Fig 2: Single-celled embryos at the blastocyst stage.(A) Representative images of long-term time-lapse of WT and single-celled mMyh9;mMyh10 embryos at the onset of fluid accumulation (Figure 8—video 1). Scale bar, 20 µm. (B) Embryo growth curves during fluid accumulation for WT (grey, n = 20) and multiple- (yellow, n = 13) or single-celled Myh9;Myh10 (green, n = 4) mutant embryos measured for seven continuous hours after a lumen of at least 20 µm is observed. Data show mean ± SEM. (C) Representative images of WT and single-celled mMyh9;mMyh10 embryos stained for TE and ICM markers CDX2 (magenta) and SOX2 (cyan), DAPI (yellow), and F-actin (grey). The same mutant embryos as in A are shown. Scale bar, 20 µm. (D-E) N/C ratio of CDX2 (D) and SOX2 (E) staining for outer cells from WT (grey, n = 23) and multiple- (yellow, n = 18) or single-celled Myh9;Myh10 (green, n = 6) embryos. Mann-Whitney U test p values compared to WT are indicated.

Fig 3: PHF6 activates trophectodermal genes for trophectoderm lineage determination. (A) Morphological features of EBs from WT and Phf6 KO ESCs on Day 7 (left). Each EBs were grown and observed separately. Magnification ×10. The average areas (mm2) of the outer cell layer were compared between WT (n = 11) and Phf6 KO (n = 9) EBs (right). Statistical significance was calculated by ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001). (B, C) Immunostaining of CDX2 and PHF6 in the outer cell layer in WT EBs. Magnification ×100 (B) and ×200 (C). (D) qRT-PCR analysis of the trophectodermal genes during EB differentiation in WT and Phf6 KO ESCs. WT and Phf6 KO EBs were maintained without LIF and harvested at the indicated days. mRNA levels of each gene were determined as relative values for Gapdh or ß-actin and relatively compared based on WT day 0. Statistical significance was calculated by ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 4: Representative images showing immunostaining of chimeric blastocysts injected with mCherry-labelled NELFAhigh and NELFAlow mESCs following 2-DG treatment. NELFAhigh chimeric blastocysts showed integration of injected cells in both the inner cells mass (ICM) (open arrows) and trophectoderm (filled arrows) whereas all NELFAlow chimeric blastocysts contributed exclusively to the ICM. Trophectoderm is marked by Cdx2 (magenta). DAPI nuclear staining is shown in blue. Scale bars: 20 µm.

Fig 5: Evaluation of CDX2 and MUC2 in gastric mucosa. a Representative immunohistochemical staining of CDX2 in the mouse gastric mucosa. b Analysis of gastric mucosa total protein of CDX2. c Representative immunohistochemical staining of MUC2 in the mouse gastric mucosa. d Analysis of gastric mucosa total protein of MUC2. Data are expressed as the mean ± SD. ** indicates P < 0.01 compared with the control group. # indicates P < 0.05 and ## indicates P < 0.01 compared with the model group (IHC, 400×). n = 5 in each group