Ets1 functions partially redundantly with Etv2 to promote embryonic vasculogenesis and angiogenesis in zebrafish.
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ABSTRACT:
SUBMITTER: Casie Chetty S
PROVIDER: S-EPMC7484174 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundC-lectin family 14 Member A (Clec14a) is a transmembrane protein specifically expressed in vascular endothelial cells during embryogenesis. Previous in vitro and in vivo studies have provided conflicting data regarding Clec14a role in promoting or inhibiting angiogenesis, therefore its functional role in vascular development remains poorly understood.ResultsHere we have generated a novel clec14a mutant allele in zebrafish embryos using TALEN genome editing. clec14a mutant embryos exhibit partial defects and delay in the sprouting of intersegmental vessels. These defects in angiogenesis are greatly increased upon the knockdown of a structurally related C1qr protein. Furthermore, a partial knockdown of an ETS transcription factor Etv2 results in a synergistic effect with the clec14a mutation and inhibits expression of early vascular markers in endothelial progenitor cells, arguing that clec14a is involved in promoting vasculogenesis. In addition, Clec14a genetically interacts with Vegfa signaling. A partial knockdown of Vegfaa function in the clec14a mutant background resulted in a synergistic inhibition of intersegmental vessel sprouting.ConclusionsThese results argue that clec14a is involved in both vasculogenesis and angiogenesis, and suggest that Clec14a genetically interacts with Etv2 and Vegf signaling during vascular development in zebrafish embryos.
| S-EPMC6451255 | biostudies-literature
Project description:OBJECTIVE:The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis. APPROACH AND RESULTS:Zebrafish fli1a and fli1b mutants were obtained using transposon-mediated gene trap approach. Individual fli1a and fli1b homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older etv2 mutant embryos does not occur in embryos lacking both etv2 and fli1b. Etv2 and fli1b double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast, fli1a mutation did not affect the recovery of etv2 mutant phenotype. Overexpression analyses indicate that both etv2 and fli1b, but not fli1a, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites. CONCLUSIONS:Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.
| S-EPMC4427907 | biostudies-literature
Project description:During embryonic development, multiple signaling pathways control specification, migration, and differentiation of the vascular endothelial cell precursors, angioblasts. No single gene responsible for the commitment of mesenchymal cells to the angioblast cell fate has been identified as yet. Here we report characterization and functional studies of Etsrp, a novel zebrafish ETS domain protein. etsrp embryonic expression is only restricted to vascular endothelial cells and their earliest precursors. Morpholino knockdown of Etsrp protein function resulted in the complete absence of circulation in zebrafish embryos. Angioblasts in etsrp-morpholino-injected embryos (morphants) failed to undergo migration and differentiation and did not coalesce into functional blood vessels. Expression of all vascular endothelial molecular markers tested was severely reduced in etsrp morphants, whereas hematopoietic markers were not affected. Overexpression of etsrp RNA caused multiple cell types to express vascular endothelial markers. etsrp RNA restored expression of vascular markers in cloche mutants, defective in hematopoietic and endothelial cell formation, arguing that etsrp functions downstream of cloche in angioblast formation. etsrp gene function was also required for endothelial marker induction by the vascular endothelial growth factor (vegf) and stem cell leukemia (scl/tal1). These results demonstrate that Etsrp is necessary and sufficient for the initiation of vasculogenesis.
