Argonaute 2 Is Required for Extra-embryonic Endoderm Differentiation of Mouse Embryonic Stem Cells

Abstract

In mouse, although four Argonaute (AGO) proteins with partly overlapping functions in small-RNA pathways exist, only Ago2 deficiency causes embryonic lethality. To investigate the role of AGO2 during mouse early development, we generated Ago2-deficient mouse embryonic stem cells (mESCs) and performed a detailed characterization of their differentiation potential. Ago2 disruption caused a global reduction of microRNAs, which resulted in the misregulation of only a limited number of transcripts. We demonstrated, both in vivo and in vitro, that AGO2 is dispensable for the embryonic germ-layer formation. However, Ago2-deficient mESCs showed a specific defect during conversion into extra-embryonic endoderm cells. We proved that this defect is cell autonomous and can be rescued by both a catalytically active and an inactive Ago2, but not by Ago2 deprived of its RNA binding capacity or by Ago1 overexpression. Overall, our results suggest a role for AGO2 in stem cell differentiation.

Keywords: Argonaute 2; XEN cells; differentiation; extra-embryonic endoderm; miRNA; mouse embryonic stem cells; transcriptomics.

Graphical abstract

Figure 1

Characterization of Ago2 Knockout mESCs and Impact on Gene Expression (A) Top: schematic representation of the CRISPR/Cas9-mediated Ago2_KO strategy. Bottom: representative pictures of the wild-type (WT) and Ago2_KO mESC clones. Scale bars, 50 μm. (B) Relative expression of Ago2 mRNA in WT, Ago2_KO1, and Ago2_KO2 mESCs, measured by RT-qPCR. The error bars show the SD of three independent experiments. (C) Western blot analysis of AGO2 and OCT4 protein levels in WT and Ago2_KO mESCs. TUBULIN is used as loading control. (D) Relative expression of Oct4, Nanog, and Sox2 mRNAs in WT and Ago2_KO cells, measured by RT-qPCR. The error bars show the SD of three independent experiments. (E) Cell-cycle analysis of WT, Ago2_KO1, Ago2_KO2, Ago1_KO1, and Ago1_KO2. Stacked bars represent the percentage of cells in G2/M, S, or G1 phase of the cell cycle. The values were obtained from three independent experiments. (F) Boxplots depicting the global levels of miRNAs in WT, Ago2_KO, Ago1_KO, and Dicer_KO mESCs measured by small-RNA-seq. The normalized miRNA reads are represented as the mean of two replicates (except for Dicer_KO) in counts per 10 million (CPTM). The significance (^∗∗∗∗p < 0.0001) was assessed using non-parametrical Mann-Whitney ranked test. The fold change (FC) relative to WT cells was calculated by comparing the mean miRNA levels in each condition. (G) Scatterplot representing the miRNA levels in CPTM after AGO1 and AGO2 immunoprecipitation compared with WT mESCs. AGO2- and AGO1-loaded miRNAs in WT cells are represented in blue and green. The AGO1-loaded miRNAs in Ago2_KO mESCs from groups 1 and 2 are represented in red. Group 2 defines miRNAs 2-fold less enriched in Ago2_KO compared with WT mESCs. The remaining miRNAs are defined as group 1. Four miRNAs from group 2, validated by northern blot (Figure S1E), are highlighted. (H) Volcano plots representing the differentially expressed genes in Ago2_KO (left) and Dicer_KO (right) cells compared with WT mESCs. RNA-seq data represent the mean expression of two independent biological replicates. Significantly (fold change >2 and adjusted p value <0.01) up- and downregulated genes are highlighted in red and blue, respectively. See also Figure S1.

Figure 2

Embryoid Body Differentiation of Ago2_KO mESCs (A) Schematic representation of the EB differentiation protocol. LIF, leukemia inhibitory factor. (B) Western blot showing the expression of AGO2, FOXA2, OCT4, GATA6, and NANOG proteins after 0, 2, 4, 6, and 10 days of EB differentiation of WT and Ago2_KO1 mESCs. TUBULIN is shown as loading control. (C) Relative gene expression of ectoderm, mesoderm, and endoderm markers in WT, Ago2_KO1, and Ago2_KO2 mESCs during EB differentiation measured by RT-qPCR. The levels at day 6 (d6) and day 10 (d10) are represented relative to the levels in WT cells at day 0 (d0). Error bars represent the SD of three biological replicates. The dashed line shows levels at d0. (D) Immunofluorescence on EB sections after 10 days of EB differentiation of WT, Ago2_KO1, and Ago2_KO2 cells. The fluorescence signal of three different sections is shown for each of the proteins: NANOG, GATA6, SOX17, GATA4, and DAB2. The nuclei are stained with Hoechst 33342. Scale bar, 50 μm. See also Figure S2.

