Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory

Chimera Colonization and Lineage Potential of Dnmt3a/3b-Deficient ES Cells.

We examined the ability of Dnmt3a and Dnmt3b double knockout (Dnmt3dKO) ES cells (15) to contribute to chimeric embryos. Compound Dnmt3a/3b mutant embryos are reportedly normal until somitogenesis (13). However, after blastocyst injection of Dnmt3dKO cells genetically labeled with constitutive mKO2, we found very sparse contributions in presomite-stage embryos at E7.5 (Fig. 1A). Even at E6.5 contributions were reduced compared with typical ES cell chimeras (Fig. 1B). Furthermore, some mutant donor cells were located in the extraembryonic ectoderm, a rare occurrence with wild-type (WT) ES cells (16).

Fig. 1.

Dnmt3a/b deficiency compromises chimera contribution and somatic lineage commitment. (A–C) Chimeric embryos obtained with parental, Dnmt3dKO (A and B), and hBCL2-expressing (C) mKO2 reporter ES cells. The 30% opacity brightfield images are overlaid onto fluorescent images. (Scale bars, 1 mm in A and 500 µm in B and C.) (D) qRT-PCR analysis of marker during culture in trophoblast medium (20). (E) qRT-PCR analysis of undifferentiated ES cells, Rex1::GFP^Lo sorted AFK cells, and further differentiated cells. (F) Cell morphology of WT and Dnmt3dKO cells in trophoblast stem cell (TS) cell medium (20) for 8 d. (Scale bars, 50 µm.) (G) DNA content quantified by propidium iodide (PI) staining at day 8 in TS cell medium. qRT-PCR data were normalized to beta-actin. Error bars are SD from technical duplicates.

To improve survival of Dnmt3dKO ES cells in chimeras, we introduced a constitutive BCL2 transgene (17). In E6.5 embryos we observed higher contributions to epiblast, comparable to wild-type ES cells (Fig. 1C). However, we also saw donor cells in extraembryonic regions. We confirmed localization in the extraembryonic ectoderm and ectoplacental cone in five out of six chimeras examined by confocal microscopy (SI Appendix, Fig. S1A). We inspected blastocyst-stage chimeras to test whether Dnmt3a/b deficiency or BCL2 expression enabled primary trophectoderm colonization. However, donor cells were correctly localized to the inner cell mass (ICM) and did not contribute to trophectoderm (SI Appendix, Fig. S1B). Thus, the presence of Dnmt3dKO cells in postimplantation trophoblast likely arises by displacement from the epiblast rather than differentiation via trophectoderm.

The poor and aberrant colonization behavior of Dnmt3dKO cells prompted us to investigate in vitro differentiation competence. In response to mesendoderm induction, mutant cells up-regulated T and Foxa2 but to a lower level than parental cells (SI Appendix, Fig. S1C). In permissive conditions for neural induction (18), Dnmt3dKO cells showed only weak up-regulation of Sox1 and Pax6 (SI Appendix, Fig. S1D) but displayed substantial and sustained up-regulation of Ascl2, Hand1, and Tpbpa, genes associated with the trophoblast lineage (SI Appendix, Fig. S1 C and D). To assess whether Dnmt3dKO cells adopt trophoblast identity, we applied two culture conditions for trophoblast cells (19, 20). Unlike parental cells, Dnmt3dKO cells showed no or low up-regulation of formative pluripotency factors Otx2 and Oct6, but instead gained expression of trophoblast markers (Fig. 1D and SI Appendix, Fig. S1E).

We also subjected single Dnmt KO ES cells (15) to lineage induction (SI Appendix, Fig. S1F). Expression of germ layer markers was reduced in both mutants, and in neural conditions trophoblast genes were up-regulated (SI Appendix, Fig. S1F). The phenotype was more marked in Dnmt3b KO cells, with higher trophoblast gene induction and lower neural and mesendodermal gene activation. We introduced expression constructs for Dnmt3a and Dnmt3b into Dnmt3dKO cells (SI Appendix, Fig. S1G). Rescued cells displayed normal differentiation with suppression of trophoblast genes (SI Appendix, Fig. S1H).

