Fig. 5. Time-dependent exposure to culture medium with or without exogenous FGF9 reveals distinct effects of FGF9 on Sertoli and germ cells. Cultured gonads were labeled with antibodies against SOX9 to detect Sertoli cells (green) and PECAM to detect germ cells and vasculature (red). Blue and purple arrows indicate stages at which samples were placed into culture. (A) Uncultured XY Fgf9^–/– control with few germ cells, no SOX9-positive cells and no male-specific vasculature. (B) Fgf9^+/– 12.5 dpc XY gonad cultured without FGF9 for comparison. (C) XY Fgf9^–/– gonad cultured without FGF9 from 11.25 to 12.5 dpc shows few germ cells and many SOX9-positive cells. Some male structures are rescued including testis cord-like organization (arrow) and male-specific vasculature (arrowheads); however, germ cell numbers are not rescued compared with Fgf9^+/– (B). (D) When 25 ng/ml FGF9 is added to the culture medium of Fgf9^–/– gonads explanted at 11.25 dpc and cultured to 12.5 dpc, germ cells are rescued as well as testis cord formation (arrow) and male-specific vasculature (arrowhead). (E) XY Fgf9^–/– gonad cultured from 11.75 to 12.5 dpc shows few germ cells, few SOX9-positive cells and no male-specific vasculature or cord formation. (F) XY Fgf9^–/– gonad cultured from 11.75 to 12.5 dpc in the presence of 25 ng/ml FGF9. Germ cell numbers are rescued but other features of testis development are not. SOX9-positive cells are reduced or absent. Figures representative of three independent experiments, n5 each. Scale bar: 50 µm.