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• W2224876166 abstract "Article21 December 2015Open Access The bimodally expressed microRNA miR-142 gates exit from pluripotency Hanna L Sladitschek Hanna L Sladitschek Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Pierre A Neveu Corresponding Author Pierre A Neveu Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Hanna L Sladitschek Hanna L Sladitschek Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Pierre A Neveu Corresponding Author Pierre A Neveu Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Author Information Hanna L Sladitschek1 and Pierre A Neveu 1 1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany *Corresponding author. Tel: +49 6221 387 8336; E-mail: [email protected] Molecular Systems Biology (2015)11:850https://doi.org/10.15252/msb.20156525 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 A stem cell's decision to self-renew or differentiate is thought to critically depend on signaling cues provided by its environment. It is unclear whether stem cells have the intrinsic capacity to control their responsiveness to environmental signals that can be fluctuating and noisy. Using a novel single-cell microRNA activity reporter, we show that miR-142 is bimodally expressed in embryonic stem cells, creating two states indistinguishable by pluripotency markers. A combination of modeling and quantitative experimental data revealed that mESCs switch stochastically between the two miR-142 states. We find that cells with high miR-142 expression are irresponsive to differentiation signals while cells with low miR-142 expression can respond to differentiation cues. We elucidate the molecular mechanism underpinning the bimodal regulation of miR-142 as a double-negative feedback loop between miR-142 and KRAS/ERK signaling and derive a quantitative description of this bistable system. miR-142 switches the activation status of key intracellular signaling pathways thereby locking cells in an undifferentiated state. This reveals a novel mechanism to maintain a stem cell reservoir buffered against fluctuating signaling environments. Synopsis The bimodally expressed microRNA miR-142 defines two stochastically interconverting states of pluripotent embryonic stem cells: a state competent to differentiate and a state irresponsive to differentiation signals akin to a stem cell reservoir. Bimodal miR-142 expression creates two functionally distinct pluripotent states miR-142 regulates ERK and AKT signaling miR-142 expression locks embryonic stem cells in an undifferentiated state Stochastic miR-142 state switching maintains a pluripotent stem cell reservoir Introduction Stem cells respond to internal and external cues by self-renewal or commitment to a differentiated fate (North et al, 2007; Jiang et al, 2009; Medema & Vermeulen, 2011; Kueh et al, 2013; Blanpain & Fuchs, 2014). Current models suggest that this balance is controlled in vivo by stem cell niches (Scadden, 2006; Voog & Jones, 2010; Simons & Clevers, 2011) and in vitro by an appropriate growth factor environment (Murry & Keller, 2008; Pera & Tam, 2010). Mouse embryonic stem cells (mESCs) constitute a powerful system to study the molecular mechanism of fate decisions in controlled in vitro environment (Rué & Martinez Arias, 2015). mESCs are continuous cell lines derived from the inner cell mass of the blastocyst (Evans & Kaufman, 1981; Martin, 1981). These cells can be propagated indefinitely in vitro while maintaining their pluripotency, that is the capacity to give rise to derivatives of all three germ layers and germ cells both in vitro and in vivo. microRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression (Bartel, 2009). A growing body of evidence suggests that miRNAs act as key players in stem cell homeostasis (Neumüller et al, 2008; Foronda et al, 2014) and cell fate decisions (Chen et al, 2004; Johnston et al, 2005; Li & Carthew, 2005; Wang et al, 2007; Yi et al, 2008; Schwamborn et al, 2009). Whereas the role of transcription factor heterogeneity in defining different pluripotent substates is well established (Chambers et al, 2007; Singh et al, 2007; Toyooka et al, 2008), it is largely unknown whether