Previous analysis of RNA sequencing (RNA-seq) data from human naive pluripotent stem cells reported multiple point "mutations" in cancer-related genes and implicated selective culture conditions. We observed, however, that those mutations were only present in co-cultures with mouse feeder cells. Inspection of reads containing the polymorphisms revealed complete identity to the mouse reference genome. After we filtered reads to remove sequences of mouse origin, the actual incidence of oncogenic polymorphisms arising in naive pluripotent stem cells is close to zero.
Resource9 March 2021Open Access Transparent process Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency Andreas Lackner Andreas Lackner orcid.org/0000-0003-1168-7947 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, AustriaThese authors contributed equally to this work as first authors. Search for more papers by this author Robert Sehlke Robert Sehlke Cologne Excellence Cluster Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work as first authors. Search for more papers by this author Marius Garmhausen Marius Garmhausen orcid.org/0000-0002-8617-388X Cologne Excellence Cluster Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work as first authors. Search for more papers by this author Giuliano Giuseppe Stirparo Giuliano Giuseppe Stirparo orcid.org/0000-0002-5911-8682 Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK Living Systems Institute, University of Exeter, Exeter, UKThese authors contributed equally to this work as first authors. Search for more papers by this author Michelle Huth Michelle Huth orcid.org/0000-0002-1152-9140 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, AustriaThese authors contributed equally to this work. Search for more papers by this author Fabian Titz-Teixeira Fabian Titz-Teixeira orcid.org/0000-0002-7007-9039 Cologne Excellence Cluster Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work. Search for more papers by this author Petra van der Lelij Petra van der Lelij orcid.org/0000-0002-7461-9645 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Julia Ramesmayer Julia Ramesmayer Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Henry F Thomas Henry F Thomas Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Meryem Ralser Meryem Ralser Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Laura Santini Laura Santini orcid.org/0000-0001-9968-2459 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Elena Galimberti Elena Galimberti Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Mihail Sarov Mihail Sarov Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author A Francis Stewart A Francis Stewart Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Biotechnology Center, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Austin Smith Austin Smith orcid.org/0000-0002-3029-4682 Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK Living Systems Institute, University of Exeter, Exeter, UK Search for more papers by this author Andreas Beyer Corresponding Author Andreas Beyer [email protected] orcid.org/0000-0002-3891-2123 Cologne Excellence Cluster Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Martin Leeb Corresponding Author Martin Leeb [email protected] orcid.org/0000-0001-5114-4782 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Andreas Lackner Andreas Lackner orcid.org/0000-0003-1168-7947 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, AustriaThese authors contributed equally to this work as first authors. Search for more papers by this author Robert Sehlke Robert Sehlke Cologne Excellence Cluster Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work as first authors. Search for more papers by this author Marius Garmhausen Marius Garmhausen orcid.org/0000-0002-8617-388X Cologne Excellence Cluster Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work as first authors. Search for more papers by this author Giuliano Giuseppe Stirparo Giuliano Giuseppe Stirparo orcid.org/0000-0002-5911-8682 Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK Living Systems Institute, University of Exeter, Exeter, UKThese authors contributed equally to this work as first authors. Search for more papers by this author Michelle Huth Michelle Huth orcid.org/0000-0002-1152-9140 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, AustriaThese authors contributed equally to this work. Search for more papers by this author Fabian Titz-Teixeira Fabian Titz-Teixeira orcid.org/0000-0002-7007-9039 Cologne Excellence