A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes
2019; Springer Nature; Volume: 38; Issue: 9 Linguagem: Inglês
10.15252/embj.2018101244
ISSN1460-2075
AutoresMagdalena M. Maslon, Ulrich Braunschweig, Stuart Aitken, Abigail R. Mann, Fiona Kilanowski, Chris Hunter, Benjamin J. Blencowe, Alberto R. Kornblihtt, Ian R. Adams, Javier F. Cáceres,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle15 April 2019Open Access Source DataTransparent process A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes Magdalena M Maslon Magdalena M Maslon MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Ulrich Braunschweig Ulrich Braunschweig Donnelly Centre, Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Stuart Aitken Stuart Aitken MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Abigail R Mann Abigail R Mann MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Fiona Kilanowski Fiona Kilanowski MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Chris J Hunter Chris J Hunter MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Benjamin J Blencowe Benjamin J Blencowe Donnelly Centre, Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Alberto R Kornblihtt Alberto R Kornblihtt Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE-UBA-CONICET) and Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina Search for more papers by this author Ian R Adams Corresponding Author Ian R Adams [email protected] orcid.org/0000-0001-8838-1271 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Javier F Cáceres Corresponding Author Javier F Cáceres [email protected] orcid.org/0000-0001-8025-6169 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Magdalena M Maslon Magdalena M Maslon MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Ulrich Braunschweig Ulrich Braunschweig Donnelly Centre, Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Stuart Aitken Stuart Aitken MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Abigail R Mann Abigail R Mann MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Fiona Kilanowski Fiona Kilanowski MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Chris J Hunter Chris J Hunter MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Benjamin J Blencowe Benjamin J Blencowe Donnelly Centre, Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Alberto R Kornblihtt Alberto R Kornblihtt Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE-UBA-CONICET) and Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina Search for more papers by this author Ian R Adams Corresponding Author Ian R Adams [email protected] orcid.org/0000-0001-8838-1271 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Javier F Cáceres Corresponding Author Javier F Cáceres [email protected] orcid.org/0000-0001-8025-6169 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Author Information Magdalena M Maslon1, Ulrich Braunschweig2, Stuart Aitken1, Abigail R Mann1, Fiona Kilanowski1, Chris J Hunter1, Benjamin J Blencowe2, Alberto R Kornblihtt3, Ian R Adams *,1 and Javier F Cáceres *,1 1MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 2Donnelly Centre, Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 3Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE-UBA-CONICET) and Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina *Corresponding author. Tel: +44 131 651 8562; E-mail: [email protected] *Corresponding author. Tel: +44 131 651 8699; E-mail: [email protected] The EMBO Journal (2019)38:e101244https://doi.org/10.15252/embj.2018101244 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 The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII. We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development. Synopsis Transcription elongation rates have a regulatory role in the expression of genes and the regulation of their alternative splicing patterns during development. A slow RNA polymerase II (RNAPII) mutant allele (R749H) causes early embryonic lethality in mice. A slow RNAPII impairs ESCs differentiation towards the neural lineage. It limits neural stem cells (NSC) renewal in vitro and preferentially affects transcription and AS of long neuronal genes. The impact of the kinetic coupling of RNAPII elongation rate with Alternative splicing (AS) is greater in ESC-differentiated neurons than in pluripotent cells. Introduction Alternative splicing (AS) is a highly regulated process that generates RNA diversity and is a major contributor to protein isoform