e-mail: [email protected] The JacksonLaboratory, 600MainStreet, BarHarbor, ME04609,USA. are encodedbytheJagged( short-range intercellularinteractions.Inmammals,theNotchligands neighboring cells.ThisrestrictstheNotchpathway toregulating interact withsingle-passtypeItransmembraneligandsexpressed on and amembrane-tetheredintracellulardomain.Notchreceptors proteolytically cleaved heterodimercomprising alarge ectodomain Each Notchfamily receptorexists atthecellsurface asa repeated copiesofanepidermalgrowth factor (EGF)-like motif. domain ofNotchfamily proteinscontainsupto36tandemly been described:NOTCH1 throughtoNOTCH4. Theextracellular (Fig.1).Inmammals,fourNotchfamily receptorshave Notch family receptorsarelarge single-passtypeItransmembrane Core componentsoftheNotchsignalingpathway and physiologyinvertebrates. that theNotchsignalingpathway playsduringvascular development diseases andcancers.Inthisreview, Iwillhighlightthemultipleroles signaling contribute tothepathogenesisofseveral inheritedhuman proliferation andcelldeath.Inaddition,perturbationsinNotch regulates boundaryformationbetweencellpopulations, cell making binarycellfate decisions.However, Notchsignalingalso Notch signalingfrequentlyplaysacrucialroleinprecursorcells development inallmetazoanorganisms intheAnimalkingdom. intercellular signalingmechanismessentialforproperembryonic The Notchsignalingpathway isanevolutionarily conserved, Introduction disease. Notch pathwaymodulationfortherapeuticapplicationsin summarize recentfindingsanddiscussthegrowingrelevanceof inherited vascularandcardiovasculardiseases.Inthisreview, I smooth musclecells.DefectsinNotchsignalingalsocause the differentiation andphysiologicalresponsesofvascular during bothnormaldevelopmentandtumorangiogenesis, muscle cells,regulationofbloodvesselsproutingandbranching artery/vein differentiation inendothelialandvascularsmooth physiology invertebrates.Theserolesincluderegulationof that playsmyriadrolesduringvasculardevelopmentand Notch signalingisanancientintercellularmechanism Thomas Gridley Notch signalinginvasculardevelopmentandphysiology (2007)doi:10.1242/dev.004184 Development 134,2709-2718 DLL3 nucleus, theNICDformsacomplex withtherecombinationsignal nuclear localizationsignalslocated intheNICD.Once fragment translocatestothenucleus becauseofthepresence complex, freestheNICDfromcellmembrane.Thiscleaved Notch heterodimer. Thefinal cleavage, catalyzedbythe proteolytic cleavages inthemembrane-tetheredfragmentof (NICD). Thereceptor-ligand interactioninducestwo additional subsequent nucleartranslocationoftheNotchintracellulardomain process involving theproteolytic cleavage ofthereceptorand Upon ligandbinding,asignalistransmittedintracellularlyby a and DLL4 genefamilies. ) JAG1 and JAG2 ) andDelta-like ( ␥ -secretase DLL1 , components ofthe ubiquitin ligaseMib1(Barsietal.,2005;Koo etal.,2005); Notch transcriptionalregulator Rbpj(Krebsetal.,2004); theE3 Dll4 (Duarteetal.,2004;GaleKrebs2004);the Uyttendaele etal.,2001);theligandsJag1(Xue1999)and et al.,2005)andNotch4(Carlson2005;Krebs2000; receptors Notch1(Huppertetal.,2000;KrebsLimbourg demonstrated aroleinembryonicvascular development includethe mutants forwhichtargeted mutagenesisandtransgenicstudieshave targeted mousemutantsinNotchpathway components.Mouse was initiallysuggestedbasedonfindings fromtheanalysisofseveral A rolefortheNotchpathway inregulating vascular development Artery-vein differentiation 2006). 2006b; IlaganandKopan, 2007;Kageyama etal.,2007;LeBorgne, recent reviews (Bray, 2006;Ehebaueretal.,2006a; biochemistry oftheNotchsignalingpathway canbefoundinseveral (Hes/Hey) family (Kageyama etal.,2007).Furtherdetailsonthe split/hairy andenhancerofsplitrelatedwithYRPWmotif (bHLH) transcriptionalrepressorsofthehairyandenhancer commonly inducedNotchtarget genesarethebasichelix-loop-helix transcriptional activation ofNotchtarget .Amongthemost (MAML1) proteinandhistoneacetyltransferases,leadingtothe complex recruitsotherproteins,suchasthemastermind-like 1 repressor complex fromtheRBPJprotein.TheNICD-RBPJ translocation oftheNICDdisplaceshistonedeacetylase–co- other componentstoformaco-repressorcomplex. Thenuclear ofthesegenesbyrecruitinghistonedeacetylasesand regulatory elementsofvarious target genesandrepresses NICD, theRBPJproteinbindstospecific DNA sequencesinthe – asequence-specific DNA-binding .Intheabsenceof binding proteinforimmunoglobulin kappaJregion (RBPJ)protein venous cell fate (Jonesetal.,2006). Indeed,recentwork has endothelial cellsatthisstageare notyetcommittedtoanarterialor pattern isestablishedpriorto theinitiationofbloodflow, but arteriovenous differentiation. Thisgeneticallydeterminedpre- by theNotchpathway, playsaprimaryrolein regulating work hasestablishedthat genetic pre-patterning,largely mediated endothelial cellsthatlineveins. However, asdescribedbelow, recent of hemodynamicflow andhigheroxygentensionsthandothe cells thatlinearteriesexperience higherbloodpressures,rates differentiation ofarteriesandveins was bloodflow. Theendothelial endothelial cells. development was toregulate thespecification ofarterialfate in that aprimaryfunctionoftheNotchpathway during vascular zebrafish embryoswithreducedNotchsignalinggave thefirst clues placenta andembryoproper(Fig.2).However, ananalysisof angiogenic vascular remodelingintheextraembryonic yolksac, exhibit asimilarphenotypecharacterizedbytheabsenceof (Fischer etal.,2004;Kokubo etal.,2005).Mostofthesemutants Notch pathway downstream effector bHLHproteinsHey1 andHey2 2003), presenilin1and2(Herremanetal.,1999);the It hadlongbeenbelieved thattheprimaryfactor thatregulates the ␥ -secretase complex, suchasnicastrin(Lietal., REVIEW 2709

