A Garden of Notch-Ly Delights Gerry Weinmaster and Raphael Kopan

CORRIGENDUM

Development 133, 4793 (2006) doi:10.1242/dev.02713

A garden of Notch-ly delights Gerry Weinmaster and Raphael Kopan

There was an error published in Development 133, 3277-3282.

There was a mistake in the ﬁrst paragraph of this meeting review, which should read as follows:

Hosted by the Cantoblanco Workshops on Biology and organized by José Luis de la Pompa, Tom Gridley and Juan Carlos Izpisúa Belmonte, the meeting covered diverse aspects of this important signaling pathway.

The authors apologize to readers for this mistake. the Notch anditstargeting toan endocytic vesicle arenecessarypriorto amino acidsrequiredforproteolysisreveal thattheubiquitinationof events occuratthecellsurface. However, closer inspectionofthe proteolysis, ithasbeenassumedthattheseactivating proteolytic mouse (previously RBPj CSL [forCbf1,Su(H),Lag1],whichisknown asRbpsuhinthe transcription throughitsinteractionswiththeDNA-binding protein as thesignaltransduceranditdirectlyregulates Notchtarget gene Fig. 1)toreleaseitsintracellulardomain(NICD).NICDfunctions intramembrane partofthereceptor(theso-calledS3cleavage, see followed bythe Tace), the regulation ofNotch proteolysisthatsuggestendocytosis Israel (CNRS,Paris, France) reportedonhismorerecentstudies *Author forcorrespondence (e-mail:[email protected]) Pharmacology, Washington University, StLouis,MO63301,USA. Development 133,3277-3282(2006)doi:10.1242/dev.02515 1 such asAdam17(alsoknown asTNF regulated proteolysisoftheNotchreceptorbyADAM proteases, Activation oftheNotchsignalingpathway involves theligand- Ubiquitination andNotchsignalingregulation disease, werethemainfocusofthismeeting. the various rolesforNotchreceptorsinvertebrate development and Mechanisms regulating Notchexpression andsignaling,aswell activate andregulate Notchsignaling(reviewed byLai,2004). jagged 2),theso-calledDSL(Delta,serrate,Lag2)ligands,that multiple vertebrate Notchligands(Delta-like 1-4,jagged1and mammary tumors(Jhappanetal.,1992).Likewise, thereare 1991) andamouseortholog(Notch4)thatisassociatedwith with T-cell acutelymphoblasticleukemia (T-ALL) (Ellisenetal., mouse, includingahumanortholog(NOTCH1) thatisassociated Notch receptors(Notch1-4)have beenidentified inhumansand stillgeneratesmuchinterest. Duringthe1990s,fourdistinct mutants, surprising thatNotch,oneoftheoldest almost every developmental decisionandprocess,itisprobablynot Given thatNotchsignaling(seeFig.1)appearstobeinvolved in Introduction this importantsignalingpathway. Carlos IzpisúaBelmonte,themeetingcovereddiverseaspectsof organized byTom Gridley, JoséLuisdelaPompaandJuan Spain. HostedbytheCantoblancoWorkshops onBiologyand signaling invertebratedevelopmentanddiseaseMadrid, lead inMaytothefirstmeetingdedicatedsolelyNotch Notch signalingcontributesatvariouslevelstohumandisease inherited andlateonsetdiseases.Therealizationthataltered development andtobeassociatedwithseveralhuman shown tobecruciallyimportantfornormalmetazoan Over thepastdecade,Notchsignalingpathwayhasbeen Gerry Weinmaster delights A garden ofNotch-ly Angeles, CA90095-1737,USA. Department ofBiologicalChemistry, DavidGeffen SchoolofMedicineatUCLA,Los ␥ -secretase cleavage ofNotch(Gupta-Rossietal.,2004).Alain 1, * andRaphaelKopan 2 ␬ Department ofMolecularBiologyand ␥ ). AsligandbindingleadstoNotch -secretase-mediated cleavage ofthe Drosophila ␣ converting enzyme, 2 developmental acute lymphoblasticleukemia (T-ALL). Theirwork identifiedtwo analysis byhisgroupofTcellsfromindividuals withsporadicT-cell (Brigham andWomen’s Hospital,Boston,MA,USA)presented resistance ofNotchproteinsintheabsencealigand.JonAster intramolecular heterodimericstructurethatmaintainstheproteolysis processing