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Journal of Molecular Y Yang et al. Mammalian early genital ridge 62:1 R47–R64 Endocrinology development 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: [email protected]

Abstract

The body of knowledge surrounding reproductive development spans the fields of Key Words genetics, anatomy, physiology and biomedicine, to build a comprehensive understanding ff gonad of the later stages of reproductive development in humans and animal models. Despite ff urogenital ridge this, there remains much to learn about the bi-potential progenitor structure that the ff disorders of sex ovary and testis arise from, known as the genital ridge (GR). This tissue forms relatively determination late in embryonic development and has the potential to form either the ovary or testis, ff infertility which in turn produce hormones required for the development of the rest of the ff molecular genetics 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 Journal of Molecular issues in the adult. Endocrinology (2019) 62, R47–R64

The distinction between sexes is one of the most obvious testicular/ovarian structures, accessory sex structures and examples of dimorphism in the animal kingdom and secondary sexual characteristics), whereby genital ridge highlights one of the most crucial fate decisions made in (GR) formation was included only as a step in gonadal sex utero to develop as a male or a female. This fate decision determination or excluded from the broader picture all to follow either developmental trajectory is an essential together. The focus of this review is formation of the GR process that determines the reproductive success of all in mice and humans, and consequences of failure of its sexually reproducing animals (Fig. 1). There are a number development, rather than sex determination, which has of different mechanisms underlying sex development been reviewed extensively elsewhere (Greenfield 2015, that can be broadly categorized as either genetically Capel 2017). sex determined or environmentally determined (Capel 2017). Typically, mammals fall within the genetically sex- Genital ridge formation determined category. Traditionally, sexual development has been viewed as two distinct processes, gonadal sex The mammalian GR is derived from intermediate meso determination (decision of the gonad to become a testis derm as paired structures that lie on either side of the dorsal or ovary) and sexual differentiation (establishment of mesentery in the coelomic cavity (Fig. 2). Formation of

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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 Figure 1 Summary of the origin of reproductive tissues within the mammalian to the endothelial cell population for the establishment of embryo. The gonad region of the GR develops into either the testis or the the male vascular system (Karl & Capel 1995, Martineau ovary depending upon expression of the Sry gene. In XY gonads, the et al. 1997, Coveney et al. 2008). Migrating into the GR 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 Figure 2 mesonephros, running in parallel to the mesonephric duct Schematic illustrations of the structures and components involved in early (Fig. 2A). This is the female equivalent to the mesonephric UGR development in mice. (A) (i) Side view of a E11.5 embryo indicating the duct, which will form the fallopian tubes, uterus and part location of the developing GR (orange bar). (ii) A ventral view of the abdominal region focuses on the mesonephros (white) and the gonadal of the vagina following female sex determination (Acien ridge (grey) that develops on the ventromedial surface. The aorta is shown in 1992). Together, the mesonephros and GR are known as red. (iii) In transverse sections the mesonephros, containing mesonephric the urogenital ridge (UGR). 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 Proliferation of the coelomic epithelial cells on the GR, from the hindgut via the dorsal mesentery. (B) Starting at E9.5, the ventromedial surface of the mesonephros creates a dense, coelomic epithelium (yellow) on the ventromedial surface of the pseudostratified epithelial cell layer Gropp( & Ohno 1966, mesonephros begins to proliferate, forming a pseudostratified epithelial layer. The basement membrane becomes fragmented, allowing GR Pelliniemi 1975, Wartenberg et al. 1991). Alongside the progenitor cells that have undergone EMT to migrate inward. These cells proliferation of the coelomic epithelium, the underlying continue to proliferate, just behind the coelomic epithelium to form the basement membrane becomes fragmented, allowing 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; many epithelial cells to ingress dorsally, towards the GR, genital ridge; HG, hindgut; M, mesonephros; MD, mesonephric duct; MT, mesonephros (Fig. 2B) (Karl & Capel 1998, Kusaka et al. mesonephric tubule; PGC, primordial germ cell; PMD, paramesonephric duct.

