- Research article
- Open Access
ERG is required for the differentiation of embryonic stem cells along the endothelial lineage
- Vesna Nikolova-Krstevski1, 2, 4,
- Lei Yuan1, 2, 4,
- Alexandra Le Bras†1, 2, 4,
- Preethi Vijayaraj†1, 2, 4,
- Maiko Kondo1, 2, 4,
- Isabel Gebauer1, 2, 4,
- Manoj Bhasin3,
- Chris V Carman2, 4 and
- Peter Oettgen1, 2, 4Email author
© Nikolova-Krstevski et al; licensee BioMed Central Ltd. 2009
- Received: 29 January 2009
- Accepted: 23 December 2009
- Published: 23 December 2009
The molecular mechanisms that govern stem cell differentiation along the endothelial lineage remain largely unknown. Ets related gene (ERG) has recently been shown to participate in the transcriptional regulation of a number of endothelial specific genes including VE-cadherin (CD144), endoglin, and von Willebrand's Factor (vWF). The specific role of the ETS factor ERG during endothelial differentiation has not been evaluated.
ERG expression and function were evaluated during the differentiation of embryonic stem cells into embryoid bodies (EB). The results of our study demonstrate that ERG is first expressed in a subpopulation of vascular endothelial growth factor receptor 2 (VEGF-R2) expressing cells that also express VE-cadherin. During ES cell differentiation, ERG expression remains restricted to cells of the endothelial lineage that eventually coalesce into primitive vascular structures within embryoid bodies. ERG also exhibits an endothelial cell (EC)-restricted pattern during embryogenesis. To further define the role of ERG during ES cell differentiation, we used a knockdown strategy to inhibit ERG expression. Delivery of three independent shRNA led to 70-85% reductions in ERG expression during ES cell differentiation compared to no change with control shRNA. ERG knockdown was associated with a marked reduction in the number of ECs, the expression of EC-restricted genes, and the formation of vascular structures.
The ETS factor ERG appears to be a critical regulator of EC differentiation.
- Embryoid Body
- Hematopoietic Lineage
- Endothelial Differentiation
- Endothelial Cell Differentiation
- Hemangioblast Development
Vasculogenesis, the development of the primary vasculature during embryogenesis, requires a highly orchestrated series of events that are spatially and temporally regulated . Intra-embryonic vasculogenesis is preceded by extra-embryonic vascular development within the yolk sac . Pioneering studies conducted in avian embryos and subsequently extended in amphibian and mammalian model systems in multiple different species have demonstrated the close association between the development of the hematopoietic and endothelial lineages .
The hemangioblast is a bipotent cell of mesodermal origin that can give rise to hematopoietic and ECs. The possible existence of a common precursor was originally suggested because of the close spatial association of hematopoietic cells and ECs in the blood islands associated with the developing embryos, and also because hematopoietic and ECs co-express a number of genes. One of the earliest markers expressed on cells of endothelial and hematopoietic origin is the VEGF receptor Flk-1 or VEGF-R2. Further support for the existence of the hemangioblast comes from the differentiation of embryonic stem cells along the endothelial and hematopoietic lineages [4, 5]. When individual cells are allowed to differentiate further, they form adherent cells that express more endothelial specific markers whereas many of the non-adherent cells, presumed to be of hematopoietic origin, express such genes as β-hemoglobin . Differentiation along the hematopoietic lineage is marked by the expression of cell surface markers including CD41 and CD45. A similar, time-dependent change in the expression of cell surface markers on cells of the hematopoietic and endothelial lineage occurs during the differentiation of embryonic stem cells into embryoid bodies.