Cluster Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work. Search for more papers by this author Petra van der Lelij Petra van der Lelij orcid.org/0000-0002-7461-9645 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Julia Ramesmayer Julia Ramesmayer Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Henry F Thomas Henry F Thomas Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Meryem Ralser Meryem Ralser Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK Search for more papers by this author Laura Santini Laura Santini orcid.org/0000-0001-9968-2459 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Elena Galimberti Elena Galimberti Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Mihail Sarov Mihail Sarov Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author A Francis Stewart A Francis Stewart Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Biotechnology Center, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Austin Smith Austin Smith orcid.org/0000-0002-3029-4682 Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK Living Systems Institute, University of Exeter, Exeter, UK Search for more papers by this author Andreas Beyer Corresponding Author Andreas Beyer [email protected] orcid.org/0000-0002-3891-2123 Cologne Excellence Cluster Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Martin Leeb Corresponding Author Martin Leeb [email protected] orcid.org/0000-0001-5114-4782 Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria Search for more papers by this author Author Information Andreas Lackner1,8, Robert Sehlke2, Marius Garmhausen2, Giuliano Giuseppe Stirparo3,4, Michelle Huth1, Fabian Titz-Teixeira2, Petra Lelij1, Julia Ramesmayer1, Henry F Thomas1, Meryem Ralser3, Laura Santini1, Elena Galimberti1, Mihail Sarov5, A Francis Stewart5,6, Austin Smith3,4, Andreas Beyer *,2,7 and Martin Leeb *,1 1Max Perutz Laboratories Vienna, University of Vienna, Vienna Biocenter, Vienna, Austria 2Cologne Excellence Cluster Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany 3Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK 4Living Systems Institute, University of Exeter, Exeter, UK 5Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 6Biotechnology Center, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Dresden, Germany 7Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany 8Present address: Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria *Corresponding author. Tel: +49 221 478 0; E-mail: [email protected] *Corresponding author. Tel: +43 1 4277 74644; E-mail: [email protected] The EMBO Journal (2021)40:e105776https://doi.org/10.15252/embj.2020105776 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 In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre- to post-implantation epiblast in utero. We identified 496 naïve state-associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition. SYNOPSIS Combining a haploid transposon-based screen and systems biology, this resource work identifies gene networks controlling the exit of mouse embryonic stem cells (ESC) from naïve pluripotency, and delineates signalling intersections involved. Transcriptional characterization of 73 differentiation-defective ESC lines defines discrete gene modules and signalling inputs essential for the exit from naïve pluripotency. Genetic depletion of specific exit genes in ESCs results in increased molecular similarity to the pre-implantation epiblast in utero. Identification of 496 naïve state-associated genes establishes an extended naïve pluripotency network conserved in primate embryos. The transcription factor Klf2 is only weakly wired into the mouse naïve pluripotency network. Introduction Mouse embryonic stem cells (ESCs) can self-renew in defined conditions in a state of naïve pluripotency (Smith, 2017). The ESC exit from naïve pluripotency provides an amenable experimental system for dissection of a cell fate decision paradigm (Buecker et al, 2014; Kalkan et al, 2017). Naïve pluripotency is under control of a gene regulatory network (GRN) containing the core pluripotency transcription factors (TFs) Pou5f1, Sox2 and naïve-specific TFs such as Nanog, Esrrb, Klf4 and others (Chen et al, 2008; Dunn et al, 2014; Niwa 2018). In defined cell culture conditions that include inhibitors against Mek1/2 (PD0325901) and Gsk3 (CHIR990201, CH; collectively termed "2i"), ESCs can be homogenously maintained in the naïve state (Ying et al, 2008). Within 24–36 h after withdrawal of 2i, ESCs transit into formative pluripotency, entirely losing naïve identity (Kalkan et