Esrrb guides naive pluripotent cells through the formative transcriptional programme

Abstract

During embryonic development, naive pluripotent epiblast cells transit to a formative state. The formative epiblast cells form a polarized epithelium, exhibit distinct transcriptional and epigenetic profiles and acquire competence to differentiate into all somatic and germline lineages. However, we have limited understanding of how the transition to a formative state is molecularly controlled. Here we used murine embryonic stem cell models to show that ESRRB is both required and sufficient to activate formative genes. Genetic inactivation of Esrrb leads to illegitimate expression of mesendoderm and extra-embryonic markers, impaired formative expression and failure to self-organize in 3D. Functionally, this results in impaired ability to generate formative stem cells and primordial germ cells in the absence of Esrrb. Computational modelling and genomic analyses revealed that ESRRB occupies key formative genes in naive cells and throughout the formative state. In so doing, ESRRB kickstarts the formative transition, leading to timely and unbiased capacity for multi-lineage differentiation.

Extended Data Fig. 1. Gene signatures of different pluripotent states

a: Line chart showing dynamics of mRNA expression based on qPCR of four pluripotency markers (Tfcp2l1, Esrrb, Sall4, Oct4) in E14 cells during monolayer differentiation (withdrawal of either 2iL or 2i for 96h) both in 2iL (purple) and 2i (green). White circles indicate the mean of n=4 independent experiments, shown as dots. P-values indicate two-sided unpaired t-test between the indicated time points. b: Heatmaps showing Z-score normalised expression of all genes of each group (defined in Fig. 1d) in E14 cells differentiating from 2iL (purple box) and 2i (green box). Integration of n=2 independent biological replicates for each time point. See also Supplementary Table 2 for the biological processes enriched in the 6 signatures.

Extended Data Fig. 2. Transcriptional response changes during commitment

a: Bar plot showing the number of AP positive colonies in the clonal assay of cells cultured in 2iL and during differentiation (purple bars) and of cells in which 2iL was re-applied for 24h at the indicated time point (yellow bars, re-induction). Bars indicate mean +/-SD of n=8 independent experiments, shown as dots. Only the sample ‘24’ was measured in n=4 independent experiments. Two-sided unpaired Student t-test. b: Heatmaps showing Z-score normalised expression of selected genes for each group (naive, formative, committed) during differentiation and re-induction. Integration of n=4 independent experiments for each time point. c: Barplots showing expression by RNAseq of Jak/Stat direct targets (Socs3 and Stat3, orange), WNT targets (Cdx2 and Axin2, green) and FGF targets (Dusp6 and Spry4, purple) in differentiating cells and after re-induction with 24h of LIF. Mean +/-SD of n=4 independent experiments. d: UCSC genome browser visualisation of normalised ATAC-seq profiles at the indicated loci. Rectangles indicate peaks found only in 2iL (green) or only at 48h (red). Integration of n=2 biological replicates. e: Volcano plot summarising published RNA-seq data of ESCs cultured in Serum+LIF (S+L) or 2iL. Data were interpolated with the six groups of genes identified in Fig. 1 (naive early and late, formative early and late, committed early and late). f: Schematic representation of experimental strategy. Cells overexpressing pluripotency genes were mixed and differentiated for 96h. The clonal assay was then performed and cells were collected after 4 days. PCR on genomic DNA was used to identify factors enriched in pluripotent colonies. g: Bar plot showing quantification of AP positive colonies of cells overexpressing an empty vector or pluripotency factors, either maintained in 2iL or differentiated for 96h. Bars indicate mean n=2 independent experiments, shown as dots.

