Induction of hematopoietic and endothelial cell program orchestrated by ETS transcription factor ER 71/ ETV 2
2015; Springer Nature; Volume: 16; Issue: 5 Linguagem: Inglês
10.15252/embr.201439939
ISSN1469-3178
AutoresFang Liu, Daofeng Li, Yik Y. L. Yu, Inyoung Kang, Min‐Ji Cha, Ju Young Kim, Changwon Park, Dennis K. Watson, Ting Wang, Kyunghee Choi,
Tópico(s)Phagocytosis and Immune Regulation
ResumoArticle23 March 2015free access Induction of hematopoietic and endothelial cell program orchestrated by ETS transcription factor ER71/ETV2 Fang Liu Fang Liu Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Daofeng Li Daofeng Li Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Yik Yeung Lawrence Yu Yik Yeung Lawrence Yu Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Inyoung Kang Inyoung Kang Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Min-Ji Cha Min-Ji Cha Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Ju Young Kim Ju Young Kim Department of Pediatrics, Children's Heart Research and Outcomes Center, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Changwon Park Changwon Park Department of Pediatrics, Children's Heart Research and Outcomes Center, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Dennis K Watson Dennis K Watson Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA Search for more papers by this author Ting Wang Corresponding Author Ting Wang Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Kyunghee Choi Corresponding Author Kyunghee Choi Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Developmental, Regenerative, and Stem Cell Biology Program, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Fang Liu Fang Liu Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Daofeng Li Daofeng Li Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Yik Yeung Lawrence Yu Yik Yeung Lawrence Yu Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Inyoung Kang Inyoung Kang Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Min-Ji Cha Min-Ji Cha Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Ju Young Kim Ju Young Kim Department of Pediatrics, Children's Heart Research and Outcomes Center, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Changwon Park Changwon Park Department of Pediatrics, Children's Heart Research and Outcomes Center, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Dennis K Watson Dennis K Watson Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA Search for more papers by this author Ting Wang Corresponding Author Ting Wang Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Kyunghee Choi Corresponding Author Kyunghee Choi Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Developmental, Regenerative, and Stem Cell Biology Program, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Author Information Fang Liu1, Daofeng Li2, Yik Yeung Lawrence Yu1, Inyoung Kang1, Min-Ji Cha1,6, Ju Young Kim3, Changwon Park3, Dennis K Watson4, Ting Wang 2 and Kyunghee Choi 1,5 1Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA 2Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA 3Department of Pediatrics, Children's Heart Research and Outcomes Center, Emory University School of Medicine, Atlanta, GA, USA 4Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA 5Developmental, Regenerative, and Stem Cell Biology Program, Washington University School of Medicine, St. Louis, MO, USA 6Present address: Catholic Kwandong University, Institute for Bio-Medical Convergence, College of Medicine, Korea *Corresponding author. Tel: +1 3142 860865; E-mail: [email protected] *Corresponding author. Tel: +1 3143 628716; E-mail: [email protected] EMBO Reports (2015)16:654-669https://doi.org/10.15252/embr.201439939 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 ETS factor ETV2 (aka ER71) is essential for the generation of the blood and vascular system, as ETV2 deficiency leads to a complete block in blood and endothelial cell formation and embryonic lethality in the mouse. However, the ETV2-mediated gene regulatory network and signaling governing hematopoietic and endothelial cell development are poorly understood. Here, we map ETV2 global binding sites and carry out in vitro differentiation of embryonic stem cells, and germ line and conditional knockout mouse studies to uncover mechanisms involved in the hemangiogenic fate commitment from mesoderm. We show that ETV2 binds to enhancers that specify hematopoietic and endothelial cell lineages. We find that the hemangiogenic progenitor population in the developing embryo can be identified as FLK1highPDGFRα−. Notably, these hemangiogenic progenitors are exclusively sensitive to ETV2-dependent FLK1 signaling. Importantly, ETV2 