DEVELOPMENT repressor certain Notchtarget genes, suchasthebHLHtranscriptional 2001). Asimilarphenotypewas observed inembryosmutantfor expression ofarterialmarkers invenous vessels (Lawson etal., phenotype: suppressionofvein-specific markers withectopic signaling hadbeenectopicallyactivated exhibited thereverse markers intonormallyarterialdomains.Embryosinwhich Notch from arterialvessels withanaccompanying expansion ofvenous exhibited alossofexpression ofarterialmarkers suchasephrinB2 and veins withoutanintervening capillarybed). Theseembryosalso accompanying arteriovenous malformations(thefusionofarteries poorly formeddorsalaortaandposteriorcardinalvein with Lawson etal.,2002).Notchsignaling-deficient embryosexhibit a was studiedfirst inzebrafish (Lawson etal.,2001; cardinal vein – thedorsalaortaand posterior blood vessels ofthetrunk– VEGFA pathways inregulating theformationoflarge axial TherolesandinterdependenceoftheNotch Welsh, 2006). (Byrne etal.,2005;CoultasShibuya andClaesson- also regulates multipleotheraspectsofbloodvessel homeostasis secreted glycoproteinthatisapotentinducerofangiogenesis (VEGFA) pathway. VEGFA isa endothelial growth factor A regulator ofvascular development andphysiology, thevascular vascular development isintertwinedwiththatofanothermajor (Vogeli etal.,2006). rise toendothelialcellprogeny thatpopulatebotharteries andveins precursor ofasubsethematopoieticandendothelialcells,cangive established that,inzebrafish, asinglehemangioblast,thebipotential 2710 et al.,2002)(Fig.3).Areduction in determining arterialandvenous cellfates inthesevessels (Lawson zebrafish embryorevealed asignalingcascadethatisresponsible for (Zhong etal.,2001;Zhong 2000). arterial expression ofvein markers. Conversely, theinjection of arterial marker expression fromthedorsalaortaandinectopic The roleoftheNotchpathway inregulating earlyembryonic This analysisoftheformation of themajortrunkvessels inthe REVIEW hey2 (also referredtoinzebrafish asthe Signal-receiving cell Signal-sending cell HDAc Repressed RBPJ vegfa CoR X activity resultsina lossof NICD JAG1,2 DLL1,3,4 NOTCH1-4 ␥ -Secretase gridlock MAML1 Activated RBPJ HAc Nucleus ) HDAc Hes/Hey differentiation iscompromisedin constricted oratretic.Itisnotknown whetherartery-vein Blood vessels, despiteformingintheseembryos,areseverely haploinsufficiency (Carmelietet al.,1996;Ferraraet1996). heterozygous fora that Vegfa isessentialforvascular development. Mouseembryos 2003). Targeted-mutagenesis studiesinmicehave alsodemonstrated endothelial cells,but notinvenous endothelialcells(Liuetal., induces pathway downstream oftheVegfa pathway. VEGFA administration the expression ofarterialmarkers (Lawson etal.,2002). activated rescue arterialmarker geneexpression, theexpression ofan mRNA intoNotchsignaling-deficient zebrafish embryoscouldnot downstream oftheVegfa pathway. Whereasinjectionof work alsodemonstratedthat,inthissetting,theNotchpathway acts shha acts upstreamof shha also exhibit alossofarterialdifferentiation, whereasinjectionof observed in hedgehog a regulated bytheexpression ofthesecretedmorphogen ephrin B2 vegfa expressing onlyVegfa 188exhibit impairedretinalarterial severe defects inretinalvascular outgrowth, whereasmice development. However, micethatexpress onlyVegfa 120exhibit express onlytheVegfa 164isoform displaynormalretinalvascular 120, Vegfa 164andVegfa 188).Genetically engineered micethat results intheproductionofseveral different proteinisoforms (Vegfa differentiation inmice. Alternative splicingofthemouse demonstrated aroleof However, othergain-of-functiontransgenicexperiments have CoR Studies inmammaliancellculturehave also placedtheNotch mRNA causestheectopicexpression ofarterialmarkers. mutant embryosrestoresnormalarterialdifferentiation. This mRNA inducesectopicexpression ofthearterialmarker notch1a NOTCH1 vegfa in theposteriorcardinalvein. ( shha -deficient embryos, vegfa transgene in ) alongtheaxialmidline.Similartowhatis and , becausetheinjectionof genes. transcriptional activationofNotchtarget to theNICD-RBPJcomplex,leading histone acetyltransferases(HAc),are recruited of anactivationcomplex,suchasMAML1and complex from theRBPJprotein. Components deacetylase (HDAc)–co-repressor (CoR) the RBPJprotein, displacingahistone nucleus (blue),where itformsacomplexwith membrane. TheNICDtranslocatestothe intracellular domain(NICD)from thecell proteolytic cleavagesthatfree theNotch ligand interactioninducestwoadditional tethered intracellulardomain.Thereceptor- of alargeectodomainandmembrane- proteolytically cleavedheterodimer consisting existsatthecellsurfaceasa cell (lowercell,showninyellow).TheNotch (NOTCH1 through toNOTCH4)onanadjacent green) interactwithNotchfamilyreceptors DLL3, DLL4)families(uppercell,shownin Jagged (JAG1andJAG2)Delta-like(DLL1, Notch signalingpathway. Fig. 1.Core componentsofthecanonical Vegfa Vegfa DLL4 in regulating arterialendothelialcell vegfa targeted mutationexhibit lethal expression inhumanarterial -deficient embryoscouldrescue shha Vegfa mutant zebrafish embryos Development 134(15) +/– vegfa vegfa mouse embryos. Ligands ofthe expression is mRNA into Vegfa vegfa sonic shha gene

