there was some evidence that this cell population was reduced in number (although it was not quantified) (Cui et al. 2004). TCF21 can cause a sex reversal-like phenotype in vitro using a rat primary ovarian cell culture system. Overexpression of Tcf21 causes these cells to express Sertoli-like gene markers, in a similar pattern to that observed with Sry overexpression (Bhandari et al. 2011). Additionally, the Tcf21 gene has proposed to be a direct target of SRY (Bhandari et al. 2011). Together, these results suggest that TCF1 acts https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R54 downstream of Sry to promote Sertoli cell expansion and repress steroidogenic cell lineages. Sine oculis-related homeobox 1/4 (Six1/Six4) Six1 and Six4 genes belong to the mammalian homolog of the Drosophila sine oculis homeobox family of transcription factors, containing a distinctive SIX domain (required for protein-protein interactions) and homeodomain (Kawakami et al. 2000). Six1 and Six4 genes are located in the same genomic regions within about 100 kb of one another, and have highly overlapping tissue expression during mouse development (Boucher et al. 1996, Esteve & Bovolenta 1999, Ozaki et al. 2001, Fougerousse et al. 2002, Laclef et al. 2003). Six1/4 double KO mice show different phenotypes compared to either Six1 or Six4 single KO mice, highlighting regions of redundant function in the development of limbs, skeletal muscle, sensory neurons and kidney (Grifone et al. 2005, Konishi et al. 2006, Giordani et al. 2007, Kobayashi et al. 2007). The suggested functional redundancy between the two genes is further exhibited as both genes share a common DNA-binding site, MEF3 (Kawakami et al. 2000, Kumar 2009). In particular, only Six1/4 double KO mice embryos display smaller gonads and adrenal glands (Kobayashi et al. 2007, Fujimoto et al. 2013). Out of the essential GR genes listed previously, only Nr5a1 gene expression is significantly reduced, a finding corroborated through reporter assays showing that SIX1/4 is able to transactivate Nr5a1 transcription (Fujimoto et al. 2013). Furthermore, it was also shown that SIX1/4 is able to activate Fog2 gene expression and that interaction, together with GATA4, regulates Sry gene expression (Fujimoto et al. 2013). Overall, this indicates that SIX1/4 is required necessary for sufficient Nr5a1 gene expression in the early gonad and, a loss of Six1/4 gene expression, reduces Nr5a1 gene expression, and this may be responsible for the Six1/4 KO undersized gonad phenotype. Nonetheless, there is still sufficient Nr5a1 gene expression to form the gonad primordium in Six1/4 KO embryos. Chromobox homolog 2 (Cbx2) (mouse polycomb group member, M33) CBX2 is a component of the polycomb group complex of regulatory proteins involved in the repression/silencing of genes. In mice, Cbx2−/−-knockout mice show XY gonadal male-to-female (testis-to-ovary) sex reversal, and XX animals have smaller or absent ovaries (Katoh-Fukui et al. 2005). Additionally, mutants show defects in adrenal and Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. splenic development, which was shown to be a result of a reduction in the expression of Nr5a1 (Katoh-Fukui et al. 2005). This has led to the suggestion that CBX2 acts as an upstream regulator of Nr5a1 gene expression. Cbx2 has also been identified as a factor contributing to the differentiation of the testis through indirect regulation of Sry (Katoh-Fukui et al. 2012). Genome-wide identification of CBX2 target genes in a human Sertoli-like cell line suggests that CBX2 acts to stimulate male-specific genes, while suppressing female-pathway genes (Eid et al. 2015). In humans, a reported case study of a 46, XY child with female internal and external genitalia and histologically normal ovaries found a compound loss-of-function mutation in the coding region of CBX2 (Biason-Lauber et al. 2009). This study further lends support to the role of CBX2 in the trans-activation of Nr5a1 and its role in both the GR and testis developmental pathways (Fig. 4A). Insulin receptor, Insr and insulin-like growth factor type 1 receptor (Igf1r) Insulin and its related growth factors IGF1 and IGF2 regulate a variety of physiological processes including metabolism, stimulation of cell proliferation, differentiation and survival (Efstratiadis 1998). Their function is mediated by two membrane-associated tyrosine kinase receptors, the insulin receptor (INSR) and the IGF type 1 receptor (IGF1R). The genes for Insr, Igf1r and insulin receptorrelated receptor (Irr) have previously been shown to be necessary for the testis determination pathway, as mutant mice lacking all three genes show male-to-female sex reversal and decreased Sry and Sox9 gene expression (Nef et al. 2003). Recently, it has been shown that Insr and Igf1r, but not Irr have roles in GR development, whereby mice lacking both Insr and Igf1r have reduced proliferation of the GR prior to sex determination but also extensive downregulation of hundreds of genes associated with adrenal, testicular and ovarian development (Pitetti et al. 2013). As a result, these mice embryos exhibit agenesis of the adrenal cortex, along with male-female sex reversal due to a delay in Sry gene upregulation. Interestingly, ovarian differentiation is also delayed in these mice, leaving the GR in an undifferentiated state until about E16.5 when the ovarian programme is eventually initiated (Pitetti et al. 2013). Among the genes downregulated are Wt1, Lhx9 and Nr5a1, indicating that genes essential for GR development are under the influence of insulin/IGF signalling (Pitetti et al. 2013), but only partially dependent on this signalling pathway, as GR development is only hindered by decreased progenitor cell numbers. https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R55 Pre-B-cell leukaemia homeobox 1 (Pbx1) Pbx1 encodes a TALE (three amino acid loop extension) class homeodomain transcription factor that has been shown to be involved in a number of process during mammalian embryogenesis including, skeletal development and patterning (Selleri et al. 2001), maintenance of haematopoiesis (DiMartino et al. 2001), pancreatic development (Kim et al. 2002) and kidney and adrenal development (Schnabel et al. 2003). The GR of Pbx1-null mice are also smaller due to decreased proliferation of the progenitor cells in the gonad primordium (Schnabel et al. 2003). Further experiments found that this is due to the downregulation of Nr5a1 gene expression; PBX1 also upregulates Nr5a1 during adrenal development (Schnabel et al. 2003). The adrenal gland primordium is initially part of the GR but then later buds off from the very rostral end around E10.5 (Schnabel et al. 2003, Pitetti et al. 2013). Therefore, perturbed Pbx1 gene expression prior to budding likely affects Nr5a1 expression in the rostral GR, if not the whole GR, causing reduced Nr5a1 expression and decreased proliferation of somatic progenitor cells. Odd-skipped related 1 (Odd1) Odd1 gene encodes a zinc finger transcription factor homologous to the Drosophila odd-skipped class of transcription factors that are involved in embryonic patterning and tissue morphogenesis (Wang et al. 2005). Targeted gene knockout of Odd1 revealed that this gene functions in both heart and intermediate mesoderm development (Wang et al. 2005). Odd1−/− embryos have severe heart malformations, and completely lack adrenal glands, kidneys and gonads, all of which derive from intermediate mesoderm (Wang et al. 2005). Although gonad development was not the focus of this study, they did show that in early development the GR was hypoplastic (Wang et al. 2005), likely due to increased apoptosis observed with the developing kidney. These hypoplastic GRs appeared to degenerate, as no visible gonad structures were observed by E15.5 (Wang et al. 2005). Although no further investigation of Odd1 with regards to urogenital development has been done since Wang et al. (2005), it is unclear if the GR phenotype was an effect of Odd1 acting either directly in the GR or indirectly, potentially acting via downregulation of Wt1 gene expression, which also has an apoptotic phenotype when deleted (Hammes et al. 2001). Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. Mammalian early genital ridge development 62 :1 R56 Genital ridge formation in humans Much of what is known of human gonadal development is based on early embryology work and case studies of individuals with abnormal characteristics stemming from improper sexual development as a result of disruption of genes involved in this process. Recently, as sequencing technologies have become more affordable and require less material, more studies are using human-derived samples to study human sex development at the molecular level, mostly through studies of cases of disorders of sexual development (DSD) (O’Shaughnessy et al. 2007, Ostrer et al. 2007, Houmard et al. 2009, Del Valle et al. 2017, Li et al. 2017). These are congenital disorders that arise as a consequence of atypical chromosomal, gonadal or anatomical sexual development (Hughes et al. 2006), including some of the aforementioned genes involved in GR development. The term DSD replaced old and ambiguous terms such as intersex, hermaphroditism and pseudo-hermaphroditism previously used to describe such disorders in humans (Dreger et al. 2005, Lees & Tuch 2006). DSD phenotypes include a broad range of conditions such as failure of gonad formation, mixed gonadal tissue (male- and female-specific cell types within the tissue), ambiguous genitalia and failure of secondary sexual characteristics to develop normally (Cools et al. 2006). There is limited data on the incidence of DSDs, but it is estimated that the overall worldwide incidence of DSDs is 1 in 5,500 (Damiani 2007, Houk & Lee 2008). In humans, the gonad develops from the coelomic epithelium (Fig. 5), and the first signs of sex differentiation are observed between 6 and 7 weeks with the formation of testicular cords (Gruenwald 1942, Wyndham 1943). Germ cells are first detected in the hindgut dorsal mesentery at 4 weeks (Carnegie Stage 12 (CS12)) and migrate into the GR by 5 weeks (CS16) (Mckay et al. 1953). Gondal sex determination occurs around 41–42 days in human embryos, signified by the upregulation of SRY gene expression in XY embryos ((Hanley et al. 2000, Del Valle et al. 2017, Mamsen et al. 2017). Genes required for steroidogenesis and secreted factors such as AMH are essential for the sex-specific development of genitalia and the reproductive tract, with their expression commencing between 54 and 57 dpc (CS23). Genes essential for GR development in mice are also expressed at similar levels in both XX and XY gonads (LHX9, EMX2, WT1 and GATA4), further supporting a role for these factors in early human gonad development (Del Valle et al. 2017, Mamsen et al. 2017). Despite their apparent early essential roles in gonad development, the loss of function of genes required for sex https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Figure 5 Histology sections through human embryos 36–44 days (Carnegie stages 14–18). At ~36 days the coelomic epithelium begins to thicken, and by 39 days a ridge of tissue is forming, facing into the gut cavity. Just prior to sex determination (~42 days), the mesenchyme has proliferated to form a gonad region that is now easily distinguished from the neighbouring mesonephros. Section images were obtained from the Virtual Human Embryo resource (https://www.prenatalorigins.org/virtual-humanembryo/). CE, coelomic epithelium; DM, dorsal mesentery; M, mesonephros; MT, mesonephric tubule; MV, mesonephric vesicle; PMD, paramesonephric duct. Scale bar = 100 µm. development typically results in a range of phenotypes from complete gonad dysgenesis to adult infertility. Mutations in some of these early gonad development genes are also responsible for some cases of human idiopathic infertility in cases with apparently normal gonad development. Like DSDs, infertility is a condition associated with severe emotional and mental stress, particularly in societies where there is a social emphasis placed upon the ideal of having biological children (Ashraf et al. 2014). The mechanisms of infertility in males and females are varied in both origin and functional impact and often these conditions are thought to have an underlying genetic component (Zorrilla & Yatsenko 2013). Only 6–18% of infertility cases have identifiable genetic causes (mainly sex-chromosome abnormalities) indicating that for many cases of infertility (and subfertility), like DSD, the underlying genetic factors are yet to be elucidated. Genes with mutations commonly identified in DSD studies include androgen receptor and synthesis genes (androgen receptor (AR), CYP17A1, SRD5A2), NR5A1(SF1), WT1, GATA4, SRY, DAX1 and CHD7 (Kremen et al. 2017). Those patients with DSD and a complete gonad dysgenesis phenotype carry mutations in SRY, MAP3K1, DHH and NR5A1 genes (Ono & Harley 2013). While these genes are also critical for mouse gonadal development, largely supporting the use of the mouse as a model for human gonad development, there are several exceptions. For example, sequencing screens have found gain-of-function mutations in the mitogen-activated protein kinase kinase kinase 1 (MAP3K1) gene, including patients with streak gonads and female genitalia (Pearlman et al. 2010, Loke et al. 2014, Baxter et al. 2015, Eggers et al. 2016, Granados et al. 2017), suggesting a requirement for MAP3K1 function in gonad development for both sexes. However, although the mouse orthologue, Map3k1, is expressed in Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. the E11.5 gonad mouse, knockout mice have only minor testicular abnormalities, indicating a minimal role in XY gonadal development (Warr et al. 2011). This stresses the importance of additionally developing mouse models that replicate human gene mutations to study phenotype, as often human gene mutations do not result in a complete loss of gene/protein function but rather gain of function. Mutations in NR5A1 were identified in 4% of patients examined with unexplained male infertility (Bashamboo et al. 2010a, Ropke et al. 2013) and in female patients within premature ovarian failure (Lourenco et al. 2009). Novel NR5A1 mutation I (R92W) leads to 46, XX ovotestis (SRY negative) (Igarashi et al. 2017, Baetens et al. 2017b). The types of NR5A1 gene mutations and phenotypes associated with these disorders are reviewed in (Ferraz-deSouza et al. 2011). WT1 mutations have also been found in male and female cases of infertility, with ‘normal’ gonadal development (Seabra et al. 2015, Nathan et al. 2017). Humans are not the only species to present with variable phenotypes; in mice, the genetic background or strain strongly influences the adult phenotype of many genes linked to gonadal development. In one such example, gene knockout of Gadd45g (growth arrest and DNA damage-inducible protein 45 g) on a mixed genetic background (129/C57BL/6), 20% of the XY homozygous mice developed as infertile males, whereas on a C57BL/6 (B6) background, 100% of XY mice were sex reversed (Johnen et al. 2013). Genetic background also influences the phenotype of Nr5a1-, Fgf9- and Wt1-null mice (Meeks et al. 2003, Brennan & Capel 2004). The B6 mouse strain is more likely to result in a male-to-female sex reversal phenotype than the 129S1/SvImJ (129S1) strain, and differences in gonadal gene expression between these strains are observed even prior to sex determination at E11.5 (Colvin et al. 2001, Munger et al. 2009). Therefore, even though studies have identified genes critical to the early steps of gonad development, a loss-of-function mutation does not necessarily lead to complete gonadal dysgenesis. In at least some cases, genetic background and possibly environmental factors determine the phenotypic consequences of loss-of-function mutations in these genes. Future directions: unravelling the molecular pathways of early gonad development The advent and affordability of high-throughput sequencing technologies and new methods of replicating organ development in vitro, together will be valuable research tools in propelling forward both genetic and cellular biology into early GR development. https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R57 The use of single-cell RNA-seq (scRNA-seq) has rapidly furthered our understanding of developmental events, particularly within heterogeneous cell populations (Shapiro et al. 2013). Given that the GR consists of a broad variety of cellular precursors for endothelial, steroidogenic and supporting cell lineages, along with maturing germ cells, it is especially suitable for scRNA-seq analysis. Li et al. undertook scRNA-seq analysis of isolated single foetal germ cells and their surrounding ‘niche’ somatic cells from 4- to 26-week-old human embryos (Li et al. 2017). Based on gene expression profiles from this study, somatic cells in the early gonad can be divided into four groups within each sex. XX gonadal cells are comprised of endothelial cells, and three types of maturing granulosa cells (early-(10 weeks), mid-(10–20 weeks) and lategranulosa (20–26 weeks)) (Li et al. 2017). In XY gonads, somatic cells group into Sertoli cells, Leydig cell precursors, differentiated Leydig cells and