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

• Published in: Molecular reproduction and development Vol. 87; no. 1; pp. 152 - 160
• Main Authors: Ortega, M. Sofia, Kelleher, Andrew M., O'Neil, Eleanore, Benne, Joshua, Cecil, Raissa, Spencer, Thomas E.
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.01.2020
• Subjects: Animals; Cattle; CDX2 protein; Cell differentiation; Cell fate; Cell Lineage - genetics; Clonal deletion; CRISPR; CRISPR-Cas Systems; Embryo, Mammalian - metabolism; Embryos; Exons; Female; Fertilization in Vitro - methods; GATA6 Transcription Factor - metabolism; Gene Expression Regulation, Developmental; Genotype; Germ Layers - metabolism; Nanog Homeobox Protein - genetics; Nanog Homeobox Protein - metabolism; Pluripotency; preimplantation development; RNA, Guide, CRISPR-Cas Systems; Transcription factors; Trophectoderm; Zygote - metabolism; preimplantation development; transcription factors; pluripotency
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/mrd.23304

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. Deletion of NANOG does not impair compaction and formation of the blastocoel, but impairs formation of a proper epiblast in the bovine embryo. In addition, when NANOG is suppressed other pluripotency markers are reduced indicating that NANOG is required for pluripotency maintenance in the bovine.