TET-mediated DNA demethylation controls gastrulation by regulating Lefty–Nodal signalling

Abstract

Mammalian genomes undergo epigenetic modifications, including cytosine methylation by DNA methyltransferases (DNMTs). Oxidation of 5-methylcytosine by the Ten-eleven translocation (TET) family of dioxygenases can lead to demethylation1,2,3. Although cytosine methylation has key roles in several processes such as genomic imprinting and X-chromosome inactivation, the functional significance of cytosine methylation and demethylation in mouse embryogenesis remains to be fully determined4,5,6,7,8,9. Here we show that inactivation of all three Tet genes in mice leads to gastrulation phenotypes, including primitive streak patterning defects in association with impaired maturation of axial mesoderm and failed specification of paraxial mesoderm, mimicking phenotypes in embryos with gain-of-function Nodal signalling10. Introduction of a single mutant allele of Nodal in the Tet mutant background partially restored patterning, suggesting that hyperactive Nodal signalling contributes to the gastrulation failure of Tet mutants. Increased Nodal signalling is probably due to diminished expression of the Lefty1 and Lefty2 genes, which encode inhibitors of Nodal signalling. Moreover, reduction in Lefty gene expression is linked to elevated DNA methylation, as both Lefty–Nodal signalling and normal morphogenesis are largely restored in Tet-deficient embryos when the Dnmt3a and Dnmt3b genes are disrupted. Additionally, a point mutation in Tet that specifically abolishes the dioxygenase activity causes similar morphological and molecular abnormalities as the null mutation. Taken together, our results show that TET-mediated oxidation of 5-methylcytosine modulates Lefty–Nodal signalling by promoting demethylation in opposition to methylation by DNMT3A and DNMT3B. These findings reveal a fundamental epigenetic mechanism featuring dynamic DNA methylation and demethylation crucial to regulation of key signalling pathways in early body plan formation.

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Figure 1: Tet-null embryos display gastrulation defects.
Figure 2: Increased Nodal expression in Tet-null embryos leads to patterning defects.
Figure 3: The Lefty1 promoter and Lefty2 enhancer are hypermethylated in Tet-null embryos.
Figure 4: Lefty–Nodal signalling is regulated by dynamic DNA methylation.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

RNA-seq and PBAT data have been deposited in the Gene Expression Omnibus database under accession number GSE76261.

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Acknowledgements

We thank A. Lashua, B. Zhou, Z. Yang and S. Li for providing probes; D. Chen for Stra8-Cre mouse; H. Wang and W. Lai for quantitative mass spectrometry analysis; D. Pei and B. Wang for karyotype analysis; Z. Xu, G. Xu and Q. Li for support with animal care and experiments; N. Zhong, J. Gao, C. Wang, Q. Xu and Q. Yang for technical assistance. We also thank C. Walsh and G. Pfeifer for critical reading of the manuscript; C. Hui, Y. Mishina, T. Chen and N. Jing for discussions. This work was supported by the National Science Foundation of China (31230039 and 31521061 to G.X., and 31429003 to S.X.), the Ministry of Sciences and Technology of China (2012CB966903 and 2014CB965200) and the ‘Key New Drug Creation and Manufacturing Program’ of China (2014ZX09507002-005) to G.X..

Author information

Affiliations

Authors

Contributions

G.X. conceived the original idea and, together with H.D., F.T. and X.S., designed the experiments. H.D. carried out mouse embryo studies. B.W. conducted bisulfite sequencing and immunostaining. L.Y. performed RNA-seq and methylome analysis. J.C. performed CRISPR–Cas9 gene editing. G.Z. performed in situ hybridization. H.D., G.X., and X.S. wrote the paper.

