Notch Signaling Regulates the Differentiation of Neural Crest From

Home , Neural crest, SNAI2

Teeatoscnrbtdeulyt hswork this to equally contributed Finland. authors Helsinki, *These FIN-00029 Hospital, Central University erlg,Boeiu tmCl ete nvriyo esni FIN-00290 Helsinki, of University Finland. Centre, Helsinki, Cell Stem Biomedicum Neurology, I-02 uk,Finland. Turku, FIN-20520 Thailand. 30000, Ratchasima Nakhon Technology, of University aiy Noisa Parinya from crest cells neural stem of pluripotent differentiation human the regulates signaling Notch ARTICLE RESEARCH eevd5Nvme 03 cetd6Fbur 2014 February 6 Accepted 2013; November 5 Received ` 3 as known can disorders 2006; stages al., et 1 congenital later (Etchevers melanoma of as at such variety cancers disruption to a contribute whereas melanocytes, to development bone neurocristopathies, skin crest lead neural alveolar of system, disruption and can Early 2007). nervous odontoblasts al., et dentin, (Dupin peripheral glial cartilage, this the and and craniofacial neurons embryos, of into vertebrate differentiate all cells can in population found cell are transient cells crest Neural INTRODUCTION signaling Notch crest, Neural induced cells, Human stem cells, pluripotent stem embryonic Human WORDS: KEY expressed pNCCs differentiation. including genes, crest Notch neural-crest-specifier of various neural self- roles the during generated investigated and signaling (hPSCs) we human cells during from stem Here, crest (pNCCs) pluripotent cells roles understood. neural neural-crest-like premigratory human poorly important renewing during is plays function development signaling its peripheral into however, and differentiating Notch craniofacial neurogenesis; of tissues. including capable types, neural are nerve the cell They between somatic epiderm. border various the the at and specified plate are cells crest Neural ABSTRACT imdcn/hsooy nvriyo esni I-01 esni Finland. Helsinki, FIN-00014 Helsinki, of University Biomedicine/Physiology, optl I-02 esni Finland. Helsinki, FIN-00029 Hospital, nrclua oan(ID)agetdteepeso fneural- of expression the augmented (NICD1) Notch1 neural-crest- activated domain of intracellular of expression ectopic expression contrast, By the the genes. during specifier suppressed signaling Notch differentiation Blocking pNCC derivatives. crest neural most as omto,mgainaddfeetaino erlcetfo hPSCs. from the crest regulates neural of signaling differentiation Notch neural- and that the migration of show formation, the and and generation the pNCCs for the for of protocol state, a generation to provide required crest we led together, their Taken neurons. signaling neural also to crest-derived Notch premigratory bind was of the to suppression activity of found Notch was maintenance regions. NICD1 and promoter genes, crest-specifier aoiaLundin Karolina uhrfrcrepnec ([email protected]) correspondence for Author uk etefrBoehooy nvriyo uk n A and Turku of University Biotechnology, for Centre Turku Suranaree Technology, Agricultural of Institute Biotechnology, of School 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,28–04doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. SNAI2 ), SOX10 6 eateto bttisadGneooy Helsinki Gynaecology, and Obstetrics of Department 1,2 and 2 aiaLund Carina , aawnChokechuwattanalert Hataiwan , 4 hlrnsHsia,Hlik nvriyCentral University Helsinki Hospital, Children’s TWIST1 5 n eeal odfeetaeinto differentiate to able were and , eerhPorm nt Molecular Unit, Programs Research 2 ate Kanduri Kartiek , ˚ SLUG oAaeiUniversity, Akademi bo 2 nttt of Institute as known (also 3 ikaLund Riikka , 2 ioOtonkoski Timo , omto ftenua ue rmgaoynua rs cells crest neural premigratory tube, neural the the of Following 1996). formation Mayor, and Mancilla 2000; Bronner-Fraser, SLUG from originate neural- the for are cells specific as the are they such that border, and genes and plate crest of plate expression 2004), the neural Bronner-Fraser, by Neural marked the and between (Meulemans 2013). border epiderm the al., at neuroectoderm et Kokta hypouemgaoynua rs el mre ythe by [marked cells crest as known neural of but migratory 2011), expression al., et produce Menendez from 2007; cells al., crest they et neural al., (Lee signaling derive available to et are Protocols hPSCs (Hay hierarchical 2010). al., development et follows embryonic Wu 2008; regulate hPSCs also that of cascades differentiation development. human The early underlying mechanisms the molecular the reported. of been of differentiation not have role(s) and lineage the specification crest neural the Nevertheless, nerve human in 2008). enteric signaling of Saga, Notch maintenance and the neural-crest-derived (Okamura and of cells muscle differentiation and the smooth formation 2012). for vascular Yutzey, crest required and neural is Mead 2012; promote Notch al., some to et (Humphreys in shown development Notch- and, been 1995). neurogenesis al., has et prevents cases, Jarriault inhibition 1996; lateral transcription al., (C/ et the mediated sequence (Hsieh activates consensus genes and target the 1994), al., of al., to et et binds the (Tun (Schroeter then T)GTGGGAA (NICD) CBF1 and complex activator protein, The transcription domain DNA-binding cleaved, 1998). a a forms are with intracellular and complex nucleus between receptors the Notch into binding Notch translocates cytoplasmic ligand–receptor the resulting Upon cells, (Perdigoto 2013). adjacent (Notch-1–Notch-4) Bardin, receptors -2) and four and Jagged-1 with and -4 interact and -3 that (Delta-1, ligands five TGF- has Garcı pathway Wnt, and FGF, pathways (Stuhlmiller BMP, signaling multiple the by including directed is ectoderm embryonic giving to away types. prior cell tissues migrate crest specialized embryonic and various to neural to rise neuroepithelium tube Migratory neural the the cells. from and from crest delaminate (EMT) neural cells transition migratory epithelial-to-mesenchymal become an undergo h erlflsadsbeunl oaieisd h dorsal cells the crest as inside neural such premigratory genes, localize These specifier tube. subsequently express neural the and within of remain folds portion cells crest neural neural the process, folding neural-tube the erlcetclsaeal ognrt ernladnon-neuronal and neuronal generate to able are cells crest neural ua lrptn tmcls(PC)aewdl sdt study to used widely are (hPSCs) cells stem pluripotent Human vivo In 3 ar La Harri , as nw as known (also CD57 h pcfcto fnua rs el rmthe from cells crest neural of specification the , 4,5 ioTuuri Timo , p75 and ¨hdesma B3GAT1 as nw as known (also DLX5 SNAI2 ´ -ato 02.TecnnclNotch canonical The 2012). a-Castro, , ¨ki MSX1 ), ] ohpeirtr n migratory and premigratory Both )]. 5,6, 3 SOX10 itaLahesmaa Riitta , n aeiRaivio Taneli and * SNAIL , MSX2 and NGFR b as nw as known (also and TWIST1 n oc pathways Notch and and ) ZIC1 ae,during Later, . LBneand (LaBonne HNK1 3 , 2,4, SNAI1 * 2083 (also , ` ),

Journal of Cell Science ee fNth nrclua oan(ID)poen but protein, increased (NICD1) an domain by intracellular shown Notch1 as of activated, was level pathway signaling and cells Tuj1 glial GFAP-positive for became positive them were 1F). of 1E). (Fig. cells few (Fig. a Wnt These and areas. without respectively peripherin, some medium Red, in spontaneously N2B27 were formed cells in neural-like detected Alizarin inhibition, BMP cultured were or and activation were osteocytes pNCCs Blue and When adipocytes Alcian chondrocytes Oil resulting whereas by by stained The O, were Red and kits. cells lipid-droplet-containing available exhibited lineage differentiation commercially directed Mesenchymal of weeks experiments. with 2 after differentiation by obtained were shown of derivatives as cells, series crest neural a of capacity differentiation typical O1 n WS1 oee,ol e fte were lines pNCC them clonal of of 78.1% derivation was the few 1D) of (Fig. efficiency a SLUG, The 1B). only PAX3, (Fig. S3). AP-2 however, for for Fig. TWIST1; positive positive also material and homogeneously can SOX10 (supplementary conditions stained identity induction crest pNCCs pNCC neural the 20 and than that maintain more genes suggesting (after marker culture passages), long-term crest in neural capacity cells sustained differentiation of stem pNCCs expression pluripotent The S2). induced the Fig. in material (supplementary observed (iPSCs) also crest neural was Similar S1). induction Fig. material (supplementary crest neural hrfr,peirtr erlcetclswudb da for 2009). of ideal specification al., the be fate. et control crest would that neural Schwarz pathways cells