Journal of Cell Science sakymdltro Cnuo eeomn yrgltn sl xrsindrcl,adidrcl hog t agtgn Hes1. gene target its through functions indirectly Rbpj and that directly, evidence expression first Ascl1 the provide regulating also results by it our development although Thus, neuron in down, Hes1. and LC of knocked transfection presence of was gene the expression modulator in vitro Hes1 key expression in when a Ascl1 our expression as on Moreover, Ascl1 effect Hes1. upregulated repressive gene Rbpj a downstream-targeted that Rbpj- exerted its conserved showed a by assays assays, counteracted electroporation ChIP was ovo and the effect EMSA and this within of analysis but sequence identified upregulation transactivation genomic was Through drastic mice. motif a knockout conditional were binding addition, of r1 r1 dorsal In dorsal the in from unchanged. observed derived dopamine- or populations and neuronal neurons, reduced other LC contrast, either early-differentiating In Phox2b-expressing neurons. and LC Phox2a- late-differentiating of tyrosine-hydroxylase-expressing number the in increase dramatic o:10.1242/jcs.102152 doi: 4320–4332 125, Science Cell of Journal 2012 May 14 Accepted ogiUiest colo eiie hnhi209,China 200092, Shanghai Medicine, of School University Tongji euaeteerysae fL pcfcto yidcn the inducing by bone specification plate embryos, LC roof of chick the stages and from early the secreted zebrafish regulate (BMPs) In proteins 2002). identified morphogenetic (Goridis have Rohrer, neurons LC studies of born and development Previous the earliest in involved 1989). 2000; the genes al., several et Trosko, among Pattyn 2006; and are al., et and Steindler (Aroca brain 1 mouse the (r) in neurons rhombomere dorsal the 2005). and al., et epilepsy Warnecke al., 2003; et Waterhouse, depression, Aston-Jones and 2001; Berridge 2004; al., disorders, disorders, neurological et (Aston-Jones of hyperactivity sleep disease Parkinson’s variety deficit and rhythm a attention to circadian (Berridge lead including system memory, stress disorders, noradrenergic and Dysfunctions the and 1998). Philippu, learning of and cycles Singewald arousal, behavioral 2003; Waterhouse, and anxiety, sleep/wake physiological is as of nociception, transmission range Noradrenaline such wide 1983). processes, al., a et (Aston-Jones with (CNS) Foote associated system 1995; to nervous projects al., central and et the brain of chief mammalian regions the the most is in nucleus in noradrenaline This located of pons. neurons source the of cluster of dense tegmentum dorsolateral a the is (LC) coeruleus locus The Introduction words: Key neuron LC in role essential and an physiological plays are of pathway, brain signaling spectrum Notch mammalian broad the a the in of in in effector inactivation implicated nuclear neurons Conditional is key mouse. noradrenergic a and the of Rbpj, brain in that development the development here the in report controlling noradrenaline We pathways of unknown. largely source genetic main However, the processes. is behavioral (LC) coeruleus locus The Summary work this to equally ` contributed authors *These 4 3 2 1 Shi coeruleus Ming locus mouse the development in the neurons noradrenergic for of required is signaling Notch–Rbpj 4320 uQagDing Yu-Qiang uhr o orsodne( correspondence for Authors eateto eia eeisadDvlpetlBooy orhMltr eia nvriy ia 102 China 710032, Xi’an University, Medical China Military 200237, Fourth Shanghai Biology, Technology, Developmental and China and Science 710032, Genetics of Neurobiology, Xi’an Medical University and University, of China Anatomy Medical Department East of Military Pharmacy, Department Fourth of and Hospital, School Hospital), Xijing (East Neurology, China of of Department Education of Ministry Arrhythmias, of Laboratory Key 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. oarnri ern nteL r eie rmpoeiosin progenitors from derived are LC the in neurons Noradrenergic 1,2, bj oc inln,Lcsceues hmoee1 e1 Ascl1 Hes1, 1, Rhombomere coeruleus, Locus signaling, Notch Rbpj, * , ` eLnHu Ze-Lan , 1, ` [email protected] 1,3, ,MnHaZheng Min-Hua *, ; [email protected] Ascl1 rmtr uieaerpre sa eeldta bjprs ol induce could se per Rbpj that revealed assay reporter luciferase A promoter. Rbpj 4, ) ,Nn-igSong Ning-Ning *, ntedra hmoee()1 hr Cnuosaebr,rsle na in resulted born, are neurons LC where 1, (r) rhombomere dorsal the in eetsuyrvae hta rhnncerrcpo Nr2f6, of neuron receptor absence the LC In nuclear for 2005). required al., et orphan also (Warnecke development is an Ascl1, of that downstream functioning revealed Vogel-Ho study 2004; recent al., et gene bHLH Hatakeyama proneural the of expression hxaadPo2 ntr ntaeteepeso of expression Pattyn the initiate 1997; hydroxylase turn al., in et Phox2b Ro Morin and Phox2a and 1998; Vogel-Hopker zebrafish al., 1997; and et al., Hirsch chick et 1999; mouse, al., the early et in the (Guo for neurons critical LC are of which differentiation activates genes, Ascl1 homeobox that size. paired-like in shown two reduced been or absent also completely Vogel-Ho has