*Author forcorrespondence (e-mail:[email protected]) Agency, CREST, Kyoto606-8507,Japan. Research,Institute forVirus KyotoUniversityandJapanScience andTechnology and intheprocessofsomitesegmentation, where in thenervous system)regulates cellproliferationanddifferentiation, suchas of cellsbychangingtheirdifferentiation competency. Without Hes progenitor cellsproliferateandsequentiallygive risetodifferent types example, inthedeveloping nervous systemofmouseembryos, progenitor cellsandbyregulating binarycellfate decisions.For essential roleinthedevelopment ofmany organs bymaintaining bHLHrepressorgenesplayan genes.Inparticular, theHes regulated bymultiplebasichelix-loop-helix(bHLH) activator and many organs, cellproliferation anddifferentiation areantagonistically for makingorgans oftherightsize,shapeandcellcomposition.In of thetimingcelldifferentiation andcellfate choicearekey issues and theirsubsequentdifferentiation intomultiplecelltypes.Regulation Embryogenesis dependsonthetimelyproliferationofprogenitorcells Introduction operate invertebrateembryogenesis. and aimstoclarifythebasicmechanismofhowgenenetworks pleiotropic rolesofHesgenesinsomedevelopmentalprocesses mediates cell-cellcommunication.Thisprimerdescribesthese of HesexpressionareregulatedbyNotchsignaling,which of biologicalevents,suchassomitesegmentation.Manyaspects display anoscillatoryexpressionpatternandcontrolthetiming state andbyregulatingbinarycellfatedecisions.Hesgenesalso processes bymaintainingprogenitorcellsinanundifferentiated helix-loop-helix repressorgenesplaycentralrolesinthese proliferation anddifferentiation. ThemammalianHesbasic Embryogenesis involvesorchestratedprocessesofcell Ryoichiro Kageyama*,Toshiyuki OhtsukaandTaeko Kobayashi orchestrate embryogenesis The Hesgenefamily:repressorsandoscillatorsthat (2007)doi:10.1242/dev.000786 Development 134,1243-1251 family ( and Hes genesaremammalianhomologsofthe Hes factors:structures andfunctions family ofgenes)(Table 1). (called theher Hesgenes Hes genes,but alsocomparethemwiththezebrafish a biologicalclock.We willmainlyfocusontherolesofmouse two well-characterizedprocesses,where namely, inthedevelopment ofthenervous anddigestive systems, detail theirrolesinsomerepresentative processes ofembryogenesis: coordinates cellularevents viacell-cellinteractions. processes, Hesgenesfunctionaseffectors ofNotchsignaling,which developmental events, suchassomitesegmentation. Inthese genes alsofunctionasbiologicalclocks,measuringtimein cell types.Thisresultsinsmallanddeformedbrainstructures.Hes they have proliferatedsufficiently andgeneratedallneuronalglial aredepletedbefore only, and differentiate intocertaintypesofneurons In thisprimer, wedescribethekey featuresofHesfactors and Enhancer ofsplit Hes1-7 ), although Hes1 [ E , however, progenitorcellsprematurely ( spl )]. Thereareseven membersintheHes Hes4 is absentinthemousegenome Hes1 Drosophila (and Hes7 Hes3 functions as genes and hairy Hes5 features asrepressorsandoscillators. (Hirata etal.,2002).Thesedomainsendow Hesfactors withunique proteasome, andthusthey have very shorthalf-lives (~20minutes) 2005): Hesfactors arepolyubiquitylatedanddegraded bythe This sequencealsoactsasapolyubiquitylationsignal(Kangetal., consists ofthetetrapeptideTrp-Arg-Pro-Trp, repressestranscription. 1995; Taelman etal.,2004).TheC-terminalWRPWdomain,which regulates theselectionofbHLHheterodimerpartners(Dawson etal., et al.,1994).TheOrangedomainhastwo amphipathichelicesand the NboxCACNAG (Akazawa etal.,1992;Sasai 1992;Ohsako factors. ThesesequencesarecalledtheclassCsiteCACG(C/A)G or highest affinity todifferent target sequencesthantheotherbHLH two groups:classAandB.However, Hesfactors bindwith region oftheirtarget genes. Thissequenceisfurtherclassified into sequence calledtheEbox(CANNTG)thatispresentinpromoter unique DNA-binding activity. MostbHLHfactors bindtoaconsensus of thebasicregion. ThisprolineisproposedtoconferHesfactors other bHLHfactors, Hesfactors have aprolineresidueinthemiddle binding andthehelix-loop-helixregion fordimerization.Unlike most bHLH domainconsistsoftwo regions: thebasic region forDNA factors: thebHLH,OrangeandWRPWdomains(Fig.1A).The Three conserved domainsconfertranscriptionalfunctionsonallHes Conserved functionaldomainsofHesfactors describe thestructuralandtranscriptionalfeaturesofHesfactors. bothactively andpassively. Inthissection,we (Table 1).Hesgenesencodenuclearproteinsthatrepress nw sTf2 –MouseGenome Informatics),whicharethe Tcfe2a known as known asAscl1– MouseGenomeInformatics)andE47(also binding bHLHactivators (passive repression). Thus, Hesfactors display adominant-negative effect onEbox- E box.However, theseheterodimerscannot bindtoDNA (Fig.1C). factors alsoformheterodimers