Extended Data Fig. 3. Characterisati on of ESC differentiation and regulation of Esrrb expression

a: Representative images of immunostaining for EpiSCs markers (Oct4 and T) in WT cells maintained in 2iL or differentiated for 96h in N2B27 or in presence of CHIR and Activin A to induce T expression. Scale bar=25μm. Similar results were obtained in n=2 independent experiments. b: Barplots showing expression by RNA-seq of key EpiSCs markers in WT cells maintained in 2iL or differentiated for 96h upon 2iL withdrawal. Mean of n=2 independent biological replicates is shown. n.d. indicates samples in which expression was undetectable or below 5 CPM. c: Violin plot showing quantification of mean intensity (arbitrary units) for ESRRB in E14 cells cultured in 2iL or differentiated for 48h, 96h or 120h (48, 96 120) or after reinduction with 2iL for 24h (48+24 and 96+24). At least 3 randomly selected fields for each sample have been measured. N=3 independent experiments were analysed. Each violin indicates an independent experiment. d: Left: Representative images of clonal assay followed by Alkaline Phosphatase staining of cells either maintained in 2iL or differentiated for 96h with or without the Gsk3 inhibitor CHIR (96+CHIR). Centre: Bar plot showing quantification of AP positive colonies. Bars indicate mean of 2 biological replicates, shown as dots. Right: Bar plot showing relative mRNA expression, measured by qPCR, for Esrrb. Bars indicate mean of 2 biological replicates, shown as dots. e: Barplot showing expression by qPCR of Esrrb in E14 cells cultured in 2iL, N2B27, ActivinA (20ng/ml), FGF2 (12.5 ng/ml) and inhibitors of TGF-beta (A83- 01, 1 μM) and FGF signalling pathways (PD173074, 0.5 μm) for 48h. Mean +/- SD of 3 independent biological replicates are shown as dots. f: ChIP-PCR analysis of E14 cells cultured in 2iL and differentiated for 24h, 48h, 72h and 96h in N2B27. Immunoprecipitation was performed using anti-ESRRB and anti-H3K27ac antibody followed by PCR with primers located on Esrrb intron or Tfcp2l1, Utf1 and Tcf15 promoter regions. Fold-enrichment over a negative region is plotted. Mean +/-SD of n=4 independent experiments, shown as dots.

Extended Data Fig. 4. Regulation of Esrrb expression

a: Genome browser snapshot of histone modifications on regulatory regions on Esrrb gene in naive (2iL, blue) and formative (48h, red) states. Integration of n=2 biological replicates. b: Top: Representative images of immunostaining for H3K27ac (green) and ESRRB (red) in E14 cells cultured in 2iL, differentiated for 84h (84) or after a pulse with 2iLIF for 24h at 84h (2iL pulse), with or without Sodium Butyrate (NaButy or H2O) treatment. Nuclei were identified by DAPI staining (blue). Scale bar: 25μm. Bottom: Barplot showing quantification of mean intensity for H3K27ac (blue) and ESRRB (red) immunostaining normalised to the 2iL H2O samples. Mean +/-SD of n=3 independent experiments, shown as dots. c: Plots showing abundance of the indicated histone modifications detected by CUT&RUN and DNA methylation in naive (2iL, left) and formative cells (48, right), on regions bound by ESRRB only in 2iL (‘2iL’), only at 48h (‘48’), or in 2iL and after 48h (‘2iL - 48’), identified in Fig. 4a-b. Integration of n=2 biological replicates. For ‘2iL’ and ‘2iL - 48’ regions we observed enrichment for H3K4me3, H3K27ac, H3K27me3 and H3K9me3 in naive cells. In formative cells, H3K4me3 and H3K9me3 decreased by ~50% while H3K27me3 was lost, while DNA methylation substantially increased. Those regions, where Esrrb binding increases at 48h (‘48’), are heavily DNA methylated and pre-decorated by H3K4me3 and H3K27ac in naive cells, while the repressive marks H3K9me3 and H3K27me3 are absent.

