Establishment of the cardiovascular system is one of theearliest events in the developing embryo (Sabin, 1917; Sabin,1920). The organization of emerging endothelial cells into avascular tree that will eventually mature into the adult cardio-vascular system is the end result of a series of interactionsbetween cells and their environment. It is a process mediated,at least in part, by a series of cell-cell and cell-matrix adhesiveevents involving specific receptors. In order to understand themolecular basis for the establishment of the cardiovascular
system it is necessary to define the adhesion moleculesmediating these interactions. This has been difficult inmammalian systems due to problems in identifying presump-tive endothelial precursors as they arise from the mesoderm.Early endothelial cell identification has been facilitated, inavian systems, by the production of monoclonal antibodies thatreact with presumptive endothelial cells as they appear in thedeveloping blastodisc (Pardanaud et al., 1987; Coffin et al.,1991b; Noden, 1991). A descriptive account of vascular devel-opment in birds (Pardanaud et al., 1989; Poole et al., 1989),using the monoclonal antibody QH1, suggests that cardiovas-
The establishment of the cardiovascular system representsan early, critical event essential for normal embryonicdevelopment. An important component of vascularontogeny is the differentiation and development of theendothelial and endocardial cell populations. This involves,at least in part, the expression and function of specific cellsurface receptors required to mediate cell-cell and cell-matrix adhesion. Platelet endothelial cell adhesionmolecule-1 (PECAM-1, CD31) may well serve such afunction. It is a member of the immunoglobulin superfam-ily expressed by the entire vascular endothelium in theadult. It is capable of mediating adhesion by a heterophilicmechanism requiring glycosaminoglycans, as well as by ahomophilic, glycosaminoglycan independent, mechanism.It has been shown to regulate the expression of otheradhesion molecules on naive T cells. This report documentsby RT-PCR and immunohistochemical analysis theexpression of PECAM-1 during early post implantationmouse embryo development. PECAM-1 was expressed byearly endothelial precursors first within the yolk sac andsubsequently within the embryo itself. Interestingly,
embryonic PECAM-1 was expressed as multiple isoformsin which one or more clusters of polypeptides were missingfrom the cytoplasmic domain. The sequence and locationof the deleted polypeptides corresponded to exons found inthe human PECAM-1 gene. The alternatively splicedisoforms were capable of mediating cell-cell adhesion whentransfected into L-cells. The isoforms differed, however, intheir sensitivity to a panel of anti-PECAM-1 monoclonalantibodies. These data suggest that changes in the cyto-plasmic domain of PECAM-1 may affect its functionduring cardiovascular development, and are consistentwith our earlier report that systematic truncation of thecytoplasmic domain of human PECAM-1 resulted inchanges in its ligand specificity, divalent cation and gly-cosaminoglycan dependence, as well as its susceptibility toadhesion blocking monoclonal antibodies. This is the firstreport of naturally occurring alternatively spliced forms ofPECAM-1 having possible functional implications.
spliced, functionally distinct isoforms expressed during mammalian
cardiovascular development
H. Scott Baldwin1,2,*, Hong Min Shen2, Horng-Chin Yan2,3, Horace M. DeLisser3, Audrey Chung1,2,Craig Mickanin1, Timothy Trask1, Nancy E. Kirschbaum4, Peter J. Newman4, Steven M. Albelda2,3
and Clayton A. Buck1,2
1Cardiology Division, Department of Pediatrics, Children’s Hospital of Philadelphia, 34th Street and Civic Centre Boulevard,Philadelphia, PA 19104, USA2The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104, USA3Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, PA19104, USA4Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI 53226, USA
*Author for correspondence at address1
2540
cular development commences in the embryo proper shortlyafter gastrulation, at about the head fold stage, as individualpresumptive endothelial cells arise from the mesoderm. Thesecells soon connect into a network that matures into majorvessels of the adult as well as the endocardium of the heart.Secondary vessels and capillary beds appear to originate fromthe larger vessels by angiogenesis. The molecular interactionsdetermining the pattern of vascular formation in this systemremain unknown.
A similar analysis of cardiovascular development in themammalian embryo has been reported using lectins and anti-factor VIII antibodies to identify endothelial cells (Coffin etal., 1991a). In general, the overview of vascular developmentprovided by these studies shows a similar pattern to that seenin birds. However, the reagents used were specific for carbo-hydrate residues present on mature endothelial cells orproducts synthesized by endothelial cells after they havebecome part of an established vascular system. Therefore,certain endothelial cell populations, such as the endocardium,were not well delineated until later stages of development.
In order to evaluate early vascular development in themammalian embryo, we initiated a search for an endothelial-specific cell adhesion molecule. We have, therefore, examinedthe possibility that platelet endothelial cell adhesion molecule(PECAM)-1 might be such a molecule. PECAM-1 was chosenbecause it is expressed by all endothelial cells in the adult(Albelda et al., 1990; Newman et al., 1990; Muller et al., 1989).It promotes cell-cell adhesion between cultured endothelialcells (Albelda et al., 1990), and between mouse L-cells trans-fected with PECAM-1 cDNA (Albelda et al., 1991; Muller etal., 1992). It is required for the movement of neutrophils andmacrophages across the vascular endothelium (Bogen et al.,1992, 1994; Muller et al., 1993; Vaporciyan et al., 1993).Finally, ligand binding or PECAM-1 clustering results in theup-regulation of integrins in lymphocytes (Tanaka et al., 1992;Piali et al., 1993) suggesting that PECAM-1 engagement canregulate the expression of other adhesion receptors. It can alsomediate adhesion by several alternative mechanisms (DeLisseret al., 1993), potentially expanding the repertoire of molecularinteractions available to endothelial cells.
We report the results of an extensive analysis of PECAM-1expression in the developing mouse embryo. The data clearlydemonstrate that PECAM-1 is one of the earliest adhesionmolecules expressed by presumptive endothelial cells. It isexpressed as multiple isoforms that appear to be the result ofalternative splicing of exons encoding portions of the cyto-plasmic domain. These isoforms differ in their sensitivity to apanel of monoclonal antibodies that block PECAM-1-mediatedcell-cell aggregation, providing evidence for functional differ-ences among naturally occurring isoforms, and supporting ourprevious speculation that the function of PECAM-1 may beregulated through a mechanism that involves modifications ofthe cytoplasmic domain (DeLisser et al., 1994).