https://jme.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/JME-17-0314 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 03:48:41AM via free access Journal of Molecular Y Yang et al. Mammalian early genital ridge 62:1 R49 Endocrinology development just prior to gondal sex determination are primordial germ cluster of cells at the anterior region of mesenchyme cells (PGC), the precursors to the germ cells that become separates from the GR primordium and moves dorso- sperm and oocytes later in life. PGCs arise near the yolk medially to form the adrenal anlage (Hatano et al. sac and travel, via the hindgut, to the GR as a result of 1996, Val & Swain 2010). From E11.5 to 12.5, neural chemotaxis (Doitsidou et al. 2002). 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, Genital ridge formation in mice form a fibrous capsule around the composite adrenal Most of the work investigating mammalian gonad primordium by E14.5 (Xing et al. 2015). development has been performed on mice. It is assumed that events in the human embryo, as well as other Development of the PGC in the Genital ridge Eutherian mammals, follow the same basic pattern, albeit with some differences in the timing and anatomy. As previously mentioned, PGCs originate from a separate The process of GR formation, starting with the location near the yolk sac, away from the mesonephros proliferation of the coelomic epithelium, begins at about and GR (Saga 2008). Mammalian PGCs are specified via E9.5 in mice (Hu et al. 2013), which equates to about an inductive system of signalling molecules, particularly 5 weeks of gestation in humans (Jost 1972). The GR BMP4. In mice, PGCs are induced around E6.5 (equating itself is not morphologically evident until about E10.0– to approximately 2 weeks gestation in humans) from 10.5 where a clear distinction between the GR and the the proximal epiblast by BMP4 signalling (Lawson et al. mesonephros can be seen under light microscopy. From 1999). These cells subsequently move to and cluster at E10.5 to 11.5, further proliferation of the GR coelomic the base of the allantois/yolk sac wall, near the forming epithelium and mesonephros expands the overall size hindgut, which can be seen in mouse embryos at about on the ventral side of the mesonephros (Fig. 2A). The E7.0 (week 3–4 human gestation) as a small population initiation, proliferation and expansion of the GR is of ~45 cells (Lawson et al. 1999). As development indistinguishable between XX and XY embryos, and proceeds, the hindgut folds and the PGCs migrate the size of the GR is determined by the length of the into the embryo proper (Hara et al. 2009, Harikae et al. embryonic trunk (Wainwright et al. 2014). At around 2013). By E9.5 the PGCs begin to migrate away from E11.5–12.0, the molecular events that determine gonad the hindgut towards the UGR and colonize the gonad fate occur, prompting the gonad to follow either a testis- between E10.0 and 11.0 (~5-week gestation in humans) or ovary-specific developmental trajectory from this (Fig. 2A; Witschi 1948, Molyneaux et al. 2001). During time-point. The first sign of sexual dimorphism of the this mass migration of PGCs, the hindgut descends into early differentiated gonad becomes evident in XY gonads the coelomic cavity and the last PGCs to migrate must at about E12.5, when testis cords can be seen (Hacker travel through the dorsal mesentery before entering the et al. 1995, Schmahl et al. 2000). gonads (Molyneaux et al. 2001). The GR is not only a progenitor tissue for the The PGCs undergo several rounds of cell division to gonad, but also the adrenal cortex, a critical endocrine achieve a population of about ~3000 cells by ~E11.5 (Tam tissue that synthesizes and secretes a variety of steroid & Snow 1981). Around this time, PGCs begin to undergo hormones to maintain body homeostasis and regulate a process known as licensing, undergoing a global the stress response (Yates et al. 2013, Walczak & change in gene expression, turning on genes required Hammer 2015). Consequently, several genes required for gametogenesis, concurrently switching off their for gonad development are also important for adrenal pluripotency genes over the course of the following days gland development (Luo et al. 1994, Gut et al. 2005, (Stebler et al. 2004, Gill et al. 2011, Rolland et al. 2011, Katoh-Fukui et al. 2005, Bandiera et al. 2013, Tevosian Seisenberger et al. 2012). Following this transition, the et al. 2015). The medulla and the cortex of the adult PGCs are referred to as gametogenesis-competent cells and adrenal gland have separate origins; the medulla is are poised to initiate either male or female differentiation, derived from neural crest cells, whereas the cortex is including meiosis, upon receiving cues from the somatic derived from cells located at the anterior-most region cells of either the forming testis or ovary and the nearby of the GR, indicating it has an intermediate mesoderm mesonephric tissue (McLaren & Southee 1997, Adams & origin (Hatano et al. 1996). Beginning at E10.5, a small McLaren 2002, Gill et al. 2011).

Genes essential for initial gonad formation factors have mainly been characterized in mice, mutations in three of these genes (NR5A1, WT1 and GATA4) have also Mutational analysis using the mouse model, with some been found in patients with DSD, indicating a conserved additional evidence from human clinical cases, have role in reproductive development (Bashamboo et al. 2010b, brought to light a number of genes that are required to Kohler et al. 2011, Lourenco et al. 2011, Swartz et al. 2017). initiate the formation and proliferation of the GR, as well However, these genes are expressed and function in many as testis/ovary differentiation (Table 1). These include developing organ systems, meaning the loss of function Wilms’ tumour suppressor 1 (WT1) (Kreidberg et al. 1993, in both mice and humans produce a range of phenotypes Hammes et al. 2001), LIM homeobox gene 9 (LHX9) beyond the reproductive tract (Ingraham et al. 1994, (Birk et al. 2000), Nuclear Receptor Subfamily 5 Group Klamt et al. 1998, Hammes et al. 2001, Tevosian et al. A member 1 (NR5A1; also called steroidogenic factor 1 2015). Genes known to be required for GR development (SF1)) (Luo et al. 1994), empty spiracles homeobox gene often show altered expression in gene knockout lines, 2 (EMX2) (Miyamoto et al. 1997) and GATA-binding indicating a co-regulatory relationship exists between protein 4 (GATA4) (Hu et al. 2013). In mouse embryos, these critical factors (discussed further below). a homozygous null mutation in any one of these genes It is evident that many of these critical early factors causes gonadal agenesis. While the function of these five discussed below have multiple roles in reproductive

Table 1 Summary of genes essential for GR formation.