Culture in activin, FGF2, and KSR (AFK) induces ES cell conversion into postimplantation formative epiblast-like cells (EpiLCs) (21). Dnmt3dKO cells showed delayed morphological change on day 1 but by day 2 appeared similar to parental cells with a comparable increase in cell number (SI Appendix, Fig. S1 I and J). The Rex1::GFPd2 (RGd2) reporter allows reliable monitoring of ES cell exit from naive pluripotency (9). Dnmt3dKO cells showed delayed down-regulation of the reporter in AFK (SI Appendix, Fig. S1K), consistent with findings in N2B27 (15). We sorted the GFP low (GFP^Lo) fraction that has exited naive pluripotency and saw that trophoblast genes Ascl2 and Tfap2c were misexpressed in mutant cells (Fig. 1E). Levels increased further upon continued culture in N2B27 (Fig. 1E) while neural markers, Sox1 and Pax6, were very lowly expressed (SI Appendix, Fig. S1L). GFP^Lo cells transferred into activin and CH gained only modest up-regulation of T and FoxA2, though they did not express most trophoblast markers.

After ongoing culture of dKO cells in N2B27, we observed additional trophoblast markers. Strikingly, however, the sequence of marker appearance differed from the in vivo developmental program. Ascl2 and Hand1, evident after 48 h in AFK, are characteristic of postimplantation trophoblast, whereas Cdx2 and Elf5, associated with primary trophectoderm, showed appreciable expression only at later stages (Fig. 1E). These results also differ from Dnmt1 mutants where up-regulation of Elf5 is thought to drive trophoblast transdifferentiation (22).

Finally, we transferred Dnmt3dKO cells from AFK into trophoblast medium (20). In contrast to parental cells, mutant cells flattened and some became very large with prominent nuclei (Fig. 1F). Propidium iodide staining showed a fraction with greater than 4N DNA content, consistent with polyploid trophoblast giant cell formation (Fig. 1G).

Transcriptome Analyses of ES Cell Differentiation Trajectory in the Absence of Dnmt3a/b.

We examined the initial misregulation of gene expression by single-cell qRT-PCR. We used Nanog (naive), Otx2 (formative), and Ascl2 (trophoblast) as representative markers (Fig. 2A and SI Appendix, Fig. S2A). The majority of parental cells down-regulated Nanog and gained Otx2 after 48 h in AFK. Dnmt3dKO cells similarly gained Otx2 but generally retained higher Nanog levels. Most strikingly, Ascl2 was up-regulated in more than half of the Dnmt3dKO cells and many of these were also positive for both Nanog and Otx2. Unexpectedly, we also detected a fraction of triple positive cells among parental cells (Fig. 2A and SI Appendix, Fig. S2A).

Fig. 2.

Dnmt3a/b-deficient ES cells adopt a deviant fate trajectory to postimplantation trophoblast. (A) Single-cell qRT-PCR analysis of ES cells and sorted Rex1::GFP^Lo AFK cells. Expression levels of Nanog (x axis), Otx2 (y axis), and Ascl2 (z axis) were normalized to beta-actin. (B) UMAP of parental and dKO ES cells and AFK 48-h cells. (C) UMAP colored to show cells coexpressing Nanog⁺Ascl2⁺ (red), Otx2⁺Ascl2⁺ (green, or Nanog⁺Otx2⁺Ascl2⁺ (blue) (Left) with enumeration (Right). (D) Pseudotime ordering. Proportions of each genotype of AFK 48-h cells in areas a– e are shown below. (E) Gene expression heatmap of 5,000 differentially expressed genes in pseudotime.