withbHLHactivators thatbind to the more efficiently thanhomodimersdo(Fig.1B)(Isoetal.,2001).Hes C siteattheirtargets withahigher affinity andrepresstranscription and Hey2. HeterodimersofHes1andHey1 orHey2 bindtotheclass bHLH ,suchasHey1 (Hes-relatedwithYRPWmotif1) only formhomodimers,but they alsoformheterodimerswithother inactivating chromatin(active repression,Fig.1B).Hesfactors not Hes–Groucho-homolog complex repressestranscriptionby by recruitinghistonedeacetylase.Thus,itislikely that the and ithasbeenshown that by histoneacetyltransferaseandinactivated byhistonedeacetylase, Grbavec andStifani, 1996).Itisknown thatchromatinisactivated groucho Drosophila E(spl) (TLE)genes/ interacts withtheco-repressorsencodedbyTransducin-like 1992). Active repressiondependsontheWRPWdomain,which (Sasaietal., mechanisms: active andpassive repression(Fig.1B,C) Hes factors represstranscriptionbyatleasttwo different Hes factorsasrepressors Direct targets forHes1includethebHLHactivators Mash1(also Groucho-related (Paroush etal.,1994;Fisher1996; Drosophila Groucho inhibitstranscription ( Grg PRIMER ), homologsof 1243

DEVELOPMENT transcription. and E47formheterodimers thatbindtotheEboxandactivate proceeds, neuroepithelial cellsbecomeradialglial cells,whichhave and Kriegstein, 2003;Götz andHuttner, 2005).Asdevelopment plate andproliferatebysymmetric celldivision (Fig.2A)(Fishell During neuraldevelopment, neuroepithelialcellsformtheneural development Hes genesregulate themaintenanceofstemcellsin neural specific transcription factor1a actively andpassively inhibitsotherbHLHactivators, suchas (Chen etal.,1997).Similarly, inthedeveloping pancreas,Hes1 to theclassCsitein Furthermore, Hes1represses DNA-binding heterodimerswiththem(passive repression,Fig.1C). whereas Hes1inhibitsMash1andE47activities byformingnon- expression bybindingtotheEbox(Fig.1D)(Johnson et al.,1992), heterodimer withE47andactivate neuronal-specific gene FlyBase) and proneural genes mammalian homologsoftheproteinsencodedby transcriptional activation.( binding heterodimers withbHLHactivatorssuchasE47andinhibit Groucho homologs.( actively repress transcriptionbyinteractingwithco-repressors, suchas homodimers (leftpanel)orheterodimers withHey(rightpanel)and repression: HesfactorsbindtotheNboxorclassCsitebyforming and WRPW(pink)domainstheirfunctionsare indicated.( domains ofHesfactors.Thebasic(blue),HLH(mauve),Orange(orange) Fig. 1.Structure andfunctionofHesfactors. 1244 the nervous anddigestive systems. effects ofbHLHactivators. Here,wedescriberolesforHesgenesin and thenormaltimingofcelldifferentiation byantagonizingthe genesregulate themaintenanceofstemcellsand progenitors Hes and progenitors Hes genesregulate the maintenanceofstemcells 2006), asdiscussedinmoredetailbelow. endocrine cellfates, respectively (Lee etal.,2001;Fukuda ( C B A Ngn3 Passive repression Active repression Structure andfunctionofHesfactors Hes1 , alsoknown as PRIMER Hes1 E box Groucho C site N box HLH b DNA binding Dimerization E47 Hes1 daughterless achaete-scute complex C ) Passiverepression: Hesfactorsformnon-DNA- Mash1 D Neurog3 Orange Selection of heterodimer partner ) Activation:bHLHactivatorssuchasMash1 , respectively. Mash1canforma Mash1 promoter (active repression;Fig.1B) ), whichspecifyexocrine and D expression bydirectlybinding Hes1 Groucho Mash1 C site N box bHLH activators (also known as ( Hey1 E box Ptf1a E47 ( A Co-repressor interaction Polyubiquitylation signal ) and ) Theconserved neurogenin 3 Drosophila Cyp4g1 B ) Active – and finally intoastrocytes (Fig. 2A)(Fujita,2003). of ,radialglialcellscan differentiate intooligodendrocytes later-born neuronsin more-superficial layers.Aftertheproduction settle indifferent layers: early-bornneuronsindeeperlayersand the mousecortex, different neuronsmigratealongradialfibers to competency andcangenerate distincttypesofneuronsover time.In 2003; GötzandHuttner, 2005).Radialglialcellscanchangetheir cell andaneuronorneuronalprecursor(FishellKriegstein, undergo asymmetriccelldivisions, giving risetoafurtherradialglial considered tobeembryonicneuralstemcells.Radialglialcells surface (Fig.2A).Bothneuroepithelialandradialglial cells are a cellbodyintheventricular zoneandaradialfiber reachingthepial such as promote formationduringlatedevelopment.Proneural genes neuroepithelial cellsandradialglialduringearlydevelopment, genesmaintain cells finallydifferentiate intoastrocytes. Hes divisions. Aftertheproduction ofdistincttypesneurons, radialglial the pialsurface.Radialglialcellsgiverisetoneurons byasymmetriccell have acellbodyintheventricularzone(VZ)andradialfiberreaching neural plate.Thesecellsgraduallydevelopintoradialglialcells,which differentiation bybHLHgenes.Neuroepithelial cellsinitiallyformthe astrocyte formationbyHesgenes. Fig. 2.Maintenanceofneuralstemcellsandpromotion of 2001)]. Scalebars:100 display radialglialcellmorphology[from Ohtsukaetal.