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processes. Npn2 crest migration through the . Interestingly, cells still condense into segmentally arranged dorsal root gang autonomously required for neural crest cells to avoid Sema3f in vitro. These data show that Npn2/Sema3f signaling guides neural and instead migrate as a uniform sheet, although somite polarity itself remains unchanged. Furthermore, Npn2 is restricted to the posterior-half somite. In ytm bt usqety irt aa ad olw defined invade only follow the anterior but not the posterior portion and of each somitic away stereotypic pathways. In the trunk, for migrate example, neural crest cells subsequently but system, During development, neural crest cells arise in the central nervous . in lineages different of variety wide a to rise gives The neural crest is a multipotent population of migratory cells that INTRODUCTION KEY WORDS:Trunk neuralcrest migration,Sclerotome, 2,Semaphorin3F, Mouse,Chick neuropilin 2 demonstrate ( that neural crest cells express the In , neural crest cells migrate only through the anterior half of each somite while avoiding the posterior ha Laura S. Gammill 2/semaphorin3Fsignaling neuropilin Guidance oftrunkneuralcrestmigrationrequires Development 133,99-106doi:10.1242/dev.02187 Accepted 27October 2005 1 et al., 2001; Davy et al., 2004; Orioli et al., 1996; Wang et al., 1998). (Adams defects migration crest neural trunk to exhibit fail examined the 1997), Anderson, Wang1997; al., et and (Krull migration crest neural trunk pattern Although previous reports suggested that Eph/ephrin signaling might debate. to open still is sclerotome anterior the through exclusively targets in the periphery. is crucial for proper wiring of the ganglia and peripheral to these ganglia relative to the , which will form the 2004; vertebrae, al., et (Kuan of segmentedarrangement The 1999). Kalcheim, and LeDouarin system nervous peripheral the of ganglia the of organization metameric the for responsible be to believed is migration crest neural of pattern segmental the Thus, 2000). 2004; al., et al., Teillet,et and Mansouri Kalcheim 2000; al., et Leitges 1989; (Bussen ganglia root dorsal crest-derived neural crest migration (Kalcheim and Teillet, 1989) and formation of fused anteroposterior somite polarity results in loss of segmental neural and Teillet, 1989). Accordingly, surgical or genetic modification of neural crest migration (Bronner-Fraser and Stern, 1991; Kalcheim sclerotome being permissive and posterior sclerotome repulsiveanterior for the with somites, the by imposed be to appears pattern segmentally arranged streams of migrating neural crest cells. This of formation the in results selectivemigration This 1990). al., et sclerotome (Bronner-Fraser, 1986; Rickman et al., 1985; Serbedzija Medicine, Baltimore, MD21205,USA. USA. *Author forcorrespondence (e-mail:[email protected]) Hopkins UniversitySchoolofMedicine, Baltimore, MD21205,USA. Division of Biology 139-74, California InstituteofTechnology,Division ofBiology139-74,California Pasadena,CA91125, The identity of the molecular cues that direct neural crest migration 2 Department ofNeuroscience, TheJohnsHopkinsUniversitySchool of nulls, suggesting that segmental neural crest migration and of the peripheral are separable 1 , Constanza Gonzalez Eph and ephrin mutant mice that havebeen that mice mutant ephrin and 3 Howard HughesMedicalInstitute,TheJohns Npn2 1 and , Chenghua Gu Sema3f mutant mice, neural crest cells lose their segmental migration pattern 2,3 ecie peiul (ikno, 92, xet ht yrdzto was hybridization that except 1992), (Wilkinson, previously described and Bronner-Fraser, 2002). Mouse in situ hybridization was performed as Chick in situ hybridization was performed as described previously (Gammill In situhybridization chain reaction (PCR) (Giger et al., 2000). at 4°C in 4% paraformaldehyde. Embryos were genotyped by polymerase overnight or temperature room at 2 hours for fixed and (PBS) saline buffered phosphate- cold ice into plug, the of day the being 0.5 day with isolated, surgically were embryos Mouse paraformaldehyde. 4% in 4°C at overnight and Hamilton, 1992). Embryos were isolated in Ringers Saline and fixed Fertile eggs were incubated at 37°C to the desired stage (Hamburger Embryos suggested to play a role (Eickholt et been al., 1999), havebut are not 3A expressed semaphorin its and 1 neuropilin Likewise, MATERIALS ANDMETHODS a segmented peripheral nervous system. ganglia, and that multiple signaling pathways are required to create migration alone does not dictate the arrangement of the peripheral well less albeit form, crest neural of pattern the that suggesting normal, than separated still ganglia root dorsal individualized Surprisingly, somites. the through migration crest neural guides cells detects a Sema3f repellant cue in the posterior sclerotome that migration. We demonstrate that the Npn2 receptor on neural crest crest neural trunk during