Gene Mouse UGR expression Mouse gonadal phenotype Human phenotype WT1 Expressed in the coelomic Degenerating gonad due to Frasier syndrome (Klamt et al. 1998) epithelium and mesonephros increased apoptosis (Kreidberg WAGR syndrome (Le Caignec et al. 2007) (Armstrong et al. 1993) et al. 1993, Hammes et al. 2001) Denys-Drash syndrome (Hastie 1992), Male WT1-KTS mice exhibit complete Meacham syndrome sex reversal (Hammes et al. 2001) Phenotypes include streak gonads, Following sex determination, roles in ambiguous genitalia, hypospadias, 46, ovary and follicle development XY sex reversal (Suri et al. 2007) (Gao et al. 2014) Nr5a1 Initially expressed in the anterior Gonad regression due to increased Range of phenotypes including 46, XY region of the coelomic epithelium. apoptosis sex reversal, gonad dysgenesis, male Expression domain expands in an Loss of adrenal development, infertility, hypospadias, adrenal anterior–posterior direction (Hu obesity and pituitary abnormalities insufficiency, gonad dysgenesis, et al. 2013) XY KO mice show complete male-to- premature ovarian failure (reviewed in female sex reversal (Luo et al. 1994, Ferraz-de-Souza et al. 2011) Shinoda et al. 1995) 46, XX sex reversal, ovotestis (Bashamboo et al. 2016, Swartz et al. 2017) Emx2 Coelomic epithelium and cells that Absent gonads Endometriosis (Daftary & Taylor 2004) move into the underlying Reduced proliferation of the coelomic Endometrial cancer (Daftary & Taylor mesenchyme epithelium (Miyamoto et al. 1997, 2004) Mesonephric duct epithelia (Hu et al. Pellegrini et al. 1997) Incomplete Müllerian fusion (Liu et al. 2013) 2015) Lhx9 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)) Gata4 Coelomic epithelium, expression Absent gonads Testicular abnormalities, ambiguous domain expands in an anterior– No expansion of the coelomic genitalia (Lourenco et al. 2011) posterior direction, precedes epithelium (Hu et al. 2013) Frequent mutation in ovarian cancers expression of Sf1 (Hu et al. 2013) Following sex determination, roles in (Cai et al. 2009) ovarian and testicular development (Efimenko et al. 2013) MAP3K1 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)

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 Figure 4 the promoter region of Nr5a1 in vitro, and together they Summary of the molecular relationships during early gonad formation. (A) Summary of relationships between the core genes necessary for GR increase reporter gene expression in a Sertoli-like (TM4) development. Relationships are based on previous studies using cell line (Wilhelm & Englert 2002), a finding replicated in knockout lines where expression changes of genes were observed. Loss Leydig-like (TM3) and C2C12 (myoblast derived) cell lines of expression from knockout indicated a positive regulatory relationship and increased expression suggests a negative relationship. If direct (Val et al. 2007, Takasawa et al. 2014). However, in primary relationships are known, through studies such as ChIP-PCR and reporter Leydig cells WT1 overexpression repressed Nr5a1 gene assays (Wilhelm & Englert 2002, Katoh-Fukui et al. 2005, Franca et al. 2013, expression. Further experiments confirmed that WT1 Chen et al. 2017), they have been indicated by a solid line. (B) Recent studies suggest a complex regulatory relationship exist between WT1 and directly binds in vivo to the Nr5a1 promoter in Sertoli Nr5a1 (Chen et al. 2017). Nr5a1 expressing cells (Nr5a1+) in the GR cells obtained from 2-week-old mice (Chen et al. 2017). contribute to the Sertoli, interstitial and Leydig cell populations following It was suggested that these conflicting results were most 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 likely due to the use of cell lines vs primary cells in these and appears to reduce its expression. In Leydig cells (no Wt1 expression), studies (Chen et al. 2017). The TM4 and TM3 cell lines Nr5a1 gene expression is significantly higher. Lhx9 is a candidate protein were derived from juvenile BALB/c mouse testis (Mather partner for WT1 (Wilhelm & Englert 2002), it is expressed in the GR, and later in interstitial cells. 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 Steroidogenic factor 1 (Sf1), nuclear receptor Leydig cells obtained from adult and juvenile mice on subfamily 5 group A member 1 (Nr5a1) a mixed background (C57Bl/6:129/SvEv), given that The Nr5a1 (Sf1) gene encodes a transcription factor that strain background can strongly influence the resulting is expressed in the adrenal glands, coelomic epithelium, phenotypes (Herzer et al. 1999, Meeks et al. 2003, Brennan hypothalamus and anterior pituitary gland during & Capel 2004, Munger et al. 2009). WT1 also may have development (Luo et al. 1994, Morohashi & Omura differing roles in the regulation of Nr5a1 gene expression, 1996). During UGR development, Nr5a1 gene expression as the gonad develops into a testis, perhaps being required is limited to the gonad primordium, where it is required for an initial activation of Nr5a1, with protein partner for the proliferation and survival of progenitor somatic LHX9 in the early gonad and, following gonadal sex cells (Luo et al. 1994). After gonadal sex determination, determination, WT1 reduces Nr5a1 expression in those Nr5a1 gene expression is restricted to testis-specific NR5A1+ cells that are fated to become Sertoli cells. In this cells, namely Leydig and Sertoli cells (Luo et al. 1994). case, the presence or absence of certain protein partners, Nr5a1−/− mice show complete failure of adrenal and for instance LHX9, would impact on how WT1 regulates gonadal development, abnormalities of the pituitary and Nr5a1 gene expression. Previous studies have shown that hypothalamus and obesity (Luo et al. 1994, Shinoda et al. WT1 is converted from an activator to a repressor protein 1995). The gonads of Nr5a1-knockout mice embryos do by the protein partners such as BASP1 (McKay et al. 1999,

