This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Japan Society for the Promotion of Science [Grant-in-Aid for Scientific Research A 22249009 (to A.Y. sharing sensitive information, make sure youre on a federal Bookshelf Knebel J, De Haro L, Janknecht R. Repression of transcription by TSGA/Jmjd1a, a novel interaction partner of the ETS protein ER71. Keywords: (J) Dil-AcLDL uptake assay. Nearly all of the ETVEC-constituting vessels allowed erythrocyte circulation through the vessels (Fig. Figure 5.. Etv deletion prevents FGF from inducing aberrant differentiation of the lens epithelium. Vascular RhoJ is an effective and selective target for tumor angiogenesis and vascular disruption. To determine which ETV2 domain is essential for the EC induction, we constructed four types of HA-tagged ETV2 truncations for lentivirus vectors (SI Appendix, Fig. S2E and S3). E, Western blot analysis of exogenous ETV2 using an anti-HA antibody with cell lysate from Dox and +Dox treated iHA-Etv2 mouse embryonic fibroblasts (MEFs). ETV2/ER71 Transcription Factor as a Therapeutic Vehicle for Cardiovascular Disease. Keywords: Rackwitz L, et al. Previous studies have shown that cell migration and proliferation are highly coordinated and coregulated processes observed in wound healing, cancer, tissue regeneration, or hypoxic environments.3740 Furthermore, effectors such as FGF2, Shh, mir-221, AMPK, YAP, and others have been shown to coregulate cell migration and cell proliferation in a context-dependent fashion.18,4144 Therefore, our studies support the hypothesis that ETV2 plays a critical role in the coregulation of hematoendothelial progenitor cell proliferation and migration mediated via YES1 and RHOJ, respectively. (D) Whole-cell lysates subjected to Western blot analysis. Red to white color and dark-blue color on the image indicate high and low perfusion signals, respectively. 2116. iHA-Etv2 MEFs were used for chromatin immunoprecipitation (ChIP) using the protocol as previously described.5 Briefly, MEFs were dissociated into single cells using 0.25% trypsin, fixed with 1% formaldehyde at room temperature for 10 minutes, and quenched in 0.125 mol/L glycine. Detecting critical transition signals from single-cell transcriptomes to infer lineage-determining transcription factors. WebHere, we describe a novel role for ETV2 (Ets variant transcription factor 2) in cell migration and provide evidence for an ETV2-Rhoj network as a mechanism responsible for this 1 [3, 4]) and (Dox+shRNA No. Before C, Quantitative polymerase chain reaction analysis of Rhoj transcripts demonstrating that the induction of ETV2 resulted in increased expression of Rhoj (n=3 replicates; **P<0.01). Here, we discuss the detailed mechanisms underlying ETV2/ER71-regulated cardiovascular lineage development. 2 Mutant Strand18 unlabeled: GGAATGCAGCttcAAACCCACGATT; Site No. Sci. Here we report that ETV2 functions as a pioneer factor that relaxes closed chromatin and regulates endothelial development. Future studies that target this cascade may serve as a platform for therapies that could benefit from increased (ie, ischemic diseases) or decreased (ie, tumorigenesis) hematoendothelial progenitor cell migration. ( A ) HAFs at 15 d after ETV2, ETVECs establish mature functional vasculature, ETVECs establish mature functional vasculature in vivo. For Sources of Funding and Disclosures, see page 2889. https://doi.org/10.1161/ATVBAHA.120.314488, National Center Data are presented as meanSEM (n=4 replicates; *P<0.05). ER71 directs mesodermal fate decisions during embryogenesis. Furthermore, our qPCR analysis using FACS-sorted cells revealed that Rhoj was robustly expressed in endothelial progenitors (angioblasts) and not in the nonendothelial lineages. Gated on 7-AADVenus+ cells. B.N. D and E, The biological process and pathways that are significantly enriched in the upregulated genes following ETV2 overexpression in the ESC/EB system. (AF) Images of Matrigel plugs extracted from NOD SCID mice 28 (AD and F) and 42 (E) days after the implantation of ETVECs (AF) and HAFs (A). Endothelial progenitors migrate early during embryogenesis to form the primary vascular plexus. By comparing engineered embryonic stem cell differentiation and reprogramming models with multi-omics techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and recruit BRG1. (G) Representative photographs. Epub 2014 Feb 26. Several murine studies have shown that although ETV2 is a master regulator of hematoendothelial development from the primitive mesoderm, its expression is transient in early murine embryos, and that mature ECs no longer express ETV2 (16). Sierra-Pagan, W. Gong, S. Das, J.W.M. 2009 Aug 1;332(1):166-76. doi: 10.1016/j.ydbio.2009.05.566. 