development has not been examined. ligands its and Npn2 of importance However,2000). al., the et Giger 2002; and al., et Cloutier 2000; al., guidance et (Chen system nervous appropriate peripheral and central the in for fasciculation required is Npn2 2002). al., et Neufeld Tessier-Lavigne,2002; and (Bagri factor growth endothelial vascular as well as 3F and 3C (Sema) semaphorins and Bronner-Fraser, 2002). Npn2 is a receptor for class 3 secreted upregulated as a consequence of neural crest induction (Gammill true regulatory molecules have not been found. neural crest mutant is due to redundancy or whether the et al., 2002). It is not clear whether the inability to identify a trunk in the mouse for appropriate migration (Kawasaki at the right time (reviewed by Kuan et al., 2004) and are not required and Marianne Bronner-Fraser Npn2 Here, we explore the role of Npn2 signaling during neural crest ( 2a1 neuropilin chick isolated We ), while its repulsive ligand semaphorin 3F ( RESEARCH ARTICLE Npn2a1 1, * ) in a screen for genes Sema3f lia in ) is lf. We 99

DEVELOPMENT (Microm) and mounted in permafluor (Thermo Electron Corporation). h mlrt o te olce poen acltd codn t the to according calculated protein collected the manufacturer’s instructions. of molarity the and Corporation) (GenHunter A Reagent Assay AP using determined was Centriplus YM-100 filter device (Millipore). a Alkaline phosphatase activity using concentrated and days 3 after collected was Strep) Pen + BSA dish according to the manufacturer’s instructions. Media (DMEM + 0.1% 20 at sectioned nitrogen, liquid in frozen sucrose, 15% embryos were infiltrated with 5% sucrose, 15% sucrose and 7.5% gelatin in Tbx18 1998), or AP empty vector using 45 293T cells were transfected with 24 Conditioned medium 0.009% hydrogen peroxide according to the manufacturer’s instructions. 3,3 mg/ml 0.1 and (VectorLaboratories) kit peroxidase ABC-horseradish the using developed and Research), Immuno (Jackson anti-mouse-Biotin 1:500 by with 1:500 anti-TUJ1 (neuron specific class III degelatinized for 20 minutes at 42°C in PBS. Dorsal root ganglia were stained 15 at sectioned nitrogen, liquid in frozen infiltrated with 5% sucrose, 15% sucrose and 7.5% gelatin in 15% sucrose, Alexa 488 secondary at 1:1000 (Molecular Probes). Unstained embryos were Immuno Research). Sema3f spots were visualized using an anti-mouse IgG Reichardt, and 1991) followed (Weskampby 1:400 anti-rabbit-Rhodamine Red X (Jackson anti-p75 1:2000 or Research) Immuno (Jackson X Red Culture; Tucker et al., 1984) followed by 1:400 anti-mouse-IgM-Rhodamine Neural crest cells with were stained with 1:50 anti-HNK-1 (American Type Immunohistochemistry Fraser,mouse 2002), digoxigenin-labeled antisense riboprobes were as follows: chick for Templates Mannheim). (Boehringer Reagent mM maleic acid, 150 mM NaCl, 0.1% Tween (pH 7.5) with 2% Blocking 100 in performed was pre-treatment antibody and 65°C, at I solution wash Embryos were washed twice in hybridization mix at 70°C, three times in yeast RNA, 0.2% Tween 20, 0.5% CHAPS and 50 performed in 50% formamide, 1.3 100 spotted region of the coverslips in 1 ml of pre-warmed neural crest complete isolated by trituration with a fire-polished pasteur pipette and plated on the with DMEM-F12 + 10% fetal bovine serum (Hyclone), neural tubes were made fresh in HBSS and 0.2 were incubated for 8 minutes at 37°C in room temperature 3 membranes lateral to the somites and removing the gut tube. Trunk pieces the trimming dissected, was somites 10 last the containing trunk the of E9.5 embryos (14-24 somites) were isolated into ice cold HBSS. The region Mouse neuraltubeculture + 1 mg/ml BSA. 4 ml of HBSS and stored overnight at 4°C in 2 ml DMEM-F12 (Invitrogen) 37°C. After aspirating the fibronectin, the coverslips were washed once with (BD Biosciences) was laid over the spots and incubated for 1.5 to 2 hours at (Chemicon), 0.2 50 with hour minutes. AP-Sema3f (75 nM) was preincubated at room temperature for 1 of spots four with plates silicone vacuum grease, and UV sterilized in a culture hood for 15 culture tissue mm 35 onto immobilized oven, 95% ethanol. Coverslips were cured for 15 minutes at 100°C in a vacuum in the 2% APTES for 10 minutes, and washed three times for 5 minutes with (25 mm; Nunc) cover slips in a wafer basket (Fluoroware) were incubated ethanol and allowed to hydrolyze for 5 minutes in a fume hood. Thermanox 95% in prepared was Sigma) APTES; (2%; Aminopropyltriethoxysilane Sema3f spotpreparation traces of HBSS had been aspirated, 150-200 remaining all Invitrogen).After (HBSS; solution Hank’sbufferedsaline 1 of ml 4 with times three washed coverslipswere the 37°C, at hour 1 and the location of the spots was marked on the underside of the dish. After Sema3f (Kraus et al., 2001) and 2001) al., et (Kraus RESEARCH ARTICLE Ј daioezdn ttayrclrd (A; im) with Sigma) (DAB; tetrahydrochloride -diaminobenzidine ␮ (Giger etal.,2000),ephrinB2(Wang