such dynamic heterogeneity exists at the level of miRNA expression. To address this gap in our knowledge, we used a single-cell miRNA activity reporter to identify miR-142 that is bimodally expressed in mESCs under pluripotency-maintaining conditions. miR-142 expression levels stratify mESCs with indistinguishable expression of pluripotency markers into two distinct subpopulations: mESCs with low miR-142 levels are amenable to signal-induced differentiation, while cells with high miR-142 levels are irresponsive to differentiation cues. Using quantitative experiments and simulations, we show that mESCs switch stochastically between the high and low miR-142 states. Dissecting the molecular mechanism, we find that miR-142 represses the activation of KRAS/ERK signaling in a double-negative feedback loop that creates a bistable system. We propose that the self-generated miR-142 two-state system functions to maintain a stem cell reservoir that is protected from differentiation signals from the environment. Results miR-142 is a new marker of mESC heterogeneity under naïve pluripotency conditions We reasoned that miRNAs that control different self-renewing mESC states should show heterogeneous expression under uniform pluripotency-maintaining conditions. To identify such miRNAs, we devised a ratiometric fluorescence sensor that can visualize miRNA activity in single cells. The reporter consists of a bidirectional promoter driving the expression of a normalizer (H2B-mCherry) and a miRNA detector (H2B-Citrine), which contains in its 3′-UTR a target sequence of the miRNA of interest (Figs 1A and EV1A). Using this reporter system, we screened 33 conserved miRNAs associated with differentiation, pluripotency or cell proliferation (Fig EV1B and C) in mESC lines stably expressing specific reporters. As expected, we found the abundant miR-294 to be highly active, whereas the differentiation-associated miRNA let-7 showed little activity (Fig EV1D and E). Most miRNA reporters displayed a normally distributed cell-to-cell variation comparable to a non-targeted control. Strikingly, however, we found a strongly variegated activity of miR-142-3p that divided clonal mESC colonies into two sectors with very different miRNA activity (Fig 1B). Also at the population level, miR-142-3p activity was clearly bimodal distinguishing two mESC populations with either a high or a low miR-142-3p activity state (in the following referred to as “high” and “low” miR-142 states, Fig 1C and D). Furthermore, this bimodal regulation was present in chemically defined media conditions that support naïve pluripotency including “2i” but was absent in primed pluripotency (Fig EV2). Thus the bimodal regulation of miR-142 represents a novel kind of mESC heterogeneity in LIF-dependent pluripotency. Figure 1. The bimodal expression of miR-142 distinguishes two states in mESCs Scheme of the experimental approach to monitor miRNA activity in single cells. Confocal section of a single-cell-derived mESC colony stably expressing the miR-142-3p activity reporter and corresponding Z-score of the reporter ratio. As individual mESCs are not motile within a colony, sister lineages are spatially clustered. Scale bar: 50 μm. Detector and normalizer expression in a clonal mESC population stably expressing the miR-142-3p reporter. Distribution of the miR-142-3p reporter ratio in a clonal mESC culture. Two log-normal distributions (“high” miR-142 activity state: red shaded area; “low’’ miR-142 activity state: green shaded area) approximated well the experimental data (black line). Distribution of the miR-142-3p reporter ratio in mir142−/− mESCs (blue line) and mir142−/− mESCs transgenic for the mir142-hosting lincRNA driven by its own promoter (mir142−/− rescue, orange line; see Materials and Methods for details). Deep sequencing analysis of miRNA expression levels in FACS-purified “high” and “low” miR-142 states. Levels of the two mature forms of miR-142, miR-142-3p and miR-142-5p are highlighted in red. Adjustment of detector mRNA expression levels as a function of miR-142-3p levels (blue dots: experimental data measured by deep sequencing in FACS-purified populations) with Hill's equation with non-cooperative binding (red line; shaded area: fit confidence interval). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Single-cell miRNA activity reporter A synthetic bidirectional promoter drove the expression of H2B-Cherry as normalizer to control for transcriptional noise and H2B-Citrine as detector of miRNA activity with a target sequence for the respective miRNA 11 bp downstream of its stop codon. Four enhancer elements of the mouse phosphoglycerate kinase Pgk1 gene (PGK) promoter were inserted between two back-to-back arranged minimal PGK-promoter fragments to create a bidirectional PGK promoter (upper panel). The bidirectional CAG-promoter was constructed by placing four CMV immediate-early enhancer elements between two back-to-back arranged fragments of the promoter, first exon and partial intron of chicken β-actin gene fused to the splice acceptor of the rabbit β-globin gene (lower panel). A positive selection cassette was included (not depicted). Intronic sequences are represented as lines. bGpA: rabbit β-globin genomic fragment containing the polyadenylation signal. For use in the Rex1-dGFP knockin mESC line, we constructed an activity reporter based on a bidirectional CAG-promoter driving the expression of H2B-2xTagBFP as normalizer and H2B-Cherry as detector. List of candidate miRNAs used in this screen. Experimental scheme for the generation and screening of clonal mESC lines stably expressing miRNA activity reporters. Reporter signal in single cell-derived mESC colonies and corresponding Z-score of the reporter ratio for a non-targeted control, a let-7a-5p and a miR-294-3p activity reporter. Scale bar: 50 μm. Reporter ratio distribution in mESCs stably expressing the activity reporters for miR-294-3p (red line), miR-142-3p (blue line), let-7a-5p (green line) and a non-targeted control (black line). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. miR-142 activity reporter in different pluripotency-sustaining media Bimodal miR-142 expression in naïve LIF-dependent pluripotency conditions. Distribution of the miR-142-3p reporter ratio in mESCs cultured in 10 ng/ml LIF supplemented with serum (LIF+serum, black line) or 10 ng/ml BMP4 (LIF+BMP4, green line) or in 1 μM PD0325901 + 3 μM CHIR99021 (LIF+2i, blue line). miR-142-3p reporter ratio distribution in primed pluripotency conditions (12 ng/ml FGF2 and 20 ng/ml Activin A, blue line) compared to naïve LIF-dependent pluripotency conditions (LIF+serum, black line). Download figure Download PowerPoint To validate that our reporter responded specifically to miR-142-3p, we generated mir142−/− mESC lines by deleting both alleles of mir142 using the CRISPR/Cas9 technology (Appendix Fig S1). As expected, the repression of the reporter was relieved in mir142−/− cells (Fig 1E). In addition, we assessed miRNA expression levels of FACS-purified “high” and “low” miR-142 state subpopulations in wild-type mESCs by deep sequencing. This analysis showed a 10-fold increase in the expression levels of miR-142-3p and miR-142-5p, the two mature forms of the miR-142 stem loop, in “high” miR-142 mESCs compared to “low” miR-142 mESCs (Fig 1F). Expression levels of all the other detected miRNAs were tightly correlated between the “high” and “low” miR-142 states (Fig 1F). Finally, we calibrated our single-cell miRNA activity reporter using expression data of miR-142-3p and mRNA reporter levels measured by deep sequencing. As expected, the signal of miR-142 activity reporter depends on miR-142-3p levels following Hill's equation with non-cooperative binding (Fig 1G). Our reporter thus specifically measures the activity of miR-142-3p. Low miR-142-3p levels correspond to large reporter ratios and high miR-142-3p levels yield small reporter ratios. The system therefore allows us to quantitate miR-142 expression changes in single living cells. The two miR-142 states are indistinguishable by pluripotency markers Previous reports of mESC heterogeneity found a metastable coexistence of a pluripotent state and a state prone to differentiate and already expressing lower levels of the pluripotency markers Nanog and Rex1 (Chambers et al, 2007; Singh et al, 2007; Toyooka et al, 2008; Singer et al, 2014). To test