(Ohtsukaal., directs theco-expression of differentiated intoneurons, whereas cells transfectedwithavectorthat glial cellshavemigratedoutoftheVZ(atbottomimage) and drives enhancedgreen fluorescent (EGFP) expression inradial . Manyofthecellstransfectedwithacontrol vectorthatonly the border oftheVZ.( Hes genes, Mash1 Hes6 , Ngn2 also promotes . Thebroken lineindicates and B ␮ ) Transfection experimentsinmouseembryonic m. Math1 Hes1 and promote neurogenesis. Unlikeother EGFP ( A ) Regulationofcell , remain intheVZand Development 134(7) Hes1 and Hes3

DEVELOPMENT are widelyexpressed byneuroepithelialcells,whereas CNS, centralnervoussystem;PNS,peripheralPSM,presomitic ;MHB,midbrain-hindbrainboundary. her3 her1 Zebrafish Hes7 Hes1 Mouse Table 1.Hesandrelated genesinmouseandzebrafish Development 134(7) 2004). there issomeredundancy betweentheHesgenes(Hatakeyama etal., Hes5 her15 her13.2 her12 her11 her9 her7 her6 her5 her4.1 Hes6 Hes5 Hes4 Hes3 Hes2 et al.,2000).However, intheabsenceof maintenance ofradialglialcellsbyinhibitingneurogenesis(Gaiano (NICD), whichisconstitutively active, alsopromotesthe (Hatakeyama etal.,2004).TheintracellulardomainofNotch upregulated, whichaccountsfortheprematureneurogenesis genes, suchas diversity aregenerated.IntheseHes-mutant mice,proneuralbHLH ensuring thatsufficient numbersofcellswithafullcell-type maintain neuralstemcellsuntillaterstagesofmousedevelopment, later-born celltypes(Hatakeyama etal.,2004).Thus,Hesgenes types ofneuronsandbecomedepletedwithoutgiving risetothe knockout mice,thesecellsprematurelydifferentiate intoearly-born formation ofneuroepithelialcellsin and prematurelydifferentiate intoneurons.Despitethecorrect In theabsenceof maintains aradialglialcellidentity(Fig.2B)(Ohtsukaetal.,2001). embryonic day(E)13.5inhibitsneuronaldifferentiation and differentiate asearlyE9.5,although noneurogenesisnormally presumptive eye regions, non-retinalneuronsprematurely diencephalon, areabsent(Hatakeyama etal.,2004).Inthe vesicles, whicharenormally formedontherightandleftsidesof and Table 1)(Ohtsukaetal.,1999). essential effectors of Notchsignalinginthenervous system(Box1 cannot inhibitneurogenesis,indicatingthat Misexpression of Strikingly, in are mainlyexpressed byradialglialcells(Fig.2A),although xrsinFnto Reference Function Expression S cci)Smt emnain(oc-eedn)(Mülleretal.,1996;Holley2002) (Besshoetal.,2001) Somitesegmentation(Notch-dependent) Somitesegmentation(Notch-dependent) (Hansetal.,2004;Bae2005) Repressionofneurogenesis(Notch-independent) CNS, MHB,Inter-proneuronal PSM (cyclic) Notknown(Notch-de Repressionofneurogenesis(bot PSM (cyclic) PSM (cyclic) CNS, PNS N,Poernldmis(Siegeretal.,2004) (Kawamuraetal.,2005) Somitesegmentation(FGF-dependent) (OatesandHo,2002) Somitesegmentation(Notch-dependent) etal.,2002) (Dunwoodie (Pasinietal.,2001;Cunliffe, 2004) (Pasinietal.,2004) Repressionofneurogenesis(Notch-dependent) (Gelingetal.,2003;Geling2004) Somitesegmentation(Notch-dependent) (Nishimura etal.,1998) Repressionofneurogenesis(Notch-independent) PSM (Pasinietal.,2004) CNS Somitesegmentation (Notch-dependent) PSM (non-cyclic) PSM CNS, Proneuronaldomains (Leveetal.,2001;Bae2005) Somitesegmentation(Notch-dependent) PSM (cyclic) Repressionofneurogenesis(Notch-independent) MHB CNS, Inter-proneuronal domains (Baeetal.,2000) (Takke etal.,1999) PSM (cyclic) PSM (non-cyclic) Promotionofneurogenesis(antagonizesHes1) Repressionofneurogenesis(Notch-dependent) CNS MHB Not known Repressionofneurogenesis PSM (non-cyclic) (Hirataetal.,2001;Hatakeyama2004) CNS, Proneuronaldomains Repressionofneurogenesis(Notch-independent) Not known CNS, PNS PSM (cyclic) CNS, PNS Exists inhumanbutnotmouse CNS, PNS CNS, PNS domains Mash1 Hes1 Hes1; Hes5 Hes1 and and Ngn2 or Hes5 Hes5 (also known as , radialglialcellsarenotmaintained double-knockout mice,the optic in thedeveloping mousebrainat Hes1; Hes3;Hes5 Hes1 Notch-dependent and-independent) and Neurog2 Hes1 Hes5 and ), arehighly , theNICD Hes1 Hes5 triple- and are pendent) (Jouve etal.,2000) (Jouve pendent) Although consists ofadistinctsetcelltypes (Kiecker andLumsden,2005). usually crossboundaries;thus, each compartmentformsaunitthat 2005) (Fig.3A).Cellsmigratewithin eachcompartmentbut donot specification ofneighboring compartments(Kiecker andLumsden, hedgehog andFgf8,respectively, andbyregulating theregional the isthmusfunctionasorganizing centersbysecretingsonic specificity ofneighboringcompartments.For example, theZliand delayed ornoneurogenesisandorganizer activities thatregulate cells, whichhave