andAnderson,1997), /l os at-ua paetl laie phosphatase alkaline placental anti- mouse g/ml ␮ l drops were spotted manually in a grid on the coverslip, Npn2 ␮ (Giger et al., 2000), al., et (Giger ␮ m filter sterilized. After rinsing several times l Lipofectamine 2000 (Invitrogen) per 10 cm Uncx4.1 ϫ ␮ SSC (pH 5), 5 mM EDTA, 50 g of AP-Sema3f (Giger et al., 2000) (Mansouri et al., 1997). Stained 1997). al., et (Mansouri ␮ Npn2 M by cryostat (Microm) and (Microm) cryostat by M ␮ ␤ l of 125 -tubulin; Babco) followed Sox10 (Gammill and Bronner- ␮ g/ml heparin at 70°C. ␮ (Kuhlbrodt et al., et (Kuhlbrodt g/ml fibronectin ␮ M by cryostat by M ␮ g/ml dispase ␮ g/ml ϫ Fig. 1. migration. To address the requirement for Npn2 during neural crest and formation crest neural influencing for candidate potential a marker p75 (Fig. 1D-F). HNK-1 mouse embryos, by identified Similarly,of 1B). sections transverse(Fig. in sclerotome somitic cells, crest neural immunoreactivity, as they migrate through the by anterior half of each expressed was revealedembryos chick that through sections Longitudinal Npn2 and Bronner-Fraser, 2002) and the mouse (Fig. 1C-F). In the trunk, on migratory neural crest in both the chick (Fig. 1A,B) (Gammill in premigratory neural crest cells in the dorsal as well as chick induction (Gammill and Bronner-Fraser, 2002). identified We posterior sclerotome in Neural crest migratesthrough anteriorand RESULTS were cultured for an additional 28-48 hours at 37°C. complete medium was added slowly down the side of the dish, and explants were tubes The allowed to stick omitted. to the coverslip was for one hour acid at 37°C, then retinoic 2 ml of and additional used was DMEM-F12 medium, prepared as described (Stemple and Anderson, 1992) except that eeomn, e sesd h ls-ffnto peoye by phenotype loss-of-function the assessed we development, sclerotome; nt,neuraltube.Scalebars:0.1 mminA-D;0.01E,F. obtained atE9.5.a,anterior;p,posterior; dm,dermomyotome;sc, Arrowheads indicatemigrating neuralcrest cells.Identicalresults were mount view. (E,F)Highermagnification viewoftheregions boxedinD. embryos showninAandC.Plane ofsectionisindicatedonthewhole- sections ( Neural crest cellswere immunostainedwithanti-HNK-1inlongitudinal somite stagechick( expression wasrevealed bywhole-mountinsituhybridization of31 The distribution of was clearly expressed in a segmental pattern in both species. both in pattern segmental a in expressed clearly was Npn2 B ) andanti-p75intransversesections ( is expressed inmigratingneuralcrest cells. Npn2 A ) andE10.0(28somite)mouse( Npn2 Npn2 expression colocalized with the neural crest neural the with colocalized expression on neural crest cells made this receptor s n al rsos t nua crest neural to response early an as Npn2 mutants D-F Development 133(1) ) through the Npn2 C ) embryos. is expressed Npn2 Npn2

DEVELOPMENT posterior; dm,dermomyotome;sc,sclerotome; nt,neuraltube.Scalebars:0.1mm. is upwards. (B,C,E,F)Longitudinalsections;BandEare sectionsthrough theembryosshowninAandDatlevelsindicated. and posterior-half sclerotomes ( for theneuralcrestmarkers mutant mice (Giger et al., 2000). In situ hybridization with probes in expression gene marker crest neural characterizing Npn2/Sema3f intrunkneuralcrest migration the anterior-half sclerotome ( Fig. 2.Npn2isrequired segmentaltrunkneuralcrest topattern migration. ye nt hw) or shown) (not type neural crest migration in mutant mice (data not shown). or specification generation of neural crest cells in wild-type, heterozygous or the on effects obvious no showed somites) 12 expressed in a pattern complementary to that of to that complementary in a pattern expressed rather than in streams (Fig. 2D), and sheet uniform a in migrated cells however,crest mice, neural neural tube (Fig. 2B,C) (Serbedzija et al., 1990). In the regionof intermediate the levelsto at adjacent sclerotome anterior-halfthe in only positivefound were cells crest neural Sox10 neural crest migration appeared as segmentally iterated blocks of form, some segmental neural crest migration was still apparent will ganglia sympathetic crest-derived neural the where aorta sclerotome (Fig. 2E,F). More ventrally, approaching the dorsal the of portions posterior and anterior both throughout present emmooa cmatet Aas t l, 1996). the in not contrast, By shown). but al., not (data E9.5 dermomyotome at sclerotome the in levels et low at detected the (Adams in only Immunostaining for vascular endothelial growth then factor was also compartment and E10, around dermomyotomal somites the in expressed Npn2 somite. the levelsof intermediate and dorsal eventsthrough guidance crest neural for required is signaling Npn2 that suggest data although ventral roots still form (data not shown). Together, these in sclerotome posterior and anterior both into normally arerestrictedtotheanteriorsclerotome,alsoproject in yrdzto ad immunohistochemistry. and hybridization situ in by molecules these of