Single-cell gene expression of the bovine blastocyst

Abstract

The first two differentiation events in the embryo result in three cell types – epiblast, trophectoderm (TE), and hypoblast. The purpose here was to identify molecular markers for each cell type in the bovine and evaluate differences in gene expression among individual cells of each lineage. The cDNA from 67 individual cells of dissociated blastocysts was used to determine transcript abundance for 93 genes implicated as cell lineage markers in other species or potentially involved in developmental processes. Clustering analysis indicated that the cells belonged to two major populations (clades A and B) with two subpopulations of clade A and four of clade B. Use of lineage-specific markers from other species indicated that the two subpopulations of clade A represented epiblast and hypoblast, respectively while the four subpopulations of clade B were TE. Among the genes upregulated in epiblast were AJAP1, DNMT3A, FGF4, H2AFZ, KDM2B, NANOG, POU5F1, SAV1 and SLIT2. Genes overexpressed in hypoblast included ALPL, FGFR2, FN1, GATA6, GJA1, HDAC1, MBNL3, PDGFRA and SOX17 while genes overexpressed in all four TE populations were ACTA2, CDX2, CYP11A1, GATA2, GATA3, IFNT, KRT8, RAC1 and SFN. The subpopulations of TE varied amongst each other for multiple genes including the prototypical TE marker IFNT. New markers for each cell type in the bovine blastocyst were identified. Results also indicate heterogeneity in gene expression among TE cells. Further studies are needed to confirm whether subpopulations of TE cells represent different stages in development of a committed TE phenotype.

OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

“…Overall, the read count of NANOG transcripts was relatively low; NANOG-specific reads were detected in one of three NT Ctrl blastocysts and in two of three IVP Ctrl blastocysts, but in none of the OCT4 KO blastocysts. There were no significant differences between the three embryo groups in the transcript levels of GATA6 or of SOX17, another HB-specific gene (22).…”

“…1E). To examine if genes specific to EPI, HB, or TE were affected by loss of OCT4, we compared the DATs in OCT4 KO embryos to sets of genes reported to be lineage specific in mouse, human, or bovine embryos (4,6,17,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Only genes with different transcript abundance in both OCT4KO tm1 vs. IVP Ctrl and OCT4KO tm1 vs. NT Ctrl, but not in NT Ctrl vs. IVP Ctrl, were considered to be affected by the loss of OCT4.…”

Adhesion molecules in gamete transport, fertilization, early embryonic development, and implantation—role in establishing a pregnancy in cattle: A review

“…The trophectoderm of bovine embryos has a polarized epithelium that develops into mononucleated and binucleate cells (Barcroft et al, 1998; Igwebuike, 2006; Negron‐Perez, 2017; Wooding, 1982; Wooding & Wathes, 1980). Bovine embryos express E‐cadherin and β‐catenin during binucleate cell differentiation (Barcroft et al, 1998; Nakano et al, 2002; Nakano, Shimada, Imai, Takahashi, & Hashizume, 2005).…”

“…Bovine embryos express E‐cadherin and β‐catenin during binucleate cell differentiation (Barcroft et al, 1998; Nakano et al, 2002; Nakano, Shimada, Imai, Takahashi, & Hashizume, 2005). However, it has yet to be conclusively shown that the cadherin–catenin system is obligatory for this differentiation to take place in cattle embryos (Negron‐Perez, 2017). The outgrowth of hatched bovine blastocysts was studied in cultures with the extracellular matrix proteins fibronectin, laminin, and vitronectin.…”

Consequences of assisted reproductive techniques on the embryonic epigenome in cattle

“…For example, at the blastocyst stage of development, a bovine embryo possesses at least three cell types (i.e. epiblast, hypoblast and trophectoderm), each with its own epigenetic program that was acquired as a result of factors driving its specification and differentiation (Negrón-Perez et al 2017). Therefore, by pooling material for analyses, we are making conclusions on averages, sometimes of multiple cell types, rather than determining absolute cellular states.…”