Biochemical and Biophysical Research Communications 276, 1089–1099 (2000)
doi:10.1006/bbrc.2000.3602, available online at http://www.idealibrary.com on
ouse flt-1 Promoter Directs Endothelial-Specificxpression in the Embyroid Bodyodel of Embryogenesis
ary Quinn, Takahiro Ochiya, Masaaki Terada, and Teruhiko Yoshida1
enetics Division, National Cancer Centre Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
eceived September 4, 2000
hematopoietic abnormalities. However, of these fourRtrdt
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The endothelial-specific receptor tyrosine kinaset-1 (VEGFR-1) is expressed early on during endothe-
ial lineage commitment both in vivo and in vitro. How-ver, the exact function of flt-1 in vascular develop-ent still remains unclear. Here we report that a
During mouse development, vascular and hemato-oietic lineage commitment is reflected in the sequen-ial activation of lineage specific genes (1, 2). flt-1 is onef the first endothelial-specific genes to be expresseduring mouse development (3, 4). With the exception ofonocytes, flt-1 expression in hematopoietic lineages
as not been reported (5). Hence flt-1 represents andeal marker for tracking endothelial differentiationuring the early stages of lineage commitment anday represent the point of divergence between hema-
opoiesis and vasculogenesis. Four endothelial-specificeceptor tyrosine kinase (RTK) genes, flt-1 (VEGF-R1),k-1 (VEGF-R2), tie-2 (tek), and tie-1, expressed duringmbryonic vasculogenesis and upregulated duringostnatal tumor angiogenesis (6–9). In utero or peri-atal lethality has been recorded in knock-out mice forll four RTKs as a result of severe vascular and/or
1 To whom correspondence and reprint requests should be ad-ressed. Fax: 181 3 3541-2685. E-mail: [email protected].
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TKs, only flk-1 has been directly implicated in endo-helial or hematopoietic lineage commitment (10) andecently it was demonstrated that flk-1 may also beispensable for endothelial and hematopoietic differen-iation (11).
flt-1 binds multiple VEGF isoforms and joins KDR/k-1 and FLT-4 in the 7-Ig-domain family of RTKsharacterized by seven immunoglobulin-like loops inhe extracellular region (7). Soluble flt-1 (sflt-1), a prod-ct of alternative flt-1 pre-mRNA splicing, can poten-ially block or reverse the action of both full-lengtht-1and flk-1 in a dominant-negative manner (12, 13).n sflt-1 the membrane-spanning carboxyl end of full-ength flt-1 is replaced by a unique 31 amino acid
oiety which is highly conserved between mouse anduman, suggesting it has a biological significance (14).ranscription of both full-length flt-1 and sflt-1 is up-egulated by VEGF121 and VEGF165 (15). Recently, how-ver, sflt-1 expression was shown to be regulated inde-endently of flt-1 in human endometrium during theenstruation cycle, and also in spongiotrophoblast
ells in the mouse placenta during gestation, indicatinghe existence of a novel post- or co-transcriptional reg-latory mechanism (16, 17).While the flt-1 gene provides a potential target for
solating endothelial progenitor cells, antibodies avail-ble at present to flt-1 recognize an intracellularpitope precluding live cell isolation. Moreover, as re-orted here, the dynamic status of flt-1 splice variantseans at any time sflt-1, which has no intracellular
egion may be the dominant species.Here we report the characterization of the mouse
t-1 promoter using the Enhanced Green Fluorescentrotein (EGFP) reporter gene. While the human flt-1romoter appears to be endothelial specific in vitro18), the mouse flt-1 promoter has not been character-zed nor functionally identified to date.
It is well established that in vitro differentiation ofurine ES cells within embryoid bodies (EBs) leads to
Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
omplex structures which can recapitulate normal de-elopmental processes of the early embryo, in particu-ar vasculogenesis and hematopoiesis (20–22). Moreecently the ability to unravel complex phenotypeshich have been observed in vivo can be attributed to
his in vitro model of endothelial and hematopoieticifferentiation (23, 24). Using this model of mouse em-ryogenesis we have demonstrated the endothelialpecificity of the 2.2-kb mouse flt-1 promoter upon sta-le genomic integration and observed the behavior ofiving EGFP-positive cells in culture. This model mayepresent an important tool for observing the earlyvents involved in endothelial and hematopoietic dif-erentiation. In addition, by analysis of flt-1 splice vari-nts, we have shown that the dominant-negative in-ibitor of VEGF signaling, sflt-1, is expressed earliesturing EB development suggesting that the primaryonsequence of flt-1 gene activation is suppression ofEGF signaling.
ATERIALS AND METHODS
Cloning of mouse flt-1 promoter and generation of constructs. TheenBank sequence AJ224863 (Accession No.) describes the 2648 bp9 flanking region of the mouse flt-1 gene. The 200-bp 39 region of theuman flt-1 promoter comprises Ets and CRE/ATF motifs which, inddition to the TATA box, are 100% conserved in the 59 region of theouse flt-1 gene (14, 19). A fragment spanning bases 21952 and206 respectively with respect to the transcriptional start site for
he full length flt-1 transcript was amplified from mouse STO cellenomic DNA using the flt-1 promoter primers (Table 1) and clonednto the promoterless EGFP vector, pEGFP-1 (Clontech). The mouse15-bp tie-1 promoter was a gift from K. Alitalo (University of Hel-inki) The mouse albumin promoter/enhancer construct, a gift from. D. Palmiter, is 2335 bp long and consists of a 28.5 to 210.4 kbnhancer fused to the 0.3-kb minimal promoter (25).