patterns expression the assessed the by revealed functions patterning the mediating was ligand which To determine 2000). al., Gluzman- et Poltorak 1997; al., et (Chen factor growth endothelial vascular and Sema3f Sema3C, ligands: different three to bind can Npn2 expression patterns Profound defects were observed,however,were defects Profound of pattern the on Npn2 staining cells. In sections through these embryos, and -null mice (Fig. 2D). Interestingly, motor , which Sema3f Npn2 exhibit complementary B Npn2 Sox10 , C E eeoyos Fg 2) embryos, 2A) (Fig. heterozygous ). Intheabsenceof , F ). Neuralcrest wasvisualizedatE9.5byinsituhybridizationfor -null mice. In the trunk of wild- , Foxd3 Sox10 and Npn2 -positive cells were Pax3 Npn2 Npn2 Sema3c mutant, we next we mutant, Npn2 at E8-8.5(4to Npn2 , segmentalmigrationislost( Sema3f (Fig. 3A,B), Npn2 mutants, s first is Sox10 mutant Npn2 -null was - hybridization for Npn2 migrate throughout thesclerotome ( bars: 0.1mm. posterior; dm,dermomyotome;sc,sclerotome; nt,neuraltube. Scale p, 2;r4,rhombomere 4;ov, oticvesicle; a,anterior; r2, LongitudinalsectionsofembryosshowninEandG.i,isthmus; (F,H) ( sclerotome anterior-half the through exclusively migration from results E9.5, thesegmentalappearanceofmigratingtrunkneuralcrest ( anterior-half somite( , branchialarches andtrunk,where ( Fig. 3. required to pattern neural crest migration. crest neural pattern to required F B ) isdisruptedin ) are expressed inreciprocal atE9.5,mostnotablyinthe patterns (A,C) or Trunk neural crest normally migrates in streams ( streams in migrates normally crest neural Trunk Sema3f D ) andneuralcrest cellsmigratethroughout bothanterior- Sema3f expression iscomplementaryto Sox10 Sema3f C Sox10 ) and (B,D). (E-H)Neuralcrest was visualizedbyinsitu . (E,G)Anterioristowards the left. mutants ( Sema3f . InA,D,anterioristowards theleft,dorsal G in theposterior-half somite( H ) becauseneuralcrest cells ). (A-D)Insituhybridizationfor RESEARCH ARTICLE Npn2 Npn2 a,anterior;p, is expressed inthe Npn2 ( A A ) and ) restricted to and Sema3f D E ) that ). At 101

DEVELOPMENT (J) ,posterior;dm,dermomyotome;sc,sclerotome; nt,neuraltube. p, hybridization andembryossectionedlongitudinally. a,anterior; Sema3f these three genesremains unchangedin Uncx4.1 sclerotome ( still posteriorlyrestricted. EphrinB2isexpressed intheposterior and mice. i.4 Somitepolarityisnormalin Fig. 4. 102 expression pattern made made pattern expression and 4A), (Fig. Npn2 Sema3f with Shy t l, 03. tiigy the Strikingly, 2003). al., et (Sahay Sema3f 2000; Giger et al., 1998). al., et development(Giger of stages later at observed been also through the trunk (Fig. 3E), Instead of segmentally arranged streams of neural crest cells in the migration phenotype was identical to that observed in for requirement the development by examining assessed next We complementary distribution of somite. Sema3f is required to restrict neural crest migration to the anterior demonstrate that signaling between the receptor Npn2 and its ligand of sclerotome uniformly distributed whereas 3F), (Fig. mice wild-type of respectively, sclerotome, negative regions were observed in the anterior- and posterior-half neural crest (Fig. 3G). In sections, alternating Uncx4.1 Sema3f Npn2 In E9.5embryos, was clearly expressed in the anterior half of half sclerotome each in the anterior expressed was clearly RESEARCH ARTICLE mutants. Geneexpression wasvisualizedbyinsitu is expressed intheposteriorsclerotome ( expression in the posterior somite (Fig. 3D). In sections, In 3D). (Fig. somite posterior the in expression mutants phenocopy also remains restricted totheposteriorsclerotome of expression in the anterior somite (Fig. 3C) mirrored by mirrored 3C) (Fig. somite anterior in the expression D in posteriorsclerotome ( Npn2 ), Tbx18 Sema3f Sema3f Sema3fF receptor during trunk neural crest migration. The is expressed intheanteriorsclerotome ( Sox10 Npn2 mutants (Fig. 3H). Together, these results Together,these 3H). (Fig. mutants nulls contained uniform sheets of migrating Sema3f Sox10 n h pseir af Fg 4) This 4B). (Fig. half posterior the in is expressed inanteriorsclerotome ( -labeled cells were seen throughout the Npn2 expression in in expression an ideal candidate for signaling B ). ( Npn2 Npn2 C Npn2 and Sema3f ) In Sema3f and Npn2 Sema3f mutants ( -null mice H Sema3f Sox10 Sema3f during neural crest neural during ). Expression of nl nua crest neural -null mutants, expression has expression -positive and - Npn2 E , mutant G mutant mice , I ). Sema3f F mutants. ) and A ), is w atrae ehnss ol epan h tuk erl crest neural trunk the explain could mechanisms migration defects observed in the alternate Two mutants is normal in Somite patterning migrating neural crest cells in vitro (Wang and Anderson, 1997), was Npn2 and 4B) (Fig. wild-type both of sclerotome posterior the in expressed posterior somite identity was retained in the mutants. mutant and anterior