al, 2017). During this transition, the naïve GRN is extinguished and expression of formative factors such as Otx2, Pou3f1, Dnmt3a/b and Fgf5 is initiated. A similar transition is evident during peri-implantation development, where the TF network maintaining naïve pluripotency dissolves between embryonic day (E) 4.5 and E5.5 (Boroviak et al, 2014; Acampora et al, 2016; Mohammed et al, 2017). The speed of the naïve to formative GRN transition is notable because (i) the cell cycle is around 12 h long; (ii) all factors that are required to establish and maintain naïve pluripotency are expressed robustly in naïve cells; and (iii) the naïve pluripotency network is recursively self-reinforcing. The rapid dissolution of naïve pluripotency implies the existence of circuit-breaking mechanisms. In recent years, we and others have identified various factors promoting ESC differentiation using screens in haploid and diploid ES cells (Guo et al, 2011; Leeb & Wutz, 2011; Betschinger et al, 2013; Leeb et al, 2014; Li et al, 2018). Robust assays employing ESCs expressing a Rex1 promoter-driven destabilised GFP reporter (Rex1::GFPd2) enable the dissection of the exit from naïve pluripotency in high resolution (Kalkan et al, 2017; Mulas et al, 2017). Rex1-GFP downregulation is initiated within 24h after 2i withdrawal (N24) and completed after 48h (N48). Nevertheless, the exact nature, mechanistic underpinnings and sequence of events during exit from naïve pluripotency remain only partially understood. In particular, we lack insight into how the different molecular components of the system co-operate to elicit proper cell fate transition. Here, we have driven a Rex1-GFP reporter screen to saturation, thus providing an extensive list of genes and pathways involved in the exit from naïve pluripotency. We utilised this information in a systems biology approach to explore regulatory principles of the exit from naïve pluripotency. To evaluate dependencies and causal relationships within the pluripotency and differentiation circuitries, we probed the response of the differentiation programme to a comprehensive series of exit factor gene knockouts. Through computational integration of molecular profiling data with regulatory networks and in vivo GRN trajectories, we expose the regulatory foundations of a cell fate choice paradigm at a pivotal junction in early mammalian development. Results Haploid ES cell saturation screen Haploid ES cells are an efficient platform for insertional mutagenesis-based screens (Elling et al, 2011; Leeb & Wutz, 2011; Kokubu & Takeda, 2014). We previously reported a medium-scale screen comprising approximately 5 × 104 mutagenic events to identify factors regulating the exit from naïve pluripotency (Leeb et al, 2014). We have now driven this approach to saturation by assaying approximately 1.2 million mutations in receptive genomic regions that cause delays in Rex1 downregulation in two independent Rex1-reporter cell lines (Fig 1A and B), utilising three different mutagenic transposon vectors in 35 independent screens (Fig 1C and D). Figure 1. Establishment of exit factor deficient KO ESC lines informed by a haploid ESC saturation screen Illustration of the Rex1-GFPd2 reporter cell line and its exit from naïve pluripotency. Rex1-GFPd2 (in short Rex1-GFP) expression is tightly linked to naïve pluripotency. Shutdown of GFP expression indicates commitment to differentiation. FACS analysis of Rex1-GFP reporter levels throughout a 72h differentiation time course after 2i withdrawal. Scheme of the screening strategy to identify candidate genes involved in the exit from naïve pluripotency. After random insertional mutagenesis using piggyBac transposon-based gene-trap vectors, haploid Rex1-GFP ESCs were released into differentiation. Cells maintaining GFP expression after exposure to differentiation conditions were isolated and the gene-trap insertion sites mapped. The cumulative number of hits (red) and the cumulative number of novel hits (blue) in 35 independent insertional mutagenesis screens in haploid ESCs are shown. Representative Rex1-GFP FACS plots showing the differentiation delays 24 h after 2i withdrawal of Tcf7l1, Rbpj, Trim71, Smg5 and Pten KOs. A Myc KO served as a negative control. Blue indicates the Rex1-GFP FACS profiles for KO, and dashed lines indicate WT. Differential expression of genes at N24 versus 2i in WT RC9 cells. Black dots show significance (FDR ≤ 0.05, H0: |log2FC| < log2(1.5)). Pluripotency genes are red dots, formative genes are orange