Corresponding authors

Correspondence to Xin Sun or Guo-Liang Xu.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks A.-K. Hadjantonakis, S. Kriaucionis and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Generation of mouse embryos deficient in the three Tet genes using germline-specific conditional knockout parents.

a, Left panel, schematics of targeted gene disruption. Coding exons are shown as filled boxes. FRT sites flanking the neo selection marker are shown as blue triangles and LoxP sites flanking the targeted region are shown as red triangles. PCR primers used for genotyping are shown as horizontal arrows. Stars indicate the exon encoding the HxD motif of the active site of TET enzymes. Gel images at right show genotyping PCR results. b, Conditional inactivation of the Tet genes in germ cells. Germ cells were isolated from (Tet1f/−Tet2f/−Tet3f/−; Stra8-Cre) ((3 × f/−; Stra8-Cre)) males and (Tet1f/−Tet2f/−Tet3f/−; Zp3-Cre) ((3 × f/−; Zp3-Cre)) females and genotyped. c, Analysis of efficiency of allele conversion from ‘f’ to ‘null’. Tet-floxed Stra8-Cre and Zp3-Cre mice with indicated genotypes were mated with wild-type mice. The resulting pups were genotyped to determine the percentage of those carrying a deleted (null) Tet allele among the total number of all pups.

Extended Data Figure 2 Normal spermatogenesis and oogenesis in Tet1/2/3 germline-specific conditional knockout mice.

(Tet1f/+Tet2f/+Tet3f/+; Stra8-Cre) ((3 × f/+; Stra8-Cre)) male and (Tet1f/+Tet2f/+Tet3f/+; Zp3-Cre) ((3 × f/+; Zp3-Cre)) female mice were used as controls. af, Normal morphology of testis (a) and ovaries (b) from adult germline conditional knockout (CKO) parents. Representative images of H&E-stained cross-sections of seminiferous tubules (c, d) and ovaries (e, f) from adult germline CKO mice. Scale bars, 2 mm (a, b), 50 μm (c, d), 200 μm (e, f). g, h, Normal litter size from (3 × f/−; Stra8-Cre) male mice (g). Reduced litter size from (3 × f/−; Zp3-Cre) female mice (h). The total number of pups examined for each cross is indicated below the x axis. The total number of deliveries during a mating term of 4 months is indicated within parentheses. Student’s t-test was performed and data shown are mean ± s.d. ***P < 0.001. NS, not significant. i, Scatter plot shows the differentially expressed genes (DEGs) of Tet-null MII oocytes compared to wild-type oocytes. nCount stands for normalized counts (mean of the counts divided by size factors; DESeq2 software package). The dashed lines indicate the twofold change threshold for identifying DEGs. Orange and blue dots denote significantly changed genes (adjusted P value < 0.05 and log2(fold change) >1 or <−1) and grey dots denote not significantly changed genes. The number of MII oocytes analysed by RNA-seq is listed in Supplementary Table 3. j, Gene ontology analysis of DEGs in Tet-null oocytes. Numbers in bars show the gene numbers of the corresponding gene ontology categories.

Extended Data Figure 3 Strategy for getting TKO embryos and analysis of Tet-null blastocysts.

a, Breeding scheme to obtain Tet-null mutants and littermate controls. Non-TKO embryos were those retaining at least one floxed Tet allele owing to incomplete deletion by Cre in the parental germline. DKO, double knockout. b, At E3.5, Tet-TKO blastocysts (c) are morphologically indistinguishable from the wild-type and controls (indicated by dashed circles) which retain at least one wild-type Tet allele (b). Representative images are shown. c, Normal karyotype of the Tet-TKO ES cell lines derived from TKO blastocysts using standard protocol. d, Scatter plot shows the differentially expressed genes (DEGs) of Tet-null blastocysts compared to wild-type blastocysts. nCount stands for normalized counts (mean of the counts divided by size factors; DESeq2 software package). The dashed lines indicate the twofold change threshold for identifying DEGs. Orange and blue dots denote significantly changed genes (adjusted P value < 0.05 and log2(fold change) >1 or <−1) and grey dots denote not significantly changed genes. The number of blastocysts analysed by RNA-seq is listed in Supplementary Table 3. e, Gene ontology analysis of DEGs in Tet-null blastocysts. Numbers in bars show the gene numbers of the corresponding gene ontology categories.

