Extended Data Fig. 7: Knockdown of m6A writers in early embryos affects the decay of 2 C mRNAs.
a, Schematic diagram of knockdown assay. b, Expression of Mettl3/14/16 and Kiaa1429 during early development. c, Knockdown efficiency measured by RT-qPCR. NC, non-target. KD, writer knockdown. Data shown represent 3 independent experiments for Mettl3 and Mettl14 KD and 2 independent experiments for Mettl16 KD; value for each biological replicate represents the mean of two or three technical replicates (shown in source data). d, Development rate of NC and KD embryos. Data are presented as mean values from two biological replicates (n = 30 each). e, Morphology of NC and KD blastocysts. Scare bar = 100 μm. f, Expression change ratio of ZGA m6A + (n = 756) and m6A- (n = 359) transcripts upon KD at middle-2-cell and morula stages. g and h, DEGs between NC and writer KD groups in middle 2-cell (g) and morula (h) stages. DEG number are indicated. Representative 2 C genes are labeled. i, Heatmap of expression (left), and m6A status (right) of DEGs in WT oocytes and embryos. j and k, Expression of Zscan4 (j) and MERVL (k) in NC and KD embryos tested by qPCR. Data are presented as mean values from two biological replicates, with 2 technical replicates for each. l, MERVL level in NC and KD embryos at middle-2-cell and morula stages. m, The decay of Zscan4 and MERVL after Actinomycin D treatment. Data are presented as mean values from two technical replicates of a single experiment. No biological replicates due to material limitation. n, The abundance of H3K9me3 at MERVL and Zscan4 genomic locus in NC and KD morula. Data are presented as mean values from two biological replicates with 2 technical replicates for each. o, The change of METTL3 protein in different KD embryos measured by immunofluorescence. Data are presented as mean values ± s.e.m. n = 10, 14, 10, 22, 16 and 10 biologically independent embryos. P-values are calculated by one-tailed unpair Student’s t test, showed above the bars. Data in f-l represent results from two biological replicates. Box plots in f,l are presented with horizontal line, median; box, IQR; whiskers, most extreme value within ±1.5 × IQR. Two-tailed unpaired Wilcoxon test was used to calculate the p-value.