endothelial cells (Li et al. 2017). Systematic examination of the expression profiles of each gonadal cell population during development will not only improve our knowledge of the in vivo mechanisms of cell differentiation but also the conditions required to induce correct cell differentiation in vitro. Organoid systems are becoming popular in vitro models of organ development (Fatehullah et al. 2016). Isolated preparations of human somatic and germ cells can selforganize into a testicular-like organoid using an artificial scaffold to aid 3D organization (Baert et al. 2017). These studies make use of cells that have already undergone sex-specific differentiation, as the cells are isolated from post-natal tissues (Baert et al. 2017). Recently, Sepponen et al., reported using human embryonic stem cells (hESCs) culture conditions to sequentially induce the primitive streak, followed by intermediate mesoderm and finally bi-potential-like gonadal cells expressing genes such as LHX9, EMX2, WT1 and GATA4 (Sepponen et al. 2017). This study found that timing and levels of BMPs (bone morphogenetic protein), WNT/β-catenin and Activin-A signalling ligands are essential to promote differentiation of gonadal cell precursors, over other mesodermal cell types (Sepponen et al. 2017). These studies, along with new sequencing resources, lay the groundwork for future research for not only modelling the early events of human gonad development, but to also examine the functional consequences of gene mutations identified in DSD patients using cultured cells engineered with the same genetic mutation. Several groups have taken a comprehensive targeted screening approach in order to identify genetic factors in DSD patients, and this in turn may lead to the identification Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. of novel genes required for GR development. Exome sequencing and targeted gene sequencing identified the genetic cause (classed as a functional gene mutation) in 28–38% of cases examined (Baxter et al. 2015, Dong et al. 2016, Fan et al. 2017, Kim et al. 2017). Deep sequencing of 64 known and 967 candidate genes improved the genetic diagnosis rate to 43% for one patient cohort (Eggers et al. 2016). Thus, despite advances in sequencing technologies, over 50% of DSD cases remain without a genetic diagnosis. While targeted sequencing and exome sequencing studies have focused on the protein coding regions of the genome, epigenetic processes, gene regulatory elements and non-coding RNAs (ncRNAs) are just as important for correct embryonic development. Errors in any of these gene regulatory mechanisms may underlie many cases of idiopathic DSD that lack mutations in proteincoding genes. Studies in vertebrates have largely focused on identifying sex-dimorphic expression of small ncRNAs called miRNAs following sex determination (Bannister et al. 2009, Real et al. 2013, Wainwright et al. 2013, Presslauer et al. 2017). There is limited information regarding how these miRNAs function in sex determination, if their function is essential, if they act to finely adjust gene expression levels during gonad development and whether these, and others, have earlier roles in the formation of the bi-potential gonad. Currently no miRNAs have been linked to human gonad development. Long ncRNAs are another important class of ncRNAs, which function to regulate gene expression (reviewed in Moran et al. 2012). With respect to sex development, these have been most thoroughly investigated for their role in X-chromosome dosage compensation (Cerase et al. 2015). It remains to be determined if autosomally encoded long ncRNAs contribute to mammalian sex determination and gonadal development but given their important roles in the development of other organ systems, it is likely they have a role in some aspects of gonadal development. Genome regulatory elements have been difficult to identify as most lie within the non-coding regions of the genome and can act over long distances to influence gene expression via chromatin folding. Mutations in regulatory elements range from single nucleotide sequence variants that prevent or reduce binding of a transcription factor to their DNA target sequence, to larger deletions, duplications and translocations resulting in structural changes that alter chromatin confirmation and regulatory interactions between enhancers and their target genes. Loss-of-function mutations in regulatory elements located near the SOX9 gene, a gene important for male sex determination, are the best-characterized regulatory https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R58 changes associated with DSD (reviewed in (Baetens et al. 2017a). As putative regulatory elements are difficult to predict, few have been mapped for human genes associated with early gonadal development. Recent genome-wide studies, including those mapping the chromatin state in specific cell lineages (Mikkelsen et al. 2007), will improve our ability to predict if DNA changes associated with DSD lie within gene regulatory regions. Epigenetic mechanisms such as DNA methylation and histone modifications have been implicated in the regulation of Sry gene in mice and dogs (Nishino et al. 2011, Jeong et al. 2016, Kuroki et al. 2017). Loss of function of the JMJD1A gene, which encodes a H3K9 demethylase enzyme, results in XY sex reversal (Kuroki et al. 2017). Some cases of XY DSD with complete sex reversal in dogs are thought to be due to persistent DNA hypermethylation of the Sry gene (Jeong et al. 2016). Therefore, it is likely that some cases of human DSD may be the result of mutations to epigenetic regulatory factors. Regulation of gene expression through epigenetic mechanisms is also especially sensitive to environmental influences and this impacts on many developmental programmes including sex determination (Feil & Fraga 2012). DNA methylation levels, determined by environmental factors, are vital to many naturally occurring forms of sex reversal and environmental sex determination in animals (Capel 2017). While difficult to study with respect to human DSD, it is possible that DNA methylation may play a role in balancing one sex developmental trajectory over another, and thus, errors in this may lead to gonad dysgenesis or sex reversal in humans. Summary The complexity of reproductive development is reflected in the difficulty in assigning a genetic diagnosis in most cases of human DSD. While we know the genetic aetiology of a small number of DSDs, up to as many as 75% of individuals with a DSD will remain without a genetic diagnosis (Arboleda et al. 2014). Even with whole genome sequencing, it is often difficult to identify functional variants and causal mutations, rendering many sequencing approaches somewhat ineffective (Fan et al. 2017). Gene expression levels, epigenetic modifiers and genetic background, along with the type of mutation and its functional consequence can all influence the resulting phenotype for both humans and mice. The future development of new technologies and improvement of existing ones will provide us with a much better understanding of the processes underlying normal Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. gonadal development in both human and mouse models, which in turn will lead to improved diagnosis in cases of DSD and infertility. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review. Funding This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector. Acknowledgements The authors would like to thank James Smith, Kathy Sircombe, Rebecca Clarke and Susie Szakats for their feedback on earlier versions of this manuscript. Yisheng Yang was supported by a University of Otago PhD scholarship award. Our work was funded by a University of Otago Research Grant to Megan Wilson. References Acien P 1992 Embryological observations on the female genital tract. Human Reproduction 7 437–445. (https://doi.org/10.1093/ oxfordjournals.humrep.a137666) Adams IR & McLaren A 2002 Sexually dimorphic development of mouse primordial germ cells: switching from oogenesis to spermatogenesis. Development 129 1155–1164. Arboleda VA, Sandberg DE & Vilain E 2014 DSDs: genetics, underlying pathologies and psychosexual differentiation. Nature Reviews Endocrinology 10 603–615. (https://doi.org/10.1038/nrendo.2014.130) 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. (https://doi.org/10.1016/0925-4773(93)90090-K) Ashraf DM, Ali D & Azadeh DM 2014 Effect of infertility on the quality of life, a cross-sectional study. Journal of Clinical and Diagnostic Research 8 OC13–OC15. Baert Y, De Kock J, Alves-Lopes JP, Soder O, Stukenborg JB & Goossens E 2017 Primary