We extended this analysis using the 10× Genomics platform for single-cell RNA-sequencing (scRNA-seq). Uniform manifold approximation and projection (UMAP) analysis clustered cells by culture condition in the first dimension and by genotype in the second dimension (Fig. 2B). We inspected markers for pluripotency states, germ layers, and trophoblast (SI Appendix, Fig. S2B). In ES cells, expression was not significantly altered between parental and mutant. However, in mutant 48-h AFK cells we observed persistent expression of multiple naive genes, lower up-regulation of formative genes, and ectopic expression of several trophoblast genes, though not Elf5 or Cdx2, which were detected in only 0.1 and 0.7% of cells, respectively. We examined Nanog, Otx2, and Ascl2 using a unique molecular identifier (UMI) count above zero to classify expression. Nanog⁺Ascl2⁺, Otx2⁺Ascl2⁺, and Nanog⁺Otx2⁺Ascl2⁺ cells were present in both parental and Dnmt3dKO cells at 48 h (Fig. 2C). However, the combined proportion of dual and triple positive cells involving Ascl2 was three times higher in the mutants, consistent with single-cell qRT-PCR.

Pseudotime analysis using Monocle 2 (23) indicated a branchpoint in differentiation trajectory (Fig. 2D). We arbitrarily partitioned cells at and after the branchpoint into five groups (a–e). Parental cells were predominantly distributed along path 1, whereas mutant cells were almost exclusively located on path 2. Notably, however, 6.4% of parental cells initiated path 2, although very few reached the endpoint. Differentially expressed genes in parental cells featured formative markers on path 1 and trophoblast genes on path 2 (SI Appendix, Fig. S2C). Differential expression analysis without considering genotypes substantiated these alternative fates (Fig. 2E).

We investigated relatedness between path 2 and in vivo trophoblast. From published data (24) we identified genes up-regulated in E3.5 trophectoderm or E6.5 trophoblast relative to ICM and postimplantation epiblast, respectively. Correlation was low for E3.5 trophectoderm-enriched genes with either pathway. In contrast, many E6.5 trophoblast-enriched genes were up-regulated on path 2 (SI Appendix, Fig. S2D).

Chromatin Accessibility and Ascl2 Misexpression in the Absence of De Novo DNA Methylation.

Chromatin is remodelled during formative transition (25). We used assay for transposase accessible chromatin coupled to deep sequencing (ATAC-seq) (26) to survey chromatin accessibility in the absence of de novo DNA methylation. We analyzed ES cells, 48-h GFP^Hi transitional cells, and GFP^Lo posttransition cells and identified loci that are more open at 48 h in Dnmt3dKO cells than parental cells (Log2 fold change >0.7, P value <0.05). We classified four groups according to opening in transitional (GFP^Hi, groups I and III) or posttransition populations (GFP^Lo, groups II and IV), and presence or absence of a CpG island (CGI), annotated by the University of California Santa Cruz (UCSC) genome browser (Fig. 3A). The strongest peaks were associated with CGIs, which were lowly methylated at all stages (Fig. 3B and SI Appendix, Fig. S3 A and B). Non-CGI peaks were weaker and within regions that are methylated in parental cells, although a short stretch of reduced methylation was apparent at many group IV peaks (Fig. 3B and SI Appendix, Fig. S3 A and B). As expected, CGI peaks are mainly associated with annotated promoters and non-CGI peaks with enhancers (SI Appendix, Fig. S3C).

Fig. 3.

Ascl2 locus opens during formative transition and remains open in Dnmt3a/3b mutants. (A) Heatmaps of ATAC-seq intensity distributions grouped according to increased accessibility in Dnmt3dKO GFP^Hi (I and III) or GFP^Lo (II and IV) presence or absence of CGI. (B) Heatmap of CpG methylation across ATAC-seq peaks in ES cells and EpiLCs (10). (C) Genome browser examples of genes that are more open in Dnmt3dKO cells. (D) Expression of Ascl2 in pseudotime. (E) Ascl2 gene locus methylation pattern during ES-to-EpiLC transition (data from ref. 10). Dashed line marks the TSS. (F) Bisulfite Sanger sequence analysis of Ascl2 TSS upstream sequence. Region 1 is within CGI and region 2 is within the CGI shore. Filled circles represent methylated cytosine and open circles represent unmethylated cytosine. At least eight clones each were sequenced.