either It is 1999; LC the 2005), Rosenthal, al., et and Hynes 2002), Rohrer, 1999; al., et Cnuoeei ntemmainbanaesilelusive. still are brain mammalian the in the neurogenesis controlling pathways to LC genetic identified development, also been LC the have were in factors critical of 2003) of be survival number al., and a maintenance Although et the neurons. in (Holm LC roles TrkB NT-4 essential receptor neurons. play and kinase to tyrosine BDNF reported the ligands LC 2001), its al., et and mature Qian 2005; (Hornbruch al., and Tlx3 et factor transcription late-differentiating homeobox the Additionally, in expressed Ascl1 nesnilfco o oarnri ergnss was neurogenesis, noradrenergic for factor essential an , 1 igHuang Ying , Dh and (Dbh) Ascl1 Hrc ta. 98 or 1998) al., et (Hirsch yoiehydroxylase tyrosine 1 agZhao Gang , he,20;Yn ta. 1998). al., et Yang 2002; ¨hrer, pe n orr 02.A 2002). Rohrer, and ¨pker Ascl1 2 eerhArticle Research Phox2a u Han Hua , b hdoyae and -hydroxylase- Goe l,1999; al., et (Guo Nr2f6 T) w genes two (TH), dopamine- and Bmps (Warnecke pe and ¨pker Phox2b 4 and Ascl1 (Guo b - , Journal of Cell Science Rbpj primordium. LC the expressing those with and overlapped were cells that found expressing and neurons, LC for markers early-differentiating h is vdneta bjfnto sanvlrgltrof regulator novel a Results as LC. function mouse the Rbpj in that neurogenesis noradrenergic provide evidence findings expression These first Hes1 interference. the RNA when by down expression showed knocked Ascl1 assays was expression upregulated gene Rbpj vivo in that and vitro in Further Hes1. of of not of but Meanwhile, downregulation neurons, significant derivatives. LC r1 of generation other the enhanced selectively Rbpj 2005). Gaiano, (Louvi and state Yoon pluripotent 2006; a and Artavanis-Tsakonas, in differentiation and progenitors neuronal neuronal inhibit have maintain studies signals help loss-of-function Notch of that group A revealed 2008). al., et (Imayoshi ie r rsn.Lcfrs eotrasyrvae htRbpj that revealed assay reporter Luciferase promoted present. are an sites identified within assay site (ChIP) Rbpj-binding immunoprecipitation chromatin and proneural of expression the as repress such which genes, 1999), Ohtsuka, rb eeldn nst yrdzto inl Fg C.In we r1, 1C). dorsal (Fig. E9.5 signals for through hybridization hybridization situ sections in transverse RNA performed situ Sense the shown). in on not (data addition, no 1B). level (Fig. low revealed r1 a probe at dorsal but the tube neural in total weak of relatively expression the and Afterwards, tube prosencephalon, neural of the 1A, tube (Fig. E9.5, r1 neural At dorsal arrowhead). the including metencephalon in and mesencephalon expression strong first with we we hybridization, r1, situ that development, in dorsal mount developing whole LC found Using the born. on is in LC expressed Rbpj the where is of Rbpj effects whether examine the progenitors explore LC the To in expressed is Rbpj aeil n ehd eto) r xrsini both in expression and Cre section). Methods and Materials Ba,20;Hne l,20) agtgnso h Rbpj–NICD signaling the Notch of include genes canonical Target complex of 2002). al., integrator activator et thus key Han Rbpj, 2006; a to (Bray, as bind serves ultimately receptors Rbpj Rbpj, Notch to All mammalian activation. binds transcriptional it four Rbpj-dependent where to nucleus, leading complex; the is thereby repressor to (NICD) a translocates Notch of of and domain part released intracellular as the DNA binding, ligand to upon bound DNA- is a Rbpj inactive, and pathway ligands Jagged2) protein J signal-binding and Notch Notch recombination mice, factor Jagged1 five transcription In canonical binding Delta4, (Notch1-4), Delta3, receptors 2001). the (Delta1, Notch al., of four et include components Scheer and established Louvi 2000; 2006; al., et Artavanis-Tsakonas, (Furukawa brain mammalian developing k et oinactivate to Next, ntepeetsuy esoe htcniinlalto of ablation conditional that showed we study, present the In oc inln ly motn oe ntenuoeei of neurogenesis the in roles important plays signaling Notch Ascl1 Rp;as nw sCF) hnNthsgaigis signaling Notch When CBF1). as known also (Rbpj; En1 Phox2b Wnt1 eei h oslr,weeL ern r born, are neurons LC where r1, dorsal the in gene Cre/+ xrsin lcrpoei oiiysitasy(EMSA) assay shift mobility Electrophoretic expression. odtoa ncot(K)and (CKO) knockout conditional Ascl1 dt o hw) ugsigta bji xrse in expressed is Rbpj that suggesting shown), not (data Rbpj ieocr serya 85i h oslr L tal., et (Li r1 dorsal the in E8.5 as early as occurs mice Ascl1 rnatvto,adti fetwsrpesdby repressed was effect this and transactivation, rncit eedtce serya E8.5 as early as detected were transcripts Rbpj Rbpj Brrn ta. 2002), al., et (Bertrand xrsinbcm bqiosthrough ubiquitous became expression Ascl1 nteL rgntr,w generated we progenitors, LC the in Hes1 Rbpj Hes1 rmtrweeHes1-binding where promoter Rbpj a tl bevdtruhthe through observed still was Rbpj and xrsinadupregulation and expression and Hes5 Rbpj eeinrsle na in resulted deletion Phox2a En1 Kgym and (Kageyama Ngn1 K ie(see mice CKO / Phox2b and Wnt1-Cre Phox2a Rbpj Ngn2 two , - eto lnso h Csoni –.