NANOG is required to form the epiblast and maintain pluripotency in the bovine embryo

Abstract

During preimplantation development, the embryo undergoes two consecutive lineages specifications. The first cell fate decision determines which cells give rise to the trophectoderm (TE) and the inner cell mass (ICM). Subsequently, the ICM differentiates into hypoblast and epiblast, the latter giving rise to the embryo proper.The transcription factors that govern these cell fate decisions have been extensively studied in the mouse, but are still poorly understood in other mammalian species. In the present study, the role of NANOG in the formation of the epiblast and maintenance of pluripotency in the bovine embryo was investigated. Using a CRISPR-Cas9 approach, guide RNAs were designed to target exon 2, resulting in a functional deletion of bovine NANOG at the zygote stage. Disruption of NANOG resulted in the embryos that form a blastocoel and an ICM composed of hypoblast cells.Furthermore, NANOG-null embryos showed lower expression of epiblast cell markers SOX2 and HA2AFZ, and hypoblast marker GATA6; without affecting the expression of TE markers CDX2 and KRT8. Results indicate that NANOG, has no apparent role in segregation or maintenance of the TE, but it is required to derive and maintain the pluripotent epiblast and during the second lineage commitment in the bovine embryo. K E Y W O R D S pluripotency, preimplantation development, transcription factors

Histone Demethylase KDM4D Could Improve the Developmental Competence of Buffalo (Bubalus Bubalis) Somatic Cell Nuclear Transfer (SCNT) Embryos

“…NANOG is another essential gene correlated with embryo developmental capacity. Former investigations revealed that in the inner cell mass of the blastocyst, NANOG -deficient might lead to failure of epiblast generation and embryo implantation in bovine (Ortega et al, 2019). At the same time, we also found that the total cell number of blastocysts was significantly increased after the microinjection of KDM4D mRNA compared with the nontreated group, and the opposite results were obtained when inhibitor KDM4D-IN-1 was used.…”

Pluripotency and Growth Factors in Early Embryonic Development of Mammals: A Comparative Approach

“…Ozawa et al [ 24 ] analyzed gene expression between the ICM and the TE in bovine embryos, and showed that Nanog and SOX2 presented similar expression patterns in bovine embryos obtained from mice and humans. Nanog expression was higher in ICM than TE in bovine expression, while Oct4 expression was similar, and Nanog is necessary for the expression of SOX2 (marker of EPI cells), GATA6 (marker of HP cells), and CDX2 [ 25 ]. Oct4 , Nanog , and SOX2 expression in bovine embryos is regulated by the exogen bone morphogenetic protein 5 (BMP5) [ 26 ].…”

Truncation of IFT80 causes early embryonic loss in cattle

“…At the end of fertilization, putative zygotes (oocytes exposed to sperm) were denuded from the surrounding cumulus cells and split in two groups: injected only with CAS9 mRNA and injected with gRNA/CAS9 mRNA against IFT80. During the injection procedure zygotes were maintained manipulation medium 22 , and then transferred to four-well dishes in groups of up to 50 zygotes in 500 µl of SOF-BE2 23 , covered with 300 µl of mineral oil per well at 38.5 °C in a humidified atmosphere of 5% (v/v) O2 and 5% (v/v) CO2. Percentage of putative zygotes that cleaved was determined at day 3 of development (day 0 = day of insemination) and blastocyst rate was estimated at day 8 of development.…”

“…The PCR products were 639-and 620 bp for exon 2 (Supplementary Figure 3) and 11 (Supplementary Figure 4), respectively. The PCR reaction, amplification (annealing temperature 53.4 °C), and visualization of bands was performed as previously described 22 . Editing efficiency was calculated from 3 separate in vitro embryo production runs, with 100 embryos per treatment group per replicate.…”