The analysis revealed chromatin regions that opened transiently in parental cells but remained accessible in Dnmt3dKO cells (groups II and IV, Fig. 3A and SI Appendix, Fig. S3B). Example genome browser tracks are shown in Fig. 3C. Genes associated with these peaks showed high correlation with differentially expressed genes in Dnmt3dKO GFP^Lo cells (SI Appendix, Fig. S3D). We also found a positive correlation with the set of E6.5 trophoblast-enriched genes (SI Appendix, Fig. S3E).

Thus, during exit from naive pluripotency, Dnmt3dKO ES cells fail to close down loci that normally open transiently during transition. These regions encompass promoters and proximal enhancers for a subset of E6.5 trophoblast genes that become misexpressed.

Transcription factor motif enrichment analysis across ATAC peaks at CGI loci in Dnmt3dKO GFP^Lo cells identified, among others, Ascl1 (SI Appendix, Fig. S3F). Ascl1 was not expressed in any of the samples studied, but the motif is shared with Ascl2, which, as noted above, is rapidly up-regulated in transitioning mutant cells (Fig. 3D). The Ascl motif was present in 531 of 609 promoter regions (−2,000 to +500 bp around the transcription start site [TSS]) of E6.5 trophoblast-enriched genes. In parental ES cells the Ascl2 locus opened during formative transition (SI Appendix, Fig. S3G) but in Dnmt3dKO cells, Ascl2 promoter accessibility increased further posttransition (SI Appendix, Fig. S3G).

Inspection of published bisulfite sequencing data (10) revealed as expected that the Ascl2 CGI is not methylated in ESCs or during naive-to-EpiLC transition. However, the flanking CGI shore gained methylation during transition (Fig. 3E). We confirmed gain of CGI shore methylation in parental cells in AFK that did not occur in dKO cells (Fig. 3F). CGI shores are thought to contribute to regulation of CGI genes (27). The CGI shore sequence (1,807 bp, within 2 kb upstream of the TSS) contains five Ascl2 motifs identified by JASPAR (28), consistent with autoactivation potential as reported in intestinal stem cells (29).

Previously it was found that ES cells deficient for Dnmt3a/3b gradually lost DNA methylation and after multiple passages could no longer form teratomas (14). That study was performed on ES cells cultured in serum, which have a hypermethylated genome compared to the early embryo. In 2iL medium used here the genome is hypomethylated, similar to the embryo (6⇓–8). To examine the acute effect of Dnmt3a/3b depletion we used selection for integration of gRNA and Cas9 expression vectors to achieve rapid enrichment for targeted cells. We transfected two ES cell lines and after 3 d, cells were transferred to AFK for a further 48 h. qRT-PCR with primers spanning the deletion registered a reduction in Dnmt3a and Dnmt3b transcripts indicating effective genome editing. The actual mutation frequency is likely to be higher due to small indels which are not detected. In the mixed populations, assayed almost immediately after Dnmt3a/3b depletion, we detected up-regulation of Rhox6, a gene previously reported to be regulated by DNA methylation (30), together with Ascl2 (SI Appendix, Fig. S3H). We carried out bisulfite sequencing of the Ascl2 locus following Dnmt3a/3b deletion as above. We detected gain of methylation in the CGI shore in parental cells after 48 h in AFK, that was reduced in the Dnmt3a/3b mutant population (SI Appendix, Fig. S3I). These findings indicate a direct effect of Dnmt3a/3b depletion on both expression and methylation of Ascl2.

