EMBO/soda-nel huggingface dataset:
Tet enzymes are essential for early embryogenesis and completion of embryonic genome activation (2021)
Figure 1

Figure 1. Tet enzyme-deficient mouse embryos arrest primarily at the 2-cell stage.

(A) Experimental setup: Mouse germinal vesicle oocytes (GVOs) were isolated and injected with Morpholinos (MOs) designed to target Tet1-3 mRNAs. After in vitro maturation (IVM), MII oocytes were fertilized by in vitro fertilization (IVF). Zygotes derived from knockdown (KD) and control-MO injected oocytes were analyzed by I) non-invasive time-lapse imaging to monitor the developmental potential of KD embryos, II) immunofluorescence to analyze changes in DNA methylation reprogramming and III) by single 2-cell embryo RNA-Seq & BS-Seq to study the impact of Tet enzymes on embryonic gene activation (EGA) and DNA methylation reprogramming in the totipotent 2-cell embryo.

(B-C) Representative time-lapse images of (B) Tet1-3 triple KD (Tet-TKD), (C) Tet3-KD and control embryos at 3.5 days post fertilization. Scale bar = 100 µm.

(D-E) Developmental rates of (D) control (control-MO injected), Tet1-, Tet2- and Tet3-single KD and Tet-TKD embryos and (E) control (control-MO + Tet2 mRNA co-injected), Tet2-KD2 (Tet2 MO2 injected), Tet2-KD2 + GFP mRNA and Tet2-KD2 + Tet2 mRNA embryos until blastocyst stage. Indicated p-values were calculated using log rank (Mantel-Cox) test against (D) control embryos or (E) Tet2-KD2 embryos or as indicated (ns = non-significant, * = p < 0.05, **** = p < 0.0001; numbers of analyzed embryos for each condition are indicated in parenthesis).
Tet enzymes are essential for early embryogenesis and completion of embryonic genome activation (2021)
Figure 2

Figure 2. Tet enzymes have distinct roles in DNA methylation reprogramming in the mouse zygote.

(A-B) Representative images of (A) 5hmC- and (B) 5mC and 5caC-IF of control (control-MO injected), Tet1+2-DKD and Tet-TKD zygotes at 12 hpf. Paternal and maternal pronuclei are indicated, Pb = polar body; Sp = sperm; Scale bar = 20 μm.

(C-E) Quantification of paternal/maternal (C) 5mC-, (D) 5hmC-, and (E) 5caC-signal ratios of 12 hpf derived zygotes normalized against DNA signal. A total of 5-16 zygotes from two to three experiments per condition were analyzed. Significance to control zygotes was calculated using ordinary one-way ANOVA with Tukey's multiple comparisons test (C, E) or Welch ANOVA with Dunnett's T3 multiple comparisons test (D) and significant differences are indicated (* = p < 0.05, ** = < 0.01, *** = p < 0.001, **** = p < 0.0001, data are represented as mean ± SD, with dots representing single zygotes).
Tet enzymes are essential for early embryogenesis and completion of embryonic genome activation (2021)
Figure 3

Figure 3. Tet enzymes are required for completion of embryonic genome activation.

(A) Principal component analysis of RNA-Seq data of genes from single Tet-TKD (purple), Tet3-KD (coral) and control (grey) 2-cell embryos. Each dot represents a single 2-cell embryo (control n=11, Tet3-KD n=11, Tet-TKD n=11).

(B) MA plots of RNA-seq data from Tet3-KD/control (left) and Tet-TKD/control (right) 2-cell embryos. Significant differentially expressed genes are colored. The amount of up- and downregulated genes is indicated. Differentially expressed genes were determined using DEseq2 (padj<0.05, |log2FC|>0.7).

(C) Venn diagrams visualizing the overlap of differentially expressed genes in Tet3-TKD, Tet-TKD and embryonically activated genes (EGA) in 2-cell embryos. EGA genes were calculated with DEseq2 comparing alpha-Amanitin treated and control 2-cell embryos (padj<0.05, log2FC>0.7).

