Development 114, 521-532 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

521

Expression of vascular endothelial growth factor during embryonic

angiogenesis and endothelial cell differentiation

GEORG BREIER, URSULA ALBRECHT, SYLVIA STERRER* and WERNER RISAU

Max-Planck-Institut filr Psychiatric, Am Klopferspitz 18A, 8033 Maninsried, FRG

•Present address: Max-Planck-Institut ftlr Biophysikahsche Chemie, Am Fassberg, 3400 GOttingen, FRG

Summary

Vascular endothelial growth factor (VEGF) is a secretedangiogenic mitogen whose target cell specificity appearsto be restricted to vascular endothelial cells. Such factorsare likely candidates for regulatory molecules involvedin endothelial growth control. We have characterizedthe murine VEGF gene and have analysed its expressionpattern in embryogenesis, particularly during brainangiogenesis. Analysis of cDNA clones predicted theexistence of three molecular forms of VEGF which differin size due to heterogeneity at the carboxy terminus ofthe protein. The predicted mature proteins consist of120,164 or 188 amino acid residues. Homodimers of thetwo lower molecular weight forms, but not of the highermolecular weight form, were secreted by COS cellstransfected with the corresponding cDNAs and wereequally potent in stimulating the growth of endothelialcells. During brain development, VEGF transcript levelswere abundant in the ventricular neuroectoderm of

embryonic and postnatal brain when endothelial cellsproliferate rapidly but were reduced in the adult whenendothelial cell proliferation has ceased. The temporaland spatial expression of VEGF is consistent with thehypothesis that VEGF is synthesized and released by theventricular neuroectoderm and may induce the ingrowthof capillaries from the perineural vascular plexus. Inaddition to the transient expression during braindevelopment, a persistent expression of VEGF wasobserved in epithelial cells adjacent to fenestratedendothelium, e.g. in choroid plexus and in kidneyglomeruli. The data are consistent with a role of VEGFas a multifunctional regulator of endothelial cell growthand differentiation.

Key words: angiogenesis, brain, endothelial cells, vascularendothelial growth factor, vascular permeability factor.

Introduction

The growth of blood vessels during embryogenesis andin the adult organism is tightly controlled. Somedeveloping organs like brain or kidney are vascularizedby sprouts branching from preexisting blood vessels(Bar, 1980; Ekblom et al., 1982). This process, termedangiogenesis, is thought to be regulated by the action ofsoluble factors. Several polypeptide growth factorshave been identified based on their ability to stimulatethe proliferation of endothelial cells (For reviews seeFolkman and Klagsbrun, 1987; Risau, 1990; Klagsbrunand D'Amore, 1991). The acidic and basic fibroblastgrowth factors, aFGF and bFGF, are potent endothelialcell mitogens both in vitro and in vivo (For review seeKlagsbrun, 1989). They therefore seemed to be likelycandidates for general mediators of angiogenesis.However, several observations cast doubt on thisproposed role in embryonic angiogenesis; first, aFGFand bFGF lack a hydrophobic signal sequence neededfor secretion and the possible mode of release from theproducer cells is unknown; second, there is no good

correlation between the temporal and spatial ex-pression of aFGF and bFGF mRNA and blood vesselgrowth during brain angiogenesis (Emoto et al., 1989;Schnurch and Risau, 1991, and unpublished results);and third, there is no evidence that FGF receptors areexpressed by endothelial cells in vivo (Heuer et al.,1990; Wanaka et al., 1991).

Recently, two angiogenic factors have been charac-terized whose mitogenic activities appear to be restric-ted to endothelial cells, a platelet-derived endothelialcell growth factor (Pd-ECGF) and a vascular endo-thelial growth factor (VEGF). Pd-ECGF is an inducerof angiogenesis in vivo and has chemotactic activity forendothelial cells (Miyazonoet al., 1987; Ishikawa et al.,1989). The presence of the factor in human platelets hasled to the suggestion that it might have a role inmaintaining the integrity of blood vessels. However,Pd-ECGF also lacks a typical hydrophobic signalsequence for secretion and it is not known by whichmechanism this factor is released in vivo. In contrast,VEGF is a secreted endothelial cell mitogen (FerTaraand Henzel, 1989; Gospodarowicz et al., 1989; Levy et