turns on other Ets genes, thereby establishing an ETS hierarchy. Consequently, the hematopoietic and endothelial cell program initiated by ETV2 is maintained partly by other ETS factors through an ETS switching mechanism. These findings highlight the critical role that transient ETV2 expression plays in the regulation of hematopoietic and endothelial cell lineage specification and stability. Synopsis ETV2 controls the gene regulatory network and signaling involved in the hemangiogenic progenitor specification from mesoderm. ETV2 induces key genes regulating hematopoietic and endothelial cell lineage specification. ETV2 function is specifically required for the generation of the FLK1highPDGFRα− cell population. ETV2 induces other Ets genes, which subsequently maintain ETV2-initiated blood and endothelial cell programs through an ETS switching mechanism. Introduction Functional blood and its conduit vascular system are the first to form during embryogenesis. Blood cells in the developing embryo are generated in close association with endothelial cells. For example, yolk sac blood islands composed of centrally located embryonic blood cells and an outer luminal layer of endothelial cells of the embryo are generated from the extra-embryonic mesoderm presumably through a hemangioblast intermediary 1234. While the hemangioblast is believed to be a common hematopoietic and endothelial cell progenitor, a recent study suggests that primitive erythroid cells in the yolk sac are developmentally distinct from the yolk sac endothelium 5. It has also been suggested that hemangioblast is not a progenitor, but represents a competent cell state that can generate either blood or endothelium depending on the surrounding signals 6. Regardless, it is now widely accepted that hemogenic endothelium within the dorsal aorta of the embryo is the cell origin of the definitive hematopoietic system 78910. Later in adult life, hematopoietic system is maintained partly through the vascular system. As such, sinusoidal endothelial cells form an important component of the niche in which hematopoietic stem cells reside 111213. It is thus noticeable that many transcription factors and signaling pathways are largely shared between blood and endothelial cells. Gene-targeting studies have shown that mutations in any of the shared genes often affect both cell lineages, supporting the notion of common genetic pathways regulating hematopoietic and endothelial cell lineage development and function. Remarkably, Vegfa- or Vegfr2 (Flk1)-deficient animals completely fail to generate blood and blood vessels and die early in embryogenesis, indicating that precise VEGFA signaling via FLK1 is critical for the proper formation of the blood and vascular systems 141516. Clearly, molecular mechanisms by which blood and vessel lineages are specified in the developing embryo need to be better elucidated. Such information in turn would be greatly useful for future applications for blood and vascular repair and regeneration as well as for obtaining hematopoietic and endothelial cells from pluripotent stem cells. ETS transcription factors have emerged as critical regulators of hematopoietic and vascular development 171819. The ETS domain, which is composed of a winged helix-turn-helix motif, binds a consensus sequence (GGAA/T) to regulate target gene expression. Many ETS factors are redundantly expressed in blood and endothelial cells. Consistently, mice or zebrafish deficient in Ets factors display differing levels of hematopoietic and vascular defects 20212223. Distinct from other ETS factors, Etv2 is transiently expressed in the primitive streak, yolk sac blood islands, and large vessels including the dorsal aorta during embryogenesis 24. Remarkably, Etv2-deficient animals display a complete block in blood and blood vessel formation, indicating that ETV2 performs a non-redundant and indispensable function in hematopoietic and vessel development 24252627. As such, Etv2 inactivation leads to similar hematopoietic and vascular defects to those of Vegfa or Flk1 deficiency. Herein, we characterized germ line and conditional Etv2 knockout mice and performed genomewide ChIP-Seq of ETV2 using in vitro differentiated embryonic stem (ES) cells to better understand how ETV2 can achieve such a non-redundant predominant role in hematopoietic and endothelial cell development. We discover that specification of the hemangiogenic program requires ETV2 activation of the blood and endothelial cell lineage-specifying genes and VEGF signaling. Moreover, ETV2 establishes an ETS hierarchy by directly activating other Ets genes, which then maintain blood and endothelial cell program