and posterior sclerotome were examined in wild-type, 2002). through the posterior sclerotome of these animals (DeBellard et al., 1997), and as a consequence, neural crest cells migrate aberrantly somite polarity is abolished anteroposterior in example, For migration. crest neural of pattern the impacting secondarily migrates, crest neural which through environment the thus and this polarity somite affect Disrupting could signaling identity. sclerotomal posterior and/or anterior anterior and posterior somite could be important for maintenance of crest migration. Alternatively, Npn2/Sema3f signaling between the somite and the Npn2 receptor on neural crest cells to guide neural posterior for the in requirement ligand repulsive a Sema3f the reflect between signaling could phenotype The mice. mutant eie gnla f h prpea nros ytm Ti pattern ensures that the ganglia and the vertebrae, This which differentiate from system. nervous peripheral the of ganglia derived is somite thought to prefigure the the segmented organization of through the neural crest- crest neural of migration segmental The Npn2 Segmentally arrangeddorsal root gangliaform in Npn2 on the neural crest during trunk neural crest migration. in the environment, and supports a cell-autonomous requirement for Npn2 receptor on neural crest cells detects a Sema3f repulsive cue or without Sema3f protein (Fig. 5B,C). This demonstrates that the mutant neuralcrestcellsmigratedequallywellonfibronectin with behavior in vivo (Kasemeier-Kulesa et al., 2004). By contrast, the with 5A), rare cells migrating onto the Sema3f (Fig. substrate, consistent with we their Sema3f avoided directly, majority of cells remaining at the spot border and only individual, cells crest crest neural neural Wild-type the in Npn2 them on fibronectin-coated substrates containing spots of Sema3f. for and wild-type explanted requirement the test To mediated repulsion Npn2 isrequired intheneuralcrest forSema3f- neural crest. the in reside to likely is signaling Npn2/Sema3f for requirement in maintained is results demonstrate that anterior and posterior sclerotomal character 4H), (Fig. wild-type of sclerotome posterior the expressedin properly (Fig. 4D) and also equivalently restricted to the posterior sclerotome of wild-type (Fig. 4F) and 4F) (Fig. 2000). al., et Mansouri 2000; al., et (Leitges Uncx4.1 and 2004) al., et and promoted by two different transcription factors, Tbx18 (Bussen , with anterior and posterior somite identity maintained mutants. in localized appropriately are molecules guidance expressed o ifrnit bten hs to osblte, akr of markers possibilities, two these between differentiate To Somite polarity is established during segmentation of the somitic Npn2 uat ie Fg 4) Ehi B, lgn ta repels that ligand a B2, Ephrin 4C). (Fig. mice mutant Tbx18 mutant mice mutant (Fig. 4I) and Sema3f Npn2 was restricted to the anterior sclerotome of wild-type of sclerotome anterior the to restricted was Npn2 Npn2 mutant embryos (Fig. 4E). Thus, two posteriorly mutant mice to determine whether anterior and mutant embryos (Fig. 4G). (Fig. embryos mutant and Npn2 Sema3f Delta1 Sema3f mutant neural tubes, and cultured and tubes, neural mutant Npn2 mutant mice (deAngelis et al., mutants, suggesting that the that suggesting mutants, mutant mice (Fig. 4J). These (Fig. 2) and Npn2 Development 133(1) Uncx4.1 and Sema3f Sema3f Sema3f was also was (Fig. 3) Npn2 Npn2 Npn2 was

DEVELOPMENT visualized usinganti-mouseIgG-Alexa488. cells were labeledwithanti-p75,whilespots(outlinedinwhite)were expressing neural crest cells in the trunk condensed into ganglia in ganglia into condensed trunk the in cells crest neural of expressing streams the E11.5, and E10.5 at embryos manipulated wild-type surgically or 2002) al., (Kalcheim and Teillet, 1989) to contain only anterior character. In et DeBellard 1997; al., non-segmentally through somites that are genetically (deAngelis et individualized ganglia. This is the case when neural crest migrates of instead form to ganglia root dorsal of mass continuous a expect form the dorsal root ganglia. As a result, in each somite, neural crest cells in the anterior sclerotome coalesce to the somites, will form in register with one another. For example, in Npn2/Sema3f intrunkneuralcrest migration the anterior somite. and its ligand Sema3f is required to restrict neural crest migration to wild-type in mice. This demonstrates that signaling between the receptor Npn2 as sclerotome anterior-half the through exclusively than rather sclerotome, posterior and anterior the both through In migration inthetrunk neuralcrestNpn2/Sema3f signalingpatterns of individualized dorsal root ganglia. found that segmental migration was not essential for the formation its and neural crest migration but not for somite Npn2 patterning. Surprisingly, we receptor crest cells, and that Npn2/Sema3f signaling is required for the segmental of trunk. We demonstrate that importance the in