DEVELOPMENT sizes. ( remodeling oftheyolksacvasculature togeneratevesselsofdifferent Dll4 the examination of of arterialmarkers andtheoccurrence ofarteriovenous (Kume etal.,2001),accompanied byreducedorabsentexpression display defectsinvascular remodelingintheyolksacandembryo and al., 2000).Mouseembryoswith compoundmutationsofthe family ofevolutionarily conserved transcriptionfactors (Kaestneret forkhead (Fox; alsoknown aswingedhelix)proteinsarealarge exhibit arteriallyrestrictedexpression inearly embryos.The target (Grego-Bessa etal.,2007). the ephrinB2genehasrecentlybeenshown tobeadirectNotch inculturedendothelialcells(Isoetal.,2006),and expressed genes,Dll4-mediatedNotchsignalinginducesephrinB2 signaling inregulating theexpression ofimportant arterially et al.,2005;Krebs2004).InsupportofadirectroleforNotch (Duarte etal.,2004;FischerGaleKokubo Hey1; Hey2 Notch signaling-deficient mouseembryos,suchas zebrafish embryos,both Similar tothatwhichisobserved inNotchsignaling-deficient heterozygous embryos(Duarteetal.,2004;Gale2004). homozygotes was similar, althoughmoresevere, thanthatof development. Similarlyto al., 2002). associated vessels inmice(Mukouyama etal.,2005;Mukouyama et required forarterialdifferentiation ofsmall-diameternerve- derived fromsensoryneurons,motorneuronsandSchwann cellsis expression inmouseprimaryembryonicendothelialcells, andVegfa gene venules (Visconti etal.,2002). Vegfa couldinduceephrinB2 while reducingthenumberofephrinreceptorB4(EphB4)-positive number ofephrinB2(Efnb2)-positive capillariesinthemouseheart 2002). Vegfa 164overexpression incardiacmuscle increasedthe development, but normalvenous development (Stalmansetal., ( Fig. 2.Vascular defectsina Development 134(15) some (Duarte etal.,2004;GaleKrebs2004).However, exhibit embryoniclethalhaploinsufficiency duetovascular defects Press. (Krebs etal.,2000).Copyright(2000)ColdSpringHarborLaboratory vascular endothelialcells.Reproduced withpermissionfrom Krebs etal. been immunostainedwithanantibodytoaprotein expressed on undergone anyangiogenicvascularremodeling. Bothyolksacshave has arrested attheprimitivevascularplexusstage,andhasnot A Extraembryonicyolksacfrom awild-typemouseembryoexhibits ) Little isknown aboutthetranscriptionalregulation ofgenesthat In mice,Dll4istheprimaryNotchligandrequiredforvascular +/– Foxc2 Dll4 B ) Yolk sacfrom a heterozygous embryosoninbredgeneticbackgrounds +/– genes, two relatedFox family transcriptionfactors, double-mutant embryos,donotexpress arterialmarkers mice areviableonanoutbredbackground,permitting Dll4 Notch1 Dll4 –/– Vegfa Notch1 embryos. Thephenotypeof -deficient embryosandothertypesof –/– mutant mouseembryo.Theyolksac +/– -null mouseembryo. heterozygous mouseembryos, Rbpj mutant and Dll4 Dll4 Foxc1 +/– –/– Notch3 both ofthesemarkers ismarkedly downregulated inarteriesof the SM22 galactosidase proteinfromarterial-specific regulatory elementsof arterial defectsoccurin smooth musclecellsofarteries,but notinthoseofveins. Marked Foxc1 andFoxc2 proteinsdirectlyactivate al., 2003).InNotchsignaling-deficient mouseembryos(e.g. visualize thepresenceofarteriovenous malformations(Sorensenet mouse embryos,injectionofinkintotheheartisaneffective way to establish ormaintaindistinctarterialandvenous vascular beds.In the formationofarteriovenous malformationsisthefailure to an arteriovenous malformation.Onemechanismthatmightexplain aberrant directcommunicationbetweenanarteryandvein istermed connect toveins onlythroughanintervening capillarybed.An embryos formarteriovenous malformations.Arteriesnormally or independentof,Vegfa signaling. is notknown whether during arteriovenous differentiation (Seoetal.,2006).However, it transcriptional regulators thatactupstreamoftheNotchpathway gene. TheseresultsdemonstratethattheFoxc proteinsarekey through aFoxc-binding elementintheupstreamregion ofthe Notch4 it shouldbenotedthatnoobvious phenotypeisobserved in vascular phenotypes(Carlsonetal.,2005;Uyttendaele2001), the expression ofanactivated embryonic period.Althoughgain-of-functioninvivo assaysusing Notch signalingtoarterializebloodvessels isnotconfined tothe expression isrepressed.Thesestudiesdemonstratethat theabilityof arterial vascular smoothmusclecellsof postnatally, becausearterialvessels fail tomature.Morphologically, wild-type arteries(Domengaetal.,2004).Thesedefectsarise with athinnervascular smoothmusclecellcoatthanisfoundin der Loopetal.,1997)andatransgeniclineexpressing the muscle cellsandnotinvenous ones.Theseincludesmoothelin(van known tobeexpressed predominantly inarterialvascular smooth those surroundingveins inwild-typemice.Onlyafew markers are smooth musclecells.The Notch signalingalsoregulates