Carpenter et al. 2004). Given that NR5A1+ cells contribute mice embryos exhibit normal PGC migration into the UGR to both cell populations, it maybe that Nr5a1 expression region, but the initial thickening of the coelomic epithelium levels determine which fate, with Sertoli cells expressing is not prominent and the GR soon degenerates (Miyamoto significantly lower levels ofNr5a1 compared to Leydig et al. 1997). These mutants completely lack gonads, genital cells (Fig. 4B; Chen et al. 2017). 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 LIM homeobox 9 (Lhx9) the basement membrane, as well as increased apoptosis The Lhx9 gene encodes a transcription factor expressed (Kusaka et al. 2010). Interestingly, Nr5a1 gene expression in a variety of regions within the developing mouse is also significantly affected inEmx2 −/− embryos (Kusaka embryo, including the brain, heart, kidney, limb buds et al. 2010), suggesting that Emx2 acts upstream of Nr5a1 in and the coelomic epithelium (Retaux et al. 1999, Birk the developmental cascade, but how Emx2 regulates Nr5a1 et al. 2000, Failli et al. 2000, Molle et al. 2004, Oshima transcription, whether directly or indirectly remains unclear. et al. 2007, Smagulova et al. 2008, Tzchori et al. 2009, Yang & Wilson 2015). Lhx9 gene expression in the UGR GATA-binding protein 4 (Gata4) is first seen in the coelomic epithelium at E9.5 and later in the gonad primordium, until sexual differentiation GATA4 is a transcription factor that is essential for the where its expression becomes restricted to the interstitial/ development of multiple organs such as the heart, foregut, mesothelial regions of testes and the cortical regions of liver, ventral pancreas and the UGR (Kuo et al. 1997, ovaries (Birk et al. 2000). Lhx9−/− mutant mice exhibit a Molkentin et al. 1997, Viger et al. 1998, Hu et al. 2013). similar gonadal phenotype to that of Wt1(−KTS) −/− mice, Gata4 gene expression in the GR was originally linked to a whereby normal GR development and PGC migration is role in testis differentiation through activating transcription observed but discrete gonads fail to form and genetically of Sry together with WT1 (Tevosian et al. 2002). Later male mice show complete male-to-female sex reversal studies revealed that Gata4 is expressed prior to any other of their secondary sex characteristics (Birk et al. 2000). gonadal factor, initially in the anterior half of the coelomic The observed underdevelopment of the GR results from epithelium at E9.5 and expanding to the posterior region disrupted proliferation of the gonad primordium (Birk by E10.2 (Hu et al. 2013). Gata4-knockout mice show no et al. 2000), as opposed to increased apoptosis observed signs of gonadal initiation, with the coelomic epithelium in the Wt1−/− and Nr5a1−/− mice (Kreidberg et al. 1993, remaining as an undifferentiated monolayer, indicating Luo et al. 1994). In addition, male- and female-knockout that Gata4 is required for the initial thickening of the offspring are infertile, with atrophic uteri, vaginas and coelomic epithelial layer (Hu et al. 2013). In these knockout oviducts, no male accessory sex organs, increased follicle- mice, Lhx9 and Nr5a1 gene expression is lost, but Wt1 and stimulating hormone levels, and undetectable levels of Emx2 gene expression is unaffected (Hu et al. 2013). This testosterone and oestrogen (Birk et al. 2000). Interestingly, suggests that GATA4 acts not only upstream of the Lhx9 Lhx9−/− mice show no other phenotypic abnormalities gene, but also Nr5a1, possibly even directly regulating both outside of gonad agenesis and male-to-female sex reversal genes, as binding sites for GATA4 have been identified in the (Birk et al. 2000, Tzchori et al. 2009), despite expression proximal promoter regions of these two genes (Tremblay & in other tissues, such as the limbs, nervous system and Viger 2001, Smagulova et al. 2008, Hu et al. 2013). pancreas, during development. This may be a result of the functional redundancy of Lhx9 with its closely related Genes with minor but essential roles in paralogue, LIM homeobox gene, Lhx2 (Jurata & Gill 1998, Genital ridge development Birk et al. 2000, Tzchori et al. 2009). Other genes have been implicated in early GR development, although mice-knockout strains for these genes exhibit a Empty spiracles homeobox 2 (Emx2) less severe phenotype than that of the null phenotype of Emx2 encodes a transcription factor that is the mouse the key genes described earlier. While the loss of function homolog of the Drosophila head gap gene empty spiracles of the following genes does not cause termination of (ems). The Emx2 transcript is expressed in the dorsal gonad development, it does result in the underdevel telencephalon, mesonephros and coelomic epithelium opment of the gonads, often in combination with sex (Miyamoto et al. 1997, Yoshida et al. 1997). Emx2−/−-knockout reversal of the secondary sex characteristics.