522 G. Breier and others

al., 1989; Conn et al., 1990a). This factor has also beentermed vascular permeability factor (VPF) because ofits ability to induce vascular leakage in vivo (Senger etal., 1983; Keck et al., 1989). Other effects of VEGFinclude the induction of the procoagulant activity ofmonocytes and endothelial cells and the stimulation ofmonocyte migration in vitro (Clauss et al., 1990).VEGF is a dimeric glycoprotein composed of presum-ably identical disulfide-linked subunits and is structur-ally related to platelet-derived growth factor (Leung etal., 1989; Keck et al., 1989; Tischer et al., 1989; Connetal., 1990b). Analysis of human cDNA clones predictedthree different forms of the growth factor which differin size due to insertions at the carboxy terminus (Leunget al., 1989). The different coding regions arise byalternative splicing of mRNA (Tischer et al., 1991).VEGF probably exerts its action via cell surfacereceptors which have been identified on endothelialcells (Plouet and Moukadiri, 1990; Vaisman et al., 1990;Connolly et al., 1989).

The properties of VEGF as a secreted endothelial-cell-specific mitogen prompted us to investigate thisfactor in more detail. We have characterized the murineVEGF gene, and we have investigated the secretorybehaviour and the mitogenic activity of the variousVEGF forms. To investigate its possible function in anintact organism, we have analysed its expressionpattern during embryonic angiogenesis. The spatial andtemporal expression pattern of VEGF during braindevelopment correlated well with the ingrowth of bloodvessels into the developing neuroectoderm. In additionto the transient VEGF expression in ventricularneuroectoderm, a constitutive VEGF expression wasobserved in epithelial cells adjacent to fenestratedendothelium of choroid plexus and of kidney glomeruli.This constitutive expression suggests that VEGF mighthave a role in establishing or maintaining the fenest-ration of organotypic endothelium. The results supportthe hypothesis that VEGF might be a regulator ofneovascularization and of endothelial cell differen-tiation.

Materials and methods

Cell culture, transfection and bioassaysCOS-1 cells were cultured in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal calf serum(FCS). Cells were transfected using the modified calciumphosphate method (Chen and Okayama, 1987). Conditionedmedia were collected 48-72 hours after transfection. Bovineaortic endothelial (BAE) cells were prepared as described bySchwartz (1978) and grown in DMEM supplemented with 5%FCS. Assays for mitogenic activity were performed asdescribed (Risau et al., 1988). In short, BAE cells (passages 9-12) were seeded in 24-well dishes at a density of 10 000 cellsper well the day before addition of the COS cell-conditionedmedia. After three days, the cells were dissociated withtrypsin and counted in a Coulter counter.

Preparation of antisera and immunoblot analysisA synthetic oligopeptide comprising 20 amino-terminalresidues of the mature murine VEGF protein (see Fig. 1) was

synthesized on a peptide synthesizer (Applied Biosystems),coupled to keyhole limpet hemacyanin (KLH) and was usedto immunize rabbits. For the immunoblot analysis, theconditioned media from COS cells were concentrated byacetone precipitation and redissolved in buffer containing 2-mercapto-ethanol. Extracts were prepared by repeatedfreeze-thawing of the cell suspension. Samples were run in12.5% polyacrylamide-SDS gels and transferred to nitrocellu-lose membrane. The filters were incubated with polyclonalanti-VEGF antiserum and subsequently with peroxidase-conjugated goat anti-rabbit secondary antibodies (Dianova).Non-immune serum and serum raised against the amino-terminal residues of human VEGF which were used ascontrols did not detect the murine VEGF polypeptides.

DNA amplification, cloning and sequence analysisFor cDNA cloning, 5 fig of poly (A)+ RNA was reversetranscribed (Krug and Berger, 1987). 20% of the cDNA weresubjected to 35 rounds of amplification using a PCR reagentkit (Perkin-Elmer Cetus). Cycles were 1 minute at 94°C, 2minutes at 55°C, 3 minutes at 72CC in a temperature cycler(Coy). Synthetic oligonucleotide primers containing in ad-dition BamHl or EcoRI restriction enzyme recognition siteswere used. The nucleotide sequences of the primers were 5'-TGGATCCATGAACnTCTGCT (5' oligonucleotide) and5'-GAATTCACCGCCTCGGCTTGTC (3'oligonucleotide).Following digestion with restriction endonucleases, theamplification products were purified and cloned into pBlues-cript vector DNA (Stratagene) cut with the same restrictionenzymes. The resulting plasmids, pVEGF-1, pVEGF-2 andpVEGF-3, were sequenced using Sequenase (USB).