arterialspecification ofvascular arteries of or is likely tobeduedisruptedregulation of malformations (Seoetal.,2006).Thisfailure ofarterialspecification independently. vascular smoothmuscle cellssurroundingthem,isspecified demonstrate thatthearterialidentity ofendothelialcells,andthe Hes1, Hey1, Hey2 andHeyl (Domengaetal.,2004).Theseresults connexin 40(alsoknown asGja5–MouseGenomeInformatics), endothelial cellarterialmarkers occurs,includingthatofephrinB2, vascular smooth muscle cells,normalexpression ofseveral malformations arereversible ifactivated uterus andskin(Carlsonetal.,2005).Surprisingly, these arteriovenous malformationsinseveral organs, includingliver, induction ofvenous expression oftheephrinB2gene,andcauses transgene inadultmiceresultsvessel arterialization, suchasthe Interestingly, theinducibleexpression ofanactivated (Fig.4)(Krebsetal.,2004). 2004; Krebsetal.,2004)form detectable byhistologicalanalysis(Duarteetal.,2004;Gale arteries in this indicatesthatvascular smoothmusclecellsthatsurround As mentionedpreviously, Notchsignaling-deficient zebrafish In additiontoregulating arterialspecification ofendothelialcells, Notch1 –/– –/– mice (Krebsetal.,2000). ␣ –/– Notch3 mice (Fig.5).Combinedwiththemorphologicaldata, Notch3 promoter (Moessleretal.,1996).Theexpression of embryos) arteriovenous malformationsthatarealso –/– –/– mice, whichdonotexpress arterialmarkers for mice have acquiredavenous fate. Notably, in Foxc1 Notch3 Notch3 and Notch4 –/– Foxc2 mice, includingenlarged arteries gene isexpressed invascular transgene cancausemutant expression isdownstream, Notch3 Dll4 Notch4 Dll4 –/– transcription. The REVIEW mice resemble transcription transgene Notch4 Dll4 2711 Dll4 +/– ␤ -

DEVELOPMENT al., 2004).Copyright(2004)ColdSpringHarborLaboratoryPress. promotion ofarterialcelldifferentiation byblocking (bluecross) ERKactivation.AandBare reproduced withpermissionfrom suppressing reception oftheVegfa signalandactivation ofNotchsignaling.Inaddition,theactivationPI3K/Aktsignaling mechanisms inhibitarterydifferentiation (bluetext).Theorphannuclearreceptor COUP-TFII(Nr2f2)suppresses neuropilin 1ex Dll4 Notch pathway(green box)andthePLC genetic regulation ofartery-veindifferentiation. (C)Duringarterydifferentiation, twoprimarysignalingpathwaysoperatedo of arterialdifferentiation. ( influencing the differentiation, migration andproliferationof regulates sproutingand branchingbehaviors inthesevessels by blood vessels whenNotch signalingisinhibited.TheNotchpathway to allofthesestudiesistheincreased sproutingandbranchingof 2006; Ridgway etal.,2006; Scehnetetal.,2007).Afinding common Lawson, 2007);andxenograft tumormodels(Noguera-Troise etal., 2007); thezebrafish embryo (Leslieetal.,2007;Siekmannand 2007; Lobov etal.,2007;Ridgway etal.,2006;Suchting model systems:themouseretinaandhindbrain(Hellstromet al., angiogenic sproutsinHUVECs,aswellseveral additional cell numbers,filopodia extension intipcellsandthebranchingof DLL4/Notch signalinghasnow beendemonstratedtoregulate tip understanding oftheroleNotchsignalingintipcellformation. bifurcation. subsequently ledtoincreasedbranchingasaresultofvessel with bothdaughtercellsbeingspecified astip cells.This sprouts. ThesuppressionofNotchsignalingledtotipcelldivision, signaling inhibitsbranchingatthetipofdeveloping angiogenic using humanumbilicalvein endothelialcells(HUVECs), Notch (Sainson etal.,2005).Inaninvitroangiogenesisculturesystem endothelial tipcells,arolethatwas first describedseveral yearsago pathway inregulating theformationandfunctionofthese 2004). Recentwork hasidentified aprimaryrolefortheNotch sprouts alongVegfa gradients(Gerhardtetal.,2003;Gerhardt the localextracellular environment, guidingthegrowth ofthese of vascular sproutsthatextend filopodia inresponsetocueswithin vessels. Tip cellsarespecializedendothelialsituatedatthetips During angiogenesis,new capillariessproutfromexisting blood Endothelial tipcelldifferentiation Fig. 3.Artery-veindifferentiation. 2712 Numerous recentpapershave confirmed andextended our gene expression, butitisunknownwhether REVIEW B ) Yolk sacfrom an ( A ␥ ) Yolk sacfrom awild-type( /MAPK pathway(pinkbox)(LamontandChilds,2006).ThetranscriptionfactorsFoxc1Foxc2induce Rbpj Foxc1 –/– mutant mouseembryo,whichdoesnotexpress theephrinB2 and Foxc2 expression isregulated byVegfa. (D)Duringveindifferentiation, twodifferent Rbpj +/+ ) mouseembryo,whichexpresses anephrinB2 reagents (Lobov etal.,2007;Ridgway etal.,2006)or (Hellstrom etal.,2007),orby administeringanti-Dll4blocking temporally-regulated al., 2007;Lobov etal., 2007; Suchtingetal.,2007)ormicewith genetically, byassessing defects areconcordantwhether Dll4/Notchsignalingisreduced defects intheearlypostnatal retinal vasculature. Theobserved vasculature, andreducedDll4/Notchsignalingleadstostriking administration ofexogenous agents. accessible bothforobservation andfortheexperimental venous vessels. Duringthesestages,theretinal vasculature is that formsinitiallyisthenremodeledintolarge andsmallarterial grows radiallytowards theperiphery. Theprimitive vascular plexus system emerges first intheregion oftheopticnerve head,andthen reproducible