Extended Data Figure 4 Morphological analysis of Tet-null embryos and confirmation of 5hmC loss.

a, b, At E6.5, Tet-TKO embryos (b) are morphologically indistinguishable from the controls which retain at least one wild-type Tet allele (a). Representative images are shown. c, d, E7.75 embryos. The control (c) is at the headfold stage. A matched mutant embryo (d) has defects in the head process (arrowhead in d), and a thickened streak (arrow in d). e, f, Lateral view of E8.5 embryos. The control (e) contains several somites (so), a forebrain (fb) and heart (ht), whereas the Tet-null embryo (f) lacks these structures. gp, The littermate mutant embryo (h) is indistinguishable from the control at E6.5 (g). epc, ectoplacental cone; epi, epiblast; exe, extraembryonic ectoderm. il, Embryos at E7.75. The control (i) is at the headfold stage. In the littermate mutant embryo (j), head mesenchyme and a morphologically distinct node are absent. Only a posterior amniotic fold forms but with no amnion or chorion. White dashed lines in (i, j) indicate the approximate level of sections shown in (k, l). Cross-sections of control (k) and mutant embryos (l) highlight the accumulation of mesoderm cells in the mutant streak. af, amniotic fold; al, allantois; am, amnion; ch, chorion; ect, ectoderm; en, endoderm; hm, head mesoderm; me, mesoderm; nd, node; nf, neural fold; ng, neural groove; np, notochordal plate; ps, primitive streak. mp, Embryos at E8.5. The control (m) contains several somites, a heart, and foregut; the littermate mutant (n) does not contain any such mesoderm- or endoderm-derived tissues. White dashed lines in (m, n) indicate the approximate level of sections shown in control (o) and mutant embryos (p). bl, blood island. Note that in TKO embryos, the amnion and chorion have formed. q, Immunofluorescent images of 5mC (green) and 5hmC (red) staining in E7.5 TKO embryos. r, Complete loss of 5hmC and marked increase in 5mC in TKO E8.0–E8.5 embryos. The frequencies of these two nucleotides in the genomic DNA were determined by quantitative mass spectrometry. n.d., not detected. Student’s t-test was performed and data shown are mean ± s.d. of 5 wild-type and 7 TKO embryos, respectively, *** P < 0.001. Scale bars, 200 μm (a, b), 500 μm (cf), 50 μm (g, h, q), 100 μm (ip).

Extended Data Figure 5 Impaired proliferation, increased apoptosis and abnormal migration in Tet-null embryos.

a, A representative image of transverse sections through the embryonic portion of E6.5 and 7.5 embryos stained with anti-phospho-histone H3 antibody (red) to detect mitotic cells. b, Quantification of the mitotic cells per section. c, A representative image of TUNEL staining (red) of transverse sections from the embryonic region of E6.5 and 7.5 embryos. d, Quantification of the apoptotic cells per section. e, f, Immunostaining detection of the distribution of E-cadherin (e) and Snail (f) in the vicinity of the streak and nascent mesoderm in E7.75 embryos. Anterior is to the left. Scale bars, 50 μm. Student’s t-test was performed and data shown are mean ± s.d. of more than 12 sections in total from 3 independent embryos, ***P < 0.001. NS, not significant. The number of sections and embryos analysed by immunostaining is listed in Supplementary Table 3.

Extended Data Figure 6 Whole-mount in situ hybridization analysis of marker gene expression in Tet-null embryos.

All embryos are oriented with the anterior to left and posterior to right. Each panel displays a control (upper) and a Tet-null (bottom) embryo. The probes and developmental stages are indicated. The number of embryos analysed by in situ hybridization is listed in Supplementary Table 3.ac, Analysis of T (a1, a2), Fgf8 (b) and Cer1 (c) expression in E6.5–E6.75 embryos. dg, Analysis of Dkk1 (d), Oct4 (e1, e2), Gsc (f) and Foxa2 (g) expression. h1, h2, T expression. Black dashed lines in (h1) show the level of transverse sections in (h2). Transverse sections show T expression (h2). Arrows point to T-expressing cells in the axial mesoderm. Red arrowheads indicate the most anterior extent of mesoderm cell migration. i1, i2, Lim1 expression. Black dashed lines in (i1) correspond to the level of transverse sections in (i2). Transverse sections show Lim1 expression (i2). Arrows point to Lim1-expressing cells in the anterior visceral endoderm. Red arrowheads as in (h2). jq, Analysis of Fgf8 (j), Mixl1 (k), Wnt3 (l), Bmp4 (m1, m2), Eomes (n), Sox17 (o1, o2), Foxa2 (p) and Shh (q) expression. r, s, Tbx6 (r) and Meox1 (s) were not detected in Tet-null embryos. t, u, Analysis of Hesx1 (t) and Otx2 (u), two anterior neural markers. v, Analysis of Gbx2 expression, a posterior neural marker. Scale bars, 200 μm in all panels.

