Figure 5: Loss of Xm-XCI is the primary cause of developmental failure immediately after implantation in most PEs.

(a) IF combined with FISH analysis of blastocysts in Egfp-PEs (upper panel) and Kdm4b-PEs (lower panel). CDX2-positive cells were identified as belonging to the trophectoderm (TE). Representative pictures of Z-sections. 4′,6-diamidino-2-phenylindole (DAPI) (blue), CDX2 (green), Xist (red) and H3K27me3 (white). Scale bars, 20 μm. The rates of cells with Xist (left) or H3K27me3 (right) in the inner cell mass (ICM) (b) and TE (c), respectively. n, number of embryos analysed. The number of cells analysed is shown in Supplementary Table 7. *P<4.3 × 10⁻²³ (Fisher’s exact test). (d) Expression states and clustering analysis of imprinted genes. Sfmbt2 important for placentation and differentially expressed genes (asterisk) are shown. The scale bar indicates normalized values of log₂. (e) Embryos with extra-embryonic tissues at E6.5 and E9.5 for Kdm4b- and Egfp-PEs, respectively. Upper and lower images indicate Egfp- and Kdm4b-PEs, respectively. Left and right column sides show E6.5 and E9.5, respectively. Scale bars, 200 μm (E6.5) and 500 μm (E9.5). (f) Summary of the developmental abilities of Kdm4b-PEs and Egfp-PEs at postimplantation stages (E6.5 and E9.5). Five and 12 independent recipients were analysed at E6.5 and E9.5, respectively. (g,h) Xist analysis in Rnf12-knockdown and control Kdm4b-PEs. Representative images of FISH analysis. Scale bars, 50 μm (g) and Xist expression states (h). (i) Expression of imprinted and X-linked genes in Rnf12-knockdown and control Kdm4b-PEs. P-values were determined using Student’s t-tests. (j) Embryos with extra-embryonic tissues at E6.5 in Rnf12-knockdown and control Kdm4b-PEs. Scale bars, 200 μm. (k) Summary of the developmental ability of Rnf12-knockdown and control Kdm4b-PEs at E6.5. Five independent recipients were analysed. The P-values were determined using Fisher’s exact test.