Immunostaining. EBs were fixed at 25°C for 20 min in 4% para-ormaldehyde in PBS, washed twice in PBS then embedded in OCTompound (Miles Scientific, Elkhart, IN). 10 mm frozen sections wereounted onto Vectabond-coated glass slides (Vector Laboratories,
List of Primers Used for PCR
Target Sense p
flt-1 promoter GTCAAATACCTGflt-1 cDNA GGACTATACGAtie-1 cDNA ATACCCTAGACPECAM-1 GTCATGGCCATVE-cadherin GGATGCAGAGGtie-2 cDNA CCTTCCTACCTGHPRT cDNA GCTGGTGAAAAEGFP cDNA GGCAAGCTGACflk-1 cDNA TCTGTGGTTCTGsflt-1 cDNA TCTTCCACTCTGfull length flt-1 TGTGGAAGGAGEGFP reversealbumin sense GGCAAACATACtie-1 sense AGTGTGTGTGCflt-1 sense CGCCTTCCTTCG
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urlingame, CA), allowed to reach room temperature for 30 min thenxed and permeabilized in methanol at 210°C for 10 min. Samplesere blocked by incubation with 1% I-Block (Tropix, Bedford, MA) inBS for 45 min to suppress non-specific binding of IgG and then
ncubated with 2 mg/ml primary antibody in 1% I-Block/PBS for 1 h.n the case of phycoerythrin (PE)-conjugated anti-PECAM-1 andnti-Sca-1 antibodies (Pharmingen), slides were mounted in Fluo-escent Mounting Medium (Dako) and viewed immediately. For anti-t-1 and anti-flk-1 primary antibodies (both rabbit anti-mouse poly-lonals from Leinco Technologies, Inc.) the slides were incubated forfurther 45 min with 2 mg/ml goat anti-rabbit IgG conjugated withexas Red (Leinco Technologies, Inc.).
Flow cytometry analysis of EGFP expression. Forty-eight hoursollowing transfection with EGFP reporter constructs, cells wererypsinized and washed twice in PBS and fixed in 2% paraformalde-yde at 25°C for 30 min. Quantitative fluorescence analysis wasarried out using a FACSCalibur flow cytometer equipped withELLQuest software (Becton–Dickinson).
Reverse transcription-polymerase chain reaction (RT-PCR) analy-is. Total RNAs were isolated using Isogen total RNA isolation kitNippongene), and the first-strand cDNA were generated usingroSTAR first-strand RT-PCR kit (Stratagene). Specific cDNA spe-ies were amplified using the primers indicated in Table 1. Thermo-ycling parameters for PCRs were 94°C for 30 s, 55°C for 30 s, 72°Cor 30 s for 25 or 30 cycles as indicated in the figure legends.xceptions were amplification of the flt-1 promoter; 94°C for 30 s,7°C for 30 s, and 72°C for 2 min for 30 cycles; and conditions formplification of the tie-2 cDNA; 94°C for 30 s, 50°C for 30 s, 72°C for0 s for 25 cycles. All samples were adjusted to yield equal amplifi-ation of hypoxanthine phosphoribosyltransferase (HPRT).
Cell culture. The mouse hemangioma cell line PmTc1 was gen-rated by immortalization of mouse endothelial cells according to theechnique described previously (4). The endothelial nature of themTc1 cell line was confirmed by von Willebrand factor staining.IH3T3, STO (both mouse embryonic fibroblast) and PmTc1 cell
ines were maintained on gelatin-coated tissue culture dishes (Iwaki)n DMEM/10% FBS (Life Technologies).
Analysis of LDL receptor expression by DiI-Ac-LDL uptake. DiI-c-LDL (Biomedical Technologies) uptake was assayed in culturedndothelial cells according to the manufacturer’s guidelines, andisualized by fluorescence microscopy.
Transfection of non-ES cell lines. Purified plasmid DNA (Qiagenaxiprep) were combined with Dosper (Boehringer) transfection
agent and used to transfect actively growing subconfluent culturesetmt
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ssentially as described by the manufacturers, For stable transfec-ants NIH3T3 cells were treated daily with medium containing 500g/ml (active base) G418 (Sigma) for 12 days beginning 24 h post-ransfection.
Growth and differentiation of ES cells. All reagents, excepthere indicated, were supplied by Life Technologies. J1 (PJ5) ES
ells were maintained on STO mouse embryonic fibroblast cells onelatin-coated tissue culture dishes in DMEM containing 20% ESell qualified FBS, 13 b-mercaptoethanol (Sigma, 1003 2 7 ml 100%tock in 10 ml PBS), 13 non-essential amino acids, 13 nucleosides1003: adenosine (8 mg/ml), guanosine (8.5 mg/ml), cytidine (7.3g/ml), uridine (7.3 mg/ml), thymidine (2.4 mg/ml)), 1000 U/mlSGRO/LIF (Amrad) and 13 penicillin/streptomycin. Induction ofB formation and ES cell differentiation was carried out as describedreviously (26). Briefly, single-cell dispersions of undifferentiated ESells were plated in 13 MCM (methyl cellulose medium), 1% IMDM,5% FBS, 450 mM MTG (Sigma) and 13 penicillin/streptomycin at aoncentration of 1000 cells/ml/plate on bacteriological grade 35-mm2
ishes (Sterilin). No medium changes were made or exogenous fac-ors added during EB differentiation. EBs were harvested by dilutionf MCM with PBS and centrifugation at 100g for 5 min. By day 10,vert globinization was seen in .50% of EBs. For partial disaggre-ation to generate primary cultures of differentiated ES cells, indi-idual EBs were first washed in PBS, then transferred to 103rypsin/EDTA for 5 min followed by plating on gelatin-coated dishesn ES (LIF-) medium.