Imprinted silencing of the paternal allele of Ascl2 in placenta does not involve DNA methylation (31). Instead, the imprinted lncRNA Kcnq1ot1 suppresses transcription in cis (32). We inspected levels of Kcnq1ot1 and found no change in Dnmt3dKO cells, indicating that loss of imprinting is not responsible for up-regulation of Ascl2 (SI Appendix, Fig. S3J).

We then investigated whether expression of Ascl2 is sufficient to impose trophoblast-like differentiation. For this we introduced an Ascl2–estrogen receptor (ER) fusion construct into parental ES cells. Upon tamoxifen (Tx) treatment in serum and LIF, cells changed morphology within 2 d, becoming larger and flattened (Fig. 4A). Oct4 was down-regulated and trophectoderm genes Elf5, Cdx2, and Tpbpa were up-regulated (Fig. 4B). Thus, misexpression of Ascl2 can initiate trophoblast-like differentiation.

Fig. 4.

Ascl2 propels transdifferentiation to trophoblast-like cells. (A) Images of ES cells cultured in serum/LIF with or without Tx induced activation of Ascl2–ER. (Scale bars, 100 µm.) (B) qRT-PCR analysis of marker expression during Ascl2–ER induction. (C) qRT-PCR analysis of trophoblast marker expression in neural differentiation culture by two clonal lines of Dnmt3dKO∆A2 cells. (D) Analysis of somatic lineage marker expression by Dnmt3dKO∆A2 cells in neural and mesendoderm induction protocol. Error bars represent SD from technical triplicates (B) or duplicates (C and D).

Finally, we tested whether activation of Ascl2 was necessary for trophoblast-like differentiation of Dnmt3dKO cells. We deleted Ascl2 to create Dnmt3dKOΔA2 cells (SI Appendix, Fig. S4A) and saw that the misregulation of trophoblast genes was abolished (Fig. 4C). Furthermore, at 24 h, Dnmt3dKOΔA2 cells expressed Otx2, Oct6, and Fgf5 formative markers (SI Appendix, Fig. S4B). However, neural genes were not subsequently up-regulated and mesendodermal differentiation remained inefficient (Fig. 4D), indicating later differentiation defects unrelated to Ascl2.

Dnmt3a/b Deficient Cells Are Outcompeted by Wild-Type Cells.

We investigated whether deletion of Ascl2 may restore contribution to chimeric epiblasts. We saw substantial colonization by Dnmt3dKOΔA2 cells in 5 of 10 epiblasts at E6.5 (Fig. 5A and SI Appendix, Fig. S5A). Moreover, we observed no donor cells in extraembryonic regions. At E7.5, contributions were reduced and appeared biased to the posterior region (Fig. 5B and SI Appendix, Fig. S5B). Immunostaining for Stella and Tfap2c showed no enrichment for primordial germ cells (PGCs) (SI Appendix, Fig. S5 C and D). By E9.5, we observed only sparse contributions in 4 of 11 embryos (Fig. 5C and SI Appendix, Fig. S5E).

Fig. 5.