( Cre F–I. and in TH shown of LC percentage the of planes express section LC the in neurons positive muoaee ihT HI and (H,I) TH with immunolabeled eeelm C ou orlu;P,prbaha ope.Saebars: Scale complex. parabrachial PB, 100 coeruleus; locus LC, cerebellum; h Caelbldb -a tP0 rohaspitt h oslr1. dorsal the in to neurons point of Arrowheads majority P10. the at and X-gal E10.5 by ( at labeled X-gal r1 are respectively. dorsal LC (F,G), the the P10 in and observed (D,E) is E10.5 staining at embryos reporter (E,G) xmn hte Cnuoswr eie from Cre derived were neurons En1 To LC generation. their knockout to whether to prior neurons examine us LC to enabling rise gives thus that region 2003), the al., et Trokovic 2002; ihwl-yeltemts(i.2–) oeaieteefcsof effects the examine To 2A–C). (Fig. littermates wild-type with nter einof region of r1 expression the the in that showed neurons hybridization LC situ of In overproduction induces inactivation Rbpj neurons. LC 1. Fig. rohas n nteL tP0(i.1,,bxdareas), boxed 1F,G, (Fig. P10 87.2 at that showed gal and LC TH with immunolabeling the double Furthermore, respectively. in and arrowheads) eut hwthat show results 95.8 b fwoemutebysadcrnlscin eeldthe revealed sections coronal and of embryos presence whole-mount of ( galactosidase tiigwspromdin performed ( was (C). staining used is probe RNA of oiatvt bjepeso nteL ycosn with crossing by LC the in mice. expression floxed used Rbpj be can inactivate lines mouse to Cre two these that suggesting neurons, LC H–I glin -gal Rbpj ; ; Rosa26 m - 9 6 Rosa26 Cre- 9 o H–I for m P10 ) .%in 3.8% Rbpj tE. A n 95()btn inli bevdwe sense a when observed is signal no but (B) E9.5 and (A) E8.5 at xrsigpoeios eto datg of advantage took we progenitors, expressing Wnt1-Cre bjrgltsteL eeomn 4321 development LC the regulates Rbpj and Wnt1-Cre sepesdi 1and r1 in expressed is ( A–C and b 0 En1 200 ; -gal b En1 gl nteCeepeso oan -a staining X-gal domain. expression Cre the in -gal) hl-on nst yrdzto hw h expression the shows hybridization situ in Whole-mount ) Wnt1 En1 Cre/+ + Rbpj ; ; Cre/+ b m Rosa26 Rosa26 6 el ntedra 1a 1. Fg 1D,E, (Fig. E10.5 at r1 dorsal the in cells gldul-aee el nteL of LC the in cells double-labeled -gal o F,G. for m Cre/+ ; D–G .%o TH of 6.1% Rosa26 -or Wnt1-Cre Wnt1 ; Rosa26 ; hl-on n ooa eto X-gal section coronal and Whole-mount ) En1 Rosa26 and K or CKO eotrmc Fg 1H–H (Fig. mice reporter eotrmc.4,fut etil;CB, ventricle; fourth 4V, mice. reporter b b En1 die r xrsini rsn in present is expression Cre -driven Wnt1 ; gl(H -gal gl(H -gal Rosa26 eotrmc Fg IJ.These 1I–J). (Fig. mice reporter + Cre/+ ern nteL co-expressed LC the in neurons J eotrmc htexpress that mice reporter -/ uniaieaayi fthe of analysis Quantitative ) Rbpj En1 9 0 ; ,I ,I Rosa26 DF and (D,F) 9 0 hwta oto TH- of most that show ) .DarmaoedpcsP10 depicts above Diagram ). epesn el iers to rise give cells -expressing En1 K iecompared mice CKO eotrmc co- mice reporter En1 Rbpj Cre/+ Wnt1 Wnt1- 99 a absent was ; Rosa26 ) and J), , - Cre Rbpj Wnt1- Rbpj -or Rbpj b b in - - Journal of Cell Science ute ofre yeaiigteepeso of expression the examining decarboxylase by were findings (753 confirmed These controls further S2). of that Fig. than material higher (supplementary times respectively 2.6 and times 2.4 were hw)adte eeue scnrl nL erncounting. in neuron LC one LC in not in neurons of (data controls numbers defects Rbpj the as that detectable used showed other results were Statistical or they neurons and LC shown) of increase no opooyadnuo ubro h C(i.2D–F), (Fig. LC not was normal the mice in CKO a LC in of of neurons development revealed defective LC number by of results caused increase the neuron Our that suggesting P10. and at morphology immunostaining TH h aedfeetainmresDhadT rmE05t E16.5. to E10.5 types, from wild and TH to and Compared E9.5–E10.5, Dbh at markers differentiation Phox2b late the and early the Phox2a of markers expression the differentiation examined we occurs, induction neuron inactivate to used were which mice, other with mice littermate E17.5 (e.g. The at 2G–R). genotyping neurons (Fig. LC 0 of (P) number day postnatal the and in increase dramatic a found and and immunohistochemistry Rbpj 4322 odtriewehrteL tefwsdfciein defective was itself S2). En1 Fig. LC material the (supplementary whether P0 determine or To E17.5 at 2002) al., et (Holm factor 2003), neurotrophic al., derived et Ma 1 2003; transporter al., et (Holm (CGRP) A type odtrietedvlpetlsae twihecsieLC excessive which at stages developmental the determine To Cre/+ Wnt1 eeino Cdvlpet epromdTH performed we development, LC on deletion Mo)(rie l,19) actnngn eae protein related gene calcitonin 1997), al., et (Arai (MaoA) ie Cnuoso hs w r iewr xmndby examined were mice Cre two these of neurons LC mice, K ie(1795 mice CKO ora fCl cec 2 (18) 125 Science Cell of Journal vlT)(aradVnBcsal,2005), Bockstaele, Van and (Barr (vGluT1) LAD)(ao ta. 1993), al., et (Eaton (L-AADC) Wnt1-Cre Phox2a 6 ; Rbpj Dbh 8 and 