In the XX gonad, two secreted ligands, WNT4 and RSPO1, are capable of activating the β-catenin canonical signaling pathway, and loss of either Wnt4
in mice results in a partial sex-reversal (5
). The existence of human sex-reversed XY males carrying chromosomal duplications suggests that overexpression of female sex-determining genes could lead to sex-reversal. Attempts to sex reverse XY mice by overexpression of Wnt4
have been unsuccessful, suggesting that Wnt4
alone is insufficient to override the male pathway (6
). However, an XY patient carrying a duplication that includes WNT4
exhibited sex-reversal (11
), leading to the hypothesis that upregulation of both of these ligands may be required to antagonize male development. Here, we show that stabilization of β-catenin, the downstream effector of WNT4 and RSPO1 signaling, was sufficient to disrupt the male pathway in XY gonads. This led to a loss of SOX9 and AMH expression, a failure of testis cord formation and the increased expression of several ovarian somatic cell markers including FOXL2, Bmp2
, suggesting that the somatic lineages have switched from a male to female fate.
Although we observed male-to-female sex-reversal of XY β-catfl.ex3
gonads, a male-specific vasculature was formed. However, when β-catenin was stabilized in XY gonads cultured in the presence of LiCl, formation of the coelomic vasculature was disrupted. This discrepancy is likely related to the timing of β-catenin stabilization relative to the time of establishment of the male program. SOX9 expression is sufficient to initiate all subsequent aspects of testis differentiation, including the migration of mesonephric endothelial cells (2
). Transient SOX9 expression in XY β-catfl.ex3
gonads may initiate the male vascular program, in contrast to the case in early LiCl-treated XY samples, where SOX9 expression is absent. However, we cannot rule out the possibility that β-catenin signaling has a more direct role in vascular development. Wnt4
has been shown to block the migration of endothelial cells into XX gonads (6
). Thus, the early stabilization of β-catenin in LiCl-treated gonads may have a more direct effect on blocking endothelial migration.
XX gonads lacking Wnt4
exhibit a partial sex-reversal and lose expression of ovarian somatic markers including Fst
). As these genes are thought to be downstream of Wnt4
signaling, we hypothesized that they would be upregulated by β-catenin signaling. Additionally, gonads lacking Rspo1
show decreased Wnt4
expression, suggesting that β-catenin signaling might also positively regulate Wnt4
). We examined expression of these genes in XY β-catfl.ex3
gonads and found that increased β-catenin signaling leads to increased expression of Wnt4
. However, Rspo1
was downregulated, suggesting that its expression is negatively correlated with high levels of β-catenin signaling. Additionally, we observed that both XX and XY β-catfl.ex3
gonads were larger than controls. This might suggest that β-catenin promotes the survival or proliferation of an ovarian somatic cell population. Consistent with this, we observed a larger number of FOXL2-expressing cells in XX β-catfl.ex3
gonads when compared with XX controls (Fig. ). In wild-type gonads, a negative-feedback loop may regulate levels of β-catenin to maintain the size of the ovary; this regulation would be lost when β-catenin is artificially stabilized, leading to the increased size. However, more work is required to test the role of β-catenin on patterning of ovarian somatic cells.
Previously, we proposed a model in which an antagonistic relationship between SOX9 and WNT4 exists within the bipotential gonad (22
). This relationship is likely to be indirect as WNT4 is an extracellular ligand and SOX9 is a nuclear transcription factor. Our data suggest that β-catenin mediates this antagonistic relationship. Similarly, Chang et al
) recently performed a similar experiment in which β-catenin was stabilized specifically in Sertoli cells. Although β-catenin stabilization occurred later than in our mouse model (~13.5 dpc), they also observed a decrease in the SOX9 expression by E14.5. Stabilized β-catenin could antagonize SOX9 in several ways. As β-catenin from the recombined β-catfl.ex3
allele cannot be degraded, β-catenin can translocate to the nucleus and could interact directly with SOX9, compete for a binding partner or target site or bind to the Sox9
promoter to negatively regulate its expression. A negative interaction between SOX9 and β-catenin has previously been documented during chondrocyte differentiation, in which heterodimerization of SOX9 and β-catenin leads to the mutual degradation of both proteins (24
Our work strongly suggests that antagonism between SOX9 and β-catenin is the molecular mechanism through which the fate of the supporting cell lineage in the gonad is established to drive male or female sex determination. This competition is pushed toward the ovarian pathway by RSPO1 and WNT4, which act to establish β-catenin as the dominant signal in XX gonads. This provides a potential mechanistic explanation for how male-to-female sex-reversal can occur in XY patients who lack mutations in genes required for the activation of the male program; stabilization of β-catenin counteracts the effects of SRY by destabilizing its only known downstream target, SOX9.