initiated by ETV2 through an ETS switching mechanism. Collectively, we provide molecular and cellular basis by which ETV2 establishes the hematopoietic and endothelial cell program. Results ETV2 ChIP-Seq and target gene identification To understand ETV2-mediated genetic program regulating hematopoietic and endothelial cell lineage development, we performed ETV2 ChIP-Seq analysis using in vitro differentiated embryonic stem (ES) cells. We previously described A2 ES cells expressing ETV2-V5 in a doxycycline (DOX)-inducible manner 2427. DOX addition from day 2 to 3.5, a time frame when Etv2 is normally expressed, in these cells robustly induced hemangioblast formation. To facilitate ETV2 target identification, we additionally generated polyclonal antibodies against ETV2200–219 peptide (ETV2-polyAbs) to pull down ETV2-associated chromatin. Two independent biological replicates from DOX-treated day 3.5 iEtv2 EB cells were subjected to ETV2-polyAbs and V5 ChIP and deep sequencing using IgG as controls. Sequencing reads were mapped to the mouse genome assembly mm9 provided by the UCSC Genome Browser 28. Using MACS2 29 at a P-value cutoff of 1E-6, we identified 8,019 ETV2 binding peaks from the ETV2-polyAbs samples and 11,892 ETV2 binding peaks from the V5 samples (Supplementary Table S1). We focused our analysis on the 3,933 peaks that overlapped between the ETV2-polyAbs samples and the V5 samples (Fig 1A and B), as we reasoned that this strategy provided the best balance between sensitivity and specificity given the genomic technology we employed. As such, the ETV2 overexpression system ensures higher sensitivity of detection of ETV2 genomewide target sites than assaying against endogenous protein. Moreover, focusing on reproducible ChIP-seq peaks based on independent antibodies ensures a higher specificity. Indeed, several initial quality assurance analyses suggested that these ETV2 binding peaks had high quality and were connected to ETV2 biology, supporting that they were bona fide biological target sites of ETV2. First, raw read densities within these ETV2 peaks were highly reproducible between experiments (cc = 0.974 for ETV2-polyAbs and 0.969 for V5 between replicates, cc = 0.993 between the two antibodies, Supplementary Fig S1A–C). ChIP-Seq signals were highly enriched in peak centers across samples when compared to surrounding genomic regions or control (Fig 1A and B). We also identified the most significant sequence motif associated with the ChIP-Seq peaks (Fig 1C). This motif, which represented ~85% of the peaks, matched perfectly with the known binding specificity of other ETS factors, FLI1, and ERG 30. We additionally identified GATA, SOX, or E-box motifs to be frequently associated with the ETV2 peaks (Supplementary Fig S1D). Genomewide distribution of these binding peaks was far from random expectation, with ~14% significant enrichment in the promoter regions and ~70% in introns or intergenic regions, suggesting that ETV2 functions by interacting with both gene promoters and distal enhancers (Fig 1D and Supplementary Fig S1F). We next subjected these ETV2 binding peaks to a GREAT analysis 31 to understand the overall ETV2-mediated biological function and found that they were strongly associated with endothelial and hematopoietic cell lineage development and differentiation (Fig 1E, for complete result see Supplementary Table S2A). Finally, we examined evolutionary conservation of sequences associated with ETV2 peaks and found that they are much more conserved than their neighboring sequences (Fig 1F). Indeed, 2,231 (56.7%) peaks overlapped with conserved elements determined based on 30-way vertebrate alignment 32 from the UCSC Genome Browser (P-value < 2.2e−16, binomial test), suggesting that the identified peaks were strongly enriched for functionally constrained sequences. As the majority of the ETV2 binding peaks were outside gene promoters, we reasoned that some of them could serve as distal enhancers. Connecting enhancers to their target genes is an extremely challenging problem, because enhancers do not necessarily regulate the nearest genes, nor do they necessarily regulate one single target genes. Therefore, we combined GREAT analysis with gene expression analysis to optimize sensitivity and specificity. Using GREAT default parameters, we defined a "basal regulatory domain" for each gene (Methods), and we associated an ETV2 ChIP-seq peak with a target gene if the peak was within the basal regulatory domain of the gene. This approach resulted in 4,580 ETV2 peak-associated genes. Figure 1. ChIP-Seq analysis of ETV2 binding regions Heatmap of read density from each replicate in a 6-kb region