Transcription factor 21 (Tcf21 or Pod1) downstream of Sry to promote Sertoli cell expansion and repress steroidogenic cell lineages. TCF21, also known as epicardin, capsuling or transcription factor 21, belongs to the basic helix-loop-helix family Sine oculis-related homeobox 1/4 (Six1/Six4) of transcription factors. It is expressed by mesodermal cell types including heart proepicardial cells, kidney Six1 and Six4 genes belong to the mammalian homolog of and visceral smooth muscle as well as the endodermal the Drosophila sine oculis homeobox family of transcription gastrointestinal tract (Quaggin et al. 1998, 1999, Lu et al. factors, containing a distinctive SIX domain (required 2000, Miyagishi et al. 2000). Tcf21-knockout mice die for protein-protein interactions) and homeodomain in the perinatal period due to lung, kidney and cardiac (Kawakami et al. 2000). Six1 and Six4 genes are located defects, but also display gastric and splenic defects (Cui in the same genomic regions within about 100 kb of one et al. 2003, Funato et al. 2003, Plotkin & Mudunuri 2008). another, and have highly overlapping tissue expression In addition, Tcf21 XX and XY KO mice have irregular during mouse development (Boucher et al. 1996, Esteve shaped gonads, the urogenital tracts of both XX and XY & Bovolenta 1999, Ozaki et al. 2001, Fougerousse et al. mice are indistinguishable and XY pups had feminized 2002, Laclef et al. 2003). Six1/4 double KO mice show genitalia (Cui et al. 2004). Consistent with this phenotype, different phenotypes compared to either Six1 or Six4 Tcf21 gene expression is initially detected throughout the single KO mice, highlighting regions of redundant GR at the bi-potential stage and continues to be expressed function in the development of limbs, skeletal muscle, in the gonads of both sexes, with expression slightly sensory neurons and kidney (Grifone et al. 2005, Konishi higher in the testes following sex determination (Tamura et al. 2006, Giordani et al. 2007, Kobayashi et al. 2007). et al. 2001). While the gonad does form in the Tcf21−/− The suggested functional redundancy between the two embryo, morphological defects such as a shorted length genes is further exhibited as both genes share a common were observed by E11, and vascular abnormalities were DNA-binding site, MEF3 (Kawakami et al. 2000, Kumar observed by E12.5 (Cui et al. 2004), suggesting a defect 2009). In particular, only Six1/4 double KO mice embryos early in gonadal development. Further studies focused on display smaller gonads and adrenal glands (Kobayashi expression analyses, these suggested a negative regulatory et al. 2007, Fujimoto et al. 2013). Out of the essential relationship exists between Tcf21 and Nr5a1, as Tcf21 KO GR genes listed previously, only Nr5a1 gene expression results in increased Nr5a1 expression and an expanded is significantly reduced, a finding corroborated through Leydig cell population (Miyagishi et al. 2000). Despite reporter assays showing that SIX1/4 is able to transactivate an increase in Leydig cell numbers, the XY KO genitalia Nr5a1 transcription (Fujimoto et al. 2013). Furthermore, it was feminized and the gonads fail to descend from an was also shown that SIX1/4 is able to activate Fog2 gene abdominal position, suggesting that testosterone levels expression and that interaction, together with GATA4, were low, either due to later apoptosis of the gonadal regulates Sry gene expression (Fujimoto et al. 2013). tissue or a not all steroidogenic enzymes were expressed Overall, this indicates that SIX1/4 is required necessary correctly (Cui et al. 2004). TCF21 also appears to repress for sufficient Nr5a1 gene expression in the early gonad Nr5a1 gene expression in the adrenal gland, with in vitro and, a loss of Six1/4 gene expression, reduces Nr5a1 gene studies revealing that TCF21 directly represses Nr5a1 gene expression, and this may be responsible for the Six1/4 expression, through binding at E-box sequences located KO undersized gonad phenotype. Nonetheless, there is in the Nr5a1 promoter (Franca et al. 2013, 2015). still sufficient Nr5a1 gene expression to form the gonad Interestingly, the Sertoli cells do differentiate in the primordium in Six1/4 KO embryos. Tcf21 KO line, but there was some evidence that this cell population was reduced in number (although it Chromobox homolog 2 (Cbx2) (mouse polycomb group was not quantified) Cui( et al. 2004). TCF21 can cause member, M33) a sex reversal-like phenotype in vitro using a rat primary ovarian cell culture system. Overexpression of Tcf21 CBX2 is a component of the polycomb group complex of causes these cells to express Sertoli-like gene markers, in regulatory proteins involved in the repression/silencing of a similar pattern to that observed with Sry overexpression genes. In mice, Cbx2−/−-knockout mice show XY gonadal (Bhandari et al. 2011). Additionally, the Tcf21 gene has male-to-female (testis-to-ovary) sex reversal, and XX proposed to be a direct target of SRY (Bhandari et al. animals have smaller or absent ovaries (Katoh-Fukui et al. 2011). Together, these results suggest that TCF1 acts 2005). Additionally, mutants show defects in adrenal and