MATERIALS AND METHODS
Cloning and sequencing of mouse PECAM-1A mouse heart cDNA library (Stratagene no. 936306, La Jolla, CA)was screened at high stringency with the full-length human PECAM-1 cDNA (Newman, 1990). A 3.2 kb fragment cloned into the
EcoRI
restriction site of the Bluescript SK II+ plasmid was identified andrescued from the
λZapII vector according to the manufacturer’s direc-tions. This clone was sequenced at least twice in both the forward (5′)and reverse (3′) direction by the didioxy-chain termination methodusing Sequenase DNA polymerase (United States Biochemical Co.,Cleveland, OH). Assembly of multiple sequence contigs, as well asnucleic acid and deduced peptide comparisons, were accomplishedusing Version 7 of the Sequence Analysis software Package byGenetics Computer, Inc (Madison, WI).
Reverse transcription polymerase chain reaction (RT-PCR)Total RNA was extracted from staged CD1 mouse embryos by a mod-ification of the method of Chomcsynski and Sacchi (1987) andPoly(A)+ mRNA was isolated using an oligo-dT spin column (MicroFast Tract, Invitrogen). Complementary cDNA was obtained byreverse transcription using a mixture of random and oligo-dT primers,mRNA, and AMV Reverse Transcriptase (cDNA Cycle Kit, Invitro-gen). PCR reactions were performed in a Perkin-Elmer-Cetusautomatic thermal cycler. Each 100 µl reaction mixture included 10mM Tris-HCl, pH 8.0, 1.5 mM MgCl2, 50 mM KCl, 0.1% gelatin,10% DMSO, 0.06% 2-mercaptoethanol, 200 µM each dNTP and 2.5units of Taq DNA polymerase (Perkin-Elmer-Cetus). Reactions werecycled (25 cycles for the extracellular domain primers and 35 cyclesfor the cytoplasmic domain primers) through a program that includedincubations at 94°C for 1 minute, 56°C for 1 minute and 72°C for 2minutes in the presence of 200 ng of each primer. For the nested PCRreactions, the initial (outside) reaction mixtures were filtered throughan Ultrafree-MC Polysufone filter (Millipore), redissolved in 20 µl ofTE, and 5 µl of this solution was used as a source of template for thesecond (inside) reaction under the conditions described above.Primers used to amplify a portion of the cDNA corresponding to theextracellular domain are delineated in Fig. 3. Primers designed toamplify the cDNA coding for a portion of the cytoplasmic domain ofmurine PECAM-1 were: 5′ primer = ccagctgctccacttctgaa and the 3′primer = gcactgccttgactgtctta. The cytoplasmic primers were selectedwith the use of the Oligo 4.0 Primer Analysis Software (National Bio-sciences) to minimize false priming and maximize primer efficiency.Reaction products utilizing the extracellular primer set were resolvedon a 0.8% agarose gel. PCR products utilizing the cytoplasmic primermixture were resolved on a 4% Nusieve GTG agarose gel (FMC Bio-products) and visualized with ethidium bromide.
Agarose gels were transferred to nitrocellulose for Southernblotting according to standard protocols (Ausubel et al., 1993). In thecase of the extracellular amplifications, 32P-labeled probe wasprepared by random priming. For the cytoplasmic amplifications, endlabeled oligonucleotide cDNA probe corresponding to exon 13 wasprepared using γ32P[ATP] and T4 polynucleotide kinase (GIBCO-BRL).
Cloning of alternatively spliced forms of PECAM-1To PCR amplify the cytoplasmic domains of all possible muPECAM-1 isoforms from the reverse transcribed cDNA, the following primerswere used: sense primer (5′-1395TATGAAAGCAAAGAGTGA1412-3′), flanking the BsteII restriction site within the extracellularimmunoglobulin-like loop 5 and antisense primer (5′-CGAATGC2253ATCCAGGAATCGGCTGCTCT-TC2235-3′), com-plementary to a region 70 bps downstream of the stop codon ofmuPECAM-1, and carrying a 5′ mutagenic NsiI recognition sequence.The PCR product was digested with BsteII and NsiI and ligated intomuPECAM-1∆12,15 in the pcDNAI/Neo vector (Invitrogen). Theresulting ligation mixture was then used to transform competent cells.DNA isolated from antibiotic resistant colonies was initially screenedfor endonuclease sensitivity. Selected clones were then characterizedby PCR analysis using the following primer pair: sense primer, 5′-1852CCAAGGCCAAACAGA1866-3′, representing a region ofmuPECAM-1 homologous to exon 10 and antisense primer, 5′-
H. S. Baldwin and others
2541PECAM-1 expression during cardiovascular development
2172AAGGGAGC-CTTCCGTTCT2157-3′, representing sequenceshomologous to exon 16 of the huPECAM-1 gene (Kirschbaum et al.,1994; Kirschbaum and Newman, 1993). DNA from colonies carryingisoforms of muPECAM-1 was sequenced using two different primerpairs in two different orientations: (sense primers, 5′-1852CCAAGGC-CAAACAGA1866-3′ and 5′-1572AAGTTTTACAAAGAAAAGGAG-GAC1593-3′; antisense primers, 5′-2172AAGGGAGCCTTC-CGTTCT2157-3′ and 5′-CGAATGC-2253ATCCAGGAATCGGCTG-CTCTTC2235-3′).
Transfection of L-cells with muPECAM-1 isoformsTo obtain L-cell transfectants expressing high levels of protein, themuPECAM-1 cDNAs that had been subcloned into the pcDNAI/Neo(Invitrogen, San Diego, CA) expression vector were transfected intoL-cells using calcium phosphate precipitation as previously described(Albelda et al., 1991) and muPECAM-1-expressing clones selectedusing G418.
Aggregation of L-cell transfectantsThe aggregation assay used in these studies has been described indetail previously (DeLisser et al., 1993). Briefly, stable L-cell trans-fectants, which had been plated (8-10×106 cells/75-cm2 flask) andgrown overnight, were non-enzymatically suspended. The cells werewashed twice with 10 mM EDTA in PBS, pH 7.2, and twice withHBSS without divalent cations. Cells were finally resuspended to aconcentration of 8×105/ml in HBSS with or without 1 mM calcium.Antibodies were added to a final concentration of 50 µg/ml. One mlaliquots of suspended cells were transferred to wells in a 24-well non-tissue culture plastic tray (Costar Corp., Cambridge, MA) that hadbeen previously incubated with 2% BSA in HBSS for at least 1 hourand washed thoroughly with HBSS immediately before use to preventnonspecific binding to the tissue culture dish. The non-tissue culturetrays containing the suspended L-cells were rotated on a gyratoryplatform (100 rpm) at 37°C for 30 minutes.