development crest the neural during Sema3f ligand repulsive examined have We DISCUSSION each between space with segmentation, sections at E11.5, TUJ1 immunoreactivity confirmed the apparent arrowheads mark the same axial levels 6D, in all (Fig. panels). somite In longitudinal the in crest neural migrating of sheet the from 6B). By E11.5, individualized ganglia appeared to have sorted out the somites, but became excluded from the somite boundaries (Fig. ganglia. At E10.5, neural crest cells were still distributed throughout in cells crest an anterior to posterior progression (Fig. 6A,C). Strikingly, neural substrates ( the Npn2receptor migrateequallywellonfibronectin andSema3f normally avoidimmobilizedSema3f( conjugated withanti-placentalalkalinephosphatase.Neuralcrest cells fibronectin andspottedwithAP-Sema-3Fconditionedmedium embryos were cultured onThermanoxcoverslipscoatedwith to avoidSema3finvitro. Npn2iscellautonomouslyrequired forneuralcrest cells Fig. 5. mutants (Giger et al., 2000). in normal are somites, the ventralsegmentalto pattern a in form which ganglia, The sympathetic was compromised. somewhat separated, suggesting that the process of gangliogenesis occurred but wild-type embryos (Fig. 6E), the mutant ganglia were not as well in those to similar morphologically Although 6F). (Fig. ganglion Npn2 and B , C Sema3f Npn2 ; n =5 spotsinthree separateexperiments).Neuralcrest mutant mice also coalesced into recognizable into coalesced also mice mutant mutant mice, trunk neural crest cells migrate Neural tubesfrom 14to24somitemouse Npn2 A is expressed in migrating neural ), whileneuralcrest cellslacking Npn2 Npn2 mutant dorsal root dorsal mutant mutants, one might Sox10 Npn2 - separated inthemutant.Scalebar:0.1mm. dorsal root gangliahadasimilarappearancebutwere lesswell heterozygous (E)andmutant(F)E11.5embryosstainedwithanti-TUJ1, type, cell a indicates anteroposterior polarity of the somites is indistinguishable in wild- This culture. in Sema3f autonomous requirement for Npn2 in the neural crest. Second, as avoid not do cells foremost, in contrast to wild-type cells, The latter mechanism seems unlikely for several reasons. First and influencing neural crest migration in a thus non-cell-autonomous manner. migrate, cells crest neural which through environment the ligand secreted by cells of the adjacent posterior sclerotome to affect receptors on anterior sclerotomal cells may interact with the Sema3f sclerotome, leading to anterior-only migration. posterior Alternatively,in Npn2 cue repulsive Sema3f a detect cells crest neural on the Npn2 in one or both cell Therefore, types. One possibility is that Npn2 receptors 4A). (Fig. sclerotome migration defects in anterior the of cells as level inallpanels.Scalebars:0.5mm.( arrowheads indicatecondensingdorsalroot gangliaatthesameaxial posteriorly, individualizeddorsalroot gangliasegregate anteriorly. Black embryos, intheabsenceofsegmentalneuralcrest migration and condenseddorsalroot gangliaanteriorly. ( segmental streams ofmigratingneuralcrest cellsintheposteriortrunk, and E11.5( segmental dorsalroot gangliain Non-segmentallymigratingneuralcrest cellsgive riseto Fig. 6. 97, hr nua cet irto hs pseudo-segmental a has migration crest neural where 1997), al., et (deAngelis lost is polarity somite which in mouse, mutant the with contrasts This borders. evensomite 3H), at (Fig. the along all present the in axis anteroposterior are cells crest neural conclusion, this of signaling does not appear to be required in this process. In support Murine Npn2 Npn2 C ) wild-typeembryos,insituhybridizationfor and is expressed by neural crest cells (Fig. 1D-F) as well Sema3f Npn2 mutant embryos (Fig. 4), Npn2/Sema3f 4), (Fig. embryos mutant mutants could reflect a requirement for Npn2 (Fig. 2E,F) and 2E,F) (Fig. Npn2 RESEARCH ARTICLE E , F ) Insectionsof mutant mice. Npn2 B , D ) In mutant neural crest Npn2 Sema3f Npn2 Sox10 In E10.5( mutant mutants Delta1 reveals 103 A )

DEVELOPMENT expressed in anterior or posterior sclerotome or in the neural crest, neural the in or sclerotome posterior or anterior in expressed matrix extracellular are that identified havebeen pairs receptor/ligand molecules,and molecules has adhesion migration cell different crest Many neural observed. first was segmental phenomenon the since field this preoccupied for basis molecular The migration. crest neural trunk pattern to required absolutely the anterior sclerotome is devoid of the repulsive cue. the posterior sclerotome, thus ensuring a sharp boundary such that rather serve as a sink to bind up any repulsive ligand diffusing from is that these receptors do not play a signaling role in the somite, but neural crest and anterior sclerotomal cells. One intriguing possibility patterning trunk neural crest migration, are also distributed on both et al., 1997), both of which have been postulated to play a role in like crest and the sclerotome through which it migrates? Interestingly, in