signaling 2012; crest the al., studying neural et premigratory for (Hari Therefore, usefulness terminally will their purposes restrict towards cells might research which crest specification types, cell neural lineage differentiated migratory progressive however, Bronner-Fraser undergo 1988); 1989; Fraser, Fraser, and and (Bronner-Fraser descendants ARTICLE RESEARCH 2084 of expression of and lack [ the genes by marker indicated neural not as but cells, pNCCs progenitor induced neural specifically protocol neural our differentiation note, crest Of S1). genes robustly Fig. material pluripotency (supplementary cells undetectable the these of expression induction, of SLUG with including days genes, medium TGF- neural-crest-specifier replacing 10 expressed withdrawing (hESC) by and After cell medium 2011), inhibition. differentiation stem al., neural embryonic et previous N2B27 (Menendez human a inhibition modified StemPro BMP we type, with naı together activation a Wnt-mediated cell used generate that To protocol differentiation crest fate. cell neural crest neural premigratory the requirements to signaling crest commitment the neural revealing for in the step of important differentiation an is the lineage of phase early the the hESCs Capturing of from of pNCCs of differentiation Derivation maintenance the the RESULTS and inhibition. fate, Notch upon state crest cells crest neural crest neural to premigratory a neural protocol to the premigratory commitment the use hPSCs in signaling we Notch of of roles Furthermore, crucial cells the hPSCs. investigate premigratory-neural-crest-like from of (pNCCs) maintenance long-term uignua rs ifrnito,w on htteNotch the that found we differentiation, crest neural During nti ae,w eotapooo o h ifrnito and differentiation the for protocol a report we paper, this In SOX1 , MSX2 fe 0dy fdfeetaini odtosta induce that conditions in differentiation of days 10 after ] , HAND2 a akro irtr erlcetcells crest neural migratory of marker a , 6 PAX6 .%( 2.4% and , 6 NGN2 SOX10 ...TeepCspsesdthe possessed pNCCs These s.d.). as nw as known (also Fg AC,weesthe whereas 1A–C), (Fig. OCT4 and PAX3 NANOG NEUROG2 , TWIST1 was ¨ve b ) , o OC1poen(i.1) nadto,w bevda observed effector Notch we the ligand, addition, of expression In the genes 1G). in (Fig. upregulation protein significant NOTCH1 not a togysprse ybt ocnrtoso DAPT, of genes neural concentrations the both of by expression the suppressed whereas strongly was 0dy Fg BC.W hncnimdteefc fNotch of effect the of confirmed expression then the silencing We by 2B,C). inhibition NGN2- for (Fig. treatment of days DAPT detection to 10 following the crest cells by neural NGN3-immunopositive shown from and explicitly switch was fate lineage The neural 2A). (Fig. upregulated was upesdb AT(i.3) ycnrs,anme fneural of number a contrast, genes By 3A). lineage (Fig. DAPT by suppressed lineage- well- Several and S4). genes 3B) lineage Fig. (Fig. crest neural material known genes Gene (supplementary number S2. Notch-related genes crest/neural-related specific and accession 3A), neural S1 (Fig. as GEO, Tables clustered genes at material were genes profiles down-regulated supplementary available expression and in up- are listed highly are most data 20 The raw GSE53203). all D; pNCCs in upregulated significantly al., et was (Ba The 1H). differentiation which (Fig. cell and induces that 2012), signaling Notch for HES1 eeepeso nlss tdy1,teepeso fneural- of expression the concentrations, 10, ( day genes different At crest-specifier two analyses. expression at gene DAPT inhibitor applied 10 chemical We a we neural either process. we First, using To differentiation, or experiments. by suppress crest (DAPT) 2004). signaling gain-of-function neural Notch to al., in and inhibited Notch et loss- reported of performed (Geling role been the neurogenesis establish has and differentiation signaling crest Notch neural of differentiation hESCs the from for indispensable is Notch ID1 gene global Notch of comparing absence or by presence (20 the inhibition cells in pNCCs crest of profiles expression neural the of of 2002). specification of repression Eisen, and expression the (Cornell genes the (2) neural-lineage of of and/or expression two activation by genes the pNCCs of neural-crest-specifier (1) formation mechanisms; the for possible that required suggest is data These activation 2F). Notch (Fig. derivatives marker, the crest neural neural addition, produce In a 2E). whereas (Fig. studied, induced neural-crest-specifier were the that all western genes of by expression lowered diminished with validated cells displayed as The 2D). expression, (Fig. protein blotting JAG1 of in level mRNA reduction the in reduction aha Fg B.A xetd efudta ubrof number a signaling that Notch found example the we for in expected, genes, implicated As Notch genes 3B). on (Fig. pathway DAPT the analyzed of we genes, effect lineage-specific Besides isolation. cell for nrae NC xrse aiu elsraemres(e.g. markers cell-surface note, gene various Of in CD44 3C). expressed differences (Fig. (supplementary pNCCs PCR observed real-time untreated applied these by confirmed of was were Some expression DAPT S4). Fig. when material upregulated were enx setie h oeo oc inln nthe in signaling Notch of role the ascertained next We m , n 20 and M KAL1 , , HES1 HES5 ITGB2 ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal JAG1 , JAG1 PAX3 , uigpC omtet(i.1H). (Fig. commitment pNCC during , and m LFNG and (NGN1 m JAG1 AT tdy1 fdfeetain(i.3A– (Fig. differentiation of 10 day at DAPT) M ,fr1 asadcletdRAsmlsfor samples RNA collected and days 10 for M, HES5 , ncdw uigtepC differentiation pNCC the during knockdown CDH1 SNAIL2 and ncdw ECcoe displayed clones hESC knockdown DLX5 , swl stegn noigaNotch a encoding gene the as well as , NGN2 i.3)ta ol eadvantageous be could that 3A) Fig. ; NCSTN , , NOTCH1 PAX3 WNT3A , AH NEUROD1 MASH1 (BMP2 eesprse pnDAPT upon suppressed were , JAG1 , JAG1 SLUG , , SOX9 edn oasubstantial a to leading , , NUMBL dfcetclsfie to failed cells -deficient CDH1 JAG1 , JAG1 SOX10 and NGN2 rca ligand crucial a , , , eeexpression gene DLX5 TWIST1 JAG1 and NGN2 and and , TWIST1 HAND1 , were ) PAX6 , NGN3 JAG2 was , 60% ) ) , ,

Journal of Cell Science ee a esrdi EC E)adpCs(C yuigra-iePR h aaaepeetda h mean the as presented Notch- are of data induction The The (H) PCR. blotting. real-time western using by shown by as (NC) NICD1, pNCCs of and presence the (ES) by hESCs proved Scal drop in was cells. lipid measured differentiation Alizarin-Red-stained as crest was red neural bar: as recognized genes during osteocytes Scale are pathway and medium. Adipocytes Notch cells neural-crest-induction pNCCs. Alcain-blue-stained the in blue of derived cultured as clonally were chondrocytes and O, from ima plates Red 500 derived Oil (left); 96-well phase-contrast osteocytes with onto Blue Representative and red hESCs. cell/well derivatives. stained chondrocytes undifferentiated 1 cells crest of adipocytes, of containing marker neural density of a to a OCT4, images at for pNCCs Phase-contrast positive plated derived (E) were were clonally cells pNCCs the of pNCCs. of Differentiation none of whereas (D) derivation cells antibody. all specific nearly in white, evident control; was staining SOX10 AP-2 and SLUG for PAX3, positive was cells the FOXC1 EERHARTICLE RESEARCH enx etdoeo h osbemcaim ywihNotch We which genes. by neural-crest-specifier mechanisms of possible expression the the augment of could one tested next We crest neural of expression genes the controls positively NICD1 Notch as such to Notch, potential by linked novel regulated be several be crest might were neural that to there during genes several these, treatment known Among DAPT that by specification. are revealed affected were that analysis signaling factors Pathway additional 3B). (Fig. treatment G cyclophilin to normalization after hESCs, to relative calculated were changes Fold differentiation. mean the the during as differentiation. time-points presented crest different neural at during activated PCR is time signaling Notch 1. Fig. and m mdl n ih) F h ifrnito fpCst erllnae a ofre yprpei,Tj n FPimnsann.()Activation (G) immunostaining. GFAP and Tuj1 peripherin, by confirmed was lineages neural to pNCCs of differentiation The (F) right). and (middle m IGFBP4 6 s.d.; Fg 3D). (Fig. n a 5 akro irtr erlcetcls C h uiyo h NCpplto a vlae yfo yoercanalyses. cytometric flow by evaluated was population pNCC the of purity The (C) cells. crest neural migratory of marker a , .()Teepeso fPX,Nsi,TIT LGadSX0wsasse yuigimnsann.Ol rcinof fraction a Only immunostaining. using by assessed was SOX10 and SLUG TWIST, Nestin, PAX3, of expression The (B) 3. CER1 , FOSB A nue xrsino erlcetseiirgnswsasse yuigreal- using by assessed was genes neural-crest-specifier of expression Induced (A) , aiu asdrn h ifrnito Fg EF.Cell 4E,F). (Fig. differentiation the during we differentiation, days at crest genes neural-crest-specifier various neural of profiles on expression effect the the followed overexpression investigate To NICD1 4C). overexpression (Fig. the of blotting confirmed hESCs western and by transfected NICD1 construct stably of this then with We iPSCs and 4B). (Fig. cells, activity 293 luciferase HEK the into activated transfected significantly NICD1 When assay. activity luciferase is osrce osiuieepeso etrfrhuman a both for performing by vector activity its expression validated HES1 and constitutive 4A) (Fig. a NICD1 constructed first rmtrasyada and assay promoter ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal CBF1 6 s.d.; epnieeeet( element responsive HES1 n 5 ;* 3; rmtrand promoter P , .5(Student’s 0.05 e fteclonal the of ges as 100 bars: e h aaare data The . CBF1RE CBF1RE 500 related let- t -test). 2085 IgG , m m m. )- m -