(D) Comparison of transcriptional profiles of Tet3-KD and Tet-TKD 2-cell embryos to a published database of early embryonic transcripts (DBTMEE, see (Park et al., 2015)). Shown are overlaps of DBTMEE categorized genes with the differentially expressed genes in Tet3-KD or Tet-TKD embryos compared to control 2-cell embryos.
Tet enzymes are essential for early embryogenesis and completion of embryonic genome activation (2021)
Figure 4

Figure 4. Tet enzyme-deficiency dysregulates specific classes of transposable elements (TEs) and 2C genes.

(A) Expression changes of different classes of TEs in Tet3-KD and Tet-TKD. Shown are log2-fold changes of KD embryos compared to control 2-cell embryos of different repetitive elements subdivided in classes of TEs. Indicated p-values were calculated using a paired two-tailed t-test (*** = p < 0.001, **** = p <0.0001, data are represented as medium smoothed violin plots with indicated median and quartiles as dotted lines).

(B-C) Expression levels of (B) maternally expressed ERV3-LTRs and (C) 2-cell embryo specific ERV3-LTRs in control, Tet3-KD and Tet-TKD 2-cell embryos (p-values were calculated using DEseq2; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, data are represented as mean ± SD, dots represent single embryos).

(D) Volcano plots showing expression changes of genes in Tet3-KD and Tet-TKD 2-cell embryos. Chimeric genes, as defined by the presence of a nearby MERVL element and upregulation in 2-cell embryos, are indicated (see (Macfarlan et al., 2012)). Dashed lines represent the cut off for differential expression: |log2FC| > 0.7 and padj < 0.05.

(E) Expression levels of important candidate factors in control, Tet3-KD and Tet-TKD 2-cell embryos, implicated in the generation of 2C-like cells and as important regulators of EGA (p-values were calculated using DEseq2; * = p < 0.05, ** = p < 0.01, **** = p < 0.0001, data are represented as mean ± SD, dots represent single embryos).
Tet enzymes are essential for early embryogenesis and completion of embryonic genome activation (2021)
Figure 5

Figure 5. Minor impact of Tet enzymes on genome-wide DNA demethylation and partial rescue of developmental arrest by non-catalytical functions

(A) % CpG methylation of 20 kb tiles in 5% bins in control or Tet-TKD 2-cell embryos. All samples from one condition were merged for this visualization.

(B) % CpG methylation of 500 bp tiles for different genomic features. All samples from one condition were merged for this visualization. Intergenic = genome excluding gene bodies and TSS -5 kb/+1 kb; Gene bodies = genes excluding TSS -5 kb/+1 kb; Promotor-CGI = CGIs overlapping TSS -5 kb/+1 kb; Non-CGI promoter = promoter (TSS -5 kb/+1 kb) not close to CGI; Non-promoter CGI = CGIs not overlapping TSS -5 kb/+1 kb. (p-values were calculated using a Wilcoxon matched-pairs signed rank test and presented in a medium smoothed violin plot with indicated median and quartiles as dotted lines, **** = p < 0.0001; mean difference is indicated above the plot).

(C) Heatmap of differentially methylated uniquely mapped 500 bp tiles. Shown are random 20% of all significantly hypo- (450 of 2253) and hyper-methylated (2063 of 10316) tiles (logistic regression function of Seqmonk, padj< 0.01, difference 20%, for details see method section).

(D) Distribution of differentially methylated tiles over promoter, gene bodies, or intergenic sequences.

(E) Correlation of significant DNA methylation changes in promoter regions and differentially expressed genes. Numbers in boxes represent number of genes.

(F) Significant methylation changes of specific TEs subdivided in different classes of TEs (calculated using logistic regression function of Seqmonk, padj < 0.05; data are represented as medium smoothed violin plot with indicated median and quartiles as dotted line).

(G) Correlation of significant DNA methylation changes in TEs (calculated with logistic regression function of Seqmonk, padj < 0.05, methylation difference >5%), which are also significant differentially expressed (DEseq2, padj < 0.05, |log2FC|>0.7).

(H) Developmental rate of embryos derived from control, Tet2-MO2, Tet2-MO2 + Tet2 full-length mRNA (as shown in Fig. 1E) and Tet2-MO2 + Tet2 truncated mRNA (no catalytical domain) injected oocytes. Indicated p-values were calculated using the log rank (Mantel-Cox) test against Tet2-MO2 KD or as indicated (* = p < 0.05, *** = p < 0.001, **** = p < 0.0001, numbers of analyzed embryos for each condition is indicated in parenthesis).

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