Tet proteins influence the balance between neuroectodermal and mesodermal fate choice by inhibiting Wnt signaling

Abstract

TET-family dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and oxidized methylcytosines in DNA. Here, we show that mouse embryonic stem cells (mESCs), either lacking Tet3 alone or with triple deficiency of Tet1/2/3, displayed impaired adoption of neural cell fate and concomitantly skewed toward cardiac mesodermal fate. Conversely, ectopic expression of Tet3 enhanced neural differentiation and limited cardiac mesoderm specification. Genome-wide analyses showed that Tet3 mediates cell-fate decisions by inhibiting Wnt signaling, partly through promoter demethylation and transcriptional activation of the Wnt inhibitor secreted frizzled-related protein 4 (Sfrp4). Tet1/2/3-deficient embryos (embryonic day 8.0-8.5) showed hyperactivated Wnt signaling, as well as aberrant differentiation of bipotent neuromesodermal progenitors (NMPs) into mesoderm at the expense of neuroectoderm. Our data demonstrate a key role for TET proteins in modulating Wnt signaling and establishing the proper balance between neural and mesodermal cell fate determination in mouse embryos and ESCs.

Keywords: DNA demethylation; TET methylcytosine oxidases; Wnt signaling; mouse embryonic stem cells; neuromesodermal progenitors.

Fig. 1.

Tet3 mediates neuroectoderm and cardiac mesoderm cell fate determination in mESCs. (A) Quantitative real-time PCR (qRT-PCR) analysis of Tet1, Tet2, and Tet3 transcript levels in mESCs and neural cells differentiated by the SFEB method and normalized to the expression levels in mESCs (set at 1). Data are shown as mean ± SD (n = 3). (B) Tet3 expression level in mESCs cultured in differentiation medium alone or in the presence of 10 ng/mL BMP4 or 15% (vol/vol) FBS. Data are shown as mean ± SD (n = 3). (C) Whole-mount in situ hybridization for Tet3 mRNA at E9.25. (D and E) qRT-PCR analysis of transcripts of neural marker genes Sox1 and Foxg1 (D) and cardiomyocyte marker genes Nkx2-5, Myh6, Myh7, and Tnnt2 (E). WT or Tet3 KO mESCs were differentiated under SFEB culture conditions for 6 or 10 d. Data are shown as mean ± SD (n = 3). (F, Left) Immunocytochemistry of WT or Tet3 KO mESCs differentiated under SFEB culture conditions for 10 d. Cells were stained with anti-Sox1 (green, neural cell marker) and anti-cTnT (red, cardiac cell marker) antibodies. Nucleus staining: DAPI (blue). (Scale bars: 100 μm.) (F, Right) Percentage of Sox1⁺ or cTnT⁺ cells in the total cell population. IHC, immunohistochemistry. (G and H) qRT-PCR analysis of transcripts of Tet3 and ectoderm (Fgf5; G) or cardiomyocyte (Nkx2-5, Myh6, Myh7, and Tnnt2; H) marker genes in mESCs transfected with empty vector or vectors encoding Tet3 or Tet3HxD^mut. The cells were cultured in differentiation medium containing 15% (vol/vol) FBS for 7 d. Data are shown as mean ± SD (n = 3). **P < 0.01.

Fig. S1.

Tet3 mediates neuroectoderm and cardiac mesoderm cell fate determination in mESCs. (A) qRT-PCR analysis of Tet1, Tet2, and Tet3 transcript levels on E6.5, E7.5, E8.5, and E9.5. For E6.5 and E7.5, three embryos are pooled. Data are shown as mean ± SD (n = 3). (B) RNA-seq data for Tet1, Tet2, and Tet3 mRNA expression in E6.75 and E7.5 embryos. RPKM, reads per kilobase of transcript per million mapped reads. (C, Left) Schematic view of targeting scheme for deletion of exon 2 in the endogenous Tet3 locus. (C, Right) Genotyping of Tet3 fl/fl and Tet3 KO mESCs. Bands for floxed (C, Upper) and Tet3 KO (C, Lower) alleles are shown. Genotyping primer sequences are provided in Dataset S4. (D) qRT-PCR analysis of Tet1, Tet2, and Tet3 transcript levels in WT and Tet3 KO mESCs. Data are shown as mean ± SD (n = 3). (E) Schematic representation of Tet3 and catalytically inactive TET3 HxD mutant. Cys-rich, cysteine-rich; DSBH, double-stranded β-helix. *P < 0.05; **P < 0.01.

Fig. 2.

