EPI, epiblast; EPI-p, EPI progenitor; TE, trophectoderm; TE-p, TE progenitor; PrE, primitive endoderm; PrE-p, PrE progenitor.
f, Average expression of selected marker genes within intermediate cell clusters in ciToti4-EMs, E3.5 NEs, and E4.5 NEs.
i, Number and percentage of intermediate cells assigned to TE, EPI, PrE, and NA identities via reference mapping.
j,k, Violin plots showing the Euclidean distances from individual intermediate cells to the TE, EPI and PrE centroids in PCA space using the full combined ciToti1–4 dataset (j; n = 2,748 intermediate cells) and in DDRTree space after proportional random downsampling of the combined dataset to 8,000 cells (k; n = 433 intermediate cells).
Within each panel, distances to all three centroids were calculated for the same set of intermediate cells.
a, UMAP plots of scRNA-seq data from ciToti structures (ciToti0-4) and natural blastocysts (E3.5, E4.5), coloured by unsupervised cluster affiliations. EPI, epiblast; EPI-p, EPI progenitor; TE, trophectoderm; TE-p, TE progenitor; PrE, primitive endoderm; PrE-p, PrE progenitor. b, Expression levels and proportions of cells expressing indicated marker genes across specified clusters and stages. c, Density ridge plots illustrating the distribution of lineage scores within the respective EPI/EPI-p, TE/TE-p, and PrE/PrE-p populations across developmental stages. Dashed arrows highlight the progressive shift in score distributions, indicating the gradual maturation of lineage-specific identities during development. d,e, Fraction of totipotent-like cells (d) and overall lineage composition (e) across the indicated stages. f, Average expression of selected marker genes within intermediate cell clusters in ciToti4-EMs, E3.5 NEs, and E4.5 NEs. g,h, Single-cell trajectory mapped along Components 1 and 2, coloured by inferred pseudotime with a grey arrow indicating developmental direction (g), and highlighting the transitional positioning of the intermediate cell population (blue arrowhead) (h). i, Number and percentage of intermediate cells assigned to TE, EPI, PrE, and NA identities via reference mapping. j,k, Violin plots showing the Euclidean distances from individual intermediate cells to the TE, EPI and PrE centroids in PCA space using the full combined ciToti1–4 dataset (j; n = 2,748 intermediate cells) and in DDRTree space after proportional random downsampling of the combined dataset to 8,000 cells (k; n = 433 intermediate cells). Within each panel, distances to all three centroids were calculated for the same set of intermediate cells. Violin plots show the distributions of individual values. Centre lines indicate the median, boxes indicate the interquartile range, and whiskers extend to 1.5× the interquartile range. l, BFM image of ciToti5-EMs in suspension culture. Scale bar, 100 μm. Representative image from 3 experiments. m, Immunofluorescence images of a ciToti5-EM stained for SOX17 (magenta), CDX2 (red), GATA3 (yellow), and DAPI (green). Scale bars, 50 μm. n = 6 ciToti5-EMs from 2 experiments. n, Sequential BFM images and schematic annotations depicting the transition to egg cylinder-like structures. PAC, pro-amniotic cavity; EPI, epiblast; ExE ecto, extraembryonic ectoderm; PrE, primitive endoderm; TE, trophectoderm. Scale bar, 100 μm. Representative images from 3 experiments. o-q, Characterization of pro-amniotic cavity (PAC) formation by confocal fluorescence imaging of ZO-1 (magenta), SOX2 (yellow), F-ACTIN (grey), and DAPI (cyan) (o), corresponding orthogonal views (p), and fluorescence intensity profiles (q), showing a PAC enclosed by SOX2+ EPI cells with apical F-ACTIN and ZO-1 enrichment. Scale bars, 50 μm. n = 5 ciToti5-EMs from 2 experiments. r,s, Apical-basal polarity of the PAC in ciToti5-EMs (r) and E5.0 NEs (s), stained for PODXL (magenta), F-ACTIN (yellow), and DAPI (cyan), with magnified views highlighting rosette-like apical organization. Scale bars, 30 μm. n = 23 ciToti5-EMs and 4 E5.0 NEs from 3 experiments. t-v, Immunofluorescence analysis of ciToti-EMs stained for SOX2 (yellow), GATA3 (magenta), and DAPI (cyan), shown as 3D reconstruction (t), orthogonal views (u), and intensity profiles (v), showing segregation of SOX2+ EPI and GATA3+ primitive extraembryonic ectoderm (pExE). Scale bars, 50 μm. n = 2 ciToti5-EMs and 2 ciToti6-EMs from 2 experiments. w, Immunofluorescence images comparing E5.0 NEs and ciToti-EMs for PODXL (magenta) and DAPI (green). Arrowheads highlight PODXL localization at apical surfaces of internal cavities and the outer surface of EMs and NEs. Scale bars, 50 μm. n = 23 ciToti5-EMs, 7 ciToti6-EMs, and 4 E5.0 NEs from 3 experiments.
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