uniquefeatures,includingslow proliferation, Boundaries areformedbyspecializedneuroepithelialorradial glial isthmus istheboundarybetweenmidbrainandhindbrain. boundary betweenthethalamusandprethalamus,whereas the intrathalamica (Zli)andtheisthmus(Fig.3A).TheZliis the compartments byboundaries,suchasthezonalimitans The developing ispartitionedintomany Hes genesregulate boundaryformation nor properlyspecified intheabsenceof al., 2004).Thus,retinalstemcells/progenitorsareneithermaintained occurs inthewild-typeopticvesicles atthis stage(Hatakeyama et 2006). Incompartments, Hes1levels arevariable: highlevels occur the modeofexpression is different inthetwo structures(Baeketal., 2A andTable 1)(Baeetal.,2000). Mash1 activity, andtherebypromotesneuronaldifferentiation (Fig. Hes1 byforminganon-functionalheterodimer, whichsupports is expressed bydifferentiating neurons.Furthermore,Hes6inhibits is uniqueamongtheHesgenes.Unlike developing nervous system,but theexpression andfunctionof Nthdpnet (Ohtsuka etal.,1999) (Notch-dependent) The otherHesgenes( (Ishibashietal.,1995 h Hes1 is expressed inbothcompartments andboundaries, (Sieger etal.,2004) (Sieger etal.,2004) Hes2 and Hes6 Hes1 Hes1 ) arealsoexpressed inthe ; Ohtsukaetal.,1999) , Hes3 and Hes5 PRIMER and . Hes5 , 1245 Hes6 Hes6

DEVELOPMENT 1246 Mash1 subsequently repress thetranscriptionofproneural genessuchas genes, suchas activators. Thus,Notchsignalingactivates thetranscriptionoftarget and recruitment oftheco- co-repressor complex displacement ofthe complex. Thistranscriptionalactivation complexisformedthrough ternary assemble ontargetDNAandform aRBP-J–NICD–MAML RBP-J andMastermind-likeproteins (MAMLfamily, MAML1-3) NICD, activator. In thisprocess, an from atranscriptionalrepressor to Drosophila Su(H)in transcription factorRBP-J(alsoknownasRbpsuh)inmice, theDNA-binding TheNICD translocates tothenucleusandassociateswith from theplasmamembrane. (NICD) domain ofNotch theintracellular release secretase activityofPresenilins 1and2,which is furtherprocessed attwoendomembranesites,S3andS4,bythe membrane-tethered formofNotch.Thetruncated product an active family, TNF- metalloprotease) oftheADAM(adisintegrinand site byanextracellularprotease at the S2 undergo successiveproteolytic cleavage.Thefirstcleavage receptors interact withNotchreceptors. Uponligandbinding,Notch the ligands(DSLfamily)expressed onthesurfaceofneighboringcells Notchsignalingistriggered when functional heterodimeric . the S1siteintotwofragmentsthatremain associatedtoformthe processed byafurin-likeconvertaseat moleculeis Notch synthesized Thenewly (Dll1),Dll3andDll4]. [Jagged1, Jagged2,Delta-like1 mammals, there are fourNotchreceptors (Notch1-4) andfiveligands (Artavanis-Tsakonas etal.,1999;SelkoeandKopan,2003).In (Delta, Serrate,Lag-2)familyoftypeItransmembraneligands fortheDSL Notch familymembersare typeItransmembranereceptors Box 1.Notchsignaling Cytoplasm Nucleus PRIMER . by RBP-J Default repression Co-R n LAG-1in , and RBP-J S2 cleavage Hes1 S1 cleavage (ligand) Delta Hes1, Hes5 and NICD Hes5 OFF C. elegans ␣ (Jarriault etal.,1995).Hesfactorsthen converting enzyme(TACE), generates (receptor) Notch Signal-sending cell sarsl,RPJisconverted Asaresult, RBP-J . Signal-receiving cell S3 cleavage S4 cleavage target activation Notch-mediated RBP-J Co-A MAML Hes1, Hes5 ON ␥ - compartment characteristics(Fig.3B). variable differentiation, raisingthepossibilitythatpersistentversus slowing cellproliferationaswelltotheinhibitionof persistent andhighlevels of Hartman etal.,2004;Baek2006).Thus,withinboundaries, S phasetransition(Castellaetal.,2000;Ström some cellcycle regulators suchasE2F-1,whichpromotestheG1- cycle, probablybecauseHes1alsorepressestheexpression of levels of p21 andp27(Murataetal.,2005).However, persistentandhigh Low levels ofHes1promotecellproliferationbydownregulating whereas theCDKinhibitorsp21andp27antagonizethisprocess. progressionbyformingcomplexes withcyclins, During theG1phase,cyclin-dependent (CDK)promotes nervous systemcouldalsobeindependentofthispathway. (Table 1),suggestingthatHesexpression inboundariesofthemouse expression of hindbrain boundary(Table 1).Ithasbeenreportedthatthe neurogenesis andcontributing totheformationofmidbrain- 2006). Inzebrafish, and thelossoforganizer activity (Hirataetal.,2001;Baek ectopically expressed inboundaries,leadingtoectopicneurogenesis (Baek etal.,2006).IntheabsenceofHesgenes,proneuralgenesare genes, suchas Hes1 versa (Fig.3B).Within boundaries,persistentandhighlevels of express highlevels ofHes1express low levels ofMash1andvice inverse correlationbetweenHes1andMash1levels: cellsthat many boundarycells(Fig.3Bb)(Baeketal.,2006).Thereisan below). Bycontrast,Hes1ispersistentlyexpressed athighlevels by