human testicular cells self-organize into organoids with testicular properties. Stem Cell Reports 8 30–38. (https://doi. org/10.1016/j.stemcr.2016.11.012) Baetens D, Mendonca BB, Verdin H, Cools M & De Baere E 2017a Noncoding variation in disorders of sex development. Clinical Genetics 91 163–172. (https://doi.org/10.1111/cge.12911) Baetens D, Stoop H, Peelman F, Todeschini AL, Rosseel T, Coppieters F, Veitia RA, Looijenga LH, De Baere E & Cools M 2017b NR5A1 is a novel disease gene for 46,XX testicular and ovotesticular disorders of sex development. Genetics in Medicine 19 367–376. (https://doi. org/10.1038/gim.2016.118) Bandiera R, Vidal VP, Motamedi FJ, Clarkson M, Sahut-Barnola I, von Gise A, Pu WT, Hohenstein P, Martinez A & Schedl A 2013 WT1 maintains adrenal-gonadal primordium identity and marks a population of AGP-like progenitors within the adrenal gland. Developmental Cell 27 5–18. (https://doi.org/10.1016/j.devcel.2013.09.003) Bannister SC, Tizard ML, Doran TJ, Sinclair AH & Smith CA 2009 Sexually dimorphic microRNA expression during chicken embryonic gonadal development. Biology of Reproduction 81 165–176. (https:// doi.org/10.1095/biolreprod.108.074005) https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R59 Barbaux S, Niaudet P, Gubler MC, Grunfeld JP, Jaubert F, Kuttenn F, Fekete CN, Souleyreau-Therville N, Thibaud E, Fellous M, et al. 1997 Donor splice-site mutations in WT1 are responsible for Frasier syndrome. Nature Genetics 17 467–470. (https://doi.org/10.1038/ ng1297-467) Bashamboo A, Ferraz-de-Souza B, Lourenco D, Lin L, Sebire NJ, Montjean D, Bignon-Topalovic J, Mandelbaum J, Siffroi JP, ChristinMaitre S, et al. 2010a Human male infertility associated with mutations in NR5A1 encoding steroidogenic factor 1. American Journal of Human Genetics 87 505–512. (https://doi.org/10.1016/j. ajhg.2010.09.009) Bashamboo A, Lourenco D, Rybczynska M, Nihoul-Fekete C, Brauner R & McElreavey K 2010b Familial case of 46, XY disorder of sex development DSD and congenital heart defects (CHD) associated with a mutation in GATA4. Endocrine Reviews 31 S1667. Bashamboo A, Donohoue PA, Vilain E, Rojo S, Calvel P, Seneviratne SN & Mulukutla SN 2016 A recurrent p. Arg92Trp variant in steroidogenic factor-1 (NR5A1) can act as a molecular switch in human sex development. Human Molecular Genetics 25 3446–3453. (https://doi.org/10.1093/hmg/ddw390) Baxter RM, Arboleda VA, Lee H, Barseghyan H, Adam MP, Fechner PY, Bargman R, Keegan C, Travers S, Schelley S, et al. 2015 Exome sequencing for the diagnosis of 46,XY disorders of sex development. Journal of Clinical Endocrinology and Metabolism 100 E333–E344. (https://doi.org/10.1210/jc.2014-2605) Beverdam A, Wilhelm D & Koopman P 2003 Molecular characterization of three gonad cell lines. Cytogenetic and Genome Research 101 242–249. (https://doi.org/10.1159/000074344) Bhandari RK, Sadler-Riggleman I, Clement TM & Skinner MK 2011 Basic helix-loop-helix transcription factor TCF21 is a downstream target of the male sex determining gene SRY. PLoS ONE 6 e19935. (https://doi. org/10.1371/journal.pone.0019935) Bhat S, Kabekkodu SP, Varghese VK, Chakrabarty S, Mallya SP, Rotti HS, Pandey D, Kushtagi P & Satyamoorthy K 2017 Aberrant gene-specific DNA methylation signature analysis in cervical cancer. Tumor Biology 39 1010428317694573. (https://doi.org/10.1177/1010428317694573) Biason-Lauber A, Konrad D, Meyer M, DeBeaufort C & Schoenle EJ 2009 Ovaries and female phenotype in a girl with 46,XY karyotype and mutations in the CBX2 gene. American Journal of Human Genetics 84 658–663. (https://doi.org/10.1016/j.ajhg.2009.03.016) Birk OS, Casiano DE, Wassif CA, Cogliati T, Zhao L, Zhao Y, Grinberg A, Huang S, Kreidberg JA, Parker KL, et al. 2000 The LIM homeobox gene Lhx9 is essential for mouse gonad formation. Nature 403 909–913. (https://doi.org/10.1038/35002622) Boucher CA, Carey N, Edwards YH, Siciliano MJ & Johnson KJ 1996 Cloning of the human SIX1 gene and its assignment to chromosome 14. Genomics 33 140–142. (https://doi.org/10.1006/geno.1996.0172) Brennan J & Capel B 2004 One tissue, two fates: molecular genetic events that underlie testis versus ovary development. Nature Reviews Genetics 5 509–521. (https://doi.org/10.1038/nrg1381) Bruening W & Pelletier J 1996 A non-AUG translational initiation event generates novel WT1 isoforms. Journal of Biological Chemistry 271 8646–8654. (https://doi.org/10.1074/jbc.271.15.8646) Cai KQ, Caslini C, Capo-Chichi CD, Slater C, Smith ER, Wu H & Xu XX 2009 Loss of GATA4 and GATA6 expression specifies ovarian cancer histological subtypes and precedes neoplastic transformation of ovarian surface epithelia. PLoS ONE 4 e6454. (https://10.1371/ journal.pone.0006454) Capel B 2017 Vertebrate sex determination: evolutionary plasticity of a fundamental switch. Nature Reviews Genetics 18 675–689. (https:// doi.org/10.1038/nrg.2017.60) Carpenter B, Hill KJ, Charalambous M, Wagner KJ, Lahiri D, James DI, Andersen JS, Schumacher V, Royer-Pokora B, Mann M, et al. 2004 BASP1 is a transcriptional cosuppressor for the Wilms’ tumor suppressor protein WT1. Molecular and Cellular Biology 24 537–549. (https://doi.org/10.1128/MCB.24.2.537-549.2004) Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. Cerase A, Pintacuda G, Tattermusch A & Avner P 2015 Xist localization and function: new insights from multiple levels. Genome Biology 16 166. (https://doi.org/10.1186/s13059-015-0733-y) Chen M, Zhang L, Cui X, Lin X, Li Y, Wang Y, Wang Y, Qin Y, Chen D, Han C, et al. 2017 Wt1 directs the lineage specification of sertoli and granulosa cells by repressing Sf1 expression. Development 144 44–53. (https://doi.org/10.1242/dev.144105) Colvin JS, Green RP, Schmahl J, Capel B & Ornitz DM 2001 Male-tofemale sex reversal in mice lacking fibroblast growth factor 9. Cell 104 875–889. (https://doi.org/10.1016/S0092-8674(01)00284-7) Conforto TL & Waxman DJ 2012 Sex-specific mouse liver gene expression: genome-wide analysis of developmental changes from pre-pubertal period to young adulthood. Biology of Sex Differences 3 9. (https://doi.org/10.1186/2042-6410-3-9) Cools M, Drop SL, Wolffenbuttel KP, Oosterhuis JW & Looijenga LH 2006 Germ cell tumors in the intersex gonad: old paths, new directions, moving frontiers. Endocrine Reviews 27 468–484. (https:// doi.org/10.1210/er.2006-0005) Coveney D, Cool J, Oliver T & Capel B 2008 Four-dimensional analysis of vascularization during primary development of an organ, the gonad. PNAS 105 7212–7217. (https://doi.org/10.1073/pnas.0707674105) Cui S, Schwartz L & Quaggin SE 2003 Pod1 is required in stromal cells for glomerulogenesis. Developmental Dynamics 226 512–522. (https:// doi.org/10.1002/dvdy.10244) 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. (https://doi.org/10.1242/dev.01266) Daftary GS & Taylor HS 2004 EMX2 gene expression in the female reproductive tract and aberrant expression in the endometrium of patients with endometriosis. Journal of Clinical Endocrinology and Metabolism 89 2390–2396. (https://doi.org/10.1210/jc.2003-031389) Dallosso AR, Hancock AL, Malik S, Salpekar A, King-Underwood L, Pritchard-Jones K, Peters J, Moorwood K, Ward A, Malik KT, et al. 2007 Alternately spliced WT1 antisense transcripts interact with WT1 sense RNA and show epigenetic and splicing defects in cancer. RNA 13 2287–2299. (https://doi.org/10.1261/rna.562907) Damiani D 2007 Disorders of sexual development – still a big challenge! Journal of Pediatric Endocrinology and Metabolism 20 749–750. Del Valle I, Buonocore F, Duncan AJ, Lin L, Barenco M, Parnaik R, Shah S, Hubank M, Gerrelli D & Achermann JC 2017 A genomic atlas of human adrenal and gonad development. Wellcome Open Research 2 25. (https://doi.org/10.12688/wellcomeopenres.11253.1) DiMartino JF, Selleri L, Traver D, Firpo MT, Rhee J, Warnke R, O’Gorman S, Weissman IL & Cleary ML 2001 The Hox cofactor and proto-oncogene Pbx1 is required for maintenance of definitive hematopoiesis in the fetal liver. Blood 98 618–626. (https://doi. org/10.1182/blood.V98.3.618) Doitsidou M, Reichman-Fried M, Stebler J, Koprunner M, Dorries J, Meyer D, Esguerra CV, Leung T & Raz E 2002 Guidance of primordial germ cell migration by the chemokine SDF-1. Cell 111 647–659. (https://doi.org/10.1016/S0092-8674(02)01135-2) Dong Y, Yi Y, Yao H, Yang Z, Hu H, Liu J, Gao C, Zhang M, Zhou L, Asan, et al. 2016 Targeted next-generation sequencing identification of mutations in patients with disorders of sex development. BMC Medical Genetics 17 23. (https://doi.org/10.1186/s12881-016-0286-2) Dreger AD, Chase C, Sousa A, Gruppuso PA & Frader J 2005 Changing the nomenclature/taxonomy for intersex: a scientific and clinical rationale. Journal