Journal of Cell Science n noeiS ln Fg E.Teepeso fteegenes these of expression The 4E). shown) (Fig. is clone clone iPSC representative one one in from in and (data cells clones NICD1-overexpressing hESC in two upregulated dramatically was EERHARTICLE RESEARCH 2086 of expression genes the neural-crest-specifier pNCCs, normal the towards differentiation were Upon 4D). hESCs (Fig. NICD1-overexpressing genes undifferentiated pluripotency-associated of in expression the and morphology 2. Fig. e etpg o legend. for page next See PAX3 , TWIST1 , SLUG and DLX5 ncnrlclsa hstm-on Fg F.Ti eutrie the raised of result 5 This TWIST1- 4F). day (Fig. as time-point soon this detected at as for cells undetectable SoxE control positive were in and proteins doubly these we SLUG NICD1 whereas were overexpression, and NICD1 without that PAX3, (i.e. addition, and cells SOX10 and cells cells control In immunopositive with at compared cells overexpression). as NICD1-overexpressing in 10 tenfold day almost amplified was ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal

Journal of Cell Science i.2 niiino oc inln rvnscmimn oteneural the to commitment prevents signaling fate. Notch crest of Inhibition 2. Fig. ARTICLE RESEARCH erlcetdfeetain ycnrs,teepeso fnua genes neural of expression of the 10 contrast, ( day By at differentiation. assessed crest as neural genes, neural-crest-specifier of expression dpcts hnrctsadotoye eie rmcnrland control from of derived images osteocytes Phase-contrast and (F) chondrocytes hESCs. adipocytes, wild-type with compared as whereas genes, neural-crest-specifier NGN2 of expression reduced significantly of ( expressing knockdown construct hESCs the a of confirmed with analyses (shJAG1) and transfected blotting NGN2 stably western became and were cells PCR that the Real-time a of (D) indicated most positive. pNCCs whereas NGN3 DAPT-treated cells, and SOX10-positive (Cont) of control loss in SOX10 and 50 NGN3 bars: Scale NC) treatment. (Cont DAPT without differentiated or were NC) that (DAPT 20 hESCs with in medium NGN3 neural-crest-induction and in NGN2 G. of cyclophilin Immunostaining to (B) normalization after hESCs undifferentiated lesandclsadotoye srdAiai-e-tie el.Scale cells. Alcain- Alizarin-Red-stained 100 blue red left as as bars: chondrocytes osteocytes O, and Red cells Oil blue-stained by stained cells droplet-containing be could in differentiation detected mesenchymal No pNCCs. hESC-derived knockdown mean NGN2 . 0 RArdcin.()Tekokonof knockdown The (E) reduction). mRNA 60% m ATfr1 as la oiiesann sosre nyafter only observed is staining positive Clear days. 10 for DAPT M 6 xrsinwsidcda a n 0o erlcetdifferentiation crest neural of 10 and 5 day at induced was expression s.d.; and A upeetto ihDP (10 DAPT with Supplementation (A) JAG1 NGN3 n 5 m ,mdl n ih 500 right and middle m, ;* 3; kokoncls dpctsaercgie srdlipid- red as recognized are Adipocytes cells. -knockdown nrae infcnl.Fl hne r eaieto relative are changes Fold significantly. increased ) P , .5(Student’s 0.05 m .()Fo yoercaayi fNGN2, of analysis cytometric Flow (C) m. t -test). JAG1 m .Tedt r rsne sthe as presented are data The m. RAadpoenexpression protein and mRNA m Mor20 JAG1 eutdin resulted JAG1 m )ihbtdthe inhibited M) shRNA JAG1 - inlfrtemgaino erlcetcls(aoe l,21) In migrate, 2011). to able al., not et were (Sato chemotactic pNCCs cells experiments, a crest wound-healing as neural tissues our of recognized various migration is in the FGF8 state for and signal cell 2008), 2012). stem al., the Mayor, et preserve (Maillard and to known Theveneau fates is 2012; cell Notch LeDouarin, of somites, and restriction developing progressive (Bronner the undergo around along simultaneously pathways migrate and lateral cells and crest medial neural the development, fetal pNCCs of early fate During the the modulates of signaling Notch activator hESCs. from direct crest These a neural of controls. as promotes differentiation thereby positive the acts and genes as NICD1 neural-crest-specifier in of used transcription that 4H). (Fig. genes were suggest hESCs neural-crest-specifier genes, results in effector of not Notch regions but bound regulatory protein pNCCs, NICD1 Significantly, the 1994). chromatin al., to et investigation (Tun complex under using 4G) NICD1–CBF1 regions the (Fig. of DNA sequence The recognition by NICD1. the contained by 5 induced found NICD1 previously the be were to that on genes of neural-crest-specifier focused of regions We regulatory capability (ChIP). the immunoprecipitation to binding investigated bind therefore the We directly genes. neural-crest-specifier might of regions NICD1 that possibility Fg A.B otat hnbt G8(0 gm)adDAPT and ng/ml) (100 FGF8 both FGF8 when ng/ml contrast, 100 with By supplemented 5A). was (Fig. medium the when even ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal rw ntepeec fDAPT. of presence the in cells grown crest neural with when compared cells crest neural in control expression lower indicates and green expression higher indicates Red differentiation. crest Notch neural by during controlled is that network signaling the showing analysis C.Tedt r rsne sthe as presented mean are data The real-time PCR. by validated was samples microarray from level comparative expression The (C) pNCCs. control Cont-NC, pNCCs; DAPT-treated DAPT-NC, transcripts; downregulated red, transcripts; upregulated Green, genes. for Notch-related results Microarray (B) genes. (NC)/neural-related crest neural for results Microarray (A) for analysis. used the were sets sample replicated pNCCs. DAPT- treated and control of expression analysis gene Global 3. Fig. 6 s.d.; HES1 n 5 .()Nthpathway Notch (D) 3. he independently Three and 9 HES5 promoter 2087 the ,

Journal of Cell Science nraewsse ntenme fHK-oiieadNGFR- and HNK1-positive 5E). the of (Fig. number days, cells the 3 positive in seen for was DAPT increase and were 5.7% the FGF8 DAPT from Upon AP-2 5B,C). with and of (Fig. alone pNCCs FGF8 number given both of was FGF8 when treatment The if not upregulated. only but were seen applied, cells, was crest neural induction migratory of Simultaneously, markers 5A). (Fig. hours 24 EERHARTICLE RESEARCH oreo esr ern Cmdmr ta. 01 abeand 2088 a Raible also 2011; are al., and et (Cimadamore (PNS) neurons system sensory nervous of peripheral source the in neurons IgG the to normalized are (ES) data (20 hESCs The in primers. performed specific with was amplified ChIP were (H) mean fragments complex. NIC DNA the NICD1–CBF1 the NICD1-bound as with the the presented region for and promoter and antibody, sequence representative value anti-NICD1 recognition a cou input an contro of the proteins in schematic with contained 5 A (NC) neural-crest-specifier me fragments day (G) pNCCs the at cells. the DNA and that (red) control as studied SOXE in indicated presented The and expression differentiation (green) are shown. with crest SLUG compared data sequence neural and as The (red), cells during PAX3 differentiation. NICD1-overexpressing (right) of and crest in ne (green) hESCs properties earlier neural of SOX10 the NICD1-overexpressing induction the TWIST1, affect and higher of during and not (left) Immunostaining cells faster (F) did hESCs showed control experiments. expression analyses three in NICD1 PCR from than NICD1 real-time ectopic level cells Quantitative of expression The (E) NICD1-overexpressing