https://jme.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/JME-17-0314 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 03:48:41AM via free access Journal of Molecular Y Yang et al. Mammalian early genital ridge 62:1 R55 Endocrinology development splenic development, which was shown to be a result of Pre-B-cell leukaemia homeobox 1 (Pbx1) a reduction in the expression of Nr5a1 (Katoh-Fukui et al. Pbx1 encodes a TALE (three amino acid loop extension) 2005). This has led to the suggestion that CBX2 acts as class homeodomain transcription factor that has been an upstream regulator of Nr5a1 gene expression. Cbx2 shown to be involved in a number of process during has also been identified as a factor contributing to the mammalian embryogenesis including, skeletal devel differentiation of the testis through indirect regulation of opment and patterning (Selleri et al. 2001), maintenance Sry (Katoh-Fukui et al. 2012). Genome-wide identification of haematopoiesis (DiMartino et al. 2001), pancreatic of CBX2 target genes in a human Sertoli-like cell line development (Kim et al. 2002) and kidney and adrenal suggests that CBX2 acts to stimulate male-specific genes, development (Schnabel et al. 2003). The GR of Pbx1-null while suppressing female-pathway genes (Eid et al. 2015). mice are also smaller due to decreased proliferation of In humans, a reported case study of a 46, XY child with the progenitor cells in the gonad primordium (Schnabel female internal and external genitalia and histologically et al. 2003). Further experiments found that this is due normal ovaries found a compound loss-of-function to the downregulation of Nr5a1 gene expression; PBX1 mutation in the coding region of CBX2 (Biason-Lauber also upregulates Nr5a1 during adrenal development et al. 2009). This study further lends support to the role of (Schnabel et al. 2003). The adrenal gland primordium is CBX2 in the trans-activation of Nr5a1 and its role in both initially part of the GR but then later buds off from the the GR and testis developmental pathways (Fig. 4A). very rostral end around E10.5 (Schnabel et al. 2003, Pitetti et al. 2013). Therefore, perturbed Pbx1 gene expression Insulin receptor, Insr and insulin-like growth factor prior to budding likely affects Nr5a1 expression in type 1 receptor (Igf1r) the rostral GR, if not the whole GR, causing reduced Nr5a1 expression and decreased proliferation of somatic Insulin and its related growth factors IGF1 and IGF2 regulate progenitor cells. a variety of physiological processes including metabolism, stimulation of cell proliferation, differentiation and survival (Efstratiadis 1998). Their function is mediated Odd-skipped related 1 (Odd1) by two membrane-associated tyrosine kinase receptors, the insulin receptor (INSR) and the IGF type 1 receptor Odd1 gene encodes a zinc finger transcription factor (IGF1R). The genes for Insr, Igf1r and insulin receptor- homologous to the Drosophila odd-skipped class of related receptor (Irr) have previously been shown to be transcription factors that are involved in embryonic necessary for the testis determination pathway, as mutant patterning and tissue morphogenesis (Wang et al. 2005). mice lacking all three genes show male-to-female sex Targeted gene knockout of Odd1 revealed that this gene reversal and decreased Sry and Sox9 gene expression (Nef functions in both heart and intermediate mesoderm et al. 2003). Recently, it has been shown that Insr and Igf1r, development (Wang et al. 2005). Odd1−/− embryos but not Irr have roles in GR development, whereby mice have severe heart malformations, and completely lacking both Insr and Igf1r have reduced proliferation lack adrenal glands, kidneys and gonads, all of which of the GR prior to sex determination but also extensive derive from intermediate mesoderm (Wang et al. 2005). downregulation of hundreds of genes associated with Although gonad development was not the focus of adrenal, testicular and ovarian development (Pitetti et al. this study, they did show that in early development 2013). As a result, these mice embryos exhibit agenesis of the GR was hypoplastic (Wang et al. 2005), likely due the adrenal cortex, along with male-female sex reversal to increased apoptosis observed with the developing due to a delay in Sry gene upregulation. Interestingly, kidney. These hypoplastic GRs appeared to degenerate, ovarian differentiation is also delayed in these mice, as no visible gonad structures were observed by E15.5 leaving the GR in an undifferentiated state until about (Wang et al. 2005). Although no further investigation E16.5 when the ovarian programme is eventually initiated of Odd1 with regards to urogenital development (Pitetti et al. 2013). Among the genes downregulated are has been done since Wang et al. (2005), it is unclear Wt1, Lhx9 and Nr5a1, indicating that genes essential for if the GR phenotype was an effect of Odd1 acting GR development are under the influence of insulin/IGF either directly in the GR or indirectly, potentially signalling (Pitetti et al. 2013), but only partially dependent acting via downregulation of Wt1 gene expression, on this signalling pathway, as GR development is only which also has an apoptotic phenotype when deleted hindered by decreased progenitor cell numbers. (Hammes et al. 2001).

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 Figure 5 technologies have become more affordable and require Histology sections through human embryos 36–44 days (Carnegie stages 14–18). At ~36 days the coelomic epithelium begins to thicken, and by less material, more studies are using human-derived 39 days a ridge of tissue is forming, facing into the gut cavity. Just prior to samples to study human sex development at the molecular sex determination (~42 days), the mesenchyme has proliferated to form a level, mostly through studies of cases of disorders of gonad region that is now easily distinguished from the neighbouring mesonephros. Section images were obtained from the Virtual Human sexual development (DSD) (O’Shaughnessy et al. 2007, Embryo resource (https://www.prenatalorigins.org/virtual-human- Ostrer et al. 2007, Houmard et al. 2009, Del Valle et al. embryo/). CE, coelomic epithelium; DM, dorsal mesentery; M, 2017, Li et al. 2017). These are congenital disorders that mesonephros; MT, mesonephric tubule; MV, mesonephric vesicle; PMD, paramesonephric duct. Scale bar = 100 µm. arise as a consequence of atypical chromosomal, gonadal or anatomical sexual development (Hughes et al. 2006), development typically results in a range of phenotypes from including some of the aforementioned genes involved complete gonad dysgenesis to adult infertility. Mutations in GR development. The term DSD replaced old and in some of these early gonad development genes are also ambiguous terms such as intersex, hermaphroditism and responsible for some cases of human idiopathic infertility pseudo-hermaphroditism previously used to describe in cases with apparently normal gonad development. such disorders in humans (Dreger et al. 2005, Lees & Like DSDs, infertility is a condition associated with severe Tuch 2006). DSD phenotypes include a broad range of emotional and mental stress, particularly in societies where conditions such as failure of gonad formation, mixed there is a social emphasis placed upon the ideal of having gonadal tissue (male- and female-specific cell types within biological children (Ashraf et al. 2014). The mechanisms the tissue), ambiguous genitalia and failure of secondary of infertility in males and females are varied in both sexual characteristics to develop normally (Cools et al. origin and functional impact and often these conditions 2006). There is limited data on the incidence of DSDs, are thought to have an underlying genetic component but it is estimated that the overall worldwide incidence (Zorrilla & Yatsenko 2013). Only 6–18% of infertility cases of DSDs is 1 in 5,500 (Damiani 2007, Houk & Lee 2008). have identifiable genetic causes (mainly sex-chromosome In humans, the gonad develops from the coelomic abnormalities) indicating that for many cases of infertility epithelium (Fig. 5), and the first signs of sex differentiation (and subfertility), like DSD, the underlying genetic factors are observed between 6 and 7 weeks with the formation of are yet to be elucidated. testicular cords (Gruenwald 1942, Wyndham 1943). Germ Genes with mutations commonly identified in DSD cells are first detected in the hindgut dorsal mesentery at studies include androgen receptor and synthesis genes 4 weeks (Carnegie Stage 12 (CS12)) and migrate into the (androgen receptor (AR), CYP17A1, SRD5A2), NR5A1(SF1), GR by 5 weeks (CS16) (Mckay et al. 1953). Gondal sex WT1, GATA4, SRY, DAX1 and CHD7 (Kremen et al. 2017). determination occurs around 41–42 days in human embryos, Those patients with DSD and a complete gonad dysgenesis signified by the upregulation ofSRY gene expression in XY phenotype carry mutations in SRY, MAP3K1, DHH and embryos ((Hanley et al. 2000, Del Valle et al. 2017, Mamsen NR5A1 genes (Ono & Harley 2013). While these genes et al. 2017). Genes required for steroidogenesis and secreted are also critical for mouse gonadal development, largely factors such as AMH are essential for the sex-specific supporting the use of the mouse as a model for human development of genitalia and the reproductive tract, with gonad development, there are several exceptions. For their expression commencing between 54 and 57 dpc example, sequencing screens have found gain-of-function (CS23). Genes essential for GR development in mice are mutations in the mitogen-activated protein kinase kinase also expressed at similar levels in both XX and XY gonads kinase 1 (MAP3K1) gene, including patients with streak (LHX9, EMX2, WT1 and GATA4), further supporting a role gonads and female genitalia (Pearlman et al. 2010, Loke for these factors in early human gonad development (Del et al. 2014, Baxter et al. 2015, Eggers et al. 2016, Granados Valle et al. 2017, Mamsen et al. 2017). et al. 2017), suggesting a requirement for MAP3K1 Despite their apparent early essential roles in gonad function in gonad development for both sexes. However, development, the loss of function of genes required for sex although the mouse orthologue, Map3k1, is expressed in

