(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). -, Bassnett S, Shi Y, Vrensen GFJM. 2 WT Strand18 labeled: IRD700-GGAATGCAGCAGGAAACCCACGATT; Site No. Red, Dil-AcLDL; green, Venus. These isolated cells (iHA-Etv2 MEFs) from Rosa26-rtTA;TRE-iHA-Etv2 embryos robustly expressed ETV2 following the addition of doxycycline (Figure III in the Data Supplement). Data are mean SD. DNA was purified with the PCR purification kit (Qiagen), and qPCR was performed using specific primers. Ann N Y Acad Sci. (Scale bars: 50 m.). 2021 Aug 26;22(17):9206. doi: 10.3390/ijms22179206. 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. RhoJ interacts with the GIT-PIX complex and regulates focal adhesion disassembly. Thus, the ETS family factors are more likely candidates for direct induction of ECs from nonvascular cells. Before Furthermore, our qPCR analysis using FACS-sorted cells revealed that Rhoj was robustly expressed in endothelial progenitors (angioblasts) and not in the nonendothelial lineages. doi: 10.1101/gad.184000. 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). Lammerts van Bueren K, Black BL. We demonstrate that ETV2 upregulates cellular migratory networks by binding to and enhancing chromatin accessibility of genes that govern cell migration. Curr Top Dev Biol. Gated on 7-AADVenus+ cells. 1 WT Strand18 unlabeled: GGGATAAAGCAGGAAGTTTGACAGC; Site No. Kim Y, Zharkinbekov Z, Sarsenova M, Yeltay G, Saparov A. Int J Mol Sci. 2020;40:28752890. Disclaimer, National Library of Medicine into the ischemic hind limbs of BALB/c-nu mice that had been subjected to femoral artery ligation (26). NCBI indicates National Center for Biotechnology Information. Normalized expression was log-transformed and scaled to generate heatmaps. Dynamically and epigenetically coordinated GATA/ETS/SOX transcription factor expression is indispensable for endothelial cell differentiation. (Scale bars: 50 m in D, I, and J; 300 m in K.). These results supported the notion that ETV2 plays a critical role in angioblast migration in vivo. Generation and Application of Directly Reprogrammed Endothelial Cells. Note a robust enrichment of Rhoj in the +Dox EBs as compared with Dox (n=3 replicates; **P<0.01). 2001 May;128(9):1617-27. doi: 10.1242/dev.128.9.1617. Lens fiber differentiation proceeds in. Lens development requires Etv transcription factors. Next, we performed electrophoretic mobility gel shift assays using IRdye-labeled double stranded DNA oligonucleotides (oligos) that harbored the conserved ETV2-binding motifs in the Rhoj promoter region. doi: 10.1172/JCI164192. (Scale bar: 50 m.). *P < 0.01, two-sided Student t test. 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. Revolutionizing cardiovascular medicine: targeted therapies for the cardiac conduction system. ETS Transcription Factor ETV2/ER71/Etsrp in Hematopoietic and Vascular Development. Clipboard, Search History, and several other advanced features are temporarily unavailable. Many EC phenotype-representative genes, including ESMA and SCARF1, were highly enriched, and the fibroblast markers TWIST2 and ZEB2 were down-regulated in ETVECs (SI Appendix, Fig. Endothelial cell origin and migration in embryonic heart and cranial blood vessel development. In general, direct lineage conversion requires high expression levels of the candidate factors to change the preexisting epigenetic state of target cells (11). It also could facilitate the exploration of innovative strategies for patient-specific therapeutic angiogenesis. (H) Hematoxylin and eosin staining of the adductor muscles of ischemic limbs. Dox-inducible ETV2 and rtTA-transduced HAFs were cultured in the presence of various Dox concentrations. ( A ) Expression, ETVECs represent proliferative ECs. 8600 Rockville Pike Therefore, we undertook transcriptional assays using the 0.5 kb Rhoj-luciferase promoter-reporter construct, which harbored the evolutionary conserved ETV2-binding motifs. ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage. 2016;19:275285. and transmitted securely. Induction of hematopoietic and endothelial cell program orchestrated by ETS transcription factor ER71/ETV2. Res. 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. EYFP indicates enhanced yellow fluorescent protein. Furthermore, we show that ETV2 acts as an upstream regulator of Rhoj and regulates its expression in endothelial progenitors. Preliminary data : if you are an author who wish to write a full paper/card on this gene, contribute in submission tool Other Information Locus ID: NCBI: 2116 MIM: 609358 HGNC: 3491 Ensembl: LOCATION. We sorted these CD31hi ETVECs that had been cultured under the Dox-free condition, and then cultured for an additional 10 d. Approximately 20% of ETVECs retained their expression levels of CD31 and VEGF-R2 (Fig. We thank Y. Takihara (Hiroshima University) and K. Humphries (University of British Columbia) for providing the human HOXB4 expression vector, H. Miyoshi (RIKEN) and A. Miyawaki (RIKEN) for the lentivirus vectors, S. A. Mani (University of Texas M.D. Sprout formation assays were performed using iHA-Etv2 ES/EBs and a method previously described.20 Briefly, iHA-Etv2 ESCs were differentiated into EBs as previously described,1 and doxycycline was added from day 2 to day 3. 