diversity. Its regulation depends not only on the interaction of trans-acting factors with regulatory RNA cis-acting sequences but also on multiple layers of regulation, which include DNA methylation, chromatin structure and modification, and transcription (Schwartz & Ast, 2010; Lev Maor et al, 2015; Naftelberg et al, 2015). The co-transcriptional nature of pre-mRNA splicing led to the suggestion that the rate of transcription elongation acts to control AS in mammalian cells (Beyer & Osheim, 1988; Roberts et al, 1998; Pandya-Jones & Black, 2009). Notably, there is a functional relationship between the transcriptional and the splicing machineries, as evidenced by the role of splicing factors, such as TCERG1, also known as CA150 (Suñé & Garcia-Blanco, 1999) and SRSF2 (Lin et al, 2008), in stimulating transcriptional elongation. Interestingly, a role for transcription elongation rate influencing splicing fidelity and co-transcriptionality was also observed in yeast (Herzel et al, 2017; Aslanzadeh et al, 2018). The elongation control of transcription can be highly regulated and have a profound effect on gene expression. Indeed, following transcription initiation, the transition of RNAPII from a paused to a productive elongation stage constitutes a major rate-limiting step in the transcription of approximately 40% of mRNA-encoding genes (Min et al, 2011; Vos et al, 2018a,b). Furthermore, transcription elongation is variable, as synthesis rates can differ between genes by several-fold and these variations in elongation rates could be associated with different gene features and epigenetic modifications. Recent studies revisited the contribution of the kinetics of RNAPII elongation to the regulation of AS, giving rise to two complementary models (Bentley, 2014; Naftelberg et al, 2015). The "window-of-opportunity" or kinetic model of AS regulation proposes that the rate of RNAPII elongation influences the outcome of alternative splicing selection. Use of a mutant form of RNAPII (C4/R749H) with a slower elongation rate leads to an increased (de la Mata et al, 2003) or decreased (Dujardin et al, 2014) inclusion of alternative cassette exons into mature mRNA. A complementary model, termed "Goldilocks", concluded, based on the study of RNAPII mutants with both slow and fast elongation rates, that an optimal rate of transcriptional elongation is required for normal co-transcriptional pre-mRNA splicing (Fong et al, 2014). In both models, recruitment of splicing regulators to cis-acting RNA sequences as well as nascent RNA folding is influenced by the elongation rate of RNAPII (Eperon et al, 1988; Buratti & Baralle, 2004; Saldi et al, 2018). The global impact of RNAPII elongation rate in the regulation of AS was confirmed with the use of drugs that inhibit RNAPII elongation (Ip et al, 2011). Exogenous agents also affect transcriptional coupling to AS. For instance, UV irradiation promotes RNAPII hyperphosphorylation with the subsequent inhibition of transcriptional elongation, leading to changes in AS, suggesting that transcriptional coupling to AS is a key feature of the DNA-damage response (Muñoz et al, 2009; Williamson et al, 2017). In plants, light regulates AS through the control of transcriptional elongation by promoting RNAPII elongation, which is negatively regulated in darkness (Godoy Herz et al, 2019). To date, all studies investigating the role of transcription elongation in pre-mRNA processing in mammalian systems have been confined to the use of cultured cells transfected with α-amanitin-resistant slow or fast RNAPII elongation mutants. Thus, the consequences of this mechanism of regulation in vivo and its effect on cellular differentiation and development remain largely unexplored. Here, we sought to address two important yet largely unexplored questions. First, how does an altered transcriptional elongation rate affect gene expression and the control of AS and impacts on mammalian development? Secondly, what are the extent and the tissue/organism phenotypic consequences of the elongation control of AS? To answer these questions, we generated mouse embryonic stem cells (ESCs) knocked in for a slow RNAPII mutant (C4/R749H). We show that an appropriate RNAPII elongation rate is essential for proper mouse development. We observed that a reduced elongation rate results in major changes in splicing and in gene expression in pluripotent ESCs and along the pathway of neuronal differentiation. The impact