of Pediatric Endocrinology and Metabolism 18 729–733. Efimenko E, Padua MB, Manuylov NL, Fox SC, Morse DA & Tevosian SG 2013 The transcription factor GATA4 is required for follicular development and normal ovarian function. Developmental Biology 381 144–158. (https://doi.org/10.1016/j.ydbio.2013.06.004) Efstratiadis A 1998 Genetics of mouse growth. International Journal of Developmental Biology 42 955–976. Eggers S, Sadedin S, van den Bergen JA, Robevska G, Ohnesorg T, Hewitt J, Lambeth L, Bouty A, Knarston IM, Tan TY, et al. 2016 https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R60 Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort. Genome Biology 17 243. (https://doi.org/10.1186/s13059-016-1105-y) Eid W, Opitz L & Biason-Lauber A 2015 Genome-wide identification of CBX2 targets: insights in the human sex development network. Molecular Endocrinology 29 247–257. (https://doi.org/10.1210/ me.2014-1339) Esteve P & Bovolenta P 1999 cSix4, a member of the six gene family of transcription factors, is expressed during placode and somite development. Mechanisms of Development 85 161–165. (https://doi. org/10.1016/S0925-4773(99)00079-9) Failli V, Rogard M, Mattei MG, Vernier P & Retaux S 2000 Lhx9 and Lhx9alpha LIM-homeodomain factors: genomic structure, expression patterns, chromosomal localization, and phylogenetic analysis. Genomics 64 307–317. (https://doi.org/10.1006/geno.2000.6123) Fan Y, Zhang X, Wang L, Wang R, Huang Z, Sun Y, Yao R, Huang X, Ye J, Han L, et al. 2017 Diagnostic application of targeted next-generation sequencing of 80 genes associated with disorders of sexual development. Scientific Reports 7 44536. (https://doi.org/10.1038/srep44536) Fatehullah A, Tan SH & Barker N 2016 Organoids as an in vitro model of human development and disease. Nature Cell Biology 18 246–254. (https://doi.org/10.1038/ncb3312) Feil R & Fraga MF 2012 Epigenetics and the environment: emerging patterns and implications. Nature Reviews Genetics 13 97–109. (https://doi.org/10.1038/nrg3142) Ferraz-de-Souza B, Lin L & Achermann JC 2011 Steroidogenic factor-1 (SF-1, NR5A1) and human disease. Molecular and Cellular Endocrinology 336 198–205. (https://doi.org/10.1016/j.mce.2010.11.006) Fougerousse F, Durand M, Lopez S, Suel L, Demignon J, Thornton C, Ozaki H, Kawakami K, Barbet P, Beckmann JS, et al. 2002 Six and Eya expression during human somitogenesis and MyoD gene family activation. Journal of Muscle Research and Cell Motility 23 255–264. (https://doi.org/10.1023/A:1020990825644) Franca MM, Ferraz-de-Souza B, Santos MG, Lerario AM, Fragoso MC, Latronico AC, Kuick RD, Hammer GD & Lotfi CF 2013 POD-1 binding to the E-box sequence inhibits SF-1 and StAR expression in human adrenocortical tumor cells. Molecular and Cellular Endocrinology 371 140–147. (https://doi.org/10.1016/j.mce.2012.12.029) Franca MM, Abreu NP, Vrechi TA & Lotfi CF 2015 POD-1/Tcf21 overexpression reduces endogenous SF-1 and StAR expression in rat adrenal cells. Brazilian Journal of Medical and Biological Research 48 1087–1094. (https://doi.org/10.1590/1414-431X20154748) Fujimoto Y, Tanaka SS, Yamaguchi YL, Kobayashi H, Kuroki S, Tachibana M, Shinomura M, Kanai Y, Morohashi K, Kawakami K, et al. 2013 Homeoproteins Six1 and Six4 regulate male sex determination and mouse gonadal development. Developmental Cell 26 416–430. (https://doi.org/10.1016/j.devcel.2013.06.018) Funato N, Ohyama K, Kuroda T & Nakamura M 2003 Basic helix-loop-helix transcription factor epicardin/capsulin/Pod-1 suppresses differentiation by negative regulation of transcription. Journal of Biological Chemistry 278 7486–7493. (https://doi.org/10.1074/jbc.M212248200) Gao F, Zhang J, Wang X, Yang J, Chen D, Huff V & Liu Y-x 2014 Wt1 functions in ovarian follicle development by regulating granulosa cell differentiation. Human Molecular Genetics 23 333–341. (https:// doi.org/10.1093/hmg/ddt423) Gill ME, Hu YC, Lin Y & Page DC 2011 Licensing of gametogenesis, dependent on RNA binding protein DAZL, as a gateway to sexual differentiation of fetal germ cells. PNAS 108 7443–7448. (https://doi. org/10.1073/pnas.1104501108) Giordani J, Bajard L, Demignon J, Daubas P, Buckingham M & Maire P 2007 Six proteins regulate the activation of Myf5 expression in embryonic mouse limbs. PNAS 104 11310–11315. (https://doi. org/10.1073/pnas.0611299104) Granados A, Alaniz VI, Mohnach L, Barseghyan H, Vilain E, Ostrer H, Quint EH, Chen M & Keegan CE 2017 MAP3K1-related gonadal dysgenesis: six new cases and review of the literature. American Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. Journal of Medical Genetics Part C: Seminars in Medical Genetics 175 253–259. (https://doi.org/10.1002/ajmg.c.31559) Greenfield A 2015 Understanding sex determination in the mouse: genetics, epigenetics and the story of mutual antagonisms. Journal of Genetics 94 585–590. (https://doi.org/10.1007/s12041-015-0565-2) Grifone R, Demignon J, Houbron C, Souil E, Niro C, Seller MJ, Hamard G & Maire P 2005 Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo. Development 132 2235–2249. (https://doi.org/10.1242/dev.01773) Gropp A & Ohno S 1966 The presence of a common embryonic blastema for ovarian and testicular parenchymal (follicular, interstitial and tubular) cells in cattle Bos taurus. Zeitschrift für Zellforschung und mikroskopische Anatomie 74 505–528. (https://doi. org/10.1007/BF00496841) Gruenwald P 1942 The development of the sex cords in the gonads of man and mammals. American Journal of Anatomy 70 359–397. (https://doi.org/10.1002/aja.1000700303) Gut P, Huber K, Lohr J, Bruhl B, Oberle S, Treier M, Ernsberger U, Kalcheim C & Unsicker K 2005 Lack of an adrenal cortex in Sf1 mutant mice is compatible with the generation and differentiation of chromaffin cells. Development 132 4611–4619. (https://doi. org/10.1242/dev.02052) Hacker A, Capel B, Goodfellow P & Lovell-Badge R 1995 Expression of Sry, the mouse sex determining gene. Development 121 1603–1614. Hammes A, Guo JK, Lutsch G, Leheste JR, Landrock D, Ziegler U, Gubler MC & Schedl A 2001 Two splice variants of the Wilms’ tumor 1 gene have distinct functions during sex determination and nephron formation. Cell 106 319–329. (https://doi.org/10.1016/ S0092-8674(01)00453-6) Hanley NA, Hagan DM, Clement-Jones M, Ball SG, Strachan T, SalasCortes L, McElreavey K, Lindsay S, Robson S, Bullen P, et al. 2000 SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development. Mechanisms of Development 91 403–407. (https://doi.org/10.1016/S0925-4773(99)00307-X) Hannema SE & Hughes IA 2007 Regulation of Wolffian duct development. Hormone Research 67 142–151. Hara K, Kanai-Azuma M, Uemura M, Shitara H, Taya C, Yonekawa H, Kawakami H, Tsunekawa N, Kurohmaru M & Kanai Y 2009 Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis. Developmental Biology 330 427–439. (https://doi.org/10.1016/j.ydbio.2009.04.012) Harikae K, Miura K & Kanai Y 2013 Early gonadogenesis in mammals: significance of long and narrow gonadal structure. Developmental Dynamics 242 330–338. (https://doi.org/10.1002/dvdy.23872) Hastie ND 1992 Dominant negative mutations in the Wilms tumour (WT1) gene cause Denys-Drash syndrome – proof that a tumour-suppressor gene plays a crucial role in normal genitourinary development. Human Molecular Genetics 1 293–295. (https://doi.org/10.1093/hmg/1.5.293) Hatano O, Takakusu A, Nomura M & Morohashi K 1996 Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF-1. Genes Cells 1 663–671. (https://doi. org/10.1046/j.1365-2443.1996.00254.x) Herzer U, Crocoll A, Barton D, Howells N & Englert C 1999 The Wilms tumor suppressor gene wt1 is required for development of the spleen. Current Biology 9 837–840. (https://doi.org/10.1016/S09609822(99)80369-8) Houk CP & Lee PA 2008 Consensus statement on terminology and management: disorders of sex development. Sexual Development 2 172–180. (https://doi.org/10.1159/000152032) Houmard B, Small C, Yang L, Naluai-Cecchini T, Cheng E, Hassold T & Griswold M 2009 Global gene expression in the human fetal testis and ovary. Biology of Reproduction 81 438–443. (https://doi. org/10.1095/biolreprod.108.075747) Hu YC, Okumura LM & Page DC 2013 Gata4 is required for formation of the genital ridge in mice. PLoS Genetics 9 e1003629. (https://doi. org/10.1371/journal.pgen.1003629) https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R61 Hughes IA, Houk C, Ahmed SF & Lee PA 2006 Consensus statement on management of intersex disorders. Journal of Pediatric Urology 2 148–162. (https://doi.org/10.1016/j.jpurol.2006.03.004) Igarashi M, Takasawa K, Hakoda A, Kanno J, Takada S, Miyado M, Baba T, Morohashi KI, Tajima T, Hata K, et al. 2017 Identical NR5A1 missense mutations