A role for Egfl7 during endothelial organization in the embryoid body model system
© Durrans and Stuhlmann. 2010
Received: 12 November 2009
Accepted: 19 February 2010
Published: 19 February 2010
Epidermal growth factor-like domain 7, Egfl7, is a largely endothelial restricted gene which is thought to have a role during the differentiation of embryonic stem cells (ESCs) along the endothelial lineage. While it has been shown that Egfl7 knock-down in zebrafish impairs endothelial cord formation, the role of the gene in mammals has been unresolved. Interpretation of mouse knockout studies has been complicated by the fact that deletion of miR-126, an intronic microRNA located within Egfl7, results in vascular defects. Here we use an siRNA knock-down approach to target specific regions of Egfl7 without affecting miR-126 expression. Egfl7 was knocked down in mouse ESCs and the effect on vascular development was assessed using the in vitro embryoid body (EB) model after either 7 or 14 days of differentiation. Knock-down of Egfl7 resulted in the formation of abnormal sheet-like CD31+ structures that were abundant within EBs after 7 days of differentiation. Only up to 60% of these sheets co-expressed basement membrane and endothelial cell junction markers. Similar CD31+ sheets were also seen as outgrowths from 7 day EBs into collagen gels. A partial remodelling occurred by 14 days of differentiation when fewer CD31+ sheets were seen both within EBs, and as outgrowths from EBs. Formation of these sheets was due, at least in part, to increased proliferation specifically of CD31+ cells. Cell death within EBs was unaffected by Egfl7 knock-down. In conclusion, our work shows that knock-down of Egfl7 causes defects in early vascular cord formation, and results in the development of CD31+ sheet-like structures. This suggests that Egfl7 is vital for the formation of endothelial cell cords, and that the gene has an important role during both vasculogenesis and angiogenesis in mammalian cells.
Epidermal growth factor-like domain 7, Egfl7, was identified in a screen for genes with restricted expression during in vitro differentiation and mouse embryogenesis . EGFL7 is expressed in undifferentiated mouse embryonic stem cells (ESCs), during early embryogenesis at sites of blood island formation and vasculogenesis, and in adults during pathological and physiological angiogenesis [1, 2] (L. Campagnolo and H. Stuhlmann, Unpublished). Expression of EGFL7 is largely restricted to endothelial cells (ECs), and is down-regulated in most adult organs with the exception of the pregnant uterus and during wound repair [1, 2]. In addition, expression has also been reported in primordial germ cells and male germ cells . Due to its early and restricted expression, Egfl7 has been proposed to have a role during the differentiation of ESCs along the endothelial lineage. EGFL7 is a secreted protein which stimulates EC migration, and knock-down of the gene in zebrafish results in a severe impairment of arterial and venous EC cord segregation leading to the formation of midline angioblast aggregates [2, 4, 5]. However, the function of Egfl7 in mammalian vascular development is still unresolved. In a mouse knockout study, Schmidt et al  showed partial embryonic lethality, delayed vascular development, and abnormal EC aggregates. In contrast, Kuhnert et al  found no phenotype in Egfl7 knockout mice, and instead proposed that the observed vascular defects could be attributed to deletion of miR-126, an endothelial microRNA located within intron 7 of Egfl7. In this study we have used an siRNA knock-down approach, enabling us to target regions of the Egfl7 gene other than intron 7. This has allowed us to specifically investigate the role of Egfl7 during vascular development, without affecting miR-126 expression.
We chose the embryoid body (EB) differentiation model to examine the effect of Egfl7 knock-down on vasculogenesis and angiogenic sprouting. Numerous studies have shown that EBs facilitate the interaction of cells of the ectodermal, mesodermal, and endodermal lineages, recapitulating the developmental kinetics of normal mouse embryonic development [8–11]. Because EBs are initially formed by differentiating ESCs, this system allows for assessment of vascular structure development and the process of ESC differentiation to be assessed. Recent work has shown that vascular structures within EBs are surrounded by a basement membrane, as is the case for blood vessels in vivo . We used EBs that were differentiated for 7 days, roughly equivalent to an early organogenesis stage, and 14 days, which is considered to be a later remodeling stage . We also looked at the effect of Egfl7 knock-down on sprouting angiogenesis using EBs in a type I collagen gel. Here we show that Egfl7 knock-down results in the formation of abnormal endothelial sheet-like structures, which form during the initial stages of in vitro vascular development. During the subsequent processes of differentiation, presumably involving remodelling, endothelial cords replace a large proportion of these sheets. Our results suggest a role for Egfl7 in EC organization, and indicate that the gene is necessary for normal vascular growth during both vasculogenesis and angiogenesis in mammalian cells.
