Embryonic Stem Cells as a Model forCardiogenesisJeffrey Robbins, Thomas Doetschman, W. Keith Jones, andAlejandro Sanchez
Embryonic stem (ES) cells are derived from the inner cell mass ofmouse blastocysts . These cells, when placed upon a suitable fibroblastfeeder layer, continue to proliferate without overt differentiation andremain totipotent. Cells in this state are competent for gene targeting viahomologous recombination . Hence, they hold the possibility ofdeveloping defined animal models of human cardiovascular disease .When removed from the feeder layer, ES cells undergo differentiationand development into large, multicellular structures, termed embryoidbodies (EBs) . Morphologic, biochemical, and molecular genetic analy-ses indicate that during EB development some early aspects ofcardiogenesis are recapitulated. Thus, EB development in culture isuseful for studying certain early cardiogenic events. (Trends CardiovascMed 1992 ;2 :44-50)
Congenital heart disease occurs with asignificant frequency, -5-1011000 livebirths in developed countries . Usuallythe disease is not life-threatening andcan be managed either by nonsurgicalmeans or by minor surgery later in life.However, in -30% of these cases, thelesions are critical and require immedi-ate attention (Tyler 1980) . The funda-mental mechanisms responsible for thesecongenital cardiac malformations, or forthe various familial and nonfamilialcardiomyopathies that develop later inlife, are unknown, although in a fewcases a primary genetic defect has been
This article is based upon the authors' presen-tation at the 1991 meeting of the InternationalSociety for Heart Research-American Section .
Jeffrey Robbins, W. Keith Jones, and Alejan-dro Sanchez are at the Department of Phar-macology and Cell Biophysics, and ThomasDoetschman is at the Department of MolecularGenetics, Biochemistry and Microbiology, Uni-versity of Cincinnati College of Medicine,Cincinnati, OH 45267-0575, USA .
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identified. For example, at least somecases of familial hypertrophic cardiomy-opathy are linked to mutations in thecardiac myosin heavy-chain genes (Geis-terfer-Lowrance et al. 1990). However,the mechanisms underlying the develop-mental lesions that occur during cardio-genesis, and the resultant pathophysiol-ogy of even the genetically defined dis-eases, remain, for the most part, com-pletely obscure. The ultimate develop-ment of prophylactic or replacementtherapies based upon the genetic etiol-ogy is dependent upon understandingboth the genetic and the developmentalbases of the disease(s) .
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Early Cardiac Development
In the human, the early development ofthe cardiac system is well defined . Acardiogenic area and pericardial cavitycan be discerned at -2 .5 weeks, and cellsin this region are already predeterminedto become myocytes. The bilaterallysymmetric embryo contains two endo-thelial heart tubes that come to lie
parallel to each other, one on each side ofthe midsagittal plane. Subsequently, acephalocaudal flexion brings the tubesclose to each other at the embryo'scephalic end, and by 22-23 days the twotubes have fused (Figure 1) . The singlecardiac tube contains an external myo-cardial layer, 1-2 cells thick . Underneathand lying between the external mantle orepicardium and the endocardium orinternal endothelial tube is the cardiacjelly, which is made up largely of glyco-conjugates. Extensive inductive interac-tions between these layers are thought tobe a driving force in the subsequentdifferentiation of the tube as it loops,grows, and develops (Mjaatvedt and Mark-wald 1989) . After looping externally, theheart presents an appearance similar tothat of the fully developed organ, andseptation begins soon after. A presentday challenge, in both the early embryo-logic events described above as well asduring the later period of differentiationand development, is to go beyond a meredescription of the process . It is impera-tive that the mechanistic underpinningsof both the normal and abnormal proc-esses be determined . If the developmentof a myocardial defect could be traced toa single gene or group of genes, then themechanistic determinants of both thenormal and pathologic processes couldbe more easily unraveled .
