Silencing of maternally expressed RNAs in Dlk1-Dio3 domain is critical for embryonic development

The mammalian imprinted Dlk1-Dio3 domain contains multiple lncRNAs, mRNAs, the largest miRNA cluster in the genome and four differentially methylated regions (DMRs), and deletion of maternal RNA within this locus results in embryonic lethality, but the mechanism by which this occurs is not clear. Here, we optimized the model of maternally expressed RNAs transcription termination in the domain and found that the cause of embryonic death was apoptosis in the embryo, particularly in the liver. We generated a mouse model of maternally expressed RNAs silencing in the Dlk1-Dio3 domain by inserting a 3×polyA termination sequence in Gtl2 locus. By analyzing mouse embryos RNA-Seq data combined with histological analysis, we found that silence of maternally expressed RNAs in the domain activated apoptosis, causing vascular rupture of fetal liver, resulting hemorrhage and injury. Mechanistically, termination of Gtl2 transcription results in the silencing of the maternally expressed RNAs and activation of the paternally expressed genes in the interval, and it is the gene itself rather than the IG-DMR and Gtl2-DMR that causes the above phenotypes. In conclusion, these findings illuminate a novel mechanism by which silencing of the maternally expressed RNAs within Dlk1-Dio3 domain leads to hepatic hemorrhage and embryonic death through activation of the apoptosis. We used Easi-CRISPR technology to insert three polyadenylation cassettes into the promoter of Gtl2 in the Dlk1-Dio3 imprinted domain to generate Gtl2 polyA knock-in mouse model. At embryonic day 12.5 (E12.5), the embryos of paternally Gtl2 polyA knock-in (PKI), maternally Gtl2 polyA knock-in (MKI), homozygous (HOMO) and wild-type (WT) mice were selected to do the RNA-sequencing.