if miR-142 heterogeneity is upstream of expression changes in pluripotency markers, we analyzed FACS-purified “high” and “low” miR-142 mESC populations. The two miR-142 states were both positive for alkaline phosphatase staining (Fig 2A). mRNA profiles measured by deep sequencing of FACS-purified “high” and “low” miR-142 state mESCs clustered together, while the mRNA profiles of mESCs with low Nanog expression clustered apart (Fig 2B–D). In addition, the miRNA expression profile of mESCs with low Nanog expression was markedly different from the miRNA expression signature of “high” and “low” miR-142 states (Appendix Fig S2). Gene set enrichment analysis (Subramanian et al, 2005) using curated gene sets (canonical pathways, BioCarta and KEGG gene sets) did not reveal any significantly dysregulated gene set between the “high” and “low” miR-142 states. Comparing the expression of genes with a highly variable expression in mESCs (Klein et al, 2015), we found only 14 genes out of 1,891 with more than twofold expression changes between the two miR-142 sates (Appendix Fig S3). This number is similar to the one obtained when comparing biological replicates. In comparison, 302 genes display more than twofold changes in expression in low Nanog cells compared to high Nanog cells. Predicted targets of miR-142-3p had significantly lower expression in “high” miR-142 mESCs compared to “low” miR-142 mESCs (P < 0.001, determined by subsampling) and removing predicted miR-142-3p targets was sufficient to abrogate the clustering of “high” and “low” miR-142 expression profiles (Appendix Fig S4). Closer examination of pluripotency factor expression showed no significant difference in the mRNA or protein expression levels of Oct4 (or Pou5f1), Nanog, Rex1 (or Zfp42) and Sox2 between the “high” and “low” miR-142 states in FACS-purified subpopulations (Fig 2E and F). Furthermore, the “high” and “low” miR-142 states showed no difference in the known heterogeneity of NANOG (Figs 2G and EV3A) or REX1 (Fig EV3B) protein expression at the single-cell level. Moreover, all cells stained positive for the pluripotency markers OCT4 and SSEA-1 irrespective of their “high” or “low” miR-142 state identities (Fig EV3C and D). Additional pluripotency markers (Ng & Surani, 2011) showed no significant difference at the mRNA expression levels (Fig EV3E). In addition, neither “high” nor “low” miR-142 state cells shared molecular markers with epiblast stem cells (Fig EV3F), that reside in a state of primed pluripotency. Thus, the “high” and “low” miR-142 states are indistinguishable in their pluripotency marker expression and did not represent a primed pluripotent state. Figure 2. “High” and “low” miR-142 cells express pluripotency markers at equal levels A. Alkaline phosphatase staining of FACS-purified “high” and “low” miR-142 state mESCs. Cells were cultured for 24 h after sorting and stained. Scale bar: 100 μm. B, C. Deep sequencing analysis of mRNA expression levels in FACS-purified “high” and “low” miR-142 mESCs (B) or mESCs with low Nanog expression (C). D. Average linkage hierarchical clustering of mRNA profiles of “high” and “low” miR-142 mESCs and of mESCs with low Nanog expression. E. mRNA expression levels of pluripotency markers in FACS-purified “high” and “low” miR-142 state mESCs (n = 2; n.s.: not significant, two-sided t-test). Data represented as mean ± SEM. F. Western blot analysis and quantification of pluripotency marker levels in FACS-purified “high” and “low” miR-142 state mESCs (n = 7; n.s.: not significant, two-sided t-test). Data represented as mean ± SEM. G. Immunostaining of OCT4 and NANOG in a clonal miR-142-3p reporter mESC colony. Quantification of NANOG levels in individual cells showed no significant difference in NANOG expression between the “high” and “low” miR-142 states (P = 0.25, Kolmogorov–Smirnov test). In the plot, the whiskers denote 1.5 times the interquartile range. Scale bar: 100 μm. H. miR-142 activity reporter ratio in a Rex1-dGFP knockin mESC line. I. Distribution of miR-142 reporter ratio in cells positive for Rex1-dGFP expression (Rex1-dGFP+, purple line) and negative for Rex1-dGFP expression (Rex1-dGFP−, black line). Gates identifying the populations are displayed in (H). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. “High” and “low” miR-142 mESCs express pluripotency markers at equal levels and do not express epiblast stem cell markers A–C. Protein expression levels of the pluripotency markers NANOG (A), REX1 (B) and OCT4 (C) in single mESCs expressing the miR-142 activity reporter. D. Expression levels of the pluripotency marker SSEA-1 in single mESCs expressing the miR-142 activity reporter. E. mRNA expression levels of additional pluripotency markers in FACS-purified “high” and “low” miR-142 state mESCs as well as cells with low Nanog expression (n = 2; data represented as mean ± SEM). F. mRNA expression levels of mESC and epiblast stem cell markers in “high” and “low” miR-142 state mESCs as well as mESCs maintained in “2i” and epiblast stem cells maintained in primed pluripotency conditions (FGF2 + Activin) (n = 2; n.s.: not significant, ***P < 0.001, two-sided t-test). Data represented as mean ± SEM. Download figure Download PowerPoint Finally, we introduced the miR-142 activity reporter in a Rex1-dGFP knockin mESC line (Wray et al, 2011) in order to compare the bimodal regulation of miR-142 to the known heterogeneity in Rex1 expression. mESCs with high Rex1-dGFP levels revealed a bimodal regulation of miR-142 activity, that is mESCs with high Rex1-dGFP reside in either the “high” miR-142 state or the “low” miR-142 state (Fig 2H and I). Moreover, cells with low Rex1-dGFP expression had a unimodal reporter distribution with a reporter ratio comparable to mir142−/− mESCs, corresponding to an absence of miR-142 expression (Fig 2H and I). Thus, the “high” miR-142 state and the “low” miR-142 state are only found together in the high Rex1 mESC compartment. This finding places miR-142 bimodality upstream of the so far described heterogeneity in pluripotency transcription factor expression. Therefore, miR-142 bimodality represents a novel kind of heterogeneity in naïve mESCs. The two miR-142 states interconvert stochastically To assess whether and how the two miR-142 states can interconvert into each other, we monitored the distribution of miR-142 activity after FACS purification of “high” or “low” miR-142 subpopulations (Fig 3A). Indeed, either state could regenerate the other within 10 days of culture under pluripotency conditions (Fig 3A). State recovery was not due to any differential growth between the two miR-142 states because “high” and “low” miR-142 cells divided at the same rate every 12 h (Fig 3B-D). To determine the interconversion rates, we quantified the fraction of the population in the “high” and “low” miR-142 states. We then fitted the population data using first order reaction kinetics (see 4 for details, Fig 3E and Appendix Fig S5A). Cells converted from “high” to “low” miR-142 states with a rate k1 = 0.072 ± 0.01 per cell division (on average one switching event every 14 divisions), while the backconversion was slightly slower occurring with a rate k−1 = 0.048 ± 0.006 per cell division (on average one switching event every 21 divisions). Investigating the reporter ratio distribution in cultures derived from FACS-purified single cells showed that cultures recovered both states whether starting from “high” or “low” miR-142 founder cells (Appendix Fig S5B; n > 160). This demonstrated that all clonogenic cells can switch between states. Single-cell live imaging of miR-142 activity revealed that switching occurred rapidly within less than a cell cycle and that after division sister lineages were not always correlated in their switching behavior (Fig 3F and G, and Movie EV1) suggesting stochastic switching events. Figure 3. Interconversion between the two miR-142 states “High” and “low” miR-142 subpopulations were FACS-purified and the temporal evolution of the miR-142 reporter ratio was measured. Shaded areas: quantification of cells in “high” or “low” miR-142 states. Scheme of the experimental design to compare proliferation rates of mESCs in the “high” and “low” miR-142 state using a dye dilution by cell division strategy. Time-lapse analysis of the fluorescence intensity of cells stained on day 0 with a commercial dye labeling free amines. The mESC