2008;2:497507. Liu F, Li D, Yu YY, Kang I, Cha MJ, Kim JY, Park C, Watson DK, Wang T, Choi K. EMBO Rep. 2015 May;16(5):654-69. doi: 10.15252/embr.201439939. Unable to load your collection due to an error, Unable to load your delegates due to an error, ETVECs establish mature functional vasculature in vivo. H and I, Quantitative polymerase chain reaction analysis of cell migratory gene expression, including Mmp9 and PlexinD1 using RNA isolated from iHA-Etv2 MEFs in the absence (Dox) or presence (+Dox) of doxycycline for 24 h. Note increased expression of these genes in the Dox-treated samples compared with Dox (n=3 replicates; **P<0.01). In addition, ETV2 alone also induced CD31+ cells from not only the human neonatal skin fibroblast cell line NB1RGB, but also primary human adult skin fibroblasts (HAFs) (SI Appendix, Figs. 1 Mutant Strand (bottom) unlabeled: GCTGTCAAACTTgaaGCTTTATCCC; Site No. 3], and Figure VI in the Data Supplement). F, Phase contrast microscopic images of iHA-Etv2 MEFs in the absence (Dox) and presence (+Dox) conditions for a 12-h period following initiation of the scratch assay. To increase the efficiency of ETVEC induction from fibroblasts, how ETV2 expression levels affect the epigenetic state of endothelial genes needs to be elucidated. Identification of a Novel Immune-Related lncRNA CTD-2288O8.1 Regulating Cisplatin Resistance in Ovarian Cancer Based on Integrated Analysis. government site. In addition, some of the Venus+ HFL-1 cells demonstrated acetylated low-density lipoprotein (AcLDL) uptake in a punctuate staining pattern characteristic of ECs (2) (SI Appendix, Fig. Nam D, Park MR, Lee H, Bae SC, Gerovska D, Arazo-Bravo MJ, Zaehres H, Schler HR, Kim JB. Quantitative RT-PCR revealed that CD31hi ETVECs that had been cultured without Dox for 20 d retained similar expression levels of ERG, FLI1, GATA2, and TAL1 as the Dox-exposed ETVECs, although CD31 cells lost the expression of these molecules (Fig. Connecting proliferation and apoptosis in development and disease. The Suppression of MAPK/JNK-MTORC1 signaling leads to premature loss of organelles and nuclei by autophagy during terminal differentiation of Lens fiber cells. Collectively, the foregoing observations demonstrate that ETVECs survive and form mature functional perfused vasculature after transplantation in vivo. The accumulative data indicate that ETV2, an ETS transcription factor, performs a potent and indispensable function in mediating vessel development. (H) Hematoxylin and eosin staining of the adductor muscles of ischemic limbs. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Open Biol. D, Schematic showing the ETV2-RHOJ network in the regulation of cell migration. Each well was imaged every 6-hour period at the same region of the well. 2016 Jan;142:92-101. doi: 10.1016/j.exer.2015.02.004. 6 B and D). YAP regulates cell proliferation, migration, and steroidogenesis in adult granulosa cell tumors. Next, we directly tested whether the overexpression of ETV2 could induce cell migration in an in vitro setting. An official website of the United States government. Sancho-Martinez I, Baek SH, Izpisua Belmonte JC. Expression profiles frame the promoter specificity dilemma of the ETS family of transcription factors. These results demonstrate that transient ETV2 expression is sufficient to directly convert part of HAFs into ETVECs that stably maintain EC properties. Careers. (. Data are representative of three independent experiments. Combined expression of multiple transcription factors specific to a particular lineage has been demonstrated to change somatic cell fate by bypassing pluripotency; for example, Gata4, Mef2c, and Tbx5 convert murine cardiac fibroblasts into functional cardiomyocytes (9), and Ascl1, Brn2, and Myt1 induce neurons from murine fibroblasts (10). Bethesda, MD 20894, Web Policies Based on the coexpression analysis of Etv2 and Rhoj, we examined whether the expression of Rhoj was regulated by ETV2 during embryogenesis. ( A ) Expression, ETVECs represent proliferative ECs. Accessibility etv2. -. 