Fig 2: Tet TKO ESCs are depleted of 5hmC but retain ESC characteristics. (A,B) While 5mC level was not globally altered by Tet TKO (A), 5hmC was undetectable (B) in Tet TKO ESCs. Bars are 5mC and 5hmC levels (relative to unmodified C) in control and TKO mESCs as measured by mass spectrometry. (C) Western blot analysis demonstrated depletion of Tet1, Tet2, and Tet3 proteins in Tet TKO cells, with Suz12 serving as a loading control for all blots. ESC nuclear extracts were used for Tet1, Tet2, and Suz12 blots, while nuclear extracts from differentiated cells (EBs) were used for the Tet3 and Suz12 blots. (D) Tet TKO mESCs exhibited relatively flatter morphology when compared with wild-type (WT) mESCs. (E) Pluripotent marker genes were expressed normally in Tet TKO mESCs. Data shown are images of wild-type and Tet TKO mESCs costained with Oct4 and Nanog antibodies. Nuclei were visualized by DAPI staining. (F) Growth curves of wild-type and Tet TKO mESCs. Data shown are mean of three biological replicates, with error bars representing standard deviation. (G) Tet TKO did not affect ESC colony formation capacity. Data shown are mean single-cell colony formation rates of three biological replicates, with error bars representing standard deviation.

Fig 3: Increase in EGC formation in Dnd1-cKO PGCs. (A), Schematic representation of the culture conditions for deriving EGCs from PGCs. (B–G), Representative images of EGC colonies derived from EGFP-positive PGCs in the testes of control (Dnd1flox/flox; Nanog-GFP) (B–D) and Dnd1-cKO (Dnd1flox/flox; Nanog-GFP; Rosa26-CreERT2) (E–G) embryos at E13.5 (B, E), E14.5 (C, F), and E15.5 (D, G). EGC colonies were stained by alkaline phosphatase (AP) activity. Scale bars: 10 mm. (H), Rate of EGC derivation from EGFP-positive PGCs in the testes of control (Dnd1flox/flox; Nanog-GFP) and Dnd1-cKO (Dnd1flox/flox; Nanog-GFP; Rosa26-CreERT2) embryos. Black bars represent the mean ± SD. **p < 0.01, ***p < 0.001 (Student’s t-test).

Fig 4: Dnd1 deletion in migrating PGCs causes testicular teratomas. (A), Mating strategy for the generation of conditional knockout mice of Dnd1. (B), Comparison of testis size between 4-week-old littermates of control (Dnd1flox/flox) and Dnd1-cKO (Dnd1flox/flox; Oct4ΔPE-CreERT2) mice. (C–G), Testis sections from 4-week-old control (C) and Dnd1-cKO (D–G) mice were stained with haematoxylin and eosin. Hair follicles, muscles, and gut-like epithelium are observed in (E, F), and (G), respectively. (H, I), Testis from control (H) and Dnd1-cKO (I) embryos with Nanog-GFP transgene at E16.5, under a fluorescence microscope. The arrowheads in (I) indicate GFP-positive foci. (J–R), Testis sections from E16.5 Dnd1-cKO embryos with Nanog-GFP transgene were immunostained with antibodies against GFP (J, M, P), NANOG (K), POU5F1 (N), and OTX2 (Q). DNA was labelled with DAPI (blue) (L, O, R). Images correspond to one of n = 3 biological replicates. S, The incidence of male embryos with at least one ECC colony in the testes was analysed in control and Dnd1-cKO embryos with Nanog-GFP transgene after injection of tamoxifen at E10.5, E11.5, and E12.5. Scale bars: 5 mm in (B), 100 μm in (C) and (D), 100 μm in (E–G), 200 μm in (H) and (I), and 100 μm in (J–R).

Fig 5: Transcriptomic analysis of PGCs in testes from the control and Dnd1-cKO embryos. (A), Expression levels of Dnd1 transcript in each PGC from five independent control and Dnd1-cKO embryos are indicated by read counts in the sequence analyses. (B), Volcano plot of RNA-seq data of PGCs from the control and Dnd1-cKO embryos at E11.5. Red: p < 0.05, fold change > 1.5; blue: p < 0.05, fold change < 1.5. (C) PCA of the transcriptomes of PGCs from control and Dnd1-cKO mice at E11.5. Each dot represents a single transcriptome. Transcriptomes from five biological replicates are plotted. (D), PCA of the transcriptomes of PGCs in testes from control (Dnd1flox/flox; Nanog-GFP) and Dnd1-cKO (Dnd1flox/flox; Nanog-GFP; Rosa26-CreERT2) embryos at E12.5 and E13.5. Each dot represents a single transcriptome. Transcriptomes from three replicates were plotted. (E, F), Heatmap representation of the expression and GO enrichment of genes upregulated (E) and downregulated (F) in PGCs from Dnd1-cKO embryos compared to those from control embryos at E12.5 and E13.5. The heatmap was generated using the ‘heatmap.2’ function in the gplot package (ver.3.1.3, https://rdocumentation.org/packages/gplots/versions/3.1.3) after performing gene clustering using ‘dist’, ‘hclust’, and ‘cutree’ functions in the stats package (ver.3.6.2, https://rdocumentation.org/packages/stats/versions/3.6.2).