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Animal Models
Animal models are critical for the studyand understanding of these developmen-tal abnormalities . The use of rodents hasproved particularly valuable in elucidat-ing certain aspects of hereditary cardio-myopathies . Animals such as the sponta-neously hypertensive rat and the cardio-myopathic Syrian hamster have provedinvaluable in determining pathophysio-logic parameters during the develop-ment of the hypertensive and cardiomy-opathic heart . Paradoxically, the pri-mary genetic defect, that is, the gene orgenes that are responsible for thesehereditary diseases, remains undeter-mined. However, this inability to definethe genetic defect(s) in animal models ofcardiovascular disease can now be cir-
cumvented. With the use of transgenicmethodologies, predetermined normalor mutated genes can be placed withinthe endogenous genetic background ; thistechnology has been applied with partic-ular emphasis to the mouse (Hanahan1990). Additionally, in the mouse, a largenumber of developmental mutations havebeen documented, and in some casestheir chromosomal sites have beenmapped (Lyon and Searle 1989 ; Green1975) .The number of transgenic animals
that have been useful in the study ofcardiac function and development islimited [reviewed by Field (1991)], but ofparticular interest to a discussion ofcardiogenesis are the "gain-of-function"studies that result in hyperplasia. Ex-pression of an oncogenic viral protein,the SV40 T antigen, in the heart resultedin the overexpression of the oncoproteinin the mouse atrium, leading to a >100-fold increase in right atrial mass, whilethe left atrium was unaffected (Field1988). This asymmetric hyperplasia iseventually lethal, owing to abnormalitiesin the conduction system . Similarly, inanother series of experiments in which atyrosine protein kinase derived from anavian oncogenic retrovirus (Fujinamisarcoma virus) was used to generate
`~~\
/ /_ ' epirnyticardial cells
4000000 ~ J ,/
endocardialfusing endocardial
tubetubes
foregut
°
-
pericardial
,
-
eprnyocardium~ f
cavity
cardiac jelly
Figure 1 . Schematic transverse sections through a part of the embryo showing developmentand fusion of the cardiac tube . Subsequently, the tube will bulge into the pericardial cavity andbegin to loop (see the text),
transgenic animals, a number of inde-pendent transgenic lines developed car-diac lesions that eventually were lethal(Yee et al . 1989; Chow et al . 1991) . Thetyrosine protein kinases are known toplay important roles in both cell prolifer-ation and development, and a directcorrelation between the extent of cardi-omegaly and the amount of the onco-gene's protein tyrosine kinase was con-firmed. With in situ immunohistochem-istry, the protein's activity was localizedto the interstitial cells of the heart, butwas not present in the cardiomyocytes(Chow et al. 1991). These and otherstudies imply that an overaccumulationof collagen can result in general hy-pertrophy as well as biventricular dilata-tion and wall remodeling . This contra-dicts the generally accepted notion thatthe anatomic changes that occur duringhypertrophy are due primarily to altera-tions in the architecture of the ventricu-lar myocytes . Although it is not yet clearwhether this transgenic animal actuallyreflects an underlying pathology that isrelevant to human hypertrophy (Anversaand Capasso 1991), the importance of ananimal model that presents a significantdefect in the heart's connective tissue,which can be traced to a single gene'saction, is undeniable . The pace of pro-
®1992, Elsevier Science Publishing Co ., 1050-17381921$5 . 0 0
ducing such transgenic models will un-doubtedly increase in the near Future-
9 Embryonic Stem Cells and TargetedGene Replacement
The above studies illustrate the use oftransgenic animals in gain-of-functionstudies, that is, when a particular proteinis overexpressed or expressed inappro-priately in the cardiovascular compart-ment, with the resultant protein elicitinga discernible phenotypic response . Ide-ally, one would also like to carry outcomplementary studies in which loss offunction is generated by mutating aspecific gene that is already present inthe animal's normal genetic complementand encodes a functionally importantprotein to the heart ; that is, only the geneof choice is ablated or "knocked out ."
Murine embryonic stem (ES) cellsoffer the possibility of genetic modifi-cations in which both gain- and loss-of-function experiments can be performed .In addition, they provide the option ofstudying the effects of these modifica-tions in the whole animal as well as in anin vitro model system for embryogenesis(Figure 2) .
