Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells
2021; Springer Nature; Volume: 40; Issue: 12 Linguagem: Inglês
10.15252/embj.2020106818
ISSN1460-2075
AutoresDaniel Olivieri, Eleonora Castelli, Yumiko Kawamura, Panagiotis Papasaikas, Ilya Lukonin, Melanie Rittirsch, Daniel Heß, Sébastien A. Smallwood, Michael Stadler, Antoine H.F.M. Peters, Joerg Betschinger,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle28 April 2021free access Source DataTransparent process Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells Daniel Olivieri Corresponding Author Daniel Olivieri [email protected] orcid.org/0000-0002-2140-7200 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Eleonora Castelli Eleonora Castelli Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Yumiko K Kawamura Yumiko K Kawamura Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Panagiotis Papasaikas Panagiotis Papasaikas Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Swiss Institute of Bioinformatics, Basel, Switzerland Search for more papers by this author Ilya Lukonin Ilya Lukonin Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Melanie Rittirsch Melanie Rittirsch Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Daniel Hess Daniel Hess Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Sébastien A Smallwood Sébastien A Smallwood Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Michael B Stadler Michael B Stadler Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Swiss Institute of Bioinformatics, Basel, Switzerland Search for more papers by this author Antoine H F M Peters Antoine H F M Peters orcid.org/0000-0002-0311-1887 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Joerg Betschinger Corresponding Author Joerg Betschinger [email protected] orcid.org/0000-0002-2627-8479 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Daniel Olivieri Corresponding Author Daniel Olivieri [email protected] orcid.org/0000-0002-2140-7200 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Eleonora Castelli Eleonora Castelli Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Yumiko K Kawamura Yumiko K Kawamura Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Panagiotis Papasaikas Panagiotis Papasaikas Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Swiss Institute of Bioinformatics, Basel, Switzerland Search for more papers by this author Ilya Lukonin Ilya Lukonin Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Melanie Rittirsch Melanie Rittirsch Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Daniel Hess Daniel Hess Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Sébastien A Smallwood Sébastien A Smallwood Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Michael B Stadler Michael B Stadler Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Swiss Institute of Bioinformatics, Basel, Switzerland Search for more papers by this author Antoine H F M Peters Antoine H F M Peters orcid.org/0000-0002-0311-1887 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Faculty of Sciences, University of Basel, Basel, Switzerland Search for more papers by this author Joerg Betschinger Corresponding Author Joerg Betschinger [email protected] orcid.org/0000-0002-2627-8479 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Author Information Daniel Olivieri *,1, Eleonora Castelli1,2, Yumiko K Kawamura1, Panagiotis Papasaikas1,3, Ilya Lukonin1, Melanie Rittirsch1, Daniel Hess1, Sébastien A Smallwood1, Michael B Stadler1,3, Antoine H F M Peters1,2 and Joerg Betschinger *,1 1Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 2Faculty of Sciences, University of Basel, Basel, Switzerland 3Swiss Institute of Bioinformatics, Basel, Switzerland *Corresponding author. Tel: +41 78 741 4008; E-mail: [email protected] *Corresponding author. Tel: +41 79 500 9513; E-mail: [email protected] The EMBO Journal (2021)40:e106818https://doi.org/10.15252/embj.2020106818 See also: A Janiszewski et al (June 2021) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network. Synopsis Embryonic stem cells (ESC) predominantly differentiate into epiblast-like fate as opposed to extraembryonic endoderm, but the molecular mechanism underlying this early developmental preference remains ill-defined. Using multi-layered expression profiling, this study identifies histone deacetylase Hdac3 and transcriptional corepressor Dax1 as key determinants of lineage barriers in mouse ESCs. Depletion of Hdac3 or Dax1 increases GATA6-dependent primitive endoderm (PrE) formation at metastable serum/LIF conditions. Dax1 