Figure 1. Rlim-independent XCI in female ESCs in vivo.

A) Development of Xist clouds in ICM of female blastocyst outgrowths lacking maternal and embryonic RLIM. RNA FISH on E4 blastocyst outgrowths cultured for 72h using Xist as probe. Embryos were generated by crossing Δ/Y males with either SC-cKO_m/Δ_p or fl/fl females. Boxed area (Δ_m/Δ_p embryo) is shown in higher magnification in the right panel. Focus in images is on cells in the ICM. Embryos were genotyped after image recording. B) Generation of female RlimKO ESC lines via CRISPR/Cas9. Western blot of undifferentiated WTp and RlimKOp lines using antibodies against RLIM (top) or β-actin (bottom). C) XCI upon RA-differentiation of ESCs is Rlim-dependent. RNA-FISH on 6d RA-differentiated ESCs using Xist (red) and Rlim (green) as probes. Note side-by-side Xist and Rlim transcription foci but no Xist clouds in RlimKO ESCs. D) XCI profile in ESCs RA-differentiated up to 12 days. Data reflect two independent experiments with >500 cells counted for each ESC line and time point. Error bars indicate standard error of the mean (SEM). E) Approx. 12–15 undifferentiated GFP-tagged ESCs were microinjected into E3.5 blastocysts, which were surgically placed into the uteri of pseudo pregnant females. MEFs were prepared from E12.5 embryos and stained with GFP antibodies. F) FACS-sorted WT and KO MEFs were stained with antibodies against H3K27me3. Arrows: H3K27me3 foci marking the Xi. G) Summary of H3K27me3 foci in F); n>150 cells each. See also Figure S1.