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Wt1 is one of numerous candidate genes comprising the hypothetical chain of gene expression essential for male sex differentiation of the bipotential indifferent gonads during embryogenesis. However, the evidence in the literature is ambivalent regarding the position of Wt1 relative to Sry in this scheme; Wt1 might act either upstream or downstream of Sry. In the present study, the effects of Sry expression upon Wt1 were investigated using M15 cells (XX karyotype), which are derived from murine embryonic mesonephros and express endogenous Wt1. In 3 stably-transformed Sry-expressing M15 cell lines, we showed that the expression levels of the mRNAs coding for all 4 isoforms of the WT1 proteins were down-regulated. Similarly, Wnt 4 expression was down-regulated in these cell lines. Silencing of Sry in the transformed cell lines using ribozymes or short hairpin RNAs (shRNAs) resulted in elevated levels of Wt1 and Wnt4 expression. These results strongly indicate that Wt1 might be under the control of Sry during gonadal differentiation in the mouse. In electrophoretic mobility shift assays (EMSA), we demonstrated that the 3.7 kb 5'-upstream DNA stretch of Wt1 containing potential Sry binding sites was capable of forming molecular complexes with nuclear protein(s) from Sry expressing cells but not with those from control non-Sry expressing cells. In summary, our present results support the notion that Wt1 is located downstream of Sry and down-regulated by the sex determining gene. Although the precise biological meaning of the present findings have yet to be clarified, it is possible that Wt1 plays a dual role during gonadal differentiation, i. e., turning on Sry expression on one hand, and being down-regulated by its product, Sry, on the other, possibly forming a type of negative feed-back mechanism. Further work is needed to substantiate this view.

Down-Regulation of Endogenous Wt1 Expression by Sry Transgene in the Murine Embryonic Mesonephros-Derived M15 Cell Line