For the analysis of RNA samples, the cDNA synthesis andamplification was performed in a one tube reaction essentiallyas described by Zafra et al. (1990) with modificationsdescribed below. The specific primers used for reversetranscription were those used for cDNA cloning. Aliquots ofpoly (A+) RNA (100 ng) were used in a 50 /A reactioncontaining 100 ng each of oligonucleotide primers, 2 U humanplacental RNAse inhibitor (Pharmacia), 2 U AMV reversetranscriptase (Pharmacia), 2 mM dNTP, 1 U Amplitaq DNApolymerase and buffer supplied by the manufacturer (PerkinElmer Cetus). As an internal standard, 1 ng of RNA obtainedby in vitro transcription of a truncated VEGF cDNA cloneusing T7 RNA polymerase (Stratagene), was added to thereaction. This clone was obtained by deleting a 249 bprestriction fragment from pVEGF-2. After 30 minutes at40°C, 20 cycles of DNA amplification were carried out asdescribed above. Samples containing 20% of the productswere separated in an agarose gel, blotted onto GeneScreenPlus membrane and hybridized with ^P-labelled VEGF-1cDNA.

For expression in COS cells, VEGF cDNAs were clonedinto pCMV5 vector DNA (Andersson et al., 1989). PlasmidDNAs were purified on Qiagen columns (Diagen).

Isolation of RNA and northern blot hybridisationRNA was prepared using the guanidinium thiocyanatemethod (Chirgwin et al., 1979) and purified by centrifugationthrough a CsCl cushion. Poly (A)+ RNA was selected byretention on oligo(dT) columns. Aliquots of poly(A)+ RNAwere fractionated on 1% agarose gels containing 6%formaldehyde and blotted onto GeneScreen Plus membrane(DuPont). Filters were hybridized at 42°C in 50% formamide,5 x SSC (750 mM sodium chloride, 75 mM sodium citrate), 5x Denhardt's (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1%BSA), 1% SDS, 10 /ig/ml yeast tRNA and 10 ng random-primed [32P]DNA probe. Washes were twice in 2 x SSC at

VEGF expression in embryonic angiogenesis 523

room temperature, once in 2 x SSC, 1% SDS at 42°C andfinally twice in 0.1 x SSC at 42°C.

In situ hybridizationThe techniques used for in situ hybridization were essentiallythose described by Hogan et al. (1986) with modificationsdescribed by Dressier et al. (1990). Mouse embryos or organs(Balb/c) were embedded in Tissue-Tek medium (Miles) andfrozen in liquid nitrogen. Sections were cut in a cryostat at—20°C, dried on subbed slides and fixed in 4% paraformal-dehyde. Sections were digested with Pronase (40 fig/ml) for 10minutes at room temperature. The digestion was stopped in0.2% glycine. Slides were rinsed with PBS, fixed in 4%paraformaldehyde and rinsed again with PBS. Sections wereacetylated with acetic anhydride diluted 1:400 with 0.1 mMtriethanolamine for 10 minutes and rinsed with PBS.Prehybridization was performed in buffer containing 50%formamide, 10% dextran sulfate, 10 mM Tris-HCl pH 7.5,10mM sodium phosphate pH 6.8, 2 x SSC, 5 mM EDTA, 150/ig/ml yeast tRNA, 0.1 mM UTP, 1 mM ADP/3S, 1 mMADPyS, 10 mM DTT and 10 mM 2-mercapto-ethanol for 1hour at 48°C. Sections were dehydrated in graded ethanol andstored at -70°C.

Single-stranded (/"SJRNA probes were generated by invitro transcription of a truncated pVEGF-2 cDNA clone. Thisclone contained 310 bp of the 3' coding region of the VEGF-2cDNA. In one experiment, a cDNA clone that contained a140 bp sequence from the 5' coding region was used a control.Both probes detected the same pattern of transcript distri-bution. The RNA probes were generated using 100 /JCI[35S]UTP and T3 or T7 RNA polymerases essentially asdescribed by the manufacturer (Stratagene). RNA probeswere purified by ethanol precipitation in the presence ofammonium acetate, dissolved in 50% formamide, 10 mMDTT and stored at -20°C.

Hybridization was performed in prehybridization buffersupplemented with 5X104 cts/min//il S-labelled RNA probe.Sections were hybridized overnight in a humid chamber at 45-

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48°C. Depending on the size of the coverslip, approximately20 /J buffer/section was used. Slides were washed in 2 x SSC,50% formamide for 2 hours at 45-48°C followed by RNAsedigestion (20 f/g/ml) for 15 min. Slides were washed in 2 xSSC, 50% formamide overnight and dried at room tempera-ture. Following dehydration in graded ethanol, slides werecoated with Kodak NTB-2 emulsion diluted 1:1 with waterand exposed for 10-14 days. Slides were developed andstained with Giemsa.