Extended Data Figure 7 Analysis of E6.5 Tet-null epiblasts by RNA-seq and generation of Nodal heterozygous embryos in the Tet-null background.

a, Scatter plot shows the differentially expressed genes (DEGs) of Tet-null epiblasts compared to wild-type epiblasts. nCount represents the normalized counts (mean of the counts divided by size factors; DESeq2 software package). The dashed lines indicate the twofold change threshold for identifying DEGs. Orange and blue dots denote significantly changed genes (adjusted P value < 0.05 and log2(fold change) >1 or <−1) and grey dots denote not significantly changed genes. The number of epiblasts analysed by RNA-seq is listed in Supplementary Table 3. b, Gene ontology analysis of DEGs in Tet-null epiblasts. Numbers in bars show the gene numbers of the corresponding GO categories. cf, Whole-mount in situ hybridization analysis of Nodal and Lefty expression. c, Analysis of Nodal (c) expression in early TKO embryos. d, Transverse sections show Nodal expression in E7.5 TKO embryos corresponding to the level indicated in Fig. 2a by asterisks. e, f, Analysis of Lefty expression in E5.75 (e) and E6.5 (f). DVE, distal visceral endoderm. g, h, CRISPR–Cas9-mediated Nodal gene disruption. The sgRNA-targeting sequence is underlined, and the protospacer-adjacent motif (PAM) sequence is labelled in red. Nucleotides of the NotI restriction site in the targeted region are capitalized. g, The Cas9–sgRNA-targeting site in Nodal exon 1 for generating Nodal heterozygous embryos. h, The partial sequencing results for Nodal heterozygous embryos show the detection of the wild-type allele and the mutated allele(s) carrying a frameshift mutation. i, Introduction of one mutant Nodal allele (+/−). Analysis of T expression in Nodal+/− embryos. There are no gross or molecular phenotypes observed in the Nodal+/− embryos we generated in the wild-type background. Scale bars, 200 μm (cf and i).

Extended Data Figure 8 Analysis of Tet-null epiblasts by PBAT whole-genome bisulfite sequencing.

a, Bar plot shows the genome-wide CpG methylation levels of E6.5 wild-type and TKO epiblasts. DNA methylation level was calculated by extracting CpG sites with at least 5× sequencing coverage. Only the single CpG sites covered by at least 5 times were used for analysis. Error bars represent ± s.d. No significant difference between wild-type and TKO epiblasts was detected. b, Scatter plot shows a global methylation comparison between wild-type and Tet-null epiblasts using 100-bp tiles. Only the tiles that were assigned at least 3 CpGs were kept for analysis. Tiles with at least 20% absolute methylation level difference between Tet-TKO and wild-type samples were defined as differentially methylated: hypermethylated tiles and hypomethylated tiles. Each point represents the methylation level of a 100-bp tile. The two triangles represent at least 20% methylation differences between wild-type and Tet-TKO samples. c, Distribution of single CpG methylation levels across the indicated genomic elements. DHS, DNase I-hypersensitive site. d, Distribution of the relative enrichment of hypermethylated and hypomethylated tiles at various genomic features in E6.5 TKO epiblasts. The relative ratio of different gene elements was calculated by dividing the corresponding hypermethylated or hypomethylated tiles ratios with the corresponding total 100-bp tiles ratios. CGI, CpG island. e, Pie chart shows the distribution of hypermethylated DMRs in E6.5 TKO epiblasts. DMRs were filtered by the DMR length (at least 400 bp), CpG number (at least 4 CpGs in a DMR) and the methylation ratio difference between wild-type and Tet-TKO (at least 20% absolute methylation level difference). For regions belonging to more than one category, the classification follows the priority of enhancer, promoter, DNase I-hypersensitive site and others. f, g, Gene ontology analysis of all genes with hypermethylated DMRs in enhancer (f) and promoter (g) regions in TKO epiblasts. Details of analysis are described in Methods. h, TET-assisted bisulfite (TAB) sequencing analysis of Lefty2 PME and promoter regions. The generation of 5hmC at these regions could be rescued by ectopic expression of Tet2 in TKO ESCs, which were derived from Tet-null blastocysts using standard protocol.