2014 Dec 15;28(24):2679-92. doi: 10.1101/gad.253443.114. Normality and variance were not tested to determine whether the applied parametric tests were appropriate for these analyses. Proceedings of the National Academy of Sciences of the United States of America, www.pnas.org/lookup/suppl/doi:10.1073/pnas.1413234112/-/DCSupplemental. Vierbuchen T, Wernig M. Molecular roadblocks for cellular reprogramming. J Biol Chem. doi: 10.1126/sciadv.abf1068. wrote the paper. Note a robust enrichment of Rhoj in the +Dox EBs as compared with Dox (n=3 replicates; **P<0.01). The mutant promoter-reporter constructs were generated using the same method but substituted mutant oligos for ETV2-binding sites. These results suggest that at least one of the 18 TFs directly converts HFL-1 cells into ECs. Federal government websites often end in .gov or .mil. Etv2 is expressed in the yolk sac hematopoietic and endothelial progenitors and regulates Lmo2 gene expression. Furthermore, ETVECs significantly improve blood flow recovery in a hind limb ischemic model using BALB/c-nu mice. Endothelial cells (ECs) form vasculature to provide vital elements, such as nutrients and oxygen, to tissues and organs in the body. (BD) Fifteen days later, Venus+ cells were subjected to quantitative RT-PCR analysis (B) and flow cytometry analysis (C and D). 2013;288:1213012141. S5), were significantly up-regulated only in the VEGF-R2+ population. These results support the capacity of ETVECs for constituting vessels in the ischemic regions. Int J Mol Sci. Etv2, which is silent in homeostatic HSCs, was transiently activated in regenerating HSPCs and was Transient ETV2 expression is sufficient to directly convert part of HAFs into ETVECs. To verify whether the rescue of perturbed migration was because of an ETV2-Rhoj network, we undertook qPCR experiments. A histone demethylase, Jmjd1a, has been shown to suppress ETV2 to activate transcription from the matrix metalloproteinase-1 promoter by directly interacting with ETV2 (21, 32). S1A). Paul and Sheila Wellstone Muscular Dystrophy Center (M.G.G., D.J.G. {"type":"entrez-geo","attrs":{"text":"GSE48980","term_id":"48980","extlink":"1"}}GSE48980). In coordination with endogenous FOXC2 in fibroblasts, transduced ETV2 elicits expression of multiple key endothelial development factors, including FLI1, ERG, and TAL1, and induces expression of endothelial functional molecules, including EGFL7 and von Willebrand factor. Figure 2.. All methods were performed in accordance with the relevant guidelines and regulations. S1 F and G), we conclude that the Venus+CD31+ HFL-1 cells are ECs (HFL-ECs). Epigenetic impact of a 1-week intensive multimodal group program for adolescents with multiple adverse childhood experiences. Unable to load your collection due to an error, Unable to load your delegates due to an error. Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis. 2014;10:11931211. Previously, we demonstrated that ETV2 is an essential transcription factor for the development of cardiac, endothelial and haematopoietic lineages. Fifteen days after ETV2 induction into HAFs, CD31+ cells that also expressed VE-cadherin were isolated, and on day 25, the expanding CD31+ cells were purified again (Fig. )]; the Core Research for Evolutional Science and Technology Program (Reprograming of immune system by modulation of intracellular signal transduction) of the Japan Science and Technology Agency (A.Y. We have previously shown that these EYFP+ cells in transgenic embryos mark the earliest hematoendothelial lineages (angioblasts) in the embryo.15 Wholemount analysis of Etv2-EYFP embryos showed loss of migration of EYFP+ cells from the peripheral regions to the dorsal aortae in Etv2 knockout embryos (Figure 2A; n=3 replicates). 