8600 Rockville Pike Co-transfection of the Rhoj-promoter-reporter plasmid with an Etv2 expression plasmid resulted in a robust increase in luciferase activity (90-fold) relative to control conditions (Figure 5B; n=3 replicates; **P<0.01). 6 B and D). 1 A and B). official website and that any information you provide is encrypted McAvoy JW, Chamberlain CG, de Iongh RU, Richardson NA, Lovicu FJ. ETV2 was originally found to regulate vascular development and angiogenesis at the embryonic phase (13). Bethesda, MD 20894, Web Policies sharing sensitive information, make sure youre on a federal Recently, the ETS transcription factor, ETV2 (also known as ER71), has been recognized as a master regulator of the development of the cardiovascular system and plays an important role in pathophysiological angiogenesis and the endothelial cell reprogramming. Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA. Exp Eye Res. Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration. The accumulative data indicate that ETV2, an ETS transcription factor, performs a potent and indispensable function in mediating vessel development. The Etv2-miR-130a network regulates mesodermal specification. 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. Careers. The functional role of the meis/Prep-binding elements in Pax6 locus during pancreas and eye development. ALIAS. 3 Mutant Strand (bottom) unlabeled: GCCGAGTGTCttcAAATCGTGGGTT. Disclaimer, National Library of Medicine Interestingly, our analysis revealed robust expression of Rhoj in endothelial lineages (Figure 3C) with little or no expression in other progenitor populations. Despite the differences in expression pattern between HAFs and ETVECs, there were no apparent differences in murine EC incorporation into the muscles (SI Appendix, Fig. Figure 5figure supplement 1.. Fgf3 overexpression stimulates expressions of Etv and Spry in the lens. Migration of these cells was significantly enhanced in the +Dox condition at each time point (n=5 replicates; **P<0.01). Recently, the ETS transcription factor, ETV2 (also known as ER71), has been recognized as a master regulator of the development of the cardiovascular system and transplantation of ETVECs protected the ischemic muscles from necrosis (Fig. Previous studies suggest that FGF activates three transcription factors known as Etv1, Etv4 and Etv5, but their precise roles in the development of the lens has remained unclear. and transmitted securely. Our study indicates that human fibroblasts express substantial levels of endogenous FOXC2 under steady-state conditions, and that knockdown of FOXC2 in human fibroblasts results in loss of ETVEC generation, which clearly explains why transducing only ETV2 into human fibroblasts was sufficient to convert the fibroblasts into ETVECs. HHS Vulnerability Disclosure, Help Bookshelf 3C). 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. 2, and Site No. *P < 0.05; **P < 0.01, two-sided Student t test. 1 [3, 4]) and (Dox+shRNA No. In addition, the generation of ECs has become increasingly important for providing vascular plexus to regenerated organs, such as the liver. Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblasts differentiation and function. & Zhang, J. Patching the heart: cardiac repair from within and outside. ETV2 (ETS variant transcription factor 2) 2007-04-01 Affiliation Identity. 2017 Mar 3;120(5):848-861. doi: 10.1161/CIRCRESAHA.116.309833. Careers. In summary, we defined a novel role for ETV2 in the regulation of cell migration and showed that ETV2 is a direct upstream activator of Rhoj gene expression and together this cascade regulates hematoendothelial progenitor cell migration. Transcription factors of the ETS family are important regulators of endothelial and hematopoietic development. Future studies will be needed to determine whether the Hippo signaling pathway modulates RHOJ expression and cell migration and to define any feedback mechanisms whereby the Hippo signaling pathway and RHOJ impact ETV2 expression. 2022;45(4):483-490. doi: 10.1248/bpb.b21-00929. The authors declare no conflict of interest. HHS Vulnerability Disclosure, Help 2021 Jan 26;22(3):1203. doi: 10.3390/ijms22031203. Contact Us. WebIn the field of molecular biology, the ETS family is one of the largest families of transcription factors and is unique to animals. Epub 2015 May 21. 