Figure 3

Generation and Analysis of Chimeric Mice (A) SSLP PCR genotyping on DNA from tissues derived from WT and Ago2_KO chimeras. The contribution of the cells to the tissues was assessed following D19mit19 and D18mit184 microsatellite length. mESCs (129/Ola strain) were injected into recipient blastocysts (C57BL/6 strain). DNA from 129/Ola and C57BL/6 mouse strains was used as control. The pictures of the tested chimeras with various degrees of coat chimerism are presented on the left side. (B) Immunofluorescence analysis of sections of pancreas extracted from adult Ago2_KO-GFP and WT-GFP chimeras. Sections from WT C57BL/6N are used as GFP-negative controls. As previously, WT and Ago2_KO2 mESCs expressing GFP were injected into recipient C57BL/6 blastocysts. Representative epifluorescence pictures show GFP-expressing cells, insulin-expressing pancreatic beta islets, and amylase-expressing acinar cells in non-fixed sections. Some co-expressing cells are indicated with a white arrow. Nuclei are stained with DAPI. (C) Flow-cytometry analysis of GFP and FOXA2 definitive endoderm marker expression in pancreatic cells derived from WT-GFP and Ago2_KO-GFP chimeras. Pancreatic cells from WT C57BL/6 mice are used as unstained control. See also Figure S3.

Figure 4

cXEN Conversion of WT, Ago2_KO, and Ago1_KO Cells (A) Schematic representation of the cXEN conversion protocol. Representative pictures of WT, Ago2_KO1, Ago2_KO2, Ago1_KO1, and Ago1_KO2 cells after 5 days of cXEN differentiation. cXEN colonies are indicated by white dashed lines. Scale bars, 50 μm. (B) Relative gene expression of ExEn markers in WT, Ago1_KO, and Ago2_KO mESCs before (ESC) and after conversion (cXEN), measured by RT-qPCR. The error bars represent the SD of three biological replicates. The significance was assessed using a two-way ANOVA followed by Dunnett's multiple comparison for the set of ExEn markers in Ago1_KO and Ago2_KO mutants compared with the WT condition after cXEN conversion. The calculated p values are WT versus Ago1_KO1 (p = 0.1655, ns), WT versus Ago1_KO1 (p = 0.1270, ns), WT versus Ago2_KO1 (^∗∗p = 0.0061), and WT versus Ago2_KO2 (^∗p = 0.0417). (C) Expression of the AGO1, AGO2, GATA6, NANOG, and OCT4 proteins before (ESC) and after (cXEN) conversion of WT, Ago1_KO1, and Ago2_KO1 cells by western blot. TUBULIN is shown as loading control for the two membranes used for blotting. (D) Western blot analysis of GATA6 and NANOG levels at several time points during cXEN conversion of WT and Ago2_KO1 cells. Coomassie staining (CM) of the membrane is shown as loading control. (E) Relative expression of ExEn markers during time series of cXEN conversion of Ago2_KO and WT cells, measured by RT-qPCR. The error bar represents the range of three independent experiments. For each gene the statistical significance of the variation caused by the absence of Ago2 over the time of differentiation (two factors) was assessed using a two-way ANOVA. The p values obtained for each gene are p < 0.0001. (F) ChIP-qPCR experiments on Gata6, Gata4, Sox17, and Foxa2 promoters before and after cXEN conversion of WT (blue/light blue) and Ago2_KO (red/light red) cells. Antibodies against SUZ12, RING1B, H3K27me3, and H3K27ac were used. The enrichment was calculated compared with a repressed (Cdx2) and an active (Smad3) promoter. An intergenic region was used as negative control. The error bars correspond to the SD of three independent experiments. See also Figure S4.