Dnmt3a/b-deficient cells are outcompeted by wild-type cells and do not persist in chimeras. (A) Maximum projection confocal images of chimeric contributions to E6.5 embryos. Red, mKO2; blue, Eomes immunostaining. (Scale bar, 100 μm.) (B) Dnmt3dKO∆A2 ES cell chimeras at E7.5. The 25% opacity brightfield images are overlaid onto fluorescent image. (Scale bar, 250 µm.) (C) Dnmt3dKO∆A2 ES cell chimeras at E9.5. (Scale bar, 1 mm.) (D) FS cells established from parental and Dnmt3dKO∆A2 ES cells. (Scale bar, 100 µm.) (E) qRT-PCR analysis of FS cell markers. Error bar represents SD from technical duplicates. (F) Percentage of GFP-labeled WT FS cells and mKO2-labeled dKO-A2 FS cells in the mixed coculture. Passage zero (p0) is 2 d after plating. Error bar represents SD from two cultures. (G) Annexin V positive cells quantified by flow cytometry after 24 h in indicated coculture. Error bars represents SD from six experiments. P < 0.05. (H) Immunofluorescence images of cMyc parental FS cells (GFP), Dnmt3dKO∆A2 FS cells (mKO2), and coculture for 24 h. Dashed regions highlight mKO2 positive cells in coculture. (I) qRT-PCR analysis of marker expression during neural and mesendoderm differentiation of BCL2-transfected ES cells. Error bar represents SD from technical duplicates. (J) BCL2-Dnmt3dKO∆A2 ES cell chimeras at E9.5. The 20% opacity brightfield images are overlaid onto fluorescent images. (Scale bar, 1 mm.) (K) BCL2-Dnmt3dKO∆A2 ES cell chimeras at E12.5. (Scale bar, 1 mm.)

Progressive dilution of mutant cells in chimeras suggested a competitive disadvantage with wild-type host epiblast cells (33). To examine this possibility, we used formative pluripotent stem (FS) cells related to E6.0 epiblasts (34). We were unable to establish FS cells from Dnmt3dKO cells, likely due to disruptive effects of Ascl2 misexpression. However, Dnmt3dKOΔA2 ES cells could be converted into stable FS cell lines that expressed the core pluripotency factor Oct4 together with formative markers (Fig. 5 D and E). We cocultured equal numbers of GFP expressing wild-type and mKO2 expressing Dnmt3dKOΔA2 FS cells. Proportions of each genotype were quantified by flow cytometry. We observed reduction in the fraction of mutant cells by passage 0 that was further increased by passage 1 (Fig. 5F). In cocultures Annexin V staining was higher for Dnmt3dKO∆A2 FS cells during self-renewal and mesendoderm induction (P < 0.05) though not neural induction (P > 0.05) (Fig. 5G and SI Appendix, Fig. S5G).

Relative level of cMyc is a key determinant in cell competition (35⇓–37). By immunostaining we saw that Dnmt3dKOΔA2 FS cells have lower levels of cMyc protein than wild-type cells (Fig. 5H). Immunoblotting confirmed that cMyc expression was reduced in mutant cells (SI Appendix, Fig. S5F). These observations suggest that cMyc-based cell competition may cause elimination of Dnmt3dKO cells mixed with wild-type cells (36) and that inactivation of Ascl2 avoids only the first round of competition. The mechanism that reduces cMyc is unclear, but we surmise is secondary to other perturbations in the absence of de novo methylation.

We introduced BCL2 into Dnmt3dKOΔA2 ES cells. Under in vitro differentiation conditions BCL2 transfectants showed up-regulation of Sox1, Pax6, T, and Foxa2, although still below the levels observed for parental transfectants (Fig. 5I). After blastocyst injection we recovered five chimeras among seven embryos at E9.5. Contributions were variable, but substantial in three of the five (Fig. 5J). We also collected embryos at E12.5 and detected mKO2 fluorescence in two of the four specimens, with donor-derived cells in neural tissues and mesenchyme (Fig. 5K and SI Appendix, Fig. S5H). These contributions were noticeably lower than at E9.5, however, suggesting ongoing loss of Dnmt3a/b-deficient cells even in the presence of a strong survival factor.

Cell Culture.