Results

Isolation and characterization of murine VEGFcDNAsMurine cDNA clones encoding VEGF sequences wereobtained by using the polymerase chain reaction (PCR)technique after reverse transcription of poly (A)+RNA from 14-day post-coital (p.c.) mouse embryos.The nucleotide sequence of the primers for theamplification reaction was derived from the humancDNA sequence (Leung et al., 1989). The codons forthe translational start and stop signals were included inorder to obtain the complete coding region. Threedifferent products were obtained, a 580 bp VEGF-1, a450 bp VEGF-2 and a 650 bp VEGF-3 cDNA (data notshown). In contrast to the VEGF-1 and VEGF-2 PCRproducts, the VEGF-3 cDNA was of very low abun-dance. DNA sequencing of all three cDNAs revealed asequence homologous to human, bovine and rat VEGF(Leung et al., 1989; Conn et al., 1990b). They wereidentical in sequence to one another except thatinsertions were present in the 3' region of the VEGF-1and VEGF-3 cDNAs (Fig. 1). Thus, the cDNAs predictthe existence of three different VEGF forms, which,after removal of a 26-residue amino-terminal hydro-

Fig. 1. DNA sequence andpredicted translation products ofmurine VEGF cDNAs. Thelocations of the oligonucleotideprimers that were used for thePCR are overlined. The putativesignal sequence and a potentialglycosylation site at Asn 74 areunderlined. The completesequence of the VEGF-3 cDNAis given. Boxed regions representthe sequences which are absentfrom the shorter VEGF-1 orVEGF-2 forms (see Fig. 2).VEGF-1 lacks a stretch of 24amino acid residues fromposition 115 to 138, VEGF-2lacks additional 44 residues up toposition 183. In both cases, Lys114 is replaced by Asn. ThecDNA sequence is shown incapital letters. Nucleotidesequences presented in lowercase were obtained from agenomic DNA clone (G. Breier,unpublished results) and differ atthe indicated positions from thesequence of the 5' PCR primer.

524 G. Breier and others

VEGF 2

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Fig. 2. Structure of murine VEGF proteins as deducedfrom cDNA sequences. The cross-hatched box at theamino terminus represents the putative 26-amino acidsignal peptide for secretion. Additional domains in VEGF-1 or VEGF-3 are represented as shaded boxes. Thenumbers of amino acids refer to the length of the matureproteins.

phobic signal sequence, would consist of 120 (VEGF-2), 164 (VEGF-1) or 188 (VEGF-3) amino acid residues(Fig. 2). The direct protein sequence analysis of theamino-terminal end of the putative murine VEGF(Plouet et al., 1989) is in agreement with the sequenceobtained from the cDNA clones. However, the aminoacid sequence differs at position 10 which might be dueto strain polymorphism. A potential N-linked glycosyl-ation site is present at asparagine 74. The insertedprotein domain in the long VEGF form (VEGF-3) ishighly basic, 11 of 24 amino acid residues being lysine orarginine. This region is homologous to a stretch ofamino acids encoded by the sixth exon of the humanPDGF A-chain gene (Betsholtz et al., 1990).Interestingly, this exon is present only in an alterna-tively spliced mRNA variant and encodes a nucleartargeting signal (Maher et al., 1989). The homologybetween VEGF and the PDGF A- and B-chains extendsto eight cysteine residues, suggesting a similar second-ary structure of the proteins (Fig. 3; Leung et al.,1989). VEGF proteins of different mammalian speciesare highly homologous (Fig. 3). The mature murine

VEGF-1 protein showed the highest degree of sequenceidentity (98%) with the rat VEGF protein. The aminoacid sequence identities between the mature murineVEGF-1 and the corresponding human and bovineproteins were 88% each. The amino-terminal se-quences of the mature proteins showed a considerabledegree of sequence diversity.