Tet3-dependent transcriptional programs during mESC differentiation. (A) GO biological process analysis of Tet3-activated genes (defined as genes with twofold or greater increase in Tet3-overexpressing cells at day 4 or 7, concomitantly with twofold or greater decrease in Tet3 KO cells at day 6 or 10, relative to WT control cells). (B) GO biological process analysis of Tet3-repressed genes (defined as genes with twofold or greater decrease in Tet3-overexpressing cells at day 4 or 7, concomitantly with twofold or greater increase in Tet3 KO cells at day 6 or 10, relative to WT control cells). (C) Genomic distribution of Tet3-binding sites relative to their nearest RefSeq genes using the cis-regulatory element annotation system. “Promoter” was defined as 3 kb upstream from the TSS. “Downstream” was defined as 3 kb downstream from the 3′ end of the gene. “Distal intergenic region” refers to all locations outside the boundaries of a gene and the 3-kb region flanking the gene on either end. (D) Histogram showing the distribution of Tet3 ChIP-seq peaks relative to the nearest TSS. The majority of sites occupied by Tet3 in the genome are near the TSS (within ∼10 kb 5′ of the TSS and ∼25 kb 3′ of the TSS). (E) The highest-ranked DNA motif conserved in Tet3-bound loci revealed by de novo motif discovery analysis. (F) GO biological process analysis of Tet3 target genes (with at least one Tet3 ChIP-seq peak within 10 kb flanking the TSS). (G) Signaling pathway analysis (using Ingenuity Pathway Analysis software) of Tet3 target genes, defined as possessing a Tet3 ChIP-seq peak within the 10 kb flanking the TSS. The statistically significant canonical pathways are listed according to their P values (−Log10) (blue bars) and the ratio of Tet3 target genes found in each pathway over the total number of genes in that pathway (ratio) is shown by orange squares. The threshold line corresponds to a P value of 0.01. Note the predominance of genes in the Wnt/β-catenin signaling pathway among Tet3 target genes.

Fig. S2.

Tet3-dependent transcriptional programs during mESC differentiation. (A) Heat map of genes related to neuroectoderm differentiation, which were significantly activated by Tet3, and genes related to mesoderm differentiation, which were significantly repressed by Tet3. Red, high expression; blue, low expression using Z-score values normalized. (B and C) Signaling pathway analysis (using Ingenuity Pathway Analysis software) of genes differentially expressed (at least twofold change) in Tet3 KO relative to WT mESCs on days 6 (B) and 10 (C) of SFEB culture. The statistically significant canonical pathways are listed according to their P value (−Log10) (blue bars), and the ratio of differentially expressed genes found in each pathway over the total number of genes in that pathway (ratio) is shown by orange squares. The threshold line corresponds to a P value of 0.01. Note the predominance of the Wnt/β-catenin signaling pathway (red asterisks) at both 6 and 10 d. (D and E) Tet3 ChIP-seq peaks in NPCs were enriched in gene promoters (D) and gene bodies (E) above the genomic background by using randomly generated peaks.

Fig. 3.

Tet3 regulates mESC differentiation by modulating Wnt/β-catenin signaling. (A and B) TOP/FOP-Flash luciferase reporter assay in vector, Tet3, or Tet3-HxD^mut transduced mESCs (A) and in WT and Tet3 KO mESCs (B) induced to differentiate by withdrawal of LIF plus addition of 0.1 μM all-trans retinoic acid (RA) for 4 d. Data are shown as mean ± SD (n = 3). (C and D) qRT-PCR analysis of transcripts of neural marker genes Sox1 and Foxg1 (C) and cardiomyocyte marker genes Nkx2-5, Myh6, Myh7, and Tnnt2 (D). WT and Tet3 KO mESCs were differentiated under SFEB culture conditions for 6 or 10 d in the absence or presence of the Wnt inhibitor Dkk1 (100 ng/mL). Data are shown as mean ± SD (n = 3). **P < 0.01.

Fig. S3.

Tet3 regulates mESC differentiation by modulating Wnt/β-catenin signaling. (A) Heat map of genes related to neuroectoderm differentiation, which were repressed in the absence of Tet3 and whose expression was restored by the addition of Dkk1 to the culture. Red, high expression; blue, low expression using Z-score values normalized. (B) Heat map of genes involved in mesoderm differentiation, which were activated in the absence of Tet3 and whose expression was inhibited by addition of Dkk1 to the culture. Red, high expression; blue, low expression using Z-score values normalized.

Fig. 4.