Extended Data Fig. 5. Esrrb KO clones characterisation

a: Left: Bar plot showing the number of AP positive colonies after clonal assay of cells with loxP sites flanking the second exon of both alleles of Esrrb (Esrrb fl/fl, dark blue) and Esrrb KO cells generated by Cre-mediated recombination (light blue), cultured in 2iL and differentiated for 24h, 48h and 72h in N2B27. Mean +/- SD of n=3 independent experiments, shown as dots. Right: Barplots showing expression measured by qPCR of Esrrb in Esrrb fl/fl (dark blue) and Esrrb KO (light blue) cells cultured in 2iL and differentiated for 24h, 48h and 72h. Mean of n=2 independent experiments. b: Schematic representation of edited alleles of 3 CRISPR-generated Esrrb KO clones. The edited genome is indicated in red. The blue sequence is an insertion. Black bars indicate deletions. c: Bright field images of 3 CRISPR-generated Esrrb KO clones, cultured in 2iL and after 48h of 2iL withdrawal. Scale bar: 300μm. d: Barplot showing expression by RNAseq of naive markers in WT E14 cells and in 3 CRISPR-generated Esrrb KO clones. WT values were set as 1. Mean of n=2 biological replicates.

Extended Data Fig. 6. Proliferation and viability analysis of Esrrb KO clones and FS differentiation

a: Left: Proliferation assay over 4 days of WT cells and Esrrb KO clones, cultured in 2iL. Mean +/-SD of n=3 independent experiments is shown. Right: Barplot showing percentage of dead cells measured by Propidium Iodide staining in two WT cell lines and 2 Esrrb KO clones. Boiled cells (95 degrees Celsius for 5 min) were used as positive control. Mean +/-SD of n=3 independent experiments is shown. P-value calculated with One-way ANOVA followed by Tukey multiple pairwise-comparisons. b: Gene Set Enrichment Analysis (GSEA) of key markers of Apoptosis and cell stress in WT and Esrrb KO cells cultured in 2iL (naive) and 48h (formative), which failed to detect any significant differences between WT and KO cells. P-values calculated by the GSEA software. c: Expression measured by qPCR of selected naive and formative genes in WT E14 cells (grey) and three Esrrb KO clones (blue) cultured in 2iL and after 24h, 48h and 72h of differentiation in N2B27. Mean of n=2 biological replicates is shown. d: Expression measured by qPCR of naive and lineage markers in Conditional Esrrb cells kept in 2iL+DOX. WT cells and Esrrb KO expressing a DOX-inducible empty vector (iEmpty) kept in 2iL+DOX are used as controls. Mean +/- SD n=3 independent experiments (dots) is shown. e: Gene expression of formative genes measured by qPCR in Conditional Esrrb cells cultured in 2iL+DOX (3^rd bar) and withdrawn of 2iL and DOX for 48h (4th bar). Esrrb KO and WT cells expressing an inducible Empty vector (iEmpty) differentiated for 48h were used as controls (2nd and 6th bars). Bars indicate mean +/-SD of n=5 independent experiments, shown as dots. One-way ANOVA followed by Tukey multiple pairwise-comparisons. f: Left: Experimental strategy used for FS cells generation from ESCs. Right: Representative images of WT cells cultures in AloXR medium for 3 passages (P1, P2 and P3). Scale bar: 25μm. Similar results were obtained in n=3 independent experiments. g: Relative mRNA expression measured by qPCR of naive and formative genes in E14 cells cultured in 2iL or AloXR medium for up to 3 passages. Mean of n=3 technical replicates.

Extended Data Fig. 7. FS cell differentiation of Esrrb KO clones

a: Gene Set Enrichment Analysis (GSEA) of key markers of Apoptosis and cell stress in WT and Esrrb KO cells cultured in 2iL (naive) and P1/48h (formative) failed to detect any significant differences between WT and KO cells. P-values calculated by the GSEA software. b: PCA of RNA sequencing data of WT and Esrrb KO cells during FS differentiation. Genes contributing to Principal Components PC1 and PC3 are indicated. N=3 independent biological replicates, shown as dots, for 2iL samples. N=4 for P1-P3 samples. N=2 for KO3 at P2 and P3. c: Heatmaps showing mean normalised relative mRNA expression measured by qPCR of naive and formative genes in WT and Esrrb KO cells cultured in 2iL or AloXR medium for up to 3 passages (P1, P2, P3). Mean of n=3 technical replicates. d: Representative images of immunostaining for TFCP2L1 and OTX2 in WT cells (left panels) and for OTX2 in Esrrb KO cells (right panels) cultured in 2iL or AloXR medium for up to 3 passages. Nuclei were identified by DAPI staining (blue). Scale bar: 25μm. Similar results were obtained in n=2 independent experiments. e: Left: Representative images of WT and Esrrb KO cells cultured in FGF2+ActivinA+XAV for at least 6 passages, to induce EpiSCs differentiation. Scale bar: 25μm. Right: Barplots showing gene expression measured by qPCR of naive (Esrrb and Klf4), general pluripotency (Oct4) and EpiSCs (Fgf5, T) markers in WT and Esrrb KO cells cultured in FGF2+ActivinA+XAV for at least 6 passages, to induce EpiSCs differentiation. Embryo-derived EpiSCs (OEC2 and GOF18) and WT E14 ESCs cultured in 2iL are used as controls. Mean +/-SD of N=4 biological replicates, shown as dots.