spatialandtemporalpattern.Theretinalvascular The mouseretinalvascular systemdevelops postnatallyinahighly Friedlander, 2006;GarianoandGardner, 2005;Uemuraetal.,2006). for theanalysisofdevelopmental angiogenesis (Dorrelland other organs, themouseretinapossessesseveral distinctadvantages 2007; Ridgway etal.,2006;Suchting2007).Comparedwith retinal vasculature inmice(Hellstrometal.,2007;Lobov etal., modulating Notchsignalingonthedifferentiation ofthedeveloping DLL4 genetic manipulationsthatblockVEGFA functionreduceboth downstream oftheVEGFA signal,becausepharmacological or of tipcellformationandangiogenicsproutingbyNotchsignalingis cell numbers,filopodia extension andvessel branching.Suppression vascular tipcells;reducedNotchsignalingleadstoincreasesin The Several oftheserecentstudieshave assessedtheeffects of expression andbloodvessel sprouting. Dll4 gene ishighlyexpressed inthedeveloping retinal Notch1 Dll4 +/– deletion intheretinalvasculature heterozygous mice(Hellstrom et -lacZ gene. ( -lacZ Development 134(15) antagonizes the wnstream ofVegfa: the Krebs etal.(Krebs et pression, thereby transgene, amarker C , D ) Modelfor ␥ -secretase

DEVELOPMENT Notch1 embryos (Leslie etal.,2007).Inaddition,the reductionofVegf sprouting andtheectopic observed in small moleculeinhibitorprevents bothnormalendothelial Siekmann andLawson, 2007).BlockingVegfa signalingwitha roles duringearlyvascular development (Leslieetal.,2007; embryos, suggestingthatadditional Notchligandsplayimportant morphant embryosaremoresevere thanthosein quiescent stalkcells.Interestingly, vascular defectsin cells intotheinitialsprout,andbyproliferationofnormally are incorporatedviaboththeincreasedmigrationofendothelial sprouts containmorecellsthanincontrols.Theseadditional an endothelialcell-specificdeletionofthe (aa) andentersthedescendingdorsalaorta(da).( visualize bloodflowasitexitsanteriorlythrough theaorticarch arteries both time-lapse confocalmicroscopy onliving embryosrevealed that,in specify endothelialcellfate insegmental arterysprouts.Theuseof indicate thatNotchsignalingisrequiredcell-autonomously to developing sprouts(SiekmannandLawson, 2007).Theseresults become locatedpreferentiallyinthedorsalaortaorbase of Conversely, transplantedcellsthatexpress activated Notch1 cardinal vein orthemostdorsalpositionofsegmental arteries. dorsal aorta,but becomelocatedpreferentiallyintheposterior transplanted cellsthatlack time-lapse confocalmicroscopy. Mosaicanalysishasrevealed that system forhigh-resolutionfluorescentmicroscopy, includinglive proteins inendothelialcellsmakes thisanimalmodelanideal availability ofseveral transgeniclinesthatexpress fluorescent Lawson, 2007).Theopticalclarityoftheseembryos andthe vasculature ofzebrafish embryos(Leslieetal.,2007;Siekmannand and behavior have beenobtained fromtheanalysisof angiogenic sprouts. signaling restrictstheacquisitionofanendothelialtipcellfate in reduced Dll4/Notchsignaling.Thesedataindicatethat and unc-5homologB( tip cells,suchasplateletderived growth factor receptorbeta( vascular plexus fusetoformsyncytial sinuses.Markers specific for front, andintheinteriorofplexus. Furthermore,portions ofthe with increasedfilopodial extensions bothat the growing vascular The vascular plexus hasanincreasedcapillarydensityanddiameter, vasculature inthesemicedisplayssevere patterningdefects(Fig.6). inhibitors (Hellstrometal.,2007;Suchting2007).Theretinal mice. Fig. 4.ArteriovenousmalformationsinNotchpathwaymutant Development 134(15) and Kopan(Weinmaster andKopan,2006). Krebs inmylaboratory;reproduced withpermissionfrom Weinmaster leak directly intothevenoussystem.Originalimagesprovided byLuke Additional insightsintoendothelialtipcellformation,migration dll4 ( A flox/– ) Inawild-typeembryo,Indiainkisinjectedintotheheartto morphant and ), arteriovenousmalformationspermittheinjectedinkto Unc5b rbpj morphant embryos,segmental artery ), arealsoupregulated inmicewith function donotcontribute tothe Notch1 B gene ( ) Inanembryowith dll4 dll4 Tek-Cre/+; morphant morphant Pdgfrb rbpj ) chain (smmhc,green) expression inwild-typeand Notch3 nuclei are stainedwithDAPI,blue)expression inwild-typeand staining (blue)oftailsfrom wild-typeand for propervascular morphogenesis. negative regulator ofVegfa-induced angiogenesis,andisessential and thezebrafish embryo indicatethatNotchsignalingactsasa Lawson, 2007).Taken together, thestudiesinbothmouseretina behavior indeveloping segmental arterysprouts(Siekmann and normally repressVegf receptor3andthus limitangiogeniccell knockdown phenotype, suggestingthatNotchactivation might levels in receptor 3(alsoknown asFlt4–Zebrafish InformationNetwork) (B) Arteriesofthe vascular smoothmusclecells. Fig. 5.Notchsignalingspecifiesarterialdifferentiation of reduced inthe smmhc inthe levels. Arrows highlightectopicvascularsmoothmusclecellsexpressing Laboratory Press. (Domenga etal.,2004).Copyright(2004)ColdSpringHarbor Notch3 vascular smoothmusclecellsincontrol miceandismarkedlyreduced in for a vein (v)demonstratethat (arrows) inthetailand(G,H)microscopic viewthrough artery(a)and SM22 –/– –/– arteries. Reproduced withpermissionfrom Domenga etal. tail arteries.Smoothelinexpression levelsare markedly ␣ rbpj -lacZ Notch3 Notch3 morphant embryospartiallyrescuesthe transgene. (E,F)Whole-mountviewofcaudalartery Notch3 –/– –/– mutant artery. ( mutant artery(D).( –/– ␤ -galactosidase stainingisrestricted toarterial mouse exhibitnormalsmmhcexpression ( A , B ) Smoothmusclemyosinheavy C , Notch3 D ) Smoothelin(red) (cell E-H ) –/– ␤ Notch3 -galactosidase mice heterozygous REVIEW –/– tail arteries. 2713 rbpj -