inhibits mESC transdifferentiation by controlling nuclear receptor Nr5a2 activity. Hdac3 acts with corepressors Ncor1/2 to target a single enhancer upstream of the PrE-specifying transcription factor gene Gata6. Hdac3 or Dax1 mutant mESCs give rise to bona fide PrE in blastocysts. Introduction Binary cell fate decisions generate two distinct daughter cell types from one common progenitor. Once specified, cells need to prevent de-differentiation and transdifferentiation in order to stay fate-committed. The processes of lineage specification and maintenance can employ different mechanisms (Holmberg & Perlmann, 2012), yet the regulatory principles underlying these differences are not well understood. A well-studied developmental binary cell fate decision is the differentiation of the mouse embryonic day (E) 3.5 inner cell mass (ICM) into extraembryonic primitive endoderm (PrE) and pluripotent epiblast (EPI) (Hermitte & Chazaud, 2014). This is mediated by the lineage-specifying transcription factors (TFs) Nanog and Gata6 that are co-expressed in the ICM. Positive auto-regulation and mutual inhibition of Nanog and Gata6, modulated by extrinsic fibroblast growth factor (FGF) signaling, drives segregation into Gata6-expressing PrE and Nanog-expressing EPI at E4.5 (Simon et al, 2018). Developmental potency of the EPI is captured in vitro by mouse embryonic stem cells (mESCs) that predominantly give rise to embryonic cell types when injected into blastocysts (Beddington & Robertson, 1989). Although the lineage barrier toward extraembryonic endoderm is not as strictly maintained in mESCs in vitro (Canham et al, 2010; Niakan et al, 2010; Cho et al, 2012; Nigro et al, 2017; Rivron et al, 2018), the molecular mechanisms that impede PrE and favor embryonic differentiation are not well understood. Expression of Gata6 enables differentiation into extraembryonic endoderm stem cells (XEN cells) (Shimosato et al, 2007), raising the possibility that the Nanog-Gata6 antagonism that segregates EPI and PrE during mouse pre-implantation development maintains lineage separation in mESCs (Graf & Enver, 2009). Nanog is, however, largely dispensable for post-implantation development and mESC self-renewal (Chambers et al, 2007), suggesting operation of additional mechanisms that govern fate restriction (Holmberg & Perlmann, 2012). Different mESC states are stabilized by specific extrinsic signaling conditions (Smith, 2017): A naïve pluripotent ground state in the presence of 2 inhibitors and leukemia inhibitory factor (LIF) (2iL), and a metastable pluripotent state by fetal calf serum (S) and LIF (SL). Although both states are interconvertible, mESCs in SL heterogeneously express differentiation and pluripotency markers (Smith, 2017), inefficiently differentiate into Epiblast-like cells (EpiLCs) in vitro (Hayashi et al, 2011), and are thought to recapitulate a developmentally more advanced state than mESCs in naïve 2iL conditions (Schröter et al, 2015; Gonzalez et al, 2016). Here, we exploit the transition from the naïve to the metastable mESC state (Schröter et al, 2015) to define the mechanism of lineage restriction. We identify inhibitors of PrE differentiation, define their interaction with pluripotent TFs, and describe how they enact competition between PrE and EPI fate. Our findings reveal that silencing of a Gata6 enhancer by transcriptional repressors antagonizes lineage plasticity of the mESC gene regulatory network (GRN) and secures the pluripotent lineage. Results Hdac3 inhibits PrE differentiation of mESCs While working on a putative Hdac3 interactor, we genetically deleted Hdac3 in naïve TNG-A mESCs that express GFP under the control of the endogenous Nanog locus (Chambers et al, 2007) (Fig EV1A). Compared with wild-type (WT) controls, Hdac3−/− cells expressed higher levels of the Nanog reporter in 2iL, but rapidly downregulated Nanog when converted to SL (Fig 1A) and were lost upon further passaging, indicating undue differentiation specifically in metastable conditions. Click here to expand this figure. Figure EV1. Transcriptomics of Hdac3−/− cells A. Anti-Hdac3 Western blot of naïve Hdac3−/− TNG-A clones. Specific Hdac3 band is indicated. B, C. Pairwise Pearson correlations similar to Fig 1B, but using different embryo RNAseq samples (Mohammed et al, 2017) (B) or in vitro cell types (Anderson et al, 2017) (D). Primitive streak (PS) Extraembryonic endoderm cell states (nEnd, XEN), anterior definitive endoderm (ADE). D. Similar to Fig 1C with a magnified view of cluster 5. Canonical PrE marker genes are highlighted in red. E. GO terms enriched in cluster 5. Modified Fisher exact P-values as determined by DAVID (Huang et al, 2008) are shown. F–H. Indicated mRNA levels relative to WT cells of indicated genotypes and treatments after 3 days in SLRA (F) and 2 days in SL (H). Average and standard deviation (SD) of at least two independent clones. Indicated PrE marker mRNA expression relative to negative control siRNA transfection of cells transfected with indicated siRNAs after 2 days in SL (G). I. Similar to Fig 1C with a magnified view of a panel of naïve and general pluripotency, and post-implantation markers. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Naïve Hdac3−/− mESCs differentiate into PrE Nanog>GFP intensity of WT and Hdac3−/− TNG-A cells in 2iL and at d2 and d5 of SL exposure. Pairwise Pearson correlations of in vitro TNG-A and in vivo embryo (Boroviak et al, 2015) RNAseq samples. k-means clustering of gene expression changes relative to naïve WT TNG-A mESCs. Source data are available online for this figure. Source Data for Figure 1 [embj2020106818-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint To determine the transcriptional changes underlying this phenotype, we performed RNA sequencing (RNAseq) of WT and Hdac3−/− cells in the naïve ESC state, and after 1 and 2 days (d) in SL and epiblast-like cell (EpiLC) (Hayashi et al, 2011) differentiation conditions. Contrasting these results with existing datasets from the early embryo (Boroviak et al, 2015; Mohammed et al, 2017) using pairwise correlation (Figs 1B and EV1B) revealed that mESCs in 2iL are most comparable to the embryonic day E3.5 ICM (Gonzalez et al, 2016). It further showed that SL and EpiLC conditions drive naïve WT mESCs into cell states that are similar to the embryonic E4.5—E6.5 pre- and post-implantation EPI. In contrast, differentiating Hdac3−/− cells, in particular in SL, transcriptionally resembled the primitive and visceral (VE), but not definitive endoderm (Anderson et al, 2017) (Fig EV1C). k-means clustering identified a class of genes (cluster 5) that was selectively induced in differentiating mutant cells (Fig 1C). Cluster 5 is enriched for endoderm regulators by gene ontology analysis and includes the TFs Gata4, Gata6, and Sox17 that are required for PrE development in vivo (Hermitte & Chazaud, 2014) (Figs 1C and EV1D and E). Hdac3 therefore represses differentiation of mESCs into a cell state resembling the PrE upon transition from naïve to metastable conditions. We made use of the Hdac3-specific inhibitor RGFP966 to test the role of Hdac3 enzymatic activity. Exposure to RGFP966 did not increase transcription of the PrE markers Gata4, Gata6, Sox7, Sox17, and Pdgfrα after release from 2iL (Fig EV1F). RGFP966 may, however, only ineffectively block Hdac3 (Phelps et al, 2016) or lack specificity (Jia et al, 2016). We therefore engineered naïve mESCs to express catalytically inactive Hdac3 (Hdac3Y118F/Y118F), or depleted Ncor1 and Ncor2 by siRNA transfection and compound knockout (Sun et al, 2013) (Table EV1). PrE markers were upregulated in these cells after SL conversion, similar to Hdac3−/− mESCs (Fig EV1G and H). Hdac3, thus, acts as a deacetylase in complex with Ncor1/2 nuclear corepressors. Hdac3 and Dax1 shield mESCs from PrE differentiation in response to extrinsic developmental signals The induction of post-implantation EPI markers in Hdac3−/− cells was overall unperturbed (Fig EV1I), suggesting simultaneous activation of embryonic and extraembryonic gene expression. To test whether this is due to population heterogeneity, we deleted Hdac3 in a TNG-A-derived mESC line (G6C18) that in addition to Nanog reports endogenous transcription of Gata6 (Fig EV2A). After 3 days in the presence of SL, around 10% of mutant cells were positive for the Gata6 reporter (Fig EV2B). Addition of minimal amounts (1 nM) of retinoic acid (SLRA) increased this fraction to around 30% (Fig 2A and B). Intracellular flow cytometry analysis showed that Gata6 reporter-positive cells homogeneously expressed the PrE markers Sox17, Dab2, and Lama1 (Hermitte & Chazaud, 2014) (Fig EV2C). Furthermore, reporter activation required LIF and FGF but not BMP signaling (Fig 2B), suggesting that PrE differentiation of Hdac3−/− cells is driven by the same pathways that control progression of ICM into PrE in vivo (Hermitte & Chazaud, 2014; Morgani & Brickman, 2015). To explore PrE differentiation further, we turned to a 3D culture system. After 3 days in SLRA, single Hdac3−/− but not WT cells formed