Fig. 2. Epiblast/stem cell and PGC-markers in Smad5^m1/m1 embryos. Transverse sister sections through hindgut region (A,E,I,M) and head region (B,F,J,N) of an E8.5 wild-type embryo, and of an E8.5 Smad5^m1/m1 littermate with multiple aggregates of cells on the amnion (C,D,G,H,K,L,O,P). The arrow points to the amnion. (A-D) Anti-Oct4 antibody staining demonstrates the presence of PGCs in the hindgut (A; open arrowheads). Oct4⁺ cells are absent in control (B) and non-thickened mutant amnion (C,D). Ectopic Oct4⁺ cells can be observed regionally in some aggregates in mutant amnion. Oct4⁺ cells have an ectoderm oriented localization (C,D). (E-H) PGCs in the hindgut are marked by their fragilis expression (E; open arrowheads). Ectopic fragilis⁺ cells can be observed within the aggregate of mutant amnion, but not in the non-thickened amnion (G,H). (I-L) Stella is expressed in PGCs in the hindgut (I; open arrowheads). Stella expression was never observed in wild-type (J) or mutant (K,L) amnion, also not in regions where Oct4⁺, SSEA-1⁺ and fragilis⁺ cells are residing (K,L). (M-P) Anti-SSEA-1 antibody staining demonstrates the presence of SSEA-1 in surface ectoderm, visceral endoderm and the neuroectoderm (N). At this stage of development PGCs in the hindgut are SSEA-1 negative (M). There is intense SSEA-1 staining in the mutant amnion ectoderm, and weaker staining can be observed in the aggregates. (Q) Schematic representation of the different regions that can be recognized within the mutant amnion. Scale bars: 100 µm.