ES cells were maintained in 2iL medium on gelatin-coated plates as described (18). 2iL medium consists of 10 ng/mL of mouse LIF, 1 µM of Mek inhibitor PD0325901, and 3 µM of GSK3 inhibitor CHIR99021 in N2B27 basal medium. EpiLCs were induced by plating 2 × 10⁵ ES cells in 20 ng/mL Activin A, 12.5 ng/mL bFgf, and 1% knockout serum replacement (KSR), in N2B27 medium on fibronectin-coated six-well plates. GFP high or low fractions were collected using MoFlo (Beckman Coulter) or FACS Fusion (BD) instruments. Neural differentiation was induced by plating 1 × 10⁵ cells in N2B27 basal medium on laminin-coated six-well plates. Mesendoderm induction was performed by plating 1 × 10⁵ cells in 10 ng/mL Activin A and 3 μM CHIR99021 in N2B27 medium on fibronectin-coated six-well plates. Trophoblast culture media were formulated as described previously (19, 20). For chimera studies, cells were stably transfected with pPBCAG-mKO2-IP plasmid by TransIT LT1 (Mirus) with pCAG-PBase plasmid and selected with 1 µg/mL puromycin. Human BCL2 was introduced by pT2PyCAG-hBCL2-IH by TransIT LT1 with pCAG-T2ase plasmid (43) and selection with 100 µg/mL of hygromycin B. To establish tamoxifen-inducible Ascl2 expression lines, pPBCAG-Ascl2ER-IN plasmid was cotransfected with pCAG-PBase plasmid into E14Tg2a ES cells and clones picked after selection with 250 µg/mL G418. DnmtdKO∆A2 FS cells were established by conversion of ES cells and maintained in 3 ng/mL of Activin A, 2 µM XAV939, and 1 µM BMS493 in N2B27 medium on fibronectin-coated plates as described (34). Alexa Fluor 647 conjugated Annexin V (BioLegend, Cat. No. 640911) was used for apoptotic cell detection by FACS Fortessa (BD).

Gene Targeting.

Dnmt3a or Dnmt3b single KO and double KO cells were established previously using CRISPR/Cas9 and gRNAs designed to excise sequences encoding the catalytic domain (15). Knockout cells were used within 15 passages and compared with wild-type cells of similar passage. To assess the effect of gene knockout acutely, gRNAs were cloned into a piggyBac vector with puromycin resistance cassette, pCML32 (34), and cotransfected with pPB-Cas9-IN and pCAG-PBase using TransIT-LT1 (Mirus) followed by selection with 1 µg/mL puromycin and 300 µg/mL G418. To rescue Dnmt KO cells, pPB-CAG-Dnmt3a-IP and pPBCAG-Dnmt3b-IZ were cotransfected with pCAG-PBase using TransIT-LT1 with selection in 1 µg/mL puromycin and 40 µg/mL zeocin. Ascl2 gRNAs were designed to excise exon2, which encodes the full-length protein. Clones were screened by genomic PCR. gRNAs and primers are listed in SI Appendix, Table S1.

Chimeric Embryo Experiments.

Reporter-expressing ES cells were dissociated and 10 to 15 cells injected into each blastocyst. Injected embryos were transferred to the uteri of pseudopregnant females or cultured in vitro for 24 h in M2 medium (Sigma-Aldrich) in 7% CO₂ at 37 °C.

Immunostaining.

Embryos and culture cells were fixed with 4% paraformaldehyde (PFA) at room temperature (for 15 min for cells and up to E7.5 stage embryos, 60 min for E9.5 and E12.5). Whole E6.5 embryos were stained with rabbit anti-Eomes antibody (Abcam, Cat. No. ab23345). Blastocysts were stained with rabbit anti-GFP (Invitrogen, Cat. No. A-11122) and rat anti-Sox2 (eBioscience, Cat. No. 14-9811-82). After the fixation, E9.5 and E12.5 embryos were incubated with 20% sucrose/phosphate buffered saline (PBS) overnight at 4 °C, then embedded in OCT cryo-embedding compound (Sakura, Cat. No. 4583). Sections were stained with rat anti-Sox2 (eBioscience) and fluorescein isothiocyanate (FITC)-conjugated mouse anti-cTnT antibodies (Abcam, Cat. No. ab105439) (E9.5 only). Nuclei were stained with DAPI. Embryos and sections were imaged by either a Leica SP5 or Zeiss LSM880 confocal microscope. E7.5 PGC were stained with anti-Tfap2c (Santa Cruz, Cat. No. sc-8977), anti-Stella (Abcam, Cat. No. ab19878), and rat anti-Sox2 (eBioscience, Cat. No. 14-9811-82). FS cells were stained with rabbit anti-cMyc antibody (Abcam, Cat. No. ab30272).