in two unrelated 46,XX individuals with testicular tissues. Human Mutation 38 39–42. (https://doi.org/10.1002/humu.23116) Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachtigal MW, Abbud R, Nilson JH & Parker KL 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes and Development 8 2302–2312. (https://doi.org/10.1101/gad.8.19.2302) Ito M, Yokouchi K, Yoshida K, Kano K, Naito K, Miyazaki J & Tojo H 2006 Investigation of the fate of Sry-expressing cells using an in vivo Cre/loxP system. Development, Growth and Differentiation 48 41–47. (https://doi.org/10.1111/j.1440-169X.2006.00844.x) Jeong YH, Lu H, Park CH, Li M, Luo H, Kim JJ, Liu S, Ko KH, Huang S, Hwang IS, et al. 2016 Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer. Scientific Reports 6 31088. (https://doi.org/10.1038/srep31088) Johnen H, Gonzalez-Silva L, Carramolino L, Flores JM, Torres M & Salvador JM 2013 Gadd45g is essential for primary sex determination, male fertility and testis development. PLoS ONE 8 e58751. (https://doi.org/10.1371/journal.pone.0058751) Jost A 1972 A new look at the mechanisms controlling sex differentiation in mammals. Johns Hopkins Medical Journal 130 38–53. Jurata LW & Gill GN 1998 Structure and function of LIM domains. Current Topics in Microbiology and Immunology 228 75–113. Karl J & Capel B 1995 Three-dimensional structure of the developing mouse genital ridge. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 350 235–242. (https://doi. org/10.1098/rstb.1995.0157) Karl J & Capel B 1998 Sertoli cells of the mouse testis originate from the coelomic epithelium. Developmental Biology 203 323–333. (https:// doi.org/10.1006/dbio.1998.9068) Katoh-Fukui Y, Owaki A, Toyama Y, Kusaka M, Shinohara Y, Maekawa M, Toshimori K & Morohashi K 2005 Mouse Polycomb M33 is required for splenic vascular and adrenal gland formation through regulating Ad4BP/SF1 expression. Blood 106 1612–1620. (https://doi. org/10.1182/blood-2004-08-3367) Katoh-Fukui Y, Miyabayashi K, Komatsu T, Owaki A, Baba T, Shima Y, Kidokoro T, Kanai Y, Schedl A, Wilhelm D, et al. 2012 Cbx2, a polycomb group gene, is required for Sry gene expression in mice. Endocrinology 153 913–924. (https://doi.org/10.1210/en.2011-1055) Kawakami K, Sato S, Ozaki H & Ikeda K 2000 Six family genes--structure and function as transcription factors and their roles in development. BioEssays 22 616–626. (https://doi.org/10.1002/15211878(200007)22:7<616::AID-BIES4>3.0.CO;2-R) Kim SK, Selleri L, Lee JS, Zhang AY, Gu X, Jacobs Y & Cleary ML 2002 Pbx1 inactivation disrupts pancreas development and in Ipf1deficient mice promotes diabetes mellitus. Nature Genetics 30 430–435. (https://doi.org/10.1038/ng860) Kim JH, Kang E, Heo SH, Kim GH, Jang JH, Cho EH, Lee BH, Yoo HW & Choi JH 2017 Diagnostic yield of targeted gene panel sequencing to identify the genetic etiology of disorders of sex development. Molecular and Cellular Endocrinology 444 19–25. (https://doi. org/10.1016/j.mce.2017.01.037) Klamt B, Koziell A, Poulat F, Wieacker P, Scambler P, Berta P & Gessler M 1998 Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms. Human Molecular Genetics 7 709–714. (https://doi.org/10.1093/hmg/7.4.709) Kobayashi H, Kawakami K, Asashima M & Nishinakamura R 2007 Six1 and Six4 are essential for Gdnf expression in the metanephric mesenchyme and ureteric bud formation, while Six1 deficiency alone causes mesonephric-tubule defects. Mechanisms of Development 124 290–303. (https://doi.org/10.1016/j.mod.2007.01.002) Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. Kohler B, Biebermann H, Friedsam V, Gellermann J, Maier RF, Pohl M, Wieacker P, Hiort O, Gruters A & Krude H 2011 Analysis of the Wilms’ tumor suppressor gene (WT1) in patients 46,XY disorders of sex development. Journal of Clinical Endocrinology and Metabolism 96 E1131–E1136. (https://doi.org/10.1210/jc.2010-2804) Konishi Y, Ikeda K, Iwakura Y & Kawakami K 2006 Six1 and Six4 promote survival of sensory neurons during early trigeminal gangliogenesis. Brain Research 1116 93–102. (https://doi. org/10.1016/j.brainres.2006.07.103) Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D & Jaenisch R 1993 WT-1 is required for early kidney development. Cell 74 679–691. (https://doi.org/10.1016/0092-8674(93)90515-R) Kremen J, Chan YM & Swartz JM 2017 Recent findings on the genetics of disorders of sex development. Current Opinion in Urology 27 1–6. (https://doi.org/10.1097/MOU.0000000000000353) Kumar JP 2009 The sine oculis homeobox (SIX) family of transcription factors as regulators of development and disease. Cellular and Molecular Life Sciences 66 565–583. (https://doi.org/10.1007/s00018-008-8335-4) Kuo CT, Morrisey EE, Anandappa R, Sigrist K, Lu MM, Parmacek MS, Soudais C & Leiden JM 1997 GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes and Development 11 1048–1060. (https://doi.org/10.1101/gad.11.8.1048) Kuroki S, Okashita N, Baba S, Maeda R, Miyawaki S, Yano M, Yamaguchi M, Kitano S, Miyachi H, Itoh A, et al. 2017 Rescuing the aberrant sex development of H3K9 demethylase Jmjd1a-deficient mice by modulating H3K9 methylation balance. PLoS Genetics 13 e1007034. (https://doi.org/10.1371/journal.pgen.1007034) Kusaka M, Katoh-Fukui Y, Ogawa H, Miyabayashi K, Baba T, Shima Y, Sugiyama N, Sugimoto Y, Okuno Y, Kodama R, et al. 2010 Abnormal epithelial cell polarity and ectopic epidermal growth factor receptor (EGFR) expression induced in Emx2 KO embryonic gonads. Endocrinology 151 5893–5904. (https://doi.org/10.1210/en.2010-0915) Laclef C, Hamard G, Demignon J, Souil E, Houbron C & Maire P 2003 Altered myogenesis in Six1-deficient mice. Development 130 2239–2252. (https://doi.org/10.1242/dev.00440) Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP & Hogan BL 1999 Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes and Development 13 424–436. (https://doi.org/10.1101/gad.13.4.424) Le Caignec C, Delnatte C, Vermeesch JR, Boceno M, Joubert M, Lavenan, F & Rival JM 2007 Complete sex reversal in a WAGR syndrome patient. American Journal of Medical Genetics Part A 143 2692–2695. (https://doi.org/10.1002/ajmg.a.31997) Lees JG & Tuch BE 2006 Conversion of embryonic stem cells into pancreatic beta-cell surrogates guided by ontogeny. Regenerative Medicine, 1327–336. (https://doi.org/10.2217/17460751.1.3.327) Li L, Dong J, Yan L, Yong J, Liu X, Hu Y, Fan X, Wu X, Guo H, Wang X, et al. 2017 Single-cell RNA-seq analysis maps development of human germline cells and gonadal niche interactions. Cell Stem Cell 20 858. e854–873.e854. (https://doi.org/10.1016/j.stem.2017.05.009) Liu S, Gao X, Qin Y, Liu W, Huang T, Ma J & Chen ZJ 2015 Nonsense mutation of EMX2 is potential causative for uterus didelphysis: first molecular explanation for isolated incomplete müllerian fusion. Fertility and Sterility 103 769–774. (https://doi.org/10.1016/j. fertnstert.2014.11.03) Loke J, Pearlman A, Radi O, Zuffardi O, Giussani U, Pallotta R, Camerino G & Ostrer H 2014 Mutations in MAP3K1 tilt the balance from SOX9/FGF9 to WNT/beta-catenin signaling. Human Molecular Genetics 23 1073–1083. (https://doi.org/10.1093/hmg/ddt502) Lourenco D, Brauner R, Lin L, De Perdigo A, Weryha G, Muresan M, Boudjenah R, Guerra-Junior G, Maciel-Guerra AT, Achermann JC, et al. 2009 Mutations in NR5A1 associated with ovarian insufficiency. New England Journal of Medicine 360 1200–1210. (https://doi.org/10.1056/NEJMoa0806228) Lourenco D, Brauner R, Rybczynska M, Nihoul-Fekete C, McElreavey K & Bashamboo A 2011 Loss-of-function mutation in GATA4 causes https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R62 anomalies of human testicular development. PNAS 108 1597–1602. (https://doi.org/10.1073/pnas.1010257108) Lu J, Chang P, Richardson JA, Gan L, Weiler H & Olson EN 2000 The basic helix-loop-helix transcription factor capsulin controls spleen organogenesis. PNAS 97 9525–9530. (https://doi.org/10.1073/ pnas.97.17.9525) Luo X, Ikeda Y & Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77 481–490. (https://doi.org/10.1016/00928674(94)90211-9) Maekawa F, Tsukahara S, Kawashima T, Nohara K & Ohki-Hamazaki H 2014 The mechanisms underlying sexual differentiation of behavior and physiology in mammals and birds: relative contributions of sex steroids and sex chromosomes. Frontiers in Neuroscience 8 242. Mamsen LS, Ernst EH, Borup R, Larsen A, Olesen RH, Ernst E, Anderson RA, Kristensen SG & Andersen CY 2017 Temporal expression pattern of genes during the period of sex differentiation in human embryonic gonads. Scientific Reports 7 15961. (https://doi. org/10.1038/s41598-017-15931-3) Martineau J, Nordqvist K, Tilmann C, Lovell-Badge R & Capel B 1997 Male-specific cell migration into the developing gonad. Current