2022 Jan 14;14(2):413. doi: 10.3390/cancers14020413. In contrast, i.m. More recently, ETV2 has been reported to transdifferentiate skeletal muscle into functional ECs in zebrafish (30). Recent Advances in Gene Therapy for Cardiac Tissue Regeneration. Here, by screening 18 transcription factors that are important for both endothelial and hematopoietic development, we demonstrate that ets variant 2 (ETV2) We found that TAD-fully deleted ETV2 failed to convert human fibroblasts to ECs, which supports the foregoing hypothesis because the truncated ETV2 has no capacity to interact with another factor via the TAD. H, qPCR analysis for Rhoj transcripts from Flk1/Pdgfra (other lineages), Flk1+/Pdgfra (lateral plate mesoderm), Flk1+/Pdgfra+ (cardiac mesoderm), and Flk1/Pdgfra+ (paraxial mesoderm) sorted cells using the wild-type (WT) ES/EB system. To decipher the correlation between Etv2 and Rhoj expression, we analyzed the single-cell RNAseq datasets obtained from the Etv2-EYFP progenitors at 3 embryonic stages, E7.25, E7.75, and E8.25.15 We used uniform manifold approximation and projection to visualize the single cells and found 3 distinct cell clusters: progenitor cells that are mostly from E7.25, endothelial lineages, and hematopoietic lineages that are mostly from later stages E7.75 and E8.25 (Figure 3A through 3D, Figure IV in the Data Supplement). The dotted line indicates the expected position of the dorsal aorta, which was absent in the Etv2 knockout (KO) embryo17 (n=3 replicates). For the bioinformatics analyses, significance was determined by using the Fisher Exact Test. Bottom: chromatin immunoprecipitation (ChIP) analysis of doxycycline (Dox)-inducible iHA-Etv2 mouse embryonic fibroblasts using an HA antibody (n=3 replicates; **P<0.01). Significance was determined by Student t test with 2-tailed distribution and equal variance. Disclaimer, National Library of Medicine The signal regulated transcription factors (SRTFs) control the ultimate transcriptional output of signaling pathways. Gao L, Jin N, Ye Z, Ma T, Huang Y, Li H, Du J, Li Z. Print 2022 May. Thus, the ETS family factors are more likely candidates for direct induction of ECs from nonvascular cells. Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures. Many cells contain proteins known as signal-induced transcription factors, which are poised to receive messages from the environment and then react by activating genes required for the cell to respond appropriately. 2012;363:320329. Vodyanik MA, Thomson JA, Slukvin II. government site. Thus, we used EGM-2 medium supplemented with both factors in our subsequent experiments. S1E). Epub 2014 Sep 3. 2004;32:56935702. We identified ETV2 as an upstream regulator of the Rhoj gene in the endothelial lineage during migration. (D) Microscopic images of ETVECs (Exp.1) at 32 d after ETV2 transduction. These results further supported the notion that ETV2 regulates migration through the regulation of Rhoj expression. Moreover, COL1A2 was significantly down-regulated in this population (Fig. Neg, negative; Lo, low; Inter, intermediate, Hi, high. -, Basu S, Rajakaruna S, Reyes B, Van Bockstaele E, Menko AS. Inthis study, by screening 18 transcription factors, including the ETS family factors, we demonstrate that the single ETS factor ETV2 is sufficient for directly converting primary human adult skin fibroblasts into functional ECs (ETVECs). qPCR analysis showed that Rhoj lentiviral clones No. ); the Mochida Memorial Foundation (R.M. Figure 5.. Etv deletion prevents FGF from. Epub 2015 Mar 23. Fibrosis in the lens. Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration. To confirm this hypothesis, we differentiated WT ESCs and undertook qPCR analysis using RNA isolated from FACS-sorted Flk1+/Pdgfra (lateral plate mesoderm), Flk1+/Pdgfra+ (cardiac mesoderm), Flk1/Pdgfra+ (paraxial mesoderm), and Flk1/Pdgfra (other lineages) cell populations (Figure 3G). doi: 10.4161/auto.28768. doi: 10.1172/JCI164192. Briefly, cells were lysed in RLT-lysis buffer, followed by a column-based purification process and on-column DNA digestion to remove any traces of DNA. 1 A, F, and G). Shrestha S, Lamattina A, Pacheco-Rodriguez G, Ng J, Liu X, Sonawane A, Imani J, Qiu W, Kosmas K, Louis P, Hentschel A, Steagall WK, Onishi R, Christou H, Henske EP, Glass K, Perrella MA, Moss J, Tantisira K, El-Chemaly S. Life Sci Alliance. Cell. 5C) and exhibited a cobblestone-like morphology (Fig. In the Flk1+/Pdgfra sorted cells, we found Etv2 to be highly expressed (Figure 3I; n=3 replicates; *P<0.05). Migratory assays showed that overexpression of ETV2 enhanced cell