dots, haploid screen hits are blue dots, and the 73 KO genes are green dots. t-SNE projection of the 73 KOs in 2i, based on expression of the 3068 differentially expressed genes between N24 and 2i in WT. The strength of differentiation delay observed at N24 in the respective KOs are indicated by a colour gradient and measured as average naïve marker log2 fold change (log2 FC, based on expression levels of Esrrb, Nanog, Tfcp2l1, Tbx3, Prdm14 and Klf4, Zfp42) in the respective KO at N24. Red: delayed differentiation; blue: accelerated differentiation. Similar to (G) for KOs at N24. Download figure Download PowerPoint Stringent filtering resulted in a candidate list comprising 489 genes (Dataset EV1). These screens generated a candidate inventory of the machinery that mediates exit from naïve pluripotency. Reassuringly, the known exit from naïve pluripotency regulators Tcf7l1, Fgfr2, Jarid2 and Mapk1 (Erk2) (Kalkan & Smith, 2014) were among the highest ranked genes (Appendix Fig S1A). The candidate hit genes were enriched for processes involved in transcription regulation, epigenetics and signalling-related functions (Appendix Fig S1B and C; Dataset EV1), as well as RNA-binding functions in-line with emerging evidence of RNA regulatory mechanisms in cell fate control (Ye & Blelloch, 2014). Many identified genes are not specific to pluripotency and have functions in common pathways and processes, implying that the exit from naïve pluripotency utilises widely expressed cellular machinery. Therefore, mechanisms mediating ESC transition might also be utilised in other differentiation processes. Establishment of a mutant ESC library for systematic transcriptional profiling To characterise deficiencies in naïve exit in molecular detail, we generated KO ESC lines deficient for 73 selected genes, comprising top ranked genes from the mutagenic screen. We also included components from pathways and protein complexes for which multiple members were recovered, even if just below the cut-off threshold (e.g. the Paf complex member Leo1, the mTORC1 regulator Tsc2 and the NMD component Smg6), and Mbd3, Zfp281 and L3mbtl3 as known players in the exit from naïve pluripotency (Betschinger et al, 2013). Three control genes were included that are either not expressed in ES cells (Nestin), expected to be neutral (Hprt) or whose ablation was expected to accelerate differentiation (c-Myc). Paired gRNAs were used to disrupt target genes in a diploid biparental Rex1::GFPd2 reporter ES cell line carrying a Cas9 transgene (henceforth termed RC9 cells) (Fig 1A) (Li et al, 2018). Following an efficient parallelised approach, we established passage matched and isogenic homozygous KO cell lines, thus maximising comparability (Appendix Fig S1D and E). All KOs were validated by genomic PCR, followed by Sanger sequencing when required. Full protein deficiency was validated for 14 KOs (Eed, Suz12, Jarid2, Kdm6a, Smg5, Smg6, Smg7, Tsc2, Pten, Raf1, Tcf7l1, Leo1, Nmt1 and Csnk1a1) (Appendix Fig S2A). Only heterozygote clones could be generated for Mapk1 (Erk2), resulting in reduced protein levels. Notably, increased levels of Erk1 in Erk2 heterozygous mutant cells failed to rescue the strong differentiation delay in the Mapk1het KOs. For five further genes (Alg13, Dido1, Msi2, Etv5, Jmjd1c), we confirmed the absence of the corresponding transcripts or specific out of frame deletion of an exon by RT–qPCR or Sanger sequencing of RT–PCR products. Successful rescue experiments using 3xflag-tagged transgenes for six genes (Rbpj, Etv5, Fgfr1, Jarid2, Mbd3 and Tcf7l1) established causality between the observed genotype and phenotype (Appendix Fig S2B and C). Thus, all tested knockouts showed the expected impact on RNA or protein expression. However, we cannot exclude the possibility of hypomorphic phenotypes in some cases. ESC differentiation behaviour is highly dependent on cell density and timing of medium changes. To enable robust comparison of the differentiation of multiple KO ESC lines, we performed parallel differentiations batch wise in duplicates, always including WT ESCs and negative controls across seven experiments. At N24, we assayed the differentiation status by FACS analysis (Fig 1E) and extracted RNA for transcriptome analysis. The exit machinery is already poised in 2i Batch-corrected RNA-seq data (Appendix Fig S3A and B) comprising 14 replicates of WT ES cells identified 3068 differentially expressed genes (DEGs) between 2i and N24 (H0: |log2FC| < log2(1.5), FDR ≤ 0.05; Fig 1F, Appendix Fig S4A–C and Dataset