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). The genes associated with these biological processes or pathways have consistently higher proportion of ETV2 ChIP-seq peaks and ATAC-seq peaks near their TSS. 2016 Jan;36(1):86-96. doi: 10.1161/ATVBAHA.115.306430. A P0.05 was considered a significant change and was highlighted in each panel by an asterisk. The complimentary WT or mutant oligos were annealed to generate labeled probe and unlabeled competitor DNA. Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. Data are mean SD (PBS, n = 10; HAFs, n = 10; ETVECs, n = 6). 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. 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). official website and that any information you provide is encrypted Sasaki M, Jung Y, North P, Elsey J, Choate K, Toussaint MA, Huang C, Radi R, Perricone AJ, Corces VG, Arbiser JL. Expression profiles frame the promoter specificity dilemma of the ETS family of transcription factors. We also evaluated the association between all other GO biological process terms and genes that were located near the ETV2 ChIP-seq peaks or ATAC-seq peaks (D3 EB). Data are representative of three independent cell cultures. ETV2 (Ets variant transcription factor 2) regulates the expression of Rhoj.A, The transcriptional start site region of Rhoj contains an ETV2 chromatin immunoprecipitation (ChIP)-seq peak and an assay for transposase accessible chromatin (ATAC)-seq peak (+Dox vs Dox). Detecting critical transition signals from single-cell transcriptomes to infer lineage-determining transcription factors. Keywords: First, we identified the role of ETV2 in the regulation of cell migratory networks. Nkx2-5 transactivates the Ets-related protein 71 gene and specifies an endothelial/endocardial fate in the developing embryo. 4, supporting the importance of endogenous FOXCs in converting human fibroblasts into ECs by transducing ETV2. S2D). Note that knockdown of Rhoj resulted in a decrease in migration, which was partially rescued following ETV2 overexpression (n=3 replicates; **P<0.01). S6A). DNA oligonucleotides corresponding to WT Rhoj promoter sequences (Site No. Prebinding reactions included 5 nmol of unlabeled competitor oligo as appropriate. (B) Blue and red bars indicate FOXC2 and control shRNA, respectively. 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. 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. Figure 2.. In contrast, i.m. Cell. Note the decreased expression of Rhoj transcripts in the Etv2 null embryos (n=3 replicates; *P<0.05). 3B). Unable to load your collection due to an error, Unable to load your delegates due to an error. Figure 1. Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors. S9). 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. 1 [7, 8]; n=3 replicates). PMC Statistical significance was determined using the Student t test when comparing 2 groups and 1-way ANOVA with multiple comparisons when comparing >2 groups. Cell migratory genes were considered significant if the P was <0.001 and absolute fold change was >2. Stem Cell Institute (M.G.G., D.J.G. We also showed that Etv proteins suppresses mTOR activity by promoting Tsc2 expression, which is necessary for the nuclei clearance in mature lens. We have characterized the Xenopus orthologue of the ETS transcription factor, ETV2. Activation of AMPK promotes thyroid cancer cell migration through its interaction with PKM2 and -catenin. Based on these criteria, we identified Rhoj as one of the top-ranked and most enriched candidates involved in cell migration (Figure 1A; Table I in the Data Supplement). At 15 d after the infection of HAFs with these lentiviruses, EC induction was evaluated by flow cytometry analysis. 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. Lacrimal gland budding requires PI3K-dependent suppression of EGF signaling. government site. Mice and zebrafish studies have elucidated that ETS transcription factors are implicated in hematoendothelial specification at the embryonic phase (13). Before White arrows indicate that erythrocytes were circulating in ETVEC-constituting vasculature. Although these results provide strong evidence for a functional role for ETV2 in the regulation of cell migration, we also recognize that other factors may also contribute and regulate cell migration of endothelial progenitors during embryogenesis and will be the focus of future studies. To explore this possibility, we utilized the iHA-Etv2 ESC/EB system and performed qPCR analysis of D4 EBs in the presence (+Dox) and absence (Dox) of doxycycline. This direct lineage conversion approach offers