Extended Data Fig. 8. PGCLC differentiation of Esrrb KO clones

a: Normalised frequency of individual gRNAs (indicative of KO) targeting Esrrb during induction of PGCLC (CRISPR screening results from57). Dots indicate the mean of n=2 independent CRISPR screens. b: Left: Frequency of individual gRNA targeting Esrrb in EpiLC that have acquired correct formative status (Stella-) and EpiLC blocked from formative transition (Stella+). Note Esrrb gRNA (KO) are enriched in EpiLC that fail to acquire formative status, indicating a functional role for Esrrb in promoting the formative program. Right: Normalised frequency of individual gRNAs targeting Olfr568 as a representative negative control gene that should not influence the induction of PGCLC upon KO. Dots indicate the mean of n=2 independent CRISPR screens. c: Immunoblot of clonal lines derived from SGET ESC transiently transfected with Cas9 and gRNAs binding Esrrb coding sequence. Out of 5 independent Esrrb KO clones, 3 (A1.2, B2.1, A2.5) were randomly chosen for further validations. Beta-TUBULIN was used as loading control. The experiment was repeated 3 times with similar results. Esrrb KO clones do not display Esrrb protein expression, but a shorter mRNA can still be detected (Fig. 7d). d: Schematic representation of SGET activation during in vitro cell fate transitions of ESC (Stella+/Esg1+) into EpiLC (Esg1+) and early and late PGCLCs (Stella+) (adapted from Hackett et al., 2018). e: Total number of cells in SGET WT and Esrrb KO clones obtained after 3 days of PGCLC induction from EpiLC differentiation. Mean +/-SD of n=3 independent experiments (dots) is shown. f: Gene expression of selected genes in WT (grey) and n=3 independent Esrrb KO SGET lines (blue) at EpiLC, d3 and d5 PGCLC stages. g: Expression of the PGC-early (left) and PGC-late (right) geneset in EpiLC, d3 and d5 PGCLC from WT and Esrrb KO lines. Bars indicate the median, box indicates the 25th and 75th percentiles, whiskers represent median plus/minus the interquartile (25-75%) range multiplied by 2. Two-sided paired Student t-test, n.s. not significant. Integration of n=3 biological replicates for each sample. h: Gene expression of BMP direct targets in WT (grey) and n=3 independent Esrrb KO SGET lines (blue) at EpiLC, d3 and d5 PGCLC stages.

