Single-cell analysis of preimplantation embryonic development in guinea pigs

Abstract

Background: Guinea pigs exhibit numerous physiological similarities to humans, yet the details of their preimplantation embryonic development remain largely unexplored.

Results: To address this, we conducted single-cell sequencing on the transcriptomes of cells isolated from the zygote stage through preimplantation stages in guinea pigs. This study identified seven distinct cell types within guinea pig preimplantation embryos and pinpointed the timing of zygotic gene activation (ZGA). Trajectory analysis revealed a bifurcation into two lineage-specific branches, accompanied by alterations in specific pathways, including oxidative phosphorylation and vascular endothelial growth factor (VEGF). Additionally, co-expressed gene network analysis highlighted the most enriched functional modules for the epiblast (EPI), primitive endoderm (PrE), and inner cell mass (ICM). Finally, we compared the similarities and differences between human and guinea pig epiblasts (EPIs).

Conclusion: This study systematically constructs a cell atlas of guinea pig preimplantation embryonic development, offering fresh insights into mammalian embryonic development and providing alternative experimental models for studying human embryonic development.

Keywords: Early embryonic development; Guinea pigs; Single-cell sequencing.

Fig. 1

Preimplantation Embryo Sampling in Guinea Pigs (A) Embryo sampling conducted at different preimplantation stages. (B) Summary of the number of embryos and cells collected at each time point. (C) Representative bright-field microscopy images of in vivo acquired embryos. Scale bar: 50 μm

Fig. 2

Transcriptomic analysis of preimplantation embryos in guinea pigs (A) UMAP visualization of 551 single-cell transcriptomes from guinea pig preimplantation embryos. Points are colored by cell types. (B) Dot plot of selected marker genes for various cell types. (C) Visualization of the top 5 upregulated and downregulated genes in each cluster. (D) Heatmap showing the top 25 differentially expressed genes (DEGs) in each cluster. (E) UMAP plot of all cells with projected expression patterns of the following marker genes for each cluster: Zscan4 (8 C), Nanog2 (EPI), Cdx2, Gata3 (TE), and Sox17, Gata6 (PrE)

Fig. 3

The zygotic genome activation (ZGA) of guinea pigs (A) Percentage difference (Δ percent of cells) and log-fold change based on the Wilcoxon rank-sum test results for differential gene expression comparing 2 C versus zygote(Left), 4 C versus 2 C(Middle), and 8 C versus 4 C(Right). Red: upregulated genes; Blue: downregulated genes. (B) Volcano plots of differential gene expression comparing 4 C versus zygote (Left) and 8 C versus zygote (Right). (C) Gene Ontology (GO) circle plot displaying gene-annotation enrichment analysis. Red indicates upregulated gene-associated GO terms, relative to the z-score of the analysis. (D) KEGG Chord plot illustrating the relationship between the list of selected KEGG terms (upregulated genes in 8 C versus 4 C) and their corresponding genes, along with the log fold change (logFC) of the genes. The left half of the KEGG Chord plot displays whether the gene expression is upregulated, and the right half represents different KEGG terms with various colors

Fig. 4

Two rounds of lineage separation in guinea pig preimplantation embryos (A) UMAP plot showing the developmental trajectory across different sampling times. (B) UMAP plot illustrating the developmental trajectory of 8-cell stage (8 C), morula, trophectoderm (TE), inner cell mass (ICM), epiblast (EPI), and primitive endoderm (PrE) cells. (C) Bar plot representing the proportion of each cell type at different sampling times. (D) Heatmap displaying trends of differentially expressed genes as a function of inferred pseudotime. Genes in each row are grouped into six clusters based on their expression patterns. (E-G) These UMAP plots represent the distribution of pathway scores across different pseudotime points. The color gradient on the plot indicates the pathway scores, with dark blue representing low scores and light yellow representing high scores.

Fig. 5

Identification of co-expressed gene modules associated with ICM, EPI, and PrE (A) Schematic representation of the second cell fate decision for preimplantation embryos. (B) Module activities in different clusters estimated using the hdWGCNA algorithm. (C) Co-expression plots for modules M3 (EPI), M6 (ICM), and M13 (PrE). (D) Differential gene heatmap and KEGG enrichment analysis results for EPI, ICM, and PrE

Fig. 6

Comparison of preimplantation EPI in humans and guinea pigs (A) Three-dimensional Principal Component Analysis (PCA) of the EPI lineage, determined by the expressed genes among all groups of EPIs during development in humans (hu) and guinea pigs (GP). In total, 10,698 out of 13,915 annotated genes expressed in human and guinea pig cells (human, 106 cells; guinea pig, 315 cells) were used. (B) Heatmap of 952 genes that highly contributed to PC1 (greater than 0.8 standard deviation of PC1). (C) Violin plots showing expression levels of pluripotency genes during the EPI pluripotency transition in human embryos. (D) Violin plots showing expression levels of selected genes from (C) in guinea pig embryos