In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states

Abstract

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.

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Fig. 1: Naive and primed markers in the embryonic rosette.
Fig. 2: WNT and MEK signals couple successive steps in morphogenesis to pluripotency progression.
Fig. 3: WNT controls the initial phase of the naive-primed transition.
Fig. 4: MEK and WNT inhibition maintains rosette characteristics.
Fig. 5: OTX2 enables MEK-driven transition to primed pluripotency.
Fig. 6: A primed chromatin landscape in RSCs.
Fig. 7: RSCs remodel pHC.
Fig. 8: Rosette-specific formatting of pHC.

Data availability

Figures 3 and 4 have associated RNA-seq and single-cell RNA-seq data deposited in the GEO and are accessible through GEO series accession number GSE145727. Figure 6 has associated ChIP-seq data deposited in GEO with the accession number GSE112234 . Previously published RNA-seq data that were re-analysed here are available from ArrayExpress under accession codes E-MTAB-2958 and E-MTAB-5147. Previously published ChIP-seq data that were re-analysed here are available from the GEO under accession codes GSE23943 and GSE60204. Source data for Figs. 1, 2 and 48 and Extended Data Figs. 1, 36 and 8 are presented with the paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Code availability

No custom computer code was used in this study.

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Acknowledgements

We are grateful to the following colleagues for providing cell lines used in this study: B. Merrill (Tcf7l1–/–), A. Smith (RGd2), J. Wysocka (Tbx3-RFP;Oct6-GFP), A. Simeone (Otx2KO and Otx2OE) and M. Kyba (A2lox.Cre). We thank M. Muraro from Single Cell Discoveries for help with single-cell sequencing and data analysis, and R. van Albada and A. Korporaal for technical assistance. NWO ECHO.10.B1.064, TI Pharma D5-402, Marie Curie FP7-PEOPLE-2009-RG-256560, ZonMW 116006104 (all to D.t.B.), NWO-VIDI 864.12.007 (to H.M.) and FES NIRM (Dutch Innovation Award) supported this study.

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Authors

Contributions

A.N., I.E., E.v.G., L.V., D.K., J.L., J.S., M.M. and D.t.B. designed and performed experiments and analysed data. A.N., I.E., D.K., J.L., J.S., R.A.M.D., G.v.M., M.M., M.D., E.B.B., H.M. and D.t.B. prepared the manuscript. J.S., D.t.B., R.A.M.D., G.v.M. and H.M. designed, performed and analysed the ChIP-seq experiments. D.t.B., J.S., A.T.d.D., R.W.W.B. and W.F.J.v.I. performed and analysed the RNA-seq experiments. E.v.G., D.t.B. and N.C.R. performed and analysed the single-cell sequencing experiments. A.N., C.E., E.v.G. and E.B.B. prepared and analysed the chromosome spreads. A.M. assisted with the mouse experiments and Y.G. performed additional experiments. H.M. and J.H.J. designed, performed and analysed nucleoside mass spectrometry analyses. D.t.B. conceived the study and coordinated the work.

Corresponding author

Correspondence to Derk ten Berge.

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Extended data

Extended Data Fig. 1 Naive and primed markers in the embryonic rosette.

a,b, Counts of embryos of the indicated stages displaying a rosette or lumen and KLF4 (a) or OTX2 (b) expression status. a, and (b) show different litters. c, Individual colour channels for Fig. 1h (representative of 5 experiments). d, Individual colour channels for Fig. 1i (representative of 8 experiments). Scale bars 20 µm. Source data

Extended Data Fig. 2 WNT and MEK signals couple successive steps in morphogenesis to pluripotency progression.

