Extended Data Figure 6: Prdm1−/− abrogates PGCLC induction by Nanog; Nanog and the Wnt pathway act independently.
a, PGCLC induction with cytokines or Nanog (+Dox) from Prdm1−/− ESCs (Prdm1−/−; Nanog). Representative brightfield images of D4 and D6 aggregations. Scale bar, 200 μm. Related to Fig. 2a.
b, Loss of Prdm1 abrogates PGCLCs induced by NANOG as shown by qPCR analysis of mutant (Prdm1−/−; Nanog) compared with control (Prdm1+/+; Nanog) cells with Nanog (+Dox) or cytokines (+cyto). Unsorted samples were used for analysis. Note that the data shown in Fig. 2a was combined with additional qPCR data on cells at D6 of PGCLC induction. ΔΔCt mean values +/− s.d. (n=2 technical replicates each from 2 biological replicates); two-sided/unpaired t-test: **p<0.01; *p<0.05. Related to Fig. 2a.
c, Nanog does not affect cell proliferation rate of Prdm1−/−; Nanog cells. IF staining for the mitotic marker H3S10ph in Prdm1−/−; Nanog cells at D6 of PGCLC induction; +Dox for Nanog expression. Scale bar, 10μm; two-sided/unpaired t-test; n.s.=not significant; n=estimated number of cells (see Methods for details). Related to Fig. 2a.
d, Induced expression of Nanog results in an increased number of cell death of Prdm1−/−; Nanog cells. IF stainings of the DNA double strand break marker ƔH2AX in Prdm1−/−; Nanog cells at D6 of PGCLC induction; +Dox for Nanog expression. Scale bar, 10μm; two-sided/unpaired t-test; s=significant; n=estimated number of cells (see Methods for details). Related to Fig. 2a.
e, Experimental design (for f-i) to test the interdependence of Nanog and the Wnt pathway for PGCLC induction. Blimp1-GFP ESCs were sequentially differentiated into PGCLCs +/− tankyrase inhibitor XAV939, which causes the degradation of β-catenin25; +Dox for Nanog expression.
f, XAV939 does not affect the morphology and proliferation of D2 EpiLCs. Representative brightfield/GFP images of D2 EpiLCs induced from GOF-GFP ESCs with 1μM XAV939. Scale bar, 200μm.
g, qPCR of D2 EpiLCs treated with XAV939 as shown in (a, b). The expression of Nanog and of the EpiLC markers Dnmt3a and Dnmt3b are not affected by XAV939. Brachyury, the downstream target of WNT, is most efficiently repressed with 1μm XAV939. ΔΔCt mean values +/− s.d. (n=2 technical replicates each from 2 biological replicates); two-sided/unpaired t-test: **p<0.01; *p<0.05.
h, The efficiency of PGCLC induction by cytokines but not by Nanog (+Dox) is markedly reduced upon XAV939 addition. PGCLCs were induced from 1μM XAV939-treated D2 EpiLCs. Representative FACS analysis for GFP with cells at D4 of PGCLC induction.
i, XAV939 does not affect the induction of PGC marker expression in Nanog-induced PGCLCs. Gene expression analysis by qPCR with FACS-sorted Nanog-induced D4 PGCLCs +/− 1μM XAV939. Mean ΔΔCt values +/− s.d. (n=2 technical replicates each from 2 biological replicates). Two-sided/unpaired t-test: **p<0.01; *p<0.05; n.s.=not significant.