Fig. 4. Ablation of MERVL impacts the expression of a subset of two-cell genes through preimplantation development.

a, Schematic of timing for minor and major ZGA (adapted from Abe et al.⁶). b, Representative images of EU incorporation assay (green) with Hoechst 33342 counterstain (gray) in control and MERVL-KD two-cell stage embryos, from three independent experiments. Scale bars, 20 µm. c, The relative intensity of EU per nucleus, normalized to Hoechst 33342 signal. NS, not significant by two-tailed unpaired t-tests. d, Bidimensional principal-component (PC) analysis of gene expression profiles in control and MERVL-KD embryos through preimplantation development. e, RNA-seq differential gene expression analysis: MERVL-KD versus control embryos obtained at two-cell, four-cell and eight-cell stages; 938, 745 and 277 genes evinced significant changes in expression in MERVL-KD embryos (blue circle, fold change ≥ |2|, P value adjusted (Padj) < 0.05, binomial test with Benjamini–Hochberg correction). f, Stacked bar chart shows the number of upregulated (up)- and downregulated (down)-DEGs. The populations of maternally inherited RNA (as defined in DBTMEE v2; refs. ^32,33) and others (zygotically expressed genes) are highlighted in red and blue. g, Predicted factors that are upstream of all up-DEGs upon MERVL-KD, assessed by the ChEA database³⁴. ChIP-seq, chromatin immunoprecipitation with sequencing; HaCaT, cultured human keratinocyte; MEF, mouse embryonic fibroblast; MESC, mouse embryonic stem cell; LNCaP, lymph node carcinoma of the prostate. h, Bubble plot showing overlap between all DEGs in MERVL-KD embryos with the list of two-cell (2C) genes and DBTMEE v2 transcriptome categories. The bubble plot sizes show the –log₁₀[P values] derived from a hypergeometric test. i, Track views show RNA-seq signals in control and MERVL-KD embryos, on two representative 2C gene loci (as defined in Macfarlan et al.¹²). The y-axis represents normalized tag counts for total RNA-seq in each sample. Refgene, refseq gene in NCBI and UCSC. j, Representative images of immunofluorescence staining for phosphorylated p53 on serine 15 (p-S15-p53, pink) with DAPI counterstain (gray) in control and ASO-mediated MERVL-KD embryos, from three independent experiments. Scale bars, 20 µm. k, Dot plots showing the relative intensity for p-S15-p53 per nucleus in control and MERVL-KD embryos, normalized to DAPI signal. Central bars represent medians, the top and bottom lines encompass 50% of the data points. *P < 0.05 and ***P < 0.001, two-tailed unpaired t-tests. Data for panels in c–h and k are available as source data.

Source data