Extended Data Fig. 9. Differentiation of Esrrb KO clones in 2D and 3D

a: Heatmap showing Z-scored, mean-scaled, normalised gene expression, measured by RNA-seq, of master regulator genes for each of the three primary germ layers and trophoblast in WT cells and three Esrrb KO clones cultured in 2iL and after 24h, 48h and 72h of differentiation in N2B27. Integration of n=2 biological replicates for each sample. b: Representative images of WT cells cultured in N2B27 medium in matrigel for 48h, 72h or 96h, to allow 3D organisation and lumenogenesis. F-actin was labelled by Phalloidin staining (green) and immunostaining for the apical protein PODXL was performed (red). Scale bar: 30 μm. Similar results were obtained in n=5 independent experiments. c: Top: Barplot showing quantification of number of structured/field in WT and Esrrb KO cells cultured in N2B27 medium in matrigel for 48h, 72h or 96h. Bars indicate mean of 2 independent experiments, shown as dots. Centre: Violin plot showing quantification of Area (expressed in pixels) of >14 structures in WT and Esrrb KO cells. P-values calculated by two-way repeated measures ANOVA. Similar results were obtained in 3 independent experiments. Bottom: Violin plot showing quantification of the ratio of the 2 main diameters (roundness) of >17 structures in WT and Esrrb KO cells, as shown in the WT panel. P-values indicate two-sided unpaired t-test. Similar results were obtained in 3 independent experiments. Box plots show 1^st, 2^nd and 3^rd quartile, whiskers represent median plus/minus the interquartile (25-75%) range multiplied by 1.5. d: Line plots showing quantification of F-ACTIN intensity along the diameter of 3D structures obtained by culturing WT and Esrrb KO cells in N2B27 in matrigel for 48h, 72h or 96h. At least 8 structures were quantified from n=2 independent experiments. The shades indicate the SD. e: Violin plots showing quantification of OTX2 intensity in 3D structures obtained from WT and Esrrb KO cells cultured in N2B27 in matrigel for 48h. N>380 nuclei for each sample. Two independent experiments are shown (left and right). Box plots show 1^st, 2^nd and 3^rd quartile, whiskers represent median plus/minus the interquartile (25-75%) range multiplied by 1.5.

Extended Data Fig. 10. Network analysis of formative gene regulation by Esrrb

a: Barplot showing expression of Otx2 measured by qPCR in ES cells treated for 48h with ActivinA (20ng/ml), FGF2 (12.5 ng/ml) and inhibitors of TGF-beta (A83-01, 1 μM) and FGF signalling pathways (PD173074, 0.5 μm). Cells cultured in 2iL or N2B27 for 48h were used as controls. Mean +/-SD of n=3 independent biological replicates (dots) are shown. b: Genome browser snapshot of histone modifications at Otx2 enhancer (E) bound by Esrrb and promoter (P), in naive and formative cells. Profiles are the integration of n=2 biological replicates. c: ABN derived from a Pearson correlation threshold of 0.56 (see Methods). Solid black lines indicate required and definite interaction, dashed lines indicate optional interactions, red lines indicate disallowed interactions. Positive regulations are indicated by a black arrow, negative regulations are indicated by a black circle-headed line. d: Summary of 4 experimental constraints, each with initial (left column) and final (right column) conditions. Gene expression is discretized as high (blue) or low (white).

Figure 1. Transcriptional changes associated with irreversible exit from naive pluripotency.

a: Schematic representation of the first stages of exit from the naive state. Upon 2i or 2iL withdrawal, cells transit through a reversible phase before being irreversibly committed to differentiate. b: Top: Morphology and AP staining images after clonal assay of E14 cells cultured in 2iL and after 2iL withdrawal. Bottom: Barplot showing the relative number of AP positive pluripotent colonies after clonal assay of E14 cells cultured both in 2iL (purple) and 2i (green) and after the withdrawal of either 2iL or 2i every 12h for 96h. Mean +/-SD of n=3 independent experiments. Unpaired two-sided t-test ‘2iL 0’ vs ‘48’ p=0.97, ‘2iL 0’ vs ‘96’ p=0.0096. Scale bars= 30 μm. c: PCA of RNA sequencing data of cells differentiating from 2iL (purple) and from 2i (green). Genes contributing to the first two Principal Components are indicated. N=2 independent biological replicates for each time point, shown as dots. d: Top: Line plot showing expression dynamics of differentially expressed genes during differentiation, grouped by hierarchical clustering based on Pearson Correlation. Grey shades represent a 95% bootstrap confidence interval around mean values. Integration of n=2 independent biological replicates for each time point. Pie charts represent the intersection of the gene signatures with published gene sets of mouse embryo development at E4.5, E5.5 and E6.5. Bottom: Heatmaps show the sum of the log2-scaled normalised expression values of the intersection lists shown in the pie charts, averaged by different time points.