et al.,2006).Hes1levels couldbeoscillatinginthesecells(see in somecells,whereas,others,levels arelower (Fig.3Ba)(Baek same origin. foregut. Thus,thegut,pancreas,liver andbiliarysystemssharethe biliary systemsalsooriginatefromtheendodermalepitheliumof the emigrate fromtheepitheliumtoformislets(Fig.4A).Theliver and cells, whichsecretedigestive enzymes,whereasendocrinecells exocrine andendocrinecells:exocrine progenitorsbecomeacinar (Murtaugh, 2007).Thepancreaticepitheliumgives risetoboth pancreas, grow fromtheendodermalepitheliumofforegut The dorsalandventral pancreaticbuds, which fusetoformthe progenitors indigestive organs Hes1 binding tothepromoter ofthisgene.Similarly, NICDinhibitsacinar differentiation. Hes1 alsorepresses Dll1–Notch–RBP-J–Hes1 pathway inhibitsprematureendocrine of sequence-binding protein(RBP-J) (Box1),orbytheoverexpression delta-like 1(Dll1)orthe Notcheffector Recombinationsignal defects areobserved following inactivation of theNotchligand and severe pancreatichypoplasia(Jensen etal.,2000).Similar Ngn3 2002). Inactivation of 4A) (Krappetal.,1998;Gradwohl etal.,2000;Kawaguchi etal., producing) andPP(pancreaticpolypeptide-producing)cells](Fig. (glucagon-producing), promotes thedifferentiation ofallfourendocrinecelltypes[ exocrine celldifferentiation, whereasthe bHLHgene It hasbeenshown thatHes1regulates cellcycle progression. In thedeveloping pancreas,thebHLH gene Ngn3 , anaccelerationofpost-mitoticendocrinecelldifferentiation expression constitutively represstheexpression ofproneural regulates themaintenance ofstemcellsand Hes1 Hes1 (Apelqvist etal.,1999), suggesting thatthe her3 expression differentially regulates boundary versus Mash1 expression have beenshown toinhibitthecell and her3 , therebyinhibitingneurogenesis(Fig.3Bb) Hes1 her5 ␤ and (insulin-producing), her5 in miceleadstotheupregulation of does notdependonNotchsignaling Hes1 have similaractivities, inhibiting expression maycontribute to Ptf1a expression bydirectly Development 134(7) ␦ Ptf1a (somatostatin- promotes Ngn3 ␣

DEVELOPMENT cells/progenitors byantagonizing Notch-Hes1 signalingpromotes themaintenanceofpancreaticstem Murtaugh etal.,2003;Esni 2004;Fujikuraetal.,2006).Thus, cell differentiation byantagonizingPtf1afunction(Haldetal.,2003; Fig. 3.Differential Development 134(7) function asorganizingcentersbyexpressing (Zli) andtheisthmus.Theseboundariesroof andfloorplates compartments byboundaries,suchasthezonalimitansintrathalamica (CNS) inthemouseatE10.5.TheCNSispartitionedintomany boundaries. Interestingly, in or Fukuda etal.,2006). to theformationofanectopic pancreas(Sumazakietal.,2004; expressed inthecommon bileduct,stomachandduodenum,leading thalamus. ( slower proliferation andnoneurogenesis. Pth,prethalamus; Th, proliferation andneurogenesis, whereas those inboundariesundergo compartments andboundaries:( oscillatory) incompartmentsand( are high,andviceversa.Thesecellsfinallylose boundaries. Incompartments,whenHes1levelsare low, Mash1 levels versus boundarycharacteristics. difference inthe persistently expressed athighlevels,andneurogenesis isinhibited.This differentiate intoneurons. Bycontrast,inboundaries, Fgf8 (purple). Cellsinthesecompartmentsundergoactive A B b. Boundary a. Compartment B

Protein level between compartmentsandboundaries Differential Hes1expression ) The Protein level Mouse developingCNS Forebrain ( A Zli Boundary ) Lateralviewofthedevelopingcentralnervoussystem Hes1 Hes1 Hes1 Pth Hes1 ieTime Time Time expression modesmayconfercompartment Th Ros expression modeisdifferent between -null mice, Shh Floor plate expression betweencompartmentsand

Mash1 tra Hes1

Mash1 l Hes1 Ba Wnt1 Bb ) variableexpression (couldbe or ) persistentlyhighexpression in Ptf1a Ptf1a Fgf8 Protein level and Shh Midbrain Isthmus Hes1 and Roof plate Hindbrain Ngn3 Compartment (pink), expression and Ngn3 Hes1 are ectopically Mash1

Wnt1

Hes1 Caudal is (Fig. 4A). (green) specification et al.,2006).ThebHLHactivator gene bottom ofthecryptdifferentiate intoPaneth cells(Fig.4B)(Crosnier and enterocytes, whereasthosemoving downwards towards the towards thevillidifferentiate intogobletcells,enteroendocrinecells 4B): thecellsmoving upwards towards thetopofcryptand intestine, stemcellsarelocatednearthebottomofcrypts(seeFig. (antimicrobial peptide-secreting)cells(Fig.4B).Intheadult secreting), enteroendocrine(-secreting)andPaneth to fourprincipalcelltypes:enterocyte (absorptive), goblet(mucous- a complex withJak2andStat3.Thisthen inducesthedifferentiation Hes1 promotesJak2-mediatedphosphorylation ofStat3byforming Stat3by phosphorylation.Ithasbeenshown that activates theJanuskinaseJak2,whichthenactivates thedownstream (Nakashima etal.,1999;Kamakura etal.,2004).Lifsignaling