Lineage specification revealed by single-cell gene expression analysis in porcine preimplantation embryos†

Abstract

After zygotic genome activation and lineage specification, zygotes develop into late blastocysts comprising three distinct cell types. The molecular mechanisms underlying this progress are largely unknown in pigs. Here, we intended to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the porcine blastocysts. Using a quantitative microfluidics approach in single cells, we detected mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency simultaneously in 480 individual cells derived from porcine preimplantation embryos. The developmental transitions can be distinguished based on distinctive gene expression profiles, and we identified paired box 6 (PAX6) and aquaporin 3 (AQP3) expressed in early and late developmental stages, respectively. Two lineages can be segregated in porcine early and late blastocysts by the expression patterns of lineage-specific genes such as DAB2, clathrin adaptor protein (DAB2) for trophectoderm (TE), platelet derived growth factor receptor alpha (PDGFRA), Nanog homeobox (NANOG), fibronectin 1 (FN1), hepatocyte nuclear factor 4 alpha (HNF4A), goosecoid homeobox (GSC), nuclear receptor subfamily 5 group A member 2 (NR5A2), and lysine acetyltransferase 6A (KAT6A; previously known as MYST3) for inner cell mass (ICM). However, the epiblast and primitive endoderm cannot be identified in late blastocysts, and those TE or ICM lineage-specific genes were low expressed in blastomeres from the morula. Our results shed light on early cell fate determination in porcine preimplantation embryos and offer theoretical support for deriving porcine embryonic stem cells.

Identification of the Lineage Markers and Inhibition of DAB2 in In Vitro Fertilized Porcine Embryos

“…Recent studies of porcine embryo had verified a number of common markers and discovered pig-specific lineage markers [ 20 , 21 , 22 ]. We have identified the trends of the marker gene expression in the porcine embryos ( Figure 1 A).…”

“…In porcine in vivo embryo, lineage-specific characteristics were identified by single-cell analysis. Using single cell qPCR, stage- and lineage-specific genes were identified, and ICM/TE cells were sorted according to the patterns of the marker gene expression [ 20 ]. Cells were categorized into lineage groups based on the single cell RNA-seq, and it has been demonstrated that certain conventional lineage markers (e.g., CDX2 and TEAD4 ) do not correspond to markers detected in porcine embryos [ 21 , 22 ].…”

“…In cattle, CDX2 is required for TE maintenance but does not repress OCT4 [ 28 ]. However, in pigs, single cell analysis based on qPCR and RNA-seq demonstrated that the expression levels of CDX2 had no differences between the ICM and TE cells, whereas the DAB2 gene was abundantly expressed in the TE cells [ 20 , 21 ]. The role of the DAB2 gene has to be investigated to understand the lineage specialization of the porcine embryo.…”

Embryo gene expression in pig pregnancy

“…Furthermore, recent studies in cloned embryos showed the dependence of pluripotency‐related and apoptosis gene expression on epigenetic transformations (Samiec, Romanek, Lipiński, & Opiela, ). The single‐cell expression analysis technique pluripotency‐related genes in pig embryos, such as paired box 6 ( PAX6 ) and aquaporin 3 ( AQP3 ), and, in late blastocysts, clathrin adaptor protein ( DAB2 ), platelet‐derived growth factor receptor alpha ( PDGFRA ), fibronectin 1 ( FN1 ), hepatocyte nuclear factor 4 alpha ( HNF4A ), goosecoid homeobox ( GSC ), nuclear receptor subfamily 5 group A member 2 ( NR5A2 ) and lysine acetyltransferase 6A ( KAT6A ) (Wei et al, ). However, the underlying factors involved in pluripotency and its regulation require further study.…”

METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development

“…The relative levels of SOX2 and CDX2 mRNA were detected by single-cell qPCR. Then, data were further analyzed by principal component analysis (PCA) using SIMCA14.1 software [29]. Blastomeres from TE and ICM were identi ed according to the clustering analyses.…”