Aggregation was quantified by examining representative aliquotsfrom each sample on a hemocytometer grid using phase contrastoptics. The number of single cells or cells in aggregates of 3 or lessversus those present in aggregates of greater than three cells werecounted from four 1 mm squares. At least 400 cells were counted fromeach sample. Data were expressed as the percentage of the total cellspresent in aggregates.
Fluorescence activated cell sorting (FACS) analysis L-cells transfected with full-length muPECAM-1, muPECAM-1∆12,15 or muPECAM-1∆14,15 cDNAs were non-enzymaticallyremoved from T25 flasks, washed in medium containing 10% FBS,and treated with various anti-muPECAM-1 monoclonal antibodies for1 hour at 4°C. The primary antibody was then removed, the cellswashed twice with ice-cold PBS, and a 1:200 dilution of FITC-labeledgoat anti-rat secondary antibody (Cappell) added for 30 minutes at4°C. After washing in cold PBS, flow cytometry was performed usingan Ortho Cytofluorograph 50H cell sorter equipped with a 2150 datahandling system (Ortho Instruments, Westwood MA).
Anti-muPECAM-1 monoclonal antibodiesThe following anti-muPECAM-1 monoclonal antibodies were usedfor immunohistochemical staining, FACS analysis, and to block theaggregation of transfected L-cells: mAb Mec13.3, provided by DrElizabetta Dejana (Institute Marion Negri, Milan), mAb EA3 (Piali etal., 1993) provided by Drs Luca Piali and Beat Imhof, and mAb 390,a monoclonal antibody generated in the rat following immunizationwith a mouse 32D leukocyte cell line and screened againstmuPECAM-1∆12,15 (Edelman et al., unpublished data). Each ofthese antibodies immunoprecipitated the characteristic 120-130×10−3 protein from L-cells transfected with muPECAM-1∆12,15 andselectively stained murine vessels in acetone-fixed tissue sections.
Tissue preparation Timed, pregnant CD1 mice were purchased from Harlan SpragueDawley, Indianapolis, IN. The day of the appearance of the vaginalplug was considered day 0. Embryos of various gestational ages wereremoved by Cesarean section, dissected free of the desidual mass andstaged according to the method of Kaufman (1992). Embryos used forwhole-mount in situ hybridization were fixed in 4% paraformalde-hyde, washed with phosphate buffer containing Tween-20, dehy-drated stepwise in methanol, and stored at −20°C in 70% methanoluntil used. Embryos used for cross sectional immunohistochemistrywere processed through a sucrose gradient in phosphate-bufferedsaline, embedded in OCT, and frozen in liquid nitrogen-cooledisopentane.
Whole-mount in situ hybridization In situ hybridization was performed using the procedure of Conlonand Herman (1993) with slight modifications. Briefly, non-isotopicriboprobe was prepared from a 1.8 kb cDNA fragment consisting of1.6 kb of 3′ coding sequence plus 0.2 kb of the 3′ untranslatedsequence of murine PECAM-1 using RNA polymerase and a standardnucleotide mix including digoxigenin-labeled UTP (BoehringerMannheim Genius 4 System). Embryos were rehydrated, washed andbriefly digested with proteinase K. Following washing, the embryoswere re-fixed with glutaraldehyde, washed and prehybridized at 63°Cin 50% formamide, 0.75 M NaCl, 1× PE (10 mM Pipes, pH 6.8, 1mM EDTA, pH 8.0), 0.1% BSA, 0.01% heparin, and 100 µg/ml yeastRNA with 1% SDS for 2-4 hours. The embryos were then hybridizedin a similar buffer containing approximately 0.5-2 µg/ml of digoxi-genin-labeled probe overnight. After extensive washing, the embryoswere exposed to pre-immune serum followed by sheep anti-digoxi-genin antibodies conjugated with alkaline phosphatase. Antibody wasdetected using nitroblue tetrazolium salt/5-bromo-4-chloro-3-indolylphosphate.
Cross sectional immunofluorescent staining Cryostat sections (7 µm) of sucrose-embedded embryos were fixed inmethanol at 4°C and stored at −20°C. They were rehydrated in PBSfor 30 minutes, soaked in a solution containing 4% BSA and 4% goatserum in PBS for 30 minutes and exposed to the appropriate primarymonoclonal antibody for 1 hour in a humidified chamber at room tem-perature. The slides were washed three times in PBS and then coun-terstained with a FITC-conjugated goat anti-rat secondary antibody ina humidified chamber at room temperature in the dark for 1 hour. Thespecimens were washed three times in PBS, mounted in anti-quenchmixture containing DABCO and photographed using a Leica fluores-cent microscope. mAb 390, a rat anti-mouse PECAM-1 antibody wasused as the primary antibody for all PECAM-1 staining and MF20, amouse anti-muscle myosin antibody (Bader et al., 1982) provided bythe Developmental Studies Hybridoma Bank, University of Iowa, wasused as a cardiac muscle marker.
RESULTS
Multiple isoforms of muPECAM-1 are detected in amouse heart cDNA library For purposes of maximum specificity throughout these studies,mouse PECAM-1 (muPECAM-1) cDNA was isolated from anadult heart library screened with human PECAM-1(huPECAM-1) cDNA. The complete nucleotide and aminoacid sequence of the coding region of a 3 kb fragment ofmuPECAM cDNA is shown in Fig. 1. The full-lengthmuPECAM-1 cDNA encodes a protein consisting of 727amino acids. It exhibits a 60% identity (74% similarity) tohuman PECAM-1 at the amino acid level. Structurally,
2542 H. S. Baldwin and others
Murine PECAM - 1 (cDNA)
Murine PECAM - 1 (AA)
Human PECAM - 1 (AA)
2543PECAM-1 expression during cardiovascular development
Fig
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Murine PECAM - 1 (cDNA)
Murine PECAM - 1 (AA)
Murine PECAM -
∆12,15 (AA)
Murine PECAM -
∆14,15 (AA)
Human PECAM - 1 (AA)
2544
muPECAM-1, like huPECAM-1, is organized into an extra-cellular amino-terminal domain containing 6 immunoglobulin(Ig)-like repeats, followed by a short, hydrophobic transmem-brane domain and finally terminating in a cytoplasmic domain.The positions of all cysteines required for folding of the Igrepeats are conserved, as would be expected for a member ofthe Ig superfamily. In this respect, all known PECAM-1cDNAs were found to be identical. Differences were howevernoted between the cDNA sequence reported previously (Xieand Muller, 1993), and those reported here. Three of four ofthe differences noted within the cytoplasmic domain (Fig. 1B)have been resolved in agreement with those presented here(Muller, personal communications). Differences within theextracellular domain have not been resolved and may wellindicate the existence of other isomorphic forms ofmuPECAM-1. In contrast, we isolated cDNAs in whichsegments of the cytoplasmic domain sequence were absent(Fig. 1B). The size and location of the missing oligonucleotidescorresponded precisely to exons within the human PECAM-1gene (Kirschbaum and Newman, 1993; Kirschbaum et al.,1994) suggesting that they were the result of alternativesplicing. These isoforms will be designated muPECAM-1∆12,15 or muPECAM-1∆14,15 to indicate the absence ofoligonucleotides corresponding to exons 12 and 15 or 14 and15 respectively.