byfurininthetrans-Golgitoproduceastable The maturationofvertebrate Notchproteinsinvolves itsproteolytic malignant Tcells Notch proteins andtheirtargetsinnormal proteolytic activation forsignaling. ubiquitination regulates boththebasallevels ofNotch,aswellits lysosomal degradation. Thesestudiesprovide insightintohow ubiquitination bytheItch/AIP4/Su(Dx)E3ligase,followed bytheir levels ofDeltex, andpossiblyofNotch,areregulated viatheir via thelesscommonlyusedLys29. Israel’s findingsuggeststhat that regulates thelevel ofcellsurface Notch)arepolyubiquitinated chain, IsraelreportedthatNotch1andDeltex (oneoftheE3ligases of many proteinsinvolves Lys48 intheformationofubiquitin precedes Notchubiquitination.Moreover, althoughubiquitination domain (NICD,Notch of Notch(S2andS3).S3cleavage releases theNotchintracellular The activationofNotchbyitsligandtriggerstwoproteolytic cleavages proteins thatcontainextracellulararraysofEGFrepeats (rectangles). the CSLtranscriptionfactor. DeltaandNotchare transmembrane are shownandconsistofaDelta-typeligand,thereceptor Notchand TheNotchsignalingpathway. Fig. 1. permission, from Lai(Lai,2004). represses thetranscription ofNotchtargetgenes.Reproduced, with associates withaco-repressor complex(Co-R,red), whichactively Notch targetgeneactivation.Inthe absenceofnuclearNICD,CSL activator complex,containingNICD (Co-A,green), whichmediates activates CSL.TheCSLco-repressor complexisdisplacedbyaco- intra ), whichtranslocatestothenucleus andthen The keyplayersinthispathway MEETING REVIEW 3277

DEVELOPMENT of Notchisthoughttofacilitate efficient vertebrate Notchsignaling.TheAdam17(Tace)-mediated cleavage indefinitely. Notch signalinggives T-ALL cellsametaboliclicensetodivide for several rapidroundsofcelldivision, whilepersistentaberrant normal upregulation ofMycbyNotchprobablyprimes DN3acells within thethymus, indicatingthatcell-cellcommunication within (Dll1) onneighboringthymocytes cansuppressT-cell fate defects stromal cells(seebelow), whiletheoverexpression ofDelta-like1 to activate Notchontheirneighborswhengrown inthe absenceof of Notch(Manilayetal.,2005). ShereportedthatT-cells areunable T-cell fate decisionsthatarereminiscentofthosecausedbytheloss negative (DN)formofKuz inT-cell progenitors producesdefectsin presented findingsthatshow thattheexpression ofadominant- Ellen Robey (University ofCalifornia,Berkeley, CA,USA) signaling, itsessentialfunctionsinthispathway remaina mystery. mice (Hartmannetal.,2002)suggestaroleforthisproteaseinNotch the targeted deletionofADAM10 (alsocalledkuzbanianorKuz) in generation oftheNICD.Althoughphenotypesgeneratedthrough the pre-Tcellreceptor, aprocessreferredtoas poised toundergo aproliferative burst thatdependsonexpression of stage alsodependsonNotchactivation. Ofinterest,DN3acellsare as Pearhasobserved thatMycexpression inthymocytes attheDN3a T-ALL cellsappearstoreflectanormaldevelopmental relationship, growth andmetabolism.TheabilityofNotchtoupregulate Mycin cohort ofco-regulated Notch/Myctargets, mostinvolved incell presented adetailedChIPonchipanalysisthatidentifiedlarge Alfredo Ferrando(ColumbiaUniversity, New York, NY, USA) expression isextinguished, byactivating theendogenous can restoretumourgrowth whentetracycline-regulated Myc pharmacological blockof demonstrated thatMycoverexpression canbypassa T-cell lineage.Inelegant experiments, theseresearchers the USA), thatidentifyMycasakey downstream target ofNotch1in with Warren Pear(University ofPennsylvania, Pittsburgh, PA, al., 2006). mechanical forceshave aroleinNotchdissociation (Miyamotoet Magp2 (microfibril-associatedglycoprotein2),whichindicatethat activation inducedbythesecretedextracellular matrixprotein presented recentlypublishedfindingsonNotchdissociationand