Secretion and mitogenic activity of recombinantmurine VEGFThe VEGF cDNAs were cloned into a mammalianexpression vector which contained the transcriptionalcontrol elements of the human cytomegalovirus. Theresulting plasmids were then transfected into COS cells.Culture supernatants and extracts prepared from thetransfected cells were assayed by immunoblot analysisfor the presence of VEGF protein. The polyclonalantibodies used had been raised against a syntheticamino- terminal 20-mer oligopeptide. Homodimers ofapparent molecular weights of 20 x 103 Mr or 23 x 103

MT were detected in the conditioned media, corre-sponding to VEGF-1 or to VEGF-2 polypeptide chains,respectively (Fig. 4). In contrast, the VEGF-3 chainswere retained in the cells. Two major bands corre-sponding in size to 27 x 103 MT and 24 x 103 Mt, weredetected in the extract, but not in the conditionedmedium of COS cells transfected with VEGF-3 cDNA.This size heterogeneity possibly reflects differences inposttranslational processing or proteolytic degradation.The cell culture supernatants were then assayed formitogenic activity on bovine aortic endothelial (BAE)cells. The conditioned media which contained homo-dimers of VEGF-1 or VEGF-2 stimulated the prolifer-ation of BAE cells to a similar extent (Fig. 5). Mediafrom cells transfected with VEGF-3 cDNA had nosignificant mitogenic effect. Whether VEGF-3 proteincan be as active as VEGF-1 or VEGF-2 in stimulatingthe proliferation of endothelial cells remains to bedetermined.

Expression of VEGF during murine developmentThe source of the VEGF cDNAs was RNA from 14-day

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Fig. 3. Comparison of VEGFprotein sequences from mouse,human, bovine and rat. Onlythe intermediate VEGF form(=VEGF-1) was used for thecomparison because thesequences of the small andlarge forms have not beenpublished for all species. Theone-letter amino acid code isused. The complete sequenceof the precursors are shown,the amino-terminal alanine ofthe mature proteins isindicated by +1. Asterisks

indicate the location of conserved cysteine residues that are also shared by the PDGF-A and PDGF-B chains. The humanand bovine sequences were taken from Leung et al. (1989), the rat sequence is from Conn et al. (1990b). It should benoted that the five carboxy-terminal amino acids of the murine VEGF were derived from a PCR primer containing thehuman sequence and do not necessarily represent the actual mouse sequence. The amino-terminal sequence of the signalpeptide has been confirmed by a partial sequence of a genomic DNA clone (see Fig. 1).

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VEGF expression in embryonic angiogenesis 525

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-3 COS cells were transfected witheucaryotic expression vectorsthat contained the differentVEGF cDNAs. 5% of theprotein present in culturesupernatants as compared to60% of the protein of extracts,obtained from cells of two petridishes (diameter 35 mm), wereused for the analysis. The blotwas stained using a polyclonalserum raised against a syntheticpeptide of the VEGF aminoterminus. Lane 1=VEGF-1, lane2=VEGF-2, lane 3=VEGF-3,lane 4=untransfected COS cells.

p.c. mouse embryos. This already indicated that theVEGF gene is transcriptionally active during embryo-genesis. Therefore, we performed northern blot ana-lyses with RNA from 14-day p.c. and 16-day p.c.embryos. RNA from adult brain was also examined.The blots were hybridized with a 32P-labelled VEGF-1cDNA. Several transcripts ranging in size between 2.7kb and 4 kb were detected in RNA from mouseembryos and from adult brain (Fig. 6). However, thistype of analysis did not distinguish between the mRNAscoding for the different VEGF forms. In order tocompare the relative abundance of these transcripts, weperformed a semiquantitative PCR analysis. Firstly,single-stranded cDNA was generated by using VEGF-specific primers. PCR was carried out under conditions

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Fig. 5. Mitogenic activity of recombinant VEGF. Bovineaortic endothelial cells were cultured in the presence orabsence of cell culture supernatants from transfected COScells (see Fig. 4). DMEM supplemented with 10% FCSand the conditioned media from untransfected COS cells orfrom COS cells that were transfected with the vector alonewere used as a negative control. After three days, the cellswere dissociated with trypsin and counted in a cytometer.Values are means ± s.e.m. of double determinations.

that did not limit the DNA synthesis. The productswere separated by agarose gel electrophoresis andanalysed by Southern blot hybridization using 32P-labelled VEGF-1 cDNA as a probe. The 580 bp VEGF-1 cDNA and the 450 bp VEGF-2 cDNA were detectedboth in 16-day p.c. mouse embryos and in adult brain(Fig. 6). In 16-day p.c. embryos, VEGF-2 transcriptswere slightly more abundant than VEGF-1 mRNA,while in the adult brain the VEGF-1 mRNA was of thehighest abundance. In both RNA samples, VEGF-3transcripts were not detectable.

