Fig. 2. Effects of RA signaling on the expression of PITX2c, Ad4BP/SF-1 and cell proliferation in chick. (A-F) RA and RAA beads were implanted into the left (A,B) and right (C,D) presumptive gonad regions of both sexes, respectively, at stage 18-19. As controls, DMSO beads were implanted (E,F). 48 hours after implantation (stage 27), the embryos were subjected to whole-mount in situ hybridization with Ad4BP/SF-1 (A,C,E) and PITX2c (B,D,F) probes, then the embryos were sectioned. Sections of female (ZW) samples are shown. Arrowheads in A and C indicate affected expression of Ad4BP/SF-1. (G-N) The effects of implantation of RA (G) and RAA (I) beads on cell proliferation were examined at stage 27 in female embryos. DMSO beads were used as a control (H,J). BrdU was injected into the operated embryos 1 hour before fixation, then gonads were stained with antibodies for BrdU (green) and cytokeratin (red). Effects of RA (K) and RAA (M) beads on cortical thickness were examined by staining with anti-cytokeratin antibody 72 hours after the implantation (stage 29). DMSO beads were used as controls (L,N). (O-R) Cell proliferation was analyzed quantitatively in gonads of embryos implanted with RA (O,P), RAA (Q,R) and DMSO beads at stage 27. Total DAPI-stained cells (data not shown) and BrdU-positive cells in the gonadal cortex (cytokeratin-positive) (O,Q) and medulla (cytokeratin-negative) (P,R) were counted. Relative fold changes in the number of BrdU-positive cells in the cortex and medulla are plotted, with the number of BrdU-positive cells in the right cortex of DMSO-bead-implanted embryos set at 1. Scale bars: 100 µm in A-J; 50 µm in K-N.