importance ofheterodimericdissociationforNotchactivation. She USA), fromGerryWeinmaster’s group,alsohighlightedthe Alison Miyamoto(UCLASchoolofMedicine,LosAngeles,CA, within thePESTmotif,whichisassociatedwithproteinturnover. stabilizes NOTCH1 byeliminatinganovel phosphorylationsite ligand bindingcansomehow expose thisregion. Thesecondhotspot based onpreventing accesstotheADAM cleavage siteandthat observations confirmthattheresistancetoproteolysis ofNotch1is extracellular domain,resultinginconstitutive proteolysis.These distance betweentheADAM siteandtherestofNOTCH1 cleavage sitedidnotdestabilizetheHDbut insteadincreasedthe Interestingly, aduplication of 14aminoacidsneartheADAM independent activation ofNOTCH1 (Maleckietal.,2006). These mutationsresultinreceptordissociationandtheligand- that arerequiredfortheNOTCH1 heterodimericstructuretoform. HD mutationsappeartodestabilizethenon-covalent interactions heterodimerization domain(HD)ofNOTCH1; mostoftheidentified USA), they foundthatonemutationalclustermapstothe Steven Blacklow’s group(Harvard MedicalSchool,Boston,MA, hot spotsforNotch1-activating mutations.Incollaborationwith 3278 At leasttwo ADAM metalloproteases have beenimplicatedin Aster alsoreportedexciting new data,producedincollaboration MEETING REVIEW ␥ -secretase (seeBox1),andthatNICD ␥ -secretase cleavage inthe ␤ -selection. The Myc gene. do notrequireNotchsignaling.Hereportedthatinthepresenceofa Pear closedthissessionbydemonstratingthatadultmurineHSCs also respondtotheNotch/WntcombinationandadoptaT-cell fate. cell development. Importantly, hematopoieticstemcells(HSCs)can development, andtheWntantagonistdickkopf 1(Dkk1)prevents T- factor thatdrives Wnttarget geneexpression, have defective T-cell Consistent withthisresult,micedeficientforTcf,thetranscription this missingactivity, whichwas suggestedtobedueWntsignals. Bone marrow stromalcellslackingDll1expression cancomplement but cannotinduceTcellsfromstemorlymphoidprogenitors. ligands presentedby3T3cellscanonlyblockBcelldevelopment regulates theJag1-mediatedactivation ofNotch).Interestingly, Dll (Lfng) bythymicprogenitorsandstemcells(Lfngnegatively as efficient, mostprobablyowing totheexpression oflunaticfringe Although jaggedligandsareexpressed inthethymus,they arenot thymic stromalcellstodirectprogenitorsintoT-cell differentiation. are sufficient toblockBcelldevelopment andarenecessaryfor demonstrated thattheNotchligandDll1(andmorerecentlyDll4) (Sunnybrook andWomen’s ResearchInstitute,Ontario, Canada) autonomous celleffects thathave beenidentifiedforKuz inflies. could notsuppressthecellfate defects,similartothenon- thymocytes. Co-expression ofDll1withDN-Kuz inthymocytes this organ differs fromtheinteractionsthatoccurbetweenisolated can beusedforsome biochemicalstudies. recommended forusewhen biologicaloutcomeismonitored, butit inhibitor, whichcannotbeconsidered tobeselective.Itisnot been disclosed.Itisabroad chymotrypticandaspartylprotease of MG132,GSI1,isnowavailable,but itssimilaritytoMG132hasnot used toinhibitNICDformation(Kopan etal.,1996).Amodifiedform In thepast,protesome inhibitorMG132(Z-LLL-CHO)hasbeen (Krawitz etal.,2005). they inhibit or animalstudies,buttheycanalsoinhibitSPPinthesamerangethat values inthenMrange,makingthemcheapertouseforlong-term such experiments,theIC selection ofthecorrect inhibitorandregimen. Before performing expanding, anddiscussionsatthismeetingarose regarding the The useofpharmacologicalreagents toinhibitNotchsignalingis Box 1.AquickguidetoNotchinhibitors IC DAPT, arecommended Notchantagonist(Doveyetal.,2001),hasan Popular inhibitors inhibitor literature. Inselectingthedrug,selectivitywinsoverefficacy. their chosensystem,examplesofwhichaboundinthe of thedrugbefore usingitinvivoand/oruseaprotocol