Electroporation of ES cells and selection of stable transfectants.or each transgene construct FLT.EGFP, TIE.EGFP, andLB.EGFP, linearized plasmid DNA (Qiagen Maxiprep) was used tolectroporate J1 ES cells 48 h following electroporation, G418 (175g/ml) selection was commenced, and DNA was isolated from at leastix G418-resistant clones from each electroporation as previouslyescribed by Wurst and Joyner (27). Integrity of the integratedromoter/transgene cassette was confirmed by PCR. For each con-truct the flt-1 sense, tie-1 sense or albumin sense primer was pairedith the upstream EGFP reverse primer, and PCR was carried outcross the promoter/transgene junction to yield bands of approxi-ately 850, 650, and 380 bp respectively in positive clones.
ESULTS
nalysis of flt-1 Promoter Activity by TransientTransfection Reporter Gene Assay
Relative activities of pFLT.EGFP, pTIE.EGFP,ALB.EGFP and pCMV.EGFP promoter constructsere initially compared by transient transfection ofon-endothelial (NIH3T3, STO) and endothelialPmTc1) cell lines. Promoter activity was quantified byow cytometry 48 h posttransfection. Visually, EGFPuorescence was detected as early as 24 h posttrans-ection in all cell lines for each construct; the strongestignals were observed in NIH3T3 and STO cells trans-ected with pFLT.EGFP and pALB.EGFP. After 48 hLT.EGFP expression was highest in NIH3T3 cells
Fig. 1F). Congruent with the previous report, the tie-1romoter appeared equally active in all cell types an-lyzed, thus displaying lack of in vitro endothelial spec-ficity (28) (Figs. 1B and 1E). However the lack ofell-type specificity displayed by pALB.EGFP duringransient transfection (data not shown), which pro-uced high levels of fluorescence in both NIH3T3 and
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he tie-1 and albumin promoters the flt-1 promoter alsopparently lacks cell-type specificity in transientransfection assays (Figs. 1C and 1F). EGFP expres-ion from the tie-1, flt-1 and CMV promoters, as quan-ified by flow cytometry is summarized in Table 2. Theesult obtained corresponded to the direct visualizationf cells by fluorescence microscopy. In general, bothie-1 and flt-1 promoter activities were equivalent inmTc1 and also STO cells, and both promoters showedighest activity in NIH3T3 cells.Immunofluorescence staining of STO and NIH3T3
ells, failed to detect the endothelial marker proteinsECAM-1, flt-1 or flk-1, all of which were expressed inmTc1 cells (data not shown). Additionally DiI-c-LDL uptake, a characteristic feature of differenti-ted endothelial cells and monocyte-macrophages, wasot detected in either STO or NIH3T3 cells (data nothown). We next broadened our analysis by using aanel of primers for endothelial specific genes to estab-ish an expression profile for each cell line by RT-PCR.s shown in Fig. 2, PmTc1 cells express each of
he characteristic endothelial specific markersVE-Cadherin was detectable following 30 cycles ofmplification (Fig. 4)). However, both STO andIH3T3 cells were negative for each of these markers,
ncluding flt-1 and tie-1 (Fig. 3).
he flt-1 Promoter Is Silenced in Nonendothelial Cellsupon Genomic Integration
Since the flt-1 promoter did not displayndothelial-specific activity when transiently trans-ected (Table 2) we sought to ask whether simpleandom genomic integration was sufficient to restoreell-type specificity to the tie-1, albumin and, per-aps, the flt-1 promoter. NIH3T3 cells were trans-ected with the following constructs; pCMV.EGFP,TIE.EGFP, pALB.EGFP and pFLT.EGFP and sub-ected to G418 selection. After 48 h all constructs hadroduced a pattern of non-specific EGFP expressionimilar to that seen in previous transient transfec-ion experiments. However by 12 days posttransfec-ion many G418 resistant colonies displayedigh levels of uniform EGFP fluorescence indicativef stable genomic integration, in pCMV.EGFP-,TIE.EGFP- and pALB.EGFP-transfected NIH3T3 cellsFigs. 3B, 3C, and 3D). In contrast, pFLT.EGFP-ransfected G418-resistant colonies showed barely visiblend undetectable fluorescence using the same cameraxposure times as for pALB.EGFP-, pTIE.EGFP- andCMV.EGFP-transfected cells (Fig. 3A). This remarkablending demonstrates that flt-1 promoter activity is some-ow silenced upon integration into the genome of other-ise permissive non-endothelial cells.
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nalysis of Promoter Activity in EBs StablyTransfected with pFLT.EGFP and pTIE.EGFP
Approxiately 600-1000 EBs derived from ES cellstably transfected with pFLT.EGFP and pTIE.EGFPonstructs were harvested at various time points be-ween day 3 and day 10 from the beginning of EBnduction. Total RNA was extracted, and an averageevel of marker gene expression was measured byT-PCR over the course of EB development (Fig. 4).ycling parameters and cDNA concentrations were op-
imized to permit semi-quantitative comparisons of rel-tive expression patterns for each gene. PmTc1 cellsransiently transfected with pCMV.EGFP were used
TABLE 2
Transcriptional Activity of Various Promoters Quantifiedby Flow Cytometry
FIG. 1. Analysis of EGFP expression in transient transfection rephowing lack of specificity displayed by putative cell-type-specific prs represented by a shift in the proportion of FITC-fluorescent cellellular autofluorescence in mock transfected cells thinner black cxpression was observed reflecting the constitutive activity of the CGFP-positive cells clustered at lower levels of EGFP. A to C, D to F, D, and G cells are transfected with pCMV.EGFP; in B, E, and H,or each analysis, 10,000 events were recorded. Dead cells were gativen in top right corner of each panel. Flow cytometer parameters wn the mock-transfected cell population was less than 0.5%.