Figure 2. Differentiation reversibility is associated with Esrrb expression

a: UCSC genome browser visualisation of normalised ATAC-seq profiles at the indicated loci. Rectangles indicate peaks found only in 2iL (green), only at 48h, both in 2iL and 48h (purple). Integration of n=2 independent biological replicates for each time point. b: Ballon plot summarising the percentage of ATAC peaks containing a given motif, and the associated p-value, at the indicated time points. Peaks on promoters, peaks at 10Kb from TSS and all peaks were analysed and are represented in blue, green and orange respectively. Integration of n=2 independent biological replicates for each time point. c: Balloon plot summarising published ChIP-seq data of ESCs cultured in Serum+LIF from the Codex compendium. The size of each balloon indicates the fold enrichment, the colour indicates the statistical significance. d: left: Bar plot showing the number of AP positive colonies after clonal assay performed on cells overexpressing a pool of pluripotency genes and maintained in 2iL (grey bars) or differentiated for 96h (blue bars). Cells overexpressing an empty vector were used as control (empty). Bars indicate mean +/-SD of n=3 independent experiments, shown as dots. Right: Bar plot showing enrichment of genomic integrations of 8 naive genes in cells differentiated for 96h and plated for clonal assay compared to cells in 2iL. Bars indicate mean +/-SD of n=3 independent experiments, shown as dots. e: Representative images of clonal assay followed by Alkaline Phosphatase staining of cells overexpressing an empty vector or pluripotency factors, either maintained in 2iL or differentiated for 96h. N=2 independent experiments, quantified in Extended Data Fig. 2g. f: Immunostaining for ESRRB (green) in E14 cells cultured in 2iL or differentiated for 48, 96 or 120h (48, 96 120) or after reinduction with 2iL for 24h (48+24 and 96+24). Nuclei were identified by DAPI staining (blue). Scale bar: 25μm. N=3 independent experiments, quantified in Extended Data Fig. 3c.

Figure 3. Esrrb promotes the expression of formative genes

a: Schematic representation of E14 cells transfected with an inducible Esrrb-Ires-Venus vector (EIV) and differentiated for 96h with or without Doxycycline (DOX) treatment. Cells were sorted for presence or absence of Venus expression (EIV+ and EIV- respectively) and further characterised. b: Left: Representative images of Alkaline phosphatase staining after clonal assay of cells expressing EIV or an inducible Empty vector (iEmpty), cultured in 2iL or without 2iL for 96h (96), in the presence of DOX. Right: Barplot showing number of AP positive colonies in cells expressing EIV or iEmpty, cultured in 2iL or without 2iL for 96h, in the presence or absence of DOX (+D or - D). Mean +/-SD of 4 biological replicates. Two-sided unpaired Student t-test. c: Heatmaps showing mean-scaled normalised expression levels, measured by RNA-seq, of selected naive, formative and committed genes in E14 cells expressing EIV cultured in 2iL or differentiated for 96h in the presence or absence of DOX (96-DOX or 96+DOX respectively). Integration of n=2 independent experiments. d: Expression levels of selected naive and formative genes measured by qPCR in cells treated as described in Fig. 3b. Mean of n=2 independent experiments. Expression of naive genes is normalised to iEmpty cells kept in 2iL -D. Formative genes are normalised to E14 cells differentiated for 48h. e: Scatter plot showing transcriptome analysis of E14 cells expressing EIV cells differentiated for 96h in N2B27 with or without DOX. Down-regulated (Log2FC < -1 and p-value < 0.01) and Up-regulated (Log2FC > 1 and p-value < 0.01) genes are plotted on the left or right part of the panel respectively. The Y-axis indicates the mean expression on a log scale. Genes belonging to the 6 genes signatures described are represented by coloured dots. Selected genes are highlighted. Integration of n=2 independent experiments.