of the kinetic coupling of RNAPII elongation rate with AS is more predominant in ESC-differentiated neurons than in pluripotent cells, as it is essential for the expression and splicing of neuron-specific genes involved in synapse signaling. Results Generation of a slow RNAPII knock-in mutant mouse ES cells To address the consequences of an altered transcriptional elongation rate for gene expression and for the kinetic control of AS, we set out to generate an in vivo model of a slow RNAPII by introducing a heterozygous or homozygous R749H mutation into the endogenous Polr2a in mouse ESCs. This mutation is equivalent to the C4 point mutation identified in the Drosophila pol II largest subunit, which confers a lower elongation rate, is less capable of transcribing through natural elongation blocks, and causes non-lethal developmental defects in the heterozygous state (Coulter & Greenleaf, 1985; Mortin et al, 1988; Chen et al, 1996). Gene targeting in mouse ESCs was achieved by rounds of homologous recombination to introduce the R749H mutation into each allele of Polr2a to generate heterozygous and homozygous ESCs (Fig 1A, henceforth referred to as WT/slow and slow/slow ESCs). We verified the correct targeting by PCR of genomic DNA isolated from these ESCs and a diagnostic XhoI digest (Fig 1B). Ion Torrent sequencing of overlapping PCR products from ESC genomic DNA encompassing a ~14-kb region around the R749H mutation confirmed that the heterozygous WT/slow and homozygous slow/slow ESCs contained no genomic re-arrangements or additional mutations in this region relative to the parental WT/WT ESCs. We verified the expression of mutant RNAPII in these cells by cDNA sequencing (Fig 1C) and using allele-specific RT–qPCR (Fig 1D). Figure 1. Generation of slow RNAPII knock-in mutant mouse ESCs Cartoon depicting the mutagenesis strategy, including the genomic target locus, as well as the structure of the targeting vector. Arrows indicate location of primers used for genotyping. Restriction enzyme diagnostic test for the presence of the R749H mutation. Sequence trace of cDNA showing the presence of the heterozygous and homozygous R749H mutations. qRT–PCR with primers specific to both wild-type and mutant RNAPII (left panel) or to the mutant form of RNAPII (right panel), confirming that only the slow version of RNAPII is expressed in homozygous slow/slow ESCs. The sequences of the respective forward primers are shown. The "WT/slow allele" primer is complementary to the sequence in exon 14 upstream of the mutation. The "slow allele only" primer has its 3′ end matching the mutated codon 749 and does not anneal to the WT DNA sequence. The mean ± SEM is plotted, n = 2. Download figure Download PowerPoint Slow transcription elongation hinders early mouse development The WT/slow ESCs were used to generate a slow RNAPII knock-in mouse model by injection into C57BL/6 blastocysts. We obtained mouse chimeras from these injections; however, no germline transmission was observed upon breeding eight male animals with at least 30% coat color chimerism to C57BL/6 females. These chimeric animals either sired only host blastocyst-derived offspring or were infertile and lacked sperm in the epididymis. As a test, breeding of 3–4 male chimeras is typically sufficient to detect germline transmission (BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement, 2003). This indicates that ESCs with a heterozygous slow RNAPII appear to be unable to functionally contribute to spermatogenesis. To investigate the developmental consequences of the Polr2a R749H mutation further, we set out to generate R749H mutant mice using CRISPR/Cas9 (Fig 2A). Specific single guide RNAs (sgRNAs) against Polr2a were microinjected into (C57BL/6 × CBA) F2 zygotes along with the Cas9 mRNA and an oligonucleotide repair template containing the R749H mutation ("slow oligo"), and subsequently, embryos were transferred into pseudopregnant recipient mice at the two-cell stage. No live-borne mice were obtained containing homozygous or even heterozygous mutations in the Polr2a locus among the 47 pups (Fig 2B). To rule out inefficient induction of double-strand DNA breaks (DSBs) by sgRNAs, or inefficient oligonucleotide-mediated repair at this locus, we co-injected the same pair of sgRNAs with a repair template mixture containing a 1:1 ratio of a slow oligo and a silent oligo, the latter being a repair template containing silent mutations. Again, we could