Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency

Abstract

The emergence of the first three lineages during development are orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system by introducing premature codon with cytosine base editor in bovine embryos with a robust efficiency. Of interest, SOX2 is universally localized in early blastocysts but gradually restricted into the inner cell mass in cattle. SOX2 knockout results in a failure of the establishment of pluripotency. Indeed, OCT4 level is significantly reduced and NANOG was barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. Single embryo RNA-seq reveals a dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 expression is sustained in the trophectoderm in absence of CDX2 in bovine late blastocysts. Overall, we propose that SOX2 is dispensable for OCT4 and NANOG expression and disappearance of SOX2 in the trophectoderm depends on CDX2 in cattle, which are all in sharp contrast with results in mice. SignificanceThe first and second cell fate decisions of a new life are important for subsequent embryonic and plancental development. These events are finely controlled by a network of transcriptional factors, which are extensively characterized in mice. Species-specific roles of these proteins are emerging in mammals. Here, we develop a gene loss-of-function system by using cytosine base editors in bovine embryos. We find that expression pattern, functional roles, and regulation of SOX2 are all different between mouse and bovine embryos. Remarkbly, SOX2 is required for OCT4 and NANOG, two well established pluripoteny genes. Furthermore, CDX2 is required to shut down SOX2 in the trophectoderm. Given similar expression pattern of SOX2 between human and bovine blastocysts, bovine embryos represents a putative model to investigate human pluripotency regulation in vivo.