migration in mouse embryonic stem cells, embryoid bodies, and mouse embryonic fibroblasts. These results supported the notion that ETV2 plays a critical role in angioblast migration in vivo. 2022 Aug;37(8):723-737. doi: 10.14670/HH-18-458. Cancers (Basel). The VEGF-R2+ population (Fig. Bookshelf Lee D, et al. Accessibility Gene expression levels are relative to HPRT1. Bethesda, MD 20894, Web Policies Author contributions: R.M., T. Shichita, T. Sekiya, A.K., K.S., H.Y., and A.Y. ER71 acts downstream of BMP, Notch, and Wnt signaling in blood and vessel progenitor specification. S1E), ETVECs seem to be more likely a venous EC subset. WebRecent findings We recently demonstrated that the ETS transcription factor Etv2, a critical factor for haematopoietic and vascular development, is also required for haematopoietic Histol Histopathol. The amniotic cell-derived ECs can overcome PSC-derived EC issues, such as poor proliferation and lineage instability. (K) Capillary-like structure formation on Matrigel-coated plates. ETV2 mediates endothelial transdifferentiation of glioblastoma. Thus, creating ECs from nonvascular cells by transducing some transcription factors not only leads to the development of new strategies for patient-specific therapeutic angiogenesis, but also facilitates the maintenance of the solid organs that are regenerated from pluripotent stem cells. Figure 7.. Activation of mTOR signaling disrupts nuclei degradation in the Etv mutant. Circ Res. reveals an unexpected complexity in how these proteins respond to upstream signals, highlighting the importance of further dissecting these relationships. (Left) Low-magnification image. 8600 Rockville Pike PMC First, we identified the role of ETV2 in the regulation of cell migratory networks. Generation of functional hemangioblasts from human embryonic stem cells. ETS Transcription Factor ETV2/ER71/Etsrp in Hematopoietic and Vascular Development. LOCUSID. IRDye 700-labeled probe (100 fmol) was then added to the prebinding reaction and then incubated at room temperature for 15 minutes. 3, respectively. (A) HAFs at 15 d after ETV2 infection were subjected to flow cytometry analysis. Augustin HG, Koh GY, Thurston G, Alitalo K. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Our data revealed that Dox-mediated overexpression of ETV2 resulted in increased expression of Rhoj as compared with Dox condition (Figure 4E; n=3 replicates; *P<0.05). ETVECs, but not HAFs, developed human CD34+ blood vessels in the muscles of the ischemic limbs (Fig. The new PMC design is here! Title: ETV2/ER71 Transcription Factor as a Therapeutic Vehicle for Skylar-Scott MA, Huang JY, Lu A, Ng AHM, Duenki T, Liu S, Nam LL, Damaraju S, Church GM, Lewis JA. Bookshelf At 14 d after injection, the HAF-injected group lost the hind limbs by autoamputation (Fig. ETV2 is an essential transcription factor that is expressed transiently in the earliest endothelial progenitors.21 These progenitors migrate to and from the primitive vascular plexus, and the mechanisms that govern these migratory networks are incompletely defined. Epub 2015 Mar 23. and A.Y. Before The site is secure. The https:// ensures that you are connecting to the Nat Cell Biol. Before Feedback mechanisms regulate Ets variant 2 (Etv2) gene expression and hematoendothelial lineages. In contrast, ETVECs constituted vessels that were associated with -SMA+ mural cells in the Matrigel plugs, which characterizes the arteriole structure (34). Migration was significantly enhanced in the +Dox condition. Global gene expression analysis revealed that ETVECs clustered with HUVECs rather than with their HAFs of origin, as illustrated by hierarchal clustering (Fig. Takebe T, et al. In addition, these growth factors up-regulated CD31 and VEGF-R2 expression levels on ETVECs (SI Appendix, Fig. Mechanistically, we showed that ETV2 binds the promoter region of Rhoj serving as an upstream regulator of cell migration. The https:// ensures that you are connecting to the 2017 Mar 3;120(5):848-861. doi: 10.1161/CIRCRESAHA.116.309833. S11). No doxycycline and no VEGF were used as controls in these experiments. 