flanking the peak centers. Averaged read density profile of each replicate in a 6-kb region flanking the peak center. ETV2 motif logo, de novo trained by Homer software using ChIP-Seq peaks. Distribution of ETV2 ChIP-Seq peaks in different genomic features. Red line represents genomic background (expectation), and blue bar represents observed peak distribution. Functional enrichment analysis of ChIP-Seq peaks. Conservation score (phastCon) in a 6-kb region flanking peak centers of all peaks. Download figure Download PowerPoint To narrow down to a more confident list of direct target genes of ETV2, we integrated our ChIP-Seq data with gene expression pattern of FLK1+ mesoderm isolated from control and iETV2 EB cells that were generated with DOX (from day 2–3.5) as well as FLK1+ mesoderm sorted from Etv2+/+ and Etv2−/− day 3.5 EBs 27. ETV2 peak-associated genes were significantly enriched for genes that exhibited increased expression in the ETV2 overexpression system and/or reduced expression in the ETV2 knockout system (Fig 2A, P-value < 2.2E-16, hypergeometric test). On the other hand, there was no significant enrichment of genes with the opposite expression pattern (i.e., upregulated in ETV2 knockout and downregulated in ETV2 overexpression, Supplementary Fig S2A). This expression pattern suggested that ETV2 primarily functions as a transcriptional activator. We identified 425 ETV2 peak-associated genes that exhibiting the expected expression difference. They constitute a group of high confidence, direct target genes of ETV2. Functional enrichment analysis revealed that this group of genes was considerably enriched for hematopoietic and endothelial cell lineage development and differentiation, with the enrichment level markedly improved from genes identified by ChIP-seq peaks alone (Supplementary Table S2B). Many genes that exhibited expression changes upon either ETV2 overexpression or ETV2 knockout did not associate with ETV2 binding peaks. They are potentially downstream but not direct targets of ETV2. Additionally, many ETV2 peak-associated genes did not show expression change, even though as a group they are strongly enriched for endothelial and hematopoietic cell relevant functions. These binding peaks could potentially be false positives, but could also reveal functions of ETV2 other than directly activating target genes. One such potential function could be to modulate chromatin structure to a permissive state that allows genes to express at future developmental stages. We thus examined the epigenetic landscape of ETV2 binding sites across several cell and tissue types. We took advantage of publicly available whole-genome bisulfite sequencing data of mouse embryonic stem cells, neural progenitor cells, as well as adult tissues 3334 to examine epigenetic changes co-occurring with ETV2 binding, focusing on distal sites that potentially function as lineage-specific enhancers. Intriguingly, these ETV2 binding sites were largely methylated in ES cells. The relatively high DNA methylation levels were maintained in neuronal progenitor cells and cerebellum, but reduced in heart and bone marrow (Fig 2B and Supplementary Fig S2B). Figure 2. Identification of ETV2 downstream target genes and regulatory network Venn diagram showing the overlap between ChIP-Seq peak-associated genes and ETV2 target genes identified by microarray data analysis. UP (+DOX): upregulated genes by ETV2 overexpression; DN (ETV2 KO): downregulated genes in ETV2 knockout. DNA methylation profile of ETV2 distal binding peaks. Averaged DNA methylation level was calculated using publicly available whole-genome bisulfite sequencing data from multiple cell and tissue types and plotted for a 6-kb region flanking peak centers. ETV2 gene regulatory network showing Notch/MAPK signaling, VEGF signaling/lineage specification, Rho-GTPase, and ETS transcription factor as downstream targets. Genomic snapshots depicting the ETV2 binding regions at the indicated genomic loci. Download figure Download PowerPoint ETV2 induces hematopoietic and endothelial cell lineage-specifying genes ETV2 targets can be broadly categorized into hematopoietic and endothelial cell lineage-specifying genes, VEGF, Notch, Rho-GTPase, and MAP kinase pathway and Ets factors (Fig 2C). Specifically, Flk1, Fli1, Erg, Gata2, Scl, Meis1, Lmo2, Tie2, VE-cadherin, Dll4, and Notch were among the 425 genes, which play critical roles in hematopoietic and endothelial cell development (Figs 2C and D, 3A and 5A). While some of these peaks occur on previously identified regulatory regions, such as VE-cadherin 27, currently identified ChIP-Seq peaks represented unique peaks that have not