testedfor Researchers shouldfamiliarizethemselveswiththepharmacokinetics which canvarybetweencelltypesandwhenusedinvivo). determined (theconcentrationthatinhibits50%ofNotchcleavage, activity againstthe 2000) are highlyeffective, active-site-directed inhibitorswithIC L685458 andL852647(Merck) (Lietal.,2000;Shearman of targettissues. signaling reduction isuncertainwithoutdetailedbiochemicalanalysis studies, whentoxicitymustbeavoided,thedegree ofNotch effective againstSPPwhenusedathigherconcentrations.Inanimal against SPPthanitis LY411575, aDAPTderivative(Searfossetal.,2003),islesseffective molecule (Morohashi etal.,2006). organisms (Chengetal.,2003).Itisnotanactivesite-directed been usedextensivelyincellandorganculture, andinmodel family membersatallconcentrations(Weihofen etal.,2002)andhas Elegant studiespresentedbyJuanCarlosZuniga-Pflucker 50 in mostcellsisthelow ␥ -secretase, whichcanhavephenotypicconsequences ␥ -secretase-related SPP(signalpeptidepeptidase) 50 ␥ of aninhibitorshouldalwaysbe -secretase insomecells,butmaybe ␮ M range.Ithasremarkably low Development 133(17) ␥ -secretase 50

DEVELOPMENT secretase inhibitor(GSI),maintainingtheNF Although earlyNF been targeted (McCrightetal.,2002),these findingssuggestthat Kopan reportedthatintheabsenceof function oftheseNotchreceptorsduringkidney development. MO, USA)presentedinterestingdataregarding thenon-redundant Kopan (Washington University, StLouis, (Pan etal.,2005).Rafi that, whenco-expressed, theseproteinshave redundantfunctions Conlon etal.,1995;Hamada1999).However, itwas assumed have non-redundantrolesduringdevelopment (Swaitek etal.,1994; Notch2 The embryoniclethalitycausedbythelossofeither Kidney development domain requiredforNotchtointeractwithNF to NF Osborne presenteddatathatsupportamodelinwhichNotchbinding Notch activation. Mechanistically, somecontroversy remains,as by interrogatingthetimelineofNotchandNF Massachusetts, MA,USA)hasfurtherinvestigated thispossibility transcription factor NF some Notchactivity maybederived viamodulationofthe Notch1 inT-cell development, andpresenteddatasuggestingthat Rome, Italy)presentedevidence thatNotch3acts downstream of of Notch1inTcells.IsabellaScrepanti(LaSapienzaUniversity, lineage. rounds ofsequentialtransplantation,withonlylosstheT-cell irradiated micecanstillreconstitutetheirimmunesystemaftertwo which functionsasapan-Notchinhibitorwhenexpressed inHSCs, DN formoftheNotchco-activator proteinmastermind(DN-Maml), (17) Development 133 Tom Gridley, inwhichasinglecopy ofboth Together withamousemodelofAlagillethathasbeen generatedby Notch2 mutations. Herfindingsarealso consistentwitharecentreportthat individuals withAlagille, asyndromethatisassociatedwith structures isreminiscentofthe paucityofbileductspresentin enhances bileductlossandarterialdefects.Thisof duct formation, whilelossof that lossof Notch1 andNotch2lossonliver development inmice.Shereported TN, USA)reportedpreliminaryfindingsdescribingtheaffects of Stacey Huppert(Vanderbilt University MedicalCenter, Vanderbilt, Liver development these two conserved proteins. rely onqualitative, ratherthenquantitative, differences between different Notchparalogsarepresentwithinthesamenucleusthat kidney defectssuggeststhatdifferent functional thresholdsfor Given this,theinabilityofactivated Notch1torescuetheNotch2 affinity ofNotch1andNotch2proteinsforRbpsuhwas identical. University, Baltimore,MD,USA),Kopan’s groupfoundthatthe function. IncollaborationwithDougBarrick(JohnsHopkins provides evidence that,inthesamecell,eachreceptorhasaunique cells attherighttimeandthatsignalisRbpsuhdependent.This embryos, despiteclearevidence thatNotch1isactivated intheright podocyte precursordifferentiation fails tooccurinmutantmouse indirect effect onNF in