Figure 5.

The effects of Prmt1 inhibition and R396A mutant of Klf4 on the PrE commitment. (A) Scheme of the chimeric assay. (B) Chimeric assay of ES cells with (Fur+) or without (Fur–) treatment. The ES cell line was AB2.2 cells with GFP and td-Tomato. After Fur treatment for 24 h, cells were microinjected into blastocysts. Embryos were observed at E6.5. GFP (green) and Td-Tomato (red) signals were detected. Dashed lines mark epiblasts. (C) IF assays showed the effect of Fur on mouse blastocysts. Blastocysts were cultured with (Fur+) or without (Fur–) for 24 h and then immunostained with Gata6 (red) to indicate PrE and Nanog (green) to indicate EPI. (D) Distribution of two cell types in the ICM. (E) IF assays showed the impact of Fur on the expression of Gata6 (green) and Nanog (red) in E14 cells with XEN induction. Nuclei were visualized with DAPI staining (blue). Yellow circles indicate Gata6-positive cells. (F) Fur treatment induced the expression of Gata4 and Gata6 genes using RT-qPCR, and Nestin was used as a negative control. (G) Chimeric assay of ES cells of wild type Klf4 and mutant Klf4 R396A. The AB2.2 cells were microjnjected into blastocysts. Embryos were observed at E6.5. Dashed yellow lines marked epiblasts, the arrows indicated chimeric cells outside of epiblasts. (H) IF assays showed the impact of Klf4 R396A mutation in AB2.2 cells. Nuclei were visualized with DAPI staining (blue). Yellow circles indicate Gata6-positive cells. (I) RT-qPCR assay showed that XEN induction further increased the expression of Gata4 and gata6 genes in R396A mutation in AB2.2 ES cells. (J) Schematic depiction of Klf4 methylation by Prmt1 to regulate PrE gene expression via mSin3a/HDAC recruitment in mouse ESCs, which heterogeneously prevents a bias toward PrE commitment.