Fig 2: Exogenous FGF9 is sufficient to repress FOXL2 inducibility in DT-treated Tg testis explants initiated at E12.5. (A) Schematic of exogenous addition of a candidate ligand (PDGF, AMH, DHH or FGF9) or agonist (BW245C for PGD2) to a 4-day culture of DT-treated Tg testes. (B-D) RT-qPCR analysis showing significant reductions of Foxl2 (B,C), and Gng13 and Lgr5 (D) in the presence of FGF9 (*P<0.05, **P<0.01; one-way ANOVA for FGF9, AMH and BW245C in B and C; paired Student's t-tests for others in B; unpaired Student's t-tests in D). Data are mean±s.e.m. In B, expression levels were normalized to the corresponding non-additive control of DT-treated Tg testes (broken horizontal line; set as 1.0). C shows relative Foxl2 expression levels in DT-treated Tg testes cultured with FGF9 (0, 1, 10 or 50 ng/ml). Actb was used as an endogenous reference. Numbers in parentheses are numbers of samples. (E) Anti-FOXL2 immunostaining of DT-treated Tg testis explants in the presence or absence of FGF9. FOXL2-positive signals near the surface epithelia and mesonephros are repressed in Tg testes by exogenous FGF9. Lower panels are high-magnification images of the regions surrounded by broken rectangles in the upper panels. (F,G) Morphometric analysis of FOXL2-positive cell density [number per 10,000 µm2 in serial sections (each 40 µm apart) of a whole explant; n=3 per group] in DT-treated Tg testes cultured with or without FGF9. FOXL2-positive cells in the surface subepithelial region (SE) and the non-surface regions, including the peri-mesonephric site (non-SE), are enumerated in G (total FOXL2-positive cell density is shown in F). Data are presented as a box and whisker plot (median, center line; whiskers, sample distribution range; *P<0.05 by unpaired Student's t-test). Each point represents the data of one section (Mayère et al., 2022). (H) Schematic of the partial sex reversal of E12.5 testis after Sertoli cell ablation. In E12.5 testis, Fgf9 expression in Sertoli cells, together with mesonephric/vascular cell migration, lead to testis differentiation (Harikae et al., 2013b). DT-dependent Sertoli cell ablation of E12.5 Tg testis triggers degeneration of the testis cord structure. In a 4-day culture of these testes without Sertoli cells, FOXL2-positive/NR2F2-negative cell clusters are re-recruited from the surface testis epithelia/subepithelia, similar to the ovarian cords from the female surface epithelia. FOXL2-positive/NR2F2-positive stromal cells appear in the peri-mesonephric region, independently of 3βHSD-positive Leydig cells in the interstitial region. Exogenous FGF9 represses FOXL2 expression in DT-treated Tg testes, implicating FGF9 in male differentiation of the surface epithelia/subepithelia and peri-mesonephric stromal cells. From top to bottom: cortex (C) to medulla (M) sides. MT, mesonephric tubule; SE, surface epithelium. Scale bars: 100 μm in E, upper panels; 25 µm in E, lower panels.