not detect the slow mutation in any of the 51 pups born; however, we obtained two homozygotes and four heterozygotes as a result of repair with silent oligo (Fig 2C). Taken together, the ESC chimeras and the CRISPR/Cas9 microinjections suggest that even heterozygosity for Polr2a R749H causes developmental defects in mice. Next, we investigated at what stage the Polr2a R749H mutation caused embryonic lethality. We microinjected slow oligo along with guide RNAs into zygotes, cultured the zygotes in vitro for 3 days, and analyzed the resulting embryos at the late morula/blastocyst stage. We found several slow heterozygous embryos and only 1 homozygous embryo, revealing that the R749H mutation was tolerated at the pre-implantation stage (Fig 2B and C). However, when microinjected zygotes were transferred to pseudopregnant recipient females at the 2-cell stage to allow them to implant and develop further, only one heterozygous and no homozygous slow mutations were found in mid-gestation embryos at E9.5-E11.5 (Fig 2C). Thus, we conclude that the Polr2A R749H mutation causes early embryonic lethality. Figure 2. CRISPR-/Cas-mediated generation of a slow RNAPII knock-in mutant mouse A. Cartoon depicting the mutagenesis strategy, including the genomic target locus, as well as two repair templates, either introducing a silent mutation (silent oligo) or the R749H mutation (slow oligo). Multiple repair oligo templates were tested with different composition of silent restriction sites. B, C. The number of embryos/mice of different genotypes recovered after injecting a slow RNAPII repair oligo (B) or a 1:1 mixture of slow and silent RNAP II repair oligos (C) into E0.5 zygotes is shown in each table. The RNAPII genotypes and the stages at which embryos/mice were analyzed are indicated. * indicates P < 0.01 (Fisher's exact test relative to E3.5). Download figure Download PowerPoint The R749H mutation decreases the transcription elongation rate in mouse ESCs We analyzed the effect of the slow RNAPII mutation in ESCs using 5,6-dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB) to measure RNAPII transcriptional elongation rates (Singh & Padgett, 2009). DRB inhibits P-TEFb-dependent phosphorylation of the transcription elongation factor Spt5 and of serine 2 in the carboxy-terminal domain (CTD) of RNAPII. Thus, newly initiated RNAPII cannot progress to the elongation phase; however, upon DRB removal, all initiated polymerases are released, and the appearance of selected intron–exon junctions can be monitored by qRT–PCR in a time-dependent manner. We monitored how transcription proceeded through the Itpr1 and Utrophin genes, following DRB removal. Transcription over the first exon–intron junctions did not differ between the wild-type (WT) and mutant cell lines (Fig EV1, Exon 1–Intron 1 panels). However, appreciable pre-mRNA levels at the more downstream exon–intron junctions were detected earlier in WT than in slow/slow cells. For example, the appearance of an exon–intron junction 133-kb downstream from the Itpr1 transcription start site was detected at 40 min post-DRB release for the WT, as compared to 90 min for the mutant RNAPII (Fig EV1A, see Exon 5–Intron 5 panel). An overall mean elongation rate across Itpr1 and Utrophin was estimated to be 3.3 and 5.6 kb/min, respectively, in WT cells, as compared to 1.5 and 1 kb/min in slow/slow cells. We also measured overall transcription using a reversible DRB block followed by incubation with medium containing tritiated 3H-uridine. Time-resolved accumulation of newly made RNA, as measured by the incorporation of 3H-uridine, was attenuated in slow/slow in comparison with WT ESCs (Fig EV1B). We also found that nuclear extracts isolated from slow/slow cells were less efficient in driving the production of a runoff transcript from the artificial DNA template (Fig EV1C). These results are in agreement with the previous observation that the R749H mutation in RNAPII leads to approximately a twofold decrease in the transcription elongation rate in vitro (Boireau et al, 2007) and that the elongation rate positively correlates with expression levels (Danko et al, 2013; Jonkers et al, 2014). Click here to expand this figure. Figure EV1. Validation of the transcriptional elongation rate in ES mutant cell lines (related to Fig 3) Cartoon depicting the structure of Itpr1 and utrophin pre-mRNAs, with primer pairs selected to monitor the appearance of selected intron–exon junctions