Fig. 6

Regionalized putative mesoderm formation and PGC precursor-like cell specification in ETX-embryoids. a Asymmetrical gene expression of Blimp1-mVenus in ETX-embryoids, Oct4 (red) indicates ESC-derived tissues, and the arrow indicates Blimp1-mVenus-positive tissues (green), orthogonal xz and yz views are shown. n = 6 ETX-embryoids, two experiments. Scale bar, 20 µm. b Representative E6.5 chimeric embryo with BVSC ESC-derived tissues. The arrows indicate the localization of Blimp1-mVenus-positive cells (green). n = 15 embryos, two experiments. Scale bar, 50 µm. c Localization of Blimp1-mVenus-positive tissues in EXE-embryoids. n = 20 EXE-embryoids. Three experiments. Scale bar, 20 µm. d Colors and shapes are assigned to the ESC and TSC single-cell groups isolated at 0, 24, and 84 h from ETX-embryoids. The x-axis and y-axis standed for the explained variance in a PLS-DA plot. ESC: n = 11 (0 h), 10 (24 h), 11 (84 h, BV-), and 16 (84 h, BV + ). TSC: n = 18 (0 h), 16 (24 h), 15 (84 h). e, f Violin plots of expression levels of naive ESC marker genes (Klf4, Nanog, Sox2) and EPI-specific marker genes (Fgf5, Fgf4, Fgfr1, Gdf3, Cripto) in BVSC-ESCs, respectively, which reveal that the pluripotency of ESCs decreased as the ESCs differentiation. A background of Ct = 26 was used to obtain expression levels. g Single-cell RT-qPCR analysis of the expression of PGC markers (Blimp1, Prdm14, Stella, Tfap2c, and Dnmt3b) in Blimp1-mVenus-positive and Blimp1-mVenus-negative cells from the ESC compartments of the ETX-embryoids. Box-plots are generated as described in Fig. 1h. Two-tailed Student’s t-test, n = 11 BV- and 16 BV + single cells (84 h). *P < 0.05, ***P < 0.001. h Immunostaining indicates asymmetrical expression of Cripto (gray) in ESC compartment of ETX-embryoids, White boxes, magnified regions showing Cripto negative (left, anterior) and positive (right, posterior) cells. n = 10 ETX-embryoids, two experiments. Scale bar, 20 µm. i Single-cell RT-qPCR analysis of the expression of the genes (Slc7a3, Pou3f1) in Blimp1-mVenus-positive and Blimp1-mVenus-negative cells from the ESC compartments of the ETX-embryoids. Box-plots are generated as described in Fig. 1h. Two-tailed Student’s t-test, n = 11 BV- and 16 BV + single cells (84 h). *P < 0.05