Knock-down construct and siRNA production
The siRNA sequences used for Egfl7 knock-down (KD1, KD2, KD3), and the scrambled controls (Scr1, Scr2) were as follows; KD1: 5'-UACUUGCCAGACAGAUGUU-3' (sense), 3'-UUAUGAACGGUCUGUCUAC-5' (antisense); KD2: 5'-GCAGCUGGACCGAAUUGAU-3' (sense), 3'-UUCGUCGACCUGGCUUAAC-5' (antisense); KD3: 5'-GCUCCCUGUCUAAGUGGUAA-3' (sense), 3'-UUCGAGGGACAGAUUCACCA-5' (antisense); Scr1: 5'-GCUCCCUAGGCUAGUGGUAA-3' (sense), 3'-UUCGAGGGAUCCGAUCACCA-3' (antisense); Scr2: 5'-UACUUGGACGACAGAUGUU-3' (sense), 3'-UUAUGAACCUGCUGUCUAC-5' (antisense). Sense and antisense oligonucleotides were annealed and ligated to linearised psiRNA-hH1neoG2 vector (Invitrogen) before sub-cloning into the FG12 lentiviral vector carrying an eGFP reporter sequence .
Production of lentivirus and stable embryonic stem cell knock-down clones
HEK 293T cells were co-transfected with FG12 lentiviral vectors carrying the siRNA sequence, HIV-1 lentiviral packaging constructs (pMDLg/pRRE and pRSV-REV), and pVSV-G (a plasmid coding for the G protein of the vesicular stomatitis virus) by the calcium phosphate method. Virus supernatant was collected 24-40 h after transfection and concentrated by ultracentrifugation (22,000 × g). The virus titers were determined on 3T3 cells by counting the number of eGFP+ cells under a microscope, and were 5 × 106-1.5 × 108 infectious units/ml. Mouse ESCs (W4/129S6; Taconic) were grown on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs) in DMEM supplemented with 15% FBS, 20 mM HEPES, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 100 U/ml penicillin/streptomycin, 0.3 mg/ml L-glutamine, and 103U/ml LIF (ESGRO; Chemicon). ESCs were infected in the presence of 8 μg/ml polybrene at a MOI of 1-2. Individual eGFP+ clones were isolated and assessed for expression of the knock-down construct by RT-PCR and western blot. RNA and protein was extracted from cells using the PARIS kit (Ambion) as per manufacturer's instructions. Reverse transcription was carried out using the SuperScript III First-Strand Synthesis System (Invitrogen), followed by PCR using the following primers; Egfl7: 5'-ACAGACCCAGCCGTAGAGTG-3' (forward, spanning exons 3 and 4), 5'-TCAATTCGGTCCAGCTGCTGG-3' (reverse, within exon 9); GAPDH: 5'-ACCACAGTCCATGCCATCAC-3' (forward), 5'-TCCACCACCCTGTTGCTGTA-3' (reverse). For westerns, 40 μg protein was run on a 10% bis-tris gel (Invitrogen) under reducing conditions, and transferred to a PVDF membrane which was incubated with antibodies against EGFL7  and actin (Santa Cruz), followed by HRP-conjugated secondary antibodies. Images of eGFP+ ESCs were taken with a Leica DFC340FX digital colour camera mounted on a Leica DMIL inverted microscope, using Leica Application Suite Software (Leica Microsystems), and eGFP+ EBs were visualized using a stereo Discovery. V20 microscope (Carl Zeiss) with an X-Cite 120 external fluorescent light source (EXFO Photonic Solutions Inc.)
Real-time PCR analysis of Egfl7 and miR-126 levels
For real-time PCR analysis, total RNA was isolated from ESCs using the RNAqueous-Micro Kit (Ambion) as per manufacturer's instructions. Egfl7 levels were determined by carrying out reverse transcription as described above, followed by PCR using the following primers; Egfl7 (spanning intron 8) 5'-AGAGGAGGTGTACAGGCTGCA-3' (forward), 5'-TTCGGTCCAGCTGCTGGAAGGAAT-3' (reverse); β-actin: 5'-CCATCATGAAGTGTGACGTTG-3' (forward), 5'-CAATGATCTTGATCTTCATGGTG-3' (reverse). Levels of the microRNAs miR-126-3p and miR-126-5p were determined by first carrying out reverse transcription using microRNA-specific primers and the Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems). PCR was then done using Taqman MicroRNA Assay (Applied Biosystems), and levels of expression were normalized to miR-16.
Embryonic stem cell growth rate
ESC growth rates were determined essentially as described by Udy et al . ESCs were plated on MEFs in 12 well plates at 30 cells/well. At each time point (4-8 days after plating) ESCs from triplicate wells were trypsinized, MEF-depleted, and counted using a haemocytometer. Averages of triplicate counts were compared by two-way ANOVA with repeated measures and a Bonferroni post-test using Prism4 (GraphPad Software, Inc.).