Figure 4. Esrrb promotes the expression of formative genes

a,b: ChIP-seq analysis of E14 cells cultured in 2iL and differentiated for 48h and 96h in N2B27. Time points are colour-coded in blue (2iL), cyan (48h) and yellow (96h). N=1 biological replicate. a: Venn diagram showing the intersection of significant ESSRB peaks for each given time point obtained by ChIP-seq analysis of E14 cells cultured in 2iL and differentiated for 48h and 96h in N2B27. b: Binding heatmaps displaying the read coverage density of ESSRB peaks along with average intensity. Peaks are grouped by the presence in one or multiple time points. For example, the “2iL - 48” group contains peaks found both in 2iL and after 48h of differentiation. c: Representative genome browser snapshots of selected gene loci bound by ESSRB in each given time point. Both reads distributions as line plots and peak intervals are displayed. N=1 biological replicate.

Figure 5. Esrrb coordinates the activation of naive and formative programs.

a: Left: Bar plot showing number of AP positive colonies after clonal assay of E14 cells transfected with a non-targeting control siRNA (siCo, dark grey), siGFP (light grey) or siEsrrb (blue) and cultured in 2iL or differentiated for 24h and 48h. Centre and Right: Expression analysis by qPCR of Esrrb and Tfcp2l1 genes in E14 cells transfected with siCo, siGFP and siEsrrb and differentiated for 24h or 48h. Bars indicate mean +/-SD of n=4 independent experiments, shown as dots. The 24h sample was analysed in n=3 experiments. b: Top: Immunoblot of clonal lines derived from E14 cell population stably expressing Cas9 and transfected with 2 gRNAs flanking Esrrb DNA-binding region. Three Esrrb KO clones were chosen (KO1, KO2, KO3). GAPDH was used as a loading control. Bottom: Barplot showing number of AP positive colonies after clonal assay of E14 cells (WT) and 3 Esrrb KO clonal lines cultured in 2iL and after 24h, 48h and 72h of differentiation. Mean +/-SD from n=3 independent experiments, shown as dots. c: PCA of RNA sequencing data obtained from E14 (WT) and 3 Esrrb KO clones cultured in 2iL and after 24h, 48h and 72h of differentiation. N=2 biological replicates for each data point, shown as dots. d: Transcriptome analysis of Esrrb KO cells cultured in 2iL and after 24h, 48h and 72h of differentiation in N2B27. Down-regulated and Up-regulated genes (P-value <0.05, FC >1 or <-1, compared to WT) are plotted on the left or right part of each panel. Genes belonging to the 6 genes signatures are highlighted with coloured dots. Integration of n=2 biological replicates for each cell line. Mean of the 3 independent Esrrb KO clones. e: Heatmaps showing mean-scaled normalised expression levels, measured by RNA-seq, of selected naive, formative and committed genes in WT and Esrrb KO clones. Integration of n=2 biological replicates for each cell line. Mean of the 3 independent Esrrb KO clones.

Figure 6. Esrrb is required for generation of FS cells

a: Experimental strategy used to induce Esrrb specifically at the time of activation of the formative program. b: Relative expression of Esrrb and selected formative genes measured by qPCR in Conditional Esrrb cells differentiated for 72h and treated with a pulse of DOX between 24 and 48h. Esrrb KO clones expressing an inducible Empty vector (iEmpty) served as controls. Bars indicate mean +/- SD of n=5 independent experiments for Tcf15 and n=3 for all other markers, shown as dots. c: Experimental strategy used to remove Esrrb at the time of activation of the formative program. d: Immunostaining for ESRRB in WT cells in 2iL and in Conditional Esrrb cells cultured either in the presence or absence of DOX for 48h. Esrrb KO cells expressing an Empty vector served as negative controls. Scale bar= 25 μm. Representative images from 3 independent experiments. e: Volcano plot depicting DEGs (adjusted P-value<0.05) in Esrrb conditional cells kept without DOX vs WT cells after 48h of differentiation. Genes belonging to the 6 gene signatures are highlighted with coloured dots. Total number of Formative early and Formative late genes down and up-regulated in Conditional Esrrb cells are coloured in green and purple in the bottom corners. Integration of n=5 independent experiments. f: RNAseq analysis in Esrrb conditional cells withdrawn of 2iL and DOX for 48h (3rd bar). WT cells expressing an inducible Empty vector (iEmpty) cultured either in 2iL or differentiated for 48h were used as controls (1st and 2nd bar). Bars indicate mean +/-SD of n=5 independent experiments, shown as dots. g: Representative images of WT and Esrrb KO cells cultured in AloXR for 3 or 9 passages. Scale bar: 25μm. Esrrb KO cells collapsed between passage 4 and 6 in n=3 independent attempts. h: Line plots showing gene expression during FS differentiation. Mean +/- SD of n=4 independent biological replicates, shown as dots.