Figure 5

The Transcriptome of Aberrantly Converted Ago2_KO mESCs Is Distinct from that of WT cXEN (A) Volcano plot of differentially expressed miRNAs in Ago2_KO compared with WT cXENs. Data are represented as the mean expression of two independent biological replicates. Significantly (2-fold change and adjusted p value < 0.01) up- and downregulated genes are highlighted in red and blue, respectively. (B–D) Volcano plot representing the differentially expressed genes in Ago2_KO cells compared with WT cells after cXEN conversion (B). Data are represented as the mean expression of two independent biological replicates. Significantly (2-fold change and adjusted p value <0.01) up- and downregulated genes are highlighted in red and blue, respectively. Heatmaps depicting the expression of (C) ExEn and pluripotency markers and (D) fibroblast-related genes in Ago2_KO compared with WT mESCs after cXEN conversion. Two replicates are represented before (ESC) and after differentiation (cXEN). The expression levels are represented as RPKM values (reads per kilobase of transcript per million reads mapped). (E) Bar graph representing the proportion of genes significantly misregulated in Ago2_KO compared with WT cells after 5 days of cXEN conversion. The represented genes are related to mESC-relevant signaling pathways. The gene list was extracted from the KEGG database (Hedgehog, WNT, TGF-beta, RAS, PI3K-AKT, JAK-STAT, MAPK, and pluripotency). See also Figure S5.

Figure 6

cXEN Conversion Defect of Ago2_KO mESCs Is Cell Autonomous (A) Schematic representation of the cXEN differentiation of WT and Ago2_KO cells co-cultured in a 1:1 ratio. The WT cells are GFP positive and Ago2_KO cells are GFP negative. Representative pictures of the Ago2_KO, WT-GFP, and WT-GFP/Ago2_KO cXEN cell conversion are shown. cXEN colonies are indicated by white dashed lines. Scale bar, 50 μm. (B) Immunofluorescence of WT-GFP/Ago2_KO co-cultured cells after cXEN conversion. Representative picture of GFP and FOXA2 co-stained sample is shown. The XENs that appear in the co-cultures are GFP positive. Nuclei are stained with DAPI. Ago2_KO cXEN converted cells are shown as negative control. Scale bars, 50 μm. (C) WT and Ago2_KO mESCs were converted to cXEN in the presence of 500 ng/mL FGF4 growth factor + 1 μg/mL heparin. Schematic representation of the experiment is presented. Levels of AGO2, NANOG, and GATA6 in the different conditions after conversion are shown. TUBULIN levels are shown as loading control. Undifferentiated cells (ESC) are used as controls. (D) RT-qPCR quantification of ExEn markers (Gata6, Gata4, and Dab2) in WT-GATA6 and Ago2_KO-GATA6 mESCs, expressing exogenous inducible GATA6, cultured in LIF/serum conditions for 3 days with or without the addition of doxycycline. The error bar represents the SD of three replicate experiments. See also Figure S6.

Figure 7

XEN Conversion of mESCs Requires the Small-RNA Binding Capacity of AGO2 (A) Schematic representation of the inducible constructs used for the ectopic expression of HA-tagged AGO2, catalytic AGO2 mutant (CATmut), and small-RNA-binding deficient PAZ domain AGO2 mutant (RNAmut) in Ago2_KO1 mESCs. Expression of the AGO2 constructs after 5 days of induction with doxycycline (dox) is shown. TUBULIN is used as loading control. (B) cXEN conversion of Ago2_KO complemented mutants with or without doxycycline. The relative expression of ExEn markers was measured by RT-qPCR before (ESC) and after conversion (cXEN). The error bars represent the SD of three independent experiments. (C) Representative pictures of the WT, Ago2_KO, and the complemented mutant (HA-AGO2, CATmut, and RNAmut) cells after 5 days of cXEN conversion with (+dox) or without (−dox) induction with doxycycline. cXEN colonies are indicated by white dashed lines. Scale bars, 50 μm. (D) Relative expression of miRNAs of group 1 and group 2 in the complemented mutants before and after 5 days of doxycycline, measured by RT-qPCR. The error bars correspond to the SD of three independent experiments. The dashed line represents miRNA levels in WT cells. (E) Western blot showing the inducible overexpression of AGO1 in stably transfected Ago2_KO cells (clone Ago2_KO-AGO1-A). The cells were induced 5 days with doxycycline. TUBULIN is used as loading control. (F) cXEN conversion of Ago2_KO-AGO1-A cells with or without doxycycline. The relative expression of ExEn markers was measured by RT-qPCR before (ESC) and after conversion (cXEN). The error bars represent the SD of three independent experiments. (G) Representative pictures of the Ago2_KO cells overexpressing AGO1 (Ago2_KO-AGO1-A and Ago2_KO-AGO1-B) after 5 days of cXEN conversion with (+dox) or without (−dox) induction with doxycycline. Scale bars, 50 μm. See also Figure S7.