indicated by arrows. Quantification of pre-mRNA at different time points at the beginning, in the middle, and at the end of the gene displayed relative to cells not treated with DRB in WT/WT, WT/slow, and slow/slow cells (black, gray, and purple bars, respectively). The mean ± SEM is shown, n = 3. Time course of incorporation of 3H-uridine in WT/WT or slow/slow cells. Time 0' corresponds to time of DRB wash-off. Mean ± SEM is plotted with *P < 0.05 as determined by t-test, n = 3. In vitro transcription of a linearized plasmid using nuclear extracts from either WT/WT or slow/slow ES cells. The image shown is representative of 1 of 2 reproducible experiments. Download figure Download PowerPoint Next, we analyzed RNAPII elongation rates genome-wide using metabolic labeling of newly transcribed RNAs by the uridine analogue, 4-thiouridine (4sU) (Rädle et al, 2013; Fuchs et al, 2014). Transcription was arrested with DRB for 3 h; then, DRB was removed and cells were allowed to transcribe for 5 and 15 mins. To label the newly transcribed RNA, cells were pulsed with 4sU for the last 10 min of each time point (Fig 3A). Cells not released from transcriptional block ("0 min") were also labeled with 4sU. Following biotinylation and purification, 4sU-labeled RNAs were subjected to deep sequencing. At time "0 min", which corresponds to the release from DRB inhibition, the vast majority of reads were observed over a narrow area near the promoter (Fig 3B, black line, and Fig EV2A, top panel). As time progresses, the reads from nascent RNA are observed further into the gene bodies, referred to as the transcription "wave-front" progression (Fig EV2A, wave-front progression in Notch1). On average, we observed that in WT cells, RNAPII had progressed approximately 11 kb into the gene at 5 min and up to 35.8 kb after 15 min after DRB removal. By contrast, in slow/slow cells the transcription wave-fronts reached only 8.6 kb and 26.7 kb at 5- and 15-min time points, respectively (Figs EV2B and 3B). Genome-wide, we observed an average elongation rate of 2,450 bases/min in wild-type cells, but reduced rates of 1,780 bases/min in slow/slow cells (Fig 3B and D). Previous work suggests that the speed of RNAPII differs between genes (Danko et al, 2013; Jonkers et al, 2014). The density plot of reported elongation rates demonstrates that the dynamic range of transcription rates is narrower in slow RNAPII cells, while in wild-type cells it seems to be bimodal, revealing a population of RNAPII transcribing at higher rates (Fig 3C). Indeed, most genes have a lower elongation rate in slow/slow cells in comparison with wild-type cells (e.g., Ern1 is transcribed at 4.2 and 1.9 kb/min, in wild-type and slow/slow cells, respectively (Table EV1). Interestingly, there are examples of genes that are transcribed faster in slow/slow cells. It is possible that a slower elongation rate might lead to a longer residence time, allowing more time for positive factors to bind and/or stimulate RNAPII and consequently lead to overall higher transcription rates for these genes. Finally, there is a positive correlation between elongation rate and expression levels (Fig EV2C), indicating that on average, highly expressed genes have faster elongation rates in agreement with previous reports (Danko et al, 2013; Jonkers et al, 2014). Overall, these data validate previous results obtained in cultured cells transfected with an α-amanitin-resistant RNAPII harboring the C4 mutation (de la Mata et al, 2003; Fong et al, 2014) and confirms that the endogenous knock-in of a slow RNAPII mutation affects negatively the transcriptional elongation rate in mouse ESCs. Figure 3. Global analysis of transcription elongation rate in mouse ESCs by 4sU-DRBseq A. Schematic of the 4sU-DRB-seq labeling protocol. B. Meta-gene profile of normalized 4sU-DRB-seq reads in WT/WT and slow/slow ESCs. C, D. Density and violin plot of elongation rate (bases/min) calculated for genes common in all genotypes in WT/WT, WT/slow, and slow/slow ESCs. Box and whisker plot (5th–95th percentile) indicates median. Mann–Whitney test, ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 3 [embj2018101244-sup-0007-SDataFig3.