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Given that the GR consists of a broad replicate human gene mutations to study phenotype, as variety of cellular precursors for endothelial, steroidogenic often human gene mutations do not result in a complete and supporting cell lineages, along with maturing germ loss of gene/protein function but rather gain of function. cells, it is especially suitable for scRNA-seq analysis. Li Mutations in NR5A1 were identified in 4% of patients et al. undertook scRNA-seq analysis of isolated single examined with unexplained male infertility (Bashamboo foetal germ cells and their surrounding ‘niche’ somatic et al. 2010a, Ropke et al. 2013) and in female patients cells from 4- to 26-week-old human embryos (Li et al. within premature ovarian failure (Lourenco et al. 2009). 2017). Based on gene expression profiles from this study, Novel NR5A1 mutation I (R92W) leads to 46, XX ovotestis somatic cells in the early gonad can be divided into four (SRY negative) (Igarashi et al. 2017, Baetens et al. 2017b). groups within each sex. XX gonadal cells are comprised of The types of NR5A1 gene mutations and phenotypes endothelial cells, and three types of maturing granulosa associated with these disorders are reviewed in (Ferraz-de- cells (early-(10 weeks), mid-(10–20 weeks) and late- Souza et al. 2011). WT1 mutations have also been found granulosa (20–26 weeks)) (Li et al. 2017). In XY gonads, in male and female cases of infertility, with ‘normal’ somatic cells group into Sertoli cells, Leydig cell precursors, gonadal development (Seabra et al. 2015, Nathan et al. differentiated Leydig cells and endothelial cells (Li et al. 2017). Humans are not the only species to present with 2017). Systematic examination of the expression profiles variable phenotypes; in mice, the genetic background of each gonadal cell population during development will or strain strongly influences the adult phenotype of not only improve our knowledge of the in vivo mechanisms many genes linked to gonadal development. In one such of cell differentiation but also the conditions required to example, gene knockout of Gadd45g (growth arrest and induce correct cell differentiation in vitro. DNA damage-inducible protein 45 g) on a mixed genetic Organoid systems are becoming popular in vitro models background (129/C57BL/6), 20% of the XY homozygous of organ development (Fatehullah et al. 2016). Isolated mice developed as infertile males, whereas on a C57BL/6 preparations of human somatic and germ cells can self- (B6) background, 100% of XY mice were sex reversed organize into a testicular-like organoid using an artificial (Johnen et al. 2013). Genetic background also influences scaffold to aid 3D organization (Baert et al. 2017). These the phenotype of Nr5a1-, Fgf9- and Wt1-null mice (Meeks studies make use of cells that have already undergone et al. 2003, Brennan & Capel 2004). The B6 mouse strain sex-specific differentiation, as the cells are isolated from is more likely to result in a male-to-female sex reversal post-natal tissues (Baert et al. 2017). Recently, Sepponen phenotype than the 129S1/SvImJ (129S1) strain, and et al., reported using human embryonic stem cells (hESCs) differences in gonadal gene expression between these culture conditions to sequentially induce the primitive strains are observed even prior to sex determination at streak, followed by intermediate mesoderm and finally E11.5 (Colvin et al. 2001, Munger et al. 2009). Therefore, bi-potential-like gonadal cells expressing genes such as even though studies have identified genes critical to the LHX9, EMX2, WT1 and GATA4 (Sepponen et al. 2017). early steps of gonad development, a loss-of-function This study found that timing and levels of BMPs (bone mutation does not necessarily lead to complete gonadal morphogenetic protein), WNT/β-catenin and Activin-A dysgenesis. In at least some cases, genetic background and signalling ligands are essential to promote differentiation possibly environmental factors determine the phenotypic of gonadal cell precursors, over other mesodermal cell consequences of loss-of-function mutations in these genes. 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 Future directions: unravelling the molecular gonad development, but to also examine the functional pathways of early gonad development consequences of gene mutations identified in DSD The advent and affordability of high-throughput sequenc patients using cultured cells engineered with the same ing technologies and new methods of replicating organ genetic mutation. development in vitro, together will be valuable research Several groups have taken a comprehensive targeted tools in propelling forward both genetic and cellular screening approach in order to identify genetic factors in biology into early GR development. DSD patients, and this in turn may lead to the identification

