Fig. 3

MEK1/2 signalling is required for male germline differentiation. Analysis of E12.5 XY gonad-mesonephros or E12.5 XY or XX gonad-mesonephros cultured for 72 h with DMSO, 125 or 500 nM FGFRi or MEKi. A Immunofluorescent images demonstrating DPPA4 localisation. Top panel: DAPI (blue), MVH (green), DPPA4 (red), SMA (cyan). Bottom panel: DPPA4 (grey). B DPPA4 staining intensity in germ cells determined by flow cytometry. C Immunofluorescent images demonstrating DNMT3L localisation. Top panel: DAPI (blue), MVH (green), NR2F2 (red), DNMT3L (cyan). Bottom panel: DNMT3L (grey). D,E Percentage DNMT3L-positive germ cells (D) and DNMT3L staining intensity (E) determined by flow cytometry. F Immunofluorescent images demonstrating PIWIL2 localisation. Top panel: DAPI (blue), MVH (green), SMA (red), PIWIL2 (cyan). Bottom panel: PIWIL2 (grey). Scale bars: 50 μm. Replicates: A, C, F n = 3–4, B n = 4–14, D,E n = 4–9. Statistics: B,E Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons, D Ordinary one-way ANOVA with Tukey’s multiple comparison. In B,E, Intensity is relative to E12.5 + 72 h XY DMSO control sample set at 1.0. Error bars: mean ± SEM. Significance between control and treatment: **P < 0.01, ***P < 0.001, ****P < 0.0001