Fig 1: Immunohistological staining of FOXL2-positive cells (red) in ovaries of two 9-mo-old SRY-KO pigs (upper images: SRY-KO pig 1,255, lower images: SRY-KO pig 1,262). Cell clusters of FOXL2-positive cells (indicated with white arrows) were detected in the cortical region of the porcine ovaries. SiR-Hoechst–stained nuclei (blue) are shown. The merged images revealed positive FOXL2 staining in the nuclei of the cells. The experiments were repeated three times with similar results. (Scale bars, 20 µm.) Reprinted with permission from ref. 50.
Fig 2: The monoubiquitylation-dependent function of FANCD2 was essential for PGC development.A, alkaline phosphatase staining of WT, KO, and MT whole mounts at E8.5 and E9.5, and genital ridges at E11.5. Scale bars represent 200 µm. B, counting of PGCs in WT, KO, and MT embryos at E8.5, E9.5, and E11.5. Ten embryos from each embryonic stage were included per genotype. Data are presented as mean ± SD. *p < 0.05. Immunofluorescence staining for DDX4 (germ cell marker; green) and FOXL2 (pregranulosa marker; red) in E13.5 (C) and E18.5 (E) ovaries from WT, KO, and MT embryos. Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bars represent 50 µm. Immunofluorescence staining for DDX4 (green) and SOX9 (Sertoli cell marker; red) in E13.5 (D) and E18.5 (F) testes from WT, KO, and MT embryos. Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bars represent 50 µm. KO, Fancd2-/-; MT, Fancd2K559R/K559R; PGC, primordial germ cell.
Fig 3: Vasa+ cells can be detected in CD63+_rOvaries cultured for 10 days. A GO and KEGG analysis of CD63+_GSCLCs. B Schematic of the IVDi culture. C Morphology of rOvaries in the culture system on day 2 and day 10. Scale bar = 50 µm. Pure PGC is scattered and nearly disappears on day 10. Scale bar = 50 µm. D Proportion of Vasa+ cells in different groups. Vasa+ cells can be detected in Gonad_ and CD63+_ rOvaries cultured for 10 days in vitro, but no such cells were observed in MEF_ and P6w_ rOvaries. Bars = Mean ± SEM (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001. E Distribution of Vasa, Foxl2, and Gata4 in E12.5_rOvaries. Scale bar = 20 µm. F Distribution of Vasa, Foxl2, and Gata4 in MEF_rOvaries. Scale bar = 20 µm. G Distribution of Vasa, Foxl2, and Gata4 in CD63+_rOvaries, GFP: CD63+_GSCLCs. Scale bar = 20 µm. H Distribution of Vasa, Foxl2, and Gata4 in P6w_rOvaries. Scale bar = 20 µm
Fig 4: Induction of E12.5_GSCLCs by small molecules. A Schematic illustration of in vitro chemical induction strategy. B qPCR detection of Gata4 and Foxl2 expression in GSCLCs induced by treatment with different small-molecule compounds. Bars = Mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. C qPCR detection of Gata4 and Foxl2 expression in GSCLCs induced by AM580 and V580. Bars = Mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. D Immunofluorescence staining of Gata4 (green) and Foxl2 (red) in GSCLCs induced by V580. Scale bar = 20 µm. E Protein levels of pluripotency markers (Nanog and Oct4) and E12.5_GSCs markers (Gata4 and Foxl2) in GSCLCs induced by V580 were determined via western blot analysis. ß-actin served as a loading control. F Heatmap highlighting DEGs compared with E12.5_GSCs, determined using RNA-seq. G PCA shows that V580_D6 GSCLCs and E12.5_GSCs were closer with regard to the overall transcriptome
Fig 5: Single cell analysis of E12.5_ and E13.5_ GSCs. A Schematic of PGCs and somatic cells separation. Mes: mesonephros. B tSNE plot showing the five clusters distribution of E12.5_GSCs. C Pheatmap showing marker genes of each cluster in E12.5_GSCs. D tSNE plot showing the four clusters distribution of E13.5_GSCs. E Pheatmap showing marker genes of each cluster in E13.5_GSCs. F Distribution and expression of marker genes including Foxl2 and Gata4 in E12.5_GSCs. G Distribution and expression of marker genes including Foxl2 and Gata4 in E13.5_GSCs. H Distribution of Gata4, Vasa and Foxl2 in E12.5 and E13.5 gonads. Scale bar = 20 µm
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