Western Blot.

Cells were lysed with RIPA buffer in the presence of protease/phosphatase inhibitor mixture (Invitrogen). Lysed cells were rotated for 20 min and sonicated in a Bioruptor (Diagenode). Cell lysates were cleared by centrifugation, and the supernatant was recovered. Protein concentrations were measured by the bicinchoninic acid (BCA) method (Pierce). A total of 15 µg of protein was loaded in each well. Blots were blocked with 5% BSA/TBS 0.1% Triton-X for 1 h at room temperature and incubated overnight with primary antibodies at 4 °C. Secondary antibodies were incubated for 1 h at room temperature and signals were detected with ECL Select (GE Healthcare) and Odyssey Fc (Li-Cor). NaOH (0.2 N) was used for stripping. Anti-cMyc (Abcam, Cat. No. ab30272) and anti–α-tubulin (Thermo Fisher Scientific, A-11126) were used.

qRT-PCR.

Total RNA was isolated with Relila RNA miniprep systems (Promega) and cDNA was synthesized using the GoScript Reverse Transcriptase system (Promega). qRT-PCR was performed with Taqman Gene Expression (Thermo Fisher Scientific) or Fast SYBR Green Master Mix (Thermo Fisher Scientific). Primers are listed in SI Appendix, Table S1.

Bisulfite Sanger Sequencing Analysis.

Genomic DNA was prepared with the DNeasy Blood and Tissue Kit (Qiagen). Purified genomic DNA was treated with an Imprint DNA modification kit to perform bisulfite conversion. Ascl2 promoter regions were amplified by nested PCR with the Touchdown protocol with LongAmp DNA Taq polymerase (New England Biolabs). The PCRs for both rounds were performed as follows: denaturing at 94 °C for 30 s, 10 cycles of gradient PCR, 94 °C for 15 s, 65 °C (annealing temperature reduced 1 °C per cycle) for 15 s, 65 °C for 30 s, and 35 cycle of 94 °C for 15 s, 56 °C for 15 s, and 65 °C for 30 s. A total of 2 µL of first PCR product was used for the nested PCR. PCR products were purified from agarose gel and cloned into TOPO cloning vector (Thermo Fisher Scientific). The sequence results were analyzed with QUMA (44).The primers are listed in SI Appendix, Table S1.

Single-Cell qPCR.

Custom Taqman probes were ordered for Nanog, Otx2, Ascl2, and beta-actin (Thermo Fisher Scientific). Single cells were collected directly into 96-well plates by flow cytometry (MoFlo, Beckman Coulter) and reverse transcription was performed as described previously (45). Cells that had Ct value of beta-actin >14 and no amplification were excluded from the analysis.

Single-Cell RNA-Seq.

ES cells and 48-h AFK cells were dissociated with accutase (BioLegend) and live cells were collected by flow cytometry (MoFlo, Beckman Coulter). Single-cell cDNA libraries were constructed using 10× Genomics technology and sequenced by the Genomics Core Facility, Cancer Research UK Cambridge Institute.

ATAC-Seq.

A total of 50,000 ES cells and AFK cells at 48 h were collected by flow cytometry (MoFlo, Beckman Coulter). Cells were washed with ice-cold PBS once then lysed in the buffer (10 mM Tris⋅HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂ 0.1% IGEPAL CA-630). The nuclear pellets were collected and Tn5 tagmentation and library construction were performed with the Illumina Nextera kit (FC-121-1030). DNA was purified with AMPure XP beads (Beckman Coulter).

Data Analysis.