Based upon the predicted exon arrangements in the humanPECAM-1 gene (Kirschbaum et al., 1994), alternativelyspliced isoforms would have differences in addition to thedeleted polypeptide. Splicing out exon 12 would theoreticallyresult in a change of the amino acid at the splice junction, asexon 12 is bracketed by type 1 intron inserts. Removal of exon15 would result in a truncated terminal peptide with an alteredamino sequence beginning at the splice junction joining exon14 to exon 16 due to a type I intron insert at the 5′ end of exon15 and a type 0 insert at the 3′ end. Thus, the deletion of exons14 and 15 would result in a frame shift and early terminationof the cytoplasmic domain (see Fig. 1B).
Alternatively spliced forms of muPECAM-1 arefunctionally distinctTo examine the possible functional consequences of alterna-tive splicing and premature termination, muPECAM-1 cDNAsencoding either full-length or alternatively spliced isoformswere cloned into expression vectors and transfected into L-cells. Transfected cells were examined for muPECAM-1expression by FACS analysis using the monoclonal antibody390 (Fig. 2). Control, mock transfected L-cells carrying thevector alone failed to react with this antibody. In contrast,
strong expression of muPECAM-1, at equivalent levels, wasdetected on the surface of cells transfected with each isoform.The ability of each isoform to bind to a panel of monoclonalantibodies was similarly compared by FACS analysis (Table1). By these criteria, the cDNAs encoding each of the isoformswere equally active. That is, all transfected cells expressedmuPECAM-1 or one of its isoforms at similar levels and allappeared to bind each of the mAbs to a similar extent (Table1).
We have previously shown that one of the characteristics ofhuPECAM-1 is its ability to mediate specific, mAb-sensitiveaggregation of transfected L-cells (Albelda et al., 1991;DeLisser et al., 1994). Therefore, the ability of L-cells, trans-fected with each isoform, to aggregate in a muPECAM-1dependent, mAb-sensitive manner was evaluated. The data aresummarized in Fig. 3. When compared to mock transfectedcontrols, each muPECAM-1 isoform was capable of mediatingL-cell aggregation to the same extent as full-lengthmuPECAM-1. Thus, alternative splicing did not compromisethe ability of muPECAM-1 to promote L-cell aggregation.However, interesting differences were noted in the sensitivityof the transfectants to three anti-muPECAM-1 mAbs. Aggre-gation of cells expressing either the full-length muPECAM-1or the ∆12,15 isoform was equally sensitive to inhibition by allthree mAbs. In contrast, cells transfected with the ∆14,15isoform, while remaining sensitive to mAbs Mec 13.3 andEA3, were completely resistant to inhibition by mAb 390. Thisresistance was not due to loss of reactivity, as FACS analysisrevealed that the muPECAM-1∆14,15 transfected cellscontinued to bind mAb 390 (Fig. 2, Table 1). This differential
H. S. Baldwin and others
Fig. 2. Fluorescence activated cell sorting(FACS) analysis of L-cells expressing each ofthe muPECAM-1 Isoforms. Cell surfaceexpression of (A) full-length muPECAM-1,(B) muPECAM-1∆12,15 and (C)muPECAM-1∆14,15 was assessed byfluorescence-activated cell sorting using Mab390. In every case, >95% of the cells in eachtransfected population were expressingmuPECAM-1 or the appropriate isoform.
Table 1. FACS analysis of L-cell transfectants expressingfull-length muPECAM-1, muPECAM-1∆12,15 or
muPECAM-1∆14,15 using anti-PECAM-1 polyclonal andmonoclonal antibodies
The ability of an irrelevant control mAb and three monoclonal antibodies(mAb 390, Mec 13.3 and EA-3) to bind to L-cells expressing the variousforms of muPECAM-1 was assessed by fluorescence activated cell sortinganalysis. The mean (log) fluorescence intensity for each antibody wasdetermined for each transfectant. The three anti-murine monoclonalantibodies bound equally well to each L-cell transfectant.
2545PECAM-1 expression during cardiovascular development
sensitivity to anti-muPECAM-1 mAbs indicates that changesin the cytoplasmic domain result in a selective change in theconfiguration of the extracellular, ligand-binding domain of themolecule. In addition, these data demonstrate that thismuPECAM-1 isoform is mediating L-cell aggregation by amechanism different from that of full-length or the ∆12,15isoform. Thus, alternative splicing may provide a mechanismby which the function of muPECAM-1 may be regulated.
Alternatively spliced isoforms are expressed indeveloping mouse embryosThree sets of oligonucleotide primers were designed todetermine (1) the earliest time at which muPECAM-1 mRNAcould be detected in the developing embryo and (2) thepossible existence of alternatively spliced forms. Theexpression of muPECAM-1 in staged embryos was determinedby nested RT-PCR analysis of mRNA. To avoid possible com-plications due to the presence of alternatively spliced isoforms,primers were designed to amplify a sequence encoding aportion of the extracellular domain (Fig. 4). The RT-PCRproducts were analyzed by agarose gel electrophoresis (Fig.4A). Extracts from all embryos yielded fragments identical insize to those amplified from the control plasmid vector (P)carrying muPECAM-1 cDNA. Each nested product carried anexpected DraI endonuclease site (Fig. 4; DraI). Their identi-ties were confirmed by Southern blot analysis (Fig. 4B). Theseproducts were taken as specific indicators of the presence ofmuPECAM-1 mRNA. They were of the expected size; theyhybridized with muPECAM-1 cDNA of the appropriatesequence; and no contaminating DNA was detected when thereverse transcription step was omitted or mRNA from 3T3fibroblasts was used as a source of template. It should be notedthat the fragment size would be considerably larger if contam-inated genomic DNA were amplified, as these primers spanned
multiple exons (Kirschbaum et al., 1994). Thus, mousePECAM-1 mRNA was present in the embryo immediatelyafter implantation and prior to somitogenesis as would beexpected for a molecule expressed by endothelial orhematopoietic progenitors.