Embryonic stem cell differentiation as embryoid bodies
ESCs were MEF-depleted and seeded in 30 μl hanging drops at 8 × 104 cells/ml in differentiation medium (as for ES cell media, except 20% FBS and no LIF). Two days later EBs were grown in suspension, and then harvested at either 7 or 14 days after initial seeding. Where TGF-β was used, 2.5 ng/ml recombinant human TGF-β (R&D Systems) was added to medium prior to making hanging drops, and also for subsequent feeding. In other experiments, conditioned medium from wild-type, or scrambled control, EBs was used on knock-down clones during differentiation to day 7 EBs. EBs were fixed in 4% PFA followed by 10% and 20% sucrose, and frozen in a 1:1 solution of OCT: 30% sucrose. 12 μm sections were used for indirect immunofluorescence (IF) staining.
Collagen type I sprouting angiogenesis assay
Ten individual 7 or 14 day EBs were plated onto 1.5 ml of solidified collagen type I medium in a 35 mm diameter plate and allowed to settle overnight in differentiation medium, before a second collagen layer was added. The collagen medium was made as described by Feraud et al  with a final concentration of rat tail type I collagen of 1.25 mg/ml (BD Biosciences). Recombinant growth factors were used at final concentrations known to provide maximal biological stimulation: human VEGF165, 50 ng/ml; mouse FGF basic, 100 ng/ml; mouse Epo, 20 ng/ml; human IL-6, 10 ng/ml (R&D Systems) . After nine days the gels were quickly dehydrated on glass slides using nylon linen and filter paper, and air-dried overnight before being stored at -80°C until staining.
Indirect immunofluorescence staining
EB cryosections were fixed in ice-cold acetone, or methanol (for Flk1 staining), and EBs within collagen type I gels were fixed in 4% PFA. Samples were blocked with 10% normal donkey serum and 5% non-fat dried milk, and antibodies were diluted in 5% non-fat dried milk. Collagen gels were also permeabilized using 0.5% triton X-100. For Annexin-V staining 2% BSA was used instead of milk. Sequential double-staining was carried out with the anti-CD31 antibody first, and antibodies were used as follows; rat anti-mouse CD31, 5 μg/ml (BD Biosciences), goat anti-mouse Flk1, 4 μg/ml (Santa Cruz), rabbit anti-mouse Collagen IV, 5 μg/ml (Chemicon), goat anti-mouse VE-Cadherin, 5 μg/ml (R&D Systems), rabbit anti-mouse Claudin-5, 2.5 μg/ml (Invitrogen), rabbit anti-mouse Ki67, 1.5 μg/ml (Abcam), rabbit anti-mouse Annexin-V, 2.5 μg/ml (Abcam). Rat, rabbit, and goat IgG controls were used on adjacent sections. Signals were detected with donkey anti-rat IgG conjugated with Cy3 or Cy5, and donkey anti-rabbit or -goat IgG conjugated with Cy5 or Cy3 (Jackson ImmunoResearch). Cryosections were mounted in ProLong Gold Antifade reagent with DAPI (Invitrogen). Collagen gels were incubated with Hoechst 33342 nuclear dye (Invitrogen) and mounted in Vectashield hard-set mounting medium (Vector Labs). Images were taken using an Axioplan 2 imaging microscope (Carl Zeiss), or a Leica TSC SP2 confocal laser microscope (Leica Microsystems).
Quantification of vascular structures
For quantification of EB cryosections and collagen gel-embedded EBs, image acquisition was performed with an ORCA-ER black and white camera (Hamamatsu Photonics) driven by Openlab software (Improvision, Ltd.). Relative CD31+, Ki67+, and DAPI+, areas were measured by determining the number of pixels corresponding to the fluorescent signal using the 'magic wand tool' in Photoshop (Adobe Systems Inc.). Individual EBs were also scored for the presence of CD31+ 'cords', 'sheets', or both. Cords were defined as CD31+ structures of more than two cells in length, and not more than two cells in width, determined by counting DAPI-stained nuclei in overlaid images. CD31+ sheet structures were defined as being more than four cells in diameter, and more than four cell's distance from another sheet to be counted individually. CD31+ sheets were also analyzed separately, and the Ki67+ pixels within each sheet determined. Where indicated, confocal images were captured using Leica Confocal Software. All analysis for the collagen gel-embedded EBs was done using Photoshop. Relative CD31+ sprout length was determined using the 'measure' tool and branching points were defined as where two or more CD31+ sprouts radiated from. All statistical analysis was carried out using Prism4 (GraphPad Software, Inc.).
