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

Abstract

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.

Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig

“…Mammalian early embryonic development, from zygote to blastocyst, shows broad similarities among mammals, [15][16][17] but also reveals crucial species differences in the transcriptional and spatio-temporal regulation of lineage and pluripotency. 17 In mouse, as a key trophoblast (TE) transcription factor, CDX2 can repress the transcription of OCT4 by binding to the fourth conserved region of the distal OCT4 enhancer to form TE cells.…”

Transcriptional and epigenetic control of cell fate decisions in early embryos

“…In contrast with these observations in mice, NANOG protein in human, bovine and porcine embryos is first detected in late blastocysts (Kuijk et al 2008;Cauffman et al 2009;Khan et al 2012). In human, bovine and porcine embryos, GATA6 is expressed in cleavage stages and all cells of the blastocyst, therefore it is not a suitable markers for PE segregation (Kimber et al 2008;Roode et al 2012;Cao et al 2014;Wei et al 2017). Thus, if the equivalent NANOG and GATA6 antagonistic relationship exists during epiblast and PE segregation in non-rodent mammals, it must be controlled by NANOG; however, this has not been determined.…”

“…Similarly, in the porcine, FGFR1 and FGFR2 are not detected in the ICM, but are highly expressed in the late E11 epiblast and the TE (Hall et al 2009;Valdez Magaña et al 2014). Interestingly, FGFR4 is detected in the ICM of bovine and porcine embryos (Wei et al 2017; P. Ramos Ibeas and R. Alberio, unpubl. obs.…”

“…Based on the expression of key components of the TGFB/activin pathway, it is likely that this signalling pathway is operational in early ICM cells in nonrodent mammals. Recent single-cell transcriptome studies have shown expression of NODAL, TGFB1, and ACVR1 in human, Cynomolgus monkey, bovine and porcine embryos (Cao et al 2014;Blakeley et al 2015;Nakamura et al 2016;Petropoulos et al 2016;Wei et al 2017; P. Ramos Ibeas and R. Alberio, unpubl. obs.).…”

Gene expression analysis and in vitro production procedures for bovine preimplantation embryos: Past highlights, present concepts and future prospects

“…Information of candidate genes regulated by cytoplasmic polyadenylation during oocyte maturation has been provided (Reyes, Chitwood, & Ross, ). More recently, transcript profiling of single embryonic cells for a set of candidate genes has been performed for different stages from zygote to blastocyst (Negron‐Perez, Zhang, & Hansen, ; Wei et al., ), providing new insight into lineage specification events in bovine embryos (Lavagi et al., ).…”