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s a positive control for analyses of endothelial markernd EGFP expression in EBs. PECAM-1, flt-1 and tie-2ere expressed from day 3 and showed an increase inxpression through day 10. Low levels of tie-1 and flk-1
er gene assays by flow cytometry. Forty-eight hours posttransfectionoters when transiently transfected. In each panel promoter activity
promoter/EGFP-transfected cells thicker black curve) relative toe). For the pCMV.EGFP-transfected cells a broad range of EGFPV promoter. For the pTIE.EGFP and pFLT.EGFP constructs mostd G to I represent STO, NIH3T3, and PmTc1 cells, respectively. Inh pTIE.EGFP; in C, F, and I with pFLT.EGFP reporter constructs.sing PI. Percentage of EGFP-positive cells for each experiment are
e set for each cell type such that the number of EGFP-positive cells
FIG. 2. Analysis of endothelial marker gene expression in endo-helial and fibroblast cell lines by RT-PCR. Lane 1, NIH3T3 cells;ane 2, PmTc1 cells; lane 3, STO cells. All PCRs were carried out for5 cycles and expression levels were controlled using HPRT.
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xpression were detected during the later stage of EBevelopment and VE-cadherin expression could not beetected at all. When analyzed in parallel, EGFP ex-ression was high from day 3 in pFLT.EGFP-ransfected EBs and sustained through day 10 corre-ponding temporally to endogenous flt-1 expression.owever in EBs derived from pTIE.EGFP-transfectedS cells, EGFP expression was very low corresponding
o the equally low expression of endogenous tie-1.hese results indicate that the activity of the exoge-
FIG. 3. Analysis of promoter cell-type specificity upon stable geells; frame B, pCMV.EGFP-transfected cells; frame C, pTIE.ELT.EGFP-transfected NIH3T3 cells, EGFP expression in G418-rexposure times as for frames B, C, and D. Frames E to H represent
FIG. 4. Sequential expression of endothelial-specific genes dur-ng EB maturation from day 3 to day 10 in transgenic ES cell lines.anes 1, 3, 5, and 7 show EBs derived from pFLT.EGFP-transfectedS cells. Lanes 2, 4, 6, and 8 show pTIE.EGFP-transfected ES cells.anes 1and 2, 3 and 4, 5 and 6, and 7 and 8 are derived from day 3,ay 5, day 7, and day 10 Ebs, respectively. In lane 9, the cDNA iserived from PmTc1 cells 48 h after transient transfection withCMV.EGFP. PCRs for all markers were carried out for 25 cyclesxcept those indicated by *.
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ous flt-1 promoter may parallel, at least temporally,hat of its endogenous counterpart.
ariant Specific Analysis of flt-1 Expressionduring EB Development
It has been previously reported that flt-1 is expressedarly during development and thereafter increasesoth in vitro and in vivo (3, 11). Most RT-PCR analysesf flt-1 gene expression fail to pick up soluble flt-1sflt-1) splice variant with primers specific for the full-ength flt-1 cDNA (11, 24). Since the transcriptionaltart sites for flt-1 splice variants are merely 4 bp apartt is unlikely that alternative promoters regulate flt-1ene expression (29). The primers used to detect flt-1xpression in Fig. 4 are located within the 2 kb 59egion of the flt-1 gene, which is identical in both vari-nts. Therefore the analysis of flt-1 expression shown
ic integration in NIH3T3 cells. Frame A, pFLT.EGFP-transfected-transfected cells; frame D, pALB.EGFP-transfected cells. For
ant colonies was too weak to be detected using the same cameracorresponding phase contrast photographs for A to D, respectively.
FIG. 5. Analysis of flt-1 splice variants by variant-specific PCR.anes 1 to 4 show days 3, 5, 7, and 10 Ebs, respectively, derived fromFLT.EGFP-transfected ES cells. In lane 5, the cDNA is derivedrom PmTc1 cells 48 h after transient transfection withCMV.EGFP. In the upper panel the primer pair recognizes bothull-length and soluble flt-1 variants. In the middle panel primerspecific for the unique 39 region of full-length flt-1 are used, and inhe lower panel primers specific for the unique 39 region of sflt-1 aresed. All PCRs were carried out for 30 cycles.
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n Fig. 4, from which comparisons with EGFP expres-ion are drawn, represents the combined expressionevels of both sflt-1 and full-length flt-1. But since bothariants encode proteins that potentially have distinctnd opposing biological activities we additionally as-essed the individual expression pattern of each spe-ies during EB development.
We designed a primer pair specific for the unique 39egion of full-length flt-1 and another primer pair spe-
FIG. 6. Localization of EGFP expression with respect to markLT.EGFP transgenic ES cells. 10-mm-thick frozen sections of EBs wtaining of lineage-specific marker protein. Frames B, E, H, and K shB section. Frames C, F, I, and L show simultaneous visualizatolocalization of EGFP and PECAM-1 in the same cells of a highlECAM-1 in the same cells of a poorly vascularized EB. Arrowheadsto I and J to L show the spatial relationship between EGFP and Sca
f, and appear to surround, Sca-1-positive cell clusters with a partia
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ific for the short unique 39 region of sflt-1. sflt-1, theominant-negative variant of flt-1, is expressed earli-st in developing EBs, and its expression remains con-tant through day 10. However full-length flt-1 is ex-ressed later (detectable at day 5) and increasesteadily during EB development with respect to sflt-1nd eventually overtakes sflt-1 as the dominant speciesy day 10 (Fig. 5). Both variants are expressed inmTc1 cells but the relative expression of the full-
s of endothelial and hematopoietic lineages in EBs derived fromfixed and stained. Frames A, D, G, and J show immunofluorescencethe pattern of FLT.EGFP derived fluorescence in the correspondingof EGFP and marker protein expression. Frames A to C show
ascularized EB. Frames D to F show colocalization of EGFP andint to examples of EGFP and PECAM-1 coexpressing cells. Frames
in the same EB section. EGFP-positive cells are located at the borderverlap (arrows).