ES cell lines are established from theinner-cell mass cells of the embryo (Evansand Kaufman 1981 ; Martin 1981) . Theycan be maintained indefinitely in theundifferentiated state if grown on STOcells (Evans and Kaufman 1981 ; Martin1981), primary mouse embryonic fibro-blasts (Wobus et al . 1984; Doetschman etal. 1985), BRL-cell conditioned medium(Smith and Hooper 1987), or leukemiainhibitory factor (LIF) (Williams et al .1988a). When maintained under theseconditions and then injected into blas-tocysts, they can colonize most tissues inthe developing embryo (Beddington andRobertson 1989) and adult (Gossler et al .1986; Bradley and Robertson 1986), in-eluding the germ line (Bradley et al .1984). In germ-line chimeric animals, amodified ES cell genome can be verti-cally transmitted to several generationsof progeny in a stable manner (Gossler etal. 1986) . This characteristic will permitmouse modeling of genetic disorders ifthe ES cells have the proper geneticmodifications .In chimeric animals, the ES-derived
cells can be marked and followed, andthe effects of their interactions with hostcells in the chimeric tissues can be
45
SELECTION : via G418, thenPCR
DEVELOPMENTAL STUDIES
129Svr/+
Blastocyst-derived ES cell colonieson fibroblast feeder layer
Electroporatfon with targetingsequences
RESULT : Genetically modified ESis derived from the colony andinjected into a blastocyst
In vitrodllferentlatlon Into embryoldbodies
Select desired offspring by PCRInterbreed it homorygotes are desired
•
• •
Breed with C57BL16 ; agouti coatIndicates a GERM-LINE CHIMERABreed back to 129 SvJ
Blastocyst derived from C57BL16
Inject blastocyst intopseudopregnantQ
Attains breeding age
Figure 2. Summarized are the procedures that lead to a germ-line chimera that is able totransmit a targeted trait to its progeny. The targeted or wild-type embryonic stem (ES) cells willdifferentiate into embryoid bodies (EBs) when grown in suspension culture in the absence of afibroblast feeder layer. The target DNA that is injected usually includes a gene useful forselection; in this example, treo was used, allowing selection of the transfected cells by treatmentwith the antibiotic G418. The lines of mice indicated allow selection of the chimeric animals onthe basis of coat color. Male germ-line chimeras are identified by subsequent breedingprograms.
investigated. Chimeric mice produced byblastocyst injection of transfected (orretrovirally infected) ES cells have beenused to study expression of the humantype-II collagen (Lovell-Badge et al . 1987)and chicken S-crystallin genes (Taka-hashi et al . 1988), hemangioma develop-ment induced by polyoma middle Texpression (Williams et al. 1988b), expres-
46
sion of a reporter gene directed byenhancers trapped in ES cells (Gossler etal. 1989), and cell lineages (Suemori etal. 1990) .
Loss-of-function studies using ES cellscan be accomplished if the transgenemutates an endogenous gene by randominsertional mutation (Kuehn et al. 1987).The process of introducing the exogenous
DNA sequence, the transgene, into a cell,a technique termed transfection, can alsolead to loss of function if the DNA beingintroduced has sequence homology to aspecific endogenous (target) gene . In thiscase, a low percentage of the transfec-tents will have the transfecting DNAincorporated into the target gene byhomologous recombination, that is, recom-bination between the identical sequencespresent in both the targeting and tar-geted DNAs (Thomas and Capecchi 1987 ;Doetschman et al. 1987). This approachhas now been used for many loss-of-function studies in germ-line chimerasand their transgenic progeny [reviewedby Mansour (1990)] . For example, miceproduced from ES cells in which thehomeobox-containing gene Hox-1 .5 isablated display a phenotype similar to DiGeorge's syndrome (Chisaka and Capec-chi 1991) . Together, these studies dem-onstrate that gene targeting in ES cellsmakes it possible to ablate or modifygene function, and offers the potential ofaltering genes that play significant rolesin the development and maintenance ofthe cardiovascular system .
In addition to whole animal studies,ES cells also provide an in vitro embryomodel system. When ES cells are re-moved from the differentiation-inhib-itory influence of the feeder cells or theirequivalent, they will spontaneously dif-ferentiate into developing embryolikestructures of increasing complexitycalled embryoid bodies (EBs) (Martinand Lock 1983 ; Doetschman et al. 1985) .This culture system provides one with anin vitro model for cardiogenesis (seebelow) that complements the whole ani-mal studies described above . The advan-tages of the in vitro system are (a) rapidgeneration of embryonic tissue, (b) easymanipulation of the developing hearttissue at the genetic and biochemicallevels, and (3) rapid screening for thegene modifications that will be the mostuseful to introduce at the whole animallevel. These interrelationships are sum-marized in Figure 2 .