88) BN)(ome l,20)and 2003) al., et (Holm (BDNF) flox/+ and nst yrdzto eeldan revealed hybridization situ in Rbpj Rbpj or Phox2b En1 En1 ntedra r1. dorsal the in Cre/+ K ie(1950 mice CKO Wnt1 nst hybridization, situ in oomn oxidase monoamine eiua glutamate vesicular ; Rbpj Ceor -Cre -mn acid L-amino flox/+ Wnt1 showed ) En1 Ceor -Cre brain- 6 6 c-Ret Cre/+ 68), 76) eeaino hs ernlppltos eeaie Tlx3 examined We populations. neuronal these of generation infcn nraei h ubro Cnuosi both in neurons LC of number the in increase significant eeaino Cnuosa oheryadlt mroi stages. embryonic late and early both at neurons LC of generation Thus, 3I–Z). (Fig. ueirvsiua ulu,btddntfn biu differences obvious find not did between but nucleus, vestibular superior ern nteprbaha ope,adTbr1 and complex, Calbindin parabrachial nucleus, the sensory in trigeminal neurons principal the in neurons whether Thus, asked 2005). al., et next Wang 2005; (Kim Fishell, we and cells Machold Purkinje 2008; of deep and al., of cells et array comprised granular vestibular external wide is nuclei, which a superior cerebellar primordium cerebellar to complex, sensory and rise parabrachial nucleus, principal gives including nucleus, types, r1 trigeminal cell dorsal cerebellar the and brainstem LC, the in Besides unchanged or mice reduced CKO either Rbpj are derivatives r1 Other and CKO and CKO oa bec fcrbla oito splmnaymaterial (supplementary foliation cerebellar of absence contrast, an By to S3A–C,E–G). Fig. material ern nti r iemue tE05 1. n 1.,both E16.5, and E12.5 and E10.5, At immunostaining LC mouse. of line TH Cre development wild-type this normal in in showing those neurons again to 3D,H), comparable (Fig. were controls progenitors LC in positive contrast, By S5B,F). by Fig. confirmed was increase stage Phox2b early This S5A,E). Fig. material nraei h ubro maueL ern nboth in neurons LC immature of number the in increase 1 hmoee1 T,vnrltgetlae.Saebr:200 bars: Scale area. midbrain; tegmental MB, ventral VTA, coeruleus; 1; locus rhombomere LC, r1, colliculus; inferior IC, hindbrain; ,–)scin hog h oso idtp (G–J), wild-type of pons the through sections N,P–R) ( P10. at A– immunostaining in shown r1 the Wnt1 of planes section ( depicts the C. embryo to E10.5 point an Arrowheads of mice. brain CKO the of where r1 regions in deleted is expression and –,–,–,–;500 D–F,H–J,L–N,P–R; (* controls iuhbiiainwspromdo aaeinsgta etosof sections sagittal (A), paramedian wild-type on E10.5 performed was hybridization situ of Deletion 2. Fig. 0bandpcstescinpae hw nG n O. and K G, in shown planes section the depicts brain P0 a in observed is Phox2b ern r eni the more in Significantly seen P0. are at neurons pons the through sections ( S uniiaino h ubrof number the of Quantification ) nst yrdzto Fg EG upeetr material supplementary 3E–G; (Fig. hybridization situ in D–F Rbpj Rbpj E or (E) Rbpj Rbpj N rb.Sgiiatices ntenme fL neurons LC of number the in increase Significant probe. RNA oprdt idtp D,teCeepeso rvnby driven expression Cre the (D), type wild to Compared ) En1 Wnt1 P En1 En1 , OR K ielbldwt niT antibody, anti-TH with labeled mice CKO (O–R) En1 .5.C,crblu;C,cre;F,frban HB, forebrain; FB, cortex; CX, cerebellum; CB, 0.05). Rbpj Rbpj K n idtp iea 0(supplementary P0 at mice wild-type and CKO K mro,i oprsnwt idtypes wild with comparison in embryos, CKO Rbpj F osntafc Cdvlpet srvae yTH by revealed as development, LC affect not does (F) K mro Fg AC supplementary 3A–C; (Fig. embryos CKO K ie h oe rao h oe iga of diagram lower the of area boxed The mice. CKO Rbpj xrsini bet h etuprdarmo the of diagram upper left The absent. is expression Rbpj eeinlast rmtclyenhanced dramatically to leads deletion Rbpj m Dbh G–R nr assL hyperplasia. LC causes r1 in Wnt1 Rbpj o G,K,O. for m Wnt1 Wnt1 aitl(,,)adcrnl(H–J,L– coronal and (G,K,O) Sagittal ) B and (B) nst yrdzto eelda revealed hybridization situ in and nciainas fetdthe affected also inactivation Cemice -Cre Dbh Rbpj Rbpj pstv ern ncoronal in neurons -positive En1 En1 Rbpj K hni littermate in than CKO C K embryos. CKO (C) Phox2a + nciainled inactivation Dbh ern nthe in neurons Rbpj -or ( pstv LC -positive A–C Wnt1 Rbpj Rbpj Phox2b ) Dbh Rbpj m (K–N) for m Rbpj Wnt1 Wnt1 or in + + - Journal of Cell Science n eooi 5H)o h aitlscin tP.Compared P0. at sections sagittal the TH on of (5-HT) immunostaining double serotonin r1- by and ventral neurons and serotonergic neurons derived dopaminergic midbrain-derived ventral ern,rsetvl.T eemn hte hs ern are neurons serotonergic these and which whether in dopaminergic 2003) determine altered al., of To et subpopulation respectively. Trokovic a neurons, 2000; to al., rise Davidson et 1998; give Kimmel al., 1988; et al., (Danielian r1 et mid–hindbrain ventral and midbrain and caudal organizer isthmic of independent patterning. noticeably are not r1 were of that that genes suggesting showed absence S5I–P), four E10.5 the these at by hybridization affected of situ patterns in whole- expression Our 2001a; section 2002). the al., and Joyner, and et mount (Li 2001), boundary and mid–hindbrain Bally-Cuif, of (Liu and position Wurst organizer of 2005; expression isthmic al., et the the Nakamura examined of we activity genes, patterning of al., expression mid–hindbrain et Nakamura regulated and 2001a; is Joyner, and formation 2005) (Liu r1 organizer the isthmic (supplementary the that Tbr2- by reduced Considering or were S4). Tbr1- Fig. neurons of material part cerebellar and deep neurons cell laminar relatively positive cell cerebellar the were of these although granular of S4A–D), marker) loss numbers (external Fig. cell a material Pax6 (Purkinje (supplementary to of normal Calbindin due expression and not marker) the was as defect structures, This S4A–D). Fig. Wnt1 and - Rbpj En1 Gbx2 Wnt1 K ie eeaie h ouain of populations the examined we mice, CKO die r xrsini lopeeti the in present also is expression Cre -driven and and Rbpj Otx2 Fgf8 Rbpj eeinidcdpeoye fthe of phenotypes deletion-induced w e ee o etn pthe up setting for genes key two , w e ee o h inductive the for genes key two , splmnaymtra Fig. material (supplementary ihwl ye,tedpmnri ern nteventral nuclei the raphe in dorsal in neurons in neurons reduced dopaminergic greatly serotonergic were the the and types, midbrain wild with ycnrs,tevnrlmdri-eie Islet1 midbrain-derived ventral Brn3 S3D,H). the by Fig. determined contrast, material as By (supplementary types, E17.5 wild at to immunostaining comparison in in lost mice completely developed was CKO colliculus colliculus inferior superior the the and that defectively found we midbrain, dorsal N rb lorvae h iia eut nboth in results sections similar coronal the by hybridized P0 revealed or also The 5-HT, the probe and to 4A,B). RNA (Fig. ventrally TH situated with nucleus is immunolabeled which raphe nucleus, raphe dorsal median the in Rbpj tdfeetebyncsae fo 85t 1.) h proportion The E13.5). to E8.5 (from labeling stages BrdU embryonic performed different we at events, these both period, of generation combination a prolonged is or neurons a LC neurogenesis, of enhanced number by the caused in increase the whether determine To prolonged and enhanced to neurogenesis due is hyperplasia development. to LC LC seems the are in Rbpj inhibitor regions that selective these suggesting a in as unchanged, function generated or types decreased LC, cell either the in neuronal neurons other noradrenergic of while increase dramatic a in of data results ablation these of together, that absent Taken the show S3I–P). in Fig. unchanged material were nucleus (supplementary red the in neurons Parvalbumin neurons, oculomotor and CKO uatddntovosyafce h eooegcneurons serotonergic the affected obviously not did mutant bjrgltsteL eeomn 4323 development LC the regulates Rbpj Rbpj En1 K ie(i.4–) nadto,frthe for addition, In 4C–N). (Fig. mice CKO idri;M,mdri;r,robmr .Scale 1. 100 rhombomere bars: r1, midbrain; HB, MB, forebrain; hindbrain; fourth FB, 4V, aqueduct; I–T. Aq, diagram in ventricle; in shown area planes boxed section and depicts r1, level dorsal section the the through indicates (above) E10.5– on embryo line E12.5 Dashed controls. wild-type to relative UZ eeladaai nraei h ubrof number the in increase dramatic a E16.5 reveal and (O–T) (U–Z) E12.5 (I–N), E10.5 at hybridization A–H. in shown r1 ( dorsal the through indicates level (right) section embryo the E9.5 representative on line in of unchanged absence are the but in increased greatly are neurons of numbers the Cre Cnuosi both in neurons LC hog h oslr fE. idtp (A,E), wild-type E9.5 of Rbpj r1 dorsal the through Phox2b stages. embryonic ( different at neurons LC 3. Fig. I–Z A–H Rbpj DH mro.Cmae ihtewl type, wild the with Compared embryos. (D,H) Himnsann and immunostaining TH ) Wnt1 Rbpj nst yrdzto for hybridization situ In ) Rbpj EH a efre ntases sections transverse on performed was (E–H) K (B,F), CKO m nter n h adlmidbrain caudal the and r1 the in o –;200 A–T; for m K ie(i.4,) However, 4A,B). (Fig. mice CKO eeinlast vrrdcinof overproduction to leads deletion + Phox2a rcla ern n Brn3a and neurons trochlear Rbpj Rbpj Wnt1-Cre -or Wnt1 En1 Phox2b m and o U–Z. for m K CG and (C,G) CKO Dbh Phox2a mro.Dashed embryos. Rbpj + /Parvalbumin pstv LC -positive nsitu in En1 AD or (A–D) K mice CKO L-AADC Rbpj Wnt1- Rbpj Rbpj Rbpj Wnt1 + + Journal of Cell Science in mro,btanme fBrdU wild-type of in number E11.5 a by The but ended embryos, mostly S1C,G,I). had neurons Fig. LC of material generation (supplementary controls wild-type el fe usn tE05 twihtm on rUlbln of labeling BrdU point time which at TH E10.5, at pulsing after cells aeigo TH of labeling eooiegcnuos(–)aegetyrdcdin reduced greatly are (I–N) neurons serotoninergic oimnsann o rUadT tE75 hnBd was BrdU When BrdU E17.5. E8.5, at at TH pulsed by determined and was BrdU BrdU of for pulse co-immunostaining single a after born neurons LC of 500 VTA, bar: compacta; Scale pars area. nigra raphe substantia tegmental median SNc, ventral MnR, colliculus; CB, colliculus; superior lower aqueduct; inferior SC, left Aq, IC, nucleus; on I–N. cortex; lines C–H, cerebral dashed in CX, The shown diagram cerebellum; B. planes upper and