Bovine lineage specification revealed by single-cell gene expression analysis from zygote to blastocyst.

Preimplantation embryos undergo zygotic genome activation and lineage specification resulting in three distinct cell types in the late blastocyst. The molecular mechanisms underlying this progress are largely unknown in bovines. Here, we sought to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the bovine blastocyst. Using a quantitative microfluidics approach in single cells, we analyzed mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency in parallel in 384 individual cells from bovine preimplantation embryos. The developmental transitions can be distinguished by distinctive gene expression profiles and we identified NOTCH1, expressed in early developmental stages, while T-box 3 (TBX3) and fibroblast growth factor receptor 4 (FGFR4), expressed in late developmental stages. Three lineages can be segregated in bovine expanded blastocysts based on the expression patterns of lineage-specific genes such as disabled homolog 2 (DAB2), caudal type homeobox 2 (CDX2), ATPase H+/K+ transporting non-gastric alpha2 subunit (ATP12A), keratin 8 (KRT8), and transcription factor AP-2 alpha (TFAP2A) for trophectoderm; GATA binding protein 6 (GATA6) and goosecoid homeobox (GSC) for primitive endoderm; and Nanog homeobox (NANOG), teratocarcinoma-derived growth factor 1 (TDGF1), and PR/SET domain 14 (PRDM14) for epiblast. Moreover, some lineage-specific genes were coexpressed in blastomeres from the morula. The commitment to trophectoderm and inner cell mass lineages in bovines occurs later than in the mouse, and KRT8 might be an earlier marker for bovine trophectoderm cells. We determined that TDGF1 and PRDM14 might play pivotal roles in the primitive endoderm and epiblast specification of bovine blastocysts. Our results shed light on early cell fate determination in bovine preimplantation embryos and offer theoretical support for deriving bovine embryonic stem cells.