Fig. 6.

In ovo suppression of epithelial ERα activity disrupts cortex differentiation. (A) Electroporation of a dominant-negative isoform of ERα (dnERα) to the gonadal epithelium. (Aa) Schematic of the inducible TET-ON plasmid system used to express dnERα (see Materials and Methods for details), LBD, ligand binding domain. (Ab,Ac) Whole-mount images of two ZW left gonads electroporated at D2.5 (HH15-17) and screened at D10 (HH36); targeted cells are highlighted by expression of an EGFP reporter (green). (Ad-Ai) Fluorescence images of D10 (HH36) gonad sections immunostained for P450 aromatase (AROM; red), or the germ cell marker VASA (red) and GFP (green); (see Fig. S7 for the EGFP pattern alone) from ZW control (Ad,Ag) and ZW electroporated (Ae,Af,Ah,Al) gonads. (Aj-Al) PITX2 RNA in situ expression pattern in ZW control (Aj) and ZW electroporated (Ak,Al) gonads. White dotted lines show the cortex/medulla border. (B) Electroporation of RCAS retroviral DNA expressing ERα shRNA (RCAS-shER1) to the gonadal epithelium. (Ba) Schematic of the construct: RCAS-shERα expressing ERα-specific short hairpin RNA molecules in tandem with an EGFP reporter. (Bb) Whole-mount image of a ZW left gonad electroporated with RC-shER1 at D2.5 (HH15-17) and screened at embryonic D9 (HH35-36) based on the EGFP reporter expression. (Bc-Bf) Fluorescence images of D9 (HH35-36) gonad sections immunostained for P450 aromatase (AROM; red) or the germ cell marker VASA (red) (see Fig. S7 for the EGFP pattern alone) from ZW control (Bc,Be) and ZW-RCAS-shER1 (Bd,Bf) gonads. The cortex is compromised by downregulation of ERα activity in both model systems compared with controls. Four out of eight samples from the two experiments showed the severe cortical phenotype in well-electroporated regions. See Fig. S8 for immunofluorescence results on sections from electroporation of various control vectors expressing an EGFP reporter (minimum of three replicates per control vector analysed).