Figure 7. Differentiation towards PGCs is impaired by loss of Esrrb

a: Left: Quantification of the percentage of Stella-GFP positive PGCLC at day3 (early) and day5 (late) of independent WT (grey) and Esrrb KO SGET lines (blue) (n=3 independent KO clones and matching WT controls, shown as dots). Bar indicates mean value. p-values indicate two-sided unpaired t-test. Right: Flow cytometry plots showing impaired induction of PGCLC in Esrrb-knockout (KO) cells. Percentages of Stella+ PGCLC are shown in representative plots of three independent WT and KO lines. b: PCA showing the developmental trajectory of independent Esrrb KO (blue) and matched-WT (grey) SGET lines during induction of PGCLC, based on the global transcriptome of n=3 independent KO clones and matching WT controls, shown as dots. c: Volcano plots depicting DEGs in Esrrb KO EpiLC, d3 and d5 PGCLC. Down-regulated and Up-regulated genes (adjusted p-value <0.05) are plotted on the left or right part of each panel respectively. Formative -early and -late and PGC -early and -late signatures (genesets) are highlighted with coloured dots indicating general shifts in activity of specific programs. Integration of n=3 independent KO clones and matching WT controls. d: Barplots showing gene expression of selected genes in independent WT (grey) and Esrrb KO lines (blue) at EpiLC, d3 and d5 PGCLC stages. Integration of n=3 independent KO clones and matching WT controls. See also Extended Data Fig. 8.

Figure 8. Impaired lumenogenesis in Esrrb KO 3D structures

a: Representative images of WT and Esrrb KO cells cultured in N2B27 medium in matrigel for 96h. Nuclei were identified by DAPI staining (blue). Left: Filamentous (F) ACTIN was labelled by Phalloidin staining. Right: Immunostaining for PODXL. Scale bars: 30μm. Similar results were obtained with 2 Esrrb KO clones in n=2 independent experiments. b: Barplot showing quantification of number of structures with an apically localised PODXL (polarised), with a defined central cavity (lumen) or negative for PODXL (negative) in WT and Esrrb KO cells cultured in N2B27 medium in matrigel for 48h, 72h or 96h. Bars indicate mean of n=2 independent experiments, shown as dots. c: Heatmaps showing mean normalised expression measured by qPCR of the naive gene Tfcp2l1 and formative genes in WT and Esrrb KO cells in 2iL or cultured in N2B27 in matrigel for 24h, 48h, 72h or 96h. Mean of n=3 independent experiments. Stars indicate p-value<0.05 calculated by two-sided unpaired Student t-test. d: Representative images of immunostaining for OTX2 in WT and Esrrb KO cells cultured in N2B27 in matrigel for 48h. Scale bar: 30μm. Similar results were obtained with 2 Esrrb KO clones in n=2 independent experiments. e: Heatmap showing Pearson’s correlation of naive, core and formative genes, obtained from RNAseq data of 2iL withdrawal (Fig. 1c-d) f: Left, trajectory followed by WT ESCs from the naive (step 0) to formative (step 9) to committed state (step 14) in a representative model. Right, trajectory followed by Esrrb KO cells. g: Network representation of the model used to calculate the trajectories shown in f. Black solid lines indicate interactions from active components. Grey lines indicate interactions not present, as they emanate from inactive components. Positive regulations are indicated by a black arrow, negative regulations are indicated by a black circle-headed line.