Figure 1.
Schematic representation of putative gonad promoter sequences.
All numbers shown are relative to the transcriptional start site (TSS) for each putative promoter sequence. The SF1p contains several promoter elements that have been described previously [16]. Both aromatase and AMH promoters contain TATA boxes and consensus SF1 binding sites. The AMH promoter also contains an estrogen responsive element (ERE). The WT1 promoter is TATA-less and no other binding elements were identified. All promoter sequences were cloned into the RCANBP viral vector directly upstream of the EGFP open reading frame.
Figure 2.
Wholemount fluorescent microscopy of novel gonad promoter expressed EGFP.
Tissues from E7.5 embryos infected with RCANBP viruses containing SV40 (SV40p), WT1 (WT1p), SF1 (SF1p), AMH (AMHp) and aromatase (AROMp) promoters. Dashed white lines delineate the left (Lg) and right (Rg) gonads, which sit on top of the mesonephros (Ms). A: Strong EGFP expression was evident for SV40p and AROMp, however, this was not confined to the urogenital systems. WT1p and AMHp produced low-level expression in the urogenital systems. EGFP expressed from SF1p was moderate in the urogenital system, and included the gonads. B: RCANBP-SF1p-EGFP infected E7.5 embryo; in addition to EGFP expression in the urogenital system, embryos also showed EGFP expression in the liver, forelimb (FL) and hind limb (HL).
Figure 3.
Tissue restricted EGFP expression from the SF1 promoter.
Immunostaining for EGFP (green) and the RCANBP viral antigen p27 (red), in RCANBP-SF1p-EGFP infected E7.5 embryos (magnification 10×). Control (male) gonad and forelimb tissues are negative for EGFP and p27. For both male and female embryos infected with the SF1p vector, EGFP expression is higher in gonad tissues compared to the forelimb.
Figure 4.
Cellular location of SF1 promoter expressed EGFP compared to key gonad factors.
Immunostaining for EGFP (green) and key testis and ovarian developmental proteins (red) in RCANBP-SF1p-EGFP infected E7.5 embryos (magnification 20×). A: Male genes: In male gonads, co-staining of EGFP with DMRT1 or SOX9 showed that both proteins were expressed in some cells simultaneously (white arrows). However, in female gonads DMRT1 expression did not overlap with EGFP. B: Female genes: In female gonads, co-staining of EGFP with aromatase or FOXL2 showed that both proteins were expressed in some cells simultaneously (white arrows).
Figure 5.
Cellular location of SF1 promoter expressed EGFP compared to germ cells.
Immunostaining for EGFP (green) and CVH (red) in RCANBP-SF1p-EGFP infected E7.5 embryos (magnification 20×). The expression of EGFP from SF1p did not overlap with any cells that were positive for the germ cell marker CVH.
Figure 6.
Over-expression of DMRT1 from SF1p in embryonic gonads.
Immunostaining for key testis and ovarian developmental genes (red) in RCANBP-SF1p-EGFP infected E7.5 embryos (magnification 10×). Low-level over-expression of DMRT1 was evident in the gonads of RCANBP-SF1p-DMRT1 infected embryos compared to the control female. This over-expression did not cause any change in the expression of SOX9 in the female or aromatase in the male.
Figure 7.
Over-expression of aromatase from SF1p in embryonic gonads.
A: Immunostaining for aromatase (green), fibronectin (red) and DAPI (blue) in RCANBP-SF1p-EGFP infected E7.5 embryos (magnification 40×). Low-level aromatase over-expression was evident in the male left gonad compared to the control male. B: Immunostaining for key male protein AMH (green) and DAPI (blue) in RCANBP-SF1p-EGFP infected E7.5 embryos (magnification 40×). The over-expression of aromatase in the male gonad caused a reduction and disruption of AMH expression.