Figure 3.

DIDO1 Is Essential for PE Formation

(A) (Left) shRNA-Control d4EBs with staining for PE formation (arrowheads). Top row: OCT4 (green; inside EBs), GATA4 (red; outer PE cells). Center: OCT4 (green; inside EBs) and DIDO1 (red; at apical membrane of outer PE cells). Bottom: DIDO1 (green; apical membrane) and FOXA4 (red; PE outer cell layer). (Right) All rows: lack of PE formation in shRNA-Dido1 d4EBs.

(B) (Left) Reconstitution of PE formation by ectopic HA-DIDO3 expression in Dido3ΔCT d4EBs; OCT4 (green; inside EBs) and DIDO1 (red; at apical membrane of PE cells). (Right) The same PE reconstitution pattern of Dido3ΔCT d4EBs after retinoic acid (RA) treatment.

(C) Comparison of solvent (DMSO) versus RA-treated shRNA-Co d4EBs. (Left) Top: OCT4 (green; reduction inside EBs) and DIDO1 (red; at apical membrane of PE cells). Bottom: DIDO1 (green; at apical membrane of PE cells) and FOXA2 (red; arrowheads indicate PE outer layer in DMSO-treated cells and inside EBs in RA-treated cells). (Right) shRNA-Dido1 d4EBs with lack of PE formation in DMSO- or RA-treated EBs, no DIDO1 labeling at apical membrane, and FOXA4 (red; only inside EBs).

(D) Ectopic HA-DIDO1 expression of reconstituted PE formation in Dido3ΔCT d4EBs. (Left) OCT4 (green; inside EBs) and DIDO1 (red; on apical membrane of PE cells). (Right) Co-localization of anti-HA (green) and DIDO1 (red) at apical membrane of outer PE cells.

Nuclei staining (DAPI, blue). Scale bars for EBs, 50 μm; Scale bar in boxed area of (D), 10 μm.