DEVELOPMENT the leadingedge(top)in areas ofvesselfusion(arrow) andincreased sprouting andbranchingat 2007). Copyright(2007)NationalAcademyofSciences,USA. Reproduced withpermissionfrom Suchtingetal.(Suchtingal., postnatal day5from ( from preclinical modelsintohumanclinicaltrials. However, these 2006). Similarissuesmightarise asanti-DLL4treatmentsprogress chemotherapy treatment (Ferrara andKerbel, 2005;Jainetal., benefit forpatients when itiscombinedwithconventional cancer typeshasbeenfoundto onlyprovide anoverall survival rodents, inclinicaltrials,anti-VEGFA antibody treatmentofseveral VEGFA therapiesinthetreatmentofxenograft tumormodelsin treatments reachtheclinic.For example, despite theefficacy ofanti- promising, many issuesremaintoberesolved beforeanti-DLL4 et al.,2006;Ridgway etal.,2006). tumors whencombinedwithanti-Vegfa therapies(Noguera-Troise treatments, andcouldprovide synergistic effects againstcertain effective againsttumorsthatwereresistanttoanti-VEGFA inhibition oftumorgrowth. Importantly, anti-Dll4therapiesare tumors, leadingtopoorperfusion,increasedhypoxiaandanoverall of thesevessels arenotconnectedtothevascular network inthe revealed thatthenewly inducedvessels functioninefficiently. Many fluorescent lectinsorbyassessinghypoxicregions inthesetumors network intheanti-Dll4-treatedtumorsbyperfusionassayswith despite theincreasedbloodvessel density. Analysisofthevascular tumors. Paradoxically, tumorgrowth was inhibitedinthesemice to amarked increaseintumorbloodvessel densityinthetreated treatment increasesbloodvessel sproutingandbranching, andleads to thefindings inzebrafish embryosandmouseretinas,anti-Dll4 inhibit thegrowth ofseveral different solidtumorsinmice.Similar Dll4 proteinthathave beenmodified toblockDll4/Notchsignaling, (Scehnet etal.,2007)administrationofrecombinantformsthe 2006), andthesystemic(Noguera-Troise et al., 2006)orlocalized anti-Dll4 antibodies(Noguera-Troise etal., 2006; Ridgway etal., Scehnet etal.,2007).Thesystemicadministrationofneutralizing therapeutic target (Noguera-Troise etal.,2006;Ridgway etal.,2006; several recentstudieshave identified theDll4proteinasjustsucha Hainaud etal.,2006;Mailhos2001;Patel etal., 2005),and expressed athighlevels intumorvasculature (Galeetal.,2004; might provide novel drugtargets duringtumorangiogenesis.Dll4is vascular development, proteincomponentsoftheNotchpathway 2006). Given theprominentroleofNotchpathway inregulating preclinical rodentmodels(FerraraandKerbel, 2005;Jainetal., other blockingreagentseffectively inhibitsolidtumorgrowth in express VEGFA, andtherapiesthatuseanti-Vegfa antibodiesor recruitment ofhostbloodvessels intothetumor. Many solidtumors The maintenance,growth andmetastasisofsolidtumors requirethe Tumor angiogenesis Fig. 6.Vascular defectsin 2714 Although theresultsfromstudiesdescribedabove arevery REVIEW A ) wild-type(WT)and( Dll4 Dll4 +/– +/– retinas. retina. a,artery;v, vein. Retinal vasculature at B ) Dll4 +/– mice. Notethe the cyclin-dependent kinaseinhibitors smooth musclecellproliferationassociatedwithreducedlevels of 2006) invascular smoothmusclecellsledtoincreasedvascular overexpression of proliferate atareducedratecomparedwithwild-typecells.The vascular injurywas significantly decreased in neointima (athickened layerofvascular smooth musclecells)after components duringtheresponsetovascular injury, formationofthe support ofafunctionalroleforthemodulationNotchpathway uninjured contralateralcontrolvessels at7-14daysafter injury. In following vascular injury, but isupregulated incomparisonto encoding theseproteinsisdownregulated withinthefirst 2days experimentally inducedvascular injury. Theexpression ofthegenes Notch1, Notch3,Jag1,Jag2,Hey1 andHey2, ismodulatedafter expression ofseveral Notchpathway components,including 2002; Doietal.,2005;Lindner2001;Wang etal.,2002).The pathway genesduringtheresponsetovascular injury(Camposetal., 2007). cardiac neuralcrestcellsintosmoothmuscle(Highetal., Notch signalingplaysanessentialroleinthedifferentiation of inactivated specifically inmouseneuralcrestcellsreveals that Furthermore, invivo studiesinwhichNotchsignalingwas et al.,2006)weredemonstratedtobedirectNotchtarget genes. heavy chain(Doietal.,2006)andsmoothmuscle fibroblast cellline(Doietal.,2006).Bothsmoothmusclemyosin human aorticsmoothmusclecellsandinamurineembryonic reported topromotesmoothmusclecelldifferentiation bothin 2006; Highetal.,2007);JAG1-mediated Notchsignalingis signaling inducessmoothmusclecelldifferentiation (Doietal., al., 2005).However, more-recentstudieshave indicatedthatNotch HEY2 protein(Doietal.,2005;Morrow etal.,2005a; Proweller et repressive effect islikely tobemediatedviatheinductionof cell differentiation duringinvitrocellculture, andthatthis described