activity areduetoalossofCSL-mediatedtranscriptionandan NICD (Wang etal.,2001);thus,itisuncleariftheobserved losses one thatisrequiredtostablizetheinteractionsbetweenCSLand Roles forNotchinmultipletissuesanddisease All thedatareviewed thusfar have focusedonthecanonicalrole ␬ B mayregulate thenuclearhalf-lifeofNF in micehaspreviously indicatedthattheseNotchreceptors mutations canproduceAlagille (McDanielletal.,2006). Notch2 ␬ impedes maturebileductandhepaticartery ␬ B responsesoccurinthepresenceofa B localization. ␬ Notch1 B. BarbaraOsborne(University of on a Notch2 Notch2 Jag1 ␬ , proximaltubule and ␬ B responserequired B isthesameas -null background ␬ and B. However the ␬ B activation. Notch2 Notch1 JAG1 have or ␥ of thesemutants. development, accountingfortheobserved motionlessphenotype additional musclecellscanformduringlaterstages of muscle stemcell/progenitorpoolprematurely. Asaresult,no into thelimb,but toomany cellsdifferentiate early, depleting the myogenic programinitiatesnormally, asdoesprecursormigration with very few skeletal musclefibers. Inthesemutants,the on characterization ofanew hypomorphicalleleofDll1.Whenplaced for MolecularBiology, Hannover, Germany) reportedhisgroup’s where axialmuscleprecursorsareborn.AchimGossler(Institute during somitogenesis. one orderofmagnitudetheexpression ofadditionalgenes thatcycle clusters containingNotch-andWnt-relatedgenes,increasedby phases withinoneclockcycle. Thisanalysisidentified two major staged 17embryosforacaudaltranscriptomecomparisonatall wavefront’ mechanism,Olivier’s grouppainstakinglycollectedand identify additionalmolecularcomponentsofthis‘clockand activation byacquiringrostralandcaudalsomiteidentities.To within thePSMinwhichcellsarecompetenttorespondNotch anterior) andretinoicacid(highintheacttodefineazone oscillates inthePSM,andopposinggradientsofFgf(low inthe the controlofcycling geneLfng.TheWntpathway also oscillation inlive mouseembryosexpressing short-lived GFPunder in thechickPSM,andatmeetingheshowed real-timeimagesof MO, USA)was thefirsttoobserve Notchtarget mRNA oscillation in spaceandtime.Olivier Pourquie(Stower Institute,KansasCity, oscillations andthuspatternthevertebrate musculoskeletal system (such astheWnt,Fgfandretinoicacidpathways) toregulate the neighboring cells. communication viaNotchsignalingmaintainssynchrony among genes createsanintracellularoscillatorineachcellandthatcell-cell zebrafish, basedontheideathatauto-inhibitionofNotchtarget for atheoryofthetimingmechanismgoverning theperiodicityin translation andproteinexport. Thedataprovide quantitative support in delaysthatareinherenttotheprocessesoftranscription, oscillations aregeneratedthroughnegative feedbackloopsandbuilt- presented datarefiningandtestingamathematicalmodelofhow the somites. JulianLewis (CancerResearchUK, London,UK) gene expression thatleadtotheformationofbilaterallysymmetrical unique inmaintainingaprecisepatternofperiodicoscillations or Rbpsuh(Okaetal.,1995).Thepresomiticmesoderm(PSM)is mutations ineitherNotch1(Swiateketal.,1994;Conlon1995) indicated bydefectsinsomitemorphologymicebearingtargeted The involvement oftheNotchpathway insomitogenesiswas first Somitogenesis Huppert. tree, orofjustparticularsegments, iscurrentlybeinginvestigated by Whether Notchplaysaroleintheformationofwholebiliary Notch2 signalinginducedbyJag1regulates bileductformation. the sametime,in balancingproliferationviaBmp10 modulation. differentiation via regulation ofephrinB2andneuregulin, and,at of activated Notch1.Notchisinvolved inregulating ventricular Notch signalingduringcardiogenesis byfollowing theappearance (Universidad deAutonoma,Madrid,Spain)reportedhisanalysisof multiple cardiovascular structures.JoseLuisdelaPompa Notch signalingisrequiredforthe development andmaintenanceof Cardiovascular development The dorsolateralcellsofthesomitesgoontoformmyotome, The Notchsignalingpathway integrates withadditionalpathways Dll1 -null background,thisalleleresultsinmicethatdevelop MEETING REVIEW 3279