spatially organized spheroids (Fig EV2D and E): Nanog reporter-positive cells were enriched in the inside, while Gata6 reporter-positive cells co-expressing Sox17 and showing polarized distribution of the apical PrE marker Dab2 (Hermitte & Chazaud, 2014) were on the outside. This resembles formation of the polarized epithelial PrE cell layer on the surface of the epiblast in E4.5 embryos and indicates spatial lineage segregation in Hdac3−/− spheroids. Click here to expand this figure. Figure EV2. Characterization of Hdac3−/− and Dax1−/− cells Anti-Hdac3 Western blot of naïve Hdac3−/− G6C18 clones. Specific Hdac3 band is indicated. Representative Nanog>GFP and Gata6::mCherry fluorescence intensity plots in indicated genotypes after 3 days that were used for quantifications shown in Fig 2B. Quantification of Gata6::mCherry reporter signal, background staining, and Sox17, Dab2, and Lama1 immunofluorescence intensities in single, 3 days SLRA-differentiated Hdac3−/− cells binned by Gata6 reporter activity. Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3−/− naïve mESCs. DNA counterstain is Hoechst. Dashed white lines indicate the embryonic Gata6::mCherry negative part. Scale bar: 10 μm. Similar to Fig 1D. Arrowheads indicate Dab2- and Gata6::mCherry-positive PrE cells. Scale bar: 10 μm. Anti-Dax1, anti-Esrrb, and anti-Hdac3 Western blots for the genotyping of G6C18 mutant clones. Migration behavior of targeted proteins is indicated. Representative Nanog>GFP and Gata6::mCherry fluorescence intensity plots in indicated genotypes and conditions that were used for quantifications shown in Fig 2C. Quantification of Nanog>GFP geometric mean intensities in 2iL. Average and SD of at least two independent clones. Representative Nanog>GFP and Gata6::mCherry fluorescence intensity plots in Dax1-/- cells after 3 days that were used for quantifications shown in Fig 2E. Ratios of Nanog>GFP and Gata6::mCherry intensity (periphery/center) in spheroids of indicated genotypes. Dashed lines indicate the mean in WT cells. Download figure Download PowerPoint Figure 2. Hdac3 and Dax1 are required for PrE lineage restriction in response to FGF, LIF, and RA A–E. Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3−/− (A) and WT and Dax1−/− (D) cells after 3 days in indicated conditions. Fraction of Gata6::mCherry-positive cells in indicated genotypes and conditions after 3 days (B, E) and 4 days (C). Jak(i) blocks LIF, LDN19 BMP4, and PD03 FGF signaling. Average and SD of at least two independent clones. F, G. Quantification of spatial lineage segregation in spheroids. Representative spheroid segmentations of indicated genotypes (F). Z-score-normalized fluorescence distributions in mutants compared with WT spheroids (G). Positive Z-scores indicate peripheral, outside, and negative Z-scores central, inside, enrichment. Scale bar: 25 μm. Source data are available online for this figure. Source Data for Figure 2 [embj2020106818-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint To gain a more complete understanding of lineage restriction, we set out to determine the relationship of Hdac3 with two previously described inhibitors of PrE differentiation, Dax1 and Prdm14 (Khalfallah et al, 2009; Ma et al, 2010; Zhang et al, 2014). Naïve and SLRA-exposed Prdm14−/− cells generated in the G6C18 background were indistinguishable from WT controls. In contrast to Prdm14−/− and similar to Hdac3−/− cells, Dax1 mutants showed increased Nanog reporter levels in 2iL, and approximately 30% of the cells expressed the Gata6 reporter in SLRA (Figs 2C and D, and EV2F–H, Table EV1). Also, the signaling pathway dependencies for induction of the Gata6 reporter were the same for Dax1−/− and Hdac3−/− cells (Figs 2E and EV2I). In Dax1 mutant 3D aggregates, however, peripheral enrichment of Gata6 reporter-expressing cells was perturbed (Figs2F and G, EV2J), suggesting different roles of Dax1 and Hdac3 in spheroid self-organization. We note that Dax1 knockout and knockdown mESCs in SL have been described before, reporting apart from the induction of PrE markers (Niakan et al, 2006; Zhang et al, 2014; Fujii et al, 2015), lack of viability (Yu et al, 1998), loss of pluripotency (Niakan et al, 2006; Khalfallah et al, 2009), induction of 2-cell stage-specific genes (Fujii et al, 2015), and multi-lineage differentiation (Khalfallah et al, 2009). We speculate that these discrepancies arise because the absence of Dax1 in metastable, but not naïve, conditions destabilizes pluripotency and results in or exacerbates culture