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ength variant is greater in these more differentiatedemangioma cells, a situation that is paralleled inifferentiated day 10 EBs.
GFP Expression Is Confined to PECAM-1-PositiveCells in Day 10 EBs Derived from pFLT.EGFP-Transfected ES Cells
Immunostaining was carried out on frozen sectionsf day 10 EBs to examine EGFP localization in relationo PECAM-1, a characteristic marker of the endothelialineage. A strikingly clear and reproducible colocaliza-ion of PECAM-1 and EGFP expression was seen inBs derived from pFLT.EGFP-transfected ES cells
Figs. 6A–6F). In .95% of EBs expressing EGFP,GFP-positive cells co-expressed PECAM-1. However
n a minor population (,5%) EGFP was seen in thebsence of PECAM-1. Conversely, about 30% of EBserived from pFLT.EGFP-transfected ES cells, whichtained strongly for PECAM-1, lacked EGFP. This in-icates, not surprisingly, that there is a major overlapather than absolute synchrony in the expression pro-les of these genes.
FIG. 7. The relationship between PECAM-1 and EGFP expres-ion in EBs derived from pFLT.EGFP-transfected (frames A and C)nd pTIE.EGFP-transfected (frames B and D) ES cells. In frame Ahe large EB is negative for EGFP while smaller, adjacent EBs areositive as indicated by the arrowheads. Frame C shows that theattern of PECAM-1 expression corresponds to FLT.EGFP expres-ion in both EGFP-positive and EGFP-negative EBs. Many of theseBs derived from pTIE.EGFP-transfected ES cells displayed strongECAM-1 expression (arrowheads in frame D) but no TIE.EGFP
frame B) due to silencing of the tie-1 promoter, a feature correlatingith the absence of endogenous tie-1 expression in these EBs.
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orted a variety of differentiation programs such asardiogenesis for example. EBs containing cardiomyo-ytes displayed distinct characteristics; they were gen-rally larger than other EBs, did not exhibit globiniza-ion and beat rhythmically. Neither EGFP norECAM-1 expression was detected in frozen sections ofhese EBs indicating that the flt-1 promoter was silentn the absence of endothelial differentiation, a furtheremonstration of flt-1 promoter specificity (Figs. 7And 7C).Colocalization of PECAM-1 and EGFP is particularly
lear since the former is restricted to cell borders form-ng a honeycomb-like pattern while EGFP is distrib-ted uniformly throughout the cell. Thus both markersre simultaneously discernible using a dual filterlock. Most of the EBs expressed PECAM-1, a markeror all endothelial cells and a small subset of leuko-ytes, although heterogeneity was seen in the extent ofECAM-1 expression between different EBs. This vari-tion may be attributed to the minimal culture me-ium used, which is lacking in exogenous factors thatan promote both the degree and synchrony of endo-helial and hematopoietic differentiation. However, forhe purpose of demonstrating that EGFP andECAM-1 expression are consistently colocalized inhe same cells during EB development, this heteroge-eity in EB vascularization worked to our advantage.sing this system we could demonstrate PECAM ex-ression in EGFP-positive cells in EBs ranging fromhe poorly vascularized to those with a high degree ofascularization at a single time point. EBs fromTIE.EGFP-transfected and pALB.EGFP-transfecteddata not shown) ES cells displayed a similar range ofECAM-1 expression but lacked any detectable EGFPxpression (Figs. 7B and 7D).There is a remote possibility that the heteroge-
eous pattern of EGFP expression observed mightave resulted from the aggregation of EGFP-positivend EGFP-negative undifferentiated ES in variousatios, each contributing respectively to the EGFP-ositive and EGFP-negative differentiated cells inhe EB. However, we safeguarded against this pos-ibility by plating undifferentiated ES cells in semi-olid medium at low concentrations ensuring thatBs developed in isolation from single cells, withollision and aggregation occurring rarely.
GFP-Positive Cells Circumscribe Sca-1-PositiveCells in FLT.EGFP-Transfected ES Cells
To investigate flt-1 promoter activity following he-atopoietic and endothelial lineage divergence, we
ext examined the relationship between EGFP andca-1 expression patterns. Many EBs stained positive
or Sca-1, a marker of pluripotent hematopoietic stemells, and in most of these EBs, EGFP expression was
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lso detected. However, EGFP was generally not, apartrom a minor overlap, observed in Sca-1-positive cells,ut rather in cells in the immediate vicinity of theseca-1-positive regions (Figs. 6G–6L). Hence it appearshat EGFP expression is downregulated during he-angioblast differentiation to the hematopoietic lin-
age, while the flt-1 promoter is active during endothe-ial differentiation. Therefore although we may haveaptured a narrow overlap of Sca-1 and FLT.EGFPxpression in some cells, most Sca-1-positive cells wereegative for EGFP and located within close proximityf EGFP-positive cells. When adjacent sections of theame EB were stained alternately with anti-PECAM-1nd anti-Sca-1 antibodies, we found that in all Sca-1-ositive EBs, PECAM-1 expression was localized toGFP-positive cells (data not shown).