2014 Sep 30;130(14):1168-78. doi: 10.1161/CIRCULATIONAHA.113.007727. Note a significant enrichment of Rhoj in the Flk1+/Pdgfra (lateral plate mesoderm) cells relative to the other lineages (n=3 replicates; **P<0.01). To assess the therapeutic benefit of ETVEC transplantation, we then injected the ETVECs and HAFs i.m. Meadows SM, Salanga MC, Krieg PA. Kruppel-like factor 2 cooperates with the ETS family protein ERG to activate Flk1 expression during vascular development. Etv2 is known to require its combination with FoxC for vascular gene expression in Xenopus and zebrafish embryos (13, 22), evoking the possibility of FOXC involvement in ETVEC induction. The regulatory role of pioneer factors during cardiovascular lineage specification - A mini review. These results define an ETV2-Rhoj cascade, which is important for the regulation of endothelial progenitor cell migration. Note a robust enrichment of Rhoj in the +Dox MEFs as compared with Dox (n=3 replicates; *P<0.05). 5D and SI Appendix, Fig. 2022 Feb 14;13:814291. doi: 10.3389/fgene.2022.814291. 19q13.12. We found that HFL-ECs preferentially express venous EC markers (NRP2, NR2F2, and EPHB4) at levels comparable to those in human umbilical vein ECs (HUVECs) (SI Appendix, Fig. At 28 d after implantation, ETVECs, but not HAFs, formed vasculature that was recognized not only by a human CD34-specific mAb, but also by Ulexeuropaeus agglutinin I (UEA I) that binds to human, but not murine, ECs (1, 24) (Fig. Significance was determined by 1-way ANOVA with multiple comparison. F, qPCR analysis for Rhoj transcripts from zsGreen and zsGreen+ sorted cells using the Etv2 zsGreen1-DR embryonic stem (ES)/embryoid body (EB) system at D3 and D4 of differentiation. Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular diseases and solid tumours. 6H), which protected the hind limbs from complete damage (Fig. In this review, we highlight the role of Etv2 in the To further examine the ability of ETV2 to regulate Rhoj gene expression, we undertook qPCR analysis using the iHA-Etv2 MEFs, in the presence (+Dox) or absence (Dox) of doxycycline. Front Cardiovasc Med. doi: 10.1074/jbc.M112.436865. Figure 7figure supplement 1.. 1 C and D). As we found a significant difference in the migratory properties of iHA-Etv2 MEFs following ETV2 induction (+Dox) as early as 6 hours (Figure 2G), we decided to undertake knockdown and rescue experiments at the earliest time period (ie, 6 hours) of Dox treatment to decipher whether ETV2 could rescue migration defects because of Rhoj knockdown. Functionally, ETVECs showed uptake of AcLDL and formation of capillary-like structures on Matrigel-coated plates (Fig. [1] In the field of molecular biology, the ETS ( E26 transformation-specific [2] or E-twenty-six. Korean Circ J. -. After binding to the FOX:ETS motif, ETV2 is thought to recruit cofactors to induce posttranscriptional modifications to ETV2 and/or alter DNA methylation states of the endothelial genes, resulting in facilitation of endothelial gene expression (31). Taken together, these findings indicate that ETV2 is a core factor for directly converting somatic cells into ECs. Han JK, Chang SH, Cho HJ, Choi SB, Ahn HS, Lee J, Jeong H, Youn SW, Lee HJ, Kwon YW, Cho HJ, Oh BH, Oettgen P, Park YB, Kim HS. Ieda M, et al. (A, B, F, and G) Flow cytometry analysis. 1H). Etv2 transcriptionally regulates Yes1 and promotes cell proliferation during embryogenesis. Epub 2015 Nov 19. Shi X, Richard J, Zirbes KM, Gong W, Lin G, Kyba M, Thomson JA, Koyano-Nakagawa N, Garry DJ. In vitro synthesized HA-ETV2 (1 L) was prebound with 250 ng of poly dI-dC (Sigma) in binding buffer (50 mmol/L Tris pH 7.6, 80 mmol/L NaCl, 8% glycerol) at room temperature for 10 minutes. 1A, did not express VEGF-R2 or generate CD31+ cells (Fig. Time-mated pregnant (Etv2-promoter driving EYFP [enhanced yellow fluorescent protein] transgenic lines15) mice were used for embryo harvest, imaging, and fluorescent activated cell sorting (FACS)-sorting experiments at E8.5. Similar to the bulk RNAseq dataset analysis using an ESC/EB database (Figure 1), our qPCR analysis revealed that the levels of multiple migratory gene transcripts, including Mmp9 and PlexinD1 were increased in +Dox MEFs compared with Dox controls (Figure 2H and 2I, n=3 replicates; **P<0.01). Park C, Lee TJ, Bhang SH, Liu F, Nakamura R, Oladipupo SS, Pitha-Rowe I, Capoccia B, Choi HS, Kim TM, Urao N, Ushio-Fukai M, Lee DJ, Miyoshi H, Kim BS, Lim DS, Apte RS, Ornitz DM, Choi K. Arterioscler Thromb Vasc Biol. 1 [7, 8]; n=3 replicates). 