overexpression hESCs. shown. in stable is NICD1-overexpressing genes NANOG the and and crest-specifier confirmed control OCT4 blotting of of the Western Immunostaining images hESCs. of (C) Phase-contrast undifferentiated activation construct. (D) dramatic transfected iPSCs. the the and showed of ESCs cells functionality 293 the genes. HEK showing neural-crest-specifier in of NICD1-overexpression, assays expression activity the Luciferase controls (B) directly Notch 4. Fig. m )wr de,teclsmvdars h on in wound the across moved cells the added, were M) 6 . o52.7% to 0.8 a imnpstv el usatal increased substantially cells -immunopositive nvivo In 6 . ( 2.2 erlcetclsgv ieto rise give cells crest neural , 6 6 s.d.; .. i.5) n similar a and 5D), Fig. s.d.; AP-2 n 5 ;* 3; a , P HNK1 , .5(Student’s 0.05 and NGFR t -test). , Fg B.I diin edetected we addition, In genes 6B). neuronal (Fig. the and s neurons), pNCCs of of expression DAPT-treated suppression 6A). significant 2011). (Fig. al., a et Menendez comple with 2007; was al., NICD1 et Simultaneously, (Lee previously of factors, differentiation suggested neurotrophic neuronal as with the This supplementation induce without 6C). to cells crest (Fig. way neural simple dishes a with be culture to the treated appears throughout were neurons pNCCs filamentous when Interestingly, 20 2006). Ungos, A ceai ftehmnNC1oeepeso construct. overexpression NICD1 human the of schematic A (A) m HES1 ATaoefrol as h el perdas appeared cells the days, 3 only for alone DAPT M ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal rmtradCF-epnieeeet( element CBF1-responsive and promoter DBH and PHOX2B el htwr obypstv for positive doubly were that cells MASH1 mreso neural-crest-derived of (markers HES1 eyaoihd naccordance in abolished, tely giiatyurgltdthe upregulated ignificantly , NEUROD1 and CBF1RE HES5 db detected be ld eotr by reporters ) and expression an nhuman in D1-binding NGN2 ural- l

Journal of Cell Science EERHARTICLE RESEARCH rvospooosfrgnrtn erlcetclsfrom cells and phenotypes crest cellular in neural resulted have generating cells for stem pluripotent protocols Previous 6C). (Fig. DISCUSSION phenotypes Tuj1) an 39.8% cells and BRN3A-positive proportio became PHOX2B the Quantitatively, for sensory peripheral pe showed (positive and cells BRN3A these for (positive of neuron Some peripheral 6C). neural-crest-derived (Fig. marked neurons which Tuj1, and peripherin with treatment after n whereas migrate, not did 100 pNCCs DAPT, bars: and Scale FGF8 wound. of the combination pNCCs. filled the of rapidly Without DAPT. migration cells and induces the FGF8 DAPT FGF8 or and and alone (DAPT) FGF8 FGF8 inhibitor either with Notch treated of pNCCs combination A 5. Fig. oto NC n NC rae ihFF ln rFF lsDP.Bu,IGcnrl ht,seii nioy h aaaepeetda h mean the as presented are data The antibody. specific white, control; IgG Blue, DAPT. increas plus significant FGF8 a or treatment, alone DAPT FGF8 and with FGF8 treated Following pNCCs (D) and (C). pNCCs immunocytochemistry and control (B) PCR AP-2 real-time of by shown number as cells crest neural migratory 5 ;* 3; P 6 , . n 13.4% and 3.1 .5cmae ihcnrlpCs(yStudent’s (by pNCCs control with compared 0.05 a pstv el a bevd E lwctmti nlsso SA ahS akr,p5(GR n N1wspromdfrhESCs, for performed was HNK1 and (NGFR) p75 marker), hPSC (a SSEA4 of analyses cytometric Flow (E) observed. was cells -positive 6 .,rsetvl Fg 6D). (Fig. respectively 2.7, so ATtetdpCsthat pNCCs DAPT-treated of ns HXBpstv el were cells PHOX2B-positive d ihrn n uooi neuron autonomic and ripherin) t -test). m .C-ramn ihFF n ATidcdteepeso fmre ee of genes marker of expression the induced DAPT and FGF8 with Co-treatment m. erllnaecls rmhSs yepoighESC-derived employing By hPSCs. from not cells, but lineage pNCCs, generates neural efficiently protocol differentiation 1988). Our Fraser, differentiate and to (Bronner-Fraser derivatives potential crest the a neural with towards developed maintained be have naı easily we surrounding can more study, pNCCs a the this derive In from the to 2004). protocol of al., received et differentiation instructions (Wilson sequential the tissues to peripheral by However, to according followed 2011). tube cells neural rapidly al., the et is from cells Menendez tissues crest 2007; neural crest of al., neural migration et migratory with (Lee consistent cells patterns expression gene ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal A on-eln saso nrae cnrl NC and pNCCs (control) untreated of assays Wound-healing (A) v NCfr rmhSs These hPSCs. from form pNCC ¨ve nthe in e 6 2089 s.d.;

Journal of Cell Science EERHARTICLE RESEARCH 09 udke l,20) eody eue 22,ntonly not 2090 al., N2B27, used et we Secondly, (Nakazaki 2005). proliferation al., et and Wurdak for 2009; crucial differentiation be to activin–TGF- crest shown the been neural has block pathway this not because did cell SB431542, 2011). al., we modulates et hence, addition, 2011; (Vogt and, In pathways al., signaling kinases many other et I through various inhibits physiology type (Vogt dorsomorphin of BMP receptors), activity proteins inhibiting ALK the as besides BMP known Also, (also of 1996). receptors and al., set to et binds distinct Zimmerman action specifically wider a which a Noggin, has inhibits than thus, blocks signaling and, BMP level Dorsomorphin receptor on respectively. the signaling at Bio, signaling Wnt and BMP and Noggin 1- BMP of crest and modulate dorsomorphin instead neural to Firstly, used migratory study. were this than azakenpaullone in rather that generated differences were pNCCs, major cells, two how colleagues are explain and there but Menendez could 2011), by al., reported et protocol (Menendez the from comes fate various differentiation. commitment regulates terminal early and from signaling way migration their to on Notch cells crest that neural of decisions show we pNCCs, h akoeo u erlcetdfeetainprotocol differentiation crest neural our of backbone The b aha with pathway osbyseiishSstwrsanua rs aeby fate crest neural a towards signaling Notch hESCs crest. neural specifies of induction possibly the during sustained hsi eni h nuto ftasrpino h Notch the of transcription of induction the differentiation. in crest seen effectors neural Firstly, is during specification. activated This fate is and signaling development Notch signaling the crest Notch in for neural time roles first al., during multiple the demonstrate et for hPSCs, results, (Lee of Our context 2011). pathways al., signaling et Menendez multiple 2007; of actions the modulatory of interventions. investigation therapeutic of the potential screening for both the drugs allow material and candidate (supplementary neurocristopathies now underlying well should mechanisms as and iPSCs S2), human Fig. protocol and to our humans Importantly, applicable in fate. crest is lineage hESC- neural early crest the the neural of 2012). both specification the the studying al., to for commitment et tool of important pNCCs events Ngamjariyawat an of are 1996; pNCCs survival derived Sulik, and and might maintenance that (Chen promotion, substances enriched antioxidant the an several contains modulate and B27 medium. insulin basal of the mixture to additive an as N2, h pcfcto fnua rs aefo EC requires hESCs from fate crest neural of specification The ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal HES1 and HES5 n h xrsino ID htwas that NICD1 of expression the and , R3-oiieadPHOX2B- and of BRN3A-positive percentages The (D) sensory neurons. peripheral of formation the suggesting peripherin, and BRN3A of co-staining the indicate Arrowheads PHOX2B. and BRN3A peripherin, TUJ1, GFAP, for performed cell was neural Immunostaining of lineages. mixture a the of in generation resulted DAPT days. with 3 Treatment for DAPT with were treated that pNCCs of immunostaining and imaging Phase-contrast (C) G. cyclophilin to after normalization hESCs, to were relative changes calculated fold the and real- PCR, using time by measured were levels expression Relative pNCCs. treated DAPT- in upregulated was significantly genes neural of expression The (B) DAPT. inhibitor the Notch with treatment following pNCCs genes target Notch HES1 the real- of and PCR NICD1 time of blot Western (A) signaling. Notch of inhibition upon generated are neurons Neural-crest-derived 6. Fig. (Student’s mean the presented as are data The fluorescent microscope. the using visualized from cells determined were cells positive and t HES5 -test). 6 s.d.; nhESC-derived in n 5 ;* 3; P , 0.05

