Fig. 4: Nr1h2 activation enhances blastoid implantation and multi-species blastocyst development.

a In vivo approaches depicted to examine Nr1h2 activation in blastoid implantation and blastocyst development. Created in BioRender.com. b Immunofluorescence for markers of the TE (Cdx2), ICM/EPI (Oct4) and PE (Gata6) in NrESC blastoid derived from 16th passage NrESC. Scale bar, 50 µm. 5 independent experiments were repeated with similar results. c A Umap plot showing the clustering of cells from Li et al. EPSblastoid, EPSC blastoids (current study), E4.5 blastocyst (GSE159883), and NrESC blastoids single-cell transctipome. d Umap plot showing Gata2 expression in clustered TE lineage cells from (c). e Polar bar chart displaying the percentage of Krt8/Krt18/Gata2/Tagln2 expressing cells in TE lineage for different blastoids or blastocysts. f Representative brightfield images showing the decidua formation in three mouse uteri at E10.5, as well as 7 days after NrESC-blastoid or EPSC-blastoid transfer at 3.5dpc. Red asterisks indicate deciduae. Scale bar, 1 cm. g Quantification of the decidua formation efficiency from the NrESC- or EPS-derived blastoids at 10.5dpc. Two-tailed Welch’s t-test. Data are represented as mean ± s.d.; n = 9 independent assays. h Quantification of the decidua length induced by E10.5 natural embryo, NrESC- or EPS-derived blastoid 10.5dpc. One-way ANOVA with Bonferroni correction for multiple comparisons. Data are represented as mean ± s.d.; n = 23 E10.5 natural embryo, n = 13 NrESC-derived blastoid 10.5dpc, n = 3 EPS-derived blastoid 10.5dpc. i Left, maximum projection image of immunofluorescence for GFP and TE marker (Cdx2) in chimeric E4.5 blastocysts microinjected with psinGFP NrESC. Right, single confocal plane insets showing Cdx2-GFP double positive cell and GFP single positive cells. Scale bar, 20 µm. Quantification of the developmental rate of mouse blastocysts after supplementing T09 in culture media from one-cell stage (j) or microinjection of GFP or Nr1h2 mRNA into one-cell embryo (k). Two-tailed Welch’s t-test. Data are represented as mean ± s.d.; n = 5 independent assays. l Quantification of the developmental rate of porcine SCNT blastocysts with or without T09 in culture media from one-cell stage. Two-tailed Welch’s t-test. Data are represented as mean ± s.d.; n = 3 independent assays. m Representative images of porcine SCNT morula and blastocyst in T09-treated and control group post-SCNT. Scale bar, 50 µm. 3 independent experiments were repeated with similar results. Source data are provided as a Source Data file.