Fig. 9: rRNA biogenesis is critically required at the two-cell-to-four-cell stage transition during pre-implantation embryo development.

a Expression of Pre-rRNA and 28S rRNA across different embryo developmental stages; N = 4 biologically independent experiments; Data are presented as mean values +/− SEM. SEM standard error of mean. b Expression of different subunit genes of RNA polymerase I across different embryo developmental stages using public pre-implantation mouse embryos RNA-seq data; Gene expression levels of two biological replicates in same developmental stage were averaged for plotting. GEO accession code GSE66390. c Different schemes of treatment with CX-5461. The 24 h time window for CX-5461 treatment is highlighted in red; Dpc days post-coitum. d Stacked bar plots showing fraction of embryos at different developmental stages with the different CX-5461 treatment schemes in Fig. 6c. The numbers of embryos of group A to group D were all 15 embryos. e qRT-PCR showing rRNA expression level in blastocysts, after CX-5461 treatment of morula embryos followed by in vitro culture of the treated embryos; 18S rRNA: p = 2.68E−06 (***), 28S rRNA: p = 1.98E−05 (***), Pre-rRNA: p = 2.17E−05 (***), two-way ANOVA; N = 3 biologically independent experiments; Data are presented as mean values +/− SEM. SEM standard error of mean. f qRT-PCR showing 2C marker gene expression level in blastocysts, after CX-5461 treatment of morula embryos followed by in vitro culture of the treated embryos. Dux: p = 4.09E−03(**), MERVL-pol: p = 6.82E−03(**), Zscan4: p = 2.56E−02(*), Gm12794: p = 2.82E−01, two-way ANOVA; N = 3 biologically independent experiments; Data are presented as mean values +/− SEM. SEM standard error of mean. g Violin plots demonstrating the expression level changes of stage-specific gene clusters of mouse pre-implantation embryos (as defined in Supplementary Fig. S1) in CX-5461-treated and control blastocyst embryos, C1 vs. All genes: p = 4.55E−09, C2 vs. all genes: p = 2.30E−14, two-sided, Mann–Whitney U-test, N = 2 biologically independent RNA-seq experiments. GEO accession code GSE166041. h Violin plots show expression levels of major ERV gene classes in control blastocyst embryos and CX-5461 treated blastocyst embryos; N denotes the number of sub-classes of ERV genes; MERVL: p = 5.11E−05(***), MERVL-MaLR: p = 1.65E−07(***), MERVK: p = 3.65E−07(***), MERV1: p = 3.67E−07(***), two-sided, Wilcox signed rank test; N = 2 biologically independent RNA-seq experiments. GEO accession code GSE166041. i Violin plots show expression levels of ERV gene sub-classes of MERVL-int and MT2_Mm in control blastocyst embryos and CX-5461 treated blastocyst embryos; seq_n denotes the number of annotated MERVL-int and MT2_Mm sequences; MERVL-int: p=2.65E-18(***), MT2_Mm: p = 8.51E−05(***), two-sided, Wilcox signed rank test, N = 2 biologically independent RNA-seq experiments. GEO accession code GSE166041. j Violin plots show expression levels of ERV gene sub-classes of GSAT_MM in control blastocyst embryos and CX-5461-treated blastocyst embryos; seq_n denotes the number of annotated GSAT_MM sequences; p = 3.01E−04(***), two-sided, Wilcox signed rank test, N = 2 biologically independent RNA-seq experiments. GEO accession code GSE166041. g–j The center line is the median, the bottom of the box is the 25th percentile boundary, the top of the box is the 75th, and the top and bottom of vertical line define the bounds of the data that are not considered outliers, with outliers defined as greater/lesser than ±1.5× IQR, where IQR inter-quartile range. Source data are provided as a Source Data file.