Journal of Cell Science yuigDP,idcdpCst irt n oexpress to and cells pathway migrate Notch to crest and pNCCs of neural induced inhibition DAPT, concomitant for using and by signal 2011), chemotactic al., et a (Sato FGF8, with as pNCCs pNCCs of of maturation inhibition and the migration seen the that promote hypothesized would Mourikis accordingly Notch 2008; We al., 2012). et CHARGE al., (Hilton et cells and stem tissue-specific 2008) several in al., et 2010). neurocristopathies, al., (Sznajer in et Tsai (Bajpai found Waardenburg syndromes 2012; feature as phenotypic al., a such et that 2001), (Humphreys al., fact et hypoplasia canal the semicircular by for supported role significant a suggest in revealed differentiation NDRG1 crest instance which signaling data neural of for during expression treatment – microarray Notch DAPT the by Our targets, affected of Notch is fate. identified newly layer crest several neural alternative controlling an underscoring and the in regulated instance, regulate indirectly For be Notch. can NICD1 by genes of targets neural-crest-specifier direct 4), being Besides (Fig. 2008). al., et Niessen 2008; h vrxrsino ID ln ssfiin oconvert to with al., sufficient interacts et to (Zabierowski NICD1 is cells shown and stem-like 2011), alone been crest neural NICD1 has into melanocytes of NICD1 processes. overexpression finding, Notch-related and The this neural-crest- other with regulate directly line neural-crest-specifier of In expression the genes. on NICD1 of been role positive 5 to has binds PAX3 NICD1 it DLX5, that regions Our show 2012), expression. analyses regulatory their ChIP the al., regulates and to genes et binds neural-crest-specifier of directly (Rada-Iglesias NICD1 whether cells unclear crest neural NGN2 erlcetseiirgns(i.4) lhuharcn study recent a Although 4E). (Fig. of Secondly, expression genes results the well. Our augmented neural-crest-specifier as clearly 2008). lineage differentiation NICD1 of crest al., hESC overexpression neural et the for to Yu required undifferentiated this 2006; extend is in al., now it et shown inactive that (Noggle have but normally commitment studies cells, is Previous (Endo stem 2004). signaling pluripotent signals al., Notch et extrinsic Geling that and 2002; factors al., et intrinsic both modulating ARTICLE RESEARCH 00 hmse l,20) h eutn el xrse other including expressed expected, cells as cells 2 resulting crest The neural migratory 2008). of markers al., et Thomas 2010; (i.e. genes crest neural genes of Notch expression effector SLUG the Notch which crest activate the neural by which the through to mechanism is hPSCs of possible lineage commitment towards the One fate promotes their identity. signaling switch to neural cells the a caused Notch and with expression silencing of treatment gene inhibition by by either the or induction, Thirdly, DAPT crest 2009). neural al., during et signaling (Jin genes target cis-regulatory of AP-2 by characterization occupied genome-wide elements the reports rmgaoyadmgaoynua rs el r beto able are cells (Bronner-Fraser crest derivatives non-neuronal neural and neuronal migratory generate and premigratory a oc inln scuilfrmitiigtepoeio state progenitor the maintaining for crucial is signaling Notch , HNK1 CDH6 Xslug nvivo in , and SOX10 Bmp4 splmnaymtra al 2.O oe u data our note, Of S2). Table material (supplementary akr fmgaoynua rs el Btese al., et (Betters cells crest neural migratory of markers , , ncailnua rs el Gai ta. 2004), al., et (Glavic cells crest neural cranial in NGN3 , ERBB3 OauaadSg,20) ned ramn of treatment Indeed, 2008). Saga, and (Okamura SLUG hc ntr nue h xrsinof expression the induces turn in which , APLNR and Cno n ad,20;Iaoh tal., et Imayoshi 2002; Rando, and (Conboy ) TWIST1 and and , COL1A2 JAG1 TWIST1 ITGA4 n upespoerlgns(i.e. genes proneural suppress and ) JAG1 upesdneural-crest-specifier suppressed , a Xenopus Jag1 , n RF nhESC-derived in NR2F1 and b CDX1 Fg H,sgetn direct a suggesting 4H), (Fig. ctnnt peuaeNotch upregulate to -catenin nnua rs development, crest neural in Fg 5B,C). (Fig. ncotmc display mice knockout , embryo, IFITM1 9 rmtrrgosof regions promoter HES1 nvivo In , Hairy2 LGALS1 and both , csto acts Xmsx1 NGFR PAX3 HES5 AP- and , , tmcls NC,peirtr erlcetlk el;Ns neural NPs, cells; cells. neural-crest-like progenitor pluripotent premigratory human pNCCs, of hPSCs, cells; absence differentiation. stem the neural In undergo pNCCs. of Notch hPSCs differentiation of Notch, and inhibition also maturation the and to activity population, leads Notch pNCC signaling lineage. the of crest self-renewal neural the the supports to signaling hPSCs Notch hPSCs. of of from commitment role differentiation proposed crest the neural of during summary Schematic 7. Fig. eie ern,adasbtnilnme fclsbecame cells of ( upregulation genes number Furthermore, neuron neural-crest- sympathetic 6C). substantial of into (Fig. when neurons a that, differentiated sensory found and peripheral pNCCs we phase protocol, of neurons, Finally, applied, source our derived use. migratory expandable was future by and for the DAPT potent derived self-renewing cells crest pNCCs a of less neural as 2003). into initiation serve al., therefore, differentiate after et cells (McKeown cells However, crest 1988). progenitor Fraser, neural and Bronner-Fraser migratory 1989; Fraser, and c hog h eaierglto fnuoa ee,sc as such genes, neuronal of might regulation that negative MASH1 signaling the Notch through by act prevented that suggest is differentiation findings neurogenesis our neuronal pNCC Furthermore, in 2012). inhibiting Yutzey, embryos, while crest and (Mead neural gliogenesis, mouse of and proliferation in the is cells experiments promotes study, signaling our earlier Notch in which by found as supported cells, important crest well migration The neural commitment, of 2008). the differentiation controlling al., and in et signaling Shimojo Notch al., 2007; of et role al., (Lassiter central et neurogenesis the Nelson sensory in 2010; the and found (CNS) support Notch, system of findings nervous role These differentiation 6B,C). anti-neuronal (Fig. pNCCs DAPT-treated ( genes fnua rs yatvtn h rncito factor transcription the activating by studies crest previous with neural Notch line which of in is differentiation in model crest This model in 7). neural a (Fig. during hPSCs propose roles from 2007). we distinct al., might plays findings, et signaling signaling in our (High Notch development on differentiation Notch crest altered Based that crest neural that aberrant show neural hypothesize underlie We for and iPSCs. indispensable humans, and is ESCs signaling human from and pNCCs state differentiation. progenitor neuronal the undergoing from preserves pNCCs activity prevents Notch that suggests ncnlso,w ecieapooo o h eeainof generation the for protocol a describe we conclusion, In Xenopus ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal MASH1 and mro,weeNthsgaigpristeinduction the permits signaling Notch where embryos, NGN , NEUROD1 L ta. 02.Tkntgte,ordata our together, Taken 2002). al., et (Lo s and DBH NGN2 and PHOX2B a lodtce in detected also was ) oc srqie o the for required is Notch n proneural and ) Hairy2 2091 ,

Journal of Cell Science 08]adwssblndit etrcnann h A rmtrand promoter human cassette. CAG the resistance The al., containing G418 vector et 2009). a a (Yu into 17623 al., subcloned [plasmid was Addgene and et 2008)] from (Zafarana obtained was hESCs high sequence a promoter NICD1 in have H1 to efficiency the shown previously by knockdown was driven system plasmid, shRNA This pSuperior-Neo (OligoEngine). the into cloned and GATCCTAACCGG-3 rmIvtoe)adwr otnl rpgtdb sn combination a using (all by medium propagated were complete routinely Helsinki) were StemPro of and with Invitrogen) (University plates from iPSCs Geltrex-coated Hel11.4 on and cultured (WiCell) hESCs and H9 cells stem pluripotent transfections human DNA of maintenance and Culture METHODS AND 2008). MATERIALS al., et Nichane cells 2004; al., crest et neural (Glavic undifferentiated maintains subsequently which ARTICLE RESEARCH n eewse ihPS h el eete tie niewith ice on stained then were 2092 cells The PBS. with solution washed trypsin-EDTA using were by vessels and culture from detached were Cells cytometry Flow primary with treated then were 4 cells for at The X-100 Triton overnight temperature. PBS 0.1% room antibodies in and at incubation (Sigma) hour serum by 1 bovine blocked fetal for were 10% paraformaldehyde containing proteins 4% using Non-specific by minutes. temperature room 10 at fixed were Cells Immunocytochemistry 2 differentiation with medium, the