Extended Data Figure 9 CRISPR–Cas9-mediated Tet3 and Dnmt3 gene editing in mouse embryos.

The sgRNA-targeting sequence is underlined, and the PAM sequence is labelled in red. The restriction sites at the target regions are capitalized. a, b, The Cas9/sgRNA/oligonucleotide-targeting site in Tet3 exon 9 for the ‘HD’ mutation (a). In the 130-nt oligonucleotide donor sequence, the HKD coding sequence is replaced by YRA coding sequence harbouring a SacI restriction site. The sequencing of Tet3 alleles from a mutant embryo confirms the presence of the desired mutation of HKD to YRA change (b). Star indicates the exon encoding the HKD motif of the active site of TET3 enzyme. c, Real-time PCR quantification of the mutant Tet3 transcripts in embryos generated by Tet3 gene editing in the Tet3+/− Tet1/2-DKO background (Tet1/2-DKO Tet3+/−, n = 3; Tet1/2-DKO Tet3HD/−, n = 2; Tet-TKO, n = 3). The expression levels are normalized to Gapdh and presented as mean ± s.e.m. d, Confirmation of the presence of the HD mutation in Tet3 mRNA. A cDNA fragment encoding the HKD motif region was prepared from an E8.0-E8.25 (Tet1/2-DKO Tet3HD/−) embryo and sequenced. eh, Cas9/sgRNA targeting sites were chosen upstream of the sequence encoding the PCN motif which is essential for DNA methyltransferase activity in Dnmt3a exon 18 (e) and Dnmt3b exon 19 (g). The partial sequencing results for Dnmt3a (f) and Dnmt3b (h) in a doubly mutant embryo reveal the presence of frameshift mutations. Stars indicate the exon encoding the PCN motif of the active site of DNMT3 enzymes. i, j, Analysis of Lefty (i) and Nodal (j) expression in Dnmt3a-KO or Dnmt3b-KO in wild-type background embryos. k, Analysis of T (k) expression in Dnmt3a-KO or Dnmt3b-KO or both in Tet-TKO background embryos. l, Analysis of methylation level at the Lefty2 PME region in Dnmt3a/3b DKO in wild-type background embryos. Bisulfite sequencing profiles show lack of methylation both in the Lefty2-negative (-) and positive (+) compartments. m, n, Analysis of Lefty, Nodal and T expression both in Dnmt3-DKO in Tet-TKO background (m) and Dnmt3-DKO in wild-type background (n). Scale bars, 200 μm in all panels.

Extended Data Figure 10 Functional redundancy among Tet1, Tet2 and Tet3 during gastrulation.

a, Real-time PCR analysis of Tet1/2/3 transcripts from wild-type E6.5 and E7.5 embryos. The expression levels are normalized to Gapdh and presented as mean ± s.e.m. (n = 8 embryos). epc, ectoplacental cone; epi, epiblast; exe, extraembryonic ectoderm. b, Lateral view of E8.5 double knockout embryos. Representative images are shown. Scale bar, 500 μm. c,Whole-mount in situ analysis of Tet1/2/3 expression in wild-type embryos. Black dashed lines in E7.5 embryos (antisense probe) indicate the approximate level of sections shown on the right respectively. Embryos were stained with sense and antisense probes. d, e, Whole-mount in situ analysis of Lefty2 (d) and Nodal (e) expression in three different double knockout embryos. Anterior is towards the left in all panels. Scale bars, 200 μm (ce).

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, original source images for Extended Data Figure 1a & b. (PDF 51 kb)

Supplementary Table 1

A list of DEGs in Tet TKO embryos (XLSX 9000 kb)

Supplementary Table 2

A list of DMRs in Tet TKO embryos. (XLSX 6309 kb)

Supplementary Table 3

This table shows biological replicates (XLSX 18 kb)

Supplementary Table 4

This table shows PCR primer information. (XLSX 10 kb)

Supplementary Table 5

This table contains sgRNA target sites and oligonucleotides. (XLSX 10 kb)

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Dai, HQ., Wang, BA., Yang, L. et al. TET-mediated DNA demethylation controls gastrulation by regulating Lefty–Nodal signalling. Nature 538, 528–532 (2016). https://doi.org/10.1038/nature20095

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