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. 2015 Nov 9;4:e09571. doi: 10.7554/eLife.09571

Figure 2. G9a represses germline and proliferation-related genes in the postimplantation epiblast.

(A,B) bright-field images of Ehmt2+/+and Ehmt2−/− embryos at E7.5 (A) (scale bar = 0.1 mm). At least nine embryos of each type were staged (B) (*Chi2 test p-value= <0.05). (C) Scatter plot showing transcript expression levels in Ehmt2+/+or +/−and Ehmt2−/−E6.25 epiblast. Blue points are differentially expressed genes (Log2RPKM>1, p-value<0.05, Log2(FC)>1.4). Shown is the geometric average from four biological replicates. (D) Heatmap showing expression of selected genes from enriched GO categories. (E,F) Single-cell RT-qPCR validation of RNA-seq performed on individual epiblast cells isolated from E6.25 Ehmt2+/+ or Ehmt2−/− embryos (minimum 2 embryos and 14 cells). Dot plots show levels of Ehmt2, pluripotency (Nanog, Pou5f1), germline (Asz1, Rhox5, Sohlh2) and proliferation regulators (Cdkn1a, Cd3e, Foxj1). Expression is normalised to Arbp and relative to average in Ehmt2−/− and for Ehmt2 relative to Ehmt2+/+. Statistical significance was calculated using Wilcoxon rank sum test for Pou5f1 and Nanog, where majority of WT and KO cells show detectable expression. For remaining genes a Chi2 test was used. (*p-value<0.05). Also see Figure 2—source data 1–4 and Figure 2—figure supplement 1–3. LHF: late head fold; EHF: early head fold; LB: late allantoic bud; OB: no allantoic bud; LS: late streak; PS: pre-streak. RT-qPCR: real-time quantitative polymerase chain reaction; RNA-seq: RNA sequencing; WT: wild-type: KO: knockout; GO: gene ontology; FC: fold change.

DOI: http://dx.doi.org/10.7554/eLife.09571.004

Figure 2—source data 1. List of differentially expressed genes in E6.25 Ehmt2−/− epiblast from RNA-seq analysis data is based on four individual Ehmt2−/− and control (Ehmt2+/+ or Ehmt2+/−) epiblasts.
Differentially expressed genes were identified using a minimum Log2(FC)>1.4 and maximum Fisher combined test of p-value<0.05. In the upregulated sample, expression was 1<log (RPKM). RNA-seq: RNA sequencing; RPKM: Reads Per Kilobase of transcript per Million mapped reads; FC: fold change.
DOI: http://dx.doi.org/10.7554/eLife.09571.005
elife-09571-fig2-data1.xls (126.5KB, xls)
DOI: 10.7554/eLife.09571.005
Figure 2—source data 2. List of enriched GO terms in genes upregulated in E6.25 Ehmt2−/− epiblast GO term enrichment for biological processes was calculated using DAVID software with minimum five genes in a category and EASE p-value<0.05.
EASE: Expression Analysis Systematic Explorer; GO: gene ontology.
DOI: http://dx.doi.org/10.7554/eLife.09571.006
elife-09571-fig2-data2.xls (34KB, xls)
DOI: 10.7554/eLife.09571.006
Figure 2—source data 3. List of differentially expressed genes in E6.25 Ezh2−/−epiblast from RNA-seq analysis data is based on 3 individual Ezh2−/− epiblasts and 3 individual control (Ezh2+/+ or Ezh2+/−) epiblasts.
Differentially expressed genes were identified using a minimum Log2(FC)>1.4 and maximum Fisher combined test of p-value<0.05. In the upregulated sample, expression was 1<log(RPKM). RNA-seq: RNA sequencing; RPKM: Reads Per Kilobase of transcript per Million mapped reads; FC: fold change.
DOI: http://dx.doi.org/10.7554/eLife.09571.007
elife-09571-fig2-data3.xlsx (88.9KB, xlsx)
DOI: 10.7554/eLife.09571.007
Figure 2—source data 4. List of enriched GO terms in genes upregulated in E6.25 Ezh2−/− epiblast GO term enrichment for biological processes was calculated using DAVID software with minimum five genes in a category and EASE p-value<0.05.
EASE: Expression Analysis Systematic Explorer; GO: gene ontology.
DOI: http://dx.doi.org/10.7554/eLife.09571.008
elife-09571-fig2-data4.xls (33.5KB, xls)
DOI: 10.7554/eLife.09571.008

Figure 2.

