Extended Data Fig. 10: Loss-of-function validation of potential TE and ICM regulators.
From: Genome-coverage single-cell histone modifications for embryo lineage tracing
(a) Bar charts showing the expression of target TFs in both NC and experimental groups of embryos based on single-embryo RNA-seq. P value was determined by two-side t-test. Error bars represent median values ± SEM. The embryo number for each group in 8 C is as below: NANOG KD (20), ZFX KD (15), YY2 KD (26), TCF12 KD (19), CEBPB KD (19), BBX KD (14), SMAD2 KD (15), HBP1 KD (16), CDX KD (12), SOX15 KD (14), MED1 KD (10), NC (19). The embryo number for each group in blastocyst is as below: HNF4A (19), KLF6 KD (10), ELF5 KD (20), HIF1A KD (13), and NC (15). (b) Stereomicroscopic representative images of embryos at the indicated time points with different siRNAs. Representative images out of three independent experiments are shown. Scale bar, 100 μm. hpf, hours post fertilization. (c) Immunofluorescence staining of mouse embryos at 108 hpf. Z-projection and single-section immunofluorescence images of non-target-, NANOG-, SMAD2-, HNF4A-, TCF12-, HBP1-, CDX2-, MED1- and ELF5-siRNA injected embryos, showing trophectoderm fate (CDX2, green), inner cell mass fate (SOX2, magenta), and DNA (DAPI, cyan). White arrowheads indicate the decrease in SOX2+ cells. Representative images out of three independent experiments are shown. Asterisks indicate the adjacent embryos. Scale bar, 100 μm. (d) UMAP showing individual embryos colored by specific knock-down (KD) TFs for 8 C embryos. The large colored dots indicate the median distribution of all embryos of each specific TF KD. (e-f) Violin plots showing the expression level of ICM signature genes (e), and TE signature genes (f) in 8-cell embryo RNA-seq data between non-targeting and TF KD groups. P value, two-sided Mann-Whitney test. Box plots center lines indicate the median, box limits indicate the first and third quartiles, and whiskers indicate 1.5× IQR. The embryo number for each group in 8 C is as below: NANOG KD (20), ZFX KD (15), YY2 KD (26), TCF12 KD (19), CEBPB KD (19), BBX KD (14), SMAD2 KD (15), HBP1 KD (16), CDX2 KD (12), SOX15 KD (14), and NC (19). (g) UMAP showing individual embryos colored by specific KD TFs for blastocyst embryo. The large colored dots indicate the median distribution of all embryos of each specific TF KD. (h-i) Violin plots showing the expression level of ICM signature genes (h), and TE signature genes (i) in blastocyst embryo RNA-seq data between non-targeting and TF KD groups. P value, two-sided Mann-Whitney test. Box plots center lines indicate the median, box limits indicate the first and third quartiles, and whiskers indicate 1.5× IQR. The embryo number for each group in blastocyst is as below: HNF4A (19), KLF6 KD (10), ELF5 KD (20), HIF1A KD (13), and NC (15). (j-k) Smoothed heatmap showing the SCENIC TF activity score for different groups in 8-cell (j) and blastocyst embryos(k) RNA-seq data.