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Cardiovasculogenic Events DuringEmbryoid Body Development
After 2-4 days in culture, simple EBsdevelop that exhibit discrete cellularlayers (Martin and Lock 1983; Doet-schman et al. 1985) . The outer layer ofcells is morphologically and immu-
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nohistochemically similar to embryonicendoderm and stains positive with theEndo-A-specific monoclonal antibody,TROMA-1 (Btvlet et al. 1980). Betweendays 3 and 8 of suspension culture, theEBs exhibit an inner layer of columnarepithelial cells morphologically simi-lar to the embryonic ectoderm (com-plex EBs) and, from days 8 to 12, cysticEBs develop, ranging up to 5 mm indiameter and containing thousands ofcells (Figure 3) .
Morphologic, biochemical, and im-munologic analyses have shown thatcystic EBs develop many embryonicstructures that are important in cardi-ovasculogenesis. In the visceral yolksac, the first hematopoiesis and vascu-lar network of the embryo develops . Asis the case in the embryonic yolk sac,cystic EBs express a-fetoprotein, trans-ferrin, and several lipoproteins (Doet-schman et al. 1985) . Under properculture conditions, these yolk-sac-likecystic EBs develop blood islets thatinitially contain nucleated embryonicerythrocytes expressing embryonicglobin genes (Doetschman et al . 1985) .In long-term differentiation culture,the entire globin gene set, from embry-onic to adult isoforms, is temporallyexpressed in a manner that mimics theisoform switching that occurs duringin vivo development (Lindenbaum andGrosveld 1991). EBs also contain angi-ogenic activity, and they have beenshown to express acidic fibroblastgrowth factor (Risau et al . 1988). Inthis regard, it is noteworthy that in theembryo angiogenic events are posi-tionally and temporally associated withthe first visible manifestations of car-diogenesis (Sadler 1985) .
One of the most striking develop-mental potentials of differentiating EScells is their ability to produce cardiactissue. Examination at the light-microscopic level reveals rhythmic con-tractions at -1 beat per second indistinct foci within the EBs . Transmis-sion electron microscopy shows thatthe contracting cells have the samemyofibrillar structure as myocardialcells in that intercalated disks arepresent at cell-cell borders where the Zbands from adjoining cells converge(Doetschman et al . 1985 ; Robbins etal. 1990) .The degree to which EB cardio-
mvocytes proliferate is unknown . In
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C
general, EB cardiomyocytes start beat-ing from days 6 to 10 of in vitrodifferentiation. About 3-5 days aftercontractions are first detected, thepercentage of EBs that contain con-tractile tissue is maximal and rangesfrom 20% to 50%. Both the timing ofEB development and the extent ofcardiogenic tissue vary considerably,depending upon culture conditionssuch as the serum lot, average size ofthe ES cell aggregates used to initiatethe "differentiation" cultures, frequencyof feeding, density of EBs, and serum
Figure 3 . In vitro differentiation of embryonic stem (ES) cells in suspension culture .(a) Undifferentiated ES cell colonies are derived from the inner cell mass cells of a mouseblastocyst (4-day embryo). The ES cells grow in colonies (-) on a feeder layer of primaryembryonic fibroblasts (-4) . Y90 . (b) Simple embryonic bodies (EBs) have developed after 3 daysof growth in suspension culture in the absence of feeder cells . The outer layer of cells isanalogous to the visceral endoderm of the 4 .5-day embryo, and the inner cells areundifferentiated ES cells . x440 . (c) Complex EB after 6 days of growth in suspension culture .The outer layer of cells is still analogous to the visceral endoderm, but the inner cells have nowdifferentiated into columnar epithelial cells analogous to the embryonic ectoderm of the5.5-day embryo. The cavity in the center is morphologically similar to the proamniotic cavity .x360. (d) Cystic EB after 11 days of growth in suspension culture . This structure is analogousto the yolk sac that first appears in an 8-day embryo . At this stage of differentiation in culture,beating foci can be detected and blood islets begin to appear (not shown) . x50. FromDoetschman et al. (1985) and Doetschman (1991) .
concentration. By 10 days after thefirst EBs begin contracting, nearly allhave ceased beating, as an individualEB will beat only for -7 days .