section left A depict on in diagram area shown boxed planes The section mice. indicates wild-type to relative mice CKO serotoninergic the (DR) in raphe decrease a dorsal in shows and neurons sections dopaminergic sagittal midbrain P0 on of (green) number 5-HT and (red) TH neurons. for serotoninergic raphe dorsal and 4. Fig. 4324 otne in continued oe ytesgiiatdcessi rUlbln fTH of labeling BrdU as in decreases embryos, LC significant wild-type the of in by E9.5 generation noted after The abruptly decreased S1B,F,I). neurons Fig. material (supplementary nete idtp or wild-type either in ad eosre vr27tmsmr olbldclsi h LC the in cells co-labeled more other times the of 2.7 on over E9.5, observed at we BrdU hand, applying After S1A,E,I). Fig. material rmE25o,vr e Cpoeiosicroae rUin BrdU incorporated progenitors LC few very on, E12.5 From oaiegcnuos(–)ad5H-and 5-HT- and (C–H) with neurons hybridized dopaminergic and 5-HT or TH AADC with immunostained sections coronal Rbpj + Rbpj Cpeusr a ae ncnrs,L neurogenesis LC contrast, In rare. was precursors LC N rb hwta H and TH- that show probe RNA Rbpj Wnt1 Wnt1 Rbpj ora fCl cec 2 (18) 125 Science Cell of Journal eeinlast h euto fmdri dopaminergic midbrain of reduction the to leads deletion K mro splmnaymtra i.S1D,H,I). 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Aq, ventricle; fourth 4V, 100 orientation. bar: section Scale show J–L in Insets otos tE05(,) peuaino sl xrsini o biu in obvious not in is (arrowheads) expression r1 Ascl1 dorsal of the upregulation (D,H), E10.5 At controls. K ie Fg EFaddt o hw) ossetwith Consistent shown). not of data expression and the results, 5E,F these (Fig. mid lines although the CKO controls, in wild-type upregulated to relative i.6. Fig. E8.5, At (Hirsch mice 1998). of in al., expression neurogenesis the noradrenergic et in for changes factor the essential examined we expression Next, Ascl1 upregulates deletion Rbpj n lgturglto tE05(,) 1. KO n 1. 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Fig. material in supplementary reduced were neurons cerebellar 4328 sbigrqie o Cseiiain a loexpressed also was specification, LC when r1 dorsal for the in required normally being of as expression the Moreover, ora fCl cec 2 (18) 125 Science Cell of Journal Rbpj niae hti diint aoia Notch– canonical to addition in that indicates Bmp6 and Bmp7 Nr2f6 Rbpj pe n orr 02.We 2002). Rohrer, and ¨pker eeepesdi h 1roof r1 the in expressed were Rbpj a eee.Tu,i slikely is it Thus, deleted. was eercnl identified recently gene a , Rbpj K ie(i.4and 4 (Fig. mice CKO K ie(i.5). 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(E) embryos day three embryonic least as designated was appeared eae nps-otmdy85 .,1.,1.,1. n 35 n mro were embryos and 13.5, timed-pregnant and to 12.5 11.5, intraperitoneally 10.5, delivered 9.5, 8.5, was day USA) post-coitum (60 on MO, pulse females Louis, BrdU single St a Sigma, experiments, staining birthdating TUNEL BrdU and For staining X-gal labeling, BrdU Wnt1 genotyping and breeding Mouse Methods and Materials rmtr n twspromda rvosydsrbd(oge l,2003). al., consensus et a (Santa EMSA: Rbpj (Song for with used described were transfected probes cells to previously oligonucleotide Rbpj HEK293 biotinylated as of from Four binding Cruz). prepared performed direct were a was extracts is Nuclear there it wherever and study to promoter, used was EMSA The chromatin and assay EMSA immunoprecipitation assay shift mobility Electrophoretic previously RNA antisense as mouse 1997), following performed al., The et were 2010). (Wassarman al., used: hybridization et were Shi situ 2008; probes al., in et section (Shi described and double mount BrdU immunostaining Whole described and hybridization as and situ X-gal performed 2009). In al., LC. TH were et the sections Zheng for 2008; in frozen al., cells of processed et (Shi staining BrdU-positive previously were TUNEL localize and to Sections staining below) (see E17.5. immunostaining at collected rgntr n oprtswt bji otoln the controlling in Rbpj LC with in neurons. LC expressed cooperates of specifically development to and is needed which are progenitors cofactor(s) studies Further the pancreatic 2007). early identify during al., cells is et acinar spinal (Masui of which in and development 2008) Ptf1a, interneurons al., et factor GABAergic Rbpj (Hori of transcription cord that specification shown the for have with required studies complex recent a sense, forms this In expression. eoye speiul ecie Dneine l,19;Hne l,2002; al., et Han 1998; al., inactivate crossed et To 1999). we (Danielian Soriano, 2000; al., described et Kimmel previously as genotyped Cre Rbpj ; Rbpj En1 -Cre, Otuae l,1999), al., et (Ohtsuka N_053 .0kb), 0.60 (NM_007553; ( K,rsetvl) h onn ftedyo hc h aia plug vaginal the which on day the of morning The respectively). 