Several receptor tyrosine kinases, including the VEGF-R2, VEGF-R1, Tie1, and Tie2 genes are known to be critical mediators of endothelial differentiation and vascular development. Targeted disruption of any of these genes leads to defects in vascular development and early embryonic lethality [7–9]. Comparison of the mouse and human DNA sequences within the regulatory regions of these genes has facilitated the identification of conserved binding sites for different classes of transcription factors. The Tie1 gene promoter contains conserved binding sites for ETS factors and AP2 . Mutations in most of these conserved binding sites leads to marked reductions in the ability of the Tie1 promoter to direct LacZ gene expression in transgenic animals. A similar approach has been used to identify conserved binding sites for the ETS factors, SCL/tal-1, and GATA factors in the VEGF-R2 gene. Point mutations in some of these binding sites also leads to marked reductions in the vascular specific expression directed by the VEGF-R2 regulatory regions in transgenic studies . Conserved ETS binding sites exist in the Tie2 and VEGF-R1 genes [12, 13]. The results of these studies strongly support the hypothesis that members of certain transcription factor families, including the ETS and GATA transcription factor families are involved in regulating the different stages of vascular development and endothelial differentiation by regulating endothelial-specific gene expression.
The ETS genes are a family of at least thirty members that function as transcription factors. All ETS factors share a highly conserved 80-90 amino acid long DNA binding domain, the ETS domain . Many macrophage, B cell and T cell specific genes are regulated by ETS factors. More recently several vascular-specific genes have been shown to be downstream targets of selected ETS factors. In addition to the Tie1, Tie2, VEGF-R1, and VEGF-R2 genes mentioned above, several other endothelial specific genes including von Willebrand Factor (vWF), PECAM-1, VE-cadherin, endothelial nitric oxide synthase genes have functionally important conserved binding sites for members of the ETS transcription factor family [15–18]. The ETS factor ERG has recently been shown to interact with a number of other transcription factors to regulate several endothelial-specific genes including angiopoietin-2, VE-cadherin, endoglin, and vWF [17–20]. The goal of this study was to determine the role of ERG as a transcriptional regulator of ES cell differentiation, and in particular, to determine whether it plays a role in regulating EC differentiation or hematopoiesis.
ERG expression during endothelial differentiation
Evaluation of temporal and spatial expression of ERG during ES cell differentiation
ERG expression during vascular development
Lentiviral knockdown of ERG in ES cells
Flow cytometric evaluation of ERG and control shRNA treated ES cells
FACS analysis of control and ERG shRNA treated embryoid bodies.
Identification of ERG downstream targets during ES cell differentiation
The results of several recent studies point towards an emerging role for ERG as a transcriptional regulator of EC-restricted genes [17–20]. The findings of our study support a unique role for the ETS factor ERG as a transcriptional regulator of endothelial differentiation. ERG expression was first detected in a sub-population of VEGF-R2+ expressing cells that co-express VE-cadherin, and closely correlates with the expression of VE-cadherin during ES cell differentiation. Inhibition of ERG expression, however, did not alter the number of VEGF-R2 expressing cells just prior to their differentiation along the endothelial and hematopoietic lineages suggesting that ERG is not required for the formation of the hemangioblast or hematopoiesis. A recent genome-wide analysis of ETS factors in zebrafish identified three members of the ETS transcription factor family, including ERG, Fli-1, and ETSRP, as playing a role in hemangioblast differentiation and angiogenesis . Of these ETS family members, ETSRP was expressed earliest and required for hemangioblast formation. Similarly, mice that have a mutation in the ER71, the homologue of ETSRP exhibit defects in hematopoiesis and endothelial differentiation . In contrast, neither knockdown of ERG or Fli-1 in zebrafish led to abnormalities in hemangioblast formation, which is consistent with our findings in murine ES cells.