the neural crest. null mice. For these reasons, we favor the idea that Npn2 is required Kalcheim and Teillet, 1989). However, this is not the case in migratory neural crest cells and their of derivativesnumbers (Bussen et reduced al., 2004; to somite leading migration, posteriorized crest neural impedes A sclerotome. the posteriorize most somite wouldprobably receptor in this through involved signaling of were loss patterning, Npn2/Sema3f if cells, sclerotomal in the somites themselves. Third, as This comparison suggests that, unlike sclerotome but avoiding somite boundaries (DeBellard et al., 2002). appearance, with migrating neural crest cells present throughout the 104 Brafman et al., 1999) and Npn1 (Kitsukawa et al., 1997). (Debby- F-spondin include signals such for Candidates root ganglia. dorsal of formation segmental for required are somite the al., 2000). This indicates that anteroposteriorly patterned signals in 2004; Kalcheim and Teillet, 1989; Leitges et al., 2000; Mansouri et abolished by either surgical or genetic manipulation (Bussen et al., is polarity somite anteroposterior when form ganglia root dorsal crest cells within the sclerotome. In favor of this possibility, fused anterior sclerotome, irrespective the of the starting location within of the aggregationneural promote could sclerotome posterior the in cue or a repulsive signal ‘sorting’ a cell-adhesive, either example, restricting signals neural crest migration and the pattern of ganglion aggregation. For independent be may there that is One explanation events. separable are ganglia of sequestration and subsequent migration crest neural segmental that suggests result This mutant mice, segmentally arranged dorsal root ganglia still form. cells migrate through the anterior and posterior sclerotome of of the neural crest migration pattern. Despite the fact that neural crest process crest that results in segmentation neural irrespective in unrecognized previously additional, signaling an uncovered has migration Npn2/Sema3f for requirement The formation required for segmental dorsal root ganglion Segmental neuralcrest migrationmaynotbe anterior-only migration through the sclerotome. patterning keydeterminant the clearly is signaling Npn2/Sema3f ligand receptor/ephrin Npn1/Sema3A, be involved in later steps in the process. However, Eph as such interactions, might fine tune neural crest migration, or signals, in the case of Other . requirement forany moleculebeenpreviously demonstratedinthe a has case no in But 2004). al., et (Kuan migration crest neural and in some cases they have been shown to be sufficient to direct This is the first report of a single receptor/ligand pair that is that pair receptor/ligand single a of report first the is This What is the purpose, then, of Npn2 RESEARCH ARTICLE , Npn1 (Eickholt et al., 1999) and Npn2 Npn2 Delta1 expression in both the neural the both in expression is expressed on anterior Ephb3 , the Npn2 receptors (Krull defect is not Npn2 Npn2 - that primary mechanisms are perturbed. mechanisms forformationofimportant organ systemsinthe event regulative nature of vertebrate embryos, which may have ‘back-up’ of formation the for segmented dorsal root essential ganglia. This may not not be surprising given butthe important, is migration more closely spaced than normal. Thus, the pattern of neural crest mutants, the a uniform sheet of migrating neural havecrest cells appears to could differences morphological restricted impact during gangliogenesis. In the case of segmentally the these of All in the absence of neural crest cells (see Christ et al., 2004). undergo will and 1990) al., et (Goldstein ganglia root dorsal for dense than posterior sclerotome (Christ et al., 2004), is mitogenic cell less is sclerotome Anterior 2004). al., (reviewedet Christ by sclerotome the within subdomains various the and sclerotome, space, as well as von Ebner’s fissure between anterior and posterior within the sclerotome. These include, most notably, the intersomitic differences and boundaries embryological are there posteriorly, as such cues, molecular restricted regionally itself can impose segmentation during gangliogenesis. In addition to play. argues that a combination of these two mechanisms is normally at ganglion (Kalcheim and Teillet, 1989). That they are fused, however, alternating thick and thin regions at random intervals within the giant with pseudo-segmentalappearance, exhibitsa that forms ganglia of mass giant halves,a somite posterior or anterior multiple with stochastic mechanism, when normal somites are this surgically replaced of support In unpublished). Bronner-Fraser, (M. aggregate of segmental neural crest migration simply because neurons tend to dorsal root ganglia eventually segment in the assumed for 20 years (reviewed by Kuan et al., 2004). Although the of a segmented peripheral nervous system, despite what has been crest cells through the somites itself is not