Duxbl-mediated suppression of ZGA drives totipotency to pluripotency transition in mice III

In mammalian embryos, Dux transcription factors (TF) drive a cleavage stage-specific transcriptional burst associated with zygotic genome activation (ZGA) during which hundreds of genes and endogenous retroviral (ERV) elements are transiently expressed. In mice, ZGA begins in late 1-cell (1C) embryos and is shut down within hours at the late 2-cell (2C) to 4-cell (4C) stage. While this transition is accompanied by the loss of totipotency, it is not clear whether shutdown of Dux-induced ZGA is required for this process and how ZGA is terminated. Here, we reveal an essential negative feedback axis by which the Dux family member Duxbl terminates Dux-induced ZGA from the mid 2C stage onward by direct suppression of genomic Dux target loci. Consequently, Duxbl inactivation results in sustained expression of Dux-induced ZGA leading to 4C arrest. Our study reveals the Dux/Duxbl axis that determines the timing of totipotency exit enabling the very first divergence of cell fates. Three week-old C57BL6/J female mice were superovulated by intraperitoneal injection of pregnant mare serum gonadotropin (10 IU). 48 h later, human chorion gonadotropin (HCG) (10 IU) was delivered i.p. followed by mating with C57BL6/J males. Next morning, zygotes were surigically isolated from ampulla of the oviducts and brought into culture in EmbryoMax® Advanced KSOM Embryo Medium (Sigma-Aldrich, MR-101-D) at 37 °C and 5 % CO2. WT embryos were collected for RNA-seq at early 2-cell stage (32 h post HCG) and late 2-cell stage (48 h post HCG). Embryos obtained from crossing heterozygous Duxbl-sgKO mice were harvested for RNA-seq at late 2-cell stage (48 h post HCG).