The spatial distribution of VEGF transcripts in 15-day p.c. and 17-day p.c. embryos was investigated by insitu hybridization. 6-day postnatal brain, adult brainand adult kidney were also examined. Frozen tissuesections were hybridized with single-stranded 35S-labelled RNA probes corresponding to the sense orantisense strand of a VEGF cDNA fragment. Thisprobe contained sequences common to all three VEGFforms. In addition, a probe derived from the murinehomeobox gene Hox 3.1 (Breier et al., 1988), which isalso expressed during embryogenesis, was used as apositive control. VEGF transcripts were detected invarious organs of developing embryos. In the brain of17-day p.c. embryos, VEGF transcripts were observedin choroid plexus epithelium and in the ventricular layer(Fig. 7). The signal in the adjacent neural tissue wassignificantly lower. The pattern of VEGF transcriptdistribution in the ventricular layer of 6-day postnatalbrain was similar to the pattern in embryonic brain (Figs7, 8). In adult brain, the VEGF mRNA levels in thechoroid plexus appeared slightly reduced. No specificlabelling was observed in the ependyme, which formsthe inner lining of the ventricle (Fig. 7). In the newbornand adult brain, VEGF transcripts were also detected inother brain regions in which single, unidentified cellswere labelled. VEGF transcripts were also observed inthe meningeal layer and in the area postrema of adultbrain.

In the developing kidney of 15-day p.c. embryos,VEGF transcripts accumulated in glomerular epi-

526 G. Breier and others

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Fig. 6. (A) Northern blothybridization of VEGFtranscripts in embryonic andadult tissues. Samples of polyA + RNA (5 fig) from 14-dayp.c. and 16-day p.c. mouseembryos and from adult brainwere hybridized with ^P-labelledVEGF-1 cDNA andsubsequently with /3-actin cDNA.Exposure times were 6 days forthe VEGF probe and 1 day forthe /3-actin probe. (B)Semiquantitative PCR analysis ofVEGF transcripts. cDNA wassynthesized from poly (A)+RNA samples (100 ng) fromembryonic and adult tissuesusing specific oligonucleotideprimers. VEGF sequences wereamplified by 20 cycles of PCRand analyzed by Southern blothybridization with VEGF-1cDNA probe. In vitrosynthesized transcripts from atruncated VEGF cDNA wereincluded in the reaction to serveas an internal standard andresulted in the synthesis of a 200bp cDNA fragment. Exposuretime was 16 h.

thelium (Fig. 9). This expression persisted duringdevelopment and was still high in the adult glomerulus.No specific labelling was observed in blood vessels andin mesangium in the glomerulus. Glomeralar capillariesare derived from outside blood vessels (Ekblom et al.,1982; Sariola et al., 1983) and are of the fenestratedtype (Rhodin, 1962). Thus, VEGF is expressed at thetime of embryonic kidney angiogenesis and continues tobe expressed in glomerular epithelium which is adjacentto glomerular fenestrated endothelium. Strong ex-pression of VEGF was observed in the 17-day p.c.placenta (Fig. 10) and was most prominent in thelabyrinth region of the placenta. VEGF transcriptswere localized over clusters of cell nuclei in thelabyrinth layer, most likely of the multinucleatedtrophoblast cells (Hogan et al., 1986; Theiler, 1972). Inthe labyrinth, an intimate contact between the embry-onic trophoblast and maternal blood space is formed. Inthis region, fetal blood vessels develop which areseparated by trophoblast cell layers from the maternalblood sinuses (Bjorkman et al., 1989; Kirby andBradbury, 1965). Endothelial cells of the labyrinth arealso characterized by frequent fenestrations (Jollie,1964). In several other embryonal organs VEGFtranscripts were also detected, e.g. in bronchial epi-thelium of the lung, in adrenal gland and in seminifer-ous tubules of testis (data not shown).

Discussion

VEGF has important properties that are characteristic

of a potential regulator of embryonic angiogenesis andof endothelial cell functions. It is a secreted growthfactor whose mitogenic activity is apparently restrictedto vascular endothelial cells. In addition, it may inducevascular permeability and is able to attract monocytes.As a step towards the elucidation of its physiologicalfunction in the developing organism, we have clonedthe murine VEGF gene and analysed its expressionpattern during mouse embryogenesis, particularly dur-ing brain angiogenesis.

