Review TRENDS in Endocrinology and Metabolism Vol.15 No.3 April 2004 Sex determination: a ‘window’ of DAX1 activity Louisa M. Ludbrook and Vincent R. Harley Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton, VIC 3168, Australia Traditionally, DAX1 was considered an ‘anti-testis’ gene that are probably important for male sex determination because DAX1 duplications in XY individuals cause have yet to be identified, because some 75% of sex reversal male-to-female sex reversal: dosage-sensitive sex rever- cases remain unexplained genetically [15]. Some progress sal (DSS). In DSS, two active DAX1 genes on one has been made in deciphering the roles and complex X chromosome can abrogate testis formation. By con- relationships of the known sex-determining genes during trast, mutations and deletions of DAX1 cause adrenal gonadogenesis. Here, we describe the emerging role of hypoplasia congenita (AHC). Although AHC patients DAX1 in male testis formation and discuss the possible develop testes, gonadal defects include disorganized molecular mechanisms through which DAX1 regulates testis cords and hypogonadotropic hypogonadism, this pathway. which is not completely restored with gonadotropin or androgen therapy. Recent evidence of XY sex reversal Expression of DAX1 in Dax1-deficient mice strongly supports a role for Dax1 DAX1 RNA expression is restricted to certain tissue types as a ‘pro-testis’ gene. Therefore, perhaps DAX1/Dax1 and is largely coexpressed with SF1, also crucial for both acts within a ‘window’ of activity, outside of which tes- adrenal and gonadal development [16–18]. Based on in tis formation does not occur. Here, we discuss the func- situ hybridization analyses, Sf1 and Dax1 are expressed in tion and possible mechanisms of DAX1 action in male both developing and adult adrenal, gonadal, hypothalamic gonadogenesis. and pituitary tissues [10]. During human and murine gonadal development, both DAX1 and SF1 are coexpressed Sex determination in most mammals (Box 1) is controlled in the gonads of both sexes before genital ridge differen- by the presence or absence of the sex-determining region tiation, with Sf1 expression slightly preceding that of Dax1 Y chromosome gene (SRY in humans, Sry in mice) [1]. SRY [10]. In humans, DAX1 expression levels are the same in deletions or mutations give rise to females with XY the embryonic testis and ovary [19]. In XY mice, Dax1 is gonadal dysgenesis [2]. Sry is expressed in the bipotential upregulated strongly in Sertoli cells at 12.5 days post gonad just before overt differentiation into a testis [3] and, coitum (dpc), just after Sry. Sertoli Dax1 expression drops when Sry is transgenically introduced into XX mice, they soon after, but increases in the interstitial cells between develop as males [4]. SRY is thought to initiate a genetic 13.5 and 17.5 dpc [20]. In the XX mouse gonadal cascade resulting in testis differentiation [5]. Other sex- determining genes act downstream of SRY within this pathway (Figure 1). Box 1. Sex determination in mammals Through studies of the genomes of sex-reversed The human embryo develops a sexual identity six to seven weeks patients (XY females, XX males), several sex-determining into gestation. Sex determination involves the differentiation of the genes have been identified. SOX9 mutations cause XY sex bipotential embryonic gonads into the male testes or the female ovaries. The presence of a testis enables further development of reversal in 75% of patients with campomelic dysplasia [6]. male-specific structures and external genitalia. Up to this point, the Sox9 is upregulated directly after Sry in the XYembryonic sex of an embryo appears ambiguous; a common gonadal ridge is mouse gonad [7] and XX mice transgenic for Sox9 [under present in both males and females, which contains bipotential the control of Wilms’ tumor-1 (encoded by WT1) regulatory supporting and steroidogenic cell lineages, in addition to the regions] develop as males [8]. Steroidogenic factor-1 (SF1 primordial germ cells destined to form spermatogonia or oogonia. As differentiation ensues, the supporting cell lineage gives rise to in humans; Sf1 in mice; official gene symbol NR5A1) and male Sertoli cells or female follicle cells, whereas the steroidogenic WT1 are both initially expressed before Sry in the gonad cells form testicular Leydig cells or ovarian thecal cells responsible [9,10]. In humans, SF1 mutations are responsible for XY for production of steroid sex hormones. sex reversal [11,12] and WT1 mutations cause severe Also present in the sexually indifferent embryo are the cardinal structures of the Wolffian (male) and Mullerian (female) accessory kidney and gonadal defects, including XY gonadal dysgen- reproductive ducts, the future genital tracts. Depending on gonadal esis [13]. Gene duplication of DAX1 (Dax1 in mice; encodes sex, the development of one ductal system will be promoted in the DSS–AHC on X chromosome gene 1; official gene symbol embryo and the alternate accessory structures will degenerate. Sex- NR0B1) is