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. 2020 Mar 12;9:e54756. doi: 10.7554/eLife.54756

Figure 1. Induction of RS increases the number of 2C-like cells in ESCs culture and activates the expression of 2C-specific genes in mouse embryos.

(a) Clustering of 1399 Drop-seq single-cell expression profiles into four cell populations. The plot shows a two-dimensional representation (t-SNE) of global gene expression relationship; cells are colored according to their cluster. Clusters were identified by shared nearest neighbor algorithm (see methods). (b) t-SNE plot showing Zscan4d expression level across all CNTL and APH-treated cells. (c) Heatmap showing the list of top 30 genes that are differentially expressed between cluster 4 cells (orange cluster in a) and the rest of the population (cluster 1,2, and 3). (d) Plot showing the scaled expression of 2C-specific markers and the percentage of cells expressing 2C-related genes in CNTL and APH-treated condition. Fisher's exact test was used to determine p-values. (e) FACS analysis on pZscan4-Emerald ESCs upon treatment with various RS-inducing agents. (f) Immunostaining of ESCs for ZSCAN4-Emerald, MERVL-GAG and canonical pluripotency marker POU5F1 upon treatment with APH (bar = 25 µm). (g–j) RT-qPCR analysis on blastocyst-stage embryos treated with APH for key 2C-like markers. Statistical significance compared to CNTL unless otherwise indicated. All bar-plots show mean with ± SD (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, one-way ANOVA).

Figure 1—source data 1. FACS, qPCR and Western quantification.
elife-54756-fig1-data1.xlsx (778.2KB, xlsx)

Figure 1.

Figure 1—figure supplement 1. Induction of RS increases the number of 2C-like cells in ESCs culture.

Figure 1—figure supplement 1.

(a) PCA plot on 1399 cells treated with or without APH. Cells are colored based on clusters. (b) Heatmap showing scaled expression values of top 30 genes and 100 ranked cells according to PC1. (c) Venn diagram showing the overlap of the top 100 DEGs of cluster four with those expressed in 2C-like cells (according to Eckersley-Maslin et al., 2016). (d) Plot showing the scaled expression of 2C-specific markers and the percentage of cells expressing 2C-related genes in the four clusters. (e) Immunoblot showing the expression of ZSCAN4 and p-CHK1 proteins upon treatment with APH at different time points. (f) FACS analysis for Emerald and CASPASE 3-Alexa 647 on pZscan4-Emerald ESCs upon treatment with increasing concentrations of APH. (g) β-galactosidase staining of H2O2-treated BJ fibroblast cells, untreated and APH-treated ESCs. For western blots quantification refer to Figure 1—source data 1.
Figure 1—figure supplement 2. Characterization of APH-induced 2C-like cells.

Figure 1—figure supplement 2.

(a–f) RT-qPCR analysis for 2C-like genes upon treatment with increasing concentrations of APH (g) FACS analysis on pMERVL-GFP ESCs upon treatment with APH. (h) FACS analysis showing the mean GFP signal intensity within pMERVL-GFP positive population in CNTL and APH-treated pMERVL-GFP ESCs. (i) RT-qPCR analysis for MERVL upon treatment with APH in two different MEF lines. (j) Immunostaining for 2C markers, MERVL-GAG and ZSCAN4 and ESC marker, POU5F1 in untreated and APH-treated ESCs, DAPI staining shows the lack of chromocenters in 2C-like cells (bar = 20 µm), (k–n) Immunoblot and RT-qPCR analysis of ESCs for ZSCAN4 and canonical pluripotency markers upon treatment with APH. Statistical significance compared to CNTL unless otherwise indicated. All bar plots show mean with ± SD (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, one-way ANOVA). For western blots quantification refer to Figure 1—source data 1.
Figure 1—figure supplement 3. Characterization of APH-induced 2C-like cells.

Figure 1—figure supplement 3.

(a) Cell cycle analysis of E14 and R1 ESCs upon treatment with low (0.3 μM) and high (6 μM) concentrations of APH. (b) Cell cycle analysis of E14 and R1 ESCs upon UV treatment. (c-e), (f-h) Two replicates of RT-qPCR analysis on blastocyst-stage embryos for key 2C-specific genes Dux, Gm4981 and MERVL. (i) Wide field microscope images of mouse embryos at blastocyst stage after APH treatment (bar = 25 µm) (j) Immunostaining of blastocyst-stage embryos for key ESCs marker, NANOG and the key TSC marker, CDX2 after APH treatment (bar = 50 µm). (k) Percentage of embryos developed to the blastocyst stage after APH treatment in three replicates. Statistical significance compared to CNTL unless otherwise indicated. All bar plots show mean with ± SD (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, one-way ANOVA).