Fig. 2. Wnt signaling is activated by Tex10 depletion to compromise the PGCLC specification.

(A) Volcano plots show the comparison of transcriptomic profiles of day 2 PGCLC (D2EB) vs. EpiLC stage (bottom volcano), and dTAG13-treated day 2 PGCLC (dD2EB) vs. control (D2EB; top volcano). Venn diagrams show the intersections between 1831 genes upregulated in D2EB vs. EpiLC (log₂FC > 1 & p-value < 0.05) and 1392 genes downregulated in dD2EB vs. D2EB (log₂FC < −1 & p-value < 0.05), and between 1278 genes downregulated in D2EB vs. EpiLC (log₂FC < −1 & p-value < 0.05) and 2454 genes upregulated in dD2EB vs. D2EB (log₂FC > 1 & p-value < 0.05). Enriched gene ontology biological processes are shown for the two overlapping gene sets.

(B) Heatmaps for gene expression profiles of pluripotency, PGC, ectoderm, mesoderm, and endoderm regulation markers in D2EBs vs. dD2EBs.

(C)Western blots of key PGC markers in EpiLCs, D2EBs, and dD2EBs. Data from two Tex10-degron clones (C1 & C2) are shown with dTAG13 added at the EpiLC stage and Tex10 depletion happened in dD2EBs. Quantification values for Otx2 relative to Vinculin are shown on top of bands for D2 and dD2 EB samples.

(D)Gene set enrichment analysis showing that Wnt signaling but not Bmp signaling is activated by Tex10 depletion on day 2 and day 4 of PGCLC induction.

(E)Flow cytometry analysis of PGCLC specification efficiency using cell surface markers SSEA1 and CD61. Percentages of double positive (SSEA1⁺ and CD61⁺) cells are indicated at day 2 and day 6 of PGCLC induction for clone C1. Quantification of double-positive percentages in live cells and numbers per 20,000 analyzed cells are shown with bar plots. Two cell clones C1 and C2 were used as biological replicates, and an ANOVA test was used to detect significance.