Fig. 1. Impaired allantois and PGC development in Smad5^m1/m1 embryos. (A-C) Allantois dimensions measured in embryos collected from Smad5^m1/+ crosses in F2 (C57BL/6J x CBA) background. Results at different developmental stages were expressed as means ± s.e. The numbers under the histogram represent the number of embryos in each group. A numerator indicates the embryos with an allantoic bud. Zero values are not included in the mean. (A) Allantois length (µm). (B) Allantois width (µm) was measured at the base of the allantois (right-left axis). (C) The ratio of the allantois length over the embryonic axis length. (D) Wild-type (5S) embryo with a well-developed head, hindgut entrance (open arrowhead) and heart, and an elongated allantois. The amnion is thin and smooth. (E) Representative stage-matched Smad5^m1/m1 (KO) littermate with abnormal body curvature, underdeveloped anterior structures and foregut, vestigial heart, broad base of the allantois and an aggregate on the amnion (^*). (F) Number of PGCs detected in embryos of the three Smad5 genotypes at late streak, neural plate and headfold stages. Wilcoxon's summed ranks test was used for statistical analysis (^***, P<0.001; ^**, P<0.01; ^*, P<0.05), and medians are depicted by a bar. (G,H) Dorsal view of wild-type (28S) and Smad5^m1/m1 (26S) mutant embryos dissected free from their yolk sac. The amnion is a thin, smooth membrane that envelops the embryo (G, arrowheads). A characteristic aggregate of cells is present on the mutant amnion (H, arrow). (I) Whole-mount staining for alkaline phosphatase (AP) activity in a mutant amnion (10-11S stage) with an aggregate of cells. A cluster of ectopic AP⁺ (brown; arrow) PGC-like cells projects from the ectoderm side of the amnion. (J) AP staining of a section through the aggregate of cells on the mutant Smad5^m1/m1 amnion shown in H, at the level of the hindbrain. Ectopic PGC-like cells (red) are localized in close association with the amnion ectoderm. Scale bars: 500 µm in G,H; 200 µm in K; 100 µm in C,D,J. Al, allantois; Am, amnion; AmEc, amnion ectoderm; AmMe, amnion mesoderm; EHF, early headfold; He, heart; Hf, headfold; Hg, hindgut; LNP, late neural plate; LS, late streak; NP, neural plate; Nt, neural tissue.