Results and Discussion
Lentiviral-mediated knock-down of Egfl7
Egfl7 knock-down reduces embryonic stem cell growth rate
Abnormal endothelial sheets form in Egfl7 knock-down embryoid bodies
Vascular structures within EBs were analyzed at 7 days and 14 days of differentiation, as these time points correspond approximately with early organogenesis and later remodelling stages respectively . Sections through EBs revealed the presence of two clearly distinguishable CD31+ cell structures, which we describe here as 'cords' (Figure 3; arrows) and 'sheets' (Figure 3; arrowheads). 'Cords' are defined as more than two CD31+ cells in length and a maximum of two CD31+ cells in width, and 'sheets' as aggregates of more than four CD31+ cells in length and width. These structures were viewed on 2-dimensional cryosections through EBs. Due to the spherical shape of EBs it is probable that the 'cords' are part of a larger network existing in multiple axes within the EBs, and that the 'sheets' represent one plane within 3-dimensional clusters of CD31+ cells. Similar CD31+ 'sheets' have been described in EBs derived from laminin γ1-deficient ESCs . To further characterize the CD31+ structures present, EB sections were co-stained with antibodies against other endothelial markers. Collagen IV is a major constituent of the basement membrane, and its deposition is characteristic of normal blood vessel formation and is required for subsequent angiogenesis [12, 21, 22]. Vascular endothelial (VE)-cadherin is the major transmembrane component of adherens junctions, and sustains cell-cell recognition and adhesion . Used together with CD31, which is expressed on haematopoietic cells as well as ECs, VE-cadherin is considered to be the gold standard for EC-specific markers . Claudin-5 is an endothelial-specific component of tight-junctions, which control para-cellular permeability and polarity [25, 26]. Flk1, a VEGF-A receptor, is an early marker of hematopoietic and endothelial cells .
When conditioned medium from wild-type, or scrambled control, EBs was added to cultures of knock-down ESCs during differentiation to day 7 EBs, we did not observe a rescue of the mutant phenotype (data not shown). This suggests that Egfl7 may act in a cell-autonomous manner. It has recently been shown that inhibition of the anti-proliferative transforming growth factor beta (TGF-β) during in vitro differentiation of human ESCs results in a 36-fold increase in the number of committed ECs generated . Therefore, to address whether EGFL7 and TGF-β might interact, EBs were grown in the presence of TGF-β, which resulted in no significant decrease in CD31+ area in knock-down EBs, whereas control EBs showed a more robust decrease (Figure 4c). Thus, if TGF-β is involved in the function of Egfl7, it is unlikely to have a major role in maintaining the CD31+ cell population.
CD31+ sheets show increased cell proliferation
Egfl7 knock-down is associated with endothelial sheet formation during sprouting angiogenesis
Recent work by Kuhnert et al  suggests that Egfl7-null mice are phenotypically normal, and that deletion of miR-126 causes embryonic lethality, edema, and hemorrhage, and postnatal defects in retinal and cranial angiogenesis. Thus, a possible role of Egfl7 in mammals has so far been elusive. Our studies are the first to show a clear role for Egfl7 in the formation of vascular structures in the EB in vitro differentiation model. In support, recent studies in transgenic mice show that Egfl7 overexpression results in hemorrhaging and defects in embryonic and post-natal angiogenesis (D. Nichol and H. Stuhlmann, Unpublished). The apparent discrepancy between studies using mouse knockout models, and the present work, could be explained by the fact that the Egfl7 phenotype detected in EBs is subtle, early, and transient. A strength of using the EB model system is the possibility to detect a transient and rather subtle phenotype in Egfl7 knock-down clones, which is evident at day 7 of differentiation, and then partially remodeled by day 14.
In conclusion, our results suggest that Egfl7 is vital for the organization of ECs into vascular cords and confirm that the gene has an important role during vasculogenesis and angiogenesis. We have shown that knock-down of Egfl7 results in the formation of CD31+ sheets, and our data support the notion that this is caused at least in part by the over-proliferation specifically of ECs during vasculogenesis. The CD31+ sheets appear to be abnormal endothelial structures lacking a complete basement membrane and cell junctions, which after further differentiation are accompanied by the formation of extensive endothelial cord networks. This indicates that partial remodelling occurs within the EBs, and points to an early developmental role for Egfl7. Thus, using an siRNA knock-down approach which did not affect miR-126 levels, we show here for the first time that Egfl7 has a role during endothelial cell differentiation and vascular development in mammalian cells.
We would like to thank Drs. Jan Kitajewski and Carrie Shawber at Columbia University Medical School and members of the Stuhlmann lab for helpful discussions on the project. We also thank the Molecular Cytology Core Facility at Memorial Sloan-Kettering Cancer Center for help with the confocal microscope. We thank Drs. Xiao-Feng Qin and David Baltimore (Caltech, CA) for providing us with the FG12 lentivirus vector. Funding for this work was provided in part by an American Heart Association fellowship 0525046Y to AD, and by a National Institutes of Health grant RO1 HL082098 to HS.
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