Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency (2020)
Figure 7

Figure 7

5C state is similar to the epiblast from E4.5/5.5 embryos

(A-B) Energy metabolism was analyzed with 5C-Oct4GFP+, R1 ESCs (naïve PSCs), and R1 ESCs-primed (primed PSCs) with Seahorse instrument.

(C) Expression of epithelial, mesenchymal, glycolysis and oxidative phosphorylation (OXPHOS) markers (listed in Dataset EV4) was summarized in R1 ESCs, 5C-Oct4GFP+ (naïve), 5C-Oct4GFP+, 5C-Oct4GFP+ (primed), and R1 ESCs-primed, based on the current RNA-Seq. For each marker, Log2 values of FKPM were normalized by subtracting the average of five groups. Log2 values of FKPM of each group of markers were then averaged and plotted. 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) were 5C-Oct4GFP+ cultured in naïve and primed medium, respectively, for three days.

(D) Genes with higher expression at different developmental stages were selected out from previously reported RNA-seq (Data ref: Bertone, 2015). "Mor", "ICM", "pre-EPI" and "post-EPI" represented genes that have high expression only in morula, inner cell mass, pre-implantation epiblast and post-implantation epiblast, respectively. "Mor+ICM", "ICM+pre", and "EPI" indicated genes that have high expression in both morula and inner cell mass, in both inner cell mass and pre-implantation epiblast, and in both pre-implantation and post-implantation epiblast, respectively. The results of previously reported RNA-seq were summarized in the left panel, while the expression of these genes in current RNA-seq was summarized in the right panel. Log2 values of FKPM were presented similarly as in (C).

(E) Markers of 5C, naïve and primed states were selected based on the current RNA-Seq (Dataset EV4). The expression of these markers in R1 ESCs, 5C-Oct4GFP+, and R1 ESCs-primed was listed together with the GO enrichment of each group of markers. Log2 values of FKPM were presented similarly as in (C).

(F) Markers of 5C, naïve and primed states (Dataset EV4) were used to analyze expression profiles of epiblast during the early development of mouse embryos (Data ref: Mohammed et al, 2017a) (F). Single-cell was plotted based on the average expression of each group of markers.

(G) The average expression of both naïve and primed markers was calculated based on GSE100597. The top-2 cells from E4.5 epiblast and top-5 cells from E5.5 epiblast were selected. The expression of several naïve markers (Dppa3, Esrrb, Klf4, Klf5, Tbx3, and Zfp42) and several mesenchymal markers (Fgf5, Lef1, Nodal, and Pou3f1) in these selected cells were listed (upper part). The average expression of these genes in E3.5, 4.5, 5.5 and 6.5 epiblast were also listed (lower part). These results were all normalized against the average expression of these genes in all tested cells.

(H) The expression of representative markers of 5C, naïve and primed states was summarized in E3.5, 4.5, 5.5 and 6.5 epiblast.

(I-K) Cells from E5.5 epiblast were separated into 8 groups (I-VIII) basing on the average expression of epithelial, mesenchymal, and glycolysis markers (H). The average expression of three groups of markers were summarized in (J). The expression of six important markers of 5C state was summarized in these eight groups of cells (K)

(L) Schematic illustration of different pluripotent states and developmental stages.

Data information: Experiments were independently repeated at least five times (n≥5) except sequencing. Error bars represent standard deviations. Additional statistical information was listed in Dataset EV7.