Fig. 5

Asymmetric Lefty1 expression in ETX-embryoids. a, b Representative ETX-embryoid assembled with Lefty1-mCherry XENCs, in which DVE-like tissues (red) formed at 72 and 84 h. n = 10 ETX-embryoids per group, three experiments. c, d Representative 3D max. projection view of ETX-embryoid with asymmetric Lefty1-mCherry expression (DVE-like tissues and AVE-like tissues). e Frequency of ETX-embryoids with DVE/AVE-like tissues. n = 50 ETX-embryoids per group, three experiments. Columns are means ± s.e.m. f Numbers of Lefty1-mCherry positive XEN-derived cells distributed on the left and right of midline. White line indicates the midline of the ETX-embryoids; n = 9 ETX-embryoids, two experiments. Two-tailed Student’s t-test, ***P < 0.001. Columns are means ± s.e.m. g Colors and shapes are assigned to the different XEN cell groups isolated at 0, 24, and 84 h from the ETX-embryoids. The x-axis and y-axis standed for the explained variance in a PLS-DA plot. The ellipse indicates the group membership for each sample and the confidence level of ellipse is 0.95. The colored background indicates areas of each cell group. n = 22 (0 h), 16 (24 h), 17 (84 h, LC-) and 19 (84 h, LC + ) single cells. h Representative EXE-embryoids, built from Lefty1-mCherry XENCs and cultured for 72 h, show the distribution of Lefty1-mCherry positive cells in a random manner. n = 10 EXE-embryoids per group, three experiments. i Box-plots of DVE/AVE markers expression levels. Single-cell qPCR assay revealed that DVE/AVE marker genes were upregulated in the mCherry positive cells compared with mCherry negative cells. j Box-plots of posterior markers of VE expression levels. Single-cell qPCR assay revealed that posterior marker genes were upregulated in the mCherry negative cells at 24 h compared with mCherry negative cells at 84 h. Box-plots are generated as described in Fig. 1h. A background of Ct = 26 was used to obtain expression levels. n = 16 single cells (24 h, LC-) and 19 (84 h, LC-), three experiments. Two-tailed Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 20 µm (a–g). LC, Lefty1-mCherry. Source data of e, f are provided as the Source Data file