aroleforNotchsignalinginrepressingsmoothmuscle context-, time-orcellline-dependent.Several groupshave cells. However, contradictoryresultssuggestthatitsrolemightbe differentiation, physiologyandfunctionofvascular smoothmuscle It isclearthatNotchsignalingplaysanimportantroleinthe physiology Vascular smoothmusclecelldifferentiation and VEGFA therapies. treatment, particularlyfortumorsthatareunresponsive toanti- promising studiespresentanovel therapeuticapproachforcancer cells from et al.,2004).Thecultureofprimaryaorticvascular smoothmuscle mechanical strain alsoexhibit reduced proliferation andincreased et al.,2005b).Vascular smoothmusclecellsthat areexposed to muscle celldifferentiation markers (Morrow etal.,2005a;Morrow concomitant withanincreasein theexpression ofvascular smooth reduction inNOTCH1 andNOTCH3 receptor expression, mechanical strainduringin vitro culturecausesasignificant of primaryhumanorratvascular smoothmusclecellstocyclic phenotype inresponsetolocalenvironmental changes.Theexposure differentiated andcanexhibit substantial plasticityintheir physiology. Adultvascular smoothmusclecellsarenotterminally regulating vascular smoothmusclecell differentiation and transcription (Havrda etal.,2006). et al.,2003)or protein directlyinteractswiththe Several studieshave characterized theexpression ofNotch Mechanical forcesareoneofseveral factors implicatedin Hey2 p27 –/– Hey1 kip1 mice hasrevealed thatthesemutantcells ( (Wang etal.,2003)or Cdkn1b ) (Havrda etal.,2006).TheHey2 p27 p21 kip1 waf1/cip1 Development 134(15) promoter torepress Hey2 Hey2 ( ␣ Cdkn1a –/– -actin (Noseda (Havrda etal., mice (Sakata ) (Wang

DEVELOPMENT cerebral vasoreactivity thatsuggestseitherdecreased relaxation these NOTCH3 Arg90Cys transgenicmice revealed animpaired damage tothebrainparenchyma isseen.Physiologicalstudiesof mice. However, despiteGOMaccumulation, noevidence of of bothcerebralandperipheral arteriolesisobserved inthese ectodomain andofGOMdeposits invascular smoothmusclecells al., 2003).Anage-dependent accumulationoftheNOTCH3 Arg90Cys mutation,invascular smoothmusclecells(Ruchouxet NOTCH3 this model,transgenicmiceweregeneratedthatexpress ahuman recapitulates theearly, preclinicalphaseofCADASIL. To create for thismutation.Thesecondmodelmoresuccessfully morphological orbehavioral phenotypes,even whenhomozygous al., 2005).Thesemicedonotexhibit any CADASIL-like introduced intotheendogenousmouse developed. Inonemodel,anArg142Cys knock-inmutationwas containing mutationsfoundinCADASIL patientshave been et al.,2000). constitutes partoralloftheGOMdeposits(Ishiko etal.,2006;Joutel although itiscontroversial whethertheNOTCH3 ectodomain membrane ofvascular smoothmusclecells(Jouteletal.,2000), accumulates inthecerebralmicrovasculature atthecytoplasmic In CADASIL patients,theectodomainofNOTCH3 protein that mutationscauseCADASIL arenot disease, whichiscausedbymutationsinthe affect thedevelopment andfunction oftheheart(Box1).Athird Alagille syndromeandinheritedbicuspidaorticvalve, primarily The vascular defectsassociatedwithtwo oftheseinheriteddiseases, components causeinheritedvascular orcardiovascular diseases. highlighted bythefinding thatmutationsinseveral Notchpathway The importanceofNotchsignalinginvascular development is Vascular smoothmusclecellphysiology:CADASIL Notch signaling. these effects aremediated,atleastinpart,viathe modulationof smooth musclecellgrowth whileincreasingapoptosis, andthat results indicatethatcyclic mechanicalstraininhibitsvascular apoptotic cellstothelevels observed inunstrainedcells.These such mechanicalstrainrestoredthepercentagesofproliferative or intracellular domainsinvascular smoothmuscle cells exposed to apoptosis. Overexpression oftheNOTCH1 orNOTCH3 Development 134(15) the of any examples inCADASIL patientsofmutationsordeletions characteristic natureofthesemutations,inadditiontotheabsence in theextracellular domainoftheNOTCH3 receptor. The gain orlossofacysteine residueinoneofthe34EGF-like repeats CADASIL. muscle cellsisoneofthemostdistinguishingfeatures (Kalaria etal.,2004).GOMaccumulationinvascular smooth (GOM) withinthesmoothmusclecellbasementmembrane muscle cellsandtheaccumulationofgranularosmiophilicmaterial characterized bytheprogressive degeneration ofvascular smooth cognitive decline,dementiaandprematuredeath.CADASIL is disorders, recurrentsubcorticalischemicstrokes, progressive exhibit avariety ofsymptoms,includingmigraines,mood vascular dementia(Kalariaetal.,2004).Affected individuals termed CADASIL, isthemostcommongeneticformofstroke and arteriopathy withsubcorticalinfarcts andleukoencephalopathy, affects vascular smoothmusclecells.Cerebralautosomaldominant et al.,1996),isaninheriteddegenerative vascular diseasethat Two different mousemodelsthatexpress NOTCH3 proteins All NOTCH3 NOTCH3 cDNA thatcontainsadifferent CADASIL mutation,the gene thatareobviously inactivating, stronglysuggests mutations associatedwithCADASIL resultina Notch3 NOTCH3 NOTCH3 gene (Lundkvistet -null alleles. gene (Joutel rescuing thearterialdefectsof human NOTCH3 Arg90Cys mutationwereequallyeffective in express eitherthewild-typehumanNOTCH3 proteinorthe induced dilation(Dubrocaetal.,2005).Transgenic