Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos.

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.

Zygotic Nuclear F-Actin Safeguards Embryonic Development.

After fertilization, sperm and oocyte nuclei are rapidly remodeled to form swollen pronuclei (PN) in mammalian zygotes, and the proper formation and function of PN are key to producing totipotent zygotes. However, how mature PN are formed has been unclear. We find that filamentous actin (F-actin) assembles in the PN of mouse zygotes and is required for fully functional PN. The perturbation of nuclear actin dynamics in zygotes results in the misregulation of genes related to genome integrity and abnormal development of mouse embryos. We show that nuclear F-actin ensures DNA damage repair, thus preventing the activation of a zygotic checkpoint. Furthermore, optogenetic control of cofilin nuclear localization reveals the dynamically regulated F-actin nucleoskeleton in zygotes, and its timely disassembly is needed for developmental progression. Nuclear F-actin is a hallmark of totipotent zygotic PN, and the temporal regulation of its polymerized state is necessary for normal embryonic development.