DEVELOPMENT regulated byVegf. with adelayinarterialandcapillaryremodeling,processesthatare branching andsproutingatthegrowing edgeofthearteries,coupled Dll4 heterozygotes.Hefoundthesemutantsdisplayincreasesin allowing Galetocharacterizevascular development intheretinasof mutant micematedtoICRoutbredmice,theirviabilityisrestored, identification ofDll4asatarget ofVegf signaling.When characterizing Pharmaceuticals, Tarrytown, NY, USA)andcolleaguesare also producing distinctinkflow patterns.NickGale (Regneron AVM, they appeartodosoinananatomicallydistinct manner, Interestingly, althoughbothgain andlossof injection intotheoutflow tractofmutanthearts (seeFig.2). loss ofNotchfunction,byfollowing thedistribution ofinkafterits arteriovenous malformations(AVM) associatedwithgainversus Gridley alsoidentifiedcharacteristicdifferences inthetypeof addition todefectsinvascular remodeling,branching andsprouting, resulting inlossofephrinB2,amarker ofarterialidentity. In produced thesamevascular phenotypeaslossofa single reported thatcompletedeletionofRbpsuhinendothelialcells Tom Gridley (TheJacksonLaboratory, BarHarbor, ME,USA) ligands Jag1andDll1arepresent.Extendingtheseobservations, because ofvascular systemmalformation,even thoughtheDSL is themostsevere, causingembryoniclethalityinheterozygotes mutations associatedwithvascular defects,the embryos defective inNotchsignaling.Ofallthepathway 3280 malformations. Images courtesyofLukeKrebs andTom Gridley. injected inkleaks directly into thevenoussystemviaarteriovenous with anendothelialcell-specificdeletion ofthe arch arteriesandentersthe descendingdorsalaorta.( into theheartinorder tovisualize bloodflowexitsthrough theaortic malformations. Fig. 2.Notchpathwaymutations andarteriovenous Vascular defectsaccountfortheembryoniclethalityofmouse MEETING REVIEW Dll4 ( A ) Inawild-typemouseembryo,India inkinjected mutant vascular defectsbasedontheir Notch1 Dll4 Dll4 B ) Inanembryo gene, the function cause -null mutation Dll4 allele, Dll4 phenotype. function ismostlikely toberesponsiblefortheCADASIL Notch3 retainsignalingactivity invivo. Therefore,anovel Notch3 was abletodemonstratethattheCADASIL-encoding mutationsin CADASIL mutations,andbyusingseveral geneticstrategies, Joutel these vascular defectsdonotphenocopy thoseassociatedwith unique targets mayberequiredinmousearterialSMCs.However, traditional Notch/CSLtargets weredecreasedin but displayspecificdefectsinarterialvascular SMCs.Asnoneofthe Notch orIkk Notch/p50 andNotch/Ikk controversial, but MieleconfirmsOsborne’s observations that sub-optimal NF of thesecells.Mechanistically, thiseffect ismediatedbyenhancing receptor reductionviasiRNA ortheuseofGSIs reducedtheviability context, Notchreceptorsplayapositive oncogenicrole.Notch Rb/p53 activity andhave anoveractive PI3Kpathway. Inthis studies oftransformedcervicalkeratinocytes, whichlackfunctional (Loyola University MedicalCenter, Chicago,IL,USA)reportedhis which inturnregulate p63,independentlyofNF Notch signalingtoaffect asubsetofinterferon-responsive genes, et al.,2006).Thisinteractionseemstobemediatedbytheabilityof Notch signalingandp63duringketarinocyte differentiation (Nguyen discussed hisrecentlypublishedfindingsofinteractionsbetween Paolo Dotto(University ofLausanne, Epalinges,Switzerland) Keratinocyte differentiation andoncogenesis function hasnotbeeneasytoresolve. how thesemutationsaffect Notch3activity andvascular SMC of asinglecysteine residueintheextracellular domainofNotch3, CADASIL. AlthoughallCADASIL