Analysis of the cDNA clones obtained from 14-dayp.c. mouse embryos predicted the existence of threedifferent VEGF polypeptides. These differed by theinsertion of one or two protein domains near thecarboxy terminus. Similar forms occur in bovine andhuman (Leung et al., 1989; Tischer et al., 1989), and thevarious human VEGF coding regions result fromalternative splicing of mRNA (Tischer et al., 1991). It isprobable, based upon Southern blot analysis of gen-omic DNA, that the different murine VEGF transcriptsare also derived from a single gene. We are presentlycharacterizing genomic DNA clones in order to deter-mine the structure of the murine VEGF gene. The threeVEGF forms differed with respect to their secretorybehaviour. Homodimers of the two lower molecularweight forms (VEGF-1 and VEGF-2), but not of thelarge form (VEGF-3), were secreted after transfectionof the corresponding cDNAs into COS cells. Theretention of VEGF-3 in these cells was due to theinsertion of a highly basic protein domain. Homologousdomains are present in the PDGF B-chain and in avariant form of the PDGF A-chain (Betsholtz et al.,

VEGF expression in embryonic angiogenesis 527

Fig. 7. Localisation of VEGF mRNA in embryonic, 6-day postnatal and adult brain. Serial brain sections were hybridizedin situ with 35S-labelled RNA probe. (A) Sagittal section of a 17-day p.c. embryo hybridized with antisense RNA probe.The image shows choroid plexus (CP) and ventricular layer (VL) of the third ventricle (V); (B) dark-field image of A; (C)control hybridization of a serial section with sense RNA probe. (D) Sagittal section of 6-day postnatal brain hybridizedwith antisense RNA probe, showing the choroid plexus (CP) and ventricular layer (VL) of the third ventricle (V); (E)dark-field image of D; (F) control hybridization with sense RNA. (G) Parasagittal section of adult brain hybridized withantisense RNA probe. The choroid plexus (CP) and ependyme (E) of a lateral ventricle (V) is shown; (H) dark-field imageof G; (I) control hybridization with sense RNA probe. Bar represents 25 /Mn.

1990) and directed the protein to the nucleus if thesignal sequences for secretion were deleted (Lee et al.,1987; Maher et al., 1989). In good correlation with ourresults, these basic domains function as a cell retentionsignal in COS cells (Ostmann et al., 1991). Thus, theseconserved domains of PDGF and VEGF are not onlystructurally, but also functionally similar. At present,we cannot rule out the possibility that the lack ofsecretion of VEGF-3 by COS cells is due to restrictionsin the secretory pathways of these cells and that thenatural producer cells are able to secrete this form.Alternatively, VEGF-3 might be integrated into theplasma membrane and might be involved in local cell-cell interactions. It is possible that VEGF-3 wouldinteract only with closely adjacent cell layers, whereasthe secreted forms, VEGF-1 and VEGF-2, mightfunction as diffusible factors. As a precedent, it has

been shown that the membrane-bound TGF-<* precur-sor is able to bind to its receptor on adjacent cells andmay lead to signal transduction (Wong et al., 1989).Although transcript levels specific for VEGF-3 werevery low in embryos, preliminary analyses revealedelevated transcript levels in adult liver, lung and heart,suggesting that the large VEGF form might have aspecific function in these organs. It will therefore beimportant to determine whether the structural diversityof three VEGF forms reflects a heterogeneity infunction. The only direct effect of VEGF on endothelialcells described so far is its mitogenic activity. Atpresent, one has to consider the possibility that themitogenic activity of VEGF is mediated by an indirectmechanism that involves the production of a differentendothelial mitogen by endothelial cells. The twosecreted VEGF forms, VEGF-1 and VEGF-2, were

528 G. Breier and others

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Fig. 8. Detection of VEGFmRNA in the ventricular layerof 6-day postnatal brain. (A)The ventricular layer (VL)lining the third ventricle (V)shows VEGF mRNAaccumulation, whereas specificlabeling in the adjacentneuroectoderm is significantlylower; (B) dark-field image ofA. (C) Higher magnification ofthe ventricular layer. Theventricle is at the bottom; (D)control hybridization of aserial section with a senseRNA probe. Bar represents 25jan.

equally potent in stimulating the proliferation ofendothelial cells, while a mitogenic activity has notbeen demonstrated yet for the long variant. It will alsobe of interest to analyse whether the various VEGFforms differ in other functions, such as the induction ofvascular permeability or the ability to attract mono-cytes.

