Fig 2: Bisulfite sequencing of Dnmt3L promoter. The top panel shows a diagram of the Dnmt3L promoter and its methylation positions (white circles). Methylation levels of the Dnmt3L promoter in germ cells of 0-d-old (P0) nontransgenic (Non-Tg) mice and of mice with a transgene cassette inserted at the B3 locus (Tg-B) as well as in those of 10-d-old (P10) non-Tg mice and mice with transgene cassettes inserted at the E1 locus (Tg-E) and at both the E1 and B3 loci (Tg-BE). Black and white circles indicate methylated and unmethylated CpGs, respectively.

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

Fig 4: Transgenic mice (Tg) phenotypes. (A) Schematic illustration of polymerase chain reaction (PCR) primer annealing sites to confirm Tg cassette insertion sites. “Tg internal” legends indicate the junctions between Miwi2 promoter and asDnmt3L. “Tg head” shows the junction between genomic DNA and the Miwi2 promoter at Ch18qB3 and E1 sites, and “Tg tail” shows the junction between the tail of the Tg cassette and genomic DNA at each insertion site. The primer sequences are shown in Supplemental Table S4. The PCR products were subjected to agarose gel electrophoresis with Tris-acetate EDTA buffer (right panel). (B) Morphology of testes of adult mice with transgenes inserted at the E1 locus (Tg-E), B3 locus (Tg-B), and B3 and E1 loci (Tg-BE). Black bar indicates a 2 mm scale. (C) Weights of the testes of Tg-E, Tg-B, and Tg-BE mice. Each dot represents the weight of each testis. The range of the whisker lines represents the minimum and maximum values; outliers were excluded. The box ranges indicate the interquartile range (IQR), the horizontal bars in the box indicate the median, and the “X” marks indicate the mean weights. (98.4, 90.8, 32.0, and 29.8 mg in non-Tg, Tg-E, Tg-B, and Tg-BE mice, respectively; t-test; [*] P = 0.15; [**] P < 0.01; [***] P = 0.72). (D) DNMT3L expression in mice testes at E16.5 was analyzed using western blotting. Upper panel shows DNMT3L, and lower panel shows β-actin. Each lane was loaded with protein extracts from one-half of a testis. The band intensity of DNMT3L was normalized to that of β-actin, as well as to that of DNMT3L in non-Tg mice. Analysis was performed using a GelAnalyzer 2010. (E) Hematoxylin and eosin-stained histological sections showing developmental differences between non-Tg, Tg-E, Tg-B, and Tg-BE mouse testes.

Fig 5: Bisulfite sequencing and northern blot of DNMT3L de novo DNA methylation targets. (A) Methylation levels of retrotransposon genes (L1A, LINE1 type A; L1Tf, LINE1 Tf; and IAP, IAP1Δ1) in germ cells of non-Tg and mice with transgenes inserted at the E1 locus (Tg-E), B3 locus (Tg-B), and B3 and E1 loci (Tg-BE). (B) Methylation levels of paternally imprinted genes (Rasgrf1, H19, and Dlk1). (C) Methylation levels of Snrpn, a maternally imprinted gene, representing sorted cell purity. Black and white circles indicate methylated and unmethylated CpGs, respectively. (D) Northern blotting of LINE1 and IAP in testes of 3 wk in the non-Tg and Tg mice. To compare the amount of RNA loaded, the filters were stained with methylene blue after transfer to show the amount of 28S and 18S.