mutationsinvolve lossorgain associated witharteries,whicharethevery cellsaffected in Notch3 which isthemostfrequentcauseofinheritedstroke inhumans. arteriopathy withsubcorticalinfarcts andleukoencephalopathy), pursuit oftheetiologyCADASIL (cerebralautosomaldominant Anne Joutel(INSERM,Paris, France)continuedherrelentless CADASIL hseffect ismediatedby pathway targets, asRBP-VP16 (a this apoptosis. AlthoughNICDleads tophosphorylationofAKT, turn protectedNICD-expressing cellsfromDNA damage-induced resulted inthephosphorylation andactivation ofAKT, whichin in softagarassays.Moreover, hefoundthatNICDexpression this, overexpression oftheNotchantagonistNumbblocked growth important formaintainingthemalignant phenotype.Insupportof adding hisvoice tothosesuggestingthatNotch signaling is reported thatactive NICDlevels arehighinbreastcancercelllines, and genetictools. from thosethatarenotwillrequiremoreselective pharmacological reported inNotch-overexpressing cellsarephysiologicallyrelevant identified byAster. Determiningwhichbiochemicalinteractions interestingly, thetarget sequenceonNotch1isthesame one in someepithelialtumorsthroughphosphorylationofNICD; that activated receptortyrosinekinases cansustainNotchsignaling Biological Sciences,Bangalore,India)presenteddatasuggesting nature ofNotchsignaling.SudhirKrishna(NationalCenter for cervical keratinocytes celllines,underscoringthecontext-dependent laboratory, showing atumorsuppressorfunctionforNotchinseveral addition, they disagreewithprevious findingsfromPaolo Dotto’s interactions arenotseeninnormalkeratinocytes orlymphocytes. In These responsesseemtodependonthetumorcellcontext; such Keith Brennan(University ofManchester, Manchester, UK) is expressed byvascular smoothmusclecells(SMCs) ␣ enhanced theactivity ofDNA damagingagents. ␬ B complexes. Exactlyhow thisoccurs isstill ␣ complexes canform.Silencingeither Notch3 Development 133(17) -null miceareviable ␬ B. LucioMiele Notch3 nulls,

DEVELOPMENT of Rbpsuh. convincing evidence thatSpenphenotypesdependonthepresence cases, theSpenphenotypeswere epistatictoRbpsuh,providing conditional doublenullanimals werealsogenerated,andinall Msx2-dependent functionsin theseprocesses,Spen/Rbpsuh in itsabsence,thatrepressive taskbecomeseasier. To ruleout that Notchactstoantagonizethe repressive functionofSpenand, Spen producedtheoppositeeffect tolossofRbpsuh,indicating B cells,follicularcellsandduringcorticalneurogenesis)loss of mediated transcriptionalrepression.Inseveral systems(marginal Hairless homologinmiceandthemainmediatorofRbpsuh- genetic experiments thatestablishSpen(previously Mint)asthe (Kyoto University, Kyoto, Japan)alsoreported anelegant seriesof repression andthustheeffects ofNotchsignaling.Daisuke Yabe of novel pharmaceuticalsthatcouldreverse GRG/TLE-mediated repression, thisstructuralanalysiscouldleadtothedevelopment with Grouchoco-repressorsarerequiredfortranscriptional Hes/Hey genesareactivated byNotchsignaling,andinteractions repressor proteins(Jenningsetal.,2006).Astheexpression of WRPW/YRPW motifwiththeWDofGroucho/Grg/Tle co- Hes/Hey family oftranscriptionalrepressorsinteractviaa UK, London,UK)presentedcrystalstructuresthatshow how the transcriptional repression.David Ish-Horowicz (CancerResearch Two speakers addressedthefunctionsofNotchsignalingin Notch andtranscriptionalrepression an epidermalprogramandtomaintaintheircornealfate. Notch1 isrequiredtoprevent thesestemcellsfromdefaulting into uncovered two populationsofstemcellsthatrepopulatethecornea; development. Usingembryologicalandgeneticstrategies, hehas Switzerland) discussedhisanalysisofNotchsignalingincorneal conditions remainsanunresolved issue. nucleus. Whethertheseinteractionsoccurunderphysiological joins Ikk enhance theefficacy