EV1). Interestingly, most of the 489 genes identified in the haploid screens including the 73 genes (Dataset EV6) selected for KO did not change significantly in expression between 2i and N24 and were not present in the list of DEGs (Fig 1F and Appendix Fig S4C), with only 21% of screen hits showing differential expression at N24 (6% up-, 15% downregulated). This implies that the exit machinery is already embedded in the ground state and ESCs are poised for rapid decommissioning of naïve identity and entry into differentiation (Kalkan & Smith, 2014). To facilitate interrogation of the KO gene expression datasets, we developed an interactive online tool (GENEES—Genetic Network Explorer for Embryonic Stem Cells—http://shiny.cecad.uni-koeln.de:3838/stemcell_network/). Using t-Distributed Stochastic Neighbour Embedding (t-SNE), we visualised similarities between KOs based on expression of the DEG in 2i and at N24 (Fig 1G and H). We observed clustering of members of the same complex or pathway: Eed-Suz12 (PRC2), Ptpn11-Raf1-Fgfr1-Etv5 (Fgf/ERK), Smg5-Smg6 (NMD; nonsense mediated decay), Mta3-Mbd3 (NuRD) and Pten-Tsc2 (mTORC1 signalling) (Fig 1G and H). The transcription profiles of the KO ESCs clustered by genotype, but mainly by culture condition (2i and N24) (Appendix Fig S4A). This is consistent with the observation that despite manifesting differentiation delays at N24, all of the KO ESCs ultimately departed from the naïve state during longer differentiation time courses, as measured by loss of Rex1-GFP. Furthermore, even KOs that showed extensive Rex1-GFP downregulation delays at N24 displayed transcription profiles that were globally adjusted towards differentiation. Therefore, the knockout of a single gene is not sufficient to permanently block exit from naïve pluripotency in culture, in accord with the finding that ternary depletion of Tcf7l1, Etv5 and Rbpj is required for sustained self-renewal in the absence of 2i or LIF (Kalkan et al, 2019). The exceptional role of Csnk1a1 and the involvement of compensatory mechanisms At N24, the Csnk1a1 KO clustered with 2i samples (Appendix Fig S4A), indicating a special behaviour for this mutant. However, at N48 most Rex1-GFP expression was downregulated, illustrating a strong delay but not a block in differentiation (Appendix Fig S5A). siRNA treatment as well as treatment with Epiblastin A, a chemical inhibitor of Csnk1a1 (Ursu et al, 2016), delayed the exit from naïve pluripotency without apparently affecting proliferation within the duration of the assay (Appendix Fig S5B–D). Csnk1a1 is a serine threonine kinase and a component of the beta-catenin destruction complex. Although KO of another destruction complex member Apc, or of the downstream repressor Tcf7l1 resulted in the upregulation of similar gene-sets (Appendix Fig S5E), we observed stronger differentiation defects and larger amplitude of gene deregulation in two independently derived Csnk1a1 mutants. However, these mutants also exhibited markedly impaired cell cycle profiles in N2B27-based media (Appendix Fig S5F). Upon continuous culture (~5 passages), proliferation was restored in Csnk1a1 KO cells and differentiation potential was regained, suggesting upregulation of compensatory mechanisms and a likely effect of proliferation rate on differentiation kinetics, complicating mechanistic characterisation. A second case of phenotype adaptation was observed in Pum1 mutants. Pum1 KOs showed pronounced differentiation delays during early passages (Appendix Fig S5G), as also seen for acute Pum1 depletion by siRNA and in previously generated CRISPR KO ESCs (Leeb et al, 2014). However, the phenotype was lost in later passages, and Pum1 KO cells showed WT-like Rex1-GFP expression levels at N24 (Fig 2A and Appendix Fig S5G). Figure 2. Systematic transcriptional profiling of a mutant ESC library Naïve (top) and formative (bottom) marker gene expression changes at N24 compared with WT in all 73 KO ESCs. Clustering based on naïve marker gene expression shows a wide distribution of differentiation delay phenotypes. Rex1-GFP FACS analysis of Jarid2 and Tcf7l1 KO ESCs after transfection with negative control or Klf2-specific siRNAs. Plot showing a comparison of the log2 fold change (KO versus WT) of mean naïve marker gene expression at N24 to the extent of change of the global transcriptome between 2i and N24 (defined as the correlation of log2FCs in WT differentiation and log2FCs in KOs at N24). Each dot corresponds to one of the 73 KOs. Pearson's correlation is shown in the