Tet3 regulates mESC differentiation through Sfrp4, an inhibitor of the Wnt signaling pathway. (A) University of California Santa Cruz Genome Browser snapshots showing Tet3 ChIP-seq peaks at the TSS and in the gene body of Sfrp4. The exon–intron structure of the Sfrp4 gene is shown below. The arrow shows the direction of transcription. The y axis of binding profiles denotes numbers of sequence tag reads. (B) qRT-PCR analysis of Sfrp4 transcripts in WT and Tet3 KO mESCs on day 6 of SFEB culture. Data are shown as mean ± SD (n = 3). (C) qRT-PCR analysis of Sfrp4 transcripts. Vector-, Tet3-, or Tet3HxD^mut-transduced mESCs were cultured in differentiation medium containing 15% (vol/vol) FBS for 7 d. Data are shown as mean ± SD (n = 3). (D) TOP/FOP-Flash luciferase reporter assay in vector and Sfrp4-transduced mESCs induced to differentiate by withdrawal of LIF plus addition of 0.1 μM all-trans retinoic acid for 4 d. Data are shown as mean ± SD (n = 3). (E and F) qRT-PCR analysis of transcripts of ectoderm marker gene Fgf5 (E) and cardiomyocyte maker genes Nkx2-5, Myh6, Myh7, and Tnnt2 (F). Vector or Sfrp4-transduced mESCs were cultured in differentiation medium containing 15% (vol/vol) FBS for 7 d. Data are shown as mean ± SD (n = 3). (G) Bisulfite sequencing showing the percentage of 5mC+5hmC at each CpG site in the promoter region of Sfrp4 in WT and Tet3 KO mESCs on day 6 of SFEB culture. The positions of CpG sites are indicated relative to the TSS. Data are shown as mean ± SD (n = 2). *P < 0.05; **P < 0.01.

Fig. S4.

Tet3 regulates mESC differentiation through Pcdha cluster. (A) University of California Santa Cruz Genome Browser snapshots showing Tet3 binding sites in Pcdha. Gene tracks are shown at the bottom. The y axis of binding profiles denotes the numbers of sequence tag reads. (B) qRT-PCR analysis of transcripts of Pcdha in Tet3 KO mESCs on day 6 of SFEB culture. The transcript level in WT cells was set as 1. Data are shown as mean ± SD (n = 3). All bars are <1 with P < 0.05. (C–H) BS-seq showing the percentage of 5mC+5hmC at each CpG site in the promoter regions of Pcdha4 (C), Pcdha9 (D), Pcdha12 (E), Pcdhac1 (F), Pcdha8 (G), and Pcdhac2 (H) in WT and Tet3 KO mESCs on day 6 of SFEB culture. The positions of CpG sites are indicated relative to the TSS. Data are shown as mean ± SD (n = 2). *P < 0.05. (I) TOP/FOP-Flash luciferase reporter assay after transient expression of vector, Pcdha4 or Pcdha7 in mESCs induced to differentiate by withdrawal of LIF plus addition of 0.1 μM all-trans retinoic acid (RA) for 4 d. Data are shown as mean ± SD (n = 3). **P < 0.01.

Fig. S5.

Cardiac progenitor marker Isl-1 expression is increased in the Tet3 KO heart. (A) qRT-PCR analysis of the transcript level of Isl1 of whole hearts from E18.5 WT and Tet3 KO embryos. Data are shown as mean ± SD (n = 3). (B and C) Immunostaining for Isl1 in transverse (B) and sagittal section (C) of heart from E18.5 WT and Tet3 KO embryos. Nucleus staining: DAPI (blue). **P < 0.01. (Scale bars: B, 100 μM; C, 62.5 μM.)

Fig. S6.

The phenotype of Tet3-deficient mESCs is exacerbated by concurrent deficiency of Tet1 and Tet2. (A) qRT-PCR analysis of Tet1, Tet2, and Tet3 transcript levels in WT and Tet1/2/3 TKO mESCs. Data are shown as mean ± SD (n = 3). (B) Growth curves of WT and Tet1/2/3 TKO mESCs. Cells were split every 3 d, and cells were counted. (C and D) qRT-PCR analysis of transcripts of neural maker genes Sox1 and Foxg1 (C), mesoderm marker T (Brachyury), and cardiomyocyte marker genes Nkx2-5, Myh7, and Tnnt2 (D). WT or Tet1/2/3 TKO mESCs were cultured in differentiation medium containing 15% (vol/vol) FBS for 7 d. Data are shown as the mean ± SD (n = 3). (E and F) qRT-PCR analysis of transcripts of neural marker genes Sox1 and Foxg1 (E) and cardiac mesoderm marker T (Brachyury), Nkx2-5, Myh7, and Tnnt2 (F). WT or Tet3 KO mESCs were differentiated under SFEB culture conditions for 7 d in 10% (vol/vol) KSR. Data are shown as mean ± SD (n = 3). (G–I) qRT-PCR analysis of transcripts of Sfrp4 (G), Pcdha1, Pcdha4, Pcdha9 (H), and Pcdh8 (I). WT or Tet1/2/3 TKO mESCs were differentiated under SFEB culture conditions for 7 d in 10% (vol/vol) KSR. Data are shown as mean ± SD (n = 3). **P < 0.01.

Fig. 5.