been reported yet. We additionally compared transcriptional profiling among FLK1+ cells generated by enforced Etv2 expression, Etv2-deficient FLK1+ cells 27, and genes immediately upregulated by Etv2 expression 35. There was a significant enrichment in genes involved in the VEGF and Notch signaling pathways, suggesting the involvement of these pathways in hemangiogenic lineage development (Supplementary Fig S2C). Figure 3. ETV2 directly regulates VEGF receptors and activate VEGF signaling pathway Genomic snapshots depicting the ETV2 binding peaks associated with the VEGFR genes. Numbers indicate ChIP-Seq peaks with high confidence. ChIP-PCR analysis showing ETV2 recruitment to the 15 potential binding sites in (A). ETV2 enrichments were shown by both V5 antibody and ETV2-polyAb pull-down. PCR primers and genomic locations are provided in Supplementary Table S3. Error bars represent SD, n = 4. Luciferase reporter assay for 1–15 ETV2 peak regions from (A). The indicated peak regions or selected ETV2 binding motif deletion mutants (2, 7, 10, 11, and 14) were cloned into the pGL4.24[luc2P/minP] vector (left panel). 293T cells were transfected with pGL4.24 control vector, ETV2 wild-type, or mutant luciferase reporter constructs, together with Renilla luciferase vector in the presence (pMSCV-Etv2) or absence of ETV2 expression plasmids. For each ETV2 peak reporter construct, luciferase activity was first normalized to Renilla luciferase values. Luciferase activity value obtained with ETV2 was then compared to that of the –ETV2 value. Error bars represent the SEM obtained from four biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student's t-test. Mouse VEGF signaling array using RNA from day 3.5 iEtv2 EBs (±DOX from day 2) is shown as scattered plots of fold changes (+DOX versus −DOX). Each gene was normalized to three housekeeping genes. Red dots are upregulated genes by DOX addition. RNA obtained from E8.5 Etv2+/+ and Etv2−/− embryos was subjected to VEGF signaling analysis. Key genes with significant changes in expression are shown. Mouse Cell Motility PCR array using RNA from day 3.5 iEtv2 EBs (±DOX from day 2) is shown as fold changes (+DOX versus −DOX). Venn diagram showing an overlap between downregulated genes in E8.5 Flk1−/− embryos 41 and upregulated genes by ETV2 overexpression (left) or ETV2 knockout (right) 27. Gene expression analysis of Rho-GTPase activating proteins 18, 25, and 27 in day 3 iEtv2 (±DOX on day 2) or Etv2+/+ and Etv2−/− EB cells. Genes were normalized to Gapdh. The fold change was obtained from the ratios of +DOX to −DOX or Etv2−/− to Etv2+/+. Error bars represent the SD of four independent biological samples. Download figure Download PowerPoint Previous studies have implicated the core gene regulatory network played by the ETS, GATA, and E-box motifs in hematopoietic and endothelial cell development 36. Etv2, Gata2, and Scl can independently modulate hemangioblast development 373839. Moreover, coexpression of Etv2, Gata2, and Scl during the time of hemangioblast formation stage can robustly induce hemangioblast cell population 39. Notably, GATA and E-box motifs were frequently associated with ETV2 peaks (Supplementary Fig S1D and E). Thus, we determined whether sequences representing binding sites of these factors occur in ETV2 peaks. We utilized the ChIP-Seq data of GATA2 and SCL from Wilson et al 30, which allowed us to train a positional weight matrix for each of these factors. GATA2 and SCL motifs were significantly enriched within the 3,933 peaks: 2,945, 609, and 484 co-occurrences of ETV2-SCL motifs (8-fold enrichment over random expectation, P = 0 from binomial test), ETV2-GATA2 motifs (14-fold enrichment, P < 1.11E-35), and ETV2-SCL-GATA2 motifs (15-fold enrichment, P = 0) (Supplementary Fig S2D). This observation is highly non-random, suggesting that ETV2 and these factors may interact or cobind to some of these sites. Indeed, ETV2 and GATA2 have been recently reported to form a complex to regulate hematopoietic and endothelial cell gene expression 40. ETV2 presumably specifies the hemangiogenic cell fate by collectively turning on hematopoietic and endothelial cell lineage-specifying genes. The ETS, GATA, and E-box gene regulatory network is integral to this process. ETV2 enhances VEGF signaling ChIP-Seq analysis revealed ETV2 binding to VEGF receptors and downstream signaling pathway genes including MAPK. Specifically, ETV2 targets include Flk1, Flt1, Flt4, Nrp1, Nrp2, and Mapk3 genes (Fig 3A). Rho-GTPases and adhesion molecules were also identified as potential ETV2 direct targets. We selected 15 peak regions associated with Flk1, Flt1, Nrp1, and Nrp2 genes occupied