Extended Data Fig. 7: Comparative characterization of BMP signaling across mouse implantation.
From: Distinct pathways drive anterior hypoblast specification in the implanting human embryo
a, Examples from a segregated blastocyst presented in Fig. 4a of Epiblast (EPI), Hypoblast (HYPO) and Trophectoderm (TE) cells with nuclear region of interest (ROI) outlined. The measured nuclear (Avg. Nuc) and background and calculated normalized (Nuc/1+Background) pSMAD1.5.9 intensity for individual cells is shown. b-c, Examples from a segregating blastocyst (b) and day 7 embryo (c) of cells with nuclear and background ROIs outlined. Measured nuclear and background (bg) and calculated normalized pSMAD1.5.9 intensities are shown for each cell. Note that here the raw, unprocessed pSMAD1.5.9 signal is shown for the central 3-plane z-stacks used directly for quantification. Cells that are central to the same 3-plane z-stack use the same background value for normalization. d, Mean ± S.E.M. normalized pSMAD1.5.9 intensity within each lineage of segregating (N = 9) and segregated blastocysts (N = 9), day 7 (N = 6), and day 9 embryos, separated by individual embryo. Note consistency of trends, particularly of TE versus ICM enrichment in segregating blastocysts, and of hypoblast versus epiblast in segregated blastocysts and at day 7. e, Immunofluorescence images of mouse embryos at E3.5 (N = 9 embryos), E4.5 (N = 4 embryos), E5.5 (N = 6 embryos) and E5.75 (N = 5 embryos) stained for phosphorylated (p)Smad1.5.9, DAPI and either Cdx2 or Gata6. f-i, Quantification of normalized pSmad1.5.9 fluorescence between lineages at the different stages of mouse development depicted in e. j-l, Quantification of normalized pSmad1.5.9 in epiblast (EPI) (j; n = 69 E3.5, 22 E4.5, 82 E5.5, 93 E5.75 cells), primitive endoderm/visceral endoderm (PrE/VE) (k; n = 69 E3.5, 26 E4.5, 102 E5.5, and 121 E5.75 cells) (j) or trophectoderm/extraembryonic ectoderm (TE/ExE) (l; n = 60 E3.5, 28 E4.5, 62 E5.5, and 69 E5.75 cells) across stages. For Violin plots e-h, central dotted line denotes median and dotted lines mark the 25th and 75th quartiles. Statistical tests: (f) two-sided Mann Whitney, (g-i) Kruskal Wallis with Dunn’s post-hoc; ****p < 0.0001, ***p < 0.001, *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: 100 µm.