2022 Sep 9;50(16):e91. RHOJ, a member of the Rho GTPase subfamily, is highly expressed in endothelial cells and is required for focal adhesion numbers and actomyosin contractility.12 Several studies have demonstrated that RHOJ interacts with the GIT-PIX complex to regulate focal adhesion disassembly in endothelial cells.9 Furthermore, RHOJ has been shown to regulate the activity of CDC42 and RAC1 during lamellipodia formation.13 The knockout of Rhoj results in delayed radial growth and defective vascular structures.9 Similarly, the conditional knockout of Rhoj in the endothelial lineage results in reduced growth and abnormal vascular development at E10.5.14 These studies support an important role for RHOJ in endothelial lineages, but the mechanisms regulating RHOJ expression in the endothelial lineage is unclear. Our analysis revealed that the migratory properties of iHA-Etv2 MEFs were enhanced following the overexpression of ETV2 (Figure 2F and 2G; n=5 replicates; **P<0.01). Interestingly, a defined Dox concentration (100 ng/mL) most efficiently induced ETVECs from ETV2-induced HAFs, and lower and higher concentrations than this level both reduced ETVEC induction somewhat (Fig. Unable to load your collection due to an error, Unable to load your delegates due to an error. (I) Immunofluorescence cytostaining. The method used for the bioinformatics analyses was previously described.5 Briefly, peak analysis using MACS2 version 2.2.6 was performed on our iHA-Etv2 ES/EB Flk1 positive cell sorted assay for transposase accessible chromatin (ATAC)-seq data following 3-hour doxycycline treatment with a q-value cutoff of 0.05, normalized to the no doxycycline Flk1 positive condition. Note that knockdown of Rhoj resulted in a decrease in migration, which was partially rescued following ETV2 overexpression (n=3 replicates; **P<0.01). 1-800-AHA-USA-1 Significance was determined by Student t test with 2-tailed distribution and equal variance. Significance was determined by Student t test with 2-tailed distribution and equal variance (B) and 1-way ANOVA with multiple comparison (E). S2A (C) and Tables S1 and S2]. 1-800-242-8721 Clipboard, Search History, and several other advanced features are temporarily unavailable. official website and that any information you provide is encrypted Bethesda, MD 20894, Web Policies Hi, CD31hi cells; Neg, CD31 cells. ETV2 (Ets variant transcription factor 2) promotes cell migration by inducing migratory networks in differentiating embryoid bodies, mouse angioblasts, and mouse embryonic fibroblasts. Flow cytometry analysis revealed that the CD31+ HFL-1 cells expressed multiple EC surface markers, including CD31, VEGF-R2, CD34, Tie2, neuropilin-1 (NRP1), and CXCR4, but not the hematopoietic cell marker CD45 (SI Appendix, Fig. Sorted CD31+ HFL-1 cells multiplied under EC culture conditions, and 28 d after infection, the expanding Venus+ cells exhibited a cobblestone-like morphology featuring vascular ECs (SI Appendix, Fig. These results indicated that ETV2 could promote migratory processes during differentiation by transcriptionally activating and relaxing the chromatin near the TSS of endothelial genes. Cell migratory genes were considered significant if the P was <0.001 and absolute fold change was >2. doi: 10.1093/nar/gkac452. Next, to validate the migratory role of ETV2 in endothelial progenitors, we utilized the iHA-Etv2 ES/EB system and induced ETV2 between D2 and D3 (24-hour period) and performed sprout formation assays using a Matrigel-sandwich assay supplemented with VEGF (50 ng/mL) in serum-free conditions. WebThe accumulative data indicate that ETV2, an ETS transcription factor, performs a potent and indispensable function in mediating vessel development. Etv2 expression marks the onset of the hematopoietic and vascular development and its deficiency leads to an absolute block in hematopoietic and vascular development. Nucleic Acids Res. n = 10 cultures (B) or triplicate (F and G). WebThe regulatory mechanisms that govern their migration are not completely defined. and transmitted securely. doi: 10.1098/rstb.2010.0302. American Heart Association, Inc. All rights reserved. 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