From light-microscopic observationsand in situ hybridizations using thecardiac-specific a-cardiac myosin heavychain (MHC), it appears that -10% ofthe tissue in the contracting EBs iscardiac tissue . Hence, in a differentia-tion experiment in which 50% of theEBs are beating, -5% of the cells arecardiomyocytes . Since the cardiac tis-sue is morphologically indistinguisha-
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48
Figure 4. Shown is an in situ hybridization to adjacent sections of a beating embryoid body(EB), carried out with [a-35 S]-labeled riboprobes prepared for the a-cardiac (a) and n-cardiac(b) myosin heavy-chain transcripts . The highly focused pattern of expression of the a transcriptis apparent (->). No hybridization is detectable in comparable sections prepared fromnonbeating EBs. x140.
ble from the other nonepithelial orblood islet tissue, it is not possible todissect out the cardiac compartmentfrom the EBs. If cardiac-specific sur-face antibody markers could be devel-oped, however, it would be possible toseparate cardiomyocytes from EB cellsuspensions by fluorescence-activatedcell sorting (FACS) .Recently, the EBs have been ex-
amined with molecular genetic tech-niques . A complementary DNA librarywas made by using mRNA derivedfrom native EB cultures that con-tained a high proportion of beatingEBs (Robbins et al . 1990). With themyosin heavy-chain cDNA sequenceused as a probe, the library wasscreened extensively under conditionsthat enabled detection of all striated
myosin cDNA sequences . Obtainedwere 200 positives and, when 43 werescreened with oligonucleotides spe-cific for the a-cardiac, P-cardiac, andembryonic skeletal transcripts, 42 ofthem were found to encode either thea or Q isoforms . Additional experi-ments indicated that expression of thecardiac myosin transcripts was tempo-rally controlled during EB develop-ment (Sanchez et al. 1991). The rela-tive amounts of the a and P transcriptsin the EBs (a ratio of -1:1) mimic thesituation present during early cardio-genesis (around days 9-10) in themouse (Robbins et al. 1990; Ng et al .1991). Recently, studies using the poly-merase chain reaction and in situhybridization analyses were carriedout on single, developing EBs. Thesestudies have shown that expression ofthe cardiac transcripts is associatedwith the competence of the EB to beat(Sanchez et al . 1991). With nucleicacid probes specific for the myosintranscripts that are expressed in theheart during fetal development, thespatial location of the cardiac myosinheavy-chain RNAs in the beating EBshas been determined (Figure 4) . Thea-cardiac transcript is localized todistinct foci in these EBs . The patternof (3-cardiac transcript expression ismore diffuse, but includes the foci inwhich the a-MHC transcripts are lo-cated. These data are consistent withthe situation in vivo, in which thea-cardiac transcript is localized in thecardiac compartment and the (3-cardiac transcript, while expressed athigh levels in the developing ventri-cles, also encodes the myosin isoformthat is present in the slow skeletalmuscle fibers .
Future Directions
Clearly, murine ES cells offer great po-tential for studying both the normalphysiology and the pathophysiology ofthe cardiovascular system . The ability tocreate defined, targeted mutations in thegenes that underlie cardiovascular devel-opment and function should yield directstructure-function correlates . For ex-ample, questions concerning the rolesplayed by the different contractile pro-tein isoforms can be answered directly bygene ablation studies. One such studyhas been initiated with the muscle en-
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possible to create the corresponding ani-mal model, and subject it to variousexercise and drug regimens. Similarly,with the use of appropriate transcrip-tional regulatory elements, it is nowpossible to inappropriately express aparticular contractile protein isoform,either during development, or in theincorrect cardiac compartment (VanDeursen et al. 1991). Although suchstudies can currently be performed onlyin the mouse, the ability to performphysiologic determinations on these ani-mals is being rapidly developed . Withrespect to the cardiovascular com-partment, aortic coarctation has beensuccessfully accomplished in the intactmouse (K. Chien, unpublished), and nu-merous data regarding cardiac functioncan be obtained in isolated, perfusedworking hearts (Ng et al . 1991) . Thus, inaddition to the general analyses carriedout on the transcript, protein, and cyto-logic levels, the consequences of a partic-ular mutation, ablation, or overexpressionof a protein on cardiac function can alsobe determined.