| S-EPMC1310653 | biostudies-literature
Project description:Signaling pathways controlling vasculogenesis and angiogenesis are still poorly understood. Zebrafish Ets1-related protein (Etsrp) which encodes an ETS domain transcription factor, evolutionary related to the mammalian ER71 protein subfamily, has been identified as a major regulator of vasculogenesis and myelopoiesis and functions at the hemangioblast stage affecting the formation of both lineages. In the absence of Etsrp, angioblasts do not migrate or differentiate while overexpression of Etsrp results in the expansion of vascular endothelial and myeloid lineages. To identify genes functioning downstream of Etsrp we performed microarray analysis of etsrp-overexpressing embryos. Etsrp RNA injected embryos and control uninjected embryos were frozen at the tailbud stage and analyzed for expression of more than 30,000 genes using Nimblegen expression arrays. Approximately 300 genes showed greater than two-fold induction in etsrp-overexpressing embryos. Scl, crl, egfl7, aqp8, fli1a, fli1b, lmo2, cdh5 were among the previously known hemangioblast or vasculature-specific genes which were strongly upregulated in Etsrp-overexpressing embryos. We isolated and characterized a number of genes that were novel or previously unassociated with the zebrafish vasculature formation. Eight of them which include angiotensin II type 2 receptor (agtr2), src homology 2 domain containing E (she), similar to mannose receptor C1 (mrc1), endothelial cell-specific adhesion molecule (esam), cdc42 guanine nucleotide exchange factor 9 (arhgef9), yes-related kinase (yrk), zinc finger protein, multitype 2b (zfpm2b/fog2b) and stabilin 2 (stab2) were specifically expressed in vascular endothelial cells during early embryonic development while keratin18 expression was localized to the myeloid cells among others. Identification of novel vasculature and myeloid-specific genes that are regulated by Etsrp will be important for dissecting molecular mechanisms that regulate vasculogenesis / angiogenesis and myelopoiesis. Approximately 50 pg of etsrp RNA was microinjected into zebrafish embryos at the 1-4 cell stages. Three separate batches of approximately 150 microinjected embryos and uninjected controls were frozen on dry ice at the tailbud stage. Total RNA was purified using the RNAquous-4PCR kit (Ambion). Approximately 10-20 µg RNA from each batch was sent to the NimbleGen facility, were it was processed, labeled and hybridized to standard NimbleGen 385K gene expression array. Briefly, RNA was converted into cDNA using the SuperScript II cDNA Conversion Kit (Invitrogen, Carlsbad, CA). Double-stranded cDNA was random-prime labeled with Cy3-nonamers and hybridized to the microarrays for 16 hours at 42°C. The zebrafish microarray was based on Zv6 genome assembly and contained approximately 32,000 genes with approximately 12 probes of 60mer oligonucleotides per each gene. The arrays were washed, dried, and scanned at 5μm resolution using a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA). Data were extracted from scanned images using NimbleScan software (Roche NimbleGen). Quantile normalization was performed across replicate arrays, and RMA (Robust Multichip Average) analysis was performed to generate gene expression values. Average ratios of expression values of etsrp-injected vs. control were calculated from three experiments. Results were sorted by the fold change from the highest to the lowest. Genes that displayed values of p>0.05 or showed expression levels less than 2-fold above the background level in both control and experimental samples were eliminated from further analysis.
2010-05-06 | E-GEOD-12909 | biostudies-arrayexpress
Project description:Fibroblast growth factor (Fgf) signaling regulates many processes during development. In most cases, one tissue layer secretes an Fgf ligand that binds and activates an Fgf receptor (Fgfr) expressed by a neighboring tissue. Although studies have identified the roles of specific Fgf ligands during development, less is known about the requirements for the receptors. We have generated null mutations in each of the five fgfr genes in zebrafish. Considering the diverse requirements for Fgf signaling throughout development, and that null mutations in the mouse Fgfr1 and Fgfr2 genes are embryonic lethal, it was surprising that all zebrafish homozygous mutants are viable and fertile, with no discernable embryonic defect. Instead, we find that multiple receptors are involved in coordinating most Fgf-dependent developmental processes. For example, mutations in the ligand fgf8a cause loss of the midbrain-hindbrain boundary, whereas, in the fgfr mutants, this phenotype is seen only in embryos that are triple mutant for fgfr1a;fgfr1b;fgfr2, but not in any single or double mutant combinations. We show that this apparent fgfr redundancy is also seen during the development of several other tissues, including posterior mesoderm, pectoral fins, viscerocranium, and neurocranium. These data are an essential step toward defining the specific Fgfrs that function with particular Fgf ligands to regulate important developmental processes in zebrafish.
| S-EPMC6707458 | biostudies-literature
Project description:The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1? and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.
| S-EPMC3283730 | biostudies-literature
Project description:Mice lacking the 4th-group paralog Hoxd4 display malformations of the anterior vertebral column, but are viable and fertile. Here, we report that zebrafish embryos having decreased function of the orthologous hoxd4a gene manifest striking perturbations in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. These defects are preceded by reduced expression of the hemangioblast markers scl1, lmo2 and fli1 within the posterior lateral plate mesoderm (PLM) at 13 hours post fertilization (hpf). Epistasis analysis revealed that hoxd4a acts upstream of meis1.1 but downstream of cdx4 as early as the shield stage in ventral-most mesoderm fated to give rise to hemangioblasts, leading us to propose that loss of hoxd4a function disrupts hemangioblast specification. These findings place hoxd4a high in a genetic hierarchy directing hemangioblast formation downstream of cdx1/cdx4 and upstream of meis1.1. An additional consequence of impaired hoxd4a and meis1.1 expression is the deregulation of multiple Hox genes implicated in vasculogenesis and hematopoiesis which may further contribute to the defects described here. Our results add to evidence implicating key roles for Hox genes in their initial phase of expression early in gastrulation.