ofBmptarget geneexpression. Thus,Hif1 previous reportsofNICDinteractionswithSmadproteinsthat through theseinteractions.Thesefindingsarereminiscentof Hif1 NICD throughdirectinteractionswithHif1 differentiate underhypoxiccultureconditions.Hypoxiastabilizes hypoxia isreversed byNotchinhibition:DAPT-treated myocytes myogenesis andneurogenesis.However, theblockinducedby differentiation. BothNotchsignaling andhypoxiaprevent cell transcription factor Hif1 between NICDandthewell-characterizedoxygen-sensing signaling (Gustafssonetal.,2005).Heproposesthatinteractions effects ofoxygentensiononcellgrowth anddifferentiation toNotch oxygen tension.Lendahlpresentedhispublishedstudiesthatlinkthe embryonic andneuralstemcells,canbeenhancedbygrowth inlow The isolationandcultivation ofmany different celltypes,especially Notch andstemcells degradation. abundant proteincantarget theothertoproteasome-mediated be tiltedbasedonthelevel oftheseproteins,suchthatthemore Stockholm, Sweden)findsthatthebalanceofNICDandNumbcan performed. Interestingly, Urban Lendahl(KarolinskaInstitute, NF resultneedstobeconsideredinthecontext ofthe This provide tumorcellswiththesameAKT-dependent protection. Notch-independent, transcriptionallyactive formofRbpsuh)can (17) Development 133 Freddie Radtke (LudwigInstituteforCancerResearch,Epalinges, ␬ ␣ B/Notch connection,wheresimilarexperiments shouldbe target genesandCSL-NICDtarget genesareenhanced ␣ , p50andSmadaspossiblepartnersforNotchinthe ␣ explain theimpactofhypoxiaon ␣ ; theexpression ofboth ␣ Malecki, M.J.,Sanchez-Irizarry, C.,Mitchell,J.L.,Histen,G., Xu, M.L.,Aster, Li, Y. M.,Xu,Lai,M.T., Huang, Q.,Castro, J.L.,DiMuzio-Mower, J., Jhappan, C.,Gallahan,D.,Stahle,Chu,E.,Smith,G.H.,Merlino,and Jennings, B.H.,Pickles,L.M.,Wainwright, S.M.,Roe,Pearl,L.H.and Hartmann, D.,deStrooper, L.,Craessaerts,K.,Herreman, B.,Serneels, A., Lai, E.C. Krawitz, P., Haffner, C.,Fluhrer, R.,Steiner, H.,Schmid,B.andHaass,C. Kopan, R.,Schroeter, E.H.,Weintraub, H.andNye,J.S. Hamada, Y., Kadokawa,Y., Okabe,M.,Ikawa,Coleman,J.R.and Gustafsson, M.V., Zheng,X.,Pereira, T., Gradin,K.,Jin,S.,Lundkvist,J., Gupta-Rossi, N.,Six,E.,laBail,O.,Lugaet,F., Chastagner, P., Olry, A.,Israel, Dovey, H.F., John,V., Anderson,J.P., Chen,L.Z.,Andrieu,P. D.,Fang,L.Y., Conlon, R.A.,Reaume,A.G.andRossant,J. Cheng, H.,Miner, J.,Lin,M.,Tansey, M.G.,Roth,K.A.andKopan,R. References ly delights. disease willensurethatweallcontinuetodiscover moreNotch- importance ofNotchsignalingtobothdevelopment andhuman the morewedon’t know’, seemsincrediblyapt.Nonetheless,the biological consequences,therealityofadage‘themorewelearn molecular mechanismsthatregulate Notchsignalingandits new twistsandinsightshave enhancedourunderstandingofthe roles identifiedforNotchsignalinginvertebrates. Althoughmany of EarthlyDelights’,insomeways reflectedthemyriadeffects and particular paintingdisplayedinthemuseum,Bosch’s ‘TheGarden to theMuseodelPradoexperience somenew images.One At theendofthisexciting andintensemeeting,many ofusescaped Conclusion Ellisen, L.W., Bird, J.,West, D.C.,Soreng, A.L.,Reynolds,T. C.,Smith,S.D. McCright, B.,Lozier, J.and Gridley, T. Manilay, J.O.,Anderson, A.C.,Kang,C.andRobey, E.A. classes. NOTCH1 heterodimerization domainfallintoatleasttwodistinctmechanistic J. C.andBlacklow, S.C. label presenilin 1. Photoactivated gamma-secretase inhibitorsdirected totheactivesitecovalently Harrison, T., Lellis,C.,Nadin,A.,Neduvelil,J.G.etal. Development mammary andsalivaryglands. interferes withcelldifferentiation andinducesneoplastictransformationin Callahan, R. 655. motifs bytheWDdomainofGroucho/TLE corepressor. Ish-Horowicz, D. 11 signalling butnotforalpha-secretase activityinfibroblasts. (2002). 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