We observed a very close correlation of ERG and VE-cadherin expression in the EB throughout ES cell differentiation. ERG expression also closely correlates with VE-cadherin expression during embryonic vascular development. ERG has previously been shown to regulate the VE-cadherin gene in ECs . Targeted disruption of VE-cadherin prevented the formation of vessel-like structures in embryoid bodies . During mouse development, deficiency of VE-cadherin did not affect the initial assembly of a vascular plexus but was associated with impaired vascular remodeling and maturation . Knockdown of ERG expression not only affected vascular structure formation in embryoid body, but was also associated with a marked reduction in the number of EC. This suggests that ERG must regulate additional genes involved in the formation of EC. Similar knockdown studies were conducted in zebrafish using morpholino oligonucleotides (MO). MOs directed against ERG resulted in hemorrhagic defects in the brain between days 2.5 and 3.0 without any obvious morphological defects . Similar defects were observed with MOs directed against Fli-1. Double knockdown of ERG and Fli-1 led to more severe developmental defects with disorganization of the intersomatic vessels. In contrast to our study, knockdown of ERG in zebrafish was not associated with a significant reduction in the number of ECs that would be expected to be associated with more pronounced defects in vascular development. One possible explanation for the differences observed is that the MOs may have reduced, but not abolished the levels of ERG to a point where sufficient levels of the ERG protein were present to promote EC differentiation but not later stages of vascular development. Alternatively, changes in the function of selected ETS family members may have occurred over the course of evolution, with ERG playing a more dominant role in murine vascular development than in zebrafish.
One of the earliest known markers of hematopoiesis is CD41 (glycoprotein IIb) . While CD41 expressing ES cells are capable of contributing to multiple hematopoietic lineages, they cannot differentiate into ECs. We did not observe significant expression of ERG in VEGF-R2+ cells expressing CD41. Furthermore, ERG knockdown was not associated with reductions in hematopoietic progenitor cells. This is in contrast to recent reports supporting a role for ERG in hematopoiesis . A missense mutation associated with the alteration of one amino acid in the ERG DNA binding domain (Mld2) was associated with marked defects in hematopoiesis due to reductions in transactivation but no change in DNA binding affinity. The results of this study contrast with our knockdown studies, and might be explained by the fact that a mutation in ERG could result in the formation of a dominant-negative protein with altered affinity for different target genes or changes in the affinity of ERG for different interacting proteins.
One of the major downstream targets of ERG is VE-cadherin. Within the developing embryo there are also temporal changes in the expression of VE-cadherin in hematopoietic stem cells (HSCs) . Whereas VE-cadherin is expressed in HSCs within the AGM of embryos at day 11.5, and in the yolk sac at day 12.5, there is marked down regulation of VE-cadherin expression during fetal liver colonization and complete loss in HSCs with the adult bone marrow formation, suggesting that although a subset of HSCs may be derived from a common bipotent precursor population, expression of endothelial markers is not required for maintenance of long-term HSC progenitor cells at later stages of embryonic development or in the adult. Previous studies have demonstrated that VEGF-R2+/CD144+ isolated from mouse embryos 7.5 to 9.5 days post coitum (dpc) or from human ES cells that are CD45 negative but are capable of differentiating along the endothelial and hematopoietic lineages [31, 32]. Although VE-cadherin expressing cells are capable of differentiating into hematopoietic cells, VE-cadherin negative cells exhibited a 15-20 fold greater potential of promoting definitive hematopoiesis than did those expressing VE-cadherin . Targeted disruption of the VE-cadherin gene did not significantly impair hematopoiesis, with similar numbers of erythroid, myeloid, and mixed hematopoietic progenitor cells in E8.5 embryos compared to wild type controls . This would support the overall concept of a "hemogenic" endothelium, but with a predominant mechanism of hematopoietic differentiation being independent of EC differentiation.
ERG is among a very few number of transcription factors that exhibit an EC-restricted expression pattern. For example, Vezf1 is a zinc finger transcription factor that contributes to vascular remodeling and the development of both the vascular and lymphatic endothelium . The homeobox transcription factor Hex is expressed at the hemangioblast stage and contributes to the hematopoietic and endothelial lineages . Another family of transcription factors belonging to the FoxO subset of forkhead transcription factors are expressed in ECs and function as transcriptional regulators of angiogenic growth factors and vascular stabilization . Selected transcription factors have also been identified that regulate later stages of endothelial differentiation, into arterial (Hey1 and Hey2), venous (COUP-TFII), and lymphatic endothelium (Prox-1) . ERG shRNA was associated with significant decreases in the expression of Vezf1, Hey1, and Hey2, with confirmation of at least one of these transcription factors, Hey2, as being a direct target of ERG as demonstrated by ChIP. In contrast, transcription factors known to be critical regulators of hematopoiesis, including Runx1 and HoxB4, were not significantly altered by knockdown of ERG. In summary, the results of our study provide strong evidence for a critical role for ERG as a transcriptional regulator of early EC differentiation that does not affect hematopoiesis or hemangioblast development.