TET proteins control the balanced differentiation of NMPs during early embryogenesis in vivo. (A and B) qRT-PCR analysis of transcripts of neural marker genes Sox1 and Foxg1 (A) and cardiac mesoderm marker T (Brachyury), Nkx2-5, Myh7, and Tnnt2 (B). WT or Tet1/2/3 KO (TKO) mESCs were differentiated under SFEB culture conditions for 7 d in 10% (vol/vol) KSR. Data are shown as mean ± SD (n = 3). (C) RNA-seq data for cardiac progenitor marker genes (Gata4 and Fgf10) and neural initiator gene Nnat at E6.75 of WT and Tet1/2/3 TKO embryos. (D–K) Immunocytochemistry of WT or Tet1/2/3 TKO at E8.0–E8.25 for Sox1 (D), Sox2 (E), T (Brachyury) (F), Sox2 and T (G), active β-catenin (H), Tbx6 (I), Isl1 (J), and Gata4 (K). ht, heart; no, node; NS, nonspecific; pm, paraxial mesoderm; so, somites; yc, yolk sac. Nucleus staining: DAPI (blue). (Scale bars: 100 μm.) WT (n = 3); TKO (n = 4). **P < 0.01.

Fig. S7.

Triple Tet deficiency promotes the expression of mesoderm-related genes during early embryogenesis. (A) Phase contrast images of WT (Tet1/2/3 fl/fl) and Tet1/2/3 TKO embryos at E6.75. (B) Genome browser snapshots showing RNA-seq reads for targeted exons of Tet1, Tet2, and Tet3. Reads corresponding to deleted Tet1 exons 8–10, Tet2 exons 8–10, and Tet3 exon 2 are specifically absent in Tet1/2/3 TKO embryos. (C) Hierarchical clustering of gene expression data for the 69 most significant differentially expressed genes between WT and Tet1/2/3 TKO E6.75 embryos. P < 0.0005; adjusted P value < 0.1. Red, high expression; blue, low expression using Z-score values normalized. (D) GO biological process analysis of up-regulated genes in Tet1/2/3 TKO embryos at E6.75. (E) RNA-seq data for seven mesoderm genes (Six1, Six4, Alx1, Alx3, Bin1, Hoxd13, and Crabp2) involved in regulation of embryonic limb and skeletal development. *P < 0.05; **P < 0.01.

Fig. S8.

TET proteins control the balanced differentiation of NMPs during early embryogenesis in vivo. (A) Phase contrast image of WT and Tet1/2/3 TKO embryo at E7.25–E7.5. (Scale bars: 100 μM.) (B–E) Immunocytochemistry of WT (E7.25) or Tet1/2/3 TKO (E7.5) for Sox2 (B), T (C), Sox2 and T (D), active β-catenin (E). ne, neuroectoderm; no, node; NS, nonspecific; ys, yolk sac. Nucleus staining: DAPI (blue). (Scale bars: 100 μm.) WT (n = 3); TKO (n = 3). (F) Immunocytochemistry of WT or Tet1/2/3 TKO at E8.25-E8.5 for Foxa2. Due to the failure of neural plate closure, both left and right sides of the embryo are shown in Tet1/2/3 TKO embryo sections. Nucleus staining: DAPI (blue). (Scale bars: 100 μm.) WT (n = 3); TKO (n = 3). (G) RNA-seq data for Wnt3 and Nodal expression in WT and Tet1/2/3 TKO embryos at E7.25–7.5 shown in A.

Fig. 6.

Molecular analysis of Tet1/2/3 TKO embryos at E8.25–8.5. (A–H) Immunocytochemistry of Tet1/2/3 TKO at E8.25–8.5 for Sox1 (A), Sox2 (B), T (Brachyury) (C), Sox2 and T (D), active β-catenin (E), Tbx6 (F), Isl1 (G), and Gata4 (H). Due to the failure of neural plate closure, both left and right sides of the embryo are shown in Tet1/2/3 TKO embryo sections. ht, heart; no, node; NS, nonspecific; pm, paraxial mesoderm; so, somites; yc, yolk sac. Nucleus staining: DAPI (blue). (Scale bars: 100 μm.) WT (n = 3); TKO (n = 3). (I) A model for TET function in developmental specification of NMPs in the E8.5 mouse embryo. In WT embryos, NMPs (red/green) are located in the NSB and CLE. When Wnt signaling is inhibited by Tet proteins, NMPs give rise to neural progenitors (green) which will contribute to the neural plate (NP), whereas activated Wnt signaling promotes the development of NMPs to mesoderm progenitors (red), which will contribute to paraxial mesoderm (PM). In Tet1/2/3 TKO embryos, Wnt signaling is abnormally activated in the absence of TET proteins. Overactivation of Wnt signaling in NMPs residing in the PS leads to their differentiation into mesoderm progenitors (red) and then further into lateral mesoderm (orange), resulting in abrogated development of body midline structures.