In order to test the possibility that alternatively splicedisoforms of muPECAM-1 were expressed in the embryo,primers were designed, as shown in Fig. 5, to amplifysequences that included the alternatively spliced regions of thecytoplasmic domain. Messenger RNA was isolated from 8- and12-day embryos, as well as from hearts of 8-, 12-, 16-dayembryos and adults. The RT-PCR products from each prepa-ration were surprisingly complex (Fig. 5), consisting of amixture of oligonucleotides ranging in size from approxi-mately 317 to 239 bp. The top and bottom bands seen byethidium bromide staining corresponded in size and sequence
Fig. 3. Differential sensitivity of alternatively spliced muPECAM-1isoforms to adhesion perturbing monoclonal antibodies. The abilityof three monoclonal antibodies (MEC 13.3, EA-3 and 390) to inhibitthe aggregation of L-cells expressing muPECAM-1 and its isoformswas compared. The data are expressed as a percentage of the controlaggregation. Monoclonal antibodies Mec 13.3 and EA-3 inhibitedaggregation mediated by all three isoforms, while mAb 390 did notblock muPECAM-1∆14,15-dependent aggregation. Antibodyconcentration = 50 µg/ml. The means and standard deviations werecalculated on the basis of at least two experiments done in duplicate.Background aggregation of control (mock) transfected cells was 10-15%; control aggregation was 30-40%.
Fig. 4. Detection of PECAM-1 message during development by‘nested’ RT-PCR of mRNA from staged mouse embryos.(A) Agarose gel electrophoresis of PCR products from amplifiedplasmid carrying muPECAM-1 cDNA (P) and cDNA from latepresomite embryos (0), 1-somite (1), and 8-somite (8) embryos. Nomessage was detected in the embryo after initial amplification withthe ‘outside’ primer set. However, after re-amplification with the‘inside’ primers, a PCR product corresponding to the expected sizefor the muPECAM-1 message could be detected in the RT productfrom the late presomite embryo as well as the 1- and 8-somiteembryo. (B) The identity of the amplified products as PECAM-1 wasconfirmed by enzyme digestion (DraI) and Southern blotting with aprobe specific for the 5′ end of the amplified region of PECAM. Noproduct was detected when mouse fibroblasts were used as a sourceof mRNA (a), or if the reverse transcription step was omitted inprocessing the mRNA from mouse fibroblasts (b) or 8 day embryos(c).
2546
to the PCR products amplified from plasmids carryingmuPECAM-1 or muPECAM-1∆12,15 cDNA respectively.Approximately five different bands could be resolved by thismethod (Fig. 5A). These RT-PCR products fell within a sizerange that would be predicted for a mixture of muPECAM-1cytoplasmic domain fragments missing various combinationsof predicted exons. They were too small to represent productsof genomic DNA amplified across several exons, and too largeto represent a single exon (Kirschbaum et al., 1994). Failure todetect RT-PCR products of this size upon omission of thereverse transcription step, and the absence of any detectableproducts from mouse fibroblasts further confirm the specificityof these primers and the authenticity of the RT-PCR products.
Southern blot analysis, using probes homologous to thecytoplasmic domain, confirmed the existence of multiple
muPECAM-1 isoforms (Fig. 5B). None of these bandshybridized with probes homologous to the extracellulardomain of muPECAM-1 (data not shown). The presence ofmRNA encoding multiple isoforms of muPECAM-1 wasconfirmed by sequencing ‘shot gun’ clones of RT-PCRproducts generated from these mRNA preparations usingprimers designed to span the entire cytoplasmic domain,beginning within the 3′-untranslated region of the message andextending into the extracellular domain (see Materials andMethods). These products, about 0.9 kb in size, each carried aBsteII endonuclease site on the 5′ end and a NsiI site on the 3′end to facilitate site-specific cloning into a plasmid carryingmuPECAM-1 cDNA digested with each of these enzymes.Random clones were sequenced as described in Materials andMethods. While an analysis of all clones has not beencompleted, the two isoforms whose sequences are shown inFig. 1 were among the first isolated. Multiple isoforms havebeen consistently detected at different stages of embryonicdevelopment. So far, however, no reproducible differences inrelative isoform abundance have been correlated with specificdevelopmental stages.
PECAM-1 expression correlates with theorganization of the blood islands and vasculature inthe post-implantation embryoThe sites of muPECAM-1 expression in the developingembryo were determined by in situ hybridization using digox-igenin-labeled anti-sense and sense riboprobes. Anti-digoxi-genin immunoglobulin conjugated with alkaline phosphatasewas used to detect hybridized complexes. muPECAM-1mRNA was first detected in the region of the blood islands inthe yolk sac of the pre-somite (day 7.0-7.5) mouse embryo(Fig. 6A, arrow). As development progressed, the muPECAM-1 mRNA was found in patterns consistent with it beingexpressed by the endothelium of the forming vasculature (Fig.6B,C). In the 5-somite embryo (day 7.75-8.0), the message wasfound concentrated rostral to the foregut (fg) in the region ofthe developing heart (ht), in the forming dorsal aortae (da), aswell as in the neural folds (nf) of the developing head process(Fig. 6B). By day 9.0-9.5 (Fig. 6C), the pattern of PECAM-1mRNA expression clearly reflected that of the developing vas-culature including the second and third branchial arches (2,3),the ventricles (v) and atria (a) of the developing heart and inter-segmental arteries (isa). It continued to be evident in thematuring vasculature of the head and trunk including the dorsalaortae. An example of a control embryo exposed to the ‘sense’oligonucleotide is shown in Fig. 6D. There was, at best, onlyfaint background staining of control embryos.