To examine the morphology of living EGFP-positiveells, we generated primary cultures from partiallyisaggregated day 10 EBs. Forty-eight hours later webserved EGFP fluorescence in these living cells bynverted phase fluorescence microscopy. In many casesGFP-positive cells were aligned along the lumenalorder of vessel-like structures in a highly orderedashion (Fig. 8). It is likely that these structures devel-ped under the influence of EB formation and subse-uently organized groups of vascular cells adhered enasse maintaining somewhat their original structural
FIG. 8. EGFP-positive cells line vessel-like structures in primarshow 3 examples of EGFP expression in cells lining vessel-like strushow the phase-contrast equivalent for each of these fluorescent i
ndothelial-like cells which are arranged in a distinct pattern lining
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eatures. In fact, the vessel-like structures were notound in cultures containing EGFP-positive cells orig-nating from ES cells differentiated in two dimensionsn the absence of embryoid body formation (data nothown). In the 2D system, undifferentiated ES cellines were cultured for 5 days in the absence of LIF andeeder cells on gelatin-coated culture dishes. While dis-inct clusters of EGFP-positive cells were observedithin areas of EGFP-negative cells by day 5 in 2D
ulture, the organized vessel-like structures containingGFP-positive cells were not observed.
ISCUSSION
As a pretext to identifying the factors required forndothelial and hematopoietic differentiation and theirechanisms for programming lineage commitment,
he precursor cells must be isolated. With this goal inind we have developed a strategy to attempt to iden-
ify these precursor cells (angioblast/bipotential he-angioblast) by labeling them with EGFP. Since flt-1
s expressed by the hemangioblast (3, 30) and one thearliest endothelial-specific genes expressed during de-elopment we rationalized that the promoter for thet-1 gene would be a suitable candidate for targetingGFP expression to early and pre-differentiated endo-
helium. The endothelial-specific cell adhesion mole-ule, PECAM-1, is strongly expressed in the endothe-ial lineage from an early stage during EB development1). Here we report that EGFP expression stronglyo-localizes with PECAM-1 in EBs derived from
ltures derived from FLT.EGFP-transfected EBs. Frames A, B, andres derived from primary cultures of day 10 EBs. Frames D, E, and
ges, respectively. The arrows indicate some of these EGFP-positivee vessel-like structures (v).
y cuctu
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pFLT.EGFP-transfected ES cells indicating that the2tEtpesde(
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Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.2-kb flt-1 promoter is endothelial specific. Moreover,he consistent colocalization of PECAM-1 and EGFP inBs displaying a range of vascular maturity indicates
hat there is significant overlap in their expressionrofiles. Very few EGFP-positive cells (,5%) did notxpress PECAM-1, which is an indication that expres-ion of both genes is initially temporally matched. In-eed the RT-PCR data confirms that FLT.EGFP isxpressed from an early stage in EB developmentFig. 4).
Sca-1 is a marker of pluripotent hematopoietic stemells and is detectable from E10 in the aorta-gonad-esonephros (AGM) region of the mouse fetus (31).arallel analysis of Sca-1 and EGFP expression re-ealed that EGFP is expressed in a minor subset ofca-1-positive cells while most of the Sca-1-positiveells observed occur within a lattice-like arrangementf EGFP-positive cells. Close proximity yet only partialverlap of these markers suggests that (a) both di-erged from a common bipotential precursor and (b)GFP is down regulated upon hematopoietic commit-ent. Both possibilities are compatible with the acti-
ation of the endogenous flt-1 promoter in hemangio-lasts and thereafter in the endothelial lineage (3).It is apparent that in vivo characterized putative
issue specific promoters/enhancers do not implicitlyetain their cell-type specificity in vitro, and vice versa.oth the mouse tie-1 and albumin promoters illustrate
his point well. Neither promoter is tissue-specifichen assayed by transient transfection in vitro (Fig. 1,ata not shown), yet both are highly cell-type specificn transgenic mice (25, 28). Like tie-1 and albumin, thet-1 promoter is active in transiently transfected fibro-lasts, thus displaying a lack of cell-type specificity.ut unlike them, flt-1 is silenced upon simple random
ntegration in otherwise permissive cells (Fig. 3A). It isossible that, in some instances, tissue specificity aris-ng from genomic integration is dependent merelypon the topological constraints imposed by the inte-ration event itself, while in other cases the cellularicroenvironment within the tissue (or EB) may play a
ey role.It has recently been demonstrated that flt-1 attenu-
tes hemangioblast proliferation (30), a finding that isompatible with the abnormal assembly of endothelialells into vascular channels observed in flt-12/2 mice6). Indeed deletion of the tyrosine kinase domain ofhe full-length flt-1 appears to have little effect uponither embryonic development or angiogenesis in mice32). Collectively, these studies suggest that the pri-ary function of flt-1 is to sequester VEGF familyembers in a dominant-negative manner. However,
he individual contributions made by each of the flt-1plice variants toward this phenotype remains unre-olved. Both variants are almost certainly expressedrom a common promoter yet they encode products
1097
he entire tyrosine kinase-containing region, can bindith high affinity and inhibit the activities of VEGF indominant-negative manner by heterodimerizing with
he extracellular ligand-binding domain of theembrane-bound full-length flt-1 and KDR (flk-1) (33,
4). In this regard sflt-1 is the only currently knownndogenously expressed selective inhibitor of VEGF33). To our knowledge this is the first report concern-ng the expression pattern of sflt-1 in the EB model ofmbryonic development. In our model of EB differen-iation levels of sflt-1 mRNA are altered vis-a-vis full-ength flt-1 during the course of EB development. Im-ortantly, sflt-1 is exclusively expressed in day 3 EBs.his observation suggests that the earliest role of flt-1ay be to repress VEGF signaling via sflt-1. It is worthoting that in cells lacking flk-1 expression, flt-1 acti-ation by VEGF does not induce cell proliferation, butk-1 activation by VEGF in flt-1-negative cells does35, 36).