requisite for the creation of condensation sympathetic precursors as well and (Kawasaki et al., 2002). localization for required are Sema3A of and formation the for propose we metameric dorsal root ganglia in those to similar be to likely aggregation of these cells into individualized sympathetic ganglia are anteriorly or posteriorly. The mechanisms that ultimately segmentsresult in two the as far as migrate can and position segmental their have passed through the somites, neural crest cells no longer maintain colleagues (Kasemeier-Kulesa et al., 2004) showed that, once they By organization. imaging segmented actively migrating neural their crest cells, Kasemeier-Kulesa for and migration crest neural of streams adjacent normally curtail between this movement as dorsal root ganglia condense. over cross could boundary somite the thus 2004), evenal., (Kasemeier-Kulesaet can and somite crest cells normally maintain filopodial contact across the posterior neural Migrating nervoussystem. peripheral the segmentationof positional information at the somite boundary that impacts upon creates the polarity somite that suggest data Together,these 2000). al., et Mansouri 2000; al., et Leitges 2004; al., et (Bussen form anteroposterior somite polarity is abolished but physical somites in still fused also are ganglia ganglia are fused (Kalcheim and Teillet, 1989). However, dorsal root root dorsal and non-segmentally migrates crest boundary,neural somite no have words other in sclerotome, anterior only contain to modified surgically are embryos chick Interestingly,when 6). segment into individual dorsal root ganglia at the somite border (Fig. Finally, it is also possible that the physical structure of the somite A second possibility is that dorsal root ganglia form in the absence The overriding message is that the segmental migration of neural The sympathetic ganglia also are not dependent upon the pattern Uncx4.1 Npn2 and mutants. Interestingly, Npn1 Tbx18 Npn2 Development 133(1) Sema3f mutants, they are uat, where mutants, expression Npn2

DEVELOPMENT neural crest markers specification, as no differences were noted in the expression of early Npn2 is required for neural crest migration and apparently not for 2002), Bronner-Fraser, and (Gammill crest neural premigratory (Coles et al., 2005). screen, is also important for proper emigration of neural crest cells - that the demonstration by supported is conclusion This development. crest neural demonstrates that our collection of severalgenes contains true regulators of and has profile expression gene crest neural our function validates migration Npn2 of analysis implications. First of all, the importance of Npn2 for neural crest current Our 2002). Bronner-Fraser,and (Gammill induction crest neural to response identified originally We expression profile Functional validationoftheneuralcrest gene Npn2/Sema3f intrunkneuralcrest migration Chen, H.,Bagri,A.,Zupicich,J.,Zou, Y., Stoeckli,E.,Pleasure, S., Chen, H.,Chédotal,A.,He,Z.,Goodman, C.andTessier-Lavigne, M. Bussen, M.,Petry, M.,Schuster-Gossler, K.,Leitges,M.,Gossler, A.and Bronner-Fraser, C. M.andStern, Bronner-Fraser, M. Adams, R.,Betz,H.andPüschel,A. References NS051051. protocol. ThisworkwassupportedbyUSPHSgrantsDE15309and APTES protocol, andChathuraniJayasenaforthesubstratumchoiceassay and IsabelleMiletichforhelpwithneuraltubecultures, AndyEwaldforthe Christian HochstimforadviceonNeuralCrest CompleteMedium,PatWhite the p75 antibody. Leefortipsonimmunostaining, ManythankstoVivian contributing for Reichardt Lou to and plasmids, of gifts kind for Wegner Kispert, PatriciaLabosky, Andreas Püschel,KirstenKuhlbrodt andMichael are gratefultoDrsDavidAnderson,PeterGruss,AhmedMansouri,Andreas on themanuscript,andtoJoaquinGutierrez forexceptionalanimalcare. We LeeandYorkof thiswork.SpecialthankstoVivian Marahrens forcomments Sema3f the providing for Ginty David to indebted are We Bagri, A.andTessier-Lavigne, M. Adams, R.,Diella,F., Hennig,S.,Helmbacher, F., Deutsch,U.andKlein, R. reveal the roles of additional genes in this process. to promises collection gene crest neural our of analysis Further of subset premigratory neural crest a cells (Gammill and in Bronner-Fraser, 2002). potential this activating migrate to signal a with development entails a sequential activation of migratory potential, crest neural early that model our supports This induction. crest neural of consequence a as crest clearly neural are premigratory in expressed migration for crest crucial neural genes in Regardless, organism. Npn2 chick for role levelshigher at and this expressedearlier in is it as specification, early an of possibility dorsal neural tube of the mouse. We cannot, however, rule out the hippocampal mossy fiberprojections. Neuropilin-2 regulates thedevelopment ofselectcranialandsensorynerves and W.,Lowenstein, D.,Skarnes, Chédotal,A.and Tessier-Lavigne, M. 547-559. 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