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Berlin, Medicine, Molecular for ¨ck-Center cDBH Osw ta. 2005), al., et (Ohsawa flox/flox Gbx2 En1 Msx2 ; Rbpj vGluT1 Ngn1 Cre/+ Bmp4 Tvrne l,19)and 1996) al., et (Tiveron M452;07 kb), 0.73 XM_415429; , LuadJye,2001b), Joyner, and (Liu flox/flox n oa6rpre iewr eeae and generated were mice reporter Rosa26 and N_161 .5kb), 0.65 (NM_013601; , Ngn2 iewith mice N_054 .6kb), 0.56 (NM_007554; N_893 .2k) eegnrtdby generated were kb), 0.72 (NM_182993; rgn rfre oas to (referred progeny Bmp7 Sme ta. 1996), al., et (Sommer Ascl1 Rbpj Phox2b i Rbpj N_057 .3k) chicken kb), 0.43 (NM_007557; raZisLM1 confocal LSM510 Zeiss a or Dbh Rbpj N_005 .0kb), 0.90 (NM_009035; BDNF N_053 . kb), 1.0 (NM_008553; flox/flox ( cPhox2b Mrne l,1997), al., et (Morin xrsini h oslr1, dorsal the in expression N_050 .0kb), 0.80 (NM_007540; L-AADC Hes1 Phox2b iet obtain to mice m Msx3 Bmp5 / fbd weight; body of g/g XM_001234150; , Lee l,2005), al., et (Lee Rbpj Otx2 b (NM_010836; (NM_016672; (NM_007555; Pty tal., et (Pattyn Wnt1 gl(1:1000; -gal le n C. and ¨ller (Martinez- K or CKO Wnt1- Ascl1 Msx1 Wnt1 Fgf8 Journal of Cell Science H,BrdU TH-, analysis hybridization. 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WI, Madison, (Benjanirut (Promega, Phox2a vector or pGL3-enhancer 2005) the al., into et 2006) (Arvidsson al., Ascl1 et mouse of elements upsteam bjatbd SnaCu) fe h rtiswr eoe rmDAby DNA from removed subjected was were 3 DNA proteins immunoprecipitated with purified PCR the the to K, After The proteinase Cruz). with USA). digestion anti- (Santa MA, an with Billerica, antibody precipitated (Millipore, were Rbpj complexes kit factor for lysis assay transcription used DNA-bound SDS were ChIP specific in lysates a cleared sonicated The min. and were by centrifugation 30 isolated. tissues by immunoprecipitation for the removed was was glycine, temperature organizer debris mM room The 150 isthmus buffer. at adding with formaldehyde by r1 1% stopped After in the sequence cross-linked and were DNA PBS samples ice-cold confirm in performed. were To dissected oligonucleotides experiments Rbpj. unlabeled competition of the with excess formation, molar transfected complex 100-fold with protein–DNA not the of cells specificity HEK293 was ute omlzdt h oto.Frra-ieR-C,tefloigprimer following the RT-PCR, real-time 5 For F: Ascl1, control. used: were the sequences to normalized mouse further and Abcam) to (1:1000; normalized anti-Ascl1 rabbit assay, al., after anti- blotting et western hours (Zhang real-time For previously Forty-eight quantitative described 2012). and as control. blotting assays western (RT-PCR) the to reverse-transcription-PCR subjected The as were transfection. cells before used mixed the Rbpj- were transfection, was ratio co-transfection, 1:1 vector Hes1 a For in or pCAGGS-IRES-EGFP (Invitrogen). plasmids Rbpj-VP16) siRNA 2000 Hes1 or and Lipofectamine Hes1 VP16 by (encoding plasmid plasmid each expression siRNA For the with activity. and transfected control control least at internal internal of each the transfections. an for quadruplicate independent in to as three performed were normalized used assays condition, of was was experimental ng/well pRL-SV40 activity 5 experiments, lysed vector luciferase all were expression In cells (Promega). the luciferase transfection, System after Dual-Luciferase vector DNA hours the pGL3 Thirty-six of using empty control. each), 100 The ng the with ng USA). 600 as (200 CA, used Rbpj-VP16 total and Carlsbad, was or each) (Invitrogen, NICD a ng 2000 Hes1, (400 Lipofectamine encoding with plasmid vector reporter co-transfected expression pGL3-Phox2a following the or The transiently FBS. pGL3-Ascl1 10% the were with containing supplemented cultures DMEM in plates day, 24-well in well per GACTCAAG (5 epciey h N apewsas mlfe sn x / rmr (5 primers F/R Ex1 using amplified also was sample DNA The respectively. 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Maeda, and I. Nagatsu, H., Kimura, R., Arai, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.102152/-/DC1 31170801, online Science available material Supplementary 31030034, Natural of 2012CB966904]. National numbers Technology and and 2011CB510005 the Science numbers [grant of [grant Ministry China by China and 31100788]; supported 81101026, of was Foundation work This Funding technical for Zhang providing Xiao for Joyner and A. Wang and En1 McMahon Li P. Chen, A. support, Jia-Yin thank We Acknowledgements or CKO eeaaye sn rgnr75sfwr n rsne smeans as presented and software OriginPro7.5 using analyzed were aisn . rhm . ie .adHl,R. Hill, and C. Sime, E., Graham, D., Davidson, F. Guillemot, and S. D. Castro, N., Bertrand, D. B. Waterhouse, and W. C. Berridge, and L. R. Hullinger, S., K. Kim, J., S. Hong, H., W. Wang, M., Paris, C., Benjanirut, K. Funa, R. Grzanna, and and T. H. M. Shipley, Uramoto, G., Aston-Jones, F., Watt, V., Sumantran, Y., Arvidsson, a,H,Tngk,K,Ymmt,N,Krd,K,Ysioo . aaaa T., Nakahata, M., Yoshimoto, K., Kuroda, N., Yamamoto, K., Tanigaki, H., Han, H. Rohrer, L. and C. C. Cepko, Goridis, and M. E. Morrow, Z., Z. Bao, S., Mukherjee, T., Furukawa, ot,S . lo,F .adAtnJns G. Aston-Jones, and E. F. Bloom, L., S. and Foote, M. Hadjiconstantinou, P., C. Silvia, T., Quach, P., K. Gudehithlu, J., M. Eaton, P. A. McMahon, and K. S. Michael, H., D. Rowitch, D., Muccino, S., P. Danielian, J. S. Bray, J. E. Bockstaele, Van and J. Barr, L. M. Oshinsky, and Y. Zhu, S., Chen, G., Aston-Jones, u,S,Buh . eak,H,Gdad . isn .W,Mlis .C and C. M. Mullins, W., S. Wilson, A., Goddard, H., Teraoka, J., Brush, S., Guo, a,E,Gawh,G,Csrs,S,Kgym,R n uleo,F. Guillemot, and R. Kageyama, S., Casarosa, G., Gradwohl, E., Cau, P. J. Card, and Y. 