Immunohistochemistry confirms muPECAM-1expression in endothelial cells of the developingvasculature and heartIn order to monitor message translation and to identify the cellssynthesizing muPECAM-1, cross sections of an embryo (Fig.7), including the heart, at the straight heart tube stage (Fig.7A,B; day 8.5) were immunostained using mAb 390 specificfor muPECAM-1. At this stage, the heart consists of cellsorganized into two concentric tubes. The outer layer of cellsconstitutes the developing myocardium (m) and the inner layerthe developing endocardium (e). The dorsal aortae (da) arevisible just below and lateral to the neural fold (nf). This
H. S. Baldwin and others
Fig. 5. Detection of alternatively spliced forms of muPECAM-1 instaged mouse embryos by RT-PCR analysis. Primers were designedto specifically amplify the cytoplasmic domain of muPECAM-1.(A) Agarose gel electrophoresis of PCR products from a mixture ofplasmids containing either full length murine PECAM-1 ormuPECAM-1∆12,15 cDNA (P) and RT-PCR products amplifiedfrom an 8-day embryo with the heart removed (8E), 8-day embryonicheart (8H), 12-day embryo with the heart removed (12E), 12-dayembryonic heart (12H), 16-day embryonic heart (16H), adult heart(AH) and 3T3 cells (a). Products from control reactions containingsimilarly processed mRNA from 3T3 cells (b) or 12 day embryos (c)in which the reverse transcription step was omitted are also shown.RT-PCR products corresponding to the predicted size of both fulllength muPECAM and muPECAM-1∆12,15, as well as severalintermediate size fragments, were detected at all stages ofdevelopment examined. (B) Southern hybridization using an end-labeled oligonucleotidecorresponding to exon 13 further confirms the identity of the PCRfragments as muPECAM.
2547PECAM-1 expression during cardiovascular development
Fig. 6. Developmental distribution of muPECAM-1 mRNA as determined by in situ hybridization. (A) muPECAM-1 message was firstdetected in the 7.5-day, presomite embryo, in a circumferential band of cells in the extra embryonic mesoderm of the yolk sack (ys) consistentwith the early formation of the embryonic blood islands. No message was detected in the embryo (emb). epc, ectoplacental cone. (B) In the 5-somite embryo, message was detected in the endothelial cells lining the dorsal aorta (da) and in an accumulation of endothelial cells justanterior to the foregut (fg) corresponding to the developing heart (ht). Message was also detected in the developing vasculature of the head andneural folds (nf). (C) By 9 days of gestation, message could be detected throughout the developing vasculature of the embryo including thedorsal aortae, 1st, 2nd and 3rd pharyngeal arches, intersegmental arteries (isa) and the atria (a) and ventricle (v) of the primitive heart. fb,forebrain. (D) A 9-day embryo incubated with sense riboprobe as a negative control.
2548 H. S. Baldwin and others
Fig
. 7.E
valu
atio
n of
the
deve
lopm
enta
l exp
ress
ion
of m
uPE
CA
M b
yim
mun
ohis
toch
emis
try.
(A
) In
the
8.5-
day
embr
yo, P
EC
AM
-1 w
as d
etec
ted
on th
een
doca
rdiu
m (
e) b
ut n
ot m
yoca
rdiu
m (
m)
of th
e st
raig
ht h
eart
tube
as
wel
l as
on th
een
doth
elia
l lin
ing
of th
e do
rsal
aor
ta (
da).
In
addi
tion,
PE
CA
M-1
exp
ress
ion
was
det
ecte
din
ext
ra e
mbr
yoni
c m
esod
erm
of
the
yolk
sac
(ar
row
head
s) c
orre
spon
ding
to th
e re
gion
of
bloo
d is
land
for
mat
ion.
nf,
neu
ral f
olds
. (B
) A
n en
larg
emen
t of
the
card
iac
regi
on o
f A
dem
onst
ratin
g th
e in
terc
ellu
lar
loca
lizat
ion
of P
EC
AM
-1 w
ithin
the
deve
lopi
ngen
doca
rdiu
m a
nd e
ndot
heliu
m o
f th
e do
rsal
aor
ta a
nd a
bsen
ce o
f PE
CA
M-1
sta
inin
g
with
in th
e m
yoca
rdiu
m. (
C)
A c
ross
sec
tion
thro
ugh
the
hear
t of
an 1
1.5-
day
embr
yode
mon
stra
tes
PEC
AM
exp
ress
ion
in th
e en
doca
rdiu
m o
f th
e ri
ght a
triu
m (
ra),
left
atr
ium
(la)
, tra
becu
latio
ns o
f th
e ve
ntri
cle
(v),
and
on
the
endo
thel
ium
of
endo
card
ial c
ushi
ons
(edc
) of
the
outfl
ow tr
act o
f th
e he
art.
(D)
An
enla
rgem
ent o
f C
dem
onst
ratin
g a
dow
nre
gula
tion
of P
EC
AM
-1 e
xpre
ssio
n by
the
endo
thel
ial c
ells
that
hav
e m
igra
ted
to f
orm
the
mes
ench
ymal
cel
ls o
f en
doca
rdia
l cus
hion
s. (
E)
An
adja
cent
sec
tion
to C
,D, s
tain
ed w
ith a
myo
sin
mar
ker,
MF2
0, to
del
inea
te th
e m
yoca
rdiu
m a
s a
sepa
rate
cel
l pop
ulat
ion
from
the
endo
card
ium
and
edc
mes
ench
yme.
2549PECAM-1 expression during cardiovascular development
(C)
DIC
imag
e an
d (D
) ep
ifluo
resc
ence
of
the
dem
arca
ted
regi
on a
t hig
her
mag
nific
atio
n.m
uPE
CA
M-1
sta
inin
g is
res
tric
ted
to th
e en
doth
elia
l lin
ing
of b
lood
isla
nds
with
in th
eyo
lk s
ac m
esod
erm
(ys
m)
and
no s
tain
ing
is d
etec
ted
in th
e yo
lk s
ac e
ndod
erm
(ys
e).
Fig
. 8.m
uPE
CA
M-1
exp
ress
ion
in b
lood
isla
nds.
(A
) D
IC im
age
of a
cro
ss s
ectio
nth
roug
h th
e ne
ural
fol
d (n
f) a
nd y
olk
sac
(ys)
of
an 8
-day
em
bryo
. am
n, a
mni
on.
(B)
Epi
fluor
esce
nce
of th
e sa
me
sect
ion
stai
ned
with
Mab
390
. muP
EC
AM
-1 s
tain
ing
isob
serv
ed w
ithin
the
yolk
sac
(ys
) an
d ve
ssel
s (a
rrow
hea
d) w
ithin
the
neur
al f
old
(nf)
.