associated with XY sex reversal in patients with specific development of external genitalia follows during week 8. The dosage-sensitive sex reversal (DSS) [14]. Many more genes onset of sex determination is genetically controlled, and is initiated by the Y chromosome gene SRY and its expression in the supporting cells of the gonadal ridge. Corresponding author: V.R. Harley ([email protected]). www.sciencedirect.com 1043-2760/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2004.02.002 Review TRENDS in Endocrinology and Metabolism Vol.15 No.3 April 2004 117 DAX1 MIS WT1/SF1 SRY SOX9 SF1 Testis NTF3, FGF9 Other gonad-forming No SRY Ovary genes TRENDS in Endocrinology & Metabolism Figure 1. Molecular genetic events surrounding the initiation of sex determination. Solid lines represent interactions for which experimental evidence exists; dashed lines represent suggested, but as yet undetermined interactions. In the week 7 urogenital ridge, WT1 and SF1 are expressed in both sexes. In the XY gonad, SRY is activated fol- lowed by upregulation of SOX9 by processes possibly involving DAX1, NTF3 and FGF9. SOX9 in turn activates SF1, which is proposed to activate unknown testis-forming genes. SF1 and SOX9 also activate AMH. DAX1 could act in multiple modes and at multiple steps. In the XX gonad, SRY is absent and an ovary develops independent of DAX1 expression. Abbreviations: AMH, anti-Mullerian hormone gene; DAX1, DSS–AHC on X chromosome gene 1; FGF9, fibroblast growth factor-9 gene; NTF3, neurotro- pin 3 gene; SF1, steroidogenic factor-1 gene; SOX9, SRY box-9 gene; SRY, sex-determining region Y chromosome gene; WT1, Wilms’ tumor gene-1. primordium, Dax1 is expressed between 12.5 and 14.5 dpc, DAX1 C-terminus [32,33], one of which (Alien) is after which expression decreases [20]. expressed in the testis [34]. Investigation into DAX1 function in steroidogenesis has DAX1 protein structure and function revealed several possible mechanisms for DAX1 action DAX1 encodes a member of the orphan nuclear hormone during gonadal development. In cultured cells, DAX1 receptor (NHR) family of transcriptional regulators. DAX1 represses Sf1-mediated transcriptional activation of ster- is 470 amino acids in length and has two distinct protein oidogenic genes, such as Cyp11A, Cyp17 and Cyp19 [35]. domains (Figure 2). The C-terminal domain shows Co-transfection of SF1 with DAX1 DNA into choriocarci- homology to the ligand-binding domain (LBD) of related noma cells showed a 75% repression of an SF1 artificial NHRs, although a ligand remains elusive. The N-terminal response element/luciferase reporter compared with domain represents a novel domain comprising 3.5 repeats transfection with SF1 alone [36]. This repressive effect of a 65–67-amino acid motif containing two putative zinc was dose dependent and relatively specific for SF1- fingers, and possibly defining a nucleic acid-binding mediated activity, because inhibition of the related thyroid domain [21,22]. DAX1 is present in mammalian species hormone receptor was not apparent [36]. Adrenal hypo- (mice [21], birds [23], humans and other primates [14,24]) plasia congenita (AHC) missense mutations in DAX1 (all of and non-mammalian vertebrates (reptiles, amphibians which localize to the C-terminal LBD) significantly reduce and fish [25–27]). Human and mouse DAX1 show 75% its repressive effects in cell culture assays [36,37], similarity in amino acid sequence [21]. The N-terminal implicating the LBD as a mediator of repression. Recent repeats contain conserved leucine LXXLL motifs resem- evidence indicates that many AHC mutant DAX1 proteins bling the nuclear receptor box protein interaction motifs show defective nuclear localization, accounting for the lack [28]. The closest relative of DAX1 is small heterodimer of repression observed in cell culture assays [38,39]. partner, SHP, which also lacks a traditional N-terminal Although correct protein folding and stability of the DNA-binding domain, and is able to interact with protein C-terminal LBD are crucial for DAX1 nuclear import [39], targets through LXXLL-related motifs [29,30]. DAX1 has a evidence is emerging of a direct role for the N-terminal bipartite transcriptional silencing domain (Figure 2), LXXLL motifs in mediating repression. Zhang and which has been proposed to interact directly with colleagues showed that the DAX1 LXXLL motifs interact corepressors to mediate repression [31]. At least two with the AF2 domain of ligand-bound estrogen receptors, corepressors, Alien and NCoR, interact in vitro with the ERa and ERb? and repress ER-mediated activation of target reporter genes [28]. Transient transfection assays showed Dax1-mediated transcriptional repression of an Putative DNA binding SF1 target, CYP11A1, was reduced when each of the three or protein interaction Putative ligand binding DAX1 LXXLL motifs was separately mutated, and domain domain completely removed when all three motifs were disrupted, N C presumably because the SF1–DAX1 interaction is lost LYNLL LYSNL LYSLL Silencing domains [40].