culture was analyzed by flow cytometry each day. The distribution of the dye retention in all live cells in the culture (blue line) could be well approximated by the sum (shaded gray area) of gaussian distributions (white lines outlined in black) representing distinct cell division cycles. The distribution of dye retention in mESCs in the “high” or “low” miR-142 state is outlined by a red or green line. Population growth of mESCs starting from FACS-purified “high” (red line) and “low” (green line) miR-142 subpopulations (error bars represent SEM, n = 6). Reaction kinetics model of the interconversion between “high” and “low” miR-142 cells. khigh and klow are the proliferation rates of “high” and “low” miR-142 cells, k1 the interconversion rate from “high” to “low” miR-142 state and k−1 the interconversion rate from “low” to “high” miR-142 state. Live imaging of switching events during the growth of a single-cell-derived mESC colony. Maximal projections of confocal stacks are shown at the indicated time points (h: hour). White arrowheads denote the cell with the first switching event at 41.5 h and the two resulting daughters at 48 h. Scale bar: 50 μm. See also Movie EV1. Single-cell tracks of reporter ratio in two sister lineages (green: sister lineage with activity switching, red and orange: sister lineage without activity switching, gray: all other lineages; spikes in reporter signal are artifacts due to signal saturation at mitotic divisions, black arrowheads correspond to the cells marked by white arrowheads in F). Download figure Download PowerPoint If switching were indeed stochastic, the variegated distribution of “high”/”low” miR-142 cells in a colony grown from a single cell will depend on the time when the first state switching occurred, since the states are on average stable for several cell cycles (Fig 4A). Using a stochastic switching model, we could simulate the expected fraction of cells that switched state in colonies derived from pure “high” or “low” miR-142 state single founders (Fig 4B). To test this prediction, we measured the fraction of switched cells in colonies grown from single FACS-purified “high” or “low” miR-142 cells. The stochastic switching model approximated well data from founder cells FACS-purified in the “low” miR-142 state but could not fit with the same parameters the state composition obtained in cultures derived from founder cells FACS-purified in the “high” miR-142 state (Fig 4B). We thus introduced a refined model in which cells stochastically switch between the two miR-142 states but “high” and “low” miR-142 cells can have different survival rates under clonogenic conditions while having the same proliferation rate (Fig 4C). The experimental data were recapitulated by simulations using this refined model including a survival bias for “low” miR-142 cells under clonogenic conditions (see 4 for details, Fig 4D). Interestingly, we experimentally confirmed this survival bias predicted by the model. Indeed, clonogenicity of FACS-purified “low” miR-142 cells (19.8 ± 6.6%) was higher than for “high” miR-142 cells (5.8 ± 3.1%) (Fig 4E). This gave a survival bias for “low” miR-142 cells of 6.9 (90% confidence interval: 1.8–17.8), in excellent agreement with the 8-fold survival bias predicted by the simulations. Using a genetic loss-of-function approach, we could show that the loss of mir142 expression indeed improved clonogenicity without affecting the proliferation rate (Fig 4F). In summary, we could demonstrate experimentally and theoretically that individual mESCs fluctuate stochastically between the two miR-142 states at a relatively low rate with a state switching event occurring on average every 8 cell divisions. Figure 4. mESCs switch stochastically between the two miR-142 states Stochastic switching model (left panel): Cells can switch state each cell division with probability k1 or k−1. Experimental scheme (right panel): Clonal cultures were derived from single FACS-purified “high” and “low” miR-142 mESCs. Occurrence of switching events was measured by assessing the miR-142 reporter ratio distribution in individual cultures. Simulation of state distribution after colony growth following the model shown