by ETV2, of which 14 were evolutionarily conserved (Fig 3A and Supplementary Table S3), and validated ETV2 binding in day 3.5 iEtv2 EBs using ChIP-qPCR. A significant mean enrichment was observed for ETV2 binding at these genomic locations with chromatin pulled down by V5 antibody or endogenous ETV2 antibody (ETV2-polyAbs) in iEtv2 EB cells (Dox added on day 2) (Fig 3B). Importantly, ETV2 binding at these genomic locations was confirmed in R1 wild-type EB cells using ETV2-polyAbs, validating that these are bona fide ETV2 targets (Supplementary Fig S3A). To assess the functional significance of the ETV2 binding, we tested these regions for the response to ETV2 using the luciferase reporter assay. We found that ETV2 could activate the luciferase constructs tested, approximately 5- to 100-folds, compared to the pGL4 vector control (Fig 3C). We further selectively deleted the ETV2 binding motif in several reporter vectors and found that the luciferase activity was greatly impaired when ETV2 binding motif was deleted (Fig 3C). Collectively, these regions may act as ETV2 cis-regulatory elements for VEGF receptor gene expression. Future in vivo transgenic reporter system would further solidify such notion. As ChIP-Seq analysis suggested that ETV2 could elevate FLK1 signaling activity, we determined whether ETV2 could directly modulate VEGF signaling. To this end, we performed VEGF signaling and Cell Motility PCR array, which included Rho-GTPases, adhesion and integrin genes, using RNA obtained from iEtv2 EBs (differentiated ES cells) with DOX 27. Strikingly, VEGF signaling pathway genes were upregulated by Etv2 overexpression, which were reciprocally downregulated in Etv2−/− embryos as well as Flk1−/− embryos (Fig 3D and E, Supplementary Table S4). Additionally, while Rho-GTPase and its activating protein genes were upregulated by Etv2 overexpression, they were greatly downregulated in E8.5 Flk1−/− embryos 41 (Fig 3F and G, Supplementary Table S5). These studies support the notion that hemangiogenic program specification requires ETV2-mediated FLK1 signaling activation. ETV2 is required for the formation and expansion of FLK1+ hemangiogenic progenitors Consistent with the data that ETV2 enhances VEGF signaling, previous findings suggested that blood and endothelial cell progenitors express high levels of FLK1 (FLK1high) compared to other FLK1-expressing cardiac or muscle progenitors (FLK1low) 42. To determine whether we can identify differential FLK1 activity associated with hemangiogenic cell population development in the embryo, we subjected embryos to FLK1 and PDGFRα expression analysis. As FLK1+PDGFRα− cells isolated from differentiating ES cells were enriched for the hemangioblast 27 and as FLK1+PDGFRα− cells in the developing embryo have not been characterized yet, we initially analyzed FLK1 and PDGFRα expression kinetics in developing embryos. In wild-type embryos, FLK1+PDGFRα− hemangiogenic progenitors were already detected around embryonic day (E) 7.5 and progressively expanded with time during the course of E7.5–E8.5 (Fig 4A). Notably, the mean fluorescence intensity of the FLK1 staining within the FLK1+PDGFRα− cell population became greater as embryos develop, suggesting an elevated FLK1 signaling activity within FLK1+PDGFRα− cells compared to FLK1+PDGFRα+ cells (Fig 4A). Remarkably, FLK1highPDGFRα− cell population was exclusively missing in Etv2−/− embryos, indicating that ETV2 was specifically required for the stage of FLK1highPDGFRα− cell generation in the embryo (Fig 4A, Supplementary Fig S3B and C). Figure 4. ETV2 is required for the generation and expansion of hemangioblast A. Etv2+/+ and Etv2−/− yolk sac and embryo proper were analyzed for FLK1 and PDGFRα expression at E7.5, 8, and 8.5 (n = 6 for each). Representative FACS analysis at each time point is shown. B. Gross morphology of the E10.5 Flk1Cre;Etv2 CKO embryos is shown. Note the wrinkled yolk sac (red arrow) and the swollen pericardial cavity accumulated with fluid/blood (yellow arrow) in the mutants. C. FACS analysis of E8.5 Flk1Cre;Etv2 CKO embryos for FLK1 and PDGFRα expression. D. CD31/PECAM1 whole-mount staining of the E9.5 embryos (up: embryos; down: yolk sacs). E. Hematopoietic colony assay of the E8.5 wild-type and mutant yolk sacs. Mean ± SD, n = 4 biological replicates. ***P < 0.001, two-tailed Student's t-test. F, G. Microarray analysis (F) and qRT–PCR analysis (G) of the Flk1Cre;Etv2 CKO yolk sacs. Mean ± SD, n = 3 biological replicates. *P < 0.05, ***P < 0.001, two-tailed Student's t-test. Download figure Download PowerPoint Etv2 is transiently expressed in developing embryos and ES/EBs 24. To determine wheth
Referência(s)