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Cre in of activity form gene inducible of Modification (1998). types. cell neural of specification locus rat the of terminals axon in peptides opioid coeruleus. endogenous with colocalizes Press. Academic York: New 183-214. In pp. groups. cell noradrenergic coli A7 E. and Mash1-promoter the phosphorylase. nucleoside by purine mediated cytotoxicity Neuroblastoma-specific idri eursBP G8 n h oedmi rti soulless/Phox2a. protein homeodomain the and FGF8, BMP, Neuron requires hindbrain A. Rosenthal, Mu neurons. of formation the promote cells. notch1 progenitor and Hes1, rax, ee euaesqeta tgso ergnssi h latr epithelium. olfactory the in neurogenesis of stages sequential Development regulate genes Biol. Cell pool. interneuronal possible zone: dendritic pericerulear the Neurosci. in ceruleus locus arousal. of regulation circadian Cre/+ Rbpj 24 20) oc inlig ipeptwybcmscomplex. becomes pathway simple a signalling: Notch (2006). a.Rv Neurosci. Rev. Nat. ie .Hve o niTr/ nioy n .Mu T. and antibody, anti-Tbr1/2 for Hevner R. mice, 24 7 555-566. , nt e.ADso.Ml el vl Biol. Evol. Cell. Mol. Discov. A Rec. Anat. 678-689. , En1 19) itiuino rmtcLaioai eabxls RAin mRNA decarboxylase acid L-amino aromatic of Distribution (1993). 2313-2321. , 127 K)adltemt idtp otoswr nldd l data All included. were controls wild-type littermate and CKO) 19) eeomn fnrdeegcnuosi h zebrafish the in neurons noradrenergic of Development (1999). Neuron 2323-2332. , 20) h APptwyi obnto ihBP regulates BMP2 with combination in pathway cAMP The (2006). nvs . aes .R n ule,L. Puelles, and R. F. Mateos, B., ´novas, ri Res. Brain 42 P 33-84. , , 26 20) pcfcto fctcoaiegcadserotonergic and catecholaminergic of Specification (2002). 0.05. 20) nuil eekoku ftasrpinfactor transcription of knockout gene Inducible (2002). 383-394. , 3 531-541. , a.Neurosci. Nat. 745 h a evu System Nervous Rat The Development eit.BodCancer Blood Pediatr. a.Rv Neurosci. Rev. 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Suter, M., Aguet, F., Radtke, S., ¨tolf, eretdr n t optnei epnigt oeri n ibaninducing midbrain and forebrain to responding in A. competence Simeone, its and and A. neuroectoderm Lumsden, F., Schubert, R., formation. L. A. derivatives. its and Notch1 mouse T. of Honjo, and K. Tamura, S., development. oemrhgntcpoenrcpo inln euae ergnssi the in neurogenesis regulates signaling receptor lip. protein rhombic cerebellar morphogenetic bone progenitors. neural rhombic-lip cerebellar ieg el otiuet iceecl ouain nCSarchitecture. CNS in populations cell discrete to Neurosci. contribute cells lineage dorsal the in specification fate neural versus midline. non-neural telencephalic regulate neurogenin opnn rti-muoecieclsi h a eta n eihrlnervous peripheral and central rat the in systems. cells protein-immunoreactive component cerebellum. 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Ding, and H. h ucinlt ftefrbancokadafcsnociception. affects and and impairs clock brainstem mice forebrain 625. Ear2(Nr2f6)-deficient the the in of coeruleus functionality within locus the the of lineages development Abnormal novel reveals and derivatives cerebellum. lip rhombic n neatoso h w lsl eae oebxgnsPo2 n Phox2b and Phox2a genes homeobox related closely two neurogenesis. the during of interactions and irto fnrdeegcnuosi ou orlu fmuebrain. mouse of coeruleus locus in neurons Neurosci. noradrenergic of migration 1099-1107. Phox2b. gene homeobox the 235-243. by phenotype noradrenergic ersinatvt smdae yac-erso n naoie yteEpstein-Barr the by antagonized EBNA2. (BMPs). and factor co-repressor transcription a proteins virus by mediated morphogenetic is activity repression bone on depends neurones Development (LC) coeruleus OP rmtrtruhRPJ n e1dpnetmechanisms. al. Hes1-dependent et Q. and L. RBP-J- Song, Y., through Biochem. X. promoter Ti, F., FOXP3 He, H., L. progenitors. crest neural enteric ucinfrNthi rmtn loeei ntezbaihretina. zebrafish the in gliogenesis promoting in Notch for function sensory Rnx/Tlx3. protein visceral homeodomain relay on first-order dependent and is centers neurons (nor)adrenergic brainstem of Formation osn,J,MMhn .P,Wrt .adPrae,J. response Partanen, sustained for and hindbrain signals. and W. isthmic mid- Wurst, developing to both P., in A. required independently McMahon, J., Rossant, mouse. the in days embryonic different on Genet. Nat. differentiation. R. Kageyama, in coeruleus locus Phox2a. of gene absence homeobox and the ganglia for autonomic deficient and mice sensory in Defects (1997). rncito atrPo2dlnae yatcptwy fteatnmcnervous autonomic the of pathways synaptic delineates system. 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