morphogenetic protein2)synergistically induceastrocyte formation pathway. Lif(leukemia inhibitoryfactor) and Bmp2(bone Hes1 canalsoinduceastrocyte development throughanother specification isthroughtheinhibitionofproneuralbHLH activators. mechanisms ofHes-inducedastrocyte versus neuronfate (Tomita etal.,2000; Nietoetal.,2001).Thus,oneofthe neurogenesis andshow acceleratedformationofastrocytes atE15.5 Furthermore, micelackingproneuralgenesdisplayablockade of activators ofthesegenes,suchas p300(Sunetal.,2001). inhibit astrocyte-specific geneexpression bysequesteringco- not onlyactivate neuronal-specific geneexpression, but they also Neurog1 –MouseGenomeInformatics)have dualactivities: they bHLH activators suchasneurogenin 1(Ngn1,alsoknown as the developing nervous system. It hasbeenshown thatproneural During latedevelopment, Hesgenespromoteastrocyte formationin Hes1 homolog ofthe inhibits thegenerationof the activation ofNotch-Hes1signalingrepresses development ofgoblet,enteroendocrineandPaneth cells,whereas the adultintestinecauseslossof 2005; Stangeretal.,2005).Theinactivation ofNotchsignalingin Yang etal.,2001;Suzuki2005;van Esetal.,2005;Fre stem cells/progenitorsbyrepressing 2005). Thus,theNotch-Hes1pathway regulates the maintenanceof virtually allcryptcellsintopostmitoticgoblet(van Esetal., concomitant upregulation of ( diversity (Kageyama etal.,2005). astrocyte versus aneuronalfate, therebygeneratingcelltype fate decisions.For example, they regulate thedeterminationofan In additiontomaintainingstemcells,Hesgenesregulate binarycell Hes genesregulate binarycellfatedecisions treating thesetumors(van Esetal.,2005). adenoma development, and the Notch-Hes1pathway alsoplaysanimportantroleinintestinal adenomas intocollectionsofgobletcells(van Esetal.,2005).Thus, to theupregulation of inhibits Notchsignalingbypreventing thereleaseofNICD,leads Strikingly, thetreatmentofmicewitha contributes tothistransformationofprogenitorcellsadenomas. Hes1 (Kinzler etal.,1991).Interestingly, theseadenomacellsexpress Apc In embryonicandadultintestinalepithelium,stemcellsgive rise Loss ofthetumorsuppressorgene ) isassociatedwiththedevelopment ofintestinaladenomas and at highlevels, suggestingthattheNotch-Hes1pathway Hes5 Drosophila regulate astrocyte versusneuron fate Math1 Math1 ␥ Math1 proneural gene -secretase inhibitorsmaybeusefulfor and thesubsequenttransformationof -dependent cells(Jensenetal.,2000; , leadingtothedifferentiation of Math1 adenomatosis polyposiscoli ␥ Hes1 -secretase inhibitor, which . Math1 atonal expression andthe Math1 PRIMER , amammalian , promotesthe , andthereby 1247

DEVELOPMENT stages ofdevelopment. One possibilityisthattheepigenetic status however, whyHesgenescannotinduceastrocyte formationatearly of (Kamakura etal.,2004).Itremainstobedetermined, Paneth cells. which promotes thedevelopment ofgoblet,enteroendocrine and and enterocyte versusnon-enterocyte fatechoicebyrepressing differentiate intoPanethcells. whereas thosemovingdownwards towards thecryptbottom differentiate intogobletcells,enteroendocrine cellsandenterocytes, moving upwards towards thecrypttopandtowards thevilli intestine, stemcellsare foundnearthebottomofcrypt.Thecells inhibiting theexpression of polypeptide) cells].Hes1regulates themaintenanceofprogenitors by cells [ promotes thedifferentiation ofallfourtypespancreatic endocrine gene endocrine cellsemigratefrom theepitheliumandformislets.ThebHLH both exocrineandendocrinecells;cellsformacini,whereas by Fig. 4.Regulationofpancreatic andintestinalcelldifferentiation 1248 B A Exocrine cells Hes1 Adult intestine Pancreatic cell-typespecification Acinar cell ␣ Ptf1a Progenitor Crypt (glucagon), PRIMER . ( A regulates exocrinecelldifferentiation, whereas ) Inthedevelopingpancreas, theepitheliumgivesriseto Ptf1a Villus ␤ (insulin), Hes1 lcgnIslnSmtsai Pancreatic Somatostatin Insulin Glucagon Ptf1a Ngn3 Hes1 ␦ ␣ Enterocyte (somatostatin) andPP(pancreatic and Endocrine cells regulates stemcellmaintenance Islet formation Ngn3 progenitor Proliferative Differentiated cells Stem cell ␤ . ( Paneth cell Goblet cell B ) Intheadultsmall ␦ Math1 Math1 endocrine cell Entero- Ngn3 Hes1 polypeptide Hes1 PP Math1 , expression of synchrony explains why, afterseveral cycles, theoscillatory oscillations amongcellseasily fall outofsynchrony. Thislossof period varies fromcycle tocycle withinacell,and,therefore, al., 2006).Rather, thedampeningarisesbecause theoscillation 2 days,asrevealed byreal-timeimaging analysis (Masamizuet cells, inwhich observation isnotduetodampenedoscillationwithinindividual dampened afterthreetosixcycles (6-12hours).However, this as abiologicalclock(Fig.5A).Thisoscillationistransient and a periodicityofapproximately2hours,suggestingthatHes1 acts this way, Hes1autonomouslyinitiatesoscillatoryexpression with short-lived duetotheshorthalf-lifeof mRNA andprotein.In own genebydirectlybindingtoitspromoter. Thisrepressionis 5A). Afterinduction,Hes1proteinrepressestheexpression ofits cell-autonomous anddependsonnegative autoregulation (Fig. expression isoscillatory(Hirataetal.,2002).Hes1oscillation is myoblasts andneuroblasts;however, strikingly, theinduced or Notchactivation inmany celltypes,suchasfibroblasts, In thedeveloping intestineof organs Hes1 time, correlatingwiththeirdifferential responseto Hesgenes. 