(a, b) Representative images of rosettes and aggregates generated by ESCs 48 hrs after seeding in BME in the indicated conditions and stained for the indicated markers. a, KLF4 (green) and OCT6 (red). b, OTX2 (green) and KLF4 (red). 3 independent experiments with similar results. c, Rosettes generated by ESCs 48 hrs after seeding in BME and stained for PODXL live or after fixation and permeabilization. Permeabilized samples showed PODXL staining, incorrectly suggesting presence of a lumen, while live staining revealed that the PODXL was not exposed at the cell surface and no lumen was actually present (illustrative examples, repeated > 5 times). d, Examples to illustrate criteria for calling ESC aggregates displaying a lumen, using live PODXL staining combined with phalloidin to reveal polarization (illustrative examples, repeated > 5 times). e, Individual colour channels for Fig. 2n (representative of 3 experiments). f, Individual colour channels for Fig. 2o (representative of 4 experiments). Scale bars 20 µm (a–f).

Extended Data Fig. 3 WNT controls the initial phase of the naive-primed transition.

(a, b) Immunostaining for the indicated markers of ESCs plated in EpiLC conditions (a) or N2B27 medium (b) for the indicated number of days (single experiments). c, Dispersed colonies (dashed white circles) obtained after passaging of a blastocyst grown out in the presence of IWP2, MEK inhibitor and LIF (LIM). 15 from 18 and 12 from 13 embryos yielded cell lines. d, Chimeras (brown), obtained after blastocyst injection of newly derived LIM cell lines (LIMA2, LIMB1 and LIMB3), with mate (black) and pups (only for LIMA2). Brown pups indicate that germline transmission occurred. e, RT-PCR analysis of ESCs maintained for 6 passages in L2i or MEK/WNT-inhibited conditions and of EpiSCs. n = 3 (LIF/2i and LIM) or n = 2 (EpiSC) biological replicates. Scale bars 20 µm (a,b), 100 µm (c). Source data

Extended Data Fig. 4 MEK/WNT-inhibition maintains rosette characteristics.

a, Immunostainings and western blot for the indicated markers of R1 ESCs maintained in L2i or LIM for 6 passages or as indicated (3 independent repeats with similar results; western blot 1 experiment, full blot in Source Data). b, Plating efficiencies of LIM cells in the presence and absence of LIF (n = 3 biological replicates, R1, FN3 and IB10 cells). c, Female SV8 L2i ESCs, LIM cells and EpiSCs stained for Xist by RNA-FISH. Puncta indicating Xist expression (arrowheads) are visible in both L2i and LIM cells, while only EpiSCs display Xist clouds indicating X-chromosome inactivation (arrows) (two independent experiments). d, Doubling times of the indicated cell lines in the indicated conditions. p12 and p24 indicate passage numbers of the cell lines. e, t-SNE plot of single cell RNA-Seq data, and log2 expression levels of the indicated genes plotted into the t-SNE plot. n = 159, 178, 102 (R1) and 184, 149 and 164 (IB10) cells for L2i, LIM and primed, respectively. f, RSCs were cultured for 10 days in the indicated conditions and immunostained for OTX2 (red). Representative images from 3 independent replicates. g, To determine apicobasal polarity, the Golgi apparatus was marked by Gm130 antibody staining, imaged by confocal microscopy and colorized according to z-stack level. Basal side of the cells correspond to low z-levels (green), and apical side to high levels (red). The data show apical Golgi localization in LIM cells, indicating their apicobasal polarity. Three experiments with similar results. h, Live tracking of individual cell locations during 24 hrs of L2i ESCs, LIM cells on serum-coating, and LIM cells on BME-coating. 80, 47 and 20 cells, respectively. Representative of 4 experiments. Scale bars 20 µm (a,g), 10 µm (c,f), 100 µm (h). Source data

Extended Data Fig. 5 Otx2 enables MEK-driven transition to primed pluripotency.

a, RGd2 ESCs and RSCs were plated in primed conditions and analysed at several time points for GFP by flow cytometry. n = 3 independent replicates (3rd is shown in main Fig. 5a). b, ESCs and RSCs, carrying the Tbx3-RFP;Oct6-GFP reporters, were plated in primed conditions and analysed daily for expression of the reporters. Lines go through means, n = 3 independent experiments. c, Western blots showing OCT6 (only for IB10), ERK and phospho-ERK in IB10 and R1 ESCs and RSCs upon transfer to primed medium. Two (for OCT6) and three biological replicates. Full blots in Source Data. d, Immunostaining for OTX2 in TRE-Otx2 ESCs following induction by doxycycline for the indicated duration (single experiment). Scale bar 40 µm. Source data