Figure 2—figure supplement 1. Epigenetic programming by G9a in postimplantation epiblast.

Figure 2—figure supplement 1.

(A) Cryosection IF of E6.25 Ehmt2+/+ and Ehmt2−/− embryos stained with antibodies raised against G9a and H3K9me2. Scale bar = 20 μm. (B) Top 10 enriched GO terms in genes upregulated in Ehmt2−/− E6.25 embryos. (C,D) Whole-mount IF of E6.5 Ehmt2+/+ and Ehmt2−/− embryos (C) stained with antibodies raised against cleaved Caspase 3 and Ki67, markers of apoptosis and proliferation, respectively. White arrows show Ki67 negative cells. Scale bar = 20 μm. Shown is maximum projection from 5 z-stacks. (D) Bar plot showing the number of apoptotic cells (cleaved Caspase 3 positive) and non-proliferative cells (Ki67 negative and Caspase 3 negative) dependent on the genotype. Data are represented as mean (± SEM) from five control and five KO embryos. (*Student’s t-test p-value< 0.05). Also see Figure 2—source data 1 and 2. GO: gene ontology; H3K9me2: histone H3 lysine 9 dimethylation; IF: immunofluorescence; SEM: standard error of the mean; KO, knockout.
DOI: http://dx.doi.org/10.7554/eLife.09571.009
Figure 2—figure supplement 2. Loss of G9a does not affect the exit from pluripotency and germline specification.

Figure 2—figure supplement 2.

(A,B) Whole-mount IF of E6.5 GGOF Ehmt2+/+ and Ehmt2−/− embryos stained with antibodies raised against Nanog (A), GFP and G9a (B). Shown is a sum of an equal number of z-stacks. Scale bar 20 = μm. (C,D) Whole-mount IF of E8.5 Ehmt2+/+ and Ehmt2−/− embryos stained with antibodies raised against GFP and AP2γ (D). GFP is expressed under GGOF reporter, which marks nascent PGCs. The different size of the Ehmt2+/+ and Ehmt2−/− embryos are noteworthy. Higher magnification images reveal that E8.5 PGCs show upregulation of a germline marker gene AP2γ (D) irrespective of the genotype. Scale bar 10 = μm. GGOF: ΔPE-Pou5f1-EGFP; GFP: green fluorescent protein; IF: immunofluorescence; PGCs: primordial germ cells.
DOI: http://dx.doi.org/10.7554/eLife.09571.010
Figure 2—figure supplement 3. EZH2 represses multiple developmental regulators in vivo.

Figure 2—figure supplement 3.

(A) Scatter plot showing transcript expression levels in Ezh2−/− versus Ezh2cntr E6.25 epiblast. Red points are differentially expressed genes (Log2RPKM>1, p-value<0.05, Log2(FC)>1.4). Shown is geometric average from three biological replicates. (B) RT-qPCR validation of RNA-seq at selected target genes. Data are represented as mean ( ± SEM) from three independent biological replicates (*Student’s t-test p-value< 0.05). (C) Top 10 enriched GO terms in genes upregulated in Ezh2−/− E6.25 embryos. Also see Figure 2—source data 3 and 4. GO: gene ontology; RT-qPCR: Real-time quantitative polymerase chain reaction; RNA-seq: RNA sequencing; SEM: standard error of the mean; FC: fold change.
DOI: http://dx.doi.org/10.7554/eLife.09571.011