2001). Thus,theintrinsicpropertiesofneuralstemcellschangeover promoters arehypomethylatedinlaterstemcells(Takizawa etal., specific promotersisrepressedbyhypermethylation,whereasthese neural stemcells;inearlycells,transcriptionfromastrocyte- of astrocyte-specific genesisdifferent betweentheearlyandlate single-cell level (Masamizu etal.,2006).Thus,atany given time, Hes1 levels seem tobeconstantbynorthernandwesternblotanalyses, analysis. Similarly, even instationarycultured cellswhereHes1 Expression of Hes1 biliary epithelialcells.Intheabsenceof enterocytes. downregulation ofNotchsignalingisrequiredforthematuration enterocyte versus non-enterocyte specification, althoughthe 2005). Thus,itislikely thatNotch-Hes1signalingpromotesthe enterocytes bytheinactivation ofNotchsignaling(Crosnieretal., goblet andenteroendocrinecellsareformedattheexpense of Suzuki etal.,2005;van Esetal.,2005).Similarly, inzebrafish, enteroendocrine andPaneth cells(Fig.4B)(Jensenetal.,2000; activator expression of process (DubrulleandPourquié,2004).Ithasbeenshown thatthe hours, indicatingthataninnatetimingmechanismregulates this In mouseembryos,abilateralpairofsomitesisformedevery 2 Hes genesare molecularoscillators brought aboutbyJagged-mediatedHesactivity. that thespecification ofabiliaryfate versus ahepatocytic oneis ligand syndrome, whichisassociatedwithmutationsinthehumanNotch (Kodama etal.,2004).Thisphenotypeissimilar tothatofAlagille normally, whereasintrahepaticbileductsarecompletelyabsent and mayfunctionasabiologicalclock; whereas timing ofbiologicalevents inmany celltypes,suchasfibroblasts, Liver progenitorsgive risetotwo typesofcells:hepatocytes and regulates binarycellfatedecisionsindigestive is acellularoscillator expression isfoundto be dynamicallychangingatthe jagged 1 jagged Hes7 Math1 Hes1 functions asthesegmentation . Hes1 Hes1 Hes1 (Oda etal.,1997;Li1997).Thus,itislikely is upregulated, leadingtoanincreaseingoblet, and can beinducedfollowing serumstimulation is notdetectedbynorthernor westernblot expression isstillcyclical even afternearly Hes7 oscillates withaperiodicityof2hours, Hes1 -knockout mice,thebHLH Hes1 Hes1 Development 134(7) seems toregulate the , hepatocytes form

DEVELOPMENT mouse of somites.InthemousePSM, (Palmeirim etal.,1997).Eachwave leadstothegenerationofapair of thePSM(propagationisclassified intothreephases;Fig.5Bb) initiating attheposteriorendandmoving towards theanteriorregion was first reportedthattheexpression of controlled byabiologicalclock,calledthesegmentation clock.It in mouseembryos.Ithasbeensuggestedthatthisperiodicevent is (Dubrulle andPourquié,2004).Thisevent isrepeatedevery 2hours anterior region ofthepresomiticmesoderm(PSM;Fig.5B) skeletal musclesanddermis,areformedbythesegmentation ofthe Somites, thesegmental unitsthatlatergive risetothevertebrae, ribs, Hes7 unknown. cellular events, suchasthecellcycle, but itspreciserolesare same stimulus.Hes1oscillationmayregulate thetimingof which mayenableacelltomediatedifferent response tothe Hes1 Development 134(7) in asimilarfashion toeachother(Fig.5B),but important forperiodicsomitesegmentation. Inzebrafish, fusion (Hirataetal.,2004).Thus,oscillatoryexpression isvery oscillation duetopersistent vertebrae andribs(Besshoetal.,2001).Interestingly, alackof 5Bc). Intheabsenceof Hes7 important forsomitesegmentation. Thiswave-like expression of 2000). Hes7proteinrepressestranscriptionfromthe Notch activity byglycosylation(Moloney etal.,2000;Brückner genes istheglycosyltransferase feedback (Fig.5A)(Besshoetal.,2003).Oneofitsdownstream target (Table 1). her7 Hes7 well asfromitsown, andthus negative-feedback loopson oscillation. Itisthoughtthatthecombinedeffects ofthesecoupled et al.,2005;Huppert2005),whichmayinturninfluenceHes7 generates oscillationsinNotchactivity (Daleetal.,2003;Morimoto 2003). LfngperiodicallyinhibitsNotchsignalingandthereby fusion (Evrardetal.,1998;ZhangandGridley, 1998;Serthetal., persistent expression of oscillation isalsocrucialforsegmentation, asboththe lossofand constitutively expressed inthePSM (Besshoetal.,2001). Real-time visualizationof expression