Extended Data Fig. 6 A primed chromatin landscape in rosette-like stem cells.

a, Immunofluorescence and western blot analysis for DNMT3L expression in the indicated cell types (3 independent repeats with similar results; western blot 1 experiment). b, Individual colour channels for Fig. 6b, right panel. Representative of 5 experiments. c, Representative input plot for H3K27me3 ChIP-Seq for the bivalent cluster. (d,e) H3K4me3 (d) or H3K27me3 (e) ChIP-Seq correlogram of ESCs, RSCs and EpiLCs. Two biological repeats for each condition. (f, g) Average intensity plots of H3K4me3 and H3K27me3 in the H3K4me3 (f) or H3K27me3 (g) enriched cluster. Two biological repeats for each condition. h, Screenshots from the UCSC genome browser showing H3K4me3 (blue) and H3K27me3 (red) peaks on the Pou3f1 and Fgf5 genes. Scale bars 20 µm (a,b). Source data

Extended Data Fig. 7 Rosette-like stem cells remodel pericentric heterochromatin.

(a–d) Metaphase chromosome spreads of ESCs, RSCs and primed cells stained for the indicated markers. DAPI (blue), centromeres (red). e, Female RSCs (LIMA6) were cultured for 3 days in primed conditions and immunostained for the indicated markers. Arrowheads indicate H3K27me3-positive inactive X chromosomes demonstrating transition to primed pluripotency. f, RSCs were cultured for 10 days in the indicated conditions and immunostained for the indicated markers. Five (a–d) or three (e, f) biological replicates. Scale bars 20 µm (a-d), 10 µm (e, f).

Extended Data Fig. 8 Rosette-specific formatting of pericentric heterochromatin.

a, Counts of embryos of the indicated stages displaying pericentric H3K27me3 accumulation, and in which the presence of a rosette, KLF4 or OTX2 expression is detected. Embryos are the same as those of Extended Data Fig. 1a, b. b, Confocal microscopy images of E5.1 embryo stained for H3K27me3 (green), centromeres (magenta), OTX2 (red), and DAPI (blue). Some H3K27me3-positive pHC foci are indicated by white arrows (light blue, overlap of H3K27me3 and DAPI). Representative example from the embryos analysed in Extended Data Fig. 8a. Scale bars 20 µm. Source data

Supplementary information

Reporting Summary

Supplementary Tables 1–6

Supplementary Table 1. Related to Fig. 3c,d. Excel file containing RNA-seq data of R1 ESC time course in LIM and primed conditions. Supplementary Table 2. Related to Fig. 3e–g. Excel file containing RNA-seq data of R1 and CGR8 ESCs in L2i, LIM, 35 h in primed, and 4 days in primed conditions. Differential gene expression fold change and significance were estimated from biological duplicates using DESeq2 Bioconductor package v.1.18.1. The DESeq2 package uses a Wald test to establish P values, which undergo Benjamini and Hochberg adjustment for multiple testing. Supplementary Table 3. Related to Fig. 4. Contribution to chimeras of male LIM-derived cell lines. Supplementary Table 4. Related to Fig. 4f. Excel file containing GO analysis of genes differentially expressed between L2i ESCs and LIM cells (two biological replicates). The P values were calculated from minimum hypergeometric scores from a ranked gene list using GOrilla. Supplementary Table 5. Related to Fig. 6c–h. Excel file of genes with bivalent promoters in L2i ESCs, RSCs and Epiblast-like cells. Supplementary Table 6. Oligonucleotide primers, antibodies and cell lines used in the study.

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Neagu, A., van Genderen, E., Escudero, I. et al. In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states. Nat Cell Biol 22, 534–545 (2020). https://doi.org/10.1038/s41556-020-0508-x

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