N2B27 supplemented using to by was induced a changing was which into lineage before crest Sigma) neural hours (PBS, The 24 medium. saline dish, phosphate-buffered in by Geltrex-coated previous passaged EDTA were a hESCs 0.02% from confluent, When using modified 2013). was al., et protocol (Menendez report differentiation crest neural The derivative mesenchymal and differentiation lineage neural crest, Neural of sequences target 5 The are 2008). siRNA al., et (Choi for report sequences siRNA Effective NICD1 human of knockdown shRNA-mediated Geltrex-coated applied 200 a was at selection into G418 transfection (Sigma) transfection. EDTA after Cell before 0.02% hours hours were with 48 24 cells dissociation. ratio dish confluent 1:3 as Briefly, culture mechanical a (Invitrogen) 2010). in al., 2000 and et split Lipofectamine (Noisa with previously treatment described performed IV was transfection collagenase of nti td r itdi upeetr aeilTbeS5. Table material supplementary in obtained listed were are study Wegner, this used Michael antibodies in All by Germany. Erlangen-Nuremberg, of developed University the antibodies, from 2003) al., et SOX medium (Vectorlabs). mounting minutes Vectashield DAPI 45 using with by for coverslips on antibody mounted secondary finally and labeled fluorescently with incubated ooiswr ikdidvdal oa2-elpaeadexpanded. and plate 24-well a to individually picked were colonies eedrce oad h erllnaeb h diino 20 of addition the by TrypLE pNCCs lineage confluent. neural medium. with the were differentiation towards cells same directed cells the single were the in into maintained until and dissociated day (Invitrogen) were other pNCCs every hESC-derived changed was medium 5 and Sigma) snua rs ifrnito,ecp ht1aaepuln was 1-azakenpaullone that except conditions Neural differentiation, Invitrogen), same instructions. crest the (all withdrawn. under manufacturer’s neural performed Kit was the as hESCs Differentiation from to differentiation according Adipogenesis the respectively, using StemPro Kit Differentiation by Chondrogenesis performed and StemPro was Kit, and Osteogenesis pNCCs adipocyte StemPro from Osteocyte, differentiation medium. chondrocyte neural-crest-inducing into (Sigma) 9 -AGGATAACTGTGCGAACATC-3 m -zknaloe(GSK3- 1-azakenpaullone M 9 epciey hs eune eeannealed were sequences These respectively. , ˚ .Atrwsigwt B,teclswere cells the PBS, with washing After C. E Shite l,20)adSX0(Stolt SOX10 and 2003) al., et (Schmidt JAG1 m JAG1 m osmrhn(M inhibitor, (BMP dorsomorphin M /lfr3wes n surviving and weeks, 3 for g/ml eeotie rmaprevious a from obtained were n vrxrsinof overexpression and b niio,Sga.The Sigma). inhibitor, 9 JAG1 n 5 and n scrambled and 9 -GGGCGTC- m DAPT M omlrbi g poie ihtekt,adwslf narotating a on left was and kit), 4 the at with overnight (provided wheel IgG rabbit normal 0mntsadscnayatbd o 0mntspirt FACScan for to antibody prior primary minutes 30 the for with antibody incubated secondary 4% analysis. washing then and after for with minutes were minutes 15 ethanol 30 fixed for Cells serum 100% PBS. goat were 10% with with with cells treated permeated For and sample. minutes the minutes, 2 each 15 proteins, Biosciences). for for acquired nuclear (BD paraformaldehyde were against cells software 20,000 antibodies CELLQUEST and 10,000 using Between FACScan a Biosciences) on analyzed were (BD and antigens cell-surface against antibodies ihtehgetitrurierne rsn n betclsfrteprobe the for calls absent set and probe Present the range. retaining correction, by interquartile sets R highest in background the probe package with Duplicate genefilter included the normalization. using Pre-processing removed in quantile were package 2008). and lumi al., stabilization the using variance The et pre-processed provided Illumina (Du (Agilent). was manual data with R kit the raw analysis to The Nano Illumina. according transcriptome by 6000 BeadChip the Expression RNA HT-12 for Human and processed Bioanalyzer were by assessed samples 2100 was RNAs DNA/ a the Prep of All quality using light the The using (Qiagen). by kit a samples Mini the RNA/Protein from under extracted were hours RNAs Total 48 profiles and expression Microarray 24 after observed was microscope. Confluent migration. 10- migration a cell with cell measure scored were to cells used of sheets was assay wound-healing A assay Migration sn h grs hPKta ecie ntemnfcue’ protocol manufacturer’s the performed in 2 Briefly, described were Pierce). as Scientific, Kit PCR (Thermo ChIP real-time Agarose the and using immunoprecipitation Chromatin immunoprecipitation Chromatin cDNA (Macherey-Nagel) first-strand kit 2 Briefly, from II synthesized instructions. Spin was manufacturer’s RNA the the to using according extracted was RNA PCR Total real-time and transcription Reverse is paper this in reported data expression gene GSE53203. microarray the for number be to considered adjusted was , Benjamini-Hochberg gene the A if 2004). expressed (Smyth, differentially R in moderated limma unpaired in an detection implemented using a performed had was detection it analysis if expression the present using be to by defined obtained were sets niNC1atbd Cl inln ehooy 44)ad5 and #4147) Technology, 5 Signaling with mixed (Cell was antibody chromatin Fragmented anti-NICD1 solution. stop the adding eprtr,cl xrcswr rprdadte ietdb using room by at digested minutes then 5 37 for and at M treatment prepared 0.125 (MNase) were nuclease of micrococcal extracts concentration final cell a temperature, to glycine of 0scnsad72 and seconds 30 opee,DAwsetatdadrsseddi 50 in 2 which resuspended of and buffer, extracted was DNA complexes, xrsinlvli EC a h eeec ape.Teprimer The sample). reference S3. the Table material (as supplementary in hESCs listed as are in sequences known level also housekeeping the expression G, of genes that target (cyclophilin to of values levels CT gene their expression calibrating relative by The calculated control. were negative a as used de n nuae o or t4 at hours 3 for incubated and added diino omleyeit h eimt ia ocnrto f1% of concentration 37 final at a minutes to 10 medium for the into formaldehyde of addition rncits Ivtoe)wt lg Tpie Ivtoe)i volume a in (Invitrogen) 20 primer dT of oligo with (Invitrogen) transcriptase itr otiig10 containing mixture f2 of hra ylr(ign o 0cce f95 of cycles 40 for (Qiagen) cycler thermal .5adtelgfl hnewas change fold log the and 0.05 m m rmrmx C ecin eepromdi Corbette a in performed were reactions PCR mix. primer M .Ec C nldd1 ftersligcN na20 a in cDNA resulting the of 1% included PCR Each l. ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal ˚ m o 0scns o-ees-rncie N was RNA Non-reverse-transcribed seconds. 30 for C ˚ a sdfrra-iePRaayi ihspecific with analysis PCR real-time for used was l .Atrcosikn a unhdb h addition the by quenched was crosslinking After C. ˚ .20 C. m m fSB re a ouin(im)ad5 and (Sigma) solution Taq Green SYBR of l fttlRAb sn ueSrp I reverse III SuperScript using by RNA total of g m fPoenAGpu–grs ed were beads Protein-A/G-plus–agarose of l m 6 ,2 iet i omk on,and wound, a make to tip pipette l ˚ 10 .Atreuino h immune the of elution After C. PPIG 1or 6 P vleof -value el eecosikdb the by crosslinked were cells ˚ P o 5scns 60 seconds, 15 for C n omlzn othe to normalizing and ) vle.Apoestwas set probe A -values. . ˚ o 5mntsbefore minutes 15 for C .TeGOaccession GEO The 1. , .1 Differential 0.01. m N elution DNA l P vlewas -value t ts as -test ˚ m m for C gof gof m m l l