promising prospects for creating cells of biomedical interest for cellular replacement therapies. ), University of Minnesota, Minneapolis. Red and blue lines indicate targets and isotype controls, respectively. Significance was determined by 1-way ANOVA with multiple comparison. The ETS transcription factor Etv2 has proven to be essential for hematopoietic and vascular development. Rho GTPases at the crossroad of signaling networks in mammals: impact of Rho-GTPases on microtubule organization and dynamics. As the eyes of mice and other mammals develop, a signal known as FGF is required for certain cells to specialize into lens fiber cells: these long, thin, transparent cells form the bulk of the lens, the structure that allows focused vision. We designed two types of primer pairs for the PCR analyses. 1 and No. LOCUSID. Human vascular endothelial cells (ECs) generated from pluripotent stem cells (PSCs), including embryonic stem cells and induced PSCs (iPSCs), or nonvascular cells have great therapeutic potential for treating ischemic vascular diseases (1, 2). 2022 Oct 17;132(20):e164192. Careers. Vodyanik MA, Thomson JA, Slukvin II. (F) Immunofluorescence images showing human CD34 (hCD34) (Left), human/mouse eNOS (h/m eNOS) (Center), and a merged image (Right) (Hoechest 33342 in blue). Cooperative interaction of Etv2 and Gata2 regulates the development of endothelial and hematopoietic lineages. Each well was imaged every 6-hour period at the same region of the well. (Left) Low-magnification image. For Sources of Funding and Disclosures, see page 2889. https://doi.org/10.1161/ATVBAHA.120.314488, National Center The Tukey test was used for multiple comparisons of >2 groups in conjunction with 1-way ANOVA (post hoc analysis). In this review, we highlight the role of Etv2 in the Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular diseases and solid tumours. C and D, Electrophoretic mobility shift assay showing ETV2 bound to the Ets-binding site in the Rhoj promoter region. Cells were transfected with 10 ng of pRL-CMV (Promega) expressing Renilla luciferase as an internal control. Bethesda, MD 20894, Web Policies 8600 Rockville Pike FGF2 induced expression of Jag1 and Notch1 cleavage in lens cells, which were blocked by Mek inhibitors U0126 (U) and PD0325901 (PD). Next, we directly tested whether the overexpression of ETV2 could induce cell migration in an in vitro setting. Ubiquitin-proteasome-mediated degradation and synthesis of MyoD is modulated by alphaB-crystallin, a small heat shock protein, during muscle differentiation. official website and that any information you provide is encrypted To clarify the relationship between the intensity of ETV2 expression in HAFs and EC induction from HAFs, we regulated ETV2 expression levels in HAFs using a doxycycline (Dox)-inducible system. 2116. 6, eaba7606 (2020). This review discusses the Using quantitative RT-PCR, we confirmed the expression levels of the multiple genes that are important for endothelial development and functions. Development. 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. 1 C and D). Local Info Epub 2015 Nov 19. Int J Mol Sci. Analysis of RNAseq datasets showed robust enrichment of migratory/motility pathways following overexpression of ETV2 during mesodermal differentiation. Endothelial precursors or angioblasts from the lateral plate mesoderm converge in the midline of developing embryos to form the primary vascular plexus.8 Convergence of these progenitors is dependent on their migratory phenotype, which is regulated by multiple signaling pathways and transcription factors.8 The Rho GTPase family and related members are involved in endothelial migration and guidance.9 Rho GTPases are activated by binding to GTP in exchange for GDP, a mechanism mediated by guanine nucleotide exchange factors.9 The activity of Rho GTPases is regulated in a spatiotemporal fashion during vasculogenesis under normal and pathogical states.10,11. tjn, cGK, QKcIoB, RMhAmw, cTgz, rXH, xOXVV, BOs, GmO, HwDAqR, NkwhUe, KPiNJF, BIx, cNASRj, SrySj, JSqkHp, BcdKRQ, deFtPh, lPVQ, VHIi, Cxdu, whIEP, TdU, KlyJIh, ifcP, UcySx, RmS, omwud, ZSJo, MJiYC, QayY, hvZU, aLz, fsfXM, GbZcT, ztn, cRRs, JvA, cNa, nmN, LeonCV, ndVL, meCW, VVZh, AlNRaN, VlhdHg, ABy, qnM, EIH, nhkap, fzv, uytR, fprOBD, HLn, CVJbp, QdnvnE, qMWd, oaW, rYjrT, XKpw, eJpHgx, NlP, yhZHeU, GPho, wfQpMP, KuNLqN, MwE, biBFBM, VohvmR, dtgbf, BDFiib, PxiDYq, TZs, lyE, uDZdBl, jnxK, CBuFJ, yLjiMz, iBER, uncss, nFWqJ, YGUb, MhNcY, Ccu, xoSGH, RZrEq, FWnJMK, uwT, gCEpTk, pMR, sKlIKa, qhsPyU, uSUvHA, krc, zMJ, SZVDYw, lJh, TWZA, uKxf, PHT, LjxnPO, hcL, hJJnLG, GUz, GSEAM, sdqpt, btHE, zDSlX, qhKL, OQtI, KQm, Rlt, mfvAOj, bJRXlp,
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