The results of our study demonstrate that ERG regulates the expression of a number of EC-restricted genes during ES cell differentiation. Knockdown of ERG is associated with a significant reduction in the formation of vascular structures in developing embryoid bodies and the number of endothelial cells, that may have important implications for the role of ERG in vascular development during mouse embryogenesis. ERG does not appear to be required for the development of hematopoietic precursor cells during ES cell differentiation. ERG may therefore play a unique role as a transcriptional regulator of EC differentiation that is distinct from hematopoiesis.
Methods (abbreviated; see Supplemental methods, additional file 14)
CCE mouse embryonic stem cells (ATCC) were maintained on irradiated primary mouse embryonic fibroblasts (MEFs) (Chemicon) in knockout KO-DMEM (Invitrogen/Gibco-BRL) supplemented with ES-cell grade 15% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin 1% (Invitrogen/Gibco-BRL), L-glutamine 2 mM (Invitrogen/Gibco-BRL), non-essential amino acids 0.1 mM, nucleosides 0.1 mM, 2-mercaptoethanol 0.1 mM (Sigma, St. Louis, MO), and ESGRO leukemia inhibitory factor (LIF) 1000 units/ml (Sigma). ES cells were grown on MEFs in the presence of LIF to maintain their undifferentiated state for 48 hours.
Embryoid Body Model
The embryoid body is a widely accepted model of differentiation that recapitulates many of the early events of embryogenesis including hematopoiesis and endothelial differentiation. Prior to differentiation, the ES cells are removed from the feeder cells by trypsinization, mixed to a single cell suspension and plated at density of 2 × 106 cells/10 cm dish (Fisher Scientific) containing KO-DMEM growth media without LIF to promote ES differentiation.
Real Time PCR
SYBR Green I-based real-time PCR was carried out on an Opticon Monitor (MJ Research, Inc., Waltham, MA). All PCR mixtures contained PCR buffer with final concentration: 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 2 mM MgCl2, and 0.1% TritonX-100), 250 μM deoxy-NTP (Roche), 0.5 μM of each PCR primer, 0.5× SYBR Green I, 5% DMSO, and 1 U Taq DNA polymerase with 2 μl cDNA in a 25 μl final volume of reaction mix. For each run, serial dilutions of human GAPDH plasmids were used as standards for quantitative measurement of the amount of amplified cDNA and GAPDH primers were used to measure the amount of GAPDH cDNA. For normalization of each sample, copy number was determined/10,000 copies of GAPDH in the sample.
Lentiviral shRNA for knockdown studies in ES cells
Lentivirus encoding the shRNA directed against particular gene targets of interest (Sigma) and polybrene (8 μg/ml; Sigma, Cat.# H9268) was added to the ES cells and incubated for 4 hours, after which 1 ml of ES media was added to the infected cells. Stable clones were puromycin selected and expanded.
Flow cytometry and Fluorescence-activated cell sorting (FACS)
EBs were washed twice in PBS (without Ca2+ and Mg2 and broken down into a single cell suspension. Flow cytometry runs and FACS sorting were performed on the FX5000 Flow Cytometer/FACS sorter at the Beth Israel Medical Center Flow Cytometry Facility, using the CXP analysis software.
For transcriptional profiling, the mouse genome 430 2.0 Affymetrix GeneChip, containing more than 45,000 transcripts were used. RNAs for the microarray experiments were obtained in duplicates from two separately conducted experiments using the murine embryonic stem cells.
This work was supported by NIH grant P01 HL76540 (PO), the AHA Established Investigator Award EIA0740012 (PO) and the Arthritis Foundation Arthritis Investigator Award (CVC).
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