Our EBs lacked flk-1 expression except for very lowxpression at day 10 corresponding to the absence ofk-1 protein by immunostaining of EB sections (dataot shown). Nevertheless, based upon the sequentialctivation of endothelial specific genes in these EBsndothelial differentiation apparently occurred (Fig.). This finding confirms the report that flk-1 is dis-ensable for endothelial differentiation in vitro (11).ne explanation is that the lack of exogenous growth
actors in the minimal culture medium used in thistudy suppressed flk-1 expression, conditions that nev-rtheless supported endothelial differentiation.Hematopoiesis and vasculogenesis can proceed in 2D
n the absence of the EB (37). However, in the 3D EBystem employed in this study, appropriate cell–cellnteractions are important for the coordination of an-iogenesis and perhaps the completion of vasculogen-sis, which overlaps with angiogenesis, enabling us torace the fate of differentiating endothelial cells to aurther extent. EGFP-positive cells in day 10 EBs de-ived from pFLT.EGFP-transfected ES cells were ar-anged into structures which bore a striking similarityo endothelial-lined blood vessels seen in vivo and initro (20). These structures were observed in primaryultures of partially disaggregated EBs (Fig. 8) but notn cultures of the same ES cell line which had beenifferentiated in 2D using the same growth medium.Our data indicate that the 2.2kb flt-1 promoter ac-
ivity is endothelial specific and becomes activated atn early stage during endothelial differentiation.ence this promoter fragment represents a useful tool
or directing transgene expression to developing endo-helium in the EB. While the characterization ofndothelial-specific cis-regulatory sequences of the flt-1ene will allow us to identify transcription factors reg-lating endothelial cell lineage establishment, theFLT.EGFP construct may provide the means to iden-
tify and isolate endothelial and/or hematopoietic pro-g
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Vascular endothelial growth factor VEGF and its receptors.
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Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
enitor cells (angioblast/hemangioblast).
CKNOWLEDGMENTS
We thank Dr. S. Nishikawa (Kyoto University) for useful discus-ion, and Dr. Y. Ikarashi (NCCRI) and Dr. S. Ohnishi (NCCRI) forssistance with flow cytometry. In addition we thank Dr. R. M.harrard (University of York) for advice regarding immunofluores-ence staining and EGFP applications. We thank Dr. K. AlitaloUniversity of Helsinki) for providing us with the mouse tie-1 pro-oter. This work was funded by a Foreign Research Fellowship from
he Foundation for Promotion of Cancer Research, Tokyo.
EFERENCES
1. Vittet, D., Prandini, M. H., Berthier, R., Schweitzer, A., Martin-Sisteron, H., Uzan, G., and Dejana, E. (1996) Embryonic stemcells differentiate in vitro to endothelial cells through successivematuration steps. Blood 88, 3424–3431.
2. Hatzpoulos, A. K., Folkman, J., Vasile, E., Eiselen, G. K., andRosenberg, R. D. (1998) Isolation and characterization of endo-thelial progenitor cells from mouse embryos. Development 125,1457–1468
3. Fong, G. H., Klingensmith J., Wood, C. R., Rossant, J., andBreitman, M. L. (1996) Regulation of flt-1 expression duringmouse embryogenesis suggests a role in the establishment ofvascular endothelium. Dev. Dyn. 207, 1–10.
4. Williams, R. L., Courtneidge, S. A., and Wagner, E. F. (1988)Embryonic lethalities and endothelial tumors in chimeric miceexpressing polyoma virus middle T oncogene. Cell 52, 121–131.
5. Barleon, B., Sozzani, S., Zhou, D., Weich, H. A., Mantovani, A.,and Marme, D. (1996) Migration of human monocytes in re-sponse to vascular endothelial growth factor VEGF is mediatedvia the VEGF receptor flt-1. Blood 87, 3336–3343.
6. Fong, G. H., Rossant, J., Gertsenstein, M., and Breitman, L.(1995) Role of the flt-1 receptor tyrosine in regulating the assem-bly of vascular endothelium. Nature 376, 66–70.
7. Mustonen, T., and Alitalo, K. (1995) Endothelial receptor ty-rosine kinases involved in angiogenesis. J. Cell. Biol. 129, 895–898.
8. Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu,X. F., Breitman, M. L., and Schuh, A. C. (1995) Failure of blood-island formation and vasculogenesis in flk-1-deficient mice. Na-ture 376, 62–66.
9. Sato, T. N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K.,Fujiwara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H.,Risau, W., and Qin, Y. (1995) Distinct roles of the receptortyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Na-ture 376, 70–74.
0. Shalaby, F., Ho, J., Stanford, W. L., Fischer, K. D., Schuh, A. C.,Schwartz, L., Bernstein, A., and Rossant, J. (1997). A require-ment for flk-1 in primitive and definitive hematopoiesis andvasculogenesis. Cell 89, 981–990.
1. Schuh, A. C., Faloon, P., Hu, Q. L., Bhimani, M., and Choi, K.(1999) In vitro hematopoietic and endothelial potential of flk-12/2
embryonic stem cells and embryos. Proc. Natl. Acad. Sci. USA96, 2159–2164.
2. Goldman et al. (1998). Paracrine expression of a native solublevascular endothelial growth factor receptor inhibits tumorgrowth, metastasis, and mortality rate. Proc. Natl. Acad. Sci.USA 95, 8795–8800.
3. Neufeld, G., Cohen, T., Gengrinovitch, S., and Poltorak, Z. (1999)
1098
FASEB J. 13, 9–22.4. Kondo, K., Hiratsuka, S., Subbalakshmi, E., Matsushime, H.,
and Shibuya, M. (1998) Genomic organization of the flt-1 geneencoding for vascular endothelial growth factor VEGF receptor-1suggests an intimate evolutionary relationship between the 7-Igand the 5-Ig tyrosine kinase receptors. Gene 208, 297–305
5. Barleon, B., Siemeister, G., Martiny-Baron, G., Weindel, K.,Herzog, C., and Marme, D. (1997) Vascular endothelial growthfactor up-regulates its receptor fms-like tyrosine kinase 1 flt-1and a soluble variant of flt-1 in human vascular endothelial cells.Cancer Res. 57, 5421–5425.