2550
section also included the extraembryonic membranes and theyolk sac (arrowheads). The muPECAM-1 mAb reacted onlywith the endocardial cells in the developing heart. No stainingwas detected in the outer myocardial layer. Immunoreactivitywas also noted on cells organized into two major vessels oneither side of the neural tube just above the developing foregutin the position of the dorsal aortae (da). At higher magnifica-tion (Fig. 7B), it was clear that the staining involved a singlelayer of cells surrounding the lumen of the dorsal aortae as wellas the endocardium. In both cases, the staining patternindicated a concentration of the antigen at cell-cell borders.
The anti-muPECAM-1 mAb also reacted with a single layerof cells beneath the extraembryonic endoderm of the sur-rounding yolk sac (Fig. 7A,B, arrowheads). At higher magni-fications, it is clear that muPECAM-1 was expressed only onthe cells forming the ‘wall’ of the blood islands and not theinner, presumably hematopoietic cells (Fig. 8). The extraem-bryonic mesoderm and endoderm were both negative formuPECAM-1. The position and distribution of this stainingwas consistent with the observation that muPECAM-1 mRNAwas expressed in the yolk sac in the region of the formingextraembryonic vasculature and not on cells that might be con-sidered extraembryonic hematopoietic stem cells.
muPECAM-1 expression is down regulated duringendocardial cushion formationDuring cardiac morphogenesis, the separation of the compart-ments of the heart and the partitioning of the conotruncalregion into two major vessels, the aorta and the pulmonaryartery, commence with the formation of the endocardialcushions (Markwald et al., 1984; Markwald et al., 1985). Thisinvolves a morphological transition of endocardial cells tomesenchymal cells (epithelial-mesenchymal transformation)accompanied by their migration into the underlying extracel-lular matrix to form the stroma of the endocardial cushions. Asection through the conotruncus of an 11.5-day embryonicheart, in the region of endocardial cushion formation (Fig. 7C),illustrates the specific cellular components of this process.After staining with an anti-PECAM-1 mAb, the endocardium(e) lining the ventricle (v), the right and left atria (ra; la) andoverlying the forming endocardial cushion (edc) demonstratedstrong immunoreactivity. In contrast, the stromal cells of theendocardial cushion that originated from the overlying endo-cardium were completely unreactive with the antibody. This isparticularly evident when viewed at higher magnification (Fig.7D). The absence of immunoreactive muPECAM-1 was notdue to masking of the antibody reactive site, as no reactivitywas noted using three independently isolated mAbs and nomuPECAM-1 mRNA expression was detected by in situhybridization (Baldwin, unpublished observations). Fig. 7Eshows an adjacent section of the same heart stained with MF20, a mAb specific for muscle myosin (Bader et al., 1982). Thisantibody reacts only with muscle cells. Its absence from theregion labeled endocardial cushion (edc) verifies that this is, infact, the septating conotruncus and outflow tract.
DISCUSSION
This report documents three significant new observations con-cerning the immunoglobulin superfamily molecule PECAM-1.
They are: (1) muPECAM-1 is expressed in multiple alterna-tively spliced forms in the developing mouse embryo; (2) alter-native splicing of exons within the cytoplasmic domain isaccompanied by changes in reactivity with function-blockingmAbs; (3) the temporal and spatial patterns of expression areconsistent with a role for muPECAM-1 in the initial organiz-ation of the mammalian cardiovascular system.
Multiple alternatively spliced isoforms ofmuPECAM-1 are expressed during embryonicdevelopmentEvidence for the existence of alternatively spliced mRNAencoding multiple muPECAM-1 isoforms is based upon threeobservations. First, multiple RT-PCR products were generatedusing mRNA preparations from staged embryos and primersbracketing the cytoplasmic domain (Figs 1-3). Second, com-parative sequence analysis revealed that the isoforms were allmissing segments corresponding to specific exons describedfor the huPECAM-1 gene (Kirschbaum and Newman, 1993;Kirschbaum et al., 1994). Third, the changes in oligonucleotideand polypeptide sequence at the splice sites were limited tothose that would be predicted from the exon-intron organi-zation within the homologous region of the huPECAM-1 gene.These observations have been consistent for over 30 differentmuPECAM-1 cDNAs sequenced to date (Yan, unpublisheddata). The distribution of cytoplasmic domain codons overseveral exons distinguishes PECAM-1 from other members ofthe immunoglobulin superfamily such as VCAM-1 and theICAMs normally expressed by endothelial cells. The cyto-plasmic domains of these latter molecules are encoded entirelywithin a single exon (Voraberger et al., 1991; Xu et al., 1992;Cybulsky et al., 1991, 1993). In this respect, PECAM-1 is morelike N-CAM (Owens et al., 1987) or isoforms of carcinoem-bryonic antigen (Barnett et al., 1989). The cytoplasmicdomains of both of these Ig-superfamily molecules are encodedby more than a single exon and alternatively spliced isoformswith altered cytoplasmic domains have been identified. Thefunctional significance of these isoforms is not known, but inboth cases, larger regions of the cytoplasmic domain are lostby alternative splicing than is found for muPECAM-1. Inter-estingly, alternative splicing of the cytoplasmic domains of twomembers of the CEA family result in frame shifts and andpremature termination as is reported for the two muPECAM-1 isoforms described here.
muPECAM-1 may be functionally modulated byalternative splicing of the cytoplasmic domainThe possibility that the abundance of muPECAM-1 isoformsportends functional significance assumes, of course, that alter-natively spliced isoforms retain their ability to mediate celladhesion. While this has yet to be proven in situ, alternativelyspliced isoforms were found to be as effective in mediating theaggregation of transfected L-cells as full-length human ormouse PECAM-1. However, variations in sensitivity to mAbswere noted among the isoforms tested here. L-cell aggregationmediated by either full-length muPECAM-1 or the ∆12,15isoform was equally sensitive to a panel of three mAbs. Incontrast, aggregation mediated by muPECAM-1∆14,15 wasunaffected by mAb 390 while remaining sensitive to two otheradhesion-blocking mAbs (Fig. 3). This difference was repro-ducible and cannot be explained on the basis of changes in the
H. S. Baldwin and others
2551PECAM-1 expression during cardiovascular development
level of muPECAM-1 expressed on the surface of the trans-fected cells, as they all showed similar levels of expression asmeasured by FACS analysis (Fig. 2; Table 1). In addition, allisoforms continued to localize to cell-cell borders precisely asnoted for cultured endothelial cells (Albelda et al., 1991) whentransfected cells are allowed to grow as monolayers (Baldwin,unpublished observations), supporting the contention thatalternatively spliced forms retain their ability to participate inintercellular adhesive events.