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Deep sequencing of studies, including those mapping the chromatin state in 64 known and 967 candidate genes improved the genetic specific cell lineages (Mikkelsen et al. 2007), will improve diagnosis rate to 43% for one patient cohort (Eggers et al. our ability to predict if DNA changes associated with DSD 2016). Thus, despite advances in sequencing technologies, lie within gene regulatory regions. over 50% of DSD cases remain without a genetic diagnosis. Epigenetic mechanisms such as DNA methylation While targeted sequencing and exome sequencing and histone modifications have been implicated in the studies have focused on the protein coding regions of the regulation of Sry gene in mice and dogs (Nishino et al. genome, epigenetic processes, gene regulatory elements 2011, Jeong et al. 2016, Kuroki et al. 2017). Loss of function and non-coding RNAs (ncRNAs) are just as important of the JMJD1A gene, which encodes a H3K9 demethylase for correct embryonic development. Errors in any of enzyme, results in XY sex reversal (Kuroki et al. 2017). these gene regulatory mechanisms may underlie many Some cases of XY DSD with complete sex reversal in dogs cases of idiopathic DSD that lack mutations in protein- are thought to be due to persistent DNA hypermethylation coding genes. Studies in vertebrates have largely focused of the Sry gene (Jeong et al. 2016). Therefore, it is likely on identifying sex-dimorphic expression of small ncRNAs that some cases of human DSD may be the result of called miRNAs following sex determination (Bannister et al. mutations to epigenetic regulatory factors. Regulation of 2009, Real et al. 2013, Wainwright et al. 2013, Presslauer gene expression through epigenetic mechanisms is also et al. 2017). There is limited information regarding how especially sensitive to environmental influences and this these miRNAs function in sex determination, if their impacts on many developmental programmes including function is essential, if they act to finely adjust gene sex determination (Feil & Fraga 2012). DNA methylation expression levels during gonad development and whether levels, determined by environmental factors, are vital these, and others, have earlier roles in the formation of to many naturally occurring forms of sex reversal and the bi-potential gonad. Currently no miRNAs have been environmental sex determination in animals (Capel linked to human gonad development. Long ncRNAs are 2017). While difficult to study with respect to human DSD, another important class of ncRNAs, which function to it is possible that DNA methylation may play a role in regulate gene expression (reviewed in Moran et al. 2012). balancing one sex developmental trajectory over another, With respect to sex development, these have been most and thus, errors in this may lead to gonad dysgenesis or thoroughly investigated for their role in X-chromosome sex reversal in humans. dosage compensation (Cerase et al. 2015). It remains to be determined if autosomally encoded long ncRNAs Summary contribute to mammalian sex determination and gonadal development but given their important roles in the The complexity of reproductive development is reflected development of other organ systems, it is likely they have in the difficulty in assigning a genetic diagnosis in a role in some aspects of gonadal development. most cases of human DSD. While we know the genetic Genome regulatory elements have been difficult to aetiology of a small number of DSDs, up to as many as identify as most lie within the non-coding regions of the 75% of individuals with a DSD will remain without genome and can act over long distances to influence gene a genetic diagnosis (Arboleda et al. 2014). Even with expression via chromatin folding. Mutations in regulatory whole genome sequencing, it is often difficult to identify elements range from single nucleotide sequence variants functional variants and causal mutations, rendering that prevent or reduce binding of a transcription factor many sequencing approaches somewhat ineffective to their DNA target sequence, to larger deletions, (Fan et al. 2017). Gene expression levels, epigenetic duplications and translocations resulting in structural modifiers and genetic background, along with the type changes that alter chromatin confirmation and regulatory of mutation and its functional consequence can all interactions between enhancers and their target genes. influence the resulting phenotype for both humans and Loss-of-function mutations in regulatory elements mice. The future development of new technologies and located near the SOX9 gene, a gene important for male improvement of existing ones will provide us with a much sex determination, are the best-characterized regulatory better understanding of the processes underlying normal

https://jme.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/JME-17-0314 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 03:48:41AM via free access Journal of Molecular Y Yang et al. Mammalian early genital ridge 62:1 R59 Endocrinology development gonadal development in both human and mouse models, Barbaux S, Niaudet P, Gubler MC, Grunfeld JP, Jaubert F, Kuttenn F, which in turn will lead to improved diagnosis in cases of Fekete CN, Souleyreau-Therville N, Thibaud E, Fellous M, et al. 1997 Donor splice-site mutations in WT1 are responsible for Frasier DSD and infertility. 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, Christin- Declaration of interest Maitre S, et al. 2010a Human male infertility associated with The authors declare that there is no conflict of interest that could be mutations in NR5A1 encoding steroidogenic factor 1. American perceived as prejudicing the impartiality of this review. 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 Funding development DSD and congenital heart defects (CHD) associated This work did not receive any specific grant from any funding agency in the with a mutation in GATA4. Endocrine Reviews 31 S1667. public, commercial, or not-for-profit sector. 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. Acknowledgements (https://doi.org/10.1093/hmg/ddw390) The authors would like to thank James Smith, Kathy Sircombe, Rebecca Baxter RM, Arboleda VA, Lee H, Barseghyan H, Adam MP, Fechner PY, Clarke and Susie Szakats for their feedback on earlier versions of this Bargman R, Keegan C, Travers S, Schelley S, et al. 2015 Exome manuscript. Yisheng Yang was supported by a University of Otago PhD sequencing for the diagnosis of 46,XY disorders of sex development. scholarship award. Our work was funded by a University of Otago Research Journal of Clinical Endocrinology and Metabolism 100 E333–E344. Grant to Megan Wilson. 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Received in final form 18 July 2018 Accepted 24 July 2018 Accepted Preprint published online 24 July 2018