Fig. 2: Embryogenesis on ex vivo uterus.

Time series observation of attached embryos at 24 h (A–G), 48 h (H–M), and 96 h (N–T). A, H, N. Stereoscopic findings. mTE attachment at 24 h (A), solid cell growth inside the blastocoel at 48 h (H), and embryonic expansion at 96 h (N) were observed on the endometria. n = 5 (A), 4 (H), 7 (N) biologically independent samples. B-G, I-M, O-T. Whole-mount immunofluorescence. B-G. Confocal microscopic images at 24 h. A representative overhead view of a three-dimensionally reconstructed confocal image (B) was dissected in an oblique slice (C), and the front half of the embryo was depicted as overlays (D, E) to visualize CDX2 gradience from pTE to mTE (bilateral arrow). See also Movie S1. F, G. A representative 3D overhead view (F) and a slice (G) of attached embryos stained for CDX2 and a PrE marker FOXA2. White dotted lines indicate the attachment sites (D-G). I-M. Confocal microscopic images at 48 h. A representative 3D image (I) was dissected in an oblique slice (J, K, L), showing the emergence of EPI-/ExE-like compartments (J, K) and nuclear enlargement of invading trophoblasts (arrowheads) (L). Yellow dotted lines indicate the surface of the embryo. See also Movie S2. M. A representative slice from 3D image of whole-mount immunostaining for CDX2 and a VE marker FOXA2. FOXA2-positive undifferentiated PaE-like compartment and yolk sac-like morphology (inset) was identified. See also Movie S3. O–T Confocal microscopic images at 96 h. A representative 3D image (O) was dissected in oblique slices (P, Q), showing a more organized form of EPI-/ExE-like compartments than 48 h. See also Movie S4. R-T. Representative slices (R, T) from 3D image (S) of whole-mount immunostaining for CDX2 and FOXA2 show the presence of VE-like compartment. See also Movie S5. ICM, inner cell mass; mTE, mural trophectoderm; pTE, polar trophectoderm; PrE, primitive endoderm; EPI, epiblast; ExE, extraembryonic ectoderm; VE, visceral endoderm; PaE, parietal endoderm; Em, embryo. tdTomato indicates the cell membrane of all embryonic/extraembryonic cell lineages. Scale bars: 50 μm. See also Supplementary Fig. 5 for the efficiencies and the number of independent experiments performed using different recipient animals.