Journal of Cell Science eig . lsy . atgr . Stra S., Rastegar, C., Plessy, A., Geling, thvr,H . me,J n yne,S. Lyonnet, and J. Amiel, C., H. Etchevers, Y. Wakamatsu, and N. Osumi, Y., Endo, ai . ishr . hkoa O. Shakhova, I., Miescher, L., Hari, eunigCnr tteTruCnr o itcnlg Tru iln)for Finland) (Turku, Biotechnology for Centre Turku the at Milla Pa Centre particular thank Sequencing in We Finland), Korhonen. (Helsinki, Eila Center and material Cell Mikkola Stem Biomedicum supplementary the thank We in listed Acknowledgements are rabbit sequences (normal S4. primer binding Table non-specific account The any into taking and IgG). DNA, (input) of chromatin of region total level particular the the a determine at to NICD1 used of was enrichment method fold-enrichment The primers. ARTICLE RESEARCH ui,E,Cloi . el . Gonc C., Real, G., Calloni, E., Dupin, M. S. Lin, and A. W. Kibbe, P., Du, S. J. Eisen, and A. R. Cornell, A. T. Rando, and M. I. Conboy, E. S. Fraser, and M. Bronner-Fraser, M. N. LeDouarin, and E. M. Bronner, Sundstro J., A., Helms, Kjaeldgaard, Y., Y., Xiong, Liu, E., J., Betters, Zhang, A., Rada-Iglesias, A., D. Chen, Y. Lin, R., and Y. Ge, Bajpai, Y., Yao, X., Cai, Y., Fu, N., Fu, X., Wei, L., Wu, X., Yang, K., Ba, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.145755/-/DC1 online available material Supplementary material Supplementary Finland Biocenter Foundation, Turku. Aaltonen of Emil University Funds, the and for Research Foundation, Foundation Hospital Juselius the Central Sigrid Finland, University the of Helsinki Academy the the Research, by Paediatric supported was work This Funding manuscript. the provided prepared T.O. T.R. and and T.T. R.L. performed data. K.L., H.C. P.N., analyzed and T.R. resources. K.K. and T.T. C.L., H.L., P.N., R.L., study. P.N., experiments. the designed T.R. and T.T. P.N., contributions Author interests. competing no declare authors The interests Competing support. technical lvc . iv,F,Abr .J,Bsia,F n ao,R. Mayor, and F. Bastidas, J., M. Aybar, F., Silva, A., Glavic, iaaoe . ihik . iso . ndv,K,Ctaos,G,Miller, G., Cattarossi, K., L. Gnedeva, T. E., Wang, Giusto, and K., M. Fishwick, I. F., Shih, Cimadamore, J., P. Morin, B., Davidson, K. T., J. K. Park, H., Sulik, J. Choi, and Y. S. Chen, S. Fraser, and M. Bronner-Fraser, csa rptenfco htbok ergnn n o2expression coe2 and boundary. neurogenin1 midbrain-hindbrain the blocks at neurogenesis that inhibit factor to Notch prepattern of a upstream as acts neurocristopathies. ectoderm avian during formation crest neural development. in involved are signaling mediated fnua rs ieg eeainb Wnt/ by generation L. lineage Sommer, 139 crest and N. neural Kessaris, of D., W. Richardson, 20) erlcetpoeiosadse cells. stem and progenitors crest Neural (2007). function. microarray. 1 Neurogenin repressing by crest 129 neural zebrafish of myogenesis. postnatal in determination fate Cell cell and activation cell satellite overview. an crest: neural development. crest neural human early of Analysis formation. (2010). crest neural multipotent J. control Wysocka, to and T. PBAF Swigut, with Y., Zhao, P., C. Chang, adipogenesis cells. induces stem signalling adipose-derived notch of of activation Jagged-1-mediated (2012). ewe oc inln n h oepoenXr1i eurdfrnua crest neural for embryos. required Xenopus is Xiro1 in homeoprotein induction the and signaling Notch between Development ua S-eie erlcetmdlrvasakyrl o O2i sensory in SOX2 for V. role A. key Terskikh, a and reveals M. model neurogenesis. Bronner-Fraser, crest neural M., ESC-derived L. Human Brill, S., growth Pluchino, tumor A., ovarian regulates loop juxtacrine adhesion. Notch3 and and Jagged-1 (2008). cells. crest neural in cytotoxicity situ. in cells crest neural cells. crest neural avian some of multipotency 2107-2117. , 2639-2648. , 3 397-409. , Bioinformatics 131 Development elSe Cell Stem Cell acrRes. Cancer 1993-2006. , d.Ep e.Biol. Med. Exp. Adv. Neuron e.Biol. Dev. 24 129 68 1547-1548. , 20) et/oc inln rmtsformation promotes signaling Delta/Notch (2002). 20) h euaino oc inln controls signaling Notch of regulation The (2002). 8 elProlif. Cell 5716-5723. , 538-551. , Development 863-873. , loo.Ci.Ep Res. Exp. Clin. Alcohol. 19) rerdcl n ethanol-induced and radicals Free (1996). 18) eeomna oeta faintrunk avian of potential Developmental (1989). 20) ui ieiefrpoesn Illumina processing for pipeline a lumi: (2008). 3 755-766. , avsTetn .adL oai,N M. N. Douarin, Le and A. ¸alves-Trentin, 21) eeomn n vlto fthe of evolution and Development (2012). 366 iiJniadteFnihMcora and Microarray Finnish the and Junni ¨ivi he .adBlyCi,L. Bally-Cuif, and U. ¨hle, ue,U,Ci,L,Tkt,M., Taketo, L., Chin, U., Suter, , 18) ellnaeaayi reveals analysis lineage Cell (1988). 2-9. , 589 45 20) ioa ucin fNotch- of functions Bimodal (2002). 20) oeua ae fhuman of bases Molecular (2006). 538-544. , b 213-234. , ,E n Garcı and E. m, ¨ ctnnsignaling. -catenin 131 Nature .R Biol. R. C. 347-359. , 21) H7cooperates CHD7 (2010). 21) eprlcontrol Temporal (2012). 335 e.Biol. Dev. Nature 20 161-164. , 330 1071-1076. , 20) Interplay (2004). aCsr,M I. M. ´a-Castro, 521-529. , 463 344 Development Development 20) Her5 (2004). 958-962. , 578-592. , (2011). Dev. esn .R,Hrmn .H,Gog,S . a,M .adRh .A. T. Reh, and S. M. Lan, A., S. Georgi, H., B. Hartman, R., B. Nelson, B., Mania-Farnell, V., Khanna, A., Reddy, B., Yun, W., Y. Shen, H., Nakazaki, aon,C n rne-rsr M. Bronner-Fraser, and C. LaBonne, L. R. Barnhill, and C. Lugassy, R., and Dhaybi, S. Al H. T., Park, Hung, N., V., Kim, Kokta, Y., K. Lee, N., Yang, I., Yang, H., Ki, H., Kim, H., Y. Jin, A. Israel, and R. Kopan, H., E. Schroeter, F., Logeat, C., Brou, S., Jarriault, R. Kageyama, and T. K., Ohtsuka, T., Loomes, Shimogori, C., I., Imayoshi, Brown, X., Qu, S., L. Prince, W., Zheng, R., Humphreys, Hayward, and G. M. Peterson, E., Robey, P., Salmon, T., Kronenberg, Henkel, T., J., J. Kobayashi, Hsieh, H., Zhao, S., Bai, X., Wu, X., Tu, J., M. Hilton, and S. W. Pear, S., M. Parmacek, L., Tu, A., Proweller, M., Zhang, A., F. High, Urruticoechea- D., McLean, A., Z. Hewitt, J., Fletcher, D., Zhao, C., D. Hay, astr .N,Bl,M . dm,J . rgt .T n tr,M R. M. Stark, and T. B. Wright, S., J. Adams, K., M. Ball, N., R. Lassiter, gmaiaa,A,Trav . es,N n olv,E N. E. Kozlova, and N. Welsh, K., Turpaev, A., Ngamjariyawat, S. Tajbakhsh, and R. Sambasivan, S., Gopalakrishnan, P., Mourikis, J. D. Anderson, and A. Greenwood, E., Dormand, T., L., Barberi, Lo, G., Panagiotakos, G., Shamy, Al Y., Elkabetz, H., Kim, G., Lee, ise,K,F,Y,Cag . odes .A,MFde,D n asn A. Karsan, and D. McFadden, A., P. Hoodless, L., Chang, Y., Fu, K., Niessen, Croze de M., Nichane, D., J. Lauderdale, S., S. M. Park, Bronner-Fraser, T., and D. A. Meulemans, Page, J., M. Kulik, L., Menendez, R. Mayor, and A. Mancilla, E., S. Pross, H., Sai, L., Xu, O., Shestova, A., Dumortier, U., Koch, I., Maillard, eedz . asivc,T . ni,P .adDlo,S. Dalton, and B. P. Antin, A., T. Yatskievych, L., Menendez, E. K. Yutzey, and J. T. Mead, G. P. Farlie, and F. D. Newgreen, J., S. McKeown, euainb a3adTFea inln:aptnilgn euaoynetwork regulatory development. gene S. crest potential C. a neural Mayanil, signaling: in and TGFbeta2 T. and Tomita, Pax3 by G., regulation D. McLone, S., Ichi, ersosaerqie nXnpsfrbt h nuto ftenua crest neural the of induction the both for Xenopus in 53 required of are repressors analysis an nevi: melanocytic congenital in cases. angiotropism of prevalence interaction. direct its through Notch1/NICD K. Kim, the in Notch. mammalian activated specification of downstream Signalling fate neural versus midline. syndrome non-neural telencephalic dorsal Alagille regulate neurogenin of and phenotype craniofacial the recapitulates patients. Jagged1 S. Goudy, and of S. Baldwin, S., S. Huppert, EBNA2. virus Epstein-Barr of that Biol. resembling mechanism a by genes D. S. osteoblast suppressing by al. progenitors et R. mesenchymal differentiation. Kopan, P., marrow F. bone Ross, L., maintains S. Teitelbaum, M., H. differentiation. muscle 117 smooth and development A. cardiovascular cells J. stem Epstein, vivo. embryonic al. in et human development R. Wolf, liver from A., 894-902. recapitulating J. hepatocytes Ross, markers of C., exhibiting Elcombe, differentiation R., J. Efficient Black, A., Uriguen, esr erndfeetaini euae ynthsgaigi h trigeminal the in signaling notch by regulated placode. is differentiation neuron Sensory migration. subsequent its and rnin nciaino oc inln ycrnzsdfeetaino neural of differentiation synchronizes signaling cells. Notch progenitor of inactivation Transient of cells. potential stem specification muscle temporal the skeletal maintains activity Notch Cell-autonomous cells. stem embryonic human from 1468-1475. derived cells stem crest L. Studer, and V. Tabar, cellularization. 20) lgi ietNthtre eurdfriiito fcriccushion cardiac of initiation for required target Notch direct a is Slug (2008). specification. progenitor crest neural J. Xenopus E. Bellefroid, partially protects cells stem death. crest cell neural cytokine-induced with against cells RIN5AH insulin-producing of development. and evolution crest neural cells. stem crest neural S. to Dalton, cells and pluripotent L. M. Cunningham, cells. stem hematopoietic adult of Cell maintenance al. the et for F. dispensable Radtke, is A., Bhandoola, C., J. Aster, progenitor neural cultured in functions homologs specification cells. atonal subtype and neuron achaete-scute mammalian and of differentiation neuronal generic the inln n mdptwybokd iettedfeetaino human of cells. differentiation crest the neural direct multipotent blockade to USA cells pathway stem Smad pluripotent a and signaling vivo. in development crest. neural 2 and 1 rhombomere in 255 potential lineage and migratory induction. Xslug of mechanisms 353-363. , 62-76. , 2 16 108 356-366. , Development 19) rnae amla oc1atvtsCBF1/RBPJk-repressed activates Notch1 mammalian Truncated (1996). ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal m .Dermatopathol. J. Am. 952-959. , 20) eactnnmdltstelvladtasrpinlatvt of activity transcriptional and level the modulates Beta-catenin (2009). 19240-19245. , u.Ml Genet. Mol. Hum. e.Biol. Dev. a.Med. Nat. .Cl Biol. Cell J. e.Biol. Dev. 20) nesnilrl o oc nnua rs during crest neural in Notch for role essential An (2007). 20) ar2I3itrcin lya seta oein role essential an play interactions Hairy2-Id3 (2008). 344 129 ,N,Rn . oogi . osr-uq .H and H. A. Monsoro-Burq, J., Souopgui, X., Ren, N., ´, e.Dyn. Dev. 836-848. , 1553-1567. , 14 20) slto n ietddfeetaino neural of differentiation directed and Isolation (2007). 304 182 21) oc aha euaino erlcetcell crest neural of regulation pathway Notch (2012). Development 19) erlcetfraini eou laevis: Xenopus in formation crest Neural (1996). 21 306-314. , Development n.J e.Biol. Dev. J. Int. 1374-1383. , e.Biol. Dev. 35 479-498. , 315-325. , 241 e.Biol. Dev. 180-183. , 376-389. , Pancreas 21) ietddfeetaino human of differentiation Directed (2013). 20) eergltr neatosin interactions Gene-regulatory (2004). 221 e.Cell Dev. ici.Bohs Acta Biophys. Biochim. 20) ni-eae transcriptional Snail-related (2000). 139 a.Protoc. Nat. 135 177 21) rna erlcetablation crest neural Cranial (2012). 195-205. , 4536-4548. , 20) aoia oc signaling notch Canonical (2008). 53 e.Biol. Dev. 2531-2541. , 41 580-589. , 69-79. , 20) eprlrsrcinof restriction Temporal (2003). 490-492. , 7 291-299. , Nature 20) oc signaling Notch (2008). 8 20) Transcriptional (2009). rc al cd Sci. Acad. Natl. Proc. 20) oprsnof Comparison (2002). 203-212. , 322 a.Biotechnol. Nat. 20) e genes Hes (2008). 377 355-367. , 21) Coculture (2012). tmCells Stem 1793 .Ci.Invest. Clin. J. 355-358. , 21) High (2013). 21) Wnt (2011). 290-299. , elStem Cell e.Biol. Dev. o.Cell. Mol. (2010). (2008). (2012). (2007). (1995). 2093 26 25 , ,

Journal of Cell Science zae,Y,Colde Y., Sznajer, Garcı and J. T. Stuhlmiller, Wegner, and A. Schedl, C., M. Chaboissier, E., Sock, P., Lommes, C., C. Stolt, R. Kageyama, and T. Ohtsuka, H., Shimojo, C. Ruhrberg, and K. Davidson, H., C. Maden, Q., R. Schwarz, Kopan, and A. J. Kisslinger, H., E. Schroeter, O. K., Rosorius, and Pegram, M. R., Wegner, A., J. Wernig, Decker, G., Glaser, A., K., H. Schmidt, Stadt, N., E. Kuhn, M., A. Scholl, A., Sato, M. J. Ungos, and W. D. Raible, and T. Swigut, A., S. Brugmann, S., Prescott, R., Bajpai, A., Rada-Iglesias, J. A. Bardin, and N. C. Perdigoto, Y. Saga, and Y. Okamura, os,P,UrtkexaUiun . i .adCi W. Cui, and M. Li, A., Urrutikoetxea-Uriguen, P., Noisa, G. B. Condie, and D. Weiler, A., S. Noggle, ARTICLE RESEARCH 2094 K. G. Smyth, 20) env O1 uaincuigsvr ye4Waardenburg 4 type severe causing mutation disease. SOX10 1041. Hirschsprung novo without de syndrome A (2008). induction. crest neural avian for epiblast 139 gastrula the in required cord. spinal developing M. Article3. neurons sensory organise to ganglia. essential root is dorsal guidance segmented cell into crest neural mediated domain. intracellular of 382-386. release proteolytic myogenesis. ligand-induced requires inhibits and cells satellite muscle for Chem. marker specific a is L. M. cells. Kirby, crest and neural cardiac R. M. Hutson, crest. neural the from crest. neural human of 648. regulators transcriptional J. Wysocka, cells. stem progenitors. crest neural enteric of ifrnilepeso nmcora experiments. microarray in expression differential progenitors. neural of maintenance regulate signaling neural in specifically GFP expressing lines reporter progenitors. cell stem embryonic human cells. stem embryonic human in inducible 20) h o9tasrpinfco eemnsgilft hiei the in choice fate glial determines factor transcription Sox9 The (2003). 289-300. , 278 29769-29775. , ici.Bohs Acta Biophys. Biochim. 20) iermdl n miia ae ehd o assessing for methods bayes empirical and models Linear (2004). tmCl Rev. Cell Stem a . er,F,Dlire . ehr,T n orie .L. R. Touraine, and T. Sekhara, I., Delpierre, F., Meire, C., ´a, 21) pgnmcantto fehnespredicts enhancers of annotation Epigenomic (2012). d.Ep e.Biol. Med. Exp. Adv. ee Dev. Genes 20) oc inln srqie o h maintenance the for required is signaling Notch (2008). e.Biol. Dev. aCsr,M I. M. ´a-Castro, 6 438-449. , 20) pcfcto fsnoynuo elfate cell neuron sensory of Specification (2006). 21) edn h ih inl oc and Notch signal: right the Sending (2013). Development 21) G8sgaigi hmtci for chemotactic is signaling FGF8 (2011). 1830 Development 17 354 1677-1689. , 2307-2322. , 18-30. , 20) oc inln siatv but inactive is signaling Notch (2006). tmCells Stem m .Md Genet. Med. J. Am. 589 21) G/AKsgaigis signaling FGF/MAPK (2012). 170-180. , 20) siltosi notch in Oscillations (2008). tt pl ee.Ml Biol. Mol. Genet. Appl. Stat. 135 136 19) oc- signalling Notch-1 (1998). elSe Cell Stem Cell 3555-3565. , 1785-1789. , 24 Neuron 1646-1653. , 21) eeainof Generation (2010). 20) Neuropilin- (2009). 58 52-64. , Development 146A 20) Sox8 (2003). Nature 11 1038- , .Biol. J. 633- , 393 3 , , airwk,S . abt . ie,B,L,L,Fkng-aai,M,Patel, M., Fukunaga-Kalabis, L., Li, B., Himes, V., Baubet, E., S. Zabierowski, M., Y. Li, P., Mali, A., Tokunaga, G., Chen, H., A., Hammond, Z., J. Ye, J., Fischer, Zou, X., U., Yu, Suter, P., Leveen, S., K. Lang, M., L. Ittner, H., Wurdak, D., Raha, S., Hutchison, C., Qiu, A., Szekely, P., Noisa, L., Habegger, Q., J. Wu, aaaa . vr,S . vr,K,Moe .D n nrw,P W. P. Andrews, and D. H. Moore, K., Avery, R., S. Avery, G., Zafarana, isn .M,Rcad,K . odPris .L,Pnhe,J .adMurphy, and J. J. Panthier, L., M. Ford-Perriss, L., K. Richards, M., Y. Wilson, imra,L . eJesu De B., L. Zimmerman, hvna,E n ao,R. Mayor, and E. Theveneau, ot . ryo,R n akt,G P. G. Sapkota, and R. Traynor, J., Vogt, u,T,Hmgci . asnm,N,Frkw,T,Hno .adKawaichi, and T. Honjo, T., Furukawa, N., Matsunami, Tymowska- Y., Hamaguchi, P., T., Roby, Tun, E., A. Kiernan, C., Rhodes, C., E., M. R. Speer, Hardisty, P., H., Tsai, Xu, C., Babarit, P., Wincker, M., Thomas, S., Thomas, . cad . ura . iot,P,Dhae .e al. et N. Dahmane, P., Gimotty, M., Guerra, R., lineage McDaid, trophoblastic, S., not but embryonic, for al. required et is commitment. of cells N. stem reminiscent Gaiano, embryonic defects C., multiple to Civin, leads cells L. stem Sommer, syndrome. crest DiGeorge and neural W. in Born, signaling S., short, Karlsson, by revealed cells al. sequencing. stem et paired-end embryonic and human S. long, of Weissman, differentiation H., neural during Lin, M., Egholm, ergamn fmlnctst erlcetse-ieclsb n defined one by cells stem-like crest neural to factor. melanocytes of reprogramming Development M. 1842. pmn raie inlngi id n nciae oemorphogenetic bone inactivates and binds 4. noggin protein signal organizer Spemann short inducible by cells stem interference. embryonic human RNA in hairpin OCT4 of knockdown Specific oeueihbtr fteTF n M pathways. BMP and TGFß the of inhibitors molecule B- kappa. RBP-J neuroepithelial in M. Jagged1 al. Corti. for et of D. role organ S. a the Brown, demonstrates in J., mutant patterning A. slalom Hunter, S., mouse cells. Rastan, The P., stem Mburu, Z., of Lalanne, hallmarks phenotypic and molecular Genet. Mol. display Hum. C. cells H. Etchevers, crest and M. neural Vekemans, S., Lyonnet, A., Munnich, migration. cell collective 366 to transition epithelium-to-mesenchyme from 19) eonto euneo ihycnevdDAbnigprotein binding DNA conserved highly a of sequence Recognition (1994). 20) erlcetcl ieg ergto ntemuenua tube. neural mouse the in segregation lineage cell crest Neural (2004). 34-54. , tmCells Stem ora fCl cec 21)17 0329 doi:10.1242/jcs.145755 2083–2094 127, (2014) Science Cell of Journal Cell elSe Cell Stem Cell 131 uli cd Res. Acids Nucleic 86 599-606. , 6153-6162. , 29 17 1752-1762. , 3411-3425. , ee Dev. Genes tmCells Stem -soa,J .adHrad .M. R. Harland, and M. J. s-Escobar, ´ 2 21) erlcetdlmnto n migration: and delamination crest Neural (2012). 461-471. , 20) oc inln ciaini human in activation signaling Notch (2008). u.Ml Genet. Mol. Hum. 19 rc al cd c.USA Sci. Acad. Natl. Proc. 22 530-535. , 965-971. , 27 776-782. , 21) h pcfcte fsmall of specificities The (2011). 20) nciaino TGFbeta of Inactivation (2005). 21) yai transcriptomes Dynamic (2010). 10 507-512. , el Signal. Cell. 107 20) Human (2008). 21) Direct (2011). 5254-5259. , 19) The (1996). 23 e.Biol. Dev. 1831- , (2001). (2009).

Journal of Cell Science