micethat increased pressure-inducedcontractionanddecreasedflow- 2005). Isolatedcaudalarteriesfromthetailsofthesemiceexhibit or increasedresistanceofcerebralbloodvessels (Lacombe etal., and disease? perturbations inNotchsignaling contribute tovascular pathology during normalvascular physiologyinadults,andhow do pathway function?What isthecontribution ofNotchsignaling mechanistically, ordothey representdifferent aspectsofNotch vascular development andphysiology?Aretheserolesrelated differentiation, and vessel wall formation,integrated during pathway playsin,forexample, arteriovenous patterning,tipcell in thevasculature. How arethedifferent rolesthattheNotch have muchtolearnabouttherolesthatNotchpathway plays 2007; SiekmannandLawson, 2007;Suchtingetal.,2007),westill Noguera-Troise etal.,2006;Ridgway etal.,2006;Scehnet (Hellstrom etal.,2007;LeslieLobov etal.,2007; formation andtheresultingimplicationsfortumorangiogenesis the roleofNotchsignalinginregulating endothelialtipcell As canbeseenbytherecentflurryofpublicationsthatfocus on Conclusion stages. progression ofCADASIL, particularlyduringitsearly, preclinical mouse modelshouldleadtovaluable insightsintotheonsetand Further analysisanddevelopment oftheNOTCH3 Arg90Cys NOTCH3 signalingactivity, underlietheetiologyofCADASIL. roles forCADASIL mutantproteins,ratherthancompromised (Monet etal.,2007).Thesedatasuggestthatnovel pathogenic regulates theinvivo expression ofaNotchsignalingreporter expression ofthemutantNOTCH3 Arg90Cys proteincorrectly 2004; Timmerman etal.,2004). 2004; Timmerman generation oftheheartvalves(Fischeretal.,2007;Noseda regulating theepithelial-mesenchymaltransitionrequired forthe bicuspid aorticvalvemightreflect therole ofNotchsignalingin aortic valvepatients(Mohamedetal.,2006).Theformationof 2005). families withautosomal-dominantaorticvalvedisease(Gargetal., of Alagillesyndrome are causedby at lowerpenetrance,severalotherorgans(Gridley, 2003).Mostcases by developmentalabnormalitiesoftheliver, heart,eye,skeletonand, Alagille syndrome isanautosomaldominantdisorder characterized disease Box 1.Notchsignalingandinheritedcardiovascular Heterozygous mutationsinthe transcription factorRUNX2(Garg,2006;Gargetal.,2005). Aortic valvecalcificationwaslinkedtoNotchregulation ofthe valve calcification,whichcanimpairbloodflowthrough thevalve. 2005; Garg,2006).Bicuspidaorticvalvepredisposes onetoaortic most commoncongenitalcardiac malformation(Bravermanetal., demonstrated inamousemodel(Highetal.,2007). neural crest cellsintosmoothmusclecells,whichhasbeen requirement forNotchsignaling-mediateddifferentiation ofcardiac and tetralogyofFallot.Thesedefectsare likelytobeduea pulmonary arterystenosisandhypoplasia,pulmonicvalvestenosis, The cardiac defectsassociatedwithAlagillesyndrome include NOTCH2 2006), althoughasmallnumberofAlagillesyndrome patientswith Bicuspid aorticvalveaffects 1-2%ofthepopulation,makingit NOTCH1 mutations havebeenidentified(McDanielletal.,2006). mutations are alsofoundin4%ofsporadicbicuspid NOTCH1 Notch3 JAG1 –/– mutations (Warthen etal., gene were foundintwo mice. Furthermore,the REVIEW 2715

DEVELOPMENT Doi, H.,Iso,T., Yamazaki, M.,Akiyama,H.,Kanai,Sato,Kawai- Barsi, J.C.,Rajendra,R.,Wu, J.I.andArtzt,K. References supported bytheNationalInstitutesofHealth. for someofthefigures. Work inmylaboratoryonthe Notchpathwayis constraints. IthankAnneJoutel,EichmannandLukeKrebs forimages I apologizetocolleagueswhoseworkcouldnotbeciteddirectly duetospace occupied formany yearstocome. to theseandotherquestionswillkeep Notchpathway researchers is operative, arealsolikely tooccurinthenearfuture.Theanswers developmental andphysiologicaldecisionsinwhichsuchcross-talk ephrin/Eph receptorandPI3K/Aktpathways, andinthe pathway andothersignalingpathways, suchastheTGF understanding ofthemechanismsforcross-talkbetweenNotch for sporadiccasesofvascular dementia.Significant advances inour evaluation oftherapeutictreatmentregimens bothforCADASIL and exhibited byCADASIL patientswillpermitthedevelopment and that betterrecapitulatethepathologicalandbehavioral abnormalities inherited stroke andvascular dementiainhumans,animalmodels CADASIL. BecauseCADASIL causesthemostcommonformof development andcharacterizationofbetteranimalmodelsfor degeneration anddiabeticretinopathy. We arealsolikely toseethe pathological angiogenesis,suchasneovascular age-relatedmacular angiogenesis aswellothervascular diseasescharacterized by (and possiblyothertypesof‘anti-Notch’ therapies)thattarget tumor upcoming yearsincludethedevelopment ofanti-DLL4therapies 2716 Doi, H.,Iso,T., Sato,H.,Yamazaki, M.,Matsui,H.,Tanaka, T., Manabe, I., Coultas, L.,Chawengsaksophak,K.andRossant,J. Carmeliet, P., Ferreira, V., Breier, G.,Pollefeyt,S.,Kieckens,L.,Gertsenstein, Carlson, T. R.,Yan, Y., Wu, X.,Lam,M.T., Tang, G.L.,Beverly, L.J.,Messina, Campos, A.H.,Wang, W., Pollman,M.J.andGibbons, G.H. Braverman, A.C.,Guven,H.,Beardslee, M.A.,Makan,M.,Kates,A.and Domenga, V., Fardoux, P., Lacombe,P., Monet,M.,Maciazek,J.,Krebs, L.T., Byrne, A.M.,Bouchier-Hayes,Byrne, D.J.andHarmey, J.H. Bray, S.J. Dorrell, M.I.andFriedlander, M. 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