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Analysis of elongation rate in mouse ESCs by 4sU-DRBseq (related to Fig 3) Example of wave progression in the Notch1 gene (reverse strand) following release from DRB block at 0, 5, and 15 min. Position of the 4sU-DRB-seq transcription wave-front for common genes over time. Dashed lines indicate median wave-front positions. Correlation of elongation rate measured in WT/WT, WT/slow, and slow/slow ESCs with mean expression. Correlation coefficients are 0.047, 0.003, and 0.015 (P = 2.1e-10, 0.036, and 9.4e-8), respectively. Download figure Download PowerPoint Role of transcriptional elongation during neural differentiation To assess whether a differential transcription elongation rate affects ESC differentiation, we exploited an in vitro model of neuronal development. During embryonic development, different pathways control self-renewal and differentiation capacity of neural progenitors (Doe, 2008; Aguirre et al, 2010). ESCs can differentiate into multipotent Sox1 and Nestin-positive neuronal progenitor cells (NPCs) in a serum-free adherent monolayer culture (Ying et al, 2003; Conti et al, 2005) (Fig 4A). The ESC-derived NPCs can then be used to generate neural stem cells (NSCs) by allowing these cells to form floating aggregates (AGGs) in epidermal/fibroblast growth factor 2 (EGF/FGF2)-containing medium from which a population of bipolar, self-renewing and multipotent NSCs outgrow in adherent conditions (Fig 4A). Alternatively, NPCs can be differentiated into all three neural lineages. For example, when cultured adherently on poly-ornithine/laminin in media containing cAMP and ascorbic acid, they differentiate into Tuj1+ immature neuronal cells and further into Map2-positive mature post-mitotic neurons. Figure 4. Differentiation of WT and slow ESCs along the neural lineage Schematic of the neural differentiation system used in this study, indicating the relevant markers that define different stages of differentiation. Bright-field images and analysis of NPC markers by immunofluorescence staining (Sox1 and Nestin) or RT–qPCR (Nestin and Pax6) (n = 3, mean ± SEM). Bright-field images of aggregates. Bright-field images and immunofluorescence staining for Nestin and neuronal marker Tuj1 in NSC cultures grown in EGF-/FGF-proliferating conditions. White arrows indicate NSCs and green arrows differentiated cells. Two small panels on the right are examples of Tuj1+ neuronal cells in slow/slow NSC cultures. Immunofluorescence staining for neuronal marker Tuj1 in neuronal cultures grown on poly-ornithine/laminin at 21 days of differentiation. Download figure Download PowerPoint We induced differentiation of WT ESCs and slow/slow ESCs into NPCs. We found that both wild-type and slow/slow cells generated Sox1, Pax6, and Nestin-positive NPCs (Fig 4B); however, we also observed decreased proliferation or compromised differentiation potential of slow/slow cells (see Materials and Methods). We next tested whether we could generate NSCs from slow/slow ESC-derived NPCs. Interestingly, we found that despite obtaining neural AGGs (Fig 4C), slow/slow NSCs could not be maintained in EGF-/FGF2-proliferating conditions (Fig 4D). Instead, following a few passages we noted the appearance of flattened differentiated cells in the slow/slow cultures, and subsequently, we observed overwhelming cellular death. Strikingly, among some of the remaining Nestin-positive cells in these slow/slow cultures, we observed promiscuous differentiation to Tuj1+ cells (Fig 4D). These results suggest that the balance between maintenance of the self-renewing cell state and differentiation might be perturbed in slow/slow NSCs. Indeed, Gene Ontology (GO) analysis revealed that those genes upregulated in slow/slow NPCs and in aggregates (AGG) were involved in neuronal functions (Table EV2 and Appendix Fig S1A), which might explain some of the phenotypes observed in slow/slow NSCs (Fig 4). We observed upregulation of Ascl1, Nr2f1, Crabp2, and Nr6a1 genes (Appendix Fig S1B) in slow/slow NPCs and AGGs. Their overexpression has been previously shown to suppress proliferation of progenitor cells, induce neurogenesis and neuronal maturation (Chanda et al, 2014; Gkikas et al, 2017), and could explain the premature differentiation observed in slow/slow NSCs. In parallel, we observed that the EGF receptor (EGFR) was twofold downregulated in slow/slow NPCs (Table EV2). As EGF withdrawal causes massive cell death and premature differentiation observed in slow/slow NSCs (Conti et al, 2005), decreased expression of EGFR in slow/slow NPCs could contribute to the observed lack of their self-renewal in EGF-/FGF2-prolif
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