Abstract
Specification of embryonic lineages is an important question in the field of early development. Numerous studies analyzed the expression patterns of the candidate transcripts and proteins in humans and mice and clearly determined the markers of each lineage. To overcome the limitations of human and mouse embryos, the expression of the marker transcripts in each cell has been investigated using in vivo embryos in pigs. In vitro produced embryos are more accessible, can be rapidly processed with low cost. Therefore, we analyzed the characteristics of lineage markers and the effects of the DAB2 gene (trophectoderm marker) in in vitro fertilized porcine embryos. We investigated the expression levels of the marker genes during embryonic stages and distribution of the marker proteins was assayed in day 7 blastocysts. Then, the shRNA vectors were injected into the fertilized embryos and the differences in the marker transcripts were analyzed. Marker transcripts showed diverse patterns of expression, and each embryonic lineage could be identified with localization of marker proteins. In DAB2-shRNA vectors injected embryos, HNF4A and PDGFRA were upregulated. DAB2 protein level was lower in shRNA-injected embryos without significant differences. Our results will contribute to understanding of the mechanisms of embryonic lineage specification in pigs.
Highlights
Cells are continuously divided into diverse cell types starting from a single fertilized egg to the whole organism [1]
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 1A)
Summary
Cells are continuously divided into diverse cell types starting from a single fertilized egg to the whole organism [1]. Stage blastomeres have totipotency and select their fate between inner cell mass (ICM) and trophectoderm (TE) [2]. Cells in ICM separate into two lineages, epiblast (EPI) and primitive endoderm (Pri-Endo) [3]. Understanding this lineage specification provides clues to modulate the cell’s fate. Eggs of frog and zebrafish already have molecular gradation at fertilization, and the gradation determined the position of the axis of the embryo [5]. In the case of mice, heterogeneity is observed starting from the four-cell stage; this asymmetry induces cell fate decisions at the early stages [6]. Models of the blastocyst (BL) formation have been suggested and molecules and signals that regulate the lineage specialization have been identified [7,8]
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