Biology 7 958–968. (https://doi.org/10.1016/S0960-9822(06)00415-5) Mather JP 1980 Establishment and characterization of two distinct mouse testicular epithelial cell lines. Biology of Reproduction 23 243–252. (https://doi.org/10.1095/biolreprod23.1.243) Mckay DG, Hertig AT, Adams EC & Danziger S 1953 Histochemical observations on the germ cells of human embryos. Anatomical Record 117 201–219. (https://doi.org/10.1002/ar.1091170206) McKay LM, Carpenter B & Roberts SG 1999 Regulation of the Wilms’ tumour suppressor protein transcriptional activation domain. Oncogene 18 6546–6554. (https://doi.org/10.1038/sj.onc.1203046) McLaren A & Southee D 1997 Entry of mouse embryonic germ cells into meiosis. Developmental Biology 187 107–113. (https://doi. org/10.1006/dbio.1997.8584) Meeks JJ, Weiss J & Jameson JL 2003 Dax1 is required for testis determination. Nature Genetics 34 32–33. (https://doi.org/10.1038/ ng1141) Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, et al. 2007 Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448 553–560. (https://doi.org/10.1038/nature06008) Miyagishi M, Nakajima T & Fukamizu A 2000 Molecular characterization of mesoderm-restricted basic helix-loop-helix protein, POD-1/ Capsulin. International Journal of Molecular Medicine 5 27–31. Miyamoto N, Yoshida M, Kuratani S, Matsuo I & Aizawa S 1997 Defects of urogenital development in mice lacking Emx2. Development 124 1653–1664. Molkentin JD, Lin Q, Duncan SA & Olson EN 1997 Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes and Development 11 1061–1072. (https://doi. org/10.1101/gad.11.8.1061) Molle B, Pere S, Failli V, Bach I & Retaux S 2004 Lhx9 and lhx9alpha: differential biochemical properties and effects on neuronal differentiation. DNA Cell Biol 23 761–768. (https://doi.org/10.1089/ dna.2004.23.761) Molyneaux KA, Stallock J, Schaible K & Wylie C 2001 Time-lapse analysis of living mouse germ cell migration. Developmental Biology 240 488–498. (https://doi.org/10.1006/dbio.2001.0436) Moran VA, Perera RJ & Khalil AM 2012 Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs. Nucleic Acids Research 40 6391–6400. (https://doi.org/10.1093/nar/gks296) Mork L, Maatouk DM, McMahon JA, Guo JJ, Zhang P, McMahon AP & Capel B 2012 Temporal differences in granulosa cell specification in the ovary reflect distinct follicle fates in mice. Biology of Reproduction 86 37. Morohashi KI & Omura T 1996 Ad4BP/SF-1, a transcription factor essential for the transcription of steroidogenic cytochrome P450 Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. genes and for the establishment of the reproductive function. FASEB Journal 10 1569–1577. (https://doi.org/10.1096/fasebj.10.14.9002548) Munger SC, Aylor DL, Syed HA, Magwene PM, Threadgill DW & Capel B 2009 Elucidation of the transcription network governing mammalian sex determination by exploiting strain-specific susceptibility to sex reversal. Genes and Development 23 2521–2536. (https://doi.org/10.1101/gad.1835809) Nathan A, Reinhardt P, Kruspe D, Jorss T, Groth M, Nolte H, Habenicht A, Herrmann J, Holschbach V, Toth B, et al. 2017 The Wilms tumor protein Wt1 contributes to female fertility by regulating oviductal proteostasis. Human Molecular Genetics 26 1694–1705. (https://doi.org/10.1093/hmg/ddx075) Nef S, Verma-Kurvari S, Merenmies J, Vassalli JD, Efstratiadis A, Accili D & Parada LF 2003 Testis determination requires insulin receptor family function in mice. Nature 426 291–295. (https://doi. org/10.1038/nature02059) Nishino K, Hattori N, Sato S, Arai Y, Tanaka S, Nagy A & Shiota K 2011 Non-CpG methylation occurs in the regulatory region of the Sry gene. Journal of Reproduction and Development 57 586–593. (https:// doi.org/10.1262/jrd.11-033A) Ono M & Harley VR 2013 Disorders of sex development: new genes, new concepts. Nature Reviews Endocrinology 9 79–91. (https://doi. org/10.1038/nrendo.2012.235) O’Shaughnessy PJ, Baker PJ, Monteiro A, Cassie S, Bhattacharya S & Fowler PA 2007 Developmental changes in human fetal testicular cell numbers and messenger ribonucleic acid levels during the second trimester. Journal of Clinical Endocrinology and Metabolism 92 4792–4801. Oshima Y, Noguchi K & Nakamura M 2007 Expression of Lhx9 isoforms in the developing gonads of Rana rugosa. Zoological Science 24 798–802. (https://doi.org/10.2108/zsj.24.798) Ostrer H, Huang HY, Masch RJ & Shapiro E 2007 A cellular study of human testis development. Sexual Development 1 286–292. (https:// doi.org/10.1159/000108930) Ozaki H, Watanabe Y, Takahashi K, Kitamura K, Tanaka A, Urase K, Momoi T, Sudo K, Sakagami J, Asano M, et al. 2001 Six4, a putative myogenin gene regulator, is not essential for mouse embryonal development. Molecular and Cellular Biology 21 3343–3350. (https:// doi.org/10.1128/MCB.21.10.3343-3350.2001) Pearlman A, Loke J, Le Caignec C, White S, Chin L, Friedman A, Warr N, Willan J, Brauer D, Farmer C, et al. 2010 Mutations in MAP3K1 cause 46,XY disorders of sex development and implicate a common signal transduction pathway in human testis determination. American Journal of Human Genetics 87 898–904. (https://doi.org/10.1016/j.ajhg.2010.11.003) Pellegrini M, Pantano S, Lucchini F, Fumi M & Forabosco A 1997 Emx2 developmental expression in the primordia of the reproductive and excretory systems. Anatomy and Embryology 196 427–433. (https:// doi.org/10.1007/s004290050110) Pelliniemi LJ 1975 Ultrastructure of gonadal ridge in male and female pig embryos. Anatomy and Embryology 147 20–34. Pitetti JL, Calvel P, Romero Y, Conne B, Truong V, Papaioannou MD, Schaad O, Docquier M, Herrera PL, Wilhelm D, et al. 2013 Insulin and IGF1 receptors are essential for XX and XY gonadal differentiation and adrenal development in mice. PLoS Genetics 9 e1003160. (https://doi.org/10.1371/journal.pgen.1003160) Plotkin M & Mudunuri V 2008 Pod1 induces myofibroblast differentiation in mesenchymal progenitor cells from mouse kidney. Journal of Cellular Biochemistry 103 675–690. (https://doi.org/10.1002/jcb.21441) Presslauer C, Tilahun Bizuayehu T, Kopp M, Fernandes JM & Babiak I 2017 Dynamics of miRNA transcriptome during gonadal development of zebrafish. Scientific Reports 7 43850. (https://doi. org/10.1038/srep43850) Quaggin SE, Vanden Heuvel GB & Igarashi P 1998 Pod-1, a mesodermspecific basic-helix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney. Mechanisms of Development 71 37–48. (https://doi.org/10.1016/S0925-4773(97)00201-3) https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Mammalian early genital ridge development 62 :1 R63 Quaggin SE, Schwartz L, Cui S, Igarashi P, Deimling J, Post M & Rossant J 1999 The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis. Development 126 5771–5783. Real FM, Sekido R, Lupianez DG, Lovell-Badge R, Jimenez R & Burgos M 2013 A microRNA (mmu-miR-124) prevents Sox9 expression in developing mouse ovarian cells. Biology of Reproduction 89 78. Retaux S, Rogard M, Bach I, Failli V & Besson MJ 1999 Lhx9: a novel LIM-homeodomain gene expressed in the developing forebrain. Journal of Neuroscience 19 783–793. (https://doi.org/10.1523/ JNEUROSCI.19-02-00783.1999) Rolland AD, Lehmann KP, Johnson KJ, Gaido KW & Koopman P 2011 Uncovering gene regulatory networks during mouse fetal germ cell development. Biology of Reproduction 84 790–800. (https://doi. org/10.1095/biolreprod.110.088443) Ropke A, Tewes AC, Gromoll J, Kliesch S, Wieacker P & Tuttelmann F 2013 Comprehensive sequence analysis of the NR5A1 gene encoding steroidogenic factor 1 in a large group of infertile males. European Journal of Human Genetics 21 1012–1015. (https://doi.org/10.1038/ ejhg.2012.290) Saga Y 2008 Mouse germ cell development during embryogenesis. Current Opinion in Genetics and Development 18 337–341. (https://doi. org/10.1016/j.gde.2008.06.003) Scharnhorst V, Dekker P, van der Eb AJ & Jochemsen AG 1999 Internal translation initiation generates novel WT1 protein isoforms with distinct biological properties. Journal of Biological Chemistry 274 23456–23462. (https://doi.org/10.1074/jbc.274.33.23456) Schmahl J, Eicher EM, Washburn LL & Capel B 2000 Sry induces cell proliferation in the mouse gonad. Development 127 65–73. Schnabel CA, Selleri L & Cleary ML 2003 Pbx1 is essential for adrenal development and urogenital differentiation. Genesis 37 123–130. (https://doi.org/10.1002/gene.10235) Seabra CM, Quental S, Lima AC, Carvalho F, Goncalves J, Fernandes S, Pereira I, Silva J, Marques PI, Sousa M, et al. 2015 The mutational spectrum of WT1 in male infertility. Journal of Urology 193 1709–1715. (https://doi.org/10.1016/j.juro.2014.11.004) Seisenberger S, Andrews S, Krueger F, Arand J, Walter J, Santos F, Popp C, Thienpont