isvariable, suggestingthatitoscillatesinthesecells. cells remainstobedetermined. Inmouseneuralstemcells, in many celltypes,thesignificance ofthisoscillationinnon-PSM such assomitesegmentation. Although Hes genesalsoregulate thetimingofseveral developmental events, decisions inmany organs, onlysomeofwhicharedescribedhere. of stemcells/progenitors,but they alsoregulate thebinarycellfate genesnotonlypromotethemaintenance It hasbeenshown thatHes Conclusion of thebiologicalclock. for sustainedandsynchronizedoscillationsthecorrecttiming unclear. Although Hes1 oscillationstostemcell proliferation anddifferentiation is (R.K. andT.O., unpublisheddata).However, thesignificance of cells, persistent and highlevels of Hes7 oscillation,like thatofHes1,isregulated bynegative have oscillatoryexpressions andregulate somitesegmentation expression (Besshoetal.,2003).Intheabsenceof is elicitedbyitsoscillatoryexpression ineachPSMcell (Fig. is anessentialcomponentofthesegmentationclock expression levels arevariable withinandbetweencells, Hes1 , isperiodicallypropagatedinawave-like fashion Hes1 Lfng Hes7 is requiredforthemaintenance ofstem Hes1 Hes7 has beenshown toleadsevere somite Lfng Hes7 , somitesfuse,whichresultsinfused lunatic fringe Hes1 expression supportsthis suggestion and expression oscillatesinphasewith Hes1 expression alsoleadstosomite , Lfng Hes5 chairy1 expression inhibitbothcell Hes1 expression areimportant and ( Lfng expression oscillates , achickhomologof Hes7 Hes7 ), whichregulates Lfng are expressed Hes7 promoter as is themost her1 , Lfng Hes1 Lfng and is clock. contexts, aswellin the crucialmaintenanceofbiological genes playanindispensable roleindifferent developmental development iscontinuallyimproving. Itisnow apparent thatHes networks operateincellproliferation anddifferentiation during feature ofmany cellular events. Ourunderstandingofhow , andthatthese oscillationscouldbearequiredgeneral that moregenesmaybeidentified thatdisplay oscillatory (Nelson etal.,2004;Lahav etal.,2004).Thisraises the possibility Hesgenesarenot the onlyoscillators circadian clockgenes, Furthermore, recentstudieshave revealed that,otherthanthe oscillatory expression maybeimportantforstemcellmaintenance. proliferation anddifferentiation (Baeketal.,2006).Thus, ( anterior region ofthepresomitic mesoderm(PSM,showninblue). five-somite stage.Somitesformperiodicallybysegmentationofthe in somitesegmentation.( expression of Fig. 5.Hesgenesare molecularoscillators. posterior totheanteriordirection. oscillatory expression ineachPSMcellwithaslightdelayfrom the a pairofsomites(buff). ( classified intothree phases),andeachwaveleadstothegenerationof posterior endtotheanteriorregion ofthePSM(shownbybluearrow, this way, they haveveryshorthalf-lives,allowingthenextround ofexpression. In (red). Then,both production ofHes1protein, whichrepresses expression ofitsowngene activation (green), asinducedbyNotch,forexample,inducesthe Bb B A mesoderm (PSM) Presomitic Somite a Activation ) Somite formation Hes1 autoregulation Hes7 Notch andothers Posterior Anterior Hes1

expression isperiodicallypropagated, likeawave,from the Level Hes1 expression autonomouslyoscillates.( 2h Hes1 Hes1 is regulated bynegativefeedback.Promoter Hes7 Repression gene Somite b c mRNA andprotein disappearrapidlybecause PSM Bc

Hes7 mRNA level Ba mRNA ) Thisdynamicchangeiselicitedby hs hs IPaeIIPhase I PhaseIII PhaseII Phase I ) Ventral viewofamouseembryoatthe hs hs IPhaseIII PhaseII Phase I Time Hes1 Hes1 mRNA Hes1 protein AAAA mRNA 2h ( A ) Oscillatory Proteasomal degradation 2 hours B ) Hes7oscillation Hes1 protein PRIMER Ub-Ub -Ub-Ub-Ub-Ub Phase I Time Time Posterio Anterior Region 2 Region 1 Region 2 Region 1 1249 r

DEVELOPMENT Bessho, Y., Sakata,R.,Komatsu,S.,Shiota,K.,Yamada, S.andKageyama,R. Bessho, Y., Hirata,H.,Masamizu,Y. andKageyama,R. Artavanis-Tsakonas, S.,Rand,M.D.andLake,R.J. Apelqvist, A.,Li,H.,Sommer, L.,Beatus,P., Anderson,D.J.,Honjo,T., de Baek, J.H.,Hatakeyama,J.,Sakamoto,S.,Ohtsuka,T. andKageyama,R. Cunliffe, V. T. Crosnier, C.,Stamataki,D.andLewis,J. Castella, P., Sawai,S.,Nakao,K.,Wagner, J.A.andCaudy, M. Brückner, K.,Perez, L.,Clausen,H.andCohen,S. Chen, H.,Thiagalingam,A.,Chopra,Borges,M.W., Feder, J.N.,Nelkin, M.andKageyama,R. Y., Hojo, Bessho, S., Bae, Bae, Y. K.,Shimizu,T. andHibi,M. Fisher, A.L.,Ohsako,S.and Caudy, M. Fishell, G.andKriegstein,A.R. Evrard, Y. A.,Lun,Y., Aulehla,A.,Gan,L.andJohnson,R. Esni, F., Ghosh,B.,Biankin,A.V., Lin,J.W., Albert,M.A.,Yu, X.,MacDonald, S.M. Weintraub, H.andParkhurst, Turner, D.L., Dawson, S.R., Dale, J.K.,Maroto, M.,Dequeant,M.-L.,Malapert,P., McGrew, M.and Crosnier, C.,Vargesson, N.,Gschmeissner, S.,Ariza-Mcnaughton,L., R. 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