in (A) with a founder cell in “low” miR-142 (dashed green line) or “high” miR-142 (dashed red line) state (170 colonies, 14 divisions, k1 + k−1 = 0.08 per cell division, k1 = 1.5 k−1). Solid red and green lines: experimentally measured state distribution in cultures derived from FACS-purified founder cells in “high” and “low” miR-142 states (n = 169 and n = 171). Stochastic switching model with differential survival. “High” and “low” miR-142 mESCs can have different survival rate under clonogenic conditions. Simulation of state distribution after colony growth following the stochastic switching model with differential survival shown in (C) with a founder cell in “low” miR-142 (dotted green line) or “high” miR-142 (dotted red line) state (170 colonies, 14 divisions, k1 + k−1 = 0.08 per cell division, k1 = 1.5 k−1, plow/phigh = 8). Shaded area: 95% confidence interval. Solid red and green lines: experimental data for FACS-purified founder cells in “high” and “low” miR-142 states, same data as shown in (B). Clonogenicity of single FACS-purified founder cells in “high” or “low” miR-142 state (c.f.u.: colony forming units; n = 50; P = 3 × 10−24, two-sided t-test; error bars represent SEM). Single cells were FACS-purified in 96-well plates (n represents the number of 96-well plates that were analyzed). Clonogenicity of single mir142+/+ (n = 19), mir142+/− (n = 20) or mir142−/− (n = 20) mESCs (c.f.u.: colony forming units; P = 10−8, P = 2 × 10−8 and P = 8 × 10−16, two-sided t-test; error bars represent SEM). Single cells were FACS-purified in 96-well plates (n represents the number of 96-well plates that were analyzed). Download figure Download PowerPoint Constitutive miR-142 expression locks cells in an undifferentiated state A hallmark of embryonic stem cells is the ability to generate distinct differentiated cell types. To assess whether mir142 expression affects differentiation capacity, we compared mir142 gain- or loss-of-function mESCs regarding their capabilities to differentiate toward fates of the three germ layers, that is neuroectoderm, mesoderm, and endoderm fate. Upon differentiation, mir142−/− cells stained positive for the neuronal marker Tuj1 (or βIII-tubulin), the muscle marker Desmin or the endoderm marker Foxa2 and were negative for the pluripotency marker Oct4 (Fig 5A–C and Appendix Fig S6). By contrast, mir142 gain-of-function cells retained Oct4 expression and showed no differentiation marker expression (Fig 5D–F and Appendix Fig S6). In order to understand genomewide this striking difference in response to differentiation cues, we profiled the transcriptomes of wild-type mESCs, mir142−/− and mir142-expressing mESCs during a 6 day endoderm differentiation time course. Strikingly, cells constitutively expressing mir142 always clustered with undifferentiated wild-type and mir142−/− cells at day 1 or 2 (Fig 5G), while differentiating wild-type mESCs and mir142−/− cells from day 3–6 cluster separately. Using principal component analysis allowed us to visualize the trajectory of expression profiles during 6 days of differentiation (Fig 5H). This showed that unlike wild-type and mir142−/− cells, cells constitutively expressing mir142 were essentially locked in an undifferentiated expression state (Fig 5H and Appendix Fig S7A and B) and consistently failed to up-regulate established endoderm markers (Fig 5I). Even at the end of the 6 day differentiation procedure, cells with constitutive mir142 expression proliferated normally under pluripotency conditions, exhibited the characteristic 3-dimensional morphology of undifferentiated mESC colonies and were alkaline phosphatase-positive (Fig 5J). In addition, genetic deletion of mir142 led to significantly larger changes in gene expression compared to wild-type cells as measured by projection on" @default.
• W2224876166 created "2016-06-24" @default.
• W2224876166 creator A5043326561 @default.
• W2224876166 creator A5056306608 @default.
• W2224876166 date "2015-12-01" @default.
• W2224876166 modified "2023-10-18" @default.
• W2224876166 title "The bimodally expressed micro <scp>RNA</scp> miR‐142 gates exit from pluripotency" @default.
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