Sexual development provides an ideal model to study organogenesis
since the gonads have the bipotential ability to form an ovary or a
testis. Wingless-type MMTV integration site family, member 4 (WNT4) is a member of WNT gene family, and encodes a cysteine-rich secreted protein. WNT4 plays a pivotal role in early embryogenesis, particularly in the formation of the urogenital system [1-4]. After the action of Sry in the XY gonad Wnt4 expression decreases to undetectable levels in the developing testis, but remains at high levels in the ovary .
This led to the initial hypothesis that WNT4 acts as an anti-testis
gene, blocking Leydig cell differentiation and steroidogenesis in the
developing ovary . Lack of Wnt4 results in masculinization of XX mouse embryos  and inhibits the migration of endothelial and steroidogenic cells into the developing ovary , while over-expression of Wnt4 in the developing testis interferes with testicular vascular development and decreases androgen production [5,6]. Sertoli cell differentiation was also compromised in Wnt4 mutant testes, demonstrating that Wnt4 has specific and distinct roles in both male and female gonadal development .
Wnt4 appears to regulate Dax1 [8,9], a gene believed to antagonize the function of SRY in the developing gonad. In vitro, Dax1 transcription can be activated by β-catenin, a key signal-transducing protein in the WNT pathway . Besides Dax1, Follistatin (Fst), encoding a TGF-β superfamily binding protein, may also be a downstream component of Wnt4 signalling that regulates vascular boundaries and maintains germ cell survival in the ovary . Furthermore, Wnt4 and fibroblast growth factor 9 (FGF9) act as antagonistic signals to regulate differentiation of the ovary and testis .
To date most of our knowledge about the role of Wnt4 in the mammalian
gonad has been based on studies only in the mice. In order to determine
the role of WNT4 in formation of the mammalian gonad we characterised
its expression in a distantly related mammal.
Marsupials give birth after a relatively short gestation to small
altricial young that complete their development during a long lactation
period attached to a teat, usually in a pouch. The tammar gonadal ridge
develops about 6 days before birth, but the gonads remain
undifferentiated until after birth [12-14].
Testicular differentiation begins with the formation of seminiferous
cords by day 2 post partum. The ovaries, as in all mammals,
differentiate after the testis at about day 8 post partum, almost 14
days after the initial development of the gonadal ridge. In contrast to
the tammar, the development of the mouse gonad is extremely rapid and
there is only 1 day between the formation of the gonadal ridge and the
onset of cord formation in males.
Marsupials have the classical mammalian XY sex determining mechanism  with a homologue of SRY on the Y chromosome .
However, the formation of some secondary sexual characteristics,
including the scrotum and mammary glands, are under primary genetic
control by genes on the X chromosome, and are not dependant on hormones
from the testis [14,17,18]. Several key genes in the sex determination and differentiation cascade, SRY , SOX3 , SOX9 , SF-1 , DAX1 , DMRT1 [24,25], ATRX/Y [26,27], AMH/MIS ,
have now been cloned and characterized. The endocrine control of male
sexual differentiation in the tammar has also been defined [29-35].
Taken together, these data form a primary framework for understanding
the evolution of the male sex-determining cascade in marsupials.
However, nothing is yet known of the female gonadogenesis pathway in
marsupials. This study has therefore characterized WNT4 during early
development to gain insight into the onset of ovarian differentiation
in a marsupial, and also to determine its expression during the
extended period of testicular differentiation.