Nlrp2, a Maternal Effect Gene Required for Early Embryonic Development in the Mouse

Maternal effect genes encode proteins that are produced during oogenesis and play an essential role during early embryogenesis. Genetic ablation of such genes in oocytes can result in female subfertility or infertility. Here we report a newly identified maternal effect gene, Nlrp2, which plays a role in early embryogenesis in the mouse. Nlrp2 mRNAs and their proteins (∼118 KDa) are expressed in oocytes and granulosa cells during folliculogenesis. The transcripts show a striking decline in early preimplantation embryos before zygotic genome activation, but the proteins remain present through to the blastocyst stage. Immunogold electron microscopy revealed that the NLRP2 protein is located in the cytoplasm, nucleus and close to nuclear pores in the oocytes, as well as in the surrounding granulosa cells. Using RNA interference, we knocked down Nlrp2 transcription specifically in mouse germinal vesicle oocytes. The knockdown oocytes could progress through the metaphase of meiosis I and emit the first polar body. However, the development of parthenogenetic embryos derived from Nlrp2 knockdown oocytes mainly blocked at the 2-cell stage. The maternal depletion of Nlrp2 in zygotes led to early embryonic arrest. In addition, overexpression of Nlrp2 in zygotes appears to lead to normal development, but increases blastomere apoptosis in blastocysts. These results provide the first evidence that Nlrp2 is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse

Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2-/- embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7-21% form blastocysts compared to 76.2-83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages

Delivery of CRISPR-Cas9 into Mouse Zygotes by Electroporation.

The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including the mice. In this scheme, components of the CRISPR-Cas9 system are delivered into the mouse zygote and mutant mice carrying genetic modifications derived. Although microinjection has been the technology of choice, electroporation has also emerged and been proven to be effective delivering CRISPR-Cas9 reagents into the mouse zygote. Here, we describe the experimental protocol employing electroporation to deliver CRISPR-Cas9 reagents into mouse embryos and derive gene-edited mutant mice