6. He, Y., Smith, S. K., Day K. A., Clark, D. E., Licence, D. R., andCharnock-Jones D. S. (1999) Alternative splicing of vascularendothelial growth factor VEGF-R1 flt-1 pre-mRNA is importantfor the regulation of VEGF activity. Mol. Endocrinol. 13, 537–545.
7. Krussel, J. S., Casan, E. M., Raga, F., Hirchenhain, J., Wen, Y.,Huang, H. Y., Bielfeld, P., and Polan, M. L. (1999) Expression ofmRNA for vascular endothelial growth factor transmembrane-ous receptors flt-1 and KDR, and the soluble receptor sflt-1 incycling human endometrium. Mol. Hum. Reprod. 5, 452–458.
8. Ikeda, T., Wakiya, K., and Shibuya, M. (1996) Characterizationof the promoter region for flt-1 tyrosine kinase gene, a receptorfor vascular endothelial growth factor. Growth Factors 13,151–62
9. Wakiya, K., Begue, A., Stehelin, D., and Shibuya, M. (1996) AcAMP response element and an Ets motif are involved in thetranscriptional regulation of flt-1 tyrosine kinase vascular endo-thelial growth factor receptor 1 gene. J. Biol. Chem. 271, 30823–30828.
0. Wang, R., Clark, R., and Bautch, V. L. (1992) Embryonic stemcell-derived embryoid bodies form vascular channels: An in vitromodel of blood vessel development. Development 114, 303–316.
1. Flamme, I., and Risau, W. (1992) Induction of vasculogenesisand hematopoiesis in vitro. Development 116, 435–439.
2. Keller, G., Kennedy, M., Papayannopoulou, T., and Wiles, M. V.(1993) Hematopoietic commitment during embryonic stem celldifferentiation in culture. Mol. Cell. Biol. 13, 473–486.
3. Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S.,Kabrun, N., and Keller, G. (1997) A common precursor for prim-itive erythropoiesis and definitive haematopoiesis. Nature 386,488–493.
4. Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J. C., andKeller G. (1998) A common precursor for hematopoietic andendothelial cells. Development 125, 725–732.
5. Pinkert, C. A., Ornitz, D. M., Brinster, R. L., and Palmiter R. D.(1987) An albumin enhancer located 10 kb upstream functionsalong with its promoter to direct efficient, liver-specific expres-sion in transgenic mice. Genes and Development 1, 268–276.
6. Wiles, M. V. (1993) Embryonic stem cell differentiation in vitro.Methods Enzymol. 225, 900–918.
7. Wurst, W., and Joyner, A. J. (1993) Production of targeted em-bryonic stem cell clones. In Gene Targeting A Practical Approach(Joyner, A. J., Ed.), pp. 52–61. IRL Press, Oxford.
8. Korhonen, J., Lahtinen, I., Halmekyto, M., Alhonen, L., Janne,J., Dumont, D., and Alitalo, A. (1995) Endothelial-specific geneexpression directed by the tie gene promoter in vivo. Blood 86,1828–1835.
9. Ayoubi, T. A. Y., and Van de Ven, W. J. M. (1996) Regulation ofgene expression by alternative promoters. FASEB J. 10, 453–460.
0. Fong, G. H., Zhang, L., Bryce, D. M., and Peng, J. (1999) In-creased hemangioblast commitment, not vascular disorganiza-
tion, is the primary defect in flt-1 knock-out mice. Development
Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
126, 3015–3025.1. Miles, C., Sanchez, M. J., Sinclair, A., and Dzierzak, E. (1997)
Expression of the Ly-6E.1 Sca-1 transgene in adult hematopoi-etic stem cells and the developing mouse embryo. Development124, 537–47.
2. Hiratsuka, S., Minowa, O., Kuno, J., Noda, T., and Shibuya, M.(1998) flt-1 lacking the tyrosine kinase domain is sufficient fornormal development and angiogenesis in mice. Proc. Natl. Acad.Sci. USA 95, 9349–9354.
3. Kendall, R. L., and Thomas, K. A. (1993) Inhibition of vascularendothelial cell growth factor activity by an endogenously en-coded soluble receptor. Proc. Natl. Acad. Sci. USA 90, 10705–10709.
4. Kendall, R. L., Wang, G., and Thomas, K. A. (1996) Identificationof a natural soluble form of the vascular endothelial growthfactor receptor, flt-1, and its heterodimerization with KDR. Bio-chem. Biophys. Res. Commun. 226, 324–328.
1099
and Shibuya, M. (1995) A unique signal transduction from flttyrosine kinase, a receptor for vascular endothelial growth factorVEGF. Oncogene 10, 135–147.
6. Waltenberger, J., Claessonwelsh, L., Siegbahn, A., Shibuya, M.,and Heldin, C. H. (1994) Different signal transduction propertiesof KDR and flt-1, two receptors for vascular endothelial growthfactor. J. Biol. Chem. 269, 26988–26995.
7. Nishikawa, S., Nishikawa, S., Hirashima, M., Matsuyoshi, N.,and Kodama, H. (1998) Progressive lineage analysis by cell sort-ing and culture identifies flk-11VE-cadherin1 cells at a divergingpoint of endothelial and hematopoietic lineages. Development125, 1747–1757.
8. Eichmann, A., Corber, C., Nataf, V., Vaigot, P., Breant, C., andLe Douarin, N. M. (1997) Ligand-dependent development of theendothelial and hematopoietic lineages from embryonic meso-dermal cells expressing vascular endothelial growth factor re-ceptor 2. Proc. Natl. Acad. Sci. USA 94, 5141–5146.