The ability to generate large numbersof defined mutants has, in lower organ-isms, resulted in fundamental discover-ies as to how physiologic systems de-velop, differentiate, and function . Wenow have the capability of carrying outsimilar analyses on the mammalian cardio-vascular system. These studies, coupledwith the ability to analyze the resultantphenotype in vivo and in vitro by usingthe ES-EB system, will help delineatethe structural and mechanistic under-pinnings of both normal and aberrantcardiac development and function. As invitro development of the EB is definedfurther in terms of the expression ofother genes that are active during earlymesoderm induction and cardiogenesis,in vitro manipulation and analysis of theprocesses involved will become possible.This system should become increasinglyuseful in dissecting out the factors andmechanisms that underlie mammaliancardiogenesis and maturation of thecardiac compartment .
•
Acknowledgments
This work was supported by grants fromthe National Institutes of Health (HL41496 to J.R and TD .; HL 22619 toJ.R .), the American Heart Association(J.R. and T.D .), and a Biomedical Re-
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search Science Grant (TD.) . J.R. is anEstablished Investigator of the AmericanHeart Association.
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50
Connexins and the HeartGlenn I . Fishman
Gap junctions are specialized regions of adjoining cell membranescomposed of numerous intercellular low-resistance channels. In theheart, these channels electrotonically couple adjacent myocytes andsynchronize the cardiac action potential . Signaling through gapjunction channels may also influence embryogenesis and development .Recent studies have identified the connexin gene family whose proteinproducts assemble to form gap junction channels. Studies of connexingene expression and function are providing new insights into thebehavior ofgap junction channels in the heart. (Trends Cardiovasc Med
1992;2:50-55)
Gap junctions are membrane structurescontaining numerous intercellular low-resistance channels . In excitable tissues,gap junctions are the site of electricalcoupling between neighboring cells. No-where is this coupling more importantthan in the heart, where the propagationof the action potential depends uponcurrent flowing rapidly from cell to cell .Evidence is also mounting that gap
junction channels regulate the passageof signaling molecules, which are impor-tant during early embryogenesis and
development. In an attempt to betterunderstand these ubiquitous channelsand define their role in cardiac electro-physiology and developmental biology,molecular techniques have recently beenapplied to study gap junction structureand function [for recent reviews, seeBennett and Spray (1985), Page andManjunath (1986), Hertzberg andJohnson (1988), and Bennett et al. (1991)] .A family of genes known as the connex-ins has been identified whose proteinproducts assemble to form gap junctionchannels (Beyer et al. 1990). Gap junc-tion channel behavior in the heart is
Glenn I. Fishman is at the Department ofMedicine, Cardiology Division, and the De-partment of Molecular Genetics, Albert Ein-stein College of Medicine, Bronx, NY 10461,USA.
complex, reflecting both modulation ofconnexin gene expression as well asmodification of the connexin proteinsthemselves . In this review, we describeour current understanding of gap junc-tion channels in the heart, with particu-lar emphasis on the results of recentmolecular studies .
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Structure of the GapJunction Channel
Gap junctions were first recognized nearly25 years ago by their distinctive appear-ance in thin-section electron micrographs .These studies showed regions of apposedcell membranes separated by a 1- to2-nm "gap" (Revel and Karnovsky 1967) .Analysis of isolated gap junction prepa-rations from liver and heart with x-raydiffraction and electron microscopy re-vealed an array of particles within func-tional plaques that represented numerousindividual channels (Caspar et al. 1977 ;Manjunath and Page 1985) . Based oncurrent models, each gap junction chan-nel results from the assembly of 12similar, if not identical, polypeptide sub-units known as connexins. Within eachcell, six connexins aggregate to form ahemichannel, or connexon. Connexonsfrom apposing cells align in mirror-image symmetry and join within theintercellular "gap" to form a complete