The complex expression pattern of VEGF that wasobserved in mouse embryos argues in favour of amultifunctional role of the growth factor. The temporalpattern of VEGF expression in the ventricular neuro-epithelium of the brain suggests a role of VEGF as anangiogenesis factor. The vascular system of the brain isderived from the perineural vascular plexus that coversthe neural tube (Bar, 1980). Vascular sprouts originat-

ing from this perineural plexus grow radially into theneuroectoderm, toward the ventricular layer, andbranch there. In rodents, the neovascularization of thedeveloping brain starts approximately at embryonic day11 and maximal endothelial cell proliferation is ob-served at postnatal day 6 (Robertson et al., 1985). Inthe adult organism, the vascularization of the brain, aswell as of other organs, is complete and the rate ofendothelial cells proliferation is very low (Engerman etal., 1967). Thus, the temporal and spatial expression ofVEGF in the developing brain correlates with brainvascularization and endothelial cell growth. It istherefore conceivable that VEGF is released by cells ofthe ventricular layer during brain development andpromotes angiogenesis by initiating an angiogenic

VEGF expression in embryonic angiogenesis 529

Fig. 9. Localisation of VEGF transcripts in the developing and adult kidney. (A) Parasagittal section of a 15-day p.c.embryo, hybridized with antisense RNA probe. Arrows point to glomerular epithelium; (B) dark-field image of A. (C)higher magnification of glomeruli; D control hybridization with VEGF sense RNA. (E) Section of adult kidney, hybridizedwith antisense RNA probe; (F) dark-field image of E. Bar represents 50 /im.

response of endothelial cells from the perineuralvascular plexus. A concentration gradient of thediffusible angiogenic factor may cause capillaries togrow in direction to the angiogenic stimulus. Thehypothesis that VEGF may function as an angiogenicgrowth factor for fetal blood vessels, is furthersubstantiated by the high levels of VEGF mRNA in the

early embryonic kidney, in the highly vascularizedplacenta, and by the correlation between VEGFexpression and vascularization in rat corpus luteum(Phillips et al., 1990).

In contrast to its transient expression in the ventricu-lar layer of the brain, VEGF expression in kidneyglomeruli and in choroid plexus of the brain persisted in

530 G. Breier and others

Fig. 10. Expression of VEGF in 17-day p.c. placenta. (A) In situ hybridization showing VEGF transcript accumulation inthe labyrinth layer (La) whereas no specific labeling is observed in the adjacent chorionic plate; (B) dark-field image of A.(C) Higher magnification of VEGF expressing cells in the labyrinth layer. (D) control hybridization. Bar represents 25 /an.

the adult, when the vascularization of these structures iscomplete. This indicates that in the adult organism,VEGF has an additional function which is differentfrom a role as mediator of neovascularization. As theexpression of VEGF in these structures was confined toepithelial cells that are in close contact to fenestratedendothelium, it is tempting to speculate that VEGFmay be involved in the establishment or the mainten-ance of fenestrated endothelia. We are currentlyinvestigating whether VEGF has a direct role in theinduction of fenestrae in cultured endothelial cells. Asthe endothelium of the placental labyrinth is alsofenestrated, VEGF might have a dual role in this organ,as an endothelial cell growth factor, but also as a factoraffecting the differentiation of endothelial cells. Thefenestration of endothelium reflects an increased per-meability of endothelial cells for low molecular weightsubstances (Levick and Smaje, 1987), thus facilitatingthe exchange of these substances in structures such askidney glomeruli, choroid plexus and placenta. This isconsistent with the permeability-inducing properties ofVEGF in vivo (Keck et al., 1989), although it is notknown whether this was a direct effect of VEGF on

endothelial cells or whether other factors contributed tothe observed vascular permeability.

Several lines of evidence, including protein secretion,an apparently restricted target cell specificity andregion-specific expression, suggest that VEGF mayfunction as a regulator of embryonic angiogenesis andof endothelial cell differentiation. A complete under-standing of its functions in endothelial cell growth anddifferentiation, however, will require a systematicdissection of its properties in in vitro systems, as well asgenetic evidence based on induced mutations or alteredembryonic expression patterns.

We wish to thank Hannes Drexler, Catherine Farrell,Vladimir Mironov and Harald Schniirch for various sugges-tions and comments on the manuscript, Regine Giessler fortechnical assistance and Monika SchOrnig for obtaining mouseembryos. This work was supported in part by the Bundesmi-nisterium fur Forschung und Technologie and by ProgenBiotechnik.

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{Accepted 18 November 1991)