heterogeneity. Conversion into a bona fide PrE state in vitro To compare Gata6 reporter-positive cells with embryonic PrE, we performed RNAseq of purified Nanog- and Gata6-expressing mutant cells in SLRA. Principal component analysis (Fig 3A) revealed that Gata6 reporter-positive Dax1−/− and Hdac3−/− cells clustered with the E4.5 PrE, while Nanog reporter-expressing cells were more similar to the E4.5 EPI. Unsorted heterogeneous Hdac3−/− cells (Fig 1B) resided in between. Pairwise comparison (Fig EV3A) showed that the transcriptional differences between Gata6 and Nanog reporter-expressing cells in vitro and between the E4.5 PrE and EPI in vivo correlated as strong (Pearson correlation coefficient R = 0.42–0.50) as differences between the E4.5 PrE and EPI of two independent studies (R = 0.49) (Boroviak et al, 2015; Mohammed et al, 2017). Gene expression alterations in Hdac3 and Dax1 mutants relative to WT cells correlated in 2iL (R = 0.53), and in differentiated Nanog reporter- (R = 0.77) and Gata6 reporter- (R = 0.95) positive cells, demonstrating similar transcriptional roles of Hdac3 and Dax1 in the three cell states (Fig 3B). Notably, cluster 5 genes were deregulated in 2iL and in GFP-positive cells, indicating PrE priming (Fig 3B). Gata6-positive cells are therefore transcriptionally similar to embryonic E4.5 PrE. Figure 3. Transcriptional and functional characterization of in vitro PrE cells Principal component analysis of indicated samples. GFP sorted cells express the Nanog>GFP reporter and mCherry sorted cells the Gata6::mCherry reporter. Morula (MOR). Scatter plots of log2 fold change (FC) gene expression changes (ΔmRNA) between naïve Hdac3−/− or Dax1−/− mutants and WT mESCs (left), between Nanog>GFP-expressing Hdac3−/− or Dax1−/− mutants and WT cells (middle), and between Gata6::mCherry-expressing Hdac3−/− or Dax1−/− mutants and Nanog>GFP-expressing WT cells (right). Cluster 5 genes are colored and selected PrE TFs labeled. Representative control embryo, and embryos that were injected with labeled SLRA-differentiated WT cells purified for Nanog>GFP expression and mutant cells purified for Gata6::mCherry expression. Embryos are outlined with continuous line. Dashed lines indicate embryonic EPI and PrE compartments. Arrowheads point to injected cells. Scale bar is 25 µm. Quantification of the cell fate (based on expression of Nanog and Sox17) and the localization of injected cells. Average and SD of two independent experiments with at least 5 embryos per condition and experiment. tSNE maps of scRNAseq of WT, Dax1, and Hdac3 mutants after 3.5 days in SLRA. Cells are color-coded by k-means clusters (top left), genotype (top right), and expression of indicated genesets (bottom). Expression distribution of Gata6 and cluster 5 genes in single cells of indicated scRNAseq clusters, and E4.5_EPI and E4.5_PrE (Mohammed et al, 2017). Source data are available online for this figure. Source Data for Figure 3 [embj2020106818-sup-0005-SDataFig3.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Characterization of mESC-generated PrE cells Pairwise Pearson correlation of transcriptional differences between indicated samples. Individual correlation coefficients are indicated. Experimental scheme used to determine developmental competence of subpopulations. Quantification of the cell fate (based on expression of Nanog and Sox17) and the localization of unlabeled endogenous cells. Average and SD of two independent experiments with at least 5 embryos per condition and experiment. Similar to Fig 3E. Only cells of indicated genotypes are shown. Similar to Fig 3F, showing expression distribution of indicated transcripts. Source data are available online for this figure. Download figure Download PowerPoint To functionally substantiate this similarity, we decided to determine the developmental competence of mESC-derived PrE cells. To do so, we injected labeled Nanog>GFP-positive WT, and Gata6::mCherry-positive Hdac3 and Dax1 mutant cells into E3.5 blastocysts (Fig EV3B). Upon in vitro development for 2 days, Nanog and Sox17 expression and localization of labeled cells in chimeric embryos were determined (Fig 3C and D). This revealed that mutant cells maintained Sox17 expression and did not contribute to the Nanog-positive embryonic EPI, which was in contrast to WT cells that maintained Nanog expression. Notably, the spatial distribution of labeled WT and mutant cells in embryos was similar to endogenous Nanog- and Sox17-expressing cells, respectively (Fig EV3C). The PrE state generated