plot. Comparison of the number of genes deregulated in 2i and at N24 in all KOs (FDR ≤ 0.05, H0: |log2FC| < log2(1.5)). Differentiation phenotypes are colour coded according to average naïve marker log2FCs in KOs at N24. Rex1-GFP FACS analysis of WT, Jmjd1c and Tcf7l1 KO cells at N48 cultured with and without the Gsk3 inhibitor CH. Download figure Download PowerPoint Robust feedback wiring in the naïve TF network Interestingly, the transcriptome data revealed that exit factors do not, in general, reduce naïve transcription factor (TF) expression in the ground state. However, Rbpj KO resulted in moderate but significant increases in Klf4, while the aforementioned Csnk1a1 KO ESCs showed limited upregulation of both Klf4 and Tbx3 in 2i (Fig EV1A). Ctbp2 KO significantly upregulated Nr0b1 in the ground state. Other KOs had no significant effects on factors of the naïve network in 2i. These data are consistent with robust feedback wiring in the naïve TF network (Dunn et al, 2014; Niwa, 2018) and neutralisation of most differentiation factors in 2i in culture (Martello & Smith, 2014). In contrast, we observed a more extensive impact on formative markers. Several KO cell lines showed lower baseline expression in 2i of Otx2, Fgf5, Dnmt3a/b and Pou3f1 (Oct6) (Fig EV1B). In-line with recent results, we noted that depletion of several Fgf/ERK components resulted in reduced Dnmt3a/b, Pou3f1 and Fgf5 expression in 2i (Kalkan et al, 2019). Although Fgf/ERK signalling is effectively inhibited in 2i (Ying et al, 2008), our data suggest that either residual pathway activity or potential moonlighting functions of pathway components mediate poised expression of the formative pluripotency programme in 2i. Click here to expand this figure. Figure EV1. Systematic transcriptional profiling of a mutant ESC library RNA-seq-derived fold changes relative to WT of indicated naïve marker genes in indicated KOs in 2i (colour scale shows FDR, only genes with FDR ≤ 0.05 are shown). RNA-seq-derived fold changes relative to WT of indicated formative marker genes in indicated KOs in 2i (colour scale shows FDR, only genes with FDR ≤ 0.05 are shown). Rex1-GFP FACS analysis of WT cells in 2i, at N24 and at N30, transfected with negative control or Klf2-specific siRNAs. Comparison of the number of differentially expressed genes at N24 (FDR ≤ 0.05, H0: |log2FC| < log2(1.5)) to the average naïve marker log2FC at N24 (phenotype strength). Correlation of Tcf7l1 KOs to Jmjd1c KOs regarding log2FCs at N24 between knockout and WT control. Each dot corresponds to one gene. Only genes showing significance (FDR ≤ 0.05, H0: |log2FC| < log2(1.5)) in either one of the two KOs are plotted. Red line: total least square regression; regression coefficients are shown. Download figure Download PowerPoint Clustering based on the expression of ten naïve pluripotency marker genes showed that the downregulation of the naïve pluripotency TFs during formative differentiation is defective across multiple KOs (Fig 2A). Although overall expression of the naïve TFs was highly correlated, Klf2 appeared to be an exception. Klf2 downregulation was notably impaired in Tcf7l1 KO ESCs, whereas it was unaffected by several KOs, including Jarid2, despite a comparable extent of deregulation of most other naïve marker genes. This indicates that Klf2 expression can be uncoupled from the core naïve network. Forced Klf2
Understanding how cell identity transitions occur and whether there are multiple paths between the same beginning and end states are questions of wide interest. Here we show that acquisition of naive pluripotency can follow transcriptionally and mechanistically distinct routes. Starting from post-implantation epiblast stem cells (EpiSCs), one route advances through a mesodermal state prior to naive pluripotency induction, whereas another transiently resembles the early inner cell mass and correspondingly gains greater developmental potency. These routes utilize distinct signaling networks and transcription factors but subsequently converge on the same naive endpoint, showing surprising flexibility in mechanisms underlying identity transitions and suggesting that naive pluripotency is a multidimensional attractor state. These route differences are reconciled by precise expression of Oct4 as a unifying, essential, and sufficient feature. We propose that fine-tuned regulation of this "transition factor" underpins multidimensional access to naive pluripotency, offering a conceptual framework for understanding cell identity transitions.