| S-EPMC3598951 | biostudies-literature
Project description:Signaling pathways controlling vasculogenesis and angiogenesis are still poorly understood. Zebrafish Ets1-related protein (Etsrp) which encodes an ETS domain transcription factor, evolutionary related to the mammalian ER71 protein subfamily, has been identified as a major regulator of vasculogenesis and myelopoiesis and functions at the hemangioblast stage affecting the formation of both lineages. In the absence of Etsrp, angioblasts do not migrate or differentiate while overexpression of Etsrp results in the expansion of vascular endothelial and myeloid lineages. To identify genes functioning downstream of Etsrp we performed microarray analysis of etsrp-overexpressing embryos. Etsrp RNA injected embryos and control uninjected embryos were frozen at the tailbud stage and analyzed for expression of more than 30,000 genes using Nimblegen expression arrays. Approximately 300 genes showed greater than two-fold induction in etsrp-overexpressing embryos. Scl, crl, egfl7, aqp8, fli1a, fli1b, lmo2, cdh5 were among the previously known hemangioblast or vasculature-specific genes which were strongly upregulated in Etsrp-overexpressing embryos. We isolated and characterized a number of genes that were novel or previously unassociated with the zebrafish vasculature formation. Eight of them which include angiotensin II type 2 receptor (agtr2), src homology 2 domain containing E (she), similar to mannose receptor C1 (mrc1), endothelial cell-specific adhesion molecule (esam), cdc42 guanine nucleotide exchange factor 9 (arhgef9), yes-related kinase (yrk), zinc finger protein, multitype 2b (zfpm2b/fog2b) and stabilin 2 (stab2) were specifically expressed in vascular endothelial cells during early embryonic development while keratin18 expression was localized to the myeloid cells among others. Identification of novel vasculature and myeloid-specific genes that are regulated by Etsrp will be important for dissecting molecular mechanisms that regulate vasculogenesis / angiogenesis and myelopoiesis.
2009-09-01 | GSE12909 | GEO
Project description:Although anti-angiogenic therapies (AATs) have some effects against multiple malignancies, they are limited by subsequent tumor vasculogenesis and progression. To investigate the mechanisms by which tumor vasculogenesis and progression following AATs, we transfected microRNA (miR)-9 into human umbilical vein endothelial cells (HUVECs) to mimic the tumor-associated endothelial cells in hepatocellular carcinoma and simulated the AATs in vitro and in vivo. We found that administration of the angiogenesis inhibitor vandetanib completely abolished miR-9-induced angiogenesis and promoted autophagy in HUVECs, but induced the release of vascular endothelial growth factor (VEGF)-enriched exosomes. These VEGF-enriched exosomes significantly promoted the formation of endothelial vessels and vasculogenic mimicry in hepatocellular carcinoma and its progression in mice. Anti-autophagic therapy is proposed to improve the efficacy of AATs. However, similar effects by AATs were observed with the application of anti-autophagy by 3-methyladenine. Our results revealed that tumor vasculogenesis and progression after AATs and anti-autophagic therapies were due to the cross-talk between endothelial and tumor cells via VEGF-enriched exosomes.
| S-EPMC6586113 | biostudies-literature
Project description:The ras/Raf/Mek/Erk pathway plays a central role in coordinating endothelial cell activities during angiogenesis. Transcription factors Ets1 and Ets2 are targets of ras/Erk signaling pathways that have been implicated in endothelial cell function in vitro, but their precise role in vascular formation and function in vivo remains ill-defined. In this work, mutation of both Ets1 and Ets2 resulted in embryonic lethality at midgestation, with striking defects in vascular branching having been observed. The action of these factors was endothelial cell autonomous as demonstrated using Cre/loxP technology. Analysis of Ets1/Ets2 target genes in isolated embryonic endothelial cells demonstrated down-regulation of Mmp9, Bcl-X(L), and cIAP2 in double mutants versus controls, and chromatin immunoprecipitation revealed that both Ets1 and Ets2 were loaded at target promoters. Consistent with these observations, endothelial cell apoptosis was significantly increased both in vivo and in vitro when both Ets1 and Ets2 were mutated. These results establish essential and overlapping functions for Ets1 and Ets2 in coordinating endothelial cell functions with survival during embryonic angiogenesis.
| S-EPMC2721789 | biostudies-literature