The change in mAb sensitivity associated with themuPECAM-1∆14,15 isoform and the absence of such a changein the ∆12,15 isoform indicate that the deletion of a specificportion of the cytoplasmic domain can result in secondaryalterations within the extracellular, ligand binding domain ofmuPECAM-1. That a conformational change in the extracel-lular domain resulted from the cytoplasmic domain modifica-tion is also indicated by the change in mAb sensitivity. It iswell known that antibodies recognize molecular conformationrather than peptide sequence and that most epitopes are dis-continuous and structurally distinct (Amit et al., 1986; Graemeet al., 1990). This characteristic has been exploited in severalsystems to demonstrate structural alterations accompanyingchanges in receptor function. For example, integrins on thesurface of activated platelets undergo a structural change thatis characterized by the acquisition of ligand binding ability andaccompanied by the reproducible presentation of new epitopesor a change in the affinity of mAb binding (Du et al., 1993;reviewed by Ginsberg et al., 1992). Such changes in molecularconformation are common among the integrins, and are indica-tive of changes in ligand binding specificity as well as receptoractivation. They are, however, less common among membersof the immunoglobulin superfamily that have, up until thistime, not been found to undergo transmembrane modificationsof receptor function.
These observations are consistent with our previous resultsshowing that the deletion of increasingly larger portions ofthe cytoplasmic domain of huPECAM-1 results in a moleculethat exhibits different adhesion characteristics whencompared to full-length PECAM-1 (DeLisser et al., 1994). Inthis case, L-cells transfected with full-length huPECAM-1aggregate in a divalent cation dependent, heterophilic mannerthat can be blocked with sulfated glycosaminoglycans. Incontrast, aggregation of L-cells expressing specific truncatedforms of PECAM-1 occurs by a very different mechanism. Itis divalent cation independent, homophilic, cannot beinhibited by sulfated glycosaminoglycans and exhibitsdifferent mAb sensitivities from full-length PECAM-1(DeLisser et al., 1994). Preliminary evidence suggests thatsimilar differences exist in the aggregation characteristics ofL-cells expressing isoforms represented by muPECAM-1∆14,15 (Yan et al., unpublished observations). Given thediversity of adhesive mechanisms utilized by muPECAM-1,and the ability to modify the adhesive characteristics of themolecule by manipulating the cytoplasmic domain, it ispossible that the establishment of the embryonic vasculature,and perhaps later angiogenesis, depends upon the ability toregulate the adhesive properties of muPECAM-1 through itscytoplasmic domain either by changing the polypeptidesequence, as shown here, or perhaps by phosphorylation orinteraction with cytoplasmic protein kinases or other factorsknown to promote signal transduction.
PECAM-1 is among the earliest endothelial cellspecific adhesion molecules expressed in thedeveloping mammalian embryoThe temporal and spatial patterns of muPECAM-1 expressionare consistent with it being one of the early adhesion receptorsutilized by vasculogenic cells. It is expressed in the yolk sacimmediately after implantation, and subsequently in theembryo itself. Its pattern of expression resembles that reportedin the quail embryo, using the endothelial cell-specific markerQH-1 (Pardanaud et al., 1987; Coffin et al., 1991b), except thatmuPECAM-1 was never detected on extraembryonichematopoietic cells within the forming blood islands (Fig. 8).However, like QH-1, muPECAM-1 was detected in the earlypost implantation embryo across the anterior intestinal portalin the region of the heart primordia (Baldwin et al., 1991) andextending into the head process on cells with thin, thread-likeprocesses suggestive of vascular primordia (Fig. 7). Also, aswas found for QH-1, muPECAM-1 expression proceeds in arostral to caudal fashion in a pattern consistent with it beingexpressed on endothelial cells as they emerge from themesoderm and are organized into the forming vasculature andheart. Secondary vessels appear to arise from the primaryvascular tree. This is particularly evident along the dorsalaortae where the intersegmental arteries arise as regularlyspaced lateral extensions of the main vessel (Fig. 7). The factthat muPECAM-1 was expressed by early vascular endothelialcells was further supported by the correlation of the pattern ofmuPECAM-1 expression seen by immunohistochemistry andthat noted by in situ hybridization. This expression pattern issimilar to that reported for members of the receptor proteintyrosine kinase (RTK) superfamily. These include flk-1, tekand tie-2 in the mouse (Yamaguchi et al., 1993; Oelrichs et al,1993; Dumont et al., 1992; Millauer et al., 1993; Schurch etal., 1993) and quek 1 and quek 2 in the quail (Eichmann et al.,1993). muPECAM-1 is seen at the same time and place as flk-1 (Yamaguchi et al., 1993), and is co-expressed with tie 2(Schurch et al., 1993) suggesting that the muPECAM-1 genemight be activated as a result of RTK/ligand interactions.
Transplantation experiments in which endothelial cells fromthe quail were placed in chick embryos suggest that much ofthe information required for the organization of endothelialcells into vessels is intrinsic to the cells themselves, and relieson cues from the extracellular environment (Noden, 1990).This, more than likely, is mediated through cell surfacereceptors capable of responding to the molecular compositionof the extracellular matrix as well as to neighboring cells.muPECAM-1 may well be such a molecule. The ability ofPECAM-1 to mediate adhesion via several different mecha-nisms (DeLisser et al., 1993, 1994) raises the possibility thatit may participate in a broad spectrum of ligand interactions,some resulting in adhesion and others triggering intracellularsignals necessary to stabilize other adhesive events (Tanaka etal., 1992; Piali et al., 1993). Thus, once present, PECAM-1,like other adhesion receptors, might, in addition to functioningas an adhesion molecule, initiate a signal transduction cascaderequired for vascular organization in response to a variety ofextracellular cues.
We wish to thank Ms. Irene Crichton and Ms. Catherine Buck forexcellent technical assistance and Ms. Marie Lennon for invaluablehelp in preparation of this manuscript. This work was funded by NIH
2552
HL 51533; HL 39023; HL 47670; HL 2917; CA 10815; CA 19144;W. W. Smith Charitable Trust Grant H12901 and the Robert WoodJohnson Foundation Minority Faculty Development Program.
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