by Hdac3 and Dax1 mutant mESCs in vitro is therefore functionally similar to the embryonic PrE. To explore PrE conversion in single cells, we performed single-cell RNAseq (scRNAseq) of WT, Hdac3, and Dax1 mutant cells in SLRA. k-means clustering and visualization using t-distributed stochastic neighbor embedding (tSNE) identified four scRNAseq cell clusters (Figs 3E and EV3D): Cells from all genotypes contributed to scRNAseq clusters 1 and 3. We note a shift of mutant cells along tSNE1 that likely reflects PrE priming. scRNAseq clusters 1 and 3 were enriched for the expression of genes specific to the pre- and post-implantation epiblast (Boroviak et al, 2015), respectively. scRNAseq clusters 2 and 4, in contrast, were exclusively populated by mutant cells. Consistent with the uniform expression of Sox17, Dab2, and Lama1 in Gata6 reporter-positive Hdac3 mutant cells (Fig EV2C), PrE markers, such as Gata6, Sox17, and cluster 5 genes, were homogeneously transcribed by scRNAseq cluster 2 cells, which was similar to the distribution in individual cells of the E4.5 PrE (Mohammed et al, 2017) (Figs 3F and EV3E). Moderate induction of these markers in scRNAseq cluster 4 suggests a transition state that bridges the pluripotent (scRNAseq cluster 1) and the PrE (scRNAseq cluster 2) cell states. Taken together, our transcriptional and functional experiments demonstrate that Hdac3 and Dax1 mutant mESCs convert into a bona fide PrE state in SLRA. Hdac3 and Dax1 independently restrict PrE fate and antagonize Nr5a2 and Esrrb Primitive endoderm conversion upon loss of Dax1 and Hdac3 was qualitatively and quantitatively highly similar, suggesting that Dax1 and Hdac3 may act together, potentially in a protein complex. However, affinity purification coupled to label-free quantitation by mass spectrometry (Fig 4A) revealed that Hdac3 co-immunoprecipitated subunits of the Ncor1/Ncor2 complexes (Gps2, NCor1, NCor2, Tbl1x, Tbl1xr1), but not Dax1. Vice versa, Dax1 formed a complex with the nuclear receptors Nr5a2 and Esrrb, but not with Hdac3. To test whether Hdac3 and Dax1 mechanisms of PrE repression were truly independent, we analyzed their genetic interaction by generating compound knockout cell lines (Fig EV2F, Table EV1). After 3 days in SLRA, PrE marker levels in Hdac3−/−; Dax1−/− double mutants were elevated two- to threefold compared with single mutants (Fig 4B). Notably, this additivity was due to a doubling of the fraction of cells expressing the Gata6 reporter, reaching more than 70% (Figs 4C and EV4A). Dax1 and Hdac3 therefore act in parallel pathways that threshold the probability of single cells to exit pluripotency and activate the PrE program. Notably, Gata6 reporter-positive cells were found at the rim and within Hdac3−/−; Dax1−/− spheroids (Fig EV4C and D), therefore presenting an intermediate phenotype compared with the single mutants. Although the greater number of Gata6-expressing cells in compound versus single knockout spheroids limits direct comparison, this observation is consistent with independent roles of Dax1 and Hdac3 in spheroid self-organization. Figure 4. Hdac3 and Nr5a2/Dax1 are genetically and biochemically distinct pathways repressing PrE differentiation A. Z-scores of high-confidence interactors coIPed by Hdac3, Dax1, Nr5a2, and Esrrb (indicated in red) in 2iL mESCs. B, C. Expression of PrE markers relative to Dax1−/− cells (B), and fraction of Gata6::mCherry-positive cells after 3 days in SLRA (C) in indicated genotypes. Average and SD of three independent clones. D. Gene expression changes in Dax1−/−, Nr5a2−/−, and Dax1−/−;Nr5a2−/− cells compared with WT cells in 2iL (upper) and after 2 days in SLRA (lower). Source data are available online for this figure. Source Data for Figure 4 [embj2020106818-sup-0006-SDataFig4.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Characterization of compound mutant cells Representative Nanog>GFP and Gata6::mCherry intensity plots of genotypes and conditions quantified in Fig 4C. Geometric mean intensity of Nanog>GFP reporter in 2iL. Average and SD of three independent clones. Similar to Fig 2F. Scale bar: 25 μm. Similar to Fig 2G. Similar to Fig 4D, but focusing on cluster 5 genes. Similar to Fig EV1I with a magnified view of a panel of naïve and general pluripotency, and post-implantation markers in indicated genotypes and conditions relative to naïve WT cells in 2iL. Source data are available online for this figure. Download figure Download PowerPoint Since Nr5a2
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