Abstract Heart failure (HF), the final stage of pathological cardiac hypertrophy, is a major cause of hospitalization and mortality. The role of inflammation in the pathogenesis of HF has been extensively studied, with great emphasis on proinflammatory cytokines. Yet, clinical trials targeting these cytokines failed to become a credible therapeutic strategy for HF. More recent studies are increasingly highlighting an active role for T cells in the progression of HF pathology. As a result, a number of novel immunotherapy strategies are emerging for the treatment of HF and other cardiovascular diseases, via the targeting of adaptive immunity. Here we provide an overview of the background, details, and expected outcomes of these attempts.
In development, lineage segregation of multiple lineages must be coordinated in time and space. An important example is the mammalian inner cell mass (ICM), in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the foetus). The physical mechanisms that determine this spatial segregation between EPI and PrE are still poorly understood. Here, we identify an asymmetry in cell-cell affinity, a mechanical property thought to play a significant role in tissue sorting in other systems, between EPI and PrE precursors (pEPI and pPrE). However, a computational model of cell sorting indicated that these differences alone appeared insufficient to explain the spatial segregation. We also observed significantly greater surface fluctuations in pPrE compared to pEPI. Including the enhanced surface fluctuation in pPrE in our simulation led to robust cell sorting. We identify phospho-ERM regulated membrane tension as an important mediator of the increased surface fluctuations in pPrE. Using aggregates of engineered cell lines with different surface fluctuation levels cells with higher surface fluctuations were consistently excluded to the outside of the aggregate. These cells behaved similarly when incorporated in the embryo. Surface fluctuations-driven segregation is reminiscent of activity-induced phase separation, a sorting phenomenon in colloidal physics. Together, our experiments and model identify dynamic cell surface fluctuations, in addition to static mechanical properties, as a key factor for orchestrating the correct spatial positioning of the founder embryonic lineages.
Abstract OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro . To determine how OCT4 protects the pluripotent lineage from differentiation into trophoblast, we used single cell transcriptomics and quantitative immunofluorescence on blastocysts and established differentially expressed genes and pathways between control and OCT4 null cells. Activation of most pluripotency-associated transcription factors in the early mouse inner cell mass appears independent of OCT4, whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single cell deconvolution, diffusion component and trajectory inference dissected the process of differentiation of OCT4 null cells by activating specific gene-network and transcription factors. Downregulation of glycolytic and oxidative metabolism was observed. CHIPseq analysis suggests OCT4 directly targets rate-limiting glycolytic enzymes. Concomitant with significant disruption of the STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos is likely attributable to aberrant metabolism. Highlights and novelty Major pluripotency-associated transcription factors are activated in OCT4-deficient early mouse ICM cells, coincident with ectopic expression of trophectoderm markers JAK/STAT signalling is defective in OCT4 null embryos OCT4 promotes expression of KATS enzymes by means of glycolytic production of Acetyl CoA to secure chromatin accessibility for acquisition of epiblast identity OCT4 regulates the metabolic and biophysical processes required for establishment of embryonic pluripotency
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