Review
Wnt4 action in gonadal development and sex determination

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Abstract

Wnt4 is a growth factor involved in multiple developmental processes such as the formation of the kidney, adrenal, mammary gland, pituitary and the female reproductive system. During mammalian embryogenesis, Wnt4 is expressed in the gonads of both sexes before sex determination events take place and is subsequently down-regulated in the male gonad. Inactivation of the Wnt4 gene in mice has revealed that it is involved at several steps of female reproductive development. Wnt4 is implicated in Müllerian duct regression, the formation of sex-specific vasculature, the inhibition of steroidogenesis and in sex-specific cell migration events. A mouse model of sex-reversal has partially unravelled the molecular pathways in which Wnt4 operates during the development of the female reproductive system. However, the specific molecular mechanism of action of Wnt4 during gonadal development remains unknown. This and downstream signaling pathways involved in Wnt4 action during female gonad development are reviewed and models of Wnt4 action are proposed for Müllerian duct formation, sex-specific vasculature development, and sex determination events. Further identification of critical downstream effectors of the Wnt4 signaling pathway in mouse models and in patients with sex-reversal conditions could help in understanding sex-reversal pathologies in humans.

Introduction

The development of the mammalian gonad during embryogenesis is unique because a common precursor, the bipotential gonad, will form two fundamentally different organs, either a testis or an ovary (Fig. 1). Sex is determined at the time of fertilization by the presence or absence of the Y chromosome: XY, male or XX, female (Goodfellow & Lovell-Badge, 1993) and subsequent expression of the Y-linked gene Sry will initiate a cascade of events leading ultimately to testicular development (Berta et al., 1990, Sinclair et al., 1990). In the absence of Sry, the bipotential gonad will differentiate into an ovary (Fig. 1). After the onset of Sry expression, male gonadal differentiation ensues and testicular hormones are produced, causing Müllerian duct regression and the development of the Wolffian duct into the epididymis, vas deferens and seminiferous tubules, and testicular descent. In females, in the absence of testicular hormones, the Wolffian duct regresses and the Müllerian duct develops to form the upper part of the vagina, the uterus and Fallopian tubes (reviewed in Brennan & Capel, 2004). Engineered mutations in mice have revealed several genes upstream and downstream of Sry also involved in sex determination and/or formation of internal and external genitalia. Most genes identified to date are involved in male gonadogenesis, namely Wt1 (Pritchard-Jones et al., 1990), Lhx9 (Birk et al., 2000), Sf1 (Luo, Ikeda, & Parker, 1994), Gata 4/Fog2 (Tevosian et al., 2002), Sox9 (Foster et al., 1994), Dhh (Clark, Garland, & Russell, 2000), Fgf9 (Colvin, Green, Schmahl, Capel, & Ornitz, 2001), Dax1 (Meeks, Weiss, & Jameson, 2003), M33 (Katoh-Fukui et al., 1998), Pdgfrα (Brennan, Tilmann, & Capel, 2003) and insulin receptor signaling (Nef et al., 2003). Only Dax1 (Bardoni et al., 1994), Wnt4 (Vainio, Heikkila, Kispert, Chin, & McMahon, 1999) and Stra8 (Koubova et al., 2006) have been shown to be involved in ovarian development.

Wnt4 is conserved throughout vertebrates and was originally studied in non-mammalian vertebrates such as zebrafish, chicken and xenopus (Hollyday, McMahon, & McMahon, 1995; Ungar, Kelly, & Moon, 1995). In these species, Wnt4 is expressed in the developing brain and appears to play a crucial role in inhibiting cell movement during embryogenesis (Hollyday et al., 1995, Ungar et al., 1995). In mammals, Wnt4 function was initially studied during nephrogenesis in the metanephric kidney as mice lacking Wnt4 die shortly after birth due to kidney failure (Stark, Vainio, Vassileva, & McMahon, 1994). It was later found that Wnt4 is required for mesenchymal to epithelial transformation, an essential step in kidney tubule formation (Kispert, Vainio, & McMahon, 1998). More recently, the observation that Wnt4 is also expressed in the mesonephros, the gonad and in close association with the Müllerian duct indicated that Wnt4 may be important for the development of the reproductive system. Analyses of mutant mice showed that a lack of Wnt4 leads to the masculinization of the XX embryo (Vainio et al., 1999). This suggested that the development of the female pathway is not a default process in the absence of Sry, but is a finely regulated genetic pathway. The initial description of Wnt4 knock-out mice led to a number of recent investigations of Wnt4 function in Müllerian duct formation, endothelial cell migration, steroidogenesis, development of oocytes, and testis differentiation. These functions will be reviewed here and aspects of Wnt4 signaling at the molecular level in relation to gonadal development will also be discussed.

Section snippets

The loss of Wnt4 causes partial female-to-male sex-reversal

A lack of Wnt4 in the XX female mouse embryo (Wnt4−/−) leads to masculinization, observed by the absence of Müllerian structures and the presence of the Wolffian duct. However, because no Sertoli cell markers were expressed and no testicular tissue was formed in the absence of Wnt4, it was concluded that Wnt4 was not a primary sex-determining gene. Furthermore, no masculinization of the external genitalia was observed at birth in the Wnt4−/− mutant females. This was later corroborated by the

The Wnt pathways

Wnt proteins are a family of locally acting signaling molecules required in diverse developmental events, including gastrulation, axis formation, cell polarity, stem cell differentiation, and organ development (Cadigan & Nusse, 1997). Two distinct classes of Wnt proteins have been defined based on functional assays (Du, Purcell, Christian, McGrew, & Moon, 1995; Moon et al., 1993): the canonical Wnts, including Wnt1, Wnt3A and Wnt8, which share a common molecular mechanism; the non-canonical

The regulation of Wnt4 expression during gonadogenesis

As shown by in situ hybridization in the mouse, Wnt4 is expressed before gonad formation at 9.5 dpc in the mesonephric mesenchyme in both males and females. At 10.0 dpc Wnt4 begins to be expressed in the presumptive gonad in both sexes until 11.5 dpc, after which it is specifically down-regulated in the male gonad but maintained in the female gonad (Barrionuevo et al., 2006, Vainio et al., 1999). Wnt4 is expressed in germ-cell-deficient ovaries, indicating a specific somatic cell lineage (

Building pathways

Wnt4 is involved in many developmental processes and its deficiency leads to abnormal development of kidneys, pituitary gland, mammary gland and both the male and female reproductive systems. In the reproductive system, Wnt4 is specifically involved in Müllerian duct formation, sex-specific blood vessel formation, oocyte maintenance and repression of steroidogenesis. Partial XY male-to-female sex-reversal is observed in mice lacking Wnt4 as well as in one human patient carrying a WNT4 point

Concluding remarks

Over the last 5 years tremendous progress has been made in understanding sex determination in mammals. Key genes involved in mammalian sex determination were identified by gene mapping in humans and by targeted disruption in mice (sometimes accidental) and/or transgenic approaches in mice. To date, mouse models of sex-reversal are our best tool to understand the morphological events leading to the formation of a testis or an ovary. Yet, many molecular signaling pathways remain unidentified.

Acknowledgments

We thank Stefan Bagheri-Fam, Kevin Knower, Louisa Ludbrook and Helena Sim for helpful discussions and critical reading of the manuscript. This work is supported by the National Health and Medical Research Council of Australia (Grant 334314).

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