Fig. 3. Function of PITX2 in the gonadal cortex before sexual differentiation. (A-I) Chick embryonic fibroblasts producing Pitx2- (A-C) and Pitx2-en- (D-F) encoding viruses were implanted into the right and left presumptive gonad regions of both sexes, respectively, at stage 11-12. As a control, cells producing AP-encoding virus were implanted into the right presumptive gonad regions (G-I). Distribution of the virus was examined by in situ hybridization for mouse Pitx2 (A,D) and by AP staining (Morgan and Fekete, 1996) (G). Arrowheads in A,D,G indicate viral distribution in the developing gonads. 72 hours after implantation (stage 27), embryos were subjected to whole-mount in situ hybridization for RALDH2 (B,E,H) and Ad4BP/SF-1 (C,F,I). Arrowheads in B,C,E,F indicate altered expression of RALDH2 and Ad4BP/SF-1. Sections of female (ZW) samples are shown. (J-U) The effect of exogenous Pitx2 (J), Pitx2-en (L) and AP (K,M) on cell proliferation was examined in female embryos at stage 27. BrdU was injected into the operated embryos 1 hour before fixation, and their gonads were stained with antibodies for BrdU (green) and cytokeratin (red) (overlays in J-M). The effect of misexpressed Pitx2 (N), Pitx2-en (P) and AP (O,Q) on cortical thickness was examined by staining with anti-cytokeratin antibody at stage 29. Cell proliferation was analyzed quantitatively in gonads misexpressing Pitx2 (R,S) or Pitx2-en (T,U) at stage 27. AP was used as a control. The total number of DAPI-stained cells (data not shown) and BrdU-positive cells in the gonadal cortex (cytokeratin-positive) (R,T) and medulla (cytokeratin-negative) (S,U) was counted. Relative fold changes in BrdU-positive cell numbers in the cortex and medulla are plotted with the numbers in the right cortex and medulla misexpressing AP set at 1. Scale bars: 100 µm in A-M; 50 µm in N-Q.