B, Dean W & Reik W 2012 The dynamics of genomewide DNA methylation reprogramming in mouse primordial germ cells. Molecular Cell 48 849–862. (https://doi.org/10.1016/j. molcel.2012.11.001) Selleri L, Depew MJ, Jacobs Y, Chanda SK, Tsang KY, Cheah KS, Rubenstein JL, O’Gorman S & Cleary ML 2001 Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation. Development 128 3543–3557. Sepponen K, Lundin K, Knuus K, Vayrynen P, Raivio T, Tapanainen JS & Tuuri T 2017 The role of sequential BMP signaling in directing human embryonic stem cells to bipotential gonadal cells. Journal of Clinical Endocrinology and Metabolism 102 4303–4314. Shapiro E, Biezuner T & Linnarsson S 2013 Single-cell sequencing-based technologies will revolutionize whole-organism science. Nature Reviews Genetics 14 618–630. (https://doi.org/10.1038/nrg3542) Shaw G & Renfree MB 2014 Wolffian duct development. Sexual Development 8 273–280. (https://doi.org/10.1159/000363432) Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, Shiba H, Sasaki H, Osawa Y, Ninomiya Y, Niwa O, et al. 1995 Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice. Developmental Dynamics 204 22–29. (https://doi.org/10.1002/aja.1002040104) Smagulova FO, Manuylov NL, Leach LL & Tevosian SG 2008 GATA4/ FOG2 transcriptional complex regulates Lhx9 gene expression in murine heart development. BMC Developmental Biology 8 67. (https:// doi.org/10.1186/1471-213X-8-67) Stebler J, Spieler D, Slanchev K, Molyneaux KA, Richter U, Cojocaru V, Tarabykin V, Wylie C, Kessel M & Raz E 2004 Primordial germ cell migration in the chick and mouse embryo: the role of the Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access Journal of Molecular Endocrinology Y Yang et al. chemokine SDF-1/CXCL12. Developmental Biology 272 351–361. (https://doi.org/10.1016/j.ydbio.2004.05.009) Stevant 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. (https://doi.org/10.1016/j.celrep.2018.01.043) Suri M, Kelehan P, O’Neill D, Vadeyar S, Grant J, Ahmed SF & FitzPatrick D 2007 WT1 mutations in Meacham syndrome suggest a coelomic mesothelial origin of the cardiac and diaphragmatic malformations. American Journal of Medical Genetics Part A 143 2312–2320. (https://doi.org/10.1002/ajmg.a.31924) Swartz JM, Ciarlo R, Guo MH, Abrha A, Weaver B, Diamond DA, Chan YM & Hirschhorn JN 2017 A 46,XX ovotesticular disorder of sex development likely caused by a steroidogenic factor-1 (NR5A1) variant. Hormone Research in Paediatrics 87 191–195. (https://doi. org/10.1159/000452888) Takasawa K, Kashimada K, Pelosi E, Takagi M, Morio T, Asahara H, Schlessinger D, Mizutani S & Koopman P 2014 FOXL2 transcriptionally represses Sf1 expression by antagonizing WT1 during ovarian development in mice. FASEB Journal 28 2020–2028. (https://doi.org/10.1096/fj.13-246108) Tam PP & Snow MH 1981 Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos. Journal of Embryology and Experimental Morphology 64 133–147. Tamura M, Kanno Y, Chuma S, Saito T & Nakatsuji N 2001 Pod-1/ Capsulin shows a sex- and stage-dependent expression pattern in the mouse gonad development and represses expression of Ad4BP/SF-1. Mechanisms of Development 102 135–144. (https://doi.org/10.1016/ S0925-4773(01)00298-2) Tevosian SG, Albrecht KH, Crispino JD, Fujiwara Y, Eicher EM & Orkin SH 2002 Gonadal differentiation, sex determination and normal Sry expression in mice require direct interaction between transcription partners GATA4 and FOG2. Development 129 4627–4634. Tevosian SG, Jimenez E, Hatch HM, Jiang T, Morse DA, Fox SC & Padua MB 2015 adrenal development in mice requires GATA4 and GATA6 transcription factors. Endocrinology 156 2503–2517. (https:// doi.org/10.1210/en.2014-1815) Tremblay JJ & Viger RS 2001 GATA factors differentially activate multiple gonadal promoters through conserved GATA regulatory elements. Endocrinology 142 977–986. (https://doi.org/10.1210/endo.142.3.7995) Tzchori I, Day TF, Carolan PJ, Zhao Y, Wassif CA, Li L, Lewandoski M, Gorivodsky M, Love PE, Porter FD, et al. 2009 LIM homeobox transcription factors integrate signaling events that control threedimensional limb patterning and growth. Development 136 1375–1385. (https://doi.org/10.1242/dev.026476) Val P & Swain A 2010 Gene dosage effects and transcriptional regulation of early mammalian adrenal cortex development. Molecular and Cellular Endocrinology 323 105–114. (https://doi.org/10.1016/j. mce.2009.12.010) Val P, Martinez-Barbera JP & Swain A 2007 Adrenal development is initiated by Cited2 and Wt1 through modulation of Sf-1 dosage. Development 134 2349–2358. (https://doi.org/10.1242/dev.004390) Viger RS, Mertineit C, Trasler JM & Nemer M 1998 Transcription factor GATA-4 is expressed in a sexually dimorphic pattern during mouse gonadal development and is a potent activator of the Mullerian inhibiting substance promoter. Development 125 2665–2675. Wainwright EN, Jorgensen JS, Kim Y, Truong V, Bagheri-Fam S, Davidson T, Svingen T, Fernandez-Valverde SL, McClelland KS, Taft RJ, Mammalian early genital ridge development 62 :1 R64 et al. 2013 SOX9 regulates microRNA miR-202-5p/3p expression during mouse testis differentiation. Biology of Reproduction 89 34. Wainwright EN, Svingen T, Ng ET, Wicking C & Koopman P 2014 Primary cilia function regulates the length of the embryonic trunk axis and urogenital field in mice. Developmental Biology 395 342–354. (https://doi.org/10.1016/j.ydbio.2014.08.037) Walczak EM & Hammer GD 2015 Regulation of the adrenocortical stem cell niche: implications for disease. Nature Reviews Endocrinology 11 14–28. (https://doi.org/10.1038/nrendo.2014.166) Wang Q, Lan Y, Cho ES, Maltby KM & Jiang R 2005 Odd-skipped related 1 (Odd 1) is an essential regulator of heart and urogenital development. Developmental Biology 288 582–594. (https://doi. org/10.1016/j.ydbio.2005.09.024) Wang PY, Protheroe A, Clarkson AN, Imhoff F, Koishi K & McLennan IS 2009 Mullerian inhibiting substance contributes to sex-linked biases in the brain and behavior. PNAS 106 7203–7208. (https://doi. org/10.1073/pnas.0902253106) Warr N, Bogani D, Siggers P, Brixey R, Tateossian H, Dopplapudi A, Wells S, Cheeseman M, Xia Y, Ostrer H, et al. 2011 Minor abnormalities of testis development in mice lacking the gene encoding the MAPK signalling component, MAP3K1. PLoS ONE 6 e19572. (https://doi.org/10.1371/journal.pone.0019572) Wartenberg H, Kinsky I, Viebahn C & Schmolke C 1991 Fine structural characteristics of testicular cord formation in the developing rabbit gonad. Journal of Electron Microscopy Technique 19 133–157. (https:// doi.org/10.1002/jemt.1060190203) Wilhelm D & Englert C 2002 The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1. Genes and Development 16 1839–1851. (https://doi.org/10.1101/gad.220102) Witschi E 1948 Migration of the germ cells of human embryos from the yolk sac to the primitive gonadal fold. Contributions to Embryology 32 57–80. Wittmann W & McLennan IS 2013 Anti-Mullerian hormone may regulate the number of calbindin-positive neurons in the sexually dimorphic nucleus of the preoptic area of male mice. Biology of Sex Differences 4 18. (https://doi.org/10.1186/2042-6410-4-18) Wyndham NR 1943 A morphological study of testicular descent. Journal of Anatomy 77 179-188.3. Xing Y, Lerario AM, Rainey W & Hammer GD 2015 Development of adrenal cortex zonation. Endocrinology Metabolism Clinics of North America 44 243–274. (https://doi.org/10.1016/j.ecl.2015.02.001) Yang Y & Wilson MJ 2015 Lhx9 gene expression during early limb development in mice requires the FGF signalling pathway. Gene Expression Patterns 19 45–51. (https://doi.org/10.1016/j.gep.2015.07.002) Yang X, Schadt EE, Wang S, Wang H, Arnold AP, Ingram-Drake L, Drake TA & Lusis AJ 2006 Tissue-specific expression and regulation of sexually dimorphic genes in mice. Genome Research 16 995–1004. (https://doi.org/10.1101/gr.5217506) Yates R, Katugampola H, Cavlan D, Cogger K, Meimaridou E, Hughes C, Metherell L, Guasti L & King P 2013 Adrenocortical development, maintenance, and disease. Current Topics in Developmental Biology 106 239–312. Yoshida M, Suda Y, Matsuo I, Miyamoto N, Takeda N, Kuratani S & Aizawa S 1997 Emx1 and Emx2 functions in development of dorsal telencephalon. Development 124 101–111. Zorrilla M & Yatsenko AN 2013 The genetics of infertility: current status of the field. Current Genetic Medicine Reports 1 247–260. (https://doi. org/10.1007/s40142-013-0027-1) Received in final form